The Medial Entorhinal Cortex's Role in Temporal
and Working Memory: Characterization of a
Mouse Lacking Synaptic Transmission in Medial
Entorhinal Cortex Layer III
MASSACHUSETTS INSTITUTE
by
Alexander Jay Rivest
Submitted to the Department of Brain and Cognitive Sciencesin partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY IN NEUROSCIENCE
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 2011
@ Massachusetts Institute of Technology 2011. All rights reserved.
Au th or ....................
r.........................................
Department of Brain and Cognitive Sciences
December 13, 2010
C ertified by .....
.. . . . . . .
Susumu Tonegawa, PhD
Picower Professor of Biology and Neuroscience
Thesis Supervisor
..............
Accepted by................
............................
Earl K. Miller, PhD
Picower Professor of Neuroscience
Director, BCS Graduate Program
OF TECHNOLOGY
FEB 0 9 2011
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The Medial Entorhinal Cortex's Role in Temporal and
Working Memory: Characterization of a Mouse Lacking
Synaptic Transmission in Medial Entorhinal Cortex Layer III
by
Alexander Jay Rivest
Submitted to the Department of Brain and Cognitive Sciences
on December 13, 2010, in partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY IN NEUROSCIENCE
Abstract
Declarative memory requires the integration and association of multiple input streams
within the medial temporal lobe. Understanding the role each neuronal circuit and
projection plays in learning and memory is essential to understanding how declarative
and episodic-like memories are formed. This work here addresses the role of the medial
entorhinal cortex layer III (MEC-III) to CA1 projections in episodic-like memory
formation and recall. This circuit is addressed with a triple transgenic mouse which
allows for the expression of tetanus toxin, an enzyme that disrupts synaptic vesicle
fusion, specifically in MEC-III neurons. Utilizing this triple transgenic mouse model,
which allows for the specific and reversible ablation of synaptic transmission only in
medial entorhinal cortex layer III excitatory neurons, the function of this pathway in
various learning and memory tasks is tested.
Synaptic output from the medial entorhinal cortex layer III neurons is necessary
for acquisition, but not recall of tone and contextual fear memories in trace fear conditioning, and not in delay conditioning. This is the first demonstration that acquisition
and recall of the same memory engram do not require the exact same anatomy. Additionally, this pathway is necessary for performance in a delayed nonmatch-to-place
working memory task, in which the animal must utilize memory from the previous
trial to successfully complete the following trial. Both the trace and working memory
paradigm require the integration of information across a delay, which we propose is
supported by known persistent activity in entorhinal neurons. CAl receives input
from both entorhinal layer III and CA3. We show that synaptic transmission from
CA3 is not required for tone fear memory in the trace paradigm and not required
for working memory in the same delayed nonmatch-to-place paradigm, further isolating the necessity for MEC-III inputs in both of these behaviors. Functional MEC-III
synaptic transmission is also necessary for pattern-completion contextual recall in the
pre-exposure contextual fear conditioning paradigm. Contrary to previous literature,
the MEC-II to CAl pathway is not necessary for consolidation of spatial memories
and anatomical tracings using this mouse line demonstrate that the MEC-III projects
to CA1 and not CA3. The MEC-II pathway however, does project via two pathways
to the same target in CA1, the perforant and alvear pathways. The alvear pathway
has not been reported before in mice.
Recent advances in mouse genetic tools have allowed for circuit studies of the
medial temporal lobe. We have used these tools and elucidated some of the specific
circuits involved with temporal and working memory.
Thesis Supervisor: Susumu Tonegawa, PhD
Title: Picower Professor of Biology and Neuroscience
Acknowledgments
Susumu Tonegawa has been my thesis advisor since 2004, and I am very grateful to
him for taking me under his wing and allowing me to develop as a scientist. His
constant curiosity and enthusiasm for understanding the way the brain works is well
matched by an attention to detail and rigor. These attributes are both infectious and
inspirational to those who work with him, and I have been honored to learn from
him. Outside of our common intellectual goals, I have also enjoyed cheering on the
Red Sox to two World Series victories with him.
Drs. Matt Wilson, Chip Quinn and Michael Hasselmo, my thesis committee members, have helped direct my research with invaluable conversations, suggestions and
ideas. I am truly appreciative to them for their guidance.
Dr. Carlos Lois, who
served on my committee early on was also a huge support for my early virus work.
I am grateful to Dr. Nancy Kanwisher for allowing me to rotate in her lab to attempt my "pattern completion" experiment in collaboration with Dr. Anthony Wagner. I have enjoyed my conversations with her and learned a lot about science and
the world outside of science.
I am grateful to Dr. Elly Nedivi for my rotation time in her lab, and for her
guidance early on in my graduate career. Her faith in me early on gave me a lot of
confidence to think creatively and to work hard at what I was interested in.
Dr. Shona Chattarji, who accepted me into his lab in India during my visit for
the MIT-India program and became a true friend in the process. I have appreciated
his guidance and friendship throughout my time as a graduate student.
The Tonegawa lab is a huge place, with many moving parts and even more people.
Without exception, every single person in the lab has enriched my studies and life
over the past 6 years. It has been fun to grow with this family, to share science and
ideas, but also to share births, birthdays, departures, life transitions and frustrations.
Arvind, thank you for your friendship, for your constant scientific optimism, and
for being the single person in this lab who kept me grounded at all times. Your
attention to details and the big picture at the same time, in science and every other
topic we discussed has helped shape not only my science, but the way I look at life.
Jung, it has been an honor and privilege to work with you. I can think of no
better person to collaborate and share ideas with. You have taught me a lot about
how to conduct research properly and how to present the work properly. Thank you
too for entertaining all of my crazy experimental ideas, and for challenging me at the
right points. Working with you has made this process enjoyable and intellectually
stimulating.
Gishnu, it has been fun laughing and eating keema with you.
Bridget, Emily, Josh, we as the more senior graduate students, have grown a lot
together. Since forming our graduate student group, it has been fun to push each
other scientifically. I have learned a lot and gained an enormous amount from you
all.
Emily, you and I have been bay-mates for the past 5 years and it has been fun
growing and learning with you. Through all of the UROPs, the scientific failures and
successes, and the conversations about life and politics, we have still survived.
Bridget, thank you for your friendship and support.
I think both of us have
kept each other on track, in both our lab endeavors and life endeavors. It has been
fun sharing perspectives, policies, advice and life with you. Thank you pushing me,
inspiring me, and helping me edit this document.
Toshi, I have greatly appreciated your help and advice and am very appreciative
of your hard work in making the triple transgenic model.
I am very appreciative to Carl, Madeline, and Carrie for their help with behavior
and technical help. Thank you!
Jayson, thanks for helping keep me sane and for being a great friend. Candy, your
friendship and your support in all of my endeavors has been amazing. Wenjiang and
Xioaning, thank you too.
My family, Chris, Jessy, Gail and Ron Rivest. You are my intellectual inspirations.
The fearlessness in you all allows me to dream. The knowledge that even if I fail, I
have already succeeded is the most comforting and inspiring base to have.
Chris and Jessy, I have appreciated the ways you challenge me, and am inspired
by the way you think about science. You both see science as a way to learn about
the world, and to use that knowledge to engineer a better life in that world. If we
had more people like you, we would all be a lot happier and healthier.
Mom and Dad, you have taught me that the world is what I make it. On every
intellectual and personal pursuit I have only had support from you. You have taught
me the two greatest lessons of all: that understanding and exploring life is an endless
and exciting endeavor and that the greatest joy is in being able to enable the dreams of
others. You have have given me amazing opportunities and allowed me to understand
how rare those opportunities are.
Elizabeth, my girlfriend, who I fell in love with two years ago, and who has
accepted and supported the dreams I have. I have enjoyed dreaming new dreams
with you.
I am amazingly thankful for the light you shine, when it is bright or
cloudy. You inspire creativity and boldness in me.
I would like to thank the kids in the Rwaza and Rebero orphanages in Rwanda,
and the kids in Asebu, Ghana. You have made me smile and appreciate that engaging
in scientific pursuits is a luxury. One that I will never take for granted.
Finally, I would like to thank the over 1000 mice that participated in this study.
This thesis is dedicated to my parents, Gail and Ron Rivest. You have taught me to
lovingly explore the world and to never stop asking questions.
Contents
20
1 Introduction
1.1
Contributions of This Thesis.
. . . . . . . . . . . . . . . . . . . . . .
20
1.2
Organization of Thesis . . . . . . . . . . . . . . . . . . . . . . . . . .
21
23
2 Background
2.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
24
2.2
Types of M emory . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
2.3
Episodic Memory and the Medial Temporal Lobe . . . . . . . . . . .
26
2.4
Episodic Memory in Non-Human Animals
. . . . . . . . . . . . . . .
28
2.4.1
Episodic Memory in Non-Primate Animals . . . . . . . . . . .
29
2.4.2
Episodic Memory in Rodents
. . . . . . . . . . . . . . . . . .
30
Circuits and Anatomy of Episodic Memory . . . . . . . . . . . . . . .
33
2.5
2.5.1
What is the Medial Temporal Lobe?
. . . . . . . . . . . . . .
33
2.5.2
Postrhinal and Perirhinal . . . . . . . . . . . . . . . . . . . . .
34
2.5.3
Postrhinal and Perirhinal Connections to the Entorhinal Cortex
35
2.5.4
Functional Implications Regarding Perirhinal - Lateral Entorhinal Cortex and Postrhinal - Medial Entorhinal Cortex Connec-
2.5.5
tions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
36
. . . . . . . . .
37
Entorhinal Connectivity to the Hippocampus
2.6
Human Hippocampal Complex versus Parahippocampal Corticies
40
2.7
Genetic Dissection of the Medial Temporal Lobe . . . . . . . . . . . .
41
2.7.1
CA1 Manipulations . . . . . . . . . . . . . . . . . . . . . . . .
43
2.7.2
CA3 Manipulations . . . . . . . . . . . . . . . . . . . . . . . .
43
2.7.3
Dentate Gyrus Maniplations . . . . . . . . . . . . . . . . . ..
44
2.8
Specific Lesions of Neuronal Output . . . . . . . . . . . . . . . . . . .
45
2.9
Medial Entorhinal Cortex Layer III Tetanus Toxin Mouse . . . . . . .
49
2.10 Entorhinal Cortex: What is Known about the Entorhinal Cortex . . .
49
2.10.1 Place Cells and Grid Cells . . . . . . . . . . . . . . . . . . . .
50
2.10.2 Entorhinal Cellular Physiology . . . . . . . . . . . . . . . . . .
53
2.10.3 Persistent Activity Blockage and Behavior . . . . . . . . . . .
55
2.10.4 Episodic Memory, Alzheimer's Disease and the Entorhinal Cortex 56
2.11 Animal Lesions of the Entorhinal Cortex . . . . . . . . . . . . . . . .
57
2.11.1 Hippocampal Physiology after Entorhinal Lesion . . . . . . . .
58
2.11.2 Spatial Learning in Rodents after Entorhinal Lesions . . . . .
58
2.11.3 Delayed Non-Match-to-Place Learning and Radial Arm Maze
learning in Rodents after Entorhinal Lesions . . . . . . . . . .
59
2.11.4 Hippocampally Dependent Trace in Entorhinal Cortex Lesions
60
2.11.5 Fear Conditioning . . . . . . . . . . . . . . . . . . . . . . . . .
61
2.11.6 Lesion of Temporoammonic Pathway . . . . . . . . . . . . . .
63
. . . . . . . . . . . . . . . . .
64
2.11.8 Genetic Manipulations of the EC . . . . . . . . . . . . . . . .
64
2.11.7 Conclusion About Lesion Data
2.12 Role of Medial Entorhinal Cortex Layer III in Learning and Memory
2.12.1 MEC-II Cre+ Mouse . . . . . . . . . . . . . . . . . . . . . . .
66
2.12.2 Synaptic Transmission of MEC-TeTx mice . . . . . . . . . . .
71
. . . . . . . . . . . . . . . .
71
2.12.3 Using the MEC-II TeTx mouse.
3
66
Medial Entorhinal Cortex Layer III: Characterization of Projections 75
3.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76
3.2
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
3.2.1
Projections into the Hippocampus . . . . . . . . . . . . . . . .
78
3.2.2
Alvear Pathway . . . . . . . . . . . . . . . . . . . . . . . . . .
79
D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
82
3.3
3.3.1
Medial Entorhinal Cortex Layer III projections to CA1 in Mice. 82
3.4
3.3.2
Alvear Pathway in Mice. . . . . . . . . . . . . . . . . . . . . .
83
3.3.3
Dissecting the Pathways . . . . . . . . . . . . . . . . . . . . .
83
M ethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
85
3.4.1
M ice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
85
3.4.2
Surgical Procedure . . . . . . . . . . . . . . . . . . . . . . . .
85
3.4.3
Fixation, Sectioning and Imaging . . . . . . . . . . . . . . . .
85
3.4.4
Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
86
4 The Role of Medial Entorhinal Cortex in Temporal and Working
87
Memory
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
88
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
90
4.2.1
Delay and Trace Fear Conditioning . . . . . . . . . . . . . . .
90
4.2.2
Analysis of 20 s Trace Deficit. MEC-III Output Necessary for
4.1
Persistent Activity
4.2
Behavior Results
Acquisition, Recall or Both? . . . . . . . . . . . . . . . . . . .
92
Results: Control Fear Conditioning Paradigms . . . . . . . . .
100
Working Memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110
4.3.1
Delayed Nonmatch-To-Place . . . . . . . . . . . . . . . . . . .
110
4.3.2
8-Arm Radial Maze Task . . . . . . . . . . . . . . . . . . . . . 111
4.3.3
Anxiety and Pain Sensitivity . . . . . . . . . . . . . . . . . . .
114
4.2.3
4.3
4.4
CA3 Functional Output: 20 s Trace and DNMP T-Maze
. . . . . . .
117
4.5
D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
4.5.1
Medial Entorhinal Cortex is Required for Acquisition of Tone
Memory in the 20 s Trace Paradigm . . . . . . . . . . . . . . .
4.5.2
Medial Entorhinal Cortex is Required for Pattern Completion
. . . . . . . . . . . . .
130
4.5.3
Working Memory . . . . . . . . . . . . . . . . . . . . . . . . .
133
4.5.4
Difficulty of Memory . . . . . . . . . . . . . . . . . . . . . . .
134
M ethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
138
DNMP T-Maze . . . . . . . . . . . . . . . . . . . . . . . . . .
138
of Context in the 20 s Trace Paradigm
4.6
124
4.6.1
4.6.2
Fear Conditioning . . . . . . . . . . . . . . . . . . . . . . . . .
139
4.6.3
Trace and Delay Conditioning Protocols
. . . . . . . . . . . .
140
4.6.4
8-Arm Radial Maze Task . . . . . . . . . . . . . . . . . . . . .
143
4.6.5
Hot-Plate
4.6.6
Elevated Plus Maze . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . ..
144
144
5 The Role of Medial Entorhinal Cortex in Spatial Learning, Memory
146
and Consolidation
5.1
Spatial Learning . . . . . . . . . . . . . . . . . . . . . . .. .
. .
147
5.2
Consolidation of Spatial Memories . . . . . . . . . . . . . . . . . . . .
148
5.3
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .
-.
.. .
5.3.1
Spatial Learning in the Watermaze
. . . . . . . . . . . . . . .
149
5.3.2
Morris Watermaze MWM: One-Trial Per Day Results . . . . .
152
5.3.3
Barnes Maze Result . . . . . . . . . . . . . . . . . . . . . . . .
153
5.3.4
Medial Entorhinal Cortex Layer III Output is Not Necessary
for Consolidation of Spatial Learning . . . . . . . . . . . . . .
5.3.5
5.4
5.5
149
154
Medial Entorhinal Cortex Layer III and Consolidation of Contextual or Tone Memory . . . . . . . . . . . . . . . . . . . . .
155
D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
156
5.4.1
Spatial Learning
. . . . . . . . . . . . . . . . . . . . . . . . .
156
5.4.2
Spatial Memory Consolidation . . . . . . . . . . . . . . . . . .
157
M ethods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
159
5.5.1
Morris Watermaze MWM: Consolidation . . . . . . . . . . . .
159
5.5.2
Morris Watermaze (MWM): Reversal . . . . . . . . . . . . . .
160
5.5.3
Fear Conditioning Consolidation . . . . . . . . . . . . . . . . .
160
5.5.4
One-Trial Per Day
. . . . . . . . . . . . . . . . . . . . . . . .
162
5.5.5
Barnes Maze
. . . . . . . . . . . . . . . . . . . . . . . . . . .
162
6 The Role of Medial Entorhinal Cortex Pattern Completion
171
6.1
Pattern Completion . . . . . . . . . . . . . . . . . . . . . . . . . . . .
171
6.2
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
173
6.3
6.2.1
Pre-exposure Paradigm . . . . . . .1
6.2.2
Immediate Shock Paradigm
. . . . . 173
. . . . . . . . . . . . . . . . . . . 175
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
6.3.1
Pattern Completion . . . . . . . . . . . . . . . . . . . . . . . . 175
6.3.2
Other Assessments of Pattern Completion . . . . . . . . . . . 177
6.4 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
6.4.1
.
179
Pre-exposure and Immediate Shock . . . . . . . . . . . . . . . 179
.1
Appendix A : 1-shock Delay Fear Conditioning . . . . . . . . . . . . . 181
.2
1-Shock Delay Conditioning . . . . . . . . . . . . . . . . . . . . . . . 181
.2.1
1-shock Delay Task . . . . . . . . . . . . . . . . . . . . . . . . 181
.2.2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
.3
Appendix B: Control Behaviors . . . . . . . . . . . . . . . . . . . . . 184
.4
Rota-Rod . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
.5
Open Field
.6
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
.6.1
Rota-Rod
.6.2
Open-Field
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
. . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
List of Figures
2-1
Medial Temporal Lobe Anatomy. ........................
2-2
Schematic of Perforant and Alver Pathways From Entorhinal
. 39
Cortex Layer III............................
2-3
34
Schematic of Triple Transgenic Approach to Silence Neuronal
47
Transmission in Specific Cell Types.................
50
........................
2-4
MEC-III TeTx mouse. .
2-5
Depiction of Place Cell and Grid Cell Firing Patterns in Space. 52
2-6
OxR1-Cre+/Rosa Expression shows Specific Expression in Me.
dial Entorhinal Cortex........................
2-7
Cre+ Expression and CA1 Retrograde Injection Demonstrates
Cre+ cells are Layer III.
2-8
......
68
.......................
Cre+ expression and CA1/DG Retrograde Injection Demon69
strates Cre+ cells are not Layer II... .....................
2-9
MEC-III Immunohistochemistry: Cre+ cells are Neurons but
not Inhibitory Neurons.
. 70
......................
72
2-10 Inhibition of Synaptic Transmission in MEC-III TeTx Mouse
2-11 Inhibition of Synaptic Transmission Profile
3-1
67
73
...........
Projections to the Hippocampus from MEC-III: Double Virus
A pproach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
...................
3-2
Alvear and Perforant Pathway ......
4-1
MEC-III Output is Necessary for Tone Memory in Trace but
not in Delay Conditioning. ............................
.
80
81
93
4-2
MEC-III Output is Necessary for Context Memory in Trace
94
but not Delay Conditioning. ......................
4-3
MEC-III is Necessary for Acquisition of 20 s Trace Conditioning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
4-4
MEC-III TeTx Mouse 20 s Trace/i-Month experiments with
Dox manipulations.
4-5
. 101
..............................
MEC-II TeTX mouse and Backwards Trace Conditioning. . 103
4-7 ECIII-TeTX mouse and 40 s Trace Conditioning .
4-8
105
........
MEC-III is not Required for Association of Scrambled CS-US
107
Pairings. ........................................
4-9
99
Contextual Fear Conditioning is not Disrupted in MEC-III
TeTx M ice.
4-6
.
.........................
MEC-III TeTX mouse and Reaction to Tone Alone ......
.109
4-10 MEC-III TeTx mice are Impaired in Rewarded DNMP T-Maze.112
4-11 MEC-III TeTx mice not Impaired in the 8-arm Radial Maze
Task.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4-12 MEC-III TeTx mice do not have Elevated Anxiety in Elevated
P lus M aze... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4-13 MEC-II do not Show Altered Pain Sensitivity: Hot Plate. . 117
4-14 CA3-TeTx Mouse is Normal in Tone but is Impaired in Contextual Learning in 20 s Trace Paradigm. .
............
119
4-15 CA3-TeTx Mouse Not Impaired Delay Conditioning...... .. 121
4-16 CA3-TeTx T-Maze: Rewarded DNMP ..............
123
4-17 Tone Memory Acquisition in Delay and Trace Conditioning.
128
4-18 Trace Conditioning: Tone Memory Acquisition and Recall in
MEC-II TeTx and CA3-TeTx Mice. ................
129
4-19 Pattern Completion in Trace Conditioning. ..............
132
4-20 Strength of Tone-Footshock Pairings and Hippocampal Dependency. .
................................
136
5-1
Watermaze Learning and Reversal Training. Quadrant Occu.. 165
pancy and Platform Crossings on Probe Trials..........
5-2
Trial-to-Trial Savings on Day 1 of Watermaze Reversal . ...
5-3 Watermaze: One Trial per Day .......................
..................
166
167
168
5-4
Spatial Learning: Barnes Maze. .
5-5
Spatial Memory Consolidation ...................
169
5-6
Fear Conditioning: Consolidation ....................
170
6-1
MEC-III TeTx mice are impaired with 10 s recall in PreExposure Paradigm. ................................
6-2
MEC-III TeTx mice Similar to Controls in Immediate-shock
Paradigm. .......................................
-3
174
176
MEC-III TeTx mice are impaired in 1-Shock Delay Conditioning. .........................................
183
-4
Rota-Rod Learning. ................................
184
-5
Open-Field Total Distance ......................
186
-6
Open-Field Rearing. ................................
187
List of Tables
4.1
MEC-Ill in Delay and 20 s Trace Conditioning . . . . . . . . . . . . .
92
4.2
Dox Induction Paradigms and 20 s Trace Fear Conditioning
. . . . .
100
4.3
Fear Conditioning Paradigms and Results
. . . . . . . . . . . . . . .
108
4.4
CA3 vs. MEC-III in Delay and 20 s Trace Conditioning . . . . . . . .
120
4.5
CA3 vs. MEC-III in Delay and 20 s Trace Conditioning . . . . . . . .
127
In this thesis, the novel work presented in the "Background" chapter is from
experiments completed by Dr. Junghyup Suh. All of the experiments presented
after the "Background" chapter were completed by or under direct supervision of
Alexander J. Rivest.
List of Abbreviations Part 1
AAV8
Adeno-associated virus serotype 8
AP5
2-amino-5-phosphonopropionic acid
AP
Alvear Pathway
BA
Basal Amygdala
#-Gal
#-Galactosidase
CA
Constitutively Active
CA1
Cornu Ammonis Area 1
CA2
Cornu Ammonis Area 2
CA3
Cornu Ammonis Area 3
CA3-Cre+
CA3 Cre+ Mouse (Control)
CA3-TeTx
CA3 Tetanus Toxin Expressing Mouse (Mutant)
CaMKII
Ca2+/calmodulin-dependent protein kinase II
CCh
Carbachol
ChR2
Channelrhodopsin-2
CMV
Human cytomegalovirus promoter
Cre
Cre-recombinase
Cre+
Cre Expressing
CTB
Cholera Toxin subunit b
DAPI
4',6-diamidino-2-phenylindole
DG
Dentate Gurys
DiI
DiICi 8 (3) 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
DiO
DiOC 1 (3) 3,3'-dioctadecyloxacarbocyanine perchlorate
DIO
Doublefloxed inverted open-reading-frame, Cre-dependent
DMP
Delayed Match-to-Place
DNMP
Delayed Nonmatch-to-Place
Dox
Doxycycline
EC
Entorhinal Cortex
EYFP
Enhanced Yellow Fluorescent Protein
List of Abbreviations Part 2
GAD67
Glutamate decarboxylase isoform
ISCFC
Immediate-shock Contextual Fear Conditioning
LA
Lateral Amygdala
LEA
Lateral Entorhinal Area
LEC
Lateral Entorhinal Cortex
LEC-III
Lateral Entorhinal Cortex Layer III
LTP
Long-term potentiation
mCherry
Monomeric red fluorescent protein
MEA
Medial Entorhinal Area
MEC
Medial Entorhinal Cortex
MEC-I
Medial Entorhinal Cortex Layer II
MEC-III
Medial Entorhinal Cortex Layer III
MEC-III Cre+
MEC-III Cre+ Mouse (Control)
MEC-III TeTx
MEC-III Tetanus Toxin Expressing Mouse (Mutant)
MEC-V
Medial Entorhinal Cortex Layer V
MTL
Medial Temporal Lobe
MWM
Morris Water Maze
NMDA
N-methyl-D-aspartate
NR1
N-methyl-D-aspartate Receptor Subunit 1
Oxrl
Oxidation resistance protein 1
PECFC
Pre-Exposure Contextual Fear Conditioning
PP
Perforant Pathway
SLM
Stratum Lacunosum Moleculare
SR
Stratum Radiatum
So
Stratum Oriens
Sub
Subiculum
TeTx
Tetanus Toxin
tTA
Tetracycline-controlled Trans-Activator protein
VAMP-2
I
Vesicle-associated membrane protein 2
Chapter 1
Introduction
Declarative memory, the type of memory that we humans can consciously recall requires the medial temporal lobe (MTL). Understanding how we form memories requires understanding the cellular properties that allow for the integration of information and understanding of the anatomical circuitry supporting the different kinds
of information. All mammals share similar MTL anatomy, and non-human animals
demonstrate features of declarative memory. Thus, we can start to scratch the surface
of how we form memories by understanding first how lower species process memories,
and by understanding the specific roles of brain regions and projections in animals.
This thesis describes a very specific pathway within the medial temporal lobe in
mouse: the medial entorhinal cortex Layer III (MEC-III) projection to CA1 in the
hippocampus. Using an advanced transgenic mouse model in which we can specifically
and reversibly turn off and on synaptic transmission in the MEC-III neurons, this
thesis describes the role of this pathway in memory formation and recall.
1.1
Contributions of This Thesis
The work in this thesis specifically outlines the anatomical connections of MEGIII and describes the specific role of this pathway in specific episodic-like memory
paradigms.
"
We demonstrate that MEC-III does not project to CA3 in mouse, as has been
suggested in the literature, and report for the first time, the presence of the
alvear pathway in mice.
" We demonstrate the necessity of MEC-II synaptic output in trace but not
delay conditioning. Further, we demonstrate that MEC-1II output is needed
ONLY in during trace acquisition, and not during recall. This result shows that
acquisition and recall of the same memory trace are supported by two different
neuronal circuits.
" We demonstrate that CA3 output, which synapses on the same target as the
MEC-III outputs, is not necessary for tone memory in trace conditioning, further specifying the role of MEC-III in this behavior.
" We demonstrate that MEC-II output is necessary for working memory in the
delayed nonmatch-to-place T-maze task and that CA3 output is not necessary
for this task.
" We demonstrate that MEC-III output is not necessary for consolidation of spatial memories, contrary to suggestions in the literature
" We demonstrate that the MEC-III output is necessary for pattern completion
in the pre-exposure contextual fear conditioning paradigm
1.2
Organization of Thesis
Chapter 2 gives the background literature that provides a historical and practical
framework for studying MTL circuitry in order to understand episodic memory formation. This section details the complications with lesion and pharmacological manipulations for answering specific behavioral questions, and outlines newer genetic
techniques that allow for more precise lesions. Finally, this chapter introduces and
describes the triple transgenic mouse used in this study.
Chapter 3 of this thesis defines the precise anatomical connectivity from the MEGIII. Using Cre-dependent viruses in combination with the MEC-III Cre+ mice, we
label axon projections from the MEC-III. This chapter challenges existing literature
about mouse MTL anatomy and reveals a pathway hitherto un-described in mice.
Chapter 4 describes the cellular properties of entorhinal neurons that may underlie
working memory or temporal memory tasks. In this chapter, the MEC-III TeTx mice
are run in temporal and non-temporal memory fear conditioning tasks (delay and
trace). MEC-III TeTx mice are disrupted in trace conditioning, but not in delay. This
chapter then describes the delay nonmatch-to-place working memory T-maze task,
and shows that the MEC-III TeTx mice are impaired in this task. To further specify
the MTL circuitry involved in these behaviors, the CA3-TeTX mouse's learning and
memory are tested in these same behaviors. The discussion outlines a framework for
tone fear memory acquisition and recall in the MTL. For contextual memory deficits
in the trace paradigm, we discuss a model for post-tone pattern completion during
acquisition.
Chapter 5 describes the role of MEC-IlI projections in spatial memory tasks,
and specifically tests the hypothesis proposed in the literature that this pathway is
necessary for spatial memory consolidation.
Chapter 6 describes the MEC-III TeTX mice in the pre-exposure, pattern-completion
version of contextual fear conditioning.
Appendix A describes the MEC-IlI TeTx mice in a 1-shock delay conditioning
paradigm. This experiment is considered in the discussion section of chapter 4.
Appendix B describes the MEC-Ill TeTx mice in the open field and rota-rod
control behaviors.
Chapter 2
Background
This purpose of this chapter is to provide the context of why circuit studies in mice
are interesting and important for understanding brain function. This chapter begins
by discussing the history of episodic memory research and describes that there are
brain regions dedicated to episodic memory processing that are conserved among all
mammals. We outline the methods and principles for testing episodic-like memory in
non-human animals and the specific circuitries participating in these memories. Our
goal in this discussion is to demonstrate that mammals share similar structures and
functions, and that studying one species gives insight into other species.
We then discuss modern molecular techniques that allow for region specific manipulations within the MTL and describe what these models have taught us about
memory formation. We describe the triple transgenic mouse model approach that
allows for cell-specific inhibition of synaptic transmission. This technology is applied
to our MEC-II specific mosue.
To lay the foundations for what is known about
entorhinal cellular and systems function, cellular physiology and lesion studies are
described in detail. Finally, we discuss the details of the MEC-II TeTx mouse used
for study in this thesis.
2.1
Introduction
Localizing and naming brain structures.
As humans, we have the unique abil-
ity to ask questions about how we ask questions, how we make plans, and how we
form memories. Understanding the precise nature of how our brain operates requires
an understanding of how brain regions connect and how the cells in each brain region process information. The first attempts to start systematically disassembling
the brain into definable structures began in the early 1900's when the German neurologist Korbinian Brodmann (Brodmann and translated by Laurence Garey 2005)
studied the cytoarchitecture of the brain and named the various regions. This set
a common language by which neuroscientists could discuss and probe the nature of
these brain regions. From physical lesions of brain regions in the 20th century, to the
current capabilities of modern neuroscience where technology allows for moleculespecific, temporally-controlled and spatially-specific manipulation in the brain, the
goal of understanding the contribution of each cell in our brain function remains the
same.
The ability of the mammalian brain to store information for later usage, as a
memory, allows for adapting to a specific environment, planning of future actions,
understanding probabilistic outcomes of everyday action, knowing what to avoid and
what to do to get rewarded, and, with humans, reminiscing and sharing events as
part of a social interaction. Broadly, two types of memories exist. First, "declarative" memories define what we can consciously recollect, such as what we consumed
for dinner last night. Second, "non-declarative" memories reflect a learned skill, that
we cannot explain but can perform, such as how to ride a bike. Due to the identification of brain regions specifically involved in the processing and storage of declarative
memories, we know more about declarative memories. These brain regions comprise
the structure known as the medial temporal lobe (MTL). Understanding the connectivity and functional role of the substructures within the MTL allows for a framework
to understand the formation and recall of declarative memories.
2.2
Types of Memory
Declarative and non-declarative memories.
Humans utilize two types of mem-
ory: "Declarative" memory and "non-declarative" memory (Squire et al. 1993) (Schacter et al. 1993). Non-declarative memories refer to the types of memories that cannot
be available for conscious recall. Priming, the condition in which a stimulus can influence the behavioral responses to subsequent stimuli, and procedural memory, also
known as motor skill learning, both reflect types of non-declarative memories. Nondeclarative memories guide behaviors and responses to the environment, but one
cannot explain them consciously. On the other hand, declarative memory refers to
memory available for conscious recall, such as recalling one's name, or describing the
previous evening's dinner party (Squire et al. 2004). Three distinct categories of
declarative memory exist. First, semantic memory, which represents fact knowledge
which may have no personal context or meaning. Knowledge of a date such as 79
A.D. when Mount Vesuvius erupted represents a fact that has no personal involvement (referred to as knowledge). Second, episodic memory, which represents a unique
personal experience. Knowledge of yesterday as one's birthday, and remembering the
details of the dinner party represent the fact that the event occurred (we know) and
personal details of what happened (referred to as remembering).
Third, working
memory, which represents information held consciously online for the length of time
needed to perform an action (Dickerson and Eichembaum 2010).
Remembering a
phone number for only as long as needed to dial represents working memory.
Episodic memory and time.
Endel Tulving describes episodic memory as the
memory system that allows for "mental time travel" (Tulving 2002). Episodic memory requires a sense of subjective time, autonoetic awareness (the ability to place
yourself in the subjective time of the event/place) and a sense of "self." To form a
proper episodic memory, an organism must associate the various features of the environment with its own presence in that environment. The organism's sensory neurons
processes these features and further downstream processing incorporates the element
of time as an additional feature. The representation of time in an episode stems from
the organism's perception of the event's time with respect to the age of the organism,
or from periods between stimuli within the event itself. The ability to describe the
event's features, to know the time in which it happened and to describe the context
in which the event took place are are generally described as the "what", "when" and
"where," of an episode. Best described by Tulving, episodic memory:
"receives and stores information about temporally dated episodes or
events, and temporal-spatial relationships among these events" (Tulving
1972).
Comparative studies.
Non-declarative memory systems represent a basic kind of
neuronal learning that many organisms share, as it is always evolutionary beneficial to be able to adapt to a local environment. Neurons and brain regions thus all
participate in this type of learning and memory. Thus no focal disruptions disrupt
non-declarative memory wholly, but rather disrupt non-declarative memory of that
lesioned system. Declarative memory systems, however, arise out of particular conserved brain structures, which all mammals share. Humans best utilized this system
for many environments by building on top of mechanisms and circuitry shared with
other mammals. Understanding the evolutionarily conserved mechanisms and circuitry in mice can help to better understand how we humans process memories, and
determining the differences among species can help to understand why we are who
we are as a species.
2.3
Episodic Memory and the Medial Temporal
Lobe
In this section, we see that declarative memory requires structures within the MTL
and is divided into semantic and episodic memory. This section provide evidence for
where these memories are stored and describes how they interact.
H.M. and episodic memory.
The importance of the medial temporal lobe (MTL)
in declarative memory became clear in the late 1950's when William Scoville and
Brenda Milner described the behavioral effects of hippocampal lesions in humans (Scoville and Milner 1957) (Milner 1972).
In the 1957 landmark paper, they described
ten human case studies in which various lesions to the MTL had varying effects on
memory. The most severe memory impairment came from the patient H.M. To prevent seizures, doctors removed large sections of H.M's hippocampus and surrounding
corticies. Scoville and Milner discovered H.M.'s inability to form new declarative
memories (anterograde amnesia) and his inability to remember events that occurred
three years prior to his surgery (retrograde amnesia). Surprisingly, H.M. could very
clearly remember events and facts from his childhood (Scoville and Milner 1957).
This suggested the necessity of the hippocampal region and surrounding tissue in the
formation of new declarative memories as well as for the recall of somewhat recent
memories, but that over time, memories moved to other brain regions. Other patients
with bi-lateral lesions showed similar deficits in memory formation and the extent of
the lesion within the MTL correlated with the level of memory impairment (Scoville
and Milner 1957).
Human MTL lesions.
This discovery sparked research aimed at understanding the
relevant circuits and responsible brain regions for formation and recall of declarative
memory. Studies on human patients with more focal hippocampal lesions allowed for
the first refinement of MTL function. Patient R.B. (Zola-Morgan et al. 1986) and
patients G.D, L.M, and W.H. (Rempel-Clower et al. 1996) were described as having
damage specific to CA1 or other hippocampus-specific regions bi-laterally. These
patients all showed anterograde memory impairment and some extended retrograde
impairment. These more specific lesions helped localize the more precise structures
necessary for acquisition of new declarative memories, as well as recall of recent
declarative memories. This began the process of using humans with finer and finer
lesions to understand the exact brain regions necessary for memory formation and
recall.
Episodic and semantic memory
The lesions in these patients had effects on
both episodic and semantic memory. This suggests a role of the MTL in declarative
memory, but does not distinguish between semantic and episodic memory. The first
true separation of semantic and episodic memory came from young children with
selective bi-lateral hippocampal damage (Vargha-Kadem et al. 1997). These children
suffered the lesions to their hippocami at birth, at age four and at age nine respectively
and could not demonstrate short term episodic memories. These children, however,
went to school and could obtain factual (semantic) knowledge. The authors suggest
that the hippocampus supports episodic memory and that the surrounding cortical
MTL areas could support semantic learning. Processed information culminates in
hippocampal processing before passage back to the cortex. Thus, the hippocampus
builds episodic memory representations upon the semantic knowledge represented in
the surrounding cortical areas (Tulving and Markowitsch 1998).
Autonoetic Consciousness.
Humans uniquely demonstrate episodic memory, in
part because of the "autonoetic consciousness" necessary to place oneself in the context of the unique experience (Tulving and Markowitsch 1998). To date, there no
current understanding exists of what consciousness would look like in non-human
animals, or if it can ever be tested (Griffiths et al. 1999).
2.4
Episodic Memory in Non-Human Animals
This section explains how the MTL is necessary for episodic-like memory in nonhuman mammals. We see how various forms of episodic-like memories are tested in
birds and rodents.
Monkey MTL lesions.
The discovery of episodic memory deficits in human pa-
tients with MTL damage allowed for investigation into other mammalian species based
on the notion that conservation of basic structure among different species could indicate similar function. Similar to the human findings, lesions in the MTL of rhesus
monkeys showed deficits in new episodic learning (anterograde amnesia), in recall of
pre-operative memories (retrograde amnesia) (Correll and Scoville 1965a), in delayed
match (Correll and Scoville 1965b) and in delayed alternation tasks
(Correll and
Scoville 1967). These studies were followed by a series of more specific MTL lesions
in the 1980's, in which specific hippocampal damage or perirhinal/parahippocampal
damage produced profound memory impairments (Zola-Morgan et al. 1989) (ZolaMorgan et al. 1989) respectively. MTL lesion studies in non-human primates share
the characteristic impairments of human amnesia such as enduring memory impairment, spared immediate memory, selective loss of declarative memory, and memory
impairment exacerbated by distracting stimuli (Zola-Morgan and Squire 1990). In
addition, the severity of the memory impairment directly correlates with the extent
of MTL damage (Zola-Morgan et al. 1994).
The similarities between human and
non-human primates support the argument that the MTL supports similar memory
systems in multiple mammalian species. While these studies do not address the addition of consciousness to an episodic memory, they do address episodic-like memory
of the declarative memory system.
2.4.1
Episodic Memory in Non-Primate Animals
If episodic memory not only contains the "what," "where," and "when," but also
contains the notion of the "self' within an episode, then non-human episodic memory
remains untestable. The lack of self-reporting of the episode from the animal simply
does not allow for a complete assay of episodic memory in animals. Testing episodiclike memory, however, allows the usage of non-human animals to probe this memory
system.
Scrub jays and episodic-like memory.
Studies in scrub jays provided the first
demonstration that non-human animals could combine the "what", "where" and
"when" of an episode (Clayton and Dickinson 1998).
Scrub jays, in this experi-
ment, learned the location (where) of cached peanuts or worms (what). The birds
could only retrieve these rewards after some time passed (when). Scrub jays prefer
wax worms to peanuts, but long delay periods affect the quality of the worms and
not the peanuts, so if too much time has passed, the jays prefer the peanuts to the
degraded worm. In one group of jays, the "Replenish" group, training included conditions where the experimenter inserted new worms in the place of degraded worms
after long time intervals. These birds never encountered a rotten worm. In the other
group, the "Degrade" group, the experimenter allowed the worm to rot.
Birds in both groups learned the location of a worm and a peanut (covered in
sand), and testing began a few hours after. Probe trials remove the reward (and thus
any potential odor cues etc..) and test the animals memory for where the reward was
contained. In a probe trial, 4 hours after learning, both groups dug in the location
previously containing the worm (worms do not rot in 4 hours). By demonstrating
their representation of the location of their preferred food, the scrub jays show they
remembered the "what" and "where."
To assess the "when," the experimenters ran a probe 124 hours after learning. In
the 124 hour probe, the Replenish group jays dug in the worm-containing location,
as they prefer worms, and never encountered rotten worms. The Degrade group jays,
however, first dug for the peanut. The preference for a less preferred food (peanut)
suggests the jays have some knowledge of the timeline it takes for worms to rot.
Thus the jays demonstrated a knowledge of "when," in addition to the "what" and
"where." Magpies (Zinkivskay et al. 2009) and chickadees (Freeney et al. 2009) also
demonstrate this episodic-like memory.
2.4.2
Episodic Memory in Rodents
Recent evidence demonstrates that rodents can perform tasks which fulfill the requirements to be episodic-like memory, in that they contain the what-where-when
information about single episodes (Crystal 2010). When testing animals for episodiclike memory, experimenters must avoid training regiments that require substantial
training, as the animal may not learn an episode per-se, but may simply learn the
rules of the task. This could test semantic memory formation and not episodic memory, as the animals may know the facts of how the test runs, but may not have a
representation of a single episode.
Preferential object recognition in rodents.
Rodents innately prefer to explore
novel objects (Ennaceur and Delacour 1988). When presented with a recently familiar
object and a novel object, rodents explore the novel object for a longer period of time,
referred to as novel-object-recognition (NOR). Similarly, when presented with two
familiar objects, rodents prefer to explore a recently moved object over a stationary
object (Ennaceur, Neave, and Aggleton 1997), referred to as novel-location recognition
(NLR). Rodents also demonstrate preference to remotely familiar objects, meaning
that if presented two familiar objects, the rodent tend to explore the object they had
encountered first. In this experiment, consisting of three phases, rodents were shown
novel objects on phase one. An hour later, in phase two, they were shown a new set
of novel objects. Finally, an hour later the mice were tested in a box that contained
both sets of now familiar objects, with the only difference being the length of time
that the animal was familiar with the object (remotely familiar with the objects
from phase one, and recently familiar with the objects from phase two).
Rodents
in this case, prefer to explore the remotely familiar objects (Mitchell and Laiacona
1998).
Rodents innately explore new objects (what), moved objects (where), and
longer-known objects (when).
What-Where-When memory in rodents.
Based on these innate rodent behav-
iors, Dere and colleagues designed a task to test the what-where-when memory in
rats (Kart-Teke et al. 2006). In the first phase of this experimental design, the experimenters placed four of the same novel object (Object "A") in the test box. In the
second phase, an hour later, the experimenters placed four of a second novel object
(Object "B") in the same test box. An hour later, the experimenters placed four objects in the test box: two object As and two object Bs. One of the object As remained
in the same spot as in phase one, the other moved to a novel location. Similarly with
object B, one remained in the same spot as phase two and one object moved. If
rats encode "what-when" information, they should explore the unmoved object A
more than the unmoved object B. If rats encode "what-where" information, they
should explore displaced objects more than the stationary objects. Rats did both,
demonstrating knowledge within this single unique experience for what-when-where
content and thus, episodic-like memory. Mice too demonstrate this behavior (Dere
et al. 2005) and mice with hippocampal lesions show deficits in what-where-when
preferences in this same test (DeVito and Eichembaum 2009).
8-arm task to assess what-where and when.
Rodents in a modified version of
the 8-arm radial maze task also demonstrate knowledge of what-where-when (Zhou
and Crystal 2009). Rats received training twice a day, either in the morning or the
afternoon (alternating days). Each session consisted of a first phase, a two minute
interval, and test phase. Four arms opened in first phase, three containing regular rat
chow, and one arm containing the preferred chocolate. After the two minute interval,
the arms previously containing regular chow no longer did, while the newly opened
arms did. Rats needed to visit the three new arms in order to eat the chow during this
session. To eat chocolate for the second session, the animals needed to visit the same
chocolate as earlier in the day. For an individual rat, the second phase contained
replenished chocolate only if the testing occurred on the time of day (morning or
afternoon) in which the chocolate replenishment occurred for that rat. If chocolate
was replenished, it was in the same arm as before. Thus, in the test phase, the rats
could get chocolate (what-where) only in the morning or in the afternoon (when)
but not both. Rats could learn this temporal contingency, visiting the chocolate arm
on the test phase at the correct time of day, thus demonstrating knowledge of the
what-where-when of an episodic-like memory.
The main point in these experiments is that behavioral models exist to test
episodic-like memory in rodents, and that animals can demonstrate memory of "what,"
"where," and "when" for a particular episode. This further validates using rodents
as a model for understanding declarative memory formation. Even in cases where
only a subset of the "what," "where," and "when" can be assessed, the animal's
episodic representation may contain all three. It seems possible that we simply do
not know how to properly test the animals for all three in most cases. Rodent behaviors susceptible to MTL damage thus provide good assays for episodic-like memory
and understanding the specific circuitry that contribute to the "what-where-when"
features of episodic-like memory processing remain possible.
2.5
Circuits and Anatomy of Episodic Memory
The above evidence demonstrates that declarative and epidosic-like memory require
intact and functioning MTL systems in human, non-human primates and in rodents.
This most likely reflects the anatomical homology among these species within the
MTL and the structures that connect to the MTL (Amaral and Witter 1995) (Witter
et al. 2000) (Manns and Eichenbaum 2006).
This section explains what is known about the anatomical connections within
the MTL. We see how the sensory corticies projects to the perirhinal and postrhinal
corticies, which in turn project to the entorhinal cortex, which then projects to the
hippocampus.
Additionally, this section discusses how the circuitry may support
individual elements of episodic-like memory.
2.5.1
What is the Medial Temporal Lobe?
Before one can de-construct the function of the MTL, one must understand the circuitry within it, and the exact connections. Four broad cortical regions comprise the
MTL: the perirhinal cortex, the parahippocampal (also known as postrhinal) corticies, the entorhinal cortex, and the hippocampus.
The hippocampus includes the
subiculum, ammon's horn and the dentate gyrus. Understanding the connectivity of
these structures allows for understanding the role each region plays in the declarative
memory system. Generally speaking, the postrhinal and perirhinal corticies exist as
the main interface between the MTL and the rest of the brain. These project to the
superficial layers of the entorhinal cortex, which in turn project to the hippocampus.
The hippocampus, in return, sends projections to the deep layers of the entorhinal
cortex.
..
..
..........
..I...................................................................................
. ..
. .....
....
..
..
....
..........
Olfactory/Auditory
Information
Visual/Visuospatial
Information
Hippocampus
r_1
Entorhinal
Cortex
Figure 2-1: Medial Temporal Lobe Anatomy. Diagram of the prominant projections within the MTL. (DG) Dentate Gyrus, (Sub) Subiculum, (LEC) Lateral
Entorhinal Cortex, (MEC) Medial Entorhinal Cortex, (CA1) Cornu Ammonis Area
1, (CA3) Cornu Ammonis Area 3.
2.5.2
Postrhinal and Perirhinal
As defined by their connectivity, the postrhinal and perirhinal cortices in the monkey
share homology with the rat (Burwell et al. 1995). Both the postrhinal and perirhinal
corticies serve as the interface between the MTL and the uni-modal sensory cortex.
Both, however, receive afferent fibers from mostly non-overlapping cortical areas.
"What" and "where" memory circuits.
Primarily innervated by the temporal
association cortex, the perirhinal cortex also receives substantial innervation from the
entorhinal cortex, the piriform cortex and the insular cortex (Burwell and Amaral
1998a). Eichenbaum et al. propose that this pathway stream processes information
about qualities of objects and makes up the "what" information that help defines any
episode (Eichenbaum et al. 2007). The postrhinal cortex primarily receives inputs
from the visual associational cortex and the posterior parietal cortex, a visuospatial
area (Burwell and Amaral 1998a). This pathway makes up the "where" information
that defines any episode (Eichenbaum et al. 2007).
Polymodal input into perirhinal and postrhinal corticies.
The breakdown of
poly-modal input into the postrhinal and perirhinal cortical areas serve as the major
differences. Visual and visuospatial information make up the majority of input to the
postrhinal area (> 60%) but only a fraction of the input to the perirhinal area ( 12%).
Olfactory areas comprise less than 1% of postrhinal inputs, but almost one third of
perirhinal inputs (31%). Finally, auditory inputs provide only 4% of the afferents to
the postrhinal cortex, but provide 12% of the inputs to the perirhinal cortex (Burwell
and Amaral 1998a). This highlights the role of the postrhinal cortex in processing
visual/visuospatial information and the perirhinal cortex in processing olfactory and
auditory information.
Knowledge of the anatomical connections and types of information processed by
both the postrhinal and perirhinal corticies allow better understanding about the
types and nature of information being processed downstream in the entorhinal cortex.
2.5.3
Postrhinal and Perirhinal Connections to the Entorhinal Cortex
Entorhinal cortex afferents.
Perirhinal and postrhinal cortex then project pri-
marily to the entorhinal cortex. While some reciprocal connections between these
areas exist, the majority of the connections flow into the entorhinal cortex
(Bur-
well and Amaral 1998a). The perirhinal cortex preferentially innervates the lateral
entorhinal area (LEA), and synapses mostly on layer II and layer III (For basic reference, please see 2-1 on page 34) (Burwell and Amaral 1998b). The LEA also receives
substantial input from the piriform cortex, the insular areas and the frontal cortex
(Burwell and Amaral 1998a). The postrhinal cortex, on the other hand, innervates
both the medial entorhinal area (MEA) and LEA (Burwell and Amaral 1998b). Additionally, the MEA receives input from the piriform cortex, frontal areas, occipital
areas and cingulate areas. Differential amounts of inputs from the visual areas comprise the major difference between the projections to the MEC versus the LEC. In
addition to the visual/visuospatial input from the postrhinal cortex, the MEC also
receives 12 percent of its inputs from occipital areas, whereas the LEC receives little
to none of these inputs. (Burwell and Amaral 1998b).
2.5.4
Functional Implications Regarding Perirhinal - Lateral
Entorhinal Cortex and Postrhinal - Medial Entorhinal
Cortex Connections
Spatial and non-spatial cellular responses in entorhinal cortex
As men-
tioned above, the postrhinal and MEC neurons process and represent the spatial
information ("where") of an environment or episode and that the perirhinal and the
LEC neurons process and represent characteristics of that environment ("what").
Electrodes implanted into a brain region can record action potentials of nearby neurons. Based on the task an implanted animal performs in or the objects presented
to it, assumptions can be made about what kinds of information individual neurons
process or represent. In one experiment, rats implanted with electrodes in the perirhinal, parasubiculum, LEC and MEC were recorded from in a box containing spatial
cues (Hargreaves et al. 2005). In agreement with the underlying anatomy, neurons
in the MEC showed high levels of spatial information, while perirhinal and LEC neurons demonstrated little to no spatial information. Further confirmation that of the
preferential representation of spatial information in the MEC pathway in another in
vivo rat study (Knierim et al. 2006). This this study, MEC neurons showed a high
degree of spatial tuning and LEC neurons did not. Further, postrhinal lesions disrupt
object-location recognition in rats (Gaffan et al. 2004) and in monkeys (Alvarado
and Bachevalier 2005).
The connections between the perirhinal cortex and the LEC lead to predictions
that these neurons encode object information about single stimuli (Eichenbaum et al.
2007) (Dickerson and Eichembaum 2010).
To this argument, studies shown that
individual neurons in the rat perirhinal cortex can selectively respond to single stimuli
in a recognition task (Young et al. 1997). In this study, 35% of the recorded LEC
neurons demonstrated odor-specific firing, and 11% of the perirhinal neurons showed
similar specificity. Additionally, in monkeys of the visually responsive neurons, over
94% showed selective response to a specific visual stimuli (Miller et al. 1996).
Thus, MEC neurons process spatial information and LEC neurons process object
information. The in vivo data supports the assumptions made by the anatomical
connections and underlines the importance of knowing the anatomy to make behavioral predictions and assumptions about downstream neuronal populations. If the
MEC processes spatial information, the neurons downstream also process this spatial
information.
2.5.5
Entorhinal Connectivity to the Hippocampus
The entorhinal cortex (EC) serves as the main relay station between the cortex and
the hippocampus proper. The superficial layers of the EC send projections to the
hippocampus, and the deep layers of the EC receive the outputs from the hippocampus.
Anatomical descriptions of the EC to hippocampus primarily come from research
done in the rat. The early studies of Ram6n Y Cajal (Ramon y Cajal et al. 1995)
and Lorente de N6 (Lorente de N6 1934) showed the hippocampus as the target of
entorhinal fibers. Later studies augmented and refined the details of the circuitry
with the use of autroradiogaphic tracing (Steward and Scoville 1976), retrograde and
antereograde tracers (Deller et al.
1996), and currently with tracing viruses (see
Chapter 1).
Entorhinal projections to the hippocampus.
The first detailed autoradio-
graphic studies demonstrated very systematic projections from subregions within the
entorhinal cortex to subregions within the hippocampus, and further projections to
subsections of the dendrites of specific cell populations. Oswald Steward (Steward
1976) demonstrated that both the MEC and LEC send projections to the dentate
gyrus (DG) and to CAl of the hippocampus. The MEC projections to the DG terminate on the middle third of the granule cell dendrites, while the LEC projections
terminate on the outer third of the same granule cell dendrites.
Both the MEC
and LEC send projections to the CAl, and terminate in the SLM portion of the
CAl dendrites. Projection patterns to CAl, however, show a different kind of segregation. MEC fibers terminate on the CAl dendrites near the CA2/CA1 border
(termed "proximal") while the LEC fibers terminate on the CAl cells furthest from
the CA1/CA2 border (termed "distal"). These studies did not differentiate between
any of the cortical cell layers of the entorhinal cortex.
With the use of retrograde tracers, Steward and Scoville revealed that the projections to the DG come mostly from EC layer II stellate cells and projections to CAl
come mostly from EC layer III pyramidal cells (Steward and Scoville 1976). Layer
III and layer II cells both project to the ipsilateral hippocampus, but only layer III
project to the contralateral pathway (via the alvear pathway). They also showed that
CA3 projections come from EC layer II.
Rat and Mouse MEC-III to CA3 differences.
Rats and mice share similar
MTL anatomy, with one large exception. In rats, only EC layer II projects to CA3
but one mouse study study suggests that the projection to CA3 comes from EC layer
III cells (van Groen et al. 2003).
MEC-III to CA1 pathways.
Controversy exists as to the exact anatomical routes
of the projections from EC layer III to CAl. Two distinct pathways exist. One via
the alvear pathway and one via the perforant pathway. Both terminate in the SLM
of CAl.
Cajal (1911) first demonstrated and named this pathway the temporo-
ammonic alvear pathway, as entorhinal stained fibers traveled through the alveus.
Steward (Steward 1976) demonstrated the presence of fibers from the alvear pathway
that passed through the pyramidal cell layer and terminated on th SLM of CAl.
I.............
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.........
.......
. ........
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....
..
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..............................................................................
Until recently, this pathway was considered to contain only "aberrant" fibers, largely
ignored (Witter 1989) and renamed the "perforant pathway" as it perforated the
subiculum on the way to its target. MEC-II projections to CA1, however, utilize
both the alvear and perforant path routes, depending on the septal-temporal position
in the brain (Deller et al. 1996).
A
CAI
B
CAI
CM
Perforant Pathway
ECIK
Alvear Pathway
Figure 2-2: Schematic of Perforant and Alver Pathways From Entorhinal
Cortex Layer III. (A) Perforant Pathway: Axons perforate the subiculum and
synapse in the SLM of CA1. (B) Alvear Pathway: axons travel via the alveus, and
perforate the CA1 pyramidal layer, cross SR and synapse in the SLM of CA1.
Alvear pathway. Deller et al. found that at the temporal portion of the hippocampus, the layer III to CA1 fibers predominately travel via the "perforant path" and
not the alvear pathway. At the septal portion of the hippocampus, however, the majority of the layer III to CA1 projections travel via the alvear pathway and not the
perforant pathway. More intermediate portions of the hippocampus contain mixed
alvear/perforant path projections to CA1.
Fibers from the alvear pahtway make
sharp right angled turns from the alveus, project through the CAl pyrmidal cell layer,
through the stratum radiatum layer and terminate in the SLM of the CAl cells. This
data suggests a more substantial contribution from the largely ignored (Witter et al.
2007) alvear projection. The contribution or existence of the alvear pathway in mice
remains un-described in the literature. We report it in Chapter 1.
2.6
Human Hippocampal Complex versus Parahippocampal Corticies
We see in this section how, in humans, the information in the hippocampus is built
upon information from the surrounding corticies.
Recollection and recognition memory.
One theory regarding the interactions
with the hippocampal formation and the parahippocampal formation as it relates to
declarative memory describes the hippocampal subregions as supporting episodic or
episodic-like memory while the parahippocampal formations support semantic memory. Support for this theory comes from a study by Vargha-Kadem et al. (VarghaKadem et al. 1997), in which young humans with hippocampal specific lesions could
not form new episodic memories, but semantic memory remained intact. The distinction between episodic and semantic memory can also be described as the difference
between recollection and recognition. Recognition requires some knowledge about
the stimuli (semantic perhaps), but lacks source information about where and what
it means. Recollection, on the other-hand, requires not only familiarity with the stimuli, but remembering the reasons for stimulus familiarity (and more information about
it). This includes knowledge of when the memory formed (source information). The
combination of recognition and one's personal experience makes an episodic memory.
fMRI and memory types.
Using event-related fMRI to study human recollection
and recognition memory, Davachi et al. (Davachi et al. 2003) found that activation in
the hippocampus or in the posterior para-hippocampus (postrhinal in rodents) during
a stimulus predicted later recollection of the stimuli. Activation in the perirhinal
cortex during stimuli presentation predicted latter recognition but not recollection.
This evidence points to the notion that the various sub-regions within the MTL play
very different roles in declarative memory formation.
Another theory, proposed by Eichenbaum and colleagues (Eichenbaum 1997) (Eichenbaum et al.
1996) suggests that the parahippocampal cortex alone can support
cortical memory representations, while the hippocampus organizes the relationship
among the cortical memories, and allows for binding of them when necessary. Both of
these theories require more specific inactivation techniques that allow for systematic
double-dissociation studies in order to truly understand the anatomical relationships
of semantic and episodic memory.
2.7
Genetic Dissection of the Medial Temporal Lobe
In this section, we are introduced to modern molecular techniques that allow for
ablation of specific molecules in specific neuronal populations. Discussed below are
first studies in which these techniques were used to study DG, CA1 and CA3 function
within the hippocampus.
Recent technological advances in genetic manipulation technology pioneered by
the Tonegawa lab allow for systematic study of specific neuronal populations within
the MTL
(Tonegawa et al. 2003). Controlling specific genes in specific cell popu-
lations removes the uncertainty of target manipulation that accompanies the use of
pharmacological agents and the collateral damage created by physical lesion. Additionally, these techniques remove the reproducibility issues of both pharmacological
and lesion techniques, thus allowing for more specific questions to be probed.
Transgenic mice models.
Manipulations of genes expressed in CA1, CA3, and
the DG began the molecular and systems dissection of the hippocampus. Targeted
genetic manipulations utilize the Cre/LoxP system (Sauer and Henderson 1988). Using cell-specific promoters that drive the Cre recombinase in one transgenic mouse
and breeding that mouse with another transgenic mouse in which the gene of interest
has been flanked by LoxP sites, results in the knockout of a specific gene in those specific cells (Tsien et al. 1996) (Tsien et al. 1996). Thus, by identifying a cell-specific
promoter, specific genes in those cells can be knocked out. Without any collateral
damage to other brain structures and with the specificity of a single type of neuron,
we can identify the precise brain circuitry involved in various memory and behavioral
tasks.
NR1 knockouts.
The first studies that started to tease apart specific cell-types
used cell-specific promoters to knockout the NR1 gene (Tsien et al.
1996).
The
NR1 gene is an essential component of the N-methyl-D-aspartate (NMDA) receptor (Moriyoshi et al.
1991) (Nakanishi 1992) and thus ablation of the NR1 gene
disrupts proper formation of the NMDA receptor. The NMDA receptor acts as a coincidence detector, as both post-synaptic and pre-synaptic activation is required for
this receptor's activation. Glutamate from the pre-synaptic terminal binds the NR1
2
subunit, and the depolarization of the post-synaptic neuron removes a Mg + block in
the pore of the channel (Nowak et al. 1984) (McBain and Mayer 1994), allowing for
pore-actication and Ca2+ to enter the post-synaptic neuron.
Synaptic plasticity and memory.
Long-term potentiation (LTP) describes the
physiological read-out of a long-lasting enhancement of synaptic strength between two
neurons (Bliss and Lmo 1973) (Bliss and Collingridge 1993). Expression of LTP requires activation of NMDA receptors. Application of the NMDA receptor antagonist
2-amino-5-phosphonopropionic acid (AP5) on in vitro hippocampal electrophysiological preparations ablates expression of LTP (Collingridge et al. 1983) (Zalutsky and
Nicoll 1990) (Hanse and Gustafsson 1992). Behaviorally, application of AP5 into the
hippocampus blocks spatial learning of a hidden platform in the Morris watermaze
task (Morris et al. 1986). In the watermaze task, animals are placed in a pool containing a hidden platform
(Morris 1984). The animals learn and recall the spatial
location of this platform for subsequent survival in the pool. Rats injected with AP5
showed in vivo suppression of hippocampal LTP and could not perform the watermaze task, suggesting that NMDA receptor-mediated LTP in the hippocampus plays
a role in certain types of learning.
The role the NMDA receptor plays in LTP and learning made it an obvious choice
to knock out in specific cell-types in the hippocampus.
2.7.1
CA1 Manipulations
Removing the NR1 receptor subunit specifically in CA1 disrupts synaptic plasticity by removing the NMDA receptor current and ablating LTP at synapses to CA1.
Behaviorally, removal of plasticity disrupts spatial learning in the Morris watermaze
hidden platform task (Tsien et al. 1996). The lack of the NR1 subunit in CA1 also
disrupts trace-fear conditioning, but not delay conditioning, implicating the necessity
of CA1 plasticity in forming associations in which events occur across a temporal
trace (Huerta et al. 2000). These mice also cannot solve a non-spatial transverse
patterning task, a task that requires the mice to acquire three overlapping odor discriminations (Rondi-Reig et al. 2001). Thus, proper CA1 plasticity allows for proper
performance in both spatial and non-spatial tasks.
Physiologically, individual CA1 neurons fire when the animal crosses a particular
position in space (Moser et al. 2008)(See also 2.10.1 on page 50). The term "placefield" describes that individual neuron's representation of space. In vivo physiology
of the CA1 place fields in the CA1-NR1 knockout mice showed degraded place fields
in that the initial formation of these place fields had less spatial specificity than those
place fields in controls (McHugh et al. 1996). Although degraded, the place fields were
stable, removing CA1 as the potential level for recognition of familiar environments.
2.7.2
CA3 Manipulations
Specifically removing the NR1 subunit from CA3 disrupts CA3 recurrent collateral
plasticity (Nakazawa et al. 2002). Nakazawa et al. found that removing plasticity
specifically at these recurrent collaterals disrupts associative memory recall. These
mutant mice can learn the Morris watermaze, and when probed with full cues and
no platform, spend most of their time swimming in the target quadrant. When three
cues are removed, control mice still explore the target quadrant more than other
quadrants, but the mutants perform at chance, exploring all quadrants equally. This
finding demonstrates the necessity of plasticity in the recurrent collaterals of CA3 in
"pattern-completion." Pattern completions describes the ability to use partial cues
to bring the full representation online to make behaviorally relevant actions. CA3
cells involvement in pattern-completion was originally proposed by David Marr (Marr
1971), due to the re-current collaterals. Behaviorally, CA3 plasticity is necessary for
learning of rapid, one-trial experiences (Nakazawa et al. 2003), rapid (but not longterm) recall of contextual memory (Cravens et al. 2006), and adaptive timing in the
trace eyeblink conditioned response (Kishimoto et al. 2006). CA3-NR1 knockout mice
also could not form rapid contextual representations, and trouble in delay conditioning
with competing stimuli presented at the same time (McHugh and Tonegawa 2009).
In vivo physiology of CA1 place cells in the CA3-NR1 knockout mice showed
impaired spatial specificity to a novel context, but normal spatial specificity by the
third day of familiarization to a context (Nakazawa et al. 2003).
Similar to the
partial cue behavioral phenotype, these mice showed disrupted spatial specificity and
dispersed place cells in a partial cue, but not full condition (Nakazawa et al. 2002).
These studies implicate the necessity of plasticity in CA3 for pattern-completion.
Removing plasticity in the recurrent collaterals disrupts spatial tuning in CA1 in a
partial cue environment and disrupts the animals ability to perform behavioral tasks
with partial cues.
2.7.3
Dentate Gyrus Maniplations
Specifically removing the NR1 subunit from the DG disrupts perforant path plasticity
on dentate granule cells (McHugh et al. 2007). In accordance with Marr's theory
about the function of the DG in pattern separation (Marr 1971), distinguishing two
similar contexts is impaired in the DG-NR1 knockout mice. In an experiment that
tested two similar contexts, only one of which the animals received a shock in, control
animals could distinguish between the two contexts and demonstrated this by freezing
in the shock context, but not in the un-shocked context. DG-NR1 knockout mice froze
in the both contexts, showing an inability to distinguish between similar contexts.
In control mice, there substantial differences exist in the rate of neuronal firing
in a novel chamber versus a familiar chamber. In vivo physiology of CA1 and CA3
place cells in the DG-NR1 knockout mice showed a reduction in this difference, when
animals were placed into a new context and had to re-map their place cell activity
to the new arena. CA1 spatial remapping indices were normal between control and
mutant mice, but CA3 spatial remapping was significantly lower in mutant than
in control mice. This inability to re-map could help explain the pattern-separation
phenotype.
In most of these behavioral tasks, only some components of an episodic-like memory are explicitly tested. For example, in both contextual fear-conditioning and watermaze learning and testing, the "what" and "where" components of the memory
are assessed. In fear conditioning, the animals need to recognize which context to be
afraid of (where) or, if presented with a tone-shock pair, they need to remember the
fear to the tone as well (what). In the watermaze, the animals must learn the location
of the hidden platform (what) by using the visual cues in the room as a reference
point (where).
As discussed in chapter 4, fear conditioning can also be used to assess the timing
within an episode, by adding a temporal "trace" between stimuli that the animals
need to bind together. While, in this case, the "when" is still not directly assayed,
the "what" and "where" are tested with the additional temporal element.
2.8
Specific Lesions of Neuronal Output
This section introduces the triple-transgenic mouse model that allows for reversible
ablation of synaptic transmission in a specific neuronal population. This thesis describes a mouse in which this control over synaptic transmission is used in MEC-III
neurons.
These genetic manipulations allow us to question the role of specific MTL subregions in memory acquisition and recall. We can lesion specific cell-types or specific
molecules and ask how they influence behavior. While these techniques and tools give
spatial specificity, there is still the limitation of temporal control. The technology
has recently been developed to control synaptic transmission of specific neuronal
populations in a temporally controlled, reversible manner (Nakashiba et al. 2008).
Triple transgenic mouse.
Nakashiba et al. developed a triple transgenic mouse
approach that allows for silencing of synaptic transmission of specific neuronal populations. The combination of three transgenic mice allows for this control, (see Diagram 2-3 on page 47). Transgenic mouse 1 (TG1) produces Cre recombinase under the
control of the cell-specific promoter. Transgenic mouse 2 (TG2) produces loxP-StopLoxP-tTA under the control of the forebrain-specific CaMKII promoter. With the
combination of TG1 and TG2, the cell-type specific Cre excises the "stop" sequence
and thus produce tTA. This double transgenic mouse is then bred with a third transgenic mouse (TG3), which produces tetanus toxin (TeTx) under the control of the
tet-Operator (tetO). tTA binds to the tetO and TeTx is produced. The enzyme TeTx
specifically cleaves VAMP-2 (also known as synaptobrevin), (Link et al. 1992) (Schiavo et al. 1992) a necessary component of the SNARE complex
(Baumert et al.
1989) and required for synaptic vesicle fusion and exocytosis (Sudhof 1995). Thus,
by breeding the three transgenic mice, TeTx production occurs only in the specific
neurons of choice (designated by the promoter in TG1 and TG2). Doxycycline (Dox)
allows for this system to be temporally controllable and reversible. Administration
of Dox to these triple transgenic mouse represses TeTX expression, as Dox directly
interferes with the tTA-tetO binding (Gossen et al. 1995). This system allows for a
'molecular lesion' of synaptic transmission in a specific neuronal population.
Using this approach, Nakashiba et al. used the CA3 specific promoter (KA1)
and forebrain specific CaMKII promoter to study the role of the output from CA3.
CA1 cells receive input from the tri-synaptic circuit via CA3, but also the direct
cortical input from EC layer III via the alvear/perforant/temporoammonic pathway.
By inhibiting the input from CA3 to CAl, Nakashiba et al. could test the necessity
of CA3 inputs and the sufficiency of the ECIII inputs in CAl related memory and
cellular properties.
Spatial learning without CA3 output.
Spatial learning in the hidden platform
version of the Morris watermaze task (MWM) requries CAl plasticity (Tsien et al.
1996). In the CA3-TeTX mice, however, with CA3 synaptic transmission inhibited
.
......
.
..........
A. Three Different Trangenic Mouse Lines
TGI
SpecificPromoteZ
TG2
pCaMKII
TG3
TetO
B. Cross Between TG1 xTG2
Cell-Specific Promote~r
pCaMKII
STOP
>
pCaMKII
C. Cross Between TG1 xTG2 and TG3
pCaMKII
TetO
Cox
Figure 2-3: Schematic of Triple Transgenic Approach to Silence Neuronal
Transmission in Specific Cell Types. (A) Schematic of each transgenic mouse.
Transgenic Mouse 1 (TG1) uses a cell-specific promoter to drive expression of Crerecombinase in only those cells that utilize that promoter. Transgenic Mouse 2 (TG2)
utilizes the forebrain-specific promoter CaMKII to drive expression of tTA, conditional to the Cre-recombinase removing the STOP codon sequence between the two
LoxP sites. Transgenic Mouse 3 (TG3) utilizes the tetO promoter to drive expression
of TeTx. (B) Combination of TG1 and TG2 excises the STOP site on TG2 and allow
for expression of tTA specifically in specific cells. (C) tTA expressed in specific neurons binds to the tetO and drive Tetanus toxin expression. Addition of doxycycline
(DOX) can inhibit tTA binding to tetO and thus stop expression of TeTX.
(Dox-off for at least 4 weeks), learning of this task remained intact. Learning MWM
requires CA1 (Maglakelidze et al. 2010), yet CA3 input is not necessary, suggesting
the sufficiency of the MEC-III to CA1 projection in this spatial learning task. This
does not, however, address whether the necessity of MEC-II in this task, or if both
inputs to CA1 are simply complementary and sufficient.
Pattern completion and contextual fear memory.
In a one-shock delay con-
ditioning paradigm, the CA3-TeTX mice demonstrate impaired freezing to the conditioning context (where) but normal freezing to the tone (what) as compared to
their control littermates. As suggested by the CA3-NR1 knockout mice, patterncompletion requires CA3 plasticity (Nakazawa et al. 2002). The CA3-TeTX mice
show a deficit with a fear-conditioning version of a pattern-completion task, suggesting that not only does the formation of complete representations based on partial
cues require CA3 plasticity, but also requires functional CA3 output. The CA3-TeTx
mice, when trained on-Dox with three tone-shock pairings showed intact fear memory
to both the context and tone. Tested after seven weeks off-Dox (transmission turned
off), these mice showed intact tone fear and degraded contextual fear memory, implying a the necessity for functional CA3 output in consolidation of contextual fear
memory (Nakashiba et al. 2009).
In vivo physiology of the CA3-TeTx mice show reduced spatial tuning in CAl
cells on a novel track. Reduced tuning of the CA1 output the novel environment
supports the contextual fear conditioning data that showed a learning deficit in the
rapid, one-trial conditioning paradigm. The animal may not be able to rapidly form a
spatial or contextual representation, and thus cannot bind that representation to the
foot-shock. Additionally, in connection with the contextual consolidation phenotype,
these mice showed a reduced incidence of CA1 ripples and re-activaton of CA1 cellpairs (Nakashiba et al. 2009).
The elegance of this triple transgenic technology allows for precise manipulation
of specific circuits in a living animal model. It cannot be overstated how important
an advance this represents.
Collateral damage and pharmacological manipulation
of un-intended targets, especially in such small brain structures, no longer need to
be considered as qualifiers to any resulting physiology or behavioral consequences
of those techniques. This work began with the CA3 output and demonstrated the
importance of this circuit in contextual fear and in memory recall with partial cues.
Applying this technique to other regions in the MTL helps us elucidate the role of
each circuit and neuronal type in episodic-like memory.
2.9
Medial Entorhinal Cortex Layer III Tetanus
Toxin Mouse
Triple Transgenic approach in the Entorhinal Cortex
We recently created
a triple transgenic mouse that allows for reversible control of synaptic transmission
specifically in MEC-III excitatory neurons. In our lab, Dr. Junghyup Suh discovered a
promoter specific to the dorsal and intermediate layer III of the MEC. This promoter,
oxidation resistance protein-1 (Oxrl), drives the expression of Cre-recombinase to
form transgenic mouse 1 (TG1).
TG1, bred with transgenic mouse 2 (CaMKII-
LoxP-Stop-LoxP-tTA, TG2) and transgenic mouse 3 (tetO-TeTX, TG3) forms our
triple transgenic mouse, see figure 2-3 on page 47. This genetic tool allows us to
non-invasively assess the role of this specific connection to CA1 and subiculum in
episodic-like memory.
2.10
Entorhinal Cortex:
What is Known about
the Entorhinal Cortex
With a mouse model to study MEC-II input into the hippocampus, studies and
properties of the entorhinal cortex help to guide predictions of what types of memory
and behavior this circuit participates in. This section reviews both in vivo and in
vitro studies that describe the cellular properties of entorhinal neurons.
.
Olfactory/Auditory
Information
Visual/Visuospatial
Information
...
. ............
..
.....
Hippocampus
, io
I II
Entorhinal
Cortex
Figure 2-4: MEC-III TeTx mouse. Diagram of the projections from MEC-III
to CA1 and subiculum. Using the triple transgenic approach, we express tetanus
toxin specifically in MEC-III. This allows for silencing of synapses from MEC-III.
Manipulated cell population and projections are labeled in green. (DG) Dentate
Gyrus, (Sub) Subiculum, (LEC) Lateral Entorhinal Cortex, (MEC) Medial Entorhinal
Cortex, (CA1) Cornu Ammonis Area 1, (CA3) Cornu Ammonis Area 3.
2.10.1
Place Cells and Grid Cells
Hippocampal pyramidal cells uniquely fire in a particular location in a given context,
and thus are named "place-cells." First discovered by O'Keefe and colleagues using in
vivo recordings of CA1 neurons, single hippocampal neurons respond to the animal's
location in space (O'Keefe and Dostrovsky 1971). Whenever an animal crosses a
specific location in a particular context, a single neuron can respond by continually
firing. A set of CA1 pyramidal cells can represent an entire space or context, but
currently the logic as to what cell fires in which space is not understood (O'Keefe
1976) (Wilson and McNaughton 1993). The place cell properties have also been seen
.....
in CA3 and DG (Barnes et al. 1990), making spatial representation a very prominent
feature of the hippocampus.
Grid Cells.
Given the importance of the hippocampus in episodic memory and
the formation of associations, place cells could represent the spatial portion of any
given episode, either for encoding or recall. Lee and Wilson demonstrated that while
these individual place cells may have no direct connection to their neighboring cells,
they still can associate together (Lee and Wilson 2002) to form a representation of
spatial sequence.
In a task in which a rodent runs down a linear track, one can
construct a sequence of place cells that would cover the length of the environment.
They found that during slow wave sleep (SWS), this exact sequence of place cell
firing replayed at 200x speed. This leads to a speculation regarding the role of sleep
in memory consolidation, but also underscores the notion of space representation as
an essential component of a memory trace. This leads to the question of how these
representations are generated, and of the role of spatial representation in memory
formation and representation.
Greek orators in ancient times used spatial maps to remember long stories or
speeches, in a method called the 'method of loci' (Yates 1966). With this method,
one walks (mentally or literally) through a space, and associates different parts of the
speech with parts of the path. With this method, the orator can imagine walking
though the space to recall the order and content of the story of speech. The sequential
firing of place cells may represent an animal's way of simply organizing the spatial
trajectories, or these sequences could contain other content which needs ordering.
These sequences may help generate one's internal representation of time, simply by
putting features in order.
With the entorhinal cortex as the main gateway of input into the hippocampus, the
spatial representation of place cells may generate from projections from the entorhinal
cortex. Neurons in all layers of the medial entorhinal cortex express "grid-cell" firing
patterns (See Figure 2-5 on page 52).
Unlike place cells, that fire primarily in a
single location in space, individual entorhinal neurons show multiple firing fields.
.
...........
. . ..........
.
Cen
.........
Cell
A__B
Place
Grid
_37
CA1 Neuron
1__
0
%
EC Neuron
Figure 2-5: Depiction of Place Cell and Grid Cell firing patterns in space
Red dots indicate firing within the square space environment (A) Depiction of a
hippocampal place cell's firing pattern in a square environment. Note that there is
one region within the environment where this cell preferentially fires. (B) Depiction
of an entorhinal grid cell's firing pattern in a square environment. Note that a single
cell fires in regular intervals within the space. Figure adapted from (Moser et al.
2008).
These entorhinal cells fire in a grid-like pattern and the firing fields occur at periodic
locations within a given space (Hafting et al. 2005), that map the entire space. The
firing peaks for a given grid-cell fire at geometric vertices of a triangular array that
covers an environment (see Figure 2-5). The spacing between firing locations in the
MEC increases down the dorsal-ventral axis (Hafting et al. 2005) (Solstad et al.
2007) (Brun et al. 2008).
In addition to "grid-cell" firing, individual MEC neurons, termed "head-direction"
cells, fire when the rodent is faced in a particular direction (Sargolini et al. 2006)
and other MEC neurons, termed "border cells" signal the boundaries of the environment (Solstad et al. 2008). In many cases, MEC "head-direction" cells also represent
the spatial information by firing with "grid-cell" properties as well, in what is termed
a "conjunctive cell." However, layers-specific differences exist in the MEC with regards to the input structures into the hippocampus. MEC layer II cells do not express
any head-direction firing, while 66% of all MEC layer III neurons demonstrated both
conjunctive firing (Sargolini et al. 2006).
"Border cells" have been found in all layers of the MEC, comprise less than 10%
of the neuronal populations and define the borders of an environment, whether those
borders are formed by a wall, or simply by and end of a table (Solstad et al. 2008).
Whether or not there are layer-specific differences in the representations of border
cells is yet unknown.
Place and grid cells fire within certain phases of the theta rhythm. Phase precession is a phenomenon that describes neuronal firing during progressively earlier
phases of theta while the animal is crossing through a place or grid field (O'Keefe
and Reece 1993).
Within the MEC, all layer II neurons express phase procession,
while layer III neurons express little to no phase procession (Hafting et al. 2008).
Layer II and layer III MEC neurons comprise the main input structures into the
hippocampus, initiating the tri-synaptic and mono-synaptic circuits respectively. Specific manipulations of layer II or layer III MEC may have very different predictions
based on their in vivo differences described here. Layer II neurons represent space
in the form of grid cells, represent borders with border cells but do not represent
directionality, while layer III neurons represent all three. Thus, a manipulation to
layer III may specifically disrupt the animal in tasks where directionality is important, while tasks requiring spatial integration could still be supported by the layer II
redundancies. Additionally, layer III neurons do not express phase procession, while
layer II neurons do. Thus, manipulations of layer II neurons may be more disruptive
to spatial representations of the animals trajectory compared to manipulations of
layer III.
2.10.2
Entorhinal Cellular Physiology
Persistent activity.
Neurons that continue to fire action potentials after removal
of the triggering stimulus display "persistent spiking." The phenomenon of persistent
spiking supports a proposed mechanism by which encoding or retrieval of episodic
events can happen when the episode includes features not occuring simultaneously
in time or space (Hasselmo et al. 2009) For example, for proper association of a
foot-shock and tone to occur when the two elements are separated in time by a
'trace interval.' For the rest of this document, the term 'trace' refers to a temporal
delay between two stimuli, for example, if a tone is played for 5 seconds, then 20
seconds later a foot-shock is delivered, there exists a 20 second 'trace" between the
two stimuli. The ability of the animal to associate the two features may rely on a
single neuron's persistent firing. In this way, both features could be combined in a
singular representation.
Persistent activity properties.
Entorhinal cortical neurons in medial layer II
(MEC-II) (Klink and Alonso 1997), MEC-II (Yoshida et al. 2008) (Tahvildari et al.
2007) and medial layer V (MEC-V) (Egorov et al. 2002) (Fransn et al. 2006) demonstrate persistent spiking in vitro. The MEC-II cells display self-terminating persistent
activity activated by the musarinic receptor agonist carbachol (CCh)
(Klink and
Alonso 1997). EC-V neurons display long periods of persistent activity that last at
least 13 minutes. These sustained levels of firing can be increased or decreased in
an input-specific manner. The firing rate of a given plateau can be increased in a
step-wise fashion to a higher stable firing rate by further depolarization steps. These
discrete steps to a higher firing rate reach a ceiling level after about five upward steps.
To decrease the firing rate in a stepwise fashion, hyperpolarization steps were used.
The firing is dependent on the muscarinic activation
(Egorov et al.
2002).
The
step-wise function may be relevant to the amount of information that can be held
within a representation at any given time.
Persistent activity in LEC.
Lateral entorhinal cortical layer III (LEC-III) neu-
rons display persistent activity with either CCh application or stimulation of layer
II/III perirhinal cortex, the main input to LEC (Tahvildari et al. 2007) (Tahvildari
et al. 2008). MEC-II neurons achieved persistent activity with stimulation of the
angular bundle (which project to MEC-III), but unlike the persistent activity in the
other layers, CCh did not induce persistent activity alone. These studies suggest
that, if this persistent activity exists in vivo, then these neurons may participate in
behaviors requiring working memory or in conditions where individual elements of an
episode are separated by a trace. Further, these studies suggest that the cholinergic
system, in combination with these cells may provide the mechanism working memory
uses to hold information online.
Persistent activity in MEC-III.
Group I metabotropic glutamate receptor (mGluR)
blockers inhibit and group I mGluR agonists enhance persistent activity in MEC-III
neurons (Yoshida et al. 2008). Persistent firing in these entorhinal neurons did not
require inhibitory and excitatory inputs, indicating this firing property as an intrinsic property of these entorhinal neurons. Persistent activity of a given neuron could
allow for two distinct behaviorally relevant properties. First, continued firing could
aid in working memory tasks (Goldman-Rakic 1995). Second, this continued firing
could allow for a single neuron, or population of neurons to integrate stimuli that are
separated in time.
2.10.3
Persistent Activity Blockage and Behavior
Blockage of persistent activity and trace conditioning.
Persistent activity in
the entorhinal cortex, describes an in vitro cellular property only. Under the assumption that these cellular properties extend to neurons in vivo, two studies looked at the
effect of blocking the cholinergic-muscarinic system in trace versus delay fear conditioning. One study looked at the effect of blocking supposed "persistent activity" by
blocking muscarinic receptors in the perirhinal cortex (Bang and Brown 2009). In
this study, Bang et al. infused the muscarinic receptor antagonist scopolamine into
the perirhinal cortex prior to delay and trace fear conditioning. In only the trace
conditioning, they found a severe impairment in tone fear memory. Another study
injected the muscarinic receptor (Ml) antagonist pirenzepine into the entorhinal cortex prior to both delay and trace fear conditioning (Esclassan et al. 2009). Esclasan
et al. demonstrated similar results to Bang et al. These animals did not express fear
to the tone in the trace conditioning only. These results implicate the involvement of
the parahippocampal cholinergic-muscarinic system in maintaining the representation
of the conditioned stimulus (CS) during the temporal 'trace' between the CS and the
unconditioned stimulus (US), so that the two can be associated. These results show
entorhinal and perirhinal cortical necessity in maintaining the CS representation during the trace, and implicate cholinergic-muscarinic mediated persistent activity as a
mechanism for the maintenance.
2.10.4
Episodic Memory, Alzheimer's Disease and the Entorhinal Cortex
Alzheimer's disease and the entorhinal cortex.
Episodic memories, as a mem-
ory class, begin formation late in childhood, after semantic and non-declarative memories (Perner and Ruffman 1995) and degrade first with aging (Herlitz and Forsell
1996). Aging humans commonly display declines in episodic memory (Verhaeghen
et al. 1993). Patients with Alzheimer's disease (AD) suffer with losses of episodic
memory and of spatial orientation (Backman et al. 2004).
As the most common
form of human dementia (Katzman 1986), AD the first causes acute episodic memory loss (Katzman and Karasu 1975). AD pathology and atrophy first engrosses the
entorhinal cortex (van Hoesen et al. 2004).
In a longitudinal study in humans (Rodrigue and Raz 2004), the researhers found
that age related shrinkage of the entorhinal cortex specifically, and not the hippocampus or pre-frontal cortex, predicted poorer memory performance. Volume reduction
also serves as a clinical indicator for individuals as they transition to dementia from
pre-clinicial cognitive impairments (Killiany et al. 2000) (Dickerson et al. 2001).
Thus, better understanding of the circuitry of the entorhinal cortex and the role of
each connection within the MTL could help in understanding the types of memory
loss humans suffer, and can give insight as to how to repair or prevent the onset of
memory loss.
2.11
Animal Lesions of the Entorhinal Cortex
Lesion studies in monkey, rat, mouse and rabbit provide a framework for the overall function of the EC. We see in this section how EC lesions influence behavioral
performances on a variety of tasks. Due to the nature of lesions, however, some results conflict. These conflicts most likely stem from collateral damage to structures
abutting the EC, or inconsistencies in the amounts of tissue remaining after lesions.
These lesions allow us to make inferences about global properties of the EC, but not
specific cell-layers within the EC. When trying to study more specific areas within
the EC, these results serve as good starting points, but may or may not apply to any
given neuronal population.
Entorhinal lesions in monkeys.
In vivo recordings of monkey EC neurons demon-
strate that individual neurons displayed activity in response to specific objects or
places, and this activity can be maintained during a delay on a delayed-match-toplace DMP task (Suzuki et al.
1997).
Activity during the delay phase suggests
that information about a particular object is being held in short-term working memory to make a later decision. Yet, bilateral EC aspiration lesions in monkeys show no
deficit in delayed-non-match to place (DNMP) and two-choice working memory tasks.
Rather, these lesions affect performance on a transitive inference test, demonstrating
the necessity of the EC for understanding the relational organization of stimuli (Buckmaster et al. 2004). A conflicting study of bilateral EC lesions in monkeys showed
normal learning in the DNMP task with short inter-trial intervals and impaired learning with longer intervals (Leonard et al. 1995). Interestingly, in this study, re-training
the impaired animals on the same task a year later resulted in learning of the task.
This suggests that other brain regions can compensate in this task. EC lesions also
attenuated attraction to positive stimuli and enhanced defense to negative stimuli,
suggesting a role in gating emotionally relevant stimuli in associative tasks (Meunier
et al. 2006). These lesions, done in a small number of animals, with all of the potentially confounding issues with physical manipulations, make it hard to interpret these
studies. While not clear in combination, these results do suggest EC involvement in
processing relationships of stimuli, wither across time or across other stimuli.
2.11.1
Hippocampal Physiology after Entorhinal Lesion
Bilateral radiofrequency lesions of rat EC cause a reduced CA1 place cell discharge
rate and a reduced CA1 place field size (Van Cauter et al. 2008), implicating the
necessity of EC inputs to the hippocampus in the stability of hippocampal spatial
representations.
Gamma-acetylenic GABA (GAG) injections in the EC result in specific lesions
of MEC-III (Brun et al. 1999). Using this protocol, the authors recorded from CAl
cells, which receive direct cortical input from MEC-III and CA3 neurons, and recorded
from CA3 neurons which receive MEC-I input. In this case, the CAl fields became
larger and more dispersed, and the CA3 place fields remained sharp. This suggests a
role for layer III in shaping CAl place cells. These studies suggest that the EC plays
a role in influencing spatial processing in the hippocampus.
2.11.2
Spatial Learning in Rodents after Entorhinal Lesions
If, as suggested above, the EC plays a crucial role in processing spatial information,
then lesions in the EC should disrupt spatial learning. Unfortunately, using physical
lesion techniques don't give a clear demonstration of such a hypothesized effect. In
some instances in the Morris watermaze (MWM) hidden platform learning task, EC
lesions in rats and mice cause learning deficits (Schenk and Morris 1985) (SpowartManning and van der Staay 2005) (Galani et al. 1997) (Nagahara et al. 1995), but
in other instances animals learn this task (Bannerman et al. 2001) (Hagan et al.
1992) (Pouzet et al. 1999) (Parron et al. 2006). One study aimed to separate out the
role of the perforant path from the LEC (LPP) from the role of the perforant path from
the MEC (MPP) with lesions. Lesions of the MPP, but not the LPP disrupted place
learning (Ferbinteanu et al. 1999). In this study, MPP lesioned animals displayed
normal escape latency during the learning phase, but showed reduced preference for
the target quadrant during a probe trial. Mice with unilateral EC lesions could learn
the MWM only after 70 days of training, while mice with bilateral EC lesions never
learned the task (Hardman et al.
1997).
After learning the MWM, rats lesioned
in the dorsolateral, but not the ventromedial, EC subsequently forgot the location
of the platform (disruption of consolidation). During a reversal test, the rats with
dorsolateral lesions learned the new platform location more slowly (Steffenach et al.
2005). In a working memory version of the watermaze task (2 trials per day delayedmatch-to-place (DMP), entorhinal lesioned animals swam a longer distance to reach
the platform that they learned one hour earlier (Glasier et al. 1995).
In another type of spatial discrimination task where animals learned to associate
one arm with food reward and one without, EC lesioned animals could not discriminate between the rewarded and un-rewarded arm (Cho and Jaffard 1995) (Gaskin
and White 2007).
It is unclear from these studies how important a role the EC plays in spatial
learning tasks, probably due to the lesion quality.
2.11.3
Delayed Non-Match-to-Place Learning and Radial Arm
Maze learning in Rodents after Entorhinal Lesions
Entorhinal cortex lesions and working memory. Entorhinal neurons in rats,
similarly to these neurons in monkeys (Suzuki et al. 1997), fire during the delay
phase of a delayed-non-match-to-place (DNMP) task (Young et al. 1997). In turn,
entorhinal lesioned animals consistently show deficits in learning a spatial non-matchto-place task (rewarded alternation task) (Bannerman et al. 2001) (Barnes et al.
2000) (Ramirez et al. 1996) (Ramirez et al.
2007).
Animals with both bilateral
and unilateral lesions show a deficit in learning the DNMP task, but the unilateral lesioned animals can eventually learn the task after overtraining (Loesche and Steward
1977) (Scheff and Cotman 1977).
Lesions to the entorhinal cortex disrupt learning in various versions of the radial
arm task (Olton et al. 1978) (Olton et al. 1982) (Jarrard et al. 1984) (Johnson
and Kesner 1994). Various forms of the radial arm task can test for working memory
deficits, reference memory deficits, and spatial memory deficits. Bilaterally EC-lesions
in mice caused an impairment in a spatial discrimination task in one version of the 8arm radial maze task (Cho and Jaffard 1995). Similarly, muscimol (a GABA agonist)
injected into the dorsal, but not ventral, EC disrupted learning in a spatial learning
task in which rats learned to associate food reward with one arm, and no food reward
with another arm (Gaskin and White 2007). In this task, muscimol disruption of the
dorsal, but not ventral EC disrupted learning of this spatial association task.
In summary, these experiments indicate the importance of the EC in spatial working memory tasks. In these tasks, the animal needs to utilize a representation of its
previous behavior to make a correct choice on subsequent trials.
2.11.4
Hippocampally Dependent Trace in Entorhinal Cortex Lesions
Trace conditioning separates the CS and US with a temporal gap (the trace). For
successful association of the CS and US in trace conditioning, an animals must hold
the representation of the CS (presented first) online to associate it with the US. In
delay conditioning, when the CS and US co-terminate, the neurons representing the
CS and US fire at the same time, and thus strengthen synaptically. For the trace
conditioning to work properly, the neurons representing the CS need to fire when the
representation of the US arrives. Trace conditioning and working memory similarly
share the necessity to maintain a representation of a stimuli or previous choice online
for later usage.
Entorhinal cortex lesions and trace conditioning.
In one study, rabbits trained
in a trace conditioning protocol to associate a tone (CS) and an eye air-puff (US),
showed differences with hippocampal -dependent trace intervals (Ryou et al. 2001).
In this experiment, electrodes in CA1 recorded activity during trace learning. With
a hippocampally dependent trace interval EC lesioned animals showed a 30% conditioned response (of CA1 neuronal activity) to the tone by the ninth paired association, compared with an 80% conditioned response to tone for the control animals.
The CA1 recordings during this trace showed no learning-related activity. With nonhippocampally dependent trace intervals, the EC lesioned animals learned as well as
controls, and CA1 in these animals showed learning related activity.
Entorhinal lesions clearly disrupt input into the hippocampus during the hippocampally - dependent trace paradigm and not the hippocampally-independent trace
paradigm. These behavioral results suggest the necessity of the EC for working memory tasks and tasks involving a temporal gap between stimuli.
2.11.5
Fear Conditioning
Fear conditioning allows for the study of both hippocampally dependent and hippocampally independent processes.
Typically, contextual learning (Debiec et al.
2002), and trace conditioning (when the trace is longer than 10 seconds) (Chowdhury
et al. 2005) require the hippocampus. However delay conditioning, when both stimuli
co-terminate, is usually hippocampally independent (Amorapanth et al. 2000), with
the possible exception of the pairing of weak stimuli (Quinn et al. 2008). The EC
provides the majority of inputs into the hippocampal complex and so manipulation
of the EC should disrupt fear conditioning during hippocampally dependent, and not
during hippocampally independent conditioning.
Entorhinal cortex lesions and contextual fear.
EC lesions in rats impairs con-
textual fear learning (Maren and Fanselow 1997). Lesions made 1 week prior to conditioning caused deficits in post-shock freezing during the conditioning and deficits
in freezing when tested 24 hours later, suggesting a role for entorhinal projections in
forming an association between a context and the foot-shock. In contrast, another
study showed no disruption in contextual fear conditioning with EC lesions (Phillips
and LeDoux 1995).
Entorhinal cortex lesions and context fear in delay and trace conditioning.
In a third study, entorhinal lesions impaired acquisition of contextual fear memory
when scrambled tone-shock pairings were presented during the conditioning day (Ma-
jchrzak et al. 2006). When the tone and shock overlapped (delay conditioning), EC
lesions did not effect contextual memory. However, when the tone and shock did not
overlap (trace but with variable trace durations) context memory did not form well. In
both experiments, both lesion and control animals showed intact tone memory. The
authors suggest that the entorhinal cortex is necessary for contextual conditioning
when there are other stimuli competing for association with the foot-shock.
Extinction.
Animals can learn to suppress fear memory to a conditioned stimulus,
in a process known as extinction. After an animal's initial association of a CS and
US, repeated CS presentations without the US, results in the animal learning not to
express fear to the CS. The fear memory suppressed, but not erased and re-activation
occurs very quickly with a mild CS-US pairing presentation (Bouton 2004) (Myers
and Davis 2007).
Entorhinal cortex and context-dependent fear extinction.
Context - depen-
dent extinction requires a functional entorhinal cortex (Ji and Maren 2008). On day
one of this experiment, rats learn an association between the tone and foot-shock in
"context A." On day two, animals receive multiple tone (CS) presentations in a very
different "context B" to extinguish tone fear. On day 3, tone memory in "context
B" where the extinction trials had happened is compared to tone memory in "context C" a familiar context not associated with any tone CS or US. In this paradigm,
sham-lesioned animals show reduced freezing when tested for tone fear in "context
B", but show robust tone fear when tested in "context C." These results indicate
the context-specific nature of tone extinction. EC lesions produce reduced tone fear
memory in both contexts. These rats extinguished tone fear in all contexts, and not
just tone fear in the extinguishing context.
Entorhinal cortex lesions and latent inhibition.
Latent inhibition (LI) de-
scribes the phenomena whereby repeated, non-reinforced pre-exposure to a conditioned stimulus (CS) causes a decrease in the conditioned response (CR). Entorhinal
inactivation disrupts LI (Seillier et al. 2007). In this task, infusion of tetrodotoxin
(TTX) into the EC prior to pre-exposure, prior to conditioning or before both showed
LI disruption only in rats that received TTX before pre-exposure. This demonstrates
the necessity of entorhinal cortex activity in producing this phenomena.
2.11.6
Lesion of Temporoammonic Pathway
TA lesions and spatial memory consolidation.
One group attempted to study
the role of the temporoammonic (TA) pathway in consolidation of spatial memories (Remondes and Schuman 2004).
In this rat study, Remondes et al. lesioned
the region where the perforant path sends projections to CA1, thus trying to distinguish between the inputs onto the DG and CAL. These lesions, however did extend
somewhat onto the layer II projections to the DG, and also ablated parts of the
subiculum. They found that lesions made prior to water-maze learning did not disrupt acquisition of the spatial location of the platform. When tested for memory
of the platform location 24-hours post training, both sham and TA-lesioned animals
showed a preference for the target quadrant. When tested 4 weeks later, however, the
sham lesioned animals, and not the TA-lesioned animals showed a preference for the
target quadrant. This suggested a disruption in acquisition of long-term memories or
in consolidation of the previously acquired memories. To address this issue, animals
were trained, probed at 24 hours and then lesioned (allowing for acquisition). When
probed 4 weeks later, the TA-lesioned animals showed a deficit in target quadrant
preference, suggesting a problem in consolidation.
Further, when animals trained
first, then probed tested at 24 hours, allowed to consolidate for 3 weeks, and then
lesioned, both the sham and TA-lesioned animals showed normal preference for the
target quadrant. This study has two conclusions: First, consolidation occurs over 3
weeks, and second, spatial memory consolidation requires the TA pathway.
This study represented the first to test the specific role of the perforant path
in formation and consolidation of episodic-like memory. The authors demonstrate
that direct cortical input from layer III to CAl is not necessary for acquisition of
these tasks but rather is necessary for the spatial memory consolidation. This study
has a few issues that are important to note. First, the lesions disrupted input from
the nucleus reuniens, as well as input from both MEC and LEC. Second, lesions
damage the layer II inputs onto DG and CA3, and damaged substantial portions
of the subiculum.
Thus, while the TA pathway is lesioned, other structures are
also manipulated and thus the conclusions must include the potential role of these
structures in the consolidation process. Additionally, depending on the exact location
of the lesion, the inputs from MEC-II may or may not be affected. As noted before,
inputs from layer III project to CA1 via the PP and alvear pathway (Deller et al.
1996), and thus more septal lesions may not disrupt the direct cortical inputs at
all, and thus the consolidation phenotype results from a lesion of one of the other
structures.
2.11.7
Conclusion About Lesion Data
Physical lesions studies on any particular brain structure have severe limitations.
For example, they can leave tissue intact, sparing fibers that can support various
behaviors and missing potential behavioral phenotypes that would be caused by a
complete lesion. In addition, lesions cause collateral damage to surrounding brain
regions thus resulting in behavioral phenpotyoes not specific to the brain region of
interest. Animal-to-animal differences also add to the noise in behavioral data making
interpretations more difficult. Given the intricate anatomy of the MTL, lesion work
paints some broad pictures about region-specific contributions of various behaviors,
but a more detailed, cell population-, and specific connection-targeted approach is
necessary to tease apart the MTL and its contributions to the declarative memory
system.
2.11.8
Genetic Manipulations of the EC
The first attempt to address the role of MTL subregions (outside of the hippocampus)
with genetic manipulations resulted in a transgenic mouse in which the superficial
layers (II and III) of the MEC and pre and parasubiculum could be manipulated
in an inducible manner (Yasuda and Mayford 2006).
In this study, the authors
introduced an inducible and constitutively active (CA) form of CaMKI. They found
that expression of the CA form of CaMKII in the MEC-II and MEC-III and pre
and para-subiculum disrupted 24-hour memory of the target quadrant in the watermaze task, but did not affect acquisition of the task with either a visible or hidden
platform.
Turning off the CA-CaMKII reversed the deficit.
Mice with the CA-
CaMKII turned on also showed a deficit in novel object recognition, in that they did
not explore the new object more, when compared to wild-type. The CA-CaMKII mice
showed intact contextual and cued fear conditioning. Using the inducible Tet system,
the authors found that activation of the CA form of CaMKII just after formation
of the spatial memory in the watermaze task caused a disruption in the integrity
of the spatial memory, and showed a time-specific role for CaMKII in the MEC-II
and III and pre and parasubiculum in the consolidation of the spatial memory. It
demonstrated the necessity of CaMKII regulation in the MEC-I and III as well as
the pre and parasubiculum in the integrity of spatial memories, specifically during
the post-training period. But, these studies have two major limitations. First of all,
within the MTL, this is not a cell type specific manipulation. In this study, MEC
II and III, as well as pre and parasubiculum are manipulated, making it difficult
to understand the specific role of any of the specific neurons in these behaviors. In
many regards, this manipulation was like a pharmacological intervention, as it was
reversible, but it lacked specificity. Secondly, in terms of MTL circuit dissections, this
study addressed the role of CamKII within these cells, and not the functional role of
these neurons per-se in the behavioral tasks.
2.12
Role of Medial Entorhinal Cortex Layer III
in Learning and Memory
This section introduces the triple transgenic mouse model used in the anatomical and
behavioral studies in the following chapters. We show here that the Cre+ expression in
the triple transgenic cross is confined to layer III of the MEC and that these Cre+ cells
are neurons and not inhibitory neurons. We show that four weeks off-Dox sufficiently
and specifically silences MEC-II synaptic transmission, and allowing four weeks back
on-Dox sufficiently turns synaptic transmission back on. We thus have in vivo control
over synaptic transmission specifically in MEC-II neurons.
This mouse provides
an excellent tool for behavioral characterization of this pathway in comparison to
pharmacological manipulations, which do not allow for cell-type specificity, and to
lesion work, which is irreversible.
2.12.1
MEC-II Cre+ Mouse
In the Tonegawa lab, Dr. Junghyup Suh discovered that the oxidative resistance
gene (Volkert et al. 2000), Oxrl, is specifically expressed in the superficial layers of
the EC. Using the Oxrl promoter, one particular mouse founder line demonstrated an
even tighter spatial restriction, only expressing the Cre recombinase in the MEC-III.
Cre mouse is layer III specific.
To assess the specificity of the Cre expression in
the MEC-II, the retrograde tracer cholera toxin subunit B (CTB) was injected into
CA1 of the hippocampus of Cre+/Rosa mice. Since layer III cells project to CAl, the
retrograde dye also expressed in the cell bodies of layer III cells, also Cre+ and stained
for B-gal (see (D) Figure 2-7 on page 68). This confirmed layer III expression. To
assess if these Cre+ cells were layer III specifically or whether they also included layer
II, CTB was simultaneously injected into both CAl and DG. Thus CTB expressed
in both layer III and layer II EC, see (D) Figure 2-8 on page 69, and the Cre+ cells
stained with B-gal did not overlap with the CTB expression in layer II. This confirms
that the Cre+ mouse line created is a layer III specific line.
A
B
Figure 2-6: OxR1-Cre+/Rosa Expression shows Specific Expression in Medial Entorhinal Cortex. Cross of Oxrl Cre+ mouse with Rosa mouse line (A)
Horizontal sections showing spatially restricted expression of LacZ in MEC-III. (B)
Close up of right hand panel from (A) showing expression in Layer III specifically and
fibers into Sub and CAL. Sub: subiculum, DG: Dentate Gyrus, Ctx: Cortex, CB:
Cerebellum, Str: Striatum, Th: Thalamus, Hp: Hippocampus. Work by Junghyup
Suh.
.
A
..
.. ..........
B
Ca-
OU
Figure 2-7: Cre+ Expression and CA1 Retrograde Injection Demonstrate
Cre+ cells are Layer III. (A) Cholera-toxin subunit B (CTB = Green) injection
into CA1 of the hippocampus. Blue is DAPI staining. (B) Diagram, based on known
anatomy of what cells should express retrograde signal if CAL. MEC-Il cells should
express signal. (C) Sagittal section showing B-Gal (Red) staining in Cre+/Rosa
mouse. (D) Sagittal section showing Cre+ cells in EC and retrograde signal from
CA1 injection. (E) Same as figure (D) with DAPI signal to show cytoarchitecture.
Work by Junghyup Suh.
..........
A
as
C"
C"
Figure 2-8: Cre+ expression and CA1/DG Retrograde Injection Demonstrates Cre+ cells are not Layer II. (A) Cholera-toxin subunit B (CTB = Green)
injection into CA1 and DG of the hippocampus. Blue is DAPI staining. (B) Diagram,
based on known anatomy of what cells should express retrograde signal if injected
into CA1 and DG. MEC-III (from CA1) and MEC-II (from DG) cells should express signal. (C) Sagittal section showing B-Gal(Red) staining in Cre+/Rosa mouse.
(D) Sagittal section showing Cre+ cells in EC and retrograde signal from CA1 injection. (E) Same as figure (D) with DAPI signal to show cytoarchitecture. Work by
Junghyup Suh.
..
. .......
AB
Figure 2-9: MEC-ILL Immunohistochemistry: Cre+ cells are Neurons but
not Inhibitory Neurons. Double immunohistochemistry in Oxrl-Cre mice crossed
with Rosa (Lox-stop-Lox-LacZ) mice. (A) Double IHC for beta-galactosidase (Red)
and a neuronal marker (NeuN, green) (B) Double IHC for beta-gal (red) and inhibitory neuronal marker (GAD-67). Work by Junghyup Suh.
This Oxrl-Cre line was crossed in the same manner as the CA3-TeTx mouse
mentioned earlier to create a mouse in which tetanus toxin expression specifically
expresses in MEC-III under the control of Dox.
Cre is expressed in excitatory neurons.
To assess the cell-type in MEC-III
that expressed the Oxrl-Cre, double immunohistochemistry was performed on brain
slices from the Cre+/Rosa mice. Staining for beta-galactosidase marked the cells
that express LacZ under the Cre promoter (see red staining in 2-9). Co-staining with
NeuN, which is a neuron-specific marker (Mullen et al. 1992), shows a clear overlap
with the Cre+ cells (see "A" figure 2-9 on page 70), indicating that these are neurons
and not glia. Further, co-staining with the inhibitory marker GAD67
(Kaufman
et al. 1991) (see "B" in Figure 2-9) showed no overlap, indicating that these neurons
are not inhibitory, and thus excitatory.
2.12.2
Synaptic Transmission of MEC-TeTx mice
Inhibition of synaptic transmission.
Suh determined the induction profile of
synaptic inhibition using voltage-sensitive dye (VSD) recordings in hippocampal slices
of both Cre+ mice and Cre-TeTx mice. Mice that were off-Dox for 8 weeks (see "A"
Figure 2-10 on page 72) were tested for synaptic transmission in the SLM versus the
control pathway in the SR. With stimulation in SLM, the mutant pathway showed
complete transmission inhibition at lower stimulation intensities, and highly reduced
transmission (80-90%) at the highest stimulation intensities (see "B" Figure 2-10).
In the control pathway, there was no signal difference (and thus no difference in
synaptic transmission) between the control Cre+ and MEC-II TeTx mouse (see "C"
Figure 2-10). This also indicates that eight weeks off-Dox sufficiently inhibits synaptic
transmission.
Synaptic transmission induction.
To assess the Dox-induction profile, Suh tested
synaptic transmission on MEC-III-TeTx mice off-Dox for only four weeks. In this case,
SLM stimulation in the mutant mice (tan line) showed a similar transmission profile
to the mutant mice eight weeks off Dox (red line) see "B" Figure 2-11 on page 73.
Mice four weeks off-Dox (transmission inhibited) were then placed on-Dox for four
weeks to turn back on synaptic transmission. Mutant mice four weeks back on-Dox
(tan line) displayed a similar synaptic transmission profile to the control mice eight
weeks off-Dox (blue line). These experiments demonstrate that four weeks off-Dox
is sufficient to inhibit synaptic transmission in the triple-transgenice TeTx mice, and
four weeks on-Dox is sufficient to restore synaptic transmission in these mice.
2.12.3
Using the MEC-II TeTx mouse.
This triple transgenic mouse is an ideal tool to study the role of the medial entorhinal
cortex in learning and memory. With the ability for the mice to develop normally
before the molecular lesion is turned on, we can assess the functional role of the MECIII in a system that has not adapted alternative strategies to cope with developmental
..........................
Test
A
On Dox
Off Dox
Manipulated Pathway:
Transmission via SLM
Control
* Mutant
0
8 weeks
Control Pathway:
Transmission via SR
CA3
rKll8*"n~ a bm y (OA)
Ibams bbn
a f l
Figure 2-10: Inhibition of Synaptic Transmission in MEC-III TeTx Mouse.
(A) Experimental design to test synaptic transmission 8 weeks off-Dox. (B) SLM
stimulation of control (blue) and mutant (red) mice off-Dox for 8 weeks. Black line is
fluorescence signal of Control-Mutant to show % reduction of signal in mutant mouse.
(C) SR stimulation in both control (blue) and mutant (red) mice off-Dox for 8 weeks.
Work by Junghyup Suh.
0 Control
A
Test
4weeks
Iw
On Dox
Off DoxI
* Mutant
C
4 Wel
The
4 weeks
On Dox
Off Dox
CA1
NNW
so
A
CA3
:-.*...
SR
CAS
SLM
DG
4
so
Sunu*l
*
mfnIty(uA)
Mutant: 4 weeks off DOX
IS
ii.
260
s
*sMns bitnIy (uA)
0 Mutant: 4 weeks OFF then 4 weeks ON DOX
Figure 2-11: Inhibition of Synaptic Transmission Profile. (A) Experimental
design to test synaptic transmission in mice 4 weeks off-Dox. (B) Stimulation of SLM
in mutant mouse 4 weeks off-Dox (tan line) mimics mutant mouse 8 weeks off Dox
(red line). (C) Experimental design to test synaptic transmission in mice that were
off Dox for 4 weeks, then on Dox for 4 weeks. (D) Stimulation of SLM in mutant
mouse 4 weeks on Dox after being off-Dox for 4 weeks (tan line) showed similar profile
to control mouse off-Dox for 8 weeks (blue line). Work by Junghyup Suh.
manipulations. Additionally, due to the temporal control and reversible manner, we
can target specific time periods during the experiment to assess whether deficits are
due to problems with acquisition or with recall, or both. The layer specificity of this
mouse model allows us to study the connectivity of this layer, and to ask specific
questions about the role of the functional output of the MEC-II in the mouse.
Chapter 3
Medial Entorhinal Cortex Layer
III: Characterization of Projections
To understand how specific circuitry within the MTL relates to memory formation and
recall, it is important to understand the precise circuitry that is being manipulated.
This chapter outlines the precise projection circuitry of the MEC-III TeTx mouse we
use in our behavioral studies described in the following chapters. Using the MEC-III
Cre+ mouse, which expresses Cre recombinase specifially in layer III of the medial
entorhinal cortex, in combination with a Cre-dependent, fluorophore-expressing AAV
virus, the anatomical circuits and MEC-III projections are revealed. In outlining the
circuitry, this work demonstrates that the MEC-Ill neurons in mice send projections
specifically to CA1 and the subiculum via two pathways: the perforant and the alvear
pathways.
Much of the anatomy of the hippocampal-entorhinal circuit that has been revealed
in the rat has mapped perfectly in mice as well. In the literature, the only exception
is the MEC-III to hippocampus connection, which in rats projects exclusively to the
SLM of the CAl hippocampal subregion and the subiculum. In mice, it has been
suggested that the MEC-Ill afferents project to CAl in the same manner with the
addition of a projection to the SLM of CA3. It is our discovery that mouse MEC-Ill
projections, contrary to what is reported in the literature, do not project to the SLM
of CA3, and are in concordance with the rat and monkey anatomy. Additionally, rat
studies have shown a substantial projection from MEC-II to CA1 in which the fibers
travel via the alvear pathway, in addition to the well studied fibers that travel via
the perforant pathway. We report that the alvear pathway exists in mice and carries
a substantial fraction of the MEC-II to CAl projections. These findings not only
clarify mouse MTL circuitry, but also help interpret our behavioral results.
3.1
Introduction
The entorhinal cortex serves as the major source of projections into the hippocampus, as well as the main recipient of projections from the hippocampus. Superficial
layers II and III of the rat entorhinal cortex send projections to the hippocampal complex (Amaral and Witter 1995) and the deep layers of the entorhinal cortex (layer
V) receive inputs from the hippocampal output (Dolorfo and Amaral 1998) (Kohler
1986) (Witter 1989). Given that the hippocampus is an essential structure for episodic
memory formation (Scoville and Milner 1957), it is essential to understand the precise
circuitry into and out of the hippocampus.
Rat MEC-III projects to CA1 and Subiculum.
In the rat, the DG and CA1
subregions of the hippocampal region receive projections from the medial (MEC) and
lateral entorhinal cortex (LEC)
(Steward 1976).
Layer II MEC sends projections
that terminate on the middle third of the dendrites of the DG granule cells, while the
layer II LEC projections terminate on the outer third of the same dentate granule
cells (Steward 1976) (Steward and Scoville 1976). These studies additionally demonstrated that the SLM of CA3 receive inputs from layer II EC. Projections to CAl of
the hippocampus come from layer III of both the MEC and LEC, but whereas the
layer II projections terminate on different dendritic regions of the same granule cell
populations, the projections from layer III terminate on different CAl populations.
Oswald Steward (Steward 1976) found that the MEC fibers predominately terminate
near the border of CA2/CA1 in the SLM (termed "proximal" CA1) while the LEC
fibers predominately terminate closer to the subiculum (termed "distal" CAl).
Rat MTL anatomy is similar to that in mice,
Mouse MEC-III projects to CA3?
with the exception of one study that injected retrograde tracers into CA3 and noticed
signal in layer III entorhinal cortex (van Groen et al. 2003). This study suggests that
the connectivity between the entorhinal cortex and hippcampus is different from the
rat, and that layer III EC projects to the CA3.
This discrepancy has functional
implications when manipulating cell-types or pathways within the MTL, and thus
requires a more through investigation.
Perforant and Alvear Pathway.
There are two routes by which entorhinal cor-
tical neurons project to CA1 of the hippocampus: the perforant pathway, which
"perforates" through the subiculum to the SLM (Witter 1989) and the alvear pathway. The existence of the alvear pathway was first described in 1911 by Ram6n y
Cajal (Ramon y Cajal et al. 1995), who named the pathway the "temporo-ammonic
alvear pathway." Steward noted the presence of stained fibers that arrived into the
hippocampus via the alvear pathway (Steward 1976).
He noted that these fibers
would cross over the pyramidal CA1 cell layer, cross through the stratium radiatum
and terminate in the SLM, but he did not characterize this pathway further.
The alvear pathway in rats comprises a substantial proportion of fibers arriving
into the SLM from the entorhinal cortex (Deller et al. 1996).
Depending on the
position along the septal-temporal axis, the alvear fibers are either non-existent or
exclusive. In the temporal portion of the hippocampus, layer III entorhinal cortex to
CAl fibers mostly follow the "perforant path" (PP) route, while in the septal portion
of the hippocampus, the majority of the fibers travel via the "alvear pathway" (AP).
At all points between the septal and temporal pole of the hippocampus, there is a
mix of perforant and alvear projections.
Using the MEC-III-Cre+ mouse in combination with a virus that expresses a
fluorophore only in Cre+ cells (Kuhn and Torres 2002) allows for anatomical analysis of these neurons' projection pathways and post-synaptic targets. Injecting the
AAV8-DIO-CHR2-EYFP virus into the MEC of the MEC-III Cre+ mice along with
another virus (AAV8-synaptophysin-mCherry) that was not Cre-dependent allowed
us to understand the mouse entorhinal-hippocampal connectivity.
Using these tools, we show that MEC-III does not project to CA3 in mice, as
previously suggested (van Groen et al. 2003), and that the alvear pathway does exist
in mice.
3.2
3.2.1
Results
Projections into the Hippocampus
Using this AAV8-DIO-CHR2-EYFP tool in combination with the MEC-III Cre+ mice,
we studied the nature of the projections from the MEC-III neurons to the hippocampal complex.
Two viruses were simultaneously injected into the MEC of the MEC-Ill Cre+ mice:
the AAV8-Synaptophysin-mCherry and the AAV8-DIO-CHR2-EYFP. The AAV8synaptophysin-mCherry expresses mCherry in all infected neurons, while the AAV8DIO-CHR2-EYFP requires combination with Cre recombinase to express the EYFP
fluorophore. CHR2 is a membrane channel, and thus inserts itself into the membrane
and since it is tagged with an EYFP, the axons/dendrites can be visualized. Thus,
when the AAV8-DIO-CHR2-EYFP virus is injected in the MEC-III Cre+ mouse,
EYFP expression should only show in MEC-III neurons.
MEC-III solely expresses EYFP when AAV8-DIO-ChR2-EYFP
injected
into MEC-III Cre+ mice.
As seen in Figure 3-1 on page 80, (B) and (C) the AAV8-synaptophysin-mCherry
infects both layer II and layer III MEC neurons (B) while the AAV8-DIO-CHR2EYFP infects only layer III neurons. The EYFP signal that crosses layer II and
spreads in layer I of the EC represents the dendrites of the layer III neurons (van
Haeften et al. 2003). The expression of mCherry signal in the middle third of the
dendrites of the dentate granule cells confirms injection into the medial and not lateral
entorhinal cortex (see (E) in 3-1).
MEC-III projections are to proximal CA 1 and subiculum. MEC-III does
not project to CA3.
Panel (F) from 3-1 shows the projection profile from the layer III cells expressing the EYFP, which projects to the subiculum and the proximal SLM of CA1.
These projections do not project to the SLM of CA3 or any other part of the CA3.
Panel (E), shows the projection termination of the EC neurons infected with AAV8synaptophysin-mCherry, which from (B) shows infection in layer II and layer III. The
projection profile of layer II and layer III projections into the hippocampus clearly
demonstrates projections to DG, CA3 and CA1. Comparing panel (E) and (F), which
are overlaid in (G) there is a clear distinction at the CA3-CA1 border, suggesting that
it is only the layer II cells that project to CA3. Thus, MEC-III neurons do not project
to CA3 in this line of mouse, and presumably in all mice.
3.2.2
Alvear Pathway
Combination of the AAV8-DIO-CHR2-EYFP virus with the MEC-II Cre+ mouse also
allows for analysis of the presence of the alvear pathway. MEC-III Cre+ mice were
injected with AAV8-DIO-CHR2-EYFP virus, and after 3 weeks of viral expression
animals were sacrificed.
ME C-III projections use perforant and alvear pathway to project to CA 1.
Panel (A) from Figure 3-2 on page 81 shows the hippocampus of a mouse that
was bi-laterally injected with AAV8-DIO-CHR2-EYFP in the MEC. Two distinct
pathways are illuminated, the perforant path, as well as the alvear path. Higher
resolution imaging of the CA1 area clearly shows fibers crossing the CAl pyramidal
cell layer, crossing the SR and terminating in the SLM. See white arrows from (B) and
(C) 3-2. In (C) especially, many fibers can be seen crossing the SR, and terminating in
the same proximal region of the SLM in CA1 that the perforant path fibers terminate.
It is clear from this analysis that the alvear pathway is one of the routes of synaptic
communication between the MEC and CAl.
U111111",
...............
.
..................................................................
.......
...
. . .......
............
...
.
.. ..............
Injection Site: Medial Entorhinal Cortex
eDAPI
* EYFP: From AAV8-DIO-CHR2-EYFP
*mCherry: From AAV8-synaptophysin-mCherry
Projection Topography: Hippocampus
r%
C
Figure 3-1: Projections to the Hippocampus from MEC-III: Double Virus
Approach. Two viruses were simultaneously injected into the MEC of the MEC-III Cre+ mice.
Virus 1 was AAV8-DIO-CHR2-EYFP. Virus 2 was AAV8-synaptophysin-mCherry. (A) Shows injection site of viruses DAPI stained with lines delineating cortical layers 1, 11 and IIl. These lines
represent the same cortical features in B and C. (B) Red expression shows expression of mCherry
virus associated with AAV8-synaptophysin-mCherry virus. mCherry expression is clearly in both
layer II and III. (C). EYFP expression of layer III cells (only cells containing Cre+). Layer III dendrites extend through layer II and into layer L (D) DAPI staining of sagittal hippocampus of same
animal injected with both viruses. White lines show SLM, dotted white line shows border between
CA1 and CA3. Sub = Subiculum, DG = Dentate Gyrus, SR= Stratum Radiatum, SLM=stratum
lacunosum moleculare, prox=proximal SLM and dist=distal SLM. (E) mCherry expression can be
seen in middle third of Dentate Gyrus dendrites (demonstrating MEC but not LEC injection, as
well as expression in SLM of CA3. (F) EYFP expression of layer III projections. Expression can
clearly be seen in SLM of CA1 and not CA3. (G) Composite of (E) and (F).
..........
..
....
Alvear Pathway and Perforant Pathway
O DAPI
* EYFP: From AAV8-DIO-CHR2-EYFP
Figure 3-2: Alvear and Perforant Pathways. (A) Sagittal section of hippocampus of
MEC-III Cre+ mouse that was injected with AAV8-DIO-CHR2-EYFP in MEC. Projections can be
seen to heavily terminate in subiculum and SLM of CAL. Perforant pathway and alvear pathway
both are evident. (B) Horizontal section of hippocampus of animal injected as in (A). PP= Perforant
pathway, AP is Alvear Pathway. Arrow shows axons projecting from AP to SLM. (C) Close up of
distal SLM showing axons arriving to distal SLM via both AP and PP.
3.3
Discussion
The ability to use genetic tools to reveal the precise circuitry of the medial temporal
lobe has clear advantages over former methods involving injection of retro-grade and
antero-grade tracers. Without the confound of neighboring cell groups or contamination with non-targeted cells, the usage of genetically modified mice with a viral
approach allows for clean and clear anatomical studies.
With the combination of a mouse that expresses Cre recombinase solely in layer III
neurons of the medial entorhinal cortex and a virus with a Cre-dependent fluorophore,
we have isolated and fluorescently tagged this circuit.
3.3.1
Medial Entorhinal Cortex Layer III projections to CA1
in Mice.
We find with these techniques that the MEC-III projects to the proximal SLM of
CA1, and to the subiculum, see (E) from figure 3-1, which is consistent with the
literature. We find that, in mice, these neurons do not project to CA3, a finding that
is consistent with the large body of literature in the rat, but contrary to one report in
the mouse (van Groen et al. 2003). There may be a few reasons for the discrepancy.
First, the result may be specific to the mouse line used. While we used C57BL/6NTac,
vanGroen et al used C57BL/6J. It seems unlikely for such profound differences to exist
in the brain circuitry with such subtle genetic background differences between these
two mouse lines. Secondly, while our study utilized a targeted approach, vanGroen's
method is sensitive to to contamination of non-targeted cells. This could happen in
two ways. In injecting their retrograde tracer in CA3, vanGroen et al. may have had
to cross CA1 possibly contaminating CAL. Alternatively, the tracer may have spread
after injection and contaminated CAL.
Either way, the genetic mouse combined
with the viral approach gives us a contamination free, specific way of looking at this
circuitry and thus we believe it confirms homology to the well-studied rat anatomy.
3.3.2
Alvear Pathway in Mice.
We also show, for the first time, that the alvear pathway also exists in mice. We
demonstrate that this pathway comprises a substantial portion of the fibers that
come from the MEC-II and terminate in the SLM of CA1.
This demonstration
creates more questions that need to be addressed: It is not clear if the majority of
the projections come from contralateral or ipsilateral entorhinal cortical projections,
as these animals were injected bi-laterally. From (C) in figure 3-2, it seems as though
the termination targets of the perforant pathway and the alvear pathway are similar,
but there could be a further segregation of CA1 cells. It could be that a certain type
of CA1 cells receive projections from entorhinal neurons projecting via the alvear
pathway, and another type receive projections via the perforant pathway.
Could
there be a functional significance, if these neurons do synapse on the same population
of CA1 neurons? For example, it could be used as a timing mechanism, if there were
differences in when the signals arrived on the same neurons. Could there be separate
roles for the entorhinal neurons that project via the alvear or the perforant path?
3.3.3
Dissecting the Pathways
Retrograde dyes could be utilized to test whether different populations of MECIII neurons solely project to CAl via the perforant or the alvear pathway. In this
experiment, horizontal slices of the Cre+/Rosa mice would be utilized as this is the
only plane in which the hippocampus and entorhinal cortex are seen on the same
slice (see "B" in 3-2). DiI and DiO are retrograde dyes which are taken into cells and
spread through the cell via lateral diffusion (Honig and Hume 1989), thus allowing
for tracing of the entire neuron's anatomy. Crystals of these tracers placed along the
axons of a neuron will illuminate the neuronal cell body, and can be used in fixed
tissue (Godement et al. 1987). Thus, taking a horizontal slice and carefully applying
the DiO crystals to the alvear pathway, and DiI crystals to the performant pathway,
we can assess whether or not the neurons projecting along these pathways represent
distinct sub-populations within the MEC. DiI and DiO label with different colors,
allowing for direct assessment of whether the MEC-II neurons are the same.
If there are two populations, one projecting via the alvear and one via the perforant pathways, then in the Cre+/Rosa mice, the Beta-galactosidase stained MEC-III
neurons will either label with green (DiI) or blue (DiO). If the neuronal populations
are similar, then the green and blue signals would overlap.
Understanding the precise MTL circuitry in the animal model used is crucial
for interpretation of any behavioral results and the nature of information processing
in that animal. We show here that MEC-III neurons project to CA1 and to the
subiculum, and not to CA3. In addition, we have revealed a previously undescribed
projection pathway in mice, the alvear pathway.
3.4
Methods
3.4.1
Mice
Mice used in this study were the triple transgenic mice described in the introduction,
MEC-III Cre+ TeTx. Males, aged 14-20 weeks, weighing 30-35g were used.
3.4.2
Surgical Procedure
MEC-III Cre+ mice were anesthetized with an intraperitoneal injection of 0.5-0.7
mL Avertin (20mg/ml solution prepared from tert-amyl alcohol and 2-2-2 tribromoethanol, Sigma) and placed in a stereotaxic frame. Skull was set to be flat. Opthalamic eye gel (Henry Schein) was used to maintain eye moisture during surgery.
5 pl
Hamilton Syringe (7634-01) with added needle (26 gague 1 inch 45 degree, Hamilton
7804-03) was used for all viral injections. Needle was coated with 1% BSA prior to
virus loading. In the case of only AAV8-DIO-CHR2-EYFP virus injection, 1l was
used. In the case where both the AAV8-DIO-CHR2-EYFP and AAV8-synaptophysinmCherry viruses were used, 0.5pl of each were mixed and loaded into the syringe.
Syringe was sequentially lowered bilaterally into the entorhinal cortex (EC) (anteroposterior = -3.94mm, dorsoventral = -3.55mm, mediolateral = ±3.1mm from bregma)
with the needle at a 12 degree angle (needle tilted 12 degrees posterior). Animals
received bilateral injections of virus; 1pl of virus per side was injected at a rate of
0.05pl/minute (UltramicropumplI, World Precision Instruments, Sarasota, FL). The
needle was left in place for 5 min after the injection, and the skin was closed using
a Vetbond Tissue Adhesive (3M). Animals were monitored for 3 days to make sure
there were no complications due to surgery. Animals were sacrificed 3-6 weeks post
injection.
3.4.3
Fixation, Sectioning and Imaging
Mice were transcardially perfused fixed with 4% paraformaldehyde (PFA) in 0.iM
sodium phosphate buffer (PB) and post-fxed in PFA for 12-16 hours. 50uM-thick
sections were sliced in a vibratome in cold phosphate buffered saline (PBS). Immunohistochemistry (IHC) was not necessary for visualization purposes due to the
strength of the flurorphore reporter from the viruses. Sections were incubated with
PBS containing DAPI (Invitrogen) for 15 minutes and mounted on glass slides and
imaged with Zeiss Axio Imager.Z1 with the ApoTome attachment and the AxioVision
software (Version 4.5.0.0).
3.4.4
Viruses
AAV8-synaptophysin-mCherry. The construct was made using standard molecular biology protocols. The adeno-associated virus (AAV) backbone vector had flanking ITRs, which consisted of a CMV promoter, a human growth hormone intronic
fragment and SV40 polyA fragment. The synaptophysin-mCherry, which consisted
of a fusion between synaptophysin and mcherry, was cloned in using Bam H1 and
Xhol restriction sites downstream of the promoter. The virus was packaged for the
serotype AAV8 using standard cell culture and molecular biology protocols in the
laboratory of Dr. Rachael Neve of MIT-BCS viral core facility.
AAV8-DIO-ChR2-EYFP. The construct was made using standard molecular biology protocols. The AAV backbone vector had flanking ITRs, which consisted of a
CMV enhancer and a synapsin promoter. The DIO-ChR2, which consisted of a fusion between ChR2 and EYFP, was cloned in the AAV backbone vector with flanking
EcoR1 restriction sites downstream of the enhancer and promoter fragments. The
virus was packaged for the serotype AAV8 using standard cell culture and molecular biology protocols in the laboratory of Dr. Miguel Sena-Esteves of Mass General
Hospital, Harvard Medical School.
Chapter 4
The Role of Medial Entorhinal
Cortex in Temporal and Working
Memory
In this chapter, using the MEC-III TeTx mouse as a model, we test the role of MECIII synaptic output in two types of tasks that require a short-term (i.e. seconds to
minutes) maintenance of a prior choice for a subsequent decision or for an association
of a pair of stimuli separated by a short (i.e., 20 s) temporal gap: working memory and
trace conditioning, respectively. We demonstrate that the MEC-1II mono-synaptic
input to the hippocampus is necessary for acquisition in of context and tone memory in
trace conditioning and for performance in a working memory task. Additionally, using
the CA3-TeTx mouse, we further refine the relevant circuitry underlying performance
in these tasks.
The process of working memory allows for a representation of information to be
held "online" for a period of time for later usage. The memory is then discarded
or modified. A common example of working memory is remembering a telephone
number for as long as you need to dial it, and then realizing afterwards that the
number cannot be remembered. The representation of the number was held online
for a short time and then discarded after usage. It is generally thought that neurons
that continually fire in the delay between learning and the time when that learned
information is used are representing the information, and holding that representation
online. Cells that fire in this manner allow for completion of short-term memory
tasks, but also may be used to combine newly acquired information with the previous
representation. Neurons in the EC display the ability to fire persistently for longperiods in vitro and to fire in the delay period in working memory tasks. Lesions
in the EC sometimes affect working memory tasks, but lesions to the hippocampus
always disrupt versions of working memory tasks.
4.1
Persistent Activity
Persistent activity is a cellular phenomenon whereby neurons can continue to fire
action potentials after a triggering stimulus has been removed (Egorov et al. 2002).
In vivo recordings of monkey pre-frontal cortex during working memory tasks show
stimulus-specific persistent activity after the stimulus has been presented (Miller
et al. 1996) and thus are thought to be the cellular representation of working memory (Goldman-Rakic 1995). Another feature of persistent activity could be that it
allows for the integration of multiple synaptic inputs that are separated in time (temporal memory) (Hasselmo et al. 2009).
One in vitro physiological feature of entorhinal cortical neurons is persistent activity. MEC-1I (Klink and Alonso 1997), MEC-III (Yoshida et al. 2008) (Tahvildari et al.
2007) and MEC-V (Egorov et al. 2002) (Fransn et al. 2006) all show persistent spiking in vitro. The persistent activity seen in these studies are muscarinic acetylcholine
receptor-dependent, metabotropic glutamate receptor dependent, or some combination of the two. Agonists enhance persistent activity and antagonists inhibit the
expression of it. This provides a potential cellular basis for the involvement of persistent activity in EC neurons in working and temporal memory tasks.
Consistent with this persistent activity also existing in vivo, entorhinal injection
of a muscarinic receptor (Ml) antagonist prior to delay or trace fear conditioning
has effects only on trace conditioning
(Esclassan et al.
2009).
Esclassan et al
demonstrated that blocking the muscarinic system prior to trace training disrupts
the foot-shock/tone association, and these mice freeze less when presented with a
tone. Entorhinal lesions in the rabbit disrupts hippocampal-dependent trace conditions (Ryou et al. 2001). This supports the notion that EC neurons use persistent
activity as a mechanism to integrate synaptic inputs over the trace.
Two simple forms of working memory tasks are the delayed match-to-place (DMP)
and delayed nonmatch-to-place (DNMP) tasks. In these tasks, a stimulus is presented
in one part of a TV screen (for monkeys) or in an area (for rodents) and upon a
subsequent trial, the animal is rewarded for making a saccade or movement to the
same place (match) or rewarded for choosing to saccade or move to the other option
(nonmatch). Both of these tasks require that a representation of the prior trial is
held online to perform successfully in the second trial. During DNMP tasks, in
vivo recordings in the EC show delay-period firing in monkeys (Suzuki et al. 1997),
suggesting that these neurons are holding the stimulus representation online during
the delay. Lesions in monkey EC disrupt performance on DNMP tasks (Leonard
et al. 1995). Blocking muscarinic cholinergic receptors in rhesus monkeys performing
a DMS disrupts performance in this working memory task (Penetar and McDonough
1983) (Bartus and Johnson 1976). Finally, cholinergic deafferentation of the EC in
rats disrupts performance in DNMP tasks (McGaughy et al. 2005).
These results indicate that the entorhinal cortex is necessary for certain types of
working memory tasks and for trace conditioning. Further, it seems likely that it is
the persistent activity that mediates proper performance in these behaviors. These
pharmacological manipulations have an advantage over lesions in that they can point
to molecular mechanisms within the region that mediate certain behaviors. However,
pharmacological manipulations lack in cell type specificity, and thus affect a wide
body of neurons, making it hard to understand the circuitry involved in mediating
these behaviors.
CA1 NMDA receptors are necessary for associating conditioned and unconditioned
stimuli when they are separated in time by a trace (temporal memory) (Huerta et al.
2000). CA1 is the region where novel associations are bound, and in trace conditioning, MEC-III provides the contextual or spatial component to the episode (Majchrzak
et al. 2006). The ability for CA1 to process temporal memory may be supported by
persistent firing from MEC-II neurons.
Testing MEC-Il in temporal and working memory tasks.
We employed
the MEC-II TeTx mouse to test the role that the functional output of MEC-III
plays in temporal and working memory tasks. We find that the MEC-III output is
necessary for trace conditioning.
MEC-TeTx mice show normal tone and context
memory in delay conditioning. However, both are disrupted in trace conditioning.
Utilizing the full capability of the temporal control of the MEC-II TeTx mouse, we
pinpoint the necessity of the MEC-III for the acquisition of trace conditioning but
not recall. Additionally, MEC-II TeTx mice are severely impaired in the rewarded
DNMP version of the T-maze. Within the entorhinal-hippocampal circuit, MEC-II
output is necessary for temporal and working memory tasks.
CA1 receives inputs from MEC-III, LEC-III, nucleus reuniens and CA3 (Witter
1989).
In a second set of experiments, using the CA3-TeTx mice, we tested the
role of CA3 in trace conditioning and the DNMP version of the T-maze to mimic
the experiments run with the MEC-III TeTx mice. We show that these mice have
no deficit in the DNMP verison of the T-maze. Thus, performance in the DNMP
T-maze requires the MEC-III output and not CA3 output.
Additionally, the CA3-TeTx mice, like the MEC-II TeTx mice, have disrupted
contextual fear memory in the trace conditioning. Unlike the MEC-III TeTx mice,
however, the CA3-TeTx mice have no deficit in tone fear memory in this conditioning
task. These results indicate that proper fear association with temporally displaced
tone requires the MEC-II input but not CA3 input to CA1.
4.2
4.2.1
Behavior Results
Delay and Trace Fear Conditioning
MEC-III to CA1 pathway is not necessary for delay fear conditioning.
The delay fear conditioning task was performed in mice off-Dox for at least 4 weeks
(transmission off). On day 1, in which the animals were trained, there was not a statistically significant difference in the freezing behavior between the two genotypes (Figure 4-1 D, page 93)(2-way ANOVA: Genotype x Time F(34,1258)=0.54, P=0.9852;
Time F(34,1258)=73.01, P < 0.0001; Genotype F(1,1258)=0.31, P=0.5824). On the
second day of this task, when animals were placed in a novel chamber and three tones
were presented, there was not a statistically significant difference between the genotypes over the 15 minute session (Figure 4-1 E, page 93) (2-way ANOVA: Genotype
x Time F(47,1739)=0.89, P=0.6821; Time F(47,1739)=34.72, P < 0.0001; Genotype
F(1,1739)=1.29, P=0.2637). More detailed analysis of the specific periods within day
2 allowed for analysis of generalized fear, tone memory, and freezing behavior in the
post-tone period. There were not significant differences between the genotypes in
freezing during tone, (Figure 4-1 E, page 93)(CT, 74.00% ± 2.89%; MT, 68.26% ±
2.58%; P=0.1430) post-tone period, (CT, 45.02% ± 3.77%; MT, 36.06% ± 3.00%;
P=0.0672) or in the 4 minutes prior to the first tone, an assessment for generalized
fear, (CT, 18.72% ± 4.53%; MT, 13.66% ± 3.50%; P=0.3857).
On the third day,
animals were tested for freezing to the conditioning context. There was not a statistically significant difference between the genotypes over the entire 5 minute context
exposure (Figure 4-2 B, page 94)(2-way ANOVA: Genotype x Time F(14,518)=0.85,
P=0.6168; Time F(14,518)=6.06, P < 0.0001; Genotype F(1,518)=0.67, P=0.4198).
In summary, animals run on the delay conditioning paradigm showed no deficits in
tone and contextual memory.
MEC-III to CA1 pathway is necessary for trace fear conditioning.
To test the role of the MEC-III output in trace conditioning, we ran a 20 s trace
fear conditioning protocol in mice off-Dox for at least 4 weeks (transmission inhibited). On day 1, in which the animals were trained, there was a statistically significant
difference in freezing behavior between the genotypes over the session, with mutants
freezing less over the session (Figure 4-1 A, page 93)(2-way ANOVA: Genotype x
Time F(34,2448)=2.08, P=0.0003; Time F(34,2448)=142.46, P < 0.0001; Genotype
F(1,2448)=11.95, P=0.0009).
On the second day of this task, when animals were
placed in a novel chamber and three tones were presented, there was a statistically
significant difference between the genotypes over the 15 minute session, again with
the mutants freezing less over the session (Figure 4-1 B, page 93)(2-way ANOVA:
Genotype x Time F(47,3384)=1.30, P=0.0861; Time F(47,3384)=33.82, P < 0.0001;
Genotype F(1,3384)=17.24, P < 0.0001). More detailed analysis of the specific periods within day 2 allowed for analysis of generalized fear, tone memory, and freezing
behavior in the post-tone period.
There were significant differences between the
genotypes in freezing during tone (Figure 4-1 C, page 93)(CT, 55.75% ± 1.95%; MT,
45.07% i 2.29%; P=0.0004) during the post-tone period (CT, 45.49% ± 2.08%; MT,
28.46% ± 1.92%; P < 0.0001) and during the 4 minute period prior to the first tone,
which tests for generalized fear (CT, 17.14%±2.07%; MT, 8.92%±1.89%; P=0.0048).
In all cases, the mutant froze less. On the third day, animals were tested for freezing
to the conditioning context, and here too there was a statistically significant difference between the genotypes over the entire 5 minute context exposure (Figure 4-2
A, page 4-2) (2-way ANOVA: Genotype x Time F(14,1008)=2.11, P=0.0097; Time
F(14,1008)=10.62, P < 0.0001; Genotype F(1,1008)=20.28, P < 0.0001). Thus, the
addition of the 20 s trace in the conditioning paradigm disrupts contextual and tone
fear expression.
Table 4.1: MEC-II in Delay and 20 s Trace Conditioning
Genotype
MEC-III TeTx
MEC-II TeTx
4.2.2
Paradigm Conditioning Tone
OK
OK
Delay
Deficit Deficit
Trace
Post - Tone
OK
Deficit
Context
OK
Deficit
Analysis of 20 s Trace Deficit. MEC-II Output Necessary for Acquisition, Recall or Both?
MEC-III to CA1 pathway is necessary for the acquisition of trace fear
conditioning.
. I-
I
, -
-
-:In-
__
- - -
i=
--
-::::
................
_-
"
Control
" Mutant
C
90
80
70
60
9o.
Cn
80706050-
0 0
30E
10
1V,
~e20
40
20-
C)O
I-
0-
Seconds
Seconds
Seconds
n.s.
90-k
C0
I
80
70
ffff
0Co
I
160
S401
a)0
30
S20
10
Seconds
Tone
Seconds
Seconds
Foot-Shock
Figure 4-1: MEC-III Output is Necessary for Tone Memory in Trace but
not in Delay Conditioning. Mice off-Dox for Day 1 acquisition and Day 2 tone
and post-tone testing. Blue circles are control (MEC-III Cre+ mice) and Red circles
are mutant (MEC-III TeTx mice) (A-C) 20 s Trace Conditioning. (A) off-Dox mice
trained with 3 tone-shock pairings with 20 s trace between end of tone and onset
of shock. (B) Day 2 testing in context different than training context. Three 60 s
tones presented and freezing during tone and post-tone assessed. (C) The three tone
and post-tone periods from (B) are combined and averaged. (D) Delay conditioning.
off-Dox mice trained with three pairings of tone-shock, where shock is administered
with termination of tone. (E-F) same as (B-C). Dox off-off = Dox off for AcquisitionRecall.
e Control
" Mutant
A
B
*
80
1
70
0
I
tj
6
1
II I 1T89
30
18
_
2
60
0
(A10-
0
10
20
180
240
3
100
rj0
1
70
00
ro
n.s.
80
_
0
_
_
120
k
18
0
_
_
240
300
0
_
_i_
60
120
Seconds
_
30
Seconds
Figure 4-2: MEC-III Output is Necessary for Context Memory in Trace
but not Delay Conditioning. Blue circles are control (MEC-II Cre+ mice) and
Red circles are mutant (MEC-II TeTx mice) (A) Freezing to conditioning context
for mice in the 20 s Trace Paradigm, see (A-C) in 4-1, page 93. (B) Freezing to
conditioning context for mice in the Delay Paradigm, see (D-F) 4-1. Dox off-off =
Dox off for Acquisition-Recall.
Utilizing temporal control over synaptic transmission in the MEC-III TeTx mouse,
we tested whether the deficit in trace conditioning was due to an impairment during
acquisition or recall of the tone and context memory. As shown in 2-11 on page 73,
Dox withdrawal or addition to the diet of the MEC-II TeTx mouse can inhibit or
allow proper synaptic transmission. The induction takes 4 weeks. In experiment 1,
we turned off synaptic transmission during the acquisition, and kept transmission off
during recall. For experiment 2, we turned off synaptic transmission for acquisition,
but turned on transmission during recall. In experiment 3, we turned on synaptic
transmission during acquisition, but turned it off during recall.
Experiment 1: Transmission OFF for Acquisition. Transmission OFF for
Recall.
Disruption of MEC-III output disrupts 1-month tone and context memory.
The 20 s trace fear conditioning paradigm was run in mice that were off-Dox for
at least 4 weeks prior to acquisition (Day 1). Mice were kept off-Dox for an additional
4 weeks prior to "Day 2" and "Day 3" testing. In this case, synaptic output from
MEC-II was inhibited during the entire task. This experiment was run as a control
for experiment 2 and experiment 3.
On day 1, during acquisition, there was a statistically significant difference in
the freezing behavior between the two genotypes (Figure 4-3 A, page 98)(2-way
ANOVA: Genotype x Time F(34,1020)=3.15, P < 0.0001; Time F(34,1020)=59.98,
P < 0.0001; Genotype F(1,1020)=13.87, P=0.0008). One month later, after 4 weeks
off-Dox, when then animals were placed in a novel chamber and three tones were
presented, there was a statistically significant difference between the genotypes, with
the mutants freezing less over the session (Figure 4-3 B, page 98)(2-way ANOVA:
Genotype x Time F(47,1410)=0.90, P=0.6704; Time F(47,1410)=3.76, P < 0.0001;
Genotype F(1,1410)=16.78, P=0.0003). More detailed analysis of the specific periods within day 2 allowed for analysis of generalized fear, tone memory, and freezing
behavior in the post-tone period.
There was a significant difference between the
genotypes in freezing during tone (Figure 4-3 C, page 98) (CT, 37.43% ± 2.72%; MT,
11.28% ± 1.82%; P < 0.0001) during the post-tone period (CT, 30.55% ± 3.06%; MT,
9.28% ± 2.06%; P < 0.0001) and during the 4 minutes prior to the first tone presentation (CT, 21.31% ± 4.61%; MT, 3.94% ± 1.73%; P=0.0034). The following day,
animals tested for freezing behavior to the conditioning context showed a statistically significant difference between genotypes, with the mutants freezing less over the
session (Figure 4-4 A, page 4-4) (2-way ANOVA: Genotype x Time F(14,420)=0.54,
P=0.9085; Time F(14,420)=3.09, P=0.0001; Genotype F(1,420)=8.46, P=0.0068).
MEC-II synaptic transmission is essential for tone and contextual memory during
the 20 s trace paradigm.
Experiment 2: Transmission OFF for Acquisition. Transmission ON for
Recall.
Blocking MEC-III output during acquisition but not during recall dis-
rupts tone and fear memory.
The 20-s trace fear conditioning paradigm was run in mice that were off Dox
for at least 4 weeks prior to acquisition day (Day 1), and then mice were put on
Dox for 4 weeks prior to "Day 2" and "Day 3" testing. In this experimental set-up,
MEC-III outputs were inhibited during acquisition, but functional for subsequent recall. On day 1, during acquisition, there was not a statistically significant difference
in the freezing behavior between the two genotypes (Figure 4-3 G, page 98) (2-way
ANOVA: Genotype x Time F(34,1428)=1.19, P=0.2099; Time F(34,1428)=93.16,
P < 0.0001; Genotype F(1,1428)=1.43, P=0.2381).
One month later, and after 4
weeks on Dox, when then animals were placed in a novel chamber and three tones were
presented, there was a statistically significant difference between the genotypes, with
the mutants freezing less during the session (Figure 4-3 H, page 98)(2-way ANOVA:
Genotype x Time F(47,1974)=1.45, P=0.0252; Time F(47,1974)=9.29, P < 0.0001;
Genotype F(1,1974)=10.04, P=0.0029). More detailed analysis of the specific periods within day 2 allowed for analysis of generalized fear, tone memory, and freezing
behavior in the post-tone period.
There was a significant difference between the
genotypes in freezing during tone (Figure 4-3 H, page98)(CT, 40.95% ± 2.42%; MT,
31.19% ± 2.58%; P=0.0108) and during the post-tone period (CT, 40.01% t 2.52%;
MT, 19.96% ± 2.75%; P < 0.0001) but not during the 4 minutes prior to the first
tone (CT, 24.38% ± 3.98%; MT, 14.95% ± 3.38%; P=0.0974).
The following day,
animals tested for freezing behavior to the conditioning context showed a statistically significant difference between genotypes, with the mutants freezing less over the
session (Figure 4-4 C, page 4-4)(2-way ANOVA: Genotype x Time F(14,588)=0.93,
P=0.5239; Time F(14,588)=9.53, P < 0.0001; Genotype F(1,588)=10.77, P=0.0021).
Thus, even when transmission is normal during recall, the expression of tone and contextual memory is disrupted, suggesting that the MEC-III is necessary for acquisition
and not recall of trace memory.
Experiment 3: Transmission ON for Acquisition. Transmission OFF for
Recall.
Functional MEC-III output is not necessary for recall of tone and fear
memory.
The 20 s trace fear conditioning paradigm was run in mice that were on Dox for
at least 4 weeks prior to acquisition (Day 1), and then taken off Dox for 4 weeks
prior to "Day 2" and "Day 3" testing. In this experimental set-up, MEC-II outputs
were normal during acquisiiton, but inhibited during subsequent recall. On day 1,
during acquisition, there was not a statistically significant difference in the freezing
behavior between the two genotypes (Figure 4-3 D, page 98)(2-way ANOVA: Genotype x Time F(34,1428)=1.02, P=0.4413; Time F(34,1428)=125.20, P < 0.0001;
Genotype F(1,1428)=0.92, P=0.3430).
One month later, after 4 weeks of Dox re-
moval when then animals were placed in a novel chamber and three tones were presented, there was not a statistically significant difference between the genotypes (Figure 4-3 E, page 98)(2-way ANOVA: Genotype x Time F(47,1974)=0.70, P=0.9407;
Time F(47,1974)=8.13, P < 0.0001; Genotype F(1,1974)=0.02, P=0.8827). More detailed analysis of the specific periods within day 2 allowed for analysis of generalized
fear, tone memory, and freezing behavior in the post-tone period. There was not
a significant difference between the genotypes in freezing during tone (Figure 4-3 F,
page 98)(CT, 38.21%±2.78%; MT, 38.20%±2.74%; P=0.9979), during the post-tone
period (CT, 33.00% ± 2.86%; MT, 31.16% ± 3.37%; P=0.6759) or in the 4 minutes
prior to the first tone presentation (CT, 18.80% ± 3.82%; MT, 19.43% ± 4.93%;
P=0.9196).
The following day, animals tested for freezing behavior to the condi-
tioning context showed no statistically significant difference between genotypes (Figure 4-4 B, page 4-4)(2-way ANOVA: Genotype x Time F(14,588)=1.31, P=0.1977;
Time F(14,588)=6.56, P < 0.0001; Genotype F(1,588)=0.02, P=0.8929). Thus, normal transmission during acquisition completely ameliorates the deficits seen when
transmission is off during acquisition.
....
..
............
OFF OFF
*
A
C*
908070-
00
t
6
~fIP
50
4030-
L
U
5
4
jap 3
10
20ut
Mutant
9
8
7
p11
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Control
10-
I
- i~;uaui~
0
ON OFF
Seconds
D
60
F
n.s.
9
8
7
6
5
4
ag3
2
1
%lip
r I
1
0
OFF ON
Seconds
G
120 180 240
Seconds
Seconds
60 120 180 240
Seconds
Seconds
H 9080-
0
~1~I
0
I
.
0
*Tone
Foot-Shock
Seconds
Seconds
60 120 180 240
Seconds
Figure 4-3: MEC-Ill is Necessary for Acquisition of 20 s Trace Conditioning.
Blue circles are control (MEC-II Cre+ mice) and Red circles are mutant (MEC-III
TeTx mice). In all cases conditioning day (A,D,G) was 1-month prior to testing.
(A-C) 20 s Trace Dox off-off-off (Experiment 1). (A) Conditioning in off-Dox mice.
(B-C) Freezing to tone during post-tone freezing assessed 1 month later in mice still
off Dox (C) is average of three tone/post-tone bouts in (B). (D-F) 20 s Trace Dox
on-off-off (Experiment 3). (D) Conditioning of on-Dox mice. (E-F) Freezing to tone
during post-tone freezing assessed 1 month later in mice off-Dox (F) is average of
three tone/post-tone bouts in (E). (G-I) 20 s Trace Dox off-on-on (Experiment 2).
(G) Conditioning of off-Dox mice. (H-I) Freezing to tone during post-tone freezing
assessed 1 month later in mice on-Dox, (I) is average of three tone/post-tone bouts
in (H). OFF OFF = Dox off for Acquisition - Dox off for Recall. ON OFF = Dox on
for Acquisition - Dox off for Recall. OFF ON = Dox off for Acquisition - Dox on for
Recall.
.......
...
........
....
......
.. ........
..
. . .. ............
Ae OFF OFF
80
Control
9 Mutant
701
60
00
5
40
U 9
C
30-
Tr
2007
L
100
0
60
12080
60
120
180
20
0
240
330
n0 .
800
Seconds
BON
n.s.
Dox
0
du
0
50
50-
P(N
triig
the
I
70
0
P
(U.
ON
COFF
OFF
20
40It
0
10
M
0
-
of-o8o0-onhpirt
60
120
180
Seconds
240
360
on
20
n
otx
0
training._(BFreezingt
60
120
180
240
30
Seconds
Figure 4-4: MEC-111 TeTx Mouse 20 s Trace/i-Month experiments with
Dox manipulations. (A) Freezing to conditioning context in mice off-Dox during
training, then off-Dox for 1-month prior to tone and context training. (B) Freezing to
conditioning context in mice on Dox during training, then off-Dox for 1-month prior
to tone and context training. (C) Freezing to conditioning context in mice off-Dox
during training, then on-Dox for 1-month prior to tone and context training. OFF
OFF = Dox off for Acquisition - Dox off for Recall. ON OFF = Dox on for Acquisition
- Dox off for Recall. OFF ON = Dox off for Acquisition - Dox on for Recall.
Table 4.2: Dox Induction Paradigms and 20 s Trace Fear Conditioning
Transmission
Transmission Conditioning
Tone
Post - Tone
Context
- - - -
Deficit
OK
Acquisition
OFF
Recall
OFF
-----Deficit
Deficit
--- - -Deficit
OFF
ON
ON
OFF
OK
OK
Deficit
OK
Deficit
OK
4.2.3
Deficit
Results: Control Fear Conditioning Paradigms
Functional MEC-III output is not necessary for contextual memory formation.
Although contextual fear memory forms normally in delay conditioning and in the
20 s trace paradigm when synaptic transmission is on for acquisition, it is disrupted
in each trace experiment where synaptic transmission is off during the acquisition.
We tested the MEC-III TeTx mice in a standard contextual memory conditioning
paradigm, with three shocks to match the conditioning paradigms used in the delay
and trace paradigms. We also administered five shocks to saturate the acquisition on
day 1. In this paradigm, there is no competing tone during acquisition. In both the
three and five shock paradigms, MEC-III TeTx mice could express contextual normal
fear memory on the following day.
3 Shock Contextual Fear Conditioning. Three-shock contextual fear conditioning was run in mice off-Dox for at least 4 weeks. During the conditioning on day 1,
three shocks were administered. Over the 706 s training period there was not a statistically significant difference in the freezing expression between the two genotypes (Figure 4-5 A, page 101)(2-way ANOVA: Genotype x Time F(34,748) =0.97, P=0.5140;
Time F(34,748) =35.19, P < 0.0001; Genotype F(1,748) =1.72, P =0.2034).
In
testing the fear expression to the training context on the subsequent day, there was
not a statistically significant difference between the two genotypes (Figure 4-5 B)(2way ANOVA: Genotype x Time F(14,308) =0.80, P=0.6741; Time F(14,308) =5.19,
P < 0.0001; Genotype F(1,308) =1.38, P =0.2529)
5 Shock Contextual Fear Conditioning. Five-shock contextual fear conditioning
was run in mice off-Dox for at least 4 weeks. During the conditioning on day 1, 5
100
...........
- --.-
....
....
. .......
............
*
A
Mutant
8
7
9B
80
Control
70
3-Shocks
60-
j&
20
10
20
10
0
00
Seconds
120
180
240
30
Seconds
C,
7
80
70
5-Shocks
6
20-
0
10
50
0
Foot-Shock
Seconds
60
120
180
240
300
Seconds
Figure 4-5: Contextual Fear Conditioning is not Disrupted in MEC-II TeTx
Mice. Blue circles represent Control (MEG-Ill Cre+) and Red circles represent Mutant (MEC-IJI TeTx) mice. (A-B) 3-Shock Contextual Fear Conditioning. (A) Day
1. Three shocks administered. (B) Day 2. Contextual Fear memory, assessed 24
hours later in training context. (C-D) 5-Shock Contextual Fear Conditioning. (C)
Day 1. Five shocks administered. (D) Day 2. Contextual fear memory, assessed 24
hours later in training context.
shocks were administered. Over the 706 s training period there was not a statistically
significant difference in the freezing expression between the two genotypes (Figure 45 C, page 101)(2-way ANOVA: Genotype x Time F(34,340) =0.58, P-0.9706; Time
F(341340) -69.24, P < 0.0001; Genotype F(1,340) =0.14, P =0.7201). In testing
the fear expression to the training context on the subsequent day, there was not a
statistically significant difference between the two genotypes (Figure 4-5 D) (2-way
ANOVA: Genotype x Time F(14,140) =0.97, P=0.4910; Time F(14,140) =3.77, P <
0.0001; Genotype F(1,140) =0.32, P =0.5862).
Functional MEG-Ill output is not necessary for backwards trace condi-
101
tioning.
The purpose of this experiment was to change the order of the stimulus presentation from the 20 s trace paradigm. In this case, there is still a 20 s trace between the
tone and the shock, but the shock is delivered first. This experiment was run in mice
that were off-Dox for at least 4 weeks prior to acquisition (Day 1). On day 1, during
acquisition, there was not a statistically significant difference in the freezing behavior between the two genotypes (Figure 4-6 A, page 103)(2-way ANOVA: Genotype
x Time F(34,340)=1.67, P=0.0126; Time F(34,340)=15.31, P < 0.0001; Genotype
F(1,340)=2.63, P=0.1362). On the second day of this task, 24 hours later, when animals were placed in a novel chamber and three tones were presented, there was not a
statistically significant difference between the genotypes (Figure 4-6 B, page 103)(2way ANOVA: Genotype x Time F(47,470)=1.14, P=0.2456; Time F(47,470)=5.09,
P < 0.0001; Genotype F(1,470)=0.45, P=0.5173).
More detailed analysis of the
specific periods within day 2 allowed for analysis of generalized fear, tone memory,
and freezing behavior in the post-tone period. There was not a significant difference between the genotypes in freezing during tone (Figure 4-6 C, page 103)(CT,
37.68% ± 5.55%; MT, 22.51%+± 4.485%; P=0.0526) during the post-tone period(CT,
25.98% ± 4.33%; MT, 18.83% ± 3.77%; P=0.2446) or in the 4 minute period prior
to the first tone presentation (CT, 7.32% ± 3.03%; MT, 10.29% ±5.15%; P=0.6068).
The following day, animals tested for freezing behavior to the conditioning context did not show a statistically significant difference between genotypes (Figure 4-6
D, page 103)(2-way ANOVA: Genotype x Time F(14,140)=0.90, P=0.5653; Time
F(14,140)=1.98, P=0.0234; Genotype F(1,140)=0.98, P=0.3466).
Functional MEC-III output is not necessary for 40 s trace conditioning.
We tested whether fear memory for tasks with different trace durations were affected by a silenced MEC-II output. Other brain structures, such as the prefrontal
cortex, and also involved with trace conditioning (Runyan et al. 2004) and thus the
deficit we see in the 20 s trace paradigm may or may not hold up with other trace
lengths. The purpose of this experiment was to elongate the trace period from 20 s
102
....
... .......
* Control
*Mutant
A
905
80
70.
60,
50-
0
0
0
40
3020
202
t'dfit'
1
Seconds
C
of~f
OP 4P 40
P 41
Seconds
n.s.
n.s.
8
7
6W
550
00
80
Seconds
3Tone
60
12
18
240
30
Seconds
Foot-Shock
Figure 4-6: MEG-IIl TeTX mouse and Backwards 'Ifrace Conditioning. All
mice off-Dox for acquisition and testing. Blue circles = Control (MEG-Ill Crew)
and Red circles = Mutant (MEC-IJI TeTx) mice. (A) Freezing during acquisition
paradigm. Three 2 s foot-shocks are administered followed by a 20 s trace and then
a 20 s tone. (B) Freezing in novel context during three 60 s tones and post-tone
periods. (C) Averaged freezing of combined three tone, post-tone episodes shown in
(B). (D) Freezing to training context.
103
to 40 s and see if the deficits in tone, post-tone and contextual freezing behavior were
similarly disrupted in the mutant animal.
This experiment was run in mice that were off-Dox for at least 4 weeks prior
to acquisition (Day 1). On day 1, during acquisition, there was not a statistically
significant difference in the freezing behavior between the two genotypes (Figure 47 A, page 105)(2-way ANOVA: Genotype x Time F(34,340)=0.93, P=0.5874; Time
F(34,340)=14.80, P < 0.0001; Genotype F(1,340)=0.02, P=0.8955). On the second
day of this task, 24 hours later, when animals were placed in a novel chamber and
three tones were presented, there was not a statistically significant difference between
the genotypes (Figure 4-7 B)(2-way ANOVA: Genotype x Time F(47,470)=0.70,
P=0.9371; Time F(47,470)=4.24, P < 0.0001; Genotype F(1,470)=0.00, P=0.9524).
More detailed analysis of the specific periods within day 2 allowed for analysis of generalized fear, tone memory, and freezing behavior in the post-tone period. There was
not a significant difference between the genotypes in freezing during tone (Figure 47 C)(CT, 51.39% ± 4.65%; MT, 48.13% ± 4.43%; P=0.6579), during the post-tone
period (CT, 38.97% +4.45%; MT, 39.87%
5.00%; P=0.9020), or in the 4 minute pe-
riod prior to the first tone presentation (CT, 16.19% ± 3.61%; MT, 18.50% t 3.84%;
P=0.7008).
The following day, animals tested for freezing behavior to the condi-
tioning context did not show a statistically significant difference between genotypes
(Figure 4-7 D)(2-way ANOVA: Genotype x Time F(14,140)=1.02, P=0.4327; Time
F(14,140)=3.40, P < 0.0001; Genotype F(1,140)=2.09, P=0.1790). This shows that,
for longer trace periods, context and tone fear memory can be formed and are not
dependent on MEC-III synaptic output.
MEC-III TeTx mice do not have disrupted fear memory in an un-paired
conditioning paradigm.
The purpose of this experiment was to scramble the tone-shock pairings. The
phenotype seen in the MEC-II TeTx mice in the 20 s trace paradigm may have
less to do with the trace per-se, and more to do with the mere presentation of the
the tone CS and foot-shock US presented separately during conditioning.
104
To re-
.
*
A
B
90
80
7060.6
50.5
Control
*Mutant
9
0
20.
90
a
4
W640
&30
&t
3&-,-----
Seconds
Seconds
n.s.
8
5
4
n.s.
8G
77'
70
6G
30
20
10
3
0i
2
0
60
120
180
240
300
Seconds
Seconds
Tone Foot-Shock
Figure 4-7: MEC-III TeTX mouse and 40 s Trace Conditioning. All -mice
off-Dox for acquisition, and testing. Blue circles = Control (MEC-III Cre+) and Red
circles = Mutant (MEC-III TeTx) mice. (A) Freezing during acquisition paradigm.
Three 20 s tones are played and a 2 s foot-shock is administered 40 s after termination
of tone. (B) Freezing in novel context during three 60 s tones and post-tone periods.
(C) Averaged freezing of combined three tone, post-tone episodes shown in (B). (D)
Freezing to training context.
105
. . ...............
move any consistent temporal relationship between the tone and shock, we scrambled the trace durations within the acquisition day. This experiment was run in
mice that were off-Dox for at least 4 weeks prior to acquisition (Day 1).
On day
1, during acquisition, there was not a statistically significant difference in the freezing behavior between the two genotypes (Figure 4-8 A, page 107)(2-way ANOVA:
Genotype x Time F(34,476)=0.78, P=0.8089; Time F(34,476)=40.44, P < 0.0001;
Genotype F(1,476)=0.23, P=0.6361). On the second day of this task, 24 hours after day 1, when then animals were placed in a novel chamber and three tones were
presented, there was not a statistically significant difference between the genotypes
(Figure 4-8 B)(2-way ANOVA: Genotype x Time F(47,658)=1.28, P=0.1066; Time
F(47,658)=4.33, P < 0.0001; Genotype F(1,658)=0.52, P=0.4825).
More detailed
analysis of the specific periods within day 2 allowed for analysis of generalized fear,
tone memory, and freezing behavior in the post-tone period. There was not a significant difference between the genotypes in freezing during tone (Figure 4-8 C)(CT,
39.97% i 3.69%; MT, 35.06% ± 3.82%; P=0.3653), during the post-tone period (CT,
38.13% i 5.10%; MT, 28.74% ± 4.54%; P=0.1894), or in the 4 minute period prior to
the first tone presentation (CT, 17.97% ± 4.18%; MT, 16.86% ± 6.93%; P=0.8878).
The following day, animals tested for freezing behavior to the conditioning context
did not show a statistically significant difference between genotypes (Figure 4-8 D) (2way ANOVA: Genotype x Time F(14,196)=1.65, P=0.0694; Time F(14,196)=4.06,
P < 0.0001; Genotype F(1,196)=0.01, P=0.9432).
Thus, the consistent temporal
relationship between the tone and foot-shock is a likely useful for forming tone and
contextual memories in normal mice.
Tone presentation alone does not elicit any differences between MEC-III
TeTx and control mice.
The purpose of this experiment was to see if there was a difference in freezing
between the two genotypes when presented with the tone only. This experiment
was run in mice that were off-Dox for at least 4 weeks prior to acquisition (Day
1).
On day 1, during acquisition, there was not a statistically significant differ-
106
....
......
....
............
........
.
..
....
...
........
::::::::
:::::::
::..
..:..........
:...
... .....
.
A g-B
L0.44IL
80
Control
Mutant
90*
8
70-
7
e0 Conro
500
40
S30-
20-
0
Seconds
90
Seconds
n.s.
n.s.
80
8
70
70
50
403020-
10
1
0
3 Tone
Foot-Shock
60
120
180
240
300
Seconds
SecOnds
Figure 4-8: MEC-III is not Required for Association of Scrambled CS-US
Pairings. All mice off-Dox for acquisition and testing. Blue circles = Control (MECIII Cre+) and Red circles = Mutant (MEC-III TeTx) mice. (A) Freezing during
acquisition paradigm. Three shocks (red lines) administered at varying times relative
to tone presentations. (B) Freezing in novel context during three 60 s tones and
post-tone periods. (C) Averaged freezing of combined three tone, post-tone episodes
shown in (B). (D) Freezing to acquisition context.
107
ence in the freezing behavior between the two genotypes (Figure 4-9 A, page 109)(2way ANOVA: Genotype x Time F(34,476)=1.13, P=0.2879; Time F(34,476)=2.36,
P < 0.0001; Genotype F(1,476)=0.00, P=0.9981). On the second day of this task, 24
hours later, when then animals were placed in a novel chamber and three tones were
presented, there was not a statistically significant difference between the genotypes
(Figure 4-9 B)(2-way ANOVA: Genotype x Time F(47,658)=0.78, P=0.8490; Time
More detailed
F(47,658)=2.76, P < 0.0001; Genotype F(1,658)=1.44, P=0.2497).
analysis of the specific periods within day 2 allowed for analysis of generalized fear,
tone memory, and freezing behavior in the post-tone period. There was not a significant difference between the genotypes in freezing during tone (Figure 4-9 C)(CT,
13.22% ± 2.25%; MT, 13.58% i 2.77%; P=0.9191), during the post-tone period (CT,
4.59% ± 0.77%; MT, 8.94% ± 2.28%; P=0.0531), or in the 4 minute period prior to
the first tone presentation (CT, 2.69% ± 1.28%; MT, 4.64% ± 3.21%; P=0.5485). The
following day, animals tested for freezing behavior to the conditioning context did
not show a statistically significant difference between genotypes (Figure 4-9 D) (2way ANOVA: Genotype x Time F(14,196)=0.87, P=0.5969; Time F(14,196)=2.35,
P=0.0051; Genotype F(1,196)=0.00, P=0.9770).
Thus, the differences on the con-
ditioning day during the 20 s trace paradigm when MEC-III synaptic transmission
is off are unlikely due simply to differences in reaction to the tone presented in the
environment.
Table 4.3: Fear Conditioning Paradigms and Results
ConditioningParadigm Conditioning Tone
OK
OK
Delay
N/A
OK
3-Shock Contextual
N/A
OK
5-Shock Contextual
Deficit Deficit
20 s Trace
OK
OK
40 s Trace
OK
OK
Backwards Trace
OK
OK
Un-Paired
OK
OK
Tone-Only(no shock)
108
Post - Tone
OK
N/A
N/A
Deficit
OK
OK
OK
OK
Context
OK
OK
OK
Deficit
OK
OK
OK
OK
---
_ -,
zZZ
.
.
-
.......
....
. ......
__:W
_::: -
""-
_
P
..
*
Control
Mutant
9.9
80
70
60-
90
80
70-
50
60
50
T40
h
0
2t0-
40
.
3
0
7160
Seconds
ScnsD
6
50
4
LP
5
40-
2
2
1
30
0
STone
0
60
120
180
240
300
Seconds
Seconds
Figure 4-9: MEC-III TeTX mouse and Reaction to Tone Alone All mice
off-Dox for acquisition, and testing. Blue circles = Control (MEC-II Cre+) and
Red circles = Mutant (MEC-III TeTx) mice. (A) Freezing during tone presentation
paradigm. No foot-shocks administered. (B) Freezing in novel context during three
60 s tones and post-tone periods. (C) Averaged freezing of combined three tone,
post-tone episodes shown in (B). (D) Freezing to first context.
109
4.3
4.3.1
Working Memory
Delayed Nonmatch-To-Place
The T-maze task is useful for testing working memory in mice (Deacon and Rawlins
2006). In this task, mice are placed in the base of the "T" and allowed to run to
either arm of the "T." In the first phase of the task, the "Sample" phase, the animal is
guided to choose one arm only, where it receives a reward. The mouse is then placed
back into the start of the maze, and the "Choice" phase begins. In the "Choice"
phase, the animal can run to either arm, but and is only rewarded for choosing the
opposite arm (delayed nonmatch-to-place, DNMP) or the same arm (delayed matchto-place, DMP) from the "Sample" phase. In both cases, the animal must remember
what they did on the "Sample" phase to perform correctly on the "Choice" phase.
This is called working memory (Olton et al. 1979).
In the non-rewarded version of the T-maze, mice tend to visit the arm different
from what they visited on the first trial, in what is termed "spontaneous alternation" (Dember and Richman 1989). Similarly to the rewarded tasks, the mice must
maintain a representation of what choice was made on the previous trial. The natural
tendency for mice is to spontaneously alternate. Due to the lack of reward, however,
mice only run the spontaneous alternation version of the T-maze for a small number
of trials, as they become un-interested in the task. In a rewarded version of the Tmaze in food deprived mice, normal mice can become 90% successful at choosing the
arm not visited in the first part of a trial (Deacon and Rawlins 2006) and mice run
the behavior as long as they stay interested in the reward.
Successful performance on the DNMP task is highly susceptible to hippocampal
damage (Reisel et al. 2002) (Deacon and Rawlins 2005) (Rawlins and Olton 1982).
Due to the persistent activity in EC neurons, and its implications in working memory
(Hasselmo et al. 2009), we tested the role of the functional output of MEC-II in this
task.
The MEC-III to CA1 pathway is required for working memory in the
110
DNMP T-maze.
The DNMP version of the T-maze was run in mice off-Dox for at least 4 weeks.
MEC-III TeTx were significantly impaired on the rewarded DNMP version of the Tmaze, as compared to controls. There was a significant difference between genotypes
in performance of the DNMP version of the T-Maze task as measured by fraction
of correct alternations per day, with 10 trials per day and over 12 days of the task
(Figure 4-10 A, page 112) (2-way ANOVA: Genotype x Day Block F(11,330)=2.02,
P=0.0257; Day F(11,330)=1.51, P=0.1253; Genotype F(1,330)=25.26, P < 0.0001).
Additionally, there was a significant difference between the genotypes in the time
to make the choice during the choice-phase of the task, with the mutants taking
significantly longer to make an arm choice (Figure 4-10 D)(CT, 22.05%± 1.24%; MT,
26.82% ± 1.41%; P=0.0140).
There was a significant difference for each genotype
in preferring to chose the "Right" arm over the "Left" arm (Figure 4-10 C). For
the control mouse, the proportions of choices towards the right arm were significantly
greater than the left arm choices (Right, 0.5527%±0.0127%; Left, 0.4473%i0.0127%;
P < 0.0001). For the mutant mouse, the proportions of choices towards the right arm
were significantly greater than the left arm choices as well (Right, 0.6313%±0.0127%;
Left, 0.3687%±0.0127%; P < 0.0001). Additionally, there was a significant difference
between the genotypes in choice of either left or right arm choice with mutant mice
significantly choosing the right arm more than the control mice (CT, 0.5527% ±
0.0127%; MT, 0.6313% ± 0.0127%; P < 0.0001).
4.3.2
8-Arm Radial Maze Task
The mono-synaptic pathway is not required for working memory in the
8-arm radial arm task
A more taxing working memory task is the 8-arm radial maze task. In this task,
mice are given 1 trial per day for 18 days. On each trial, the food-deprived mouse can
receive a single reward at the end of each arm. The most efficient way to complete
the task is for the mouse to visit each arm once, and to complete the task in that
manner, the mouse must remember which arms it had previously visited on that day.
111
............
-- .- . ...........
A
B
1.00-
0.75-
1.00-
Control
Mutant
0.75-
0.50-
0.50
U.
0.250
e
I.
1 2 3~ 4 5 6 789
C
025
0
10 1 12
Day
1
2
3
4
5
6
2-Day Block
3025
0.75'
20
0.50
15
0.25"
10
5-
0.00 -
Control
Mutant
Genotype
Direction
Figure 4-10: MEC-III TeTx mice are Impaired in Rewarded DNMP T-Maze.
(A) Fraction of successful alternations over 12 days of training. Fraction of successful
alternations denotes number of correct choices during choice task divided by number
of possible choices during during day. 10 trials per day. (B) Fraction of successful
alternations over 12 days, 12 days shortened into 6 2-day blocks. Blue is control
(MEC-III Cre+ mice) and Red is mutant (MEC-II TeTx mice). (C) Proportion of
right or left arm choices over all 12 days. (D) Averaged time to make choice over all
12 days. Determined from when choice trials started to when mouse chose arm.
112
Revisiting an arm that the animal has already visited and consumed the reward is
called a "working memory" error. This task is known to be hippocampally-dependent
(Olton and Papas 1979) (Becker et al. 1980), but it is questionable if mice can really
perform this task as well as rats (Mizumori et al. 1982). Additionally, with only 8
arms, C57B16 mice (the genetic background used for generation of the MEC-III Cre+
mice) make on average five working memory errors after they reach an asymptomatic
level of learning (Crusio et al. 1987). The 8-arm radial maze task was run using mice
off-Dox for at least 4 weeks.
8-Arm radial maze task: Revisiting errors and working memory errors. There was no significant difference between genotypes in revisiting errors (entering an arm that animal had already visited (Figure 4-11 A, page 115) (2-way ANOVA:
Genotype x 2-Day Block F(8,392)=0.65, P=0.7364; 2-day Block F(8,392)=17.66,
P < 0.0001; Genotype F(1,392)=0.75, P=0.3894) or in working memory errors (entering an arm that animal had already visited and consumed reward) (Figure 4-11
B)(2-way ANOVA: Genotype x 2-Day Block F(8,392)=0.89, P=0.5286; 2-day Block
F(8,392)=7.28, P < 0.0001; Genotype F(1,392)=0.84, P=0.3626).
Thus, MEC-II
TeTx mice do not show any working memory deficits in this task. Both controls and
mutants, however still do make nearly 6 working memory errors, with only 8 arms.
This task may not be testing working memory.
8-Arm radial maze task: Latency, distance, number of arms in first 8arm choices, omissions. There was no significant difference between genotypes in
omissions, which are counts of entering an arm but not eating the reward, (Figure 4-11
C, page 115)(2-way ANOVA: Genotype x 2-Day Block F(8,392)=0.63, P=0.7539; 2day Block F(8,392)=32.30, P < 0.0001; Genotype F(1,392)=0.09, P=0.7714). There
was no significant difference between the two genotypes in the total distance traveled
(Figure 4-11 D)(2-way ANOVA: Genotype x 2-Day Block F(8,392)=0.37, P=0.9375;
2-day Block F(8,392)=14.75, P < 0.0001; Genotype F(1,392)=2.71, P=0.1059).
There was no significant difference between the two genotypes in the time to finish
the task, (Figure 4-11 E)(2-way ANOVA: Genotype x 2-Day Block F(8,392)=1.01,
P=0.4273; 2-day Block F(8,392) = 21.39, P < 0.0001; Genotype F(1,392) = 0.12,
113
P=0.7331). Thus, we do not see differences in any of these analyses.
4.3.3
Anxiety and Pain Sensitivity
There are no differences in anxiety or pain sensitivity in MEC-III TeTx
and MEC-III Cre+ mice.
Elevated Plus Maze. Anxiety can enhance an animal's response to an aversive task. Genotype-specific anxiety differences could mask or enhance behavioral
results in aversive task, such as with fear conditioning so it is important to have
an independent assay of anxiety in two mouse strains before interpreting the results
of an aversive task. The elevated plus maze is used to assay an animal's anxiety
(Lister 1987). In this task, animals can explore open or closed arms on an elevated
platform. More anxious animals avoid the open arms, while less anxious animals
explore the open arms more. There was no significant differences between the two
genotypes in the percentage of time spent in the open and closed arms (Figure 4-12
A, page 116)(MT, 15.20%
5.9%; CT, 12.31%
6.2%; P=0.7388 for percentage time
in the open arms), the frequency of visits to each arm type (Figure 4-12 B)(MT,
23.66 ± 8.6; CT, 22.55 ± 7.7; P=0.9249 for the number of visits to the open arms),
the total distance traveled (Figure 4-12 C)(MT, 1962cm ± 137; CT, 1857cm ± 100;
P=0.5485), and the velocity of each genotype during the task (Figure 4-12 D)(MT,
3.44cm/sec ± 0.23; CT, 3.19cm/sec ± 0.18; P=0.4133). Thus, as assessed by the elevated plus maze, MEC-II TeTx mice do not display heightened anxiety as compared
to the MEC-III Cre+ mice.
Hot Plate. To exclude the possibility that the differences in behavioral responses
in fear memory tasks are due to a difference in pain sensitivity, we compared both
strains of mice on the hot plate (Wilson and Mogil 2001). Performance in the hot
plate test of pain sensivity was performed in mice that had been off-Dox for at least
4 weeks. There was no significant differences in the latency to lift their front paws
(MT, 7.9sec ± 0.60;CT, 7.9sec ± 0.42; P=0.9871). See 4-13 on page 117.
114
.....................
e
A
Revisiting Errors
20-'
I.
Control
* Mutant
Working Memory Errors
15'
15
I.a
10
10.1
5-
0
n
1
3
2
2-day Block
4
5
6 7
8
9
2-day Block
Total Distance Traveled
Omissions
3000-
2500-
2000
1500-0
1
2
3
4
S0 AB4ock
2
2-day Block
2-day Block
Time to Finish Task
700600I
500-
1
1
1
400,ann .
0
.
.
1
2
3
4
5
6
7 8
1
1
9
2-day Block
Figure 4-11: MEC-III TeTx mice not Impaired in the 8-arm Radial Maze
Task. All data averaged over 2-Day Blocks. Total of 18 Days of learning task.
Control (MEC-III Cre+ mice) are listed in Blue and Mutant (MEC-III TeTx) are
listed in Red. (A) Revisiting Errors: Averaged number of times mouse visited an
arm that it previously visited an arm it had already visited. (B) Working Memory
Errors: Averaged number of times mouse re-visisted an arm that it had already taken
food reward from. (C) Omissions: averaged number of times mouse visited arm but
did not take food reward. (D) Number of arm choices during the first 8 arm choices
over the length of the test. (E) Averaged distance traveled during each 2-day block.
(F) Averaged time to finish task. Maximum time per day was 900 s.
115
------....
....
W- .- ... .....
...........
Elevated Plus Maze
Frequency of visits inouter parts of arms:
% in Closed, % in Open
Elevated Plus Maze
Of time in outer parts of arms:
%inClosed, %inOpen
Control
Mutant
100,
75
50
25
Open
Arm Type
C
Closed
Arm Type
Distance in EPM
Velocity in EPM
2250
2000
1750
1500
1250
1000,
500"
70'
2500- -
Control
Control
Genotype
Genotype
Mutant
Figure 4-12: MEC-III TeTx mice do not have Elevated Anxiety in Elevated
Plus Maze. Blue Bars represent Control (MEC-II Cre+) and Red Bars represent
Mutant (MEC-III TeTx) mice. (A) Percentage of total time spent exploring open
or closed arms. (B) percentage of total visits to all arms broken down into percent
frequency of visits into the open and closed arms. (C) Average total distance (cm)
during the ten minute session. (D) Averaged velocity (cm/sec) during the ten minute
session).
116
---
--
----------------------
..............
....
..........
. ......
..
......
..
...
..
..
..............
Hot Plate
. .....
..
..........
...........
..........
----------------....
.........
_
Control
Mutant
10C00
Control
Mutant
Genotype
Figure 4-13: MEC-II do not Show Altered Pain Sensitivity: Hot Plate. Blue
Bar represents Control (MEC-III Cre+) and Red Bar represents Mutant (MEC-III
TeTx) mice. Averaged seconds to front-paw removal from hot plate for each genotype.
4.4
CA3 Functional Output: 20 s Trace and DNMP
T-Maze
The CA1 subregion of the hippocampus receives synaptic input from MEC-III, LECIII, nucleus reuniens, as well as from CA3 (Witter 1989). We have shown the importance of the input from MEC-Ill in the acquisition of tone and contextual fear
memory in the 20 s trace paradigm, as well as in performance in the DNMP T-maze.
To more throughly investigate the importance of MEC-III input in these behavioral
tasks, we tested the CA3-TeTx mice on these same paradigms.
CA 3 functional output is necessary for contextual but not tone fear memory in trace conditioning.
This experiment was run with CA3-TeTx mice. The 20 s trace fear conditioning
paradigm was run in mice that were off-Dox for at least 4 weeks prior to acquisition
(Day 1), and kept off-Dox for the following 2 days of experiments. On day 1, during
117
acquisition, there was not a statistically significant difference in the freezing behavior between the two genotypes (Figure 4-14 A, page 119)(2-way ANOVA: Genotype
x Time F(34,1292)=1.18, P=0.2261; Time F(34,1292)=84.47, P < 0.0001; Genotype F(1,1292)=0.10, P=0.7515). On the second day of this task, 24 hours after the
first day when then animals were placed in a novel chamber and three tones were
presented, there was not a statistically significant difference between the genotypes
(Figure 4-14 B)(2-way ANOVA: Genotype x Time F(47,1786)=2.06, P < 0.0001;
Time F(47,1786)=17.41, P < 0.0001; Genotype F(1,1786)=2.07, P=0.1584). More
detailed analysis of the specific periods within day 2 allowed for analysis of generalized fear, tone memory, and freezing behavior in the post-tone period. There was not
a significant difference between the genotypes in freezing during tone (Figure 4-14
C)(CT, 45.15% ± 2.98%; MT, 44.04% ± 2.77%; P=0.7863). There was a weak but
statistically significant difference in the post-tone freezing (CT, 32.12% ± 2.23%; MT,
24.68%±2.56%; P=0.0306) and not a statistically significant difference in the 4 minute
period prior to the first tone presentation (CT, 12.20% ± 1.65%; MT, 7.45% ± 1.83%;
P=0.0615).
The following day, animals tested for freezing behavior to the condi-
tioning context did show a statistically significant difference between genotypes, with
mutants freezing less during the session (Figure 4-14 D)(2-way ANOVA: Genotype
x Time F(14,532)=1.15, P=0.3136; Time F(14,532)=3.65, P < 0.0001; Genotype
F(1,532)=4.74, P=0.0358). These results show that functional CA3 output is necessary for proper contextual conditioning in the 20 s trace paradigm, but not necessary
for proper tone conditioning.
CA3 functional output is not necessary for contextual or tone fear memory in delay conditioning.
The delay fear conditioning paradigm was run in mice that were off-Dox for at
least 4 weeks prior to acquisition (Day 1), and mice were kept off Dox for the following
two days of experiments. On day 1, during acquisition, there was not a statistically
significant difference in the freezing behavior between the two genotypes (Figure 4-15
A, page 121) (2-way ANOVA: Genotype x Time F(34,204)=0.63, P=0.9470; Time
F(34,204)=16.00, P < 0.0001; Genotype F(1,204)=0.07, P=0.8049).
118
On the sec-
.....
........
- .....
..
..........................................
...
* Control
A
B90*
80
7
707
Mutant
80
6060
50
at
20
30
20.
10
120
0
240
n.S.*
360
480)
600
7200
0
70
60
50
60
5
400
20
20 -
10
0
0
0i
0
Foot-Shock
960
Seconds
9080
80
70
Tone
120 240 360 480 600 720 840
Seconds
Seconds
60
120
1
tf}
180
240
300
Seconds
Figure 4-14: CA3-TeTx Mouse is Normal in Tone but is Impaired in Contextual Learning in 20 s Trace Paradigm. All mice off-Dox for acquisition and
testing. Blue circles = Control (CA3-Cre+) and Orange circles = Mutant (CA3TeTx) mice. (A) Freezing during acquisition paradigm. Three 20 s tones are played
and a 2 s foot-shock is administered 20 s after termination of tone. (B) Freezing in
novel context during three 60s tones and post-tone periods. (C) Averaged freezing of
combined 3 tone, post-tone episodes shown in (B). (D) Freezing to training context.
119
ond day of this task, 24 hours after the first day, when the animals were placed in
a novel chamber and three tones were presented, there was not a statistically significant difference between the genotypes (Figure 4-15 B)(2-way ANOVA: Genotype
x Time F(47,282)=1.02, P=0.4420; Time F(47,282)=7.04, P < 0.0001; Genotype
F(1,282)=2.98, P=0.1351). More detailed analysis of the specific periods within day
2 allowed for analysis of generalized fear, tone memory, and freezing behavior in the
post-tone period. There was not a significant difference between the genotypes in
freezing during tone (Figure 4-15 C)(CT, 60.34% ± 4.49%; MT, 58.68% ± 2.74%;
P=0.7739), not a statistically significant difference in the post-tone freezing (CT,
32.23% ± 6.64%; MT, 19.93% ± 2.85%; P=0.1030) and not a statistically significant difference in the 4 minute period prior to the first tone presentation (CT,
10.59% ± 5.63%; MT, 2.29% ± 1.7%; P=0.2404). The following day, animals tested
for freezing behavior to the conditioning context did not show a statistically significant difference between genotypes (Figure 4-15 D) (2-way ANOVA: Genotype x Time
F(14,84)=1.79, P=0.0538; Time F(14,84)=2.83, P=0.0016; Genotype F(1,84)=0.65,
P=0.4521). These results indicate that CA3 output is not required for proper delay
conditioning. This experiment was run with only four mice of each genotype, but
importantly, this data matches delay conditioning in previous studies done in the
CA3-TeTx mice (Nakashiba et al. 2009), in which "recent" tone and contextual fear
memory was not disrupted. It is difficult to understand from these results, why the
control mice freeze more than controls in the third minute post tone (see figure 4-15
C, on page 121).
Table 4.4: CA3 vs. MEC-II in Delay and 20 s Trace Conditioning
Genotype
MEC-III TeTx
MEC-II TeTx
CA3-TeTx
CA3-TeTx
Paradigm Conditioning Tone
OK
OK
Delay
Deficit Deficit
Trace
OK
OK
Delay
OK
OK
Trace
120
Post - Tone
OK
Deficit
OK
Deficit
Context
OK
Deficit
OK
Deficit
.......................................................
................................................................................
................
"
Control
" Mutant
:II~
~d1~
i1iii
120
240
__
f] I __
0
360
480
600
720
Seconds
C
Seconds
90
80
7
F? 6
5
4
20
10
P
0
1b
Tone
Foot-Shock
60
120
180
240
300
Seconds
Seconds
Figure 4-15: CA3-TeTx Mouse Not Impaired Delay Conditioning. All mice
off-Dox for acquisition, and testing. Blue circles = Control (CA3-Cre+) and Orange
circles = Mutant (CA3-TeTx) mice. (A) Freezing during acquisition paradigm. Three
20 s tones are played and co-terminate with a 2 s foot-shock. (B) Freezing in novel
context during three 60 s tones and post-tone periods. (C) Averaged freezing of
combined 3 tone, post-tone episodes shown in (B). (D) Freezing to training context.
121
The CA3 to CA1 pathway is not required for working memory in the
DNMP T-maze
We tested the CA3-TeTx mice in the same DNMP T-maze task as the MECIII TeTx mice were run in to assess whether this deficit was MEC-III specific or, if
any manipulation to a common downstream target would disrupt this behavior. We
found that CA3 functional output is not necessary for performance in this task. Thus,
MEC-III provides the necessary input into the hippocampal complex to perform this
working memory task.
There was not a significant difference between genotypes in performance of the
DNMP version of the T-Maze task as measured by fraction of correct alternations per
day, with 10 trials per day and over 12 days of the task (Figure 4-16 A, page 123)(2way ANOVA: Genotype x Day Block F(11,154)=0.67, P=0.7672; Day F(11,154)=0.21,
P=0.9968; Genotype F(1,154)=0.27, P=0.6088). Thus functional output from CA3
is not required for performance in the DNMP T-maze task.
There were some differences between the two mice genotypes in this task, however.
There was a significant difference between the genotypes in the time to make the
choice during the choice-phase of the task, with the mutants making the choice faster
than controls (Figure 4-10 D, page 123)(CT, 20.39% ± 1.53%; MT, 9.84% ± 0.79%;
P < 0.0001). There was a significant difference for each genotype in preferring to
chose the "Right" arm over the "Left" arm (Figure 4-10 C). For the control mouse,
the proportions of choices towards the right arm were significantly greater than the
left arm choices (Right, 0.5448% ± 0.018%; Left, 0.4594% ± 0.018%; P=0.0012). For
the mutant mouse, the proportions of choices towards the right arm were significantly
greater than the left arm choices as well (Right, 0.5729% ± 0.022%; Left, 0.4271% i
0.022%; P < 0.0001). There was not a significant difference between the genotypes
in choice of either left or right arm choice. Mutant mice and control mice made
their "Right" arm choices the same fraction of the time (CT, 0.5448% ± 0.018%;
MT, 0.5729% ± 0.022%; P=0.3314). Overall, both genotypes has a similar preference
to choose the right arm over the left, over all 120 trials. Additionally, the mutant
animals ran the "choice" task in less time than controls, possibly reflecting a different
122
.
. ............
. ......
...
. .. .....
. .......
A
n.s.
II
nr
'II.
"
B
0.75
f
t I
i
11
0.250
1 2
3
4
7
0.50-
25+
10 11 12
2-Day Block
Day
30]i
ill
n.s.
1.00-
11111111
Control
" Mutant
*
0.50t-
A.
0.00,
Mutant
Control
Genotype
Right
Direction
Figure 4-16: CA3-TeTx T-Maze: Rewarded DNMP. (A) Fraction of successful
alternations over 12 days, 12 days shortened into 6 2-Day Blocks. (B) Fraction of
successful alternations over 12 days of training. Fraction of successful alternations
denotes number of correct choices during choice task divided by number of possible
choices during during day. 10 trials per day. (C) Proportion of right or left arm choices
over all 12 days. (D) Averaged time to make choice over all 12 days. Determined
from when choice trials started to when mouse chose arm. Blue is control (CA3-Cre+
mice) and Orange is mutant (CA3-TeTx mice).
strategy or altered motivation during the task. Regardless, both genotypes learned
the task over time and did not demonstrate any working memory deficits.
4.5
Discussion
In this chapter, we show three distinct phenotypes. First, MEC-III is required for
acquisition of tone memory in the 20 s trace paradigm. Second, MEC-III is required
for acquisition of contextual memory in the 20 s trace paradigm. Third, MEC-III is
required for successful performance in the rewarded DNMP version of the T-maze.
123
This section discusses all of these phenotypes independently.
4.5.1
Medial Entorhinal Cortex is Required for Acquisition
of Tone Memory in the 20 s Trace Paradigm
It is generally accepted that fear associations are encoded and stored via plasticity
at extra-amygdala to amygdala synapses. In delay conditioning, when the presentation of the tone and foot-shock co-terminate, the association of the two stimuli
happen in the lateral amygdala (LA)
(Romanski et al. 1993) (Fanselow and Kim
1994) (Fanselow and LeDoux 1999) (Maren 2005) (Kim and Jung 2006) (Cahill et al.
1999). The tone representation is sent to the amygdala by two independent pathways, one from the auditory cortex and one from the auditory thalamus (Romanski
and LeDoux 1992). It is thought that the shock representation arrives at the amygdala via other thalamic and cortical areas (LeDoux 2000). For a depiction of encoding
in delay conditioning, please see "A" in 4-17 on page 128 . Neurons representing the
tone and foot-shock are synapsing in the same area. In delay conditioning the stimuli
are presented at the same time, therefore these neurons fire-together, and by Hebbian
plasticity, they wire together (Hebb 1949). Plasticity in delay conditioning occurs at
the thamalic-LA synapses. Presentation of the tone elicits a freezing behavior (fear
memory) because it directly activates the modified thalamic-LA synapse (see "B" in
4-17). For all fear conditioning, the fear association is stored in neurons within the
the lateral or basal nuclei of the amygdala. These neurons send projections to the
central nucleus, which is required for fear expression (Amorapanth et al. 2000).
In trace conditioning, Hebbian plasticity in the amygdala cannot directly link the
tone and foot-shock, as the neurons representing these two stimuli do not fire together in the same brain region. Another brain structure is needed to hold the tone
representation so that it can be sent to the amygdala coincident to the arrival of the
shock signal there, see "C" in 4-18 on page 129. It is thought that the prefrontal cortex stores the tone representation online for later association with the foot-shock, as
lesions to the prefrontal cortex disrupt trace conditioning (McLaughlin et al. 2002),
124
but other brain structures or downstream pathways could also be involved (Esclassan
et al. 2009). One of these structures may be the hippocampus (see 4-17 In tone
memory recall, it is likely that the tone as the recall cue reaches the fear memory engram in the amygdala through the "other brain structures" modified during training,
see "D" in 4-17.
We demonstrate that disruption of synaptic transmission from the MEC-III specifically disrupts fear memory to tone in trace but not delay conditioning. Using the
temporal control of TeTx in these mice, we have revealed that the MEC-III output
is necessary specifically for acquisition of these tasks but not for recall.
Trace conditioning is hippocampal dependent (Solomon et al. 1986) (McEchron
et al. 1998) and requires plasticity in CA1 (Huerta et al. 2000). Additionally, the
basolateral-amygdala (BA) receives input from the hippocampus (Pitkanen 2000) (Sah
et al. 2003), suggesting that the plasticity at the BA-hippocampus synapses may be
involved in trace fear conditioning. If this model is correct, an attractive possibility
would be that tone input is integrated into a contextual representation in the hippocampus via the MECIII-CA1 connection during acquisition, and plasticity at the
hippocampus-amygdala synapses, to which the shock signal converges, may store the
fear association. Given that the output of MEC-III goes into the hippocampus, it
makes sense that the hippocampus is the "other brain structure" which sends the
tone representation to the amygdala for association with the foot-shock during the
trace, see "C" in 4-17.
How can the MECIII-CA1 connection keep holding the tone during the trace, as a
component of the contextual representation and send it to the amygdala in coincidence
with the arrival of the shock signal so that Hebbian plasticity will be established at
this synapse? The necessity of MEC-III during acquisition of the features in trace
conditioning fits the hypothesis that persistent activity in the EC allows for the
integration of multiple synaptic inputs that are separated in time (Hasselmo et al.
2009). In this case, the tone and foot-shock, when separated by the trace, cannot
be properly associated due to a disruption in the functional output of MEC-IJI. A
mechanism is needed to hold online the representation of the tone for association with
125
the shock, as the tone does not co-terminate with the shock. The tone representation
could be sustained by a cellular mechanism like persistent activity in MEC-IlI, which
provides a constant signal to CAl to incorporate the tone as part of its representations.
However, during recall, the MEC-III-CA1 connection is not required. We hypothesize that the tone input enters the hippocampus via the MEC-II-DG-CA3-CA1
connection. This leads to activation of the hippocampal representation in CAl, which,
in turn, activates the modified hippocampal-amygdala synapses, resulting in fear expression.
The role of the MEC-II-DG-CA3-CA1 circuit in activation of the tone representation in the hippocampus is suggested from "Experiment 3" in which synaptic
transmission in MEC-III was turned on for acquisition and turned off for recall. In
this experiment, expression of tone memory was not impaired. If MEC-Ill is required
during the acquisition, but not during recall, then successful memory retrieval is supported by a different circuit. In this model, tone memory recall can happen by one of
two pathways: Via the MEC-III-CA1-amygdala pathway, see (1) in "A" in 4-18, or
via the EC-CA3-CA1-amygdala pathway see (2) in "D" in 4-18. We believe this is
the first systems-level demonstration that encoding and retrieval of the same memory
do not require identical circuits.
To encode the tone and foot-shock association in the trace paradigm, it seems
plausible that the neurons encoding the tone representation in CAl can synapse on
neurons in the amygdala that receive the foot-shock stimulus. The CAl signal then
provides the tone representation to the amygdala during the time of the foot-shock,
and thus the tone is associated with the foot-shock. If the tone representation is
formed in CAl, then the representation is dependent on inputs from MEC-Ill and
not CA3. We demonstrated this by showing that tone memory formation was normal
in CA3-TeTx mice (see "A" in 4-18). This is because the tone representation from the
MEC-Ill is intact. In the MEC-Ill TeTx mice, tone memory formation is disrupted
(see "B" in 4-18). The MEC-Ill to CAl connection is necessary for formation of
temporal memory.
MEC-Ill is not necessary for retrieval, so another indirect circuit for tone-recall
126
is sufficient. This circuit needs to be indirect because the original binding of toneshock did not happen directly in the amygdala. In our model, to reactivate the similar
representation, the CA1 tone representation needs to be activated. The representation
in CA1 could be activated via the mono-synaptic MEC-III-CA1 synapse (see "D" in
4-18). This pathway is sufficient to elicit tone memory recall, as we demonstrated
with the CA3-TeTx mice. Additionally, CAl could be activated via the tri-synaptic
circuit, which is still intact in the MEC-III TeTx mice (see "D" in 4-18). Interestingly,
neither the CA3 or MEC-II functional output is necessary for recalling tone memory,
suggesting that each are sufficient.
In the case where both MEC-II and CA3 are sufficient to recall the tone memory,
CAl probably holds the representation of the tone memory trace. Given that both
MEC-II and CA3 have a common output structure, and it is that structure that
connects with the amygdala, it seems as though this trace an be activated by either
input. A direct test of this model would be to inactivate CA1 in the recall phase of the
trace paradigm. This could be done with pharmacological agents, such as muscimol
(a GABAA agonist), or with a CA1-TeTx mouse in which synaptic transmission was
on during acquisition and off during recall. The prediction here is that CA1 output
is necessary for tone recall in the trace paradigm, and mice would display impaired
freezing during recall.
Table 4.5: CA3 vs. MEC-III in Delay and 20 s Trace Conditioning
Genotype
Paradigm Conditioning
Tone
Post - Tone
Context
MEC-II TeTx
MEC-III TeTx
CA3-TeTx
Delay
Trace
Delay
OK
Deficit
OK
OK
Deficit
OK
OK
Deficit
OK
OK
Deficit
OK
CA3-TeTx
Trace
OK
OK
Deficit
Deficit
127
..............
......
.....
_ t ......
B
A
Delay Conditioning: Tone Encoding
Tone
Delay Conditioning: Tone Recall
Tone
-o
Amygdala
Freezing
Amygdala
Trace Conditioning: Tone Encoding
Tone
Trace Conditioning: Tone Recall
Tone
Foot-Shock
Brain Region
Holding online
Tone Representation
-+
Amygdala
Freezing
Amygdala
Figure 4-17: Tone Memory Acquisition in Delay and Trace Conditioning.
(A) Tone encoding in delay conditioning. Tone and foot-shock are directly paired in
the amygdala. (B) Tone recall in delay conditioning. Tone can elicit fear response by
direct activation of association in amygdala. (C) Tone encoding in trace conditioning.
Tone and foot-shock do not temporally overlap, so another brain region is needed to
hold online the tone representation. (D) Tone recall in trace conditioning. Tone can
not elicit memory (freeing) by direct amygdala activation. Tone must pass through
some other brain structure to then elicit tone and foot-shock pairing.
128
'--'..
'_-: 1111 - .-
A
-
___
-,A-
-_#_
- a
. .
Tone Memory Encoding
in CA3-TeTX Mice
..........
. ... ........
Tone Memory Encoding
in MEC-TeTX Mice
Tone
Tone
Foot-Shock
Tone Memory Formation OK
Foot-Shock
Tone Memory Formation Disrupted
Tone Recall in CA3-TeTX Mice
Tone Recall in MEC-TeTX Mice
Tone
Tone
-*Freezing
-oFreezing
Tone Memory Recall OK
Tone Memory Recall OK
Figure 4-18: Trace Conditioning: Tone Memory Acquisition and Recall in
MEC-III TeTx and CA3-TeTx Mice. (A) Tone memory formation in CA3-TeTx
mice. Memory formation normal, due to intact MEC-III pathway. (B) Tone memory
formation in MEC-III TeTx mice. Tone memory is disrupted. CA3 pathway not
sufficient for encoding tone-footshock association. (C) Tone recall in CA3-TeTx mice.
Memory formation was normal, and tone memory can be retrieved via the MEC-III
pathway. (D) Tone recall in MEC-III TeTx mice. Memory formation under Dox-on
(transmission ON), and recall under Dox-off conditions shows that MEC-III pathway
not necessary for recall. Tone recall could be mediated via EC-CA3-CA1-Amygdala
circuit.
129
4.5.2
Medial Entorhinal Cortex is Required for Pattern Completion of Context in the 20 s Trace Paradigm
MEC-III TeTx mice are defective in recalling the context in which they were shocked
during the trace conditioning paradigm (see panel "A" in figure 4-2 on page 94.
Interestingly, the CA3-TeTx mice are also impaired in contextual recall (see panel
"D" in figure 4-14 on page 4-14). Similar to the tone-deficit in the MEC-II TeTx
mice in the trace paradigm, the context deficit in the same paradigm is due to a
problem with acquisition (see ON-OFF context freeing in figure 4-4 B on page 99).
We believe this is a contextual pattern-completion problem that occurs during the
day 1 conditioning stage.
Contextual fear memory is normal in the delay conditioning (see "B" in Figure 42 on page 94), as well as in the contextual fear conditioning paradigm (see "B" in
Figure 4-5 on page 101), in which no tones are presented. Thus, there is something
about the 20 s between the tone and the foot-shock that is disrupting the contextual
representation from being bound to the foot-shock and causing impaired contextual
memory on day 3 after the trace fear conditioning. We believe that during this 20 s
trace, the mouse is pattern completing the pre-tone context.
What is a context? Context is a stable representation of independent features
combined together in a location (Rudy and O'Reilly 1999) (Rudy 2009) that form
the notion of "somewhere." These elements together represent a unique context. In
our set-up, to the degree which we can control the independent features, the context
contains specific lighting, specific odors, specific grid floors and roofing, as well as the
mouse's familiarity to handling.
Pattern completion is the process by which a full memory can be recalled by limited cues. It has been shown that CA3 plasticity is necessary for pattern completion
in a MWM task. CA3 output is also required for pattern completion in a pre-exposure
paradigm
(Nakashiba et al. 2008). As seen in Chapter 6, (See 6-1 on page 174),
the MEC-II TeTx mouse is also impaired in pattern completion in the pre-exposure
paradigm. MEC-II TeTX require greater than 20 s of an exposure to the chamber
130
context for the contextual representation to get associated with the foot-shock (see
20 and 60 s data on panel "A" in 6-1). When the mice are in the context for longer
than 20 s, they are either pattern completing the context from the prior experience or
forming a new representation of the context, which they then bind to the foot-shock.
In our trace conditioning paradigm, the mouse first spends 4 minutes in the chamber prior to the tone delivery, which is more than sufficient to form a contextual
representation of the chamber (see 4-5 on page 101). However, it can be speculated
that the tone disrupts this representation either by taking away all of the animal's
attention from the original context, or by robustly revising it by its inclusion into
the representation. Upon termination of the tone, the animal is back to the original
context without tone, and may start being reminded of the original contextual representation without tone. But, this period is only 20 s, so a full reactivation of the
original representation may have to resort to pattern completion for it to be associated with the foot-shock to form a contextual fear memory. Since MEC-III TeTx
mice are impaired in pattern completion as demonstrated by a deficit in the preexposure experiment (Figure 6-1 B), it makes sense that they also exhibit a deficit in
the contextual memory in the trace conditioning paradigm.
Thus, both the MEC-II TeTx and CA3-TeTx mice are impaired in the preexposure version of a pattern completion, and we believe that the impaired contextual
fear memory in the trace paradigm is due to this similar deficit. Both mice have
trouble re-acquiring the context representation during the trace period in time for it
to be bound with the foot-shock.
Outlined in figure 4-19 on page 132 is the basic components of trace conditioning
(A) and what we believe the mouse to be doing during those periods (B). In the case
of delay conditioning, the contextual representation is formed prior to the tone, and
thus when the foot-shock arrives, that contextual representation is bound with the
foot-shock. In the short-trace period, after the tone terminates, the animal is now
presented with the same environment it was in previously, which is different from
the context with the tone. In this short period the animal pattern completes the
prior contextual memory, unless there are disruptions to CA3 or MEC-II output.
131
......
. . . . . ......
A Trace Conditioning Paradigm.
Tone
Context Exploration
B
Trace
Foot-Shock
Pattern Completion States in the Trace Paradigm.
Context Encoding
Modification Recall of O d Context
of Context
C
Necessity of Pattern Completion in Delay, Short Trace, Long Trace:
I
Delay: Contextual Representation Formed Before Tone.
Contextual Memory Normal.
Context Encoding
I
I
Short Trace: Contextual Recall is Impaired.
Contextual Memory Disrupted.
Too Short for Pattern Completion
E
|1
Long Trace: New Contextual Representation Formed.
Contextual Memory Normal.
New Context Encoding
* Tone I Foot-Shock
Figure 4-19: Pattern Completion in Trace Conditioning. (A) Main components
in a trace conditioning paradigm. (B) Features of the trace paradigm described in
terms of the pattern completion model. The trace period becomes a recall of the original context. (C) Depiction of no trace (delay), short trace and long-trace paradigms
and description about how the length of the trace affects contextual memory formation. Green box = tone, Red line = foot-shock.
132
In the long-trace period, the animal may not successfully pattern complete the prior
context, but is re-forming a new representation of the context, which an successfully
bind with the foot-shock, see "C" in figure 4-19.
The MEC-III TeTx mice, in the 40 s trace paradigm, do not have disrupted
contextual fear memory, see "D" in 4-7 on page 105. In this case, 40 s is a sufficient
amount of time to form a new contextual representation that can be bound to the
foot-shock. This too could explain why there is no disruption in context memory in
the un-paired experiment, see "D" in 4-8 on page 107.
4.5.3
Working Memory
Our results also show that the MEC-II output is necessary for proper performance
in the DNMP version of the T-maze. Various manipulations of the EC have indicated
that the EC is necessary for DNMP working memory (Leonard et al. 1995) (Penetar
and McDonough 1983) (Bartus and Johnson 1976) (McGaughy et al. 2005).
We
further resolve this and show that in our visual-cued version of the DNMP T-maze,
the MEC-II is required.
A double-dissociation in different versions of the DNMP between the LEC-III and
MEC-III would be an interesting experiment. In our setup, one arm is white, while the
other arm has a strip of black tape. We assume that the mice use these distinctions
to make their "choice" decision, although we cannot rule out that a separate strategy
is used.
One possible alternate strategy is that the mice remember a rule, such
as "went-left, now go-right," rather than the visual cue memory. If the mice use
the visual information to make these distinctions, this fits well with the MEC as a
processor of visual information, and the LEC as a processor of object information
(Hafting et al. 2005) (Yoganarasimha et al. 2010) (Hargreaves et al. 2005).
It would be interesting to run the MEC-III TeTx mice on a non-visual version of
the DNMP T-maze. In such a maze, the floors would have distinct textures, different
smells, or some other non-visual feature could distinguish the two arms. With an
intact LEC-III, the MEC-III TeTx mice may be able to perform the DNMP T-maze
task. In addition, it would be nice to disrupt the LEC-Ill output. The prediction
133
would be that these mice could complete the spatial version, but not the non-spatial
version of the DNMP T-maze. At this point no LEC-II specific promoter has been
discovered, and thus this double-dissociation can not be completed as cleanly as one
would want, but LEC lesions may give some indication that this double-dissociation
exists.
Our version of the DNMP T-maze may not directly test spatial-working memory
per-se, but instead may test visual working memory. To assess whether the MEC-III
TeTx mouse model is deficient in spatial working memory, we would have to construct
a plus maze version of the DNMP task. In this modified version of the T-maze, two
opposing ends of the plus would be rewarded and the remaining two would be start
boxes. There would be extra-maze spatial cues, and no internal cues. In the sample
phase, animals would be places in one of the start boxes and directed towards the
rewarded arm. In the choice phase, the animals would be places in the a randomized
start box. If the animals were truly using a spatial working memory, and not the
"right-left" strategy or within-maze cues, then they would still be able to navigate
to the correct non-match arm, even though the arm could require the same direction
of turn. We expect that, due to the anatomy, the MEC-III TeTx mice would have a
similar disruption in this version of the task.
To better understand the necessary circuitry in our version of the DNMP T-maze,
we tested the CA3-TeTx mice on this task. We found that CA3-TeTx mice perform
as well as their controls on this task. This suggests that functional output from CA3
to CA1 is not necessary for working memory in this task. This solidifies the MEC-III
to CA1 circuit as essential for this task. We propose that persistent activity in the
MEC-III is the mechanism by which the previous "sample" representation is held
online for use in the "choice" phase.
4.5.4
Difficulty of Memory
An alternative explanation to describe the fear conditioning data in this chapter takes
into account the 'difficulty' of the training task. A recent study in the Fanselow lab
suggests that the dorsal hippocampus' role in delay fear conditioning depends on the
134
strength of the tone-shock pairings (Quinn et al. 2008). In this study the authors
trained rats in three different conditioning paradigms before lesioning the dorsal hippocampus and testing for tone and context fear memory. In the first paradigm the
animals were given many shocks at a high voltage ('Strong'), in the second paradigm,
animals were given few shocks at a a high voltage ('Weaker') and in the third conditioning paradigm the animals were given many shocks at a low voltage ('Weak').
In all cases, contextual fear memory was disrupted, which is expected for posttraining lesions of the dorsal hippocampus
(Maren and Fanselow 1997). However,
the hippocampal lesions also caused a deficit in tone fear memory when the tonefootshock association was weak. This suggests that the dorsal hippocampus does
play a role in processing these associations, typically believed to only occur in the
amygdala (Romanski et al. 1993) (Fanselow and Kim 1994) (Fanselow and LeDoux
1999).
The CA3-TeTx mice show a contextual memory deficit in a weak delay-conditioning
protocol (Nakashiba et al. 2008), but no deficit in tone memory. Our experiments
with the MEC-II TeTx mice in the 1-shock delay conditioning paradigm show memory deficits in both contextual and tone memory,(see 1-shock experiment in Appendix
A -3 on page 183) . Comparatively, and in combination with Fanselow's data, this
suggests that the tone representation is supported by the MEC-III to CA1 circuitry.
In the MEC-II TeTx mice, we show impaired tone and contextual memory in
the 1-shock delay conditioning paradigm, but normal tone and contextual memory
in the 3-shock delay conditioning paradigm. Intermediate tone-shock associations,
between the 1-shock and 3-shock delay, thus may show intermediate impairments.
The addition of the trace between the tone and shock may serve as this intermediate
'strength-of-association' data point. The differences in memory between the 3-shock
delay conditioning, and the 3-shock trace conditioning may reflect the difficulty or
strength of associations. See figure 4-20 on page 136 for details.
To further test this idea, a series of experiments would be needed to address
the strength of the tone-shock pairings between the 'weak' 1-shock delay and the
'strong' 3-shock delay. With delay conditioning, the addition of a 2-shock delay,
135
A
Hippocampally Dependent
No
Yes
Tone
Context Yes
Yes
t
t
Weak-Pairings
Strong-Pairings
MEC-Ill Dependent
Yes
Tone
Context Yes
t
1-Shock Delay
'Weak'
Yes
No
Yes
No
t
3-Shock Trace
'Intermediate'?
t
3-Shock Delay
'Strong'
Figure 4-20: Strength of Tone-Footshock Pairings and Hippocampal Dependency. (A) Adapted from (Quinn et al. 2008). Weak tone-footshock pairings require
hippocampus for tone and context memory recall. Strong tone-footshock pairings do
not require hippocampus for tone memory recall. (B) How 20 s trace phenotypes
may fit between our data suggesting that MEC-III projections are required for weak
1-shock delay conditioning, but not for 3-shock delay conditioning. 20 s trace may
represent an intermediate strength-of-association.
136
and weaker shock voltages for the 3-shock delay conditioning could help generate
a relational graph of 'strength' and freezing to tone or shock. Similarly, with this
idea, 1-shock and 2-shock trace paradigms should show larger deficits in tone and
contextual memory. Additionally, this idea would suggest that increasing the number
of trace pairings could overcome the tone and contextual deficit.
137
4.6
4.6.1
Methods
DNMP T-Maze
Both control and MEC-III TeTx mice off-Dox for at least 4 weeks were food deprived
until they reached
85% body weight. on the first two days of food depravation,
mice were fed only the sugar-pellet rewards that they would get in the task (BioServ (20mg, F0163)), and were fed a 50/50 mix of those pellets and dry food until
habituation started. Habituation Phase: Mice were habituated to the T-Maze over
two days. Cage mate mice were all placed into the T-Maze Apparatus for 4 sessions
of three minutes each for both days. Sugar pellet rewards were placed in the reward
cup at the end of each arm, and were replaced over the three minute session as they
were consumed.
Training Phase: Mice were given ten trials per day. Each trial
consisted of two phases, the Sample phase that was followed by the Choice phase. In
the sample phase, mice were placed in the stem of the T-maze and allowed to run
to the end of one arm of the maze. The reward was placed at the end of one arm,
while the other arm was closed off to force the mouse to run one way. Over the ten
trials, the Sample Arm was being Left or Right was pseudo-randomly assigned to
avoid biases, based on and adapted from (Fellows 1967). If the mouse did not run
after 120 seconds, it was gently pushed to initiate movement. once mouse reached
end of Sample Arm, it was allowed to consume the reward, and was then gently
picked up and placed back in the start box. Mouse was trapped in the start arm
while the maze was quickly wiped with 70% EtOH. The previously blocked door was
opened so that both arms of the T were opened, and the mouse was released from
the start box by raising the door. Mouse was allowed to choose which arm to visit
next. The opposite arm from the sample arm was rewarded, while the arm previously
visited was not. If animal chose previous arm, it was blocked in that arm for a 20s
punishment and was then placed back in its home cage until the next trial. If the
mouse chose the opposite arm that was rewarded, it was given time to consume the
pellet and then gently picked up and placed back in the home cage. Each mouse
was run for a trial, and then trial two started for the first mouse. The time interval
138
between trials was between 15-20 minutes. Mice were scored on the percentage of
time they made a successful alternation, how long it took them to choose an arm
on the choice phase from when the door released them from the start box, on the
% of Right/Left choices they made overall, and on the time between the sample and
choice phases. MEC-III Cre+(Control, CT) (N=14) and MEC-II TeTx (Mutant,
MT) (N=18). CA3 Cre+(Control, CT) (N=8) and CA3-TeTx (Mutant, MT) (N=8).
4.6.2
Fear Conditioning
Fear conditioning was performed with male mice between 14 and 27 weeks of age
in the animal facility during the light cycle with minor modifications of the method
described previously (McHugh et al. 2007). Experiments were performed by operators
who were blind to the genotypes.
There are two distinct contexts for these behavioral assays which are in two distinct
rooms. Context "A" is a dimly lit room with red lights. In Context "A" The chambers
had plexiglass fronts and backs and aluminum side walls with a triangular plastic
roof and measured 30 x 25 x 21 cm. The chamber floors consisted of 19 stainless
steel rods spaced 16 mm apart connected via a cable harness to a shock generator.
Chambers were cleaned prior to an introduction of an individual mouse into them with
quatricide. A 1% acetic acid solution was placed beneath the chamber floor during
the experiment to provide a dominant odor. Context "B" contains direct overhead
fluorescent lighting and distinct chambers, measuring 30 x 25 x 21 cm, with plexiglass
front and back walls, and aluminum side walls but with a flat roof. In addition, the
floors of these chambers were made up with white plastic and the odor was provided
with 0.25% benzaldehyde (in 100% ethanol). These lighting, chamber materials and
odor employed on Day 3 provided a context quite distinct from Context "A". In each
context, there are 4 conditioning chambers.
In each fear conditioning experiment, mice were transported from the colony room
to a room adjacent to the conditioning behavioral suite and were left undisturbed for
30 minutes prior to the start of the experiment.
In all of these experiments, the animals activity in the chamber was recorded using
139
FreezeFrame software. Freezing behavior was assessed from the video image of the
mouse using FreezeView software, with a minimum bout time of 1 second. Freezing
values were then averaged over mice of a particular genotype for each session.
4.6.3
Trace and Delay Conditioning Protocols
3-Shock Delay Paradigm: on Day 1, mice were placed in Context "A" and allowed
to explore for 240 seconds, at which point the first of 3 tone-shock pairings started.
The pairing consisted of a 20 s tone, 75 db, 2000Hz which co-terminated in a 2 s,
.75-mA foot-shock. The second tone-shock pair initiated at 380 s into the experiment
and the third pair initiated at 520 s. Mice remained in the conditioning chamber for a
total of 706 s at which point they were removed back into their transport (home-cage)
and to the adjacent room. 24 hours later, on Day 2, mice were placed in Context "B"
and allowed to explore for 240 s, at which point the same tone as from day 1 played
for 60 seconds, followed by 180 s of no-tone (post-tone period). This repeated two
more times and mice were then removed after 960 s in the chamber. 24 hours later,
on Day 3, mice were placed back in Context "A" for 5 minutes to assess their fear to
the training context. These experiments were carried out in Dox-off animals.
Three-Day Delay Conditioning Paradigms:
* MEC-III MEC-1II Cre+ (Control, CT (N=20)) and MEC-III-TeTX (Mutant,
MT (N=19).
" CA3 CA3-Cre+ mice (Control, CT (N=4)) and CA3-TeTx (Mutant, MT (N=4)).
20-second Trace Paradigm: On Day 1, mice were placed in Context "A" and
allowed to explore for 240 s, at which point a 20 s tone (75 db, 2000Hz) played,
followed by a 20 s trace, and then a 2 s, 0.75-mA foot-shock. This repeated two more
times, starting at 402 s and 564 s. Mice remained in the conditioning chamber for a
total of 706 s. on Day 2, mice were placed in Context "B" and allowed to explore for
240 s, at which point the same tone as from day 1 played for 60 s, followed by 180
s of no-tone (post-tone period). This repeated two more times and mice were then
removed after 960 s in the chamber. on Day 3, mice were placed back in Context "A"
140
for 5 minutes to assess their fear to the training context. In the Dox on-off-off case,
mice that had been on-Dox from birth (transmission normal, Dox food 20mg/kg) were
conditioned on Day 1 on-Dox, and then Dox food was immediately removed. One
month after Dox removal, mice were placed in Context "B" and had "Day 2" testing
as described above, and then 24 hours later placed back in Context "A" for "Day 3"
testing. In the Dox-off-on-on case, mice who had been off-Dox for at-least 4 weeks
were conditioned on Day 1, and then immediately after conditioning were put on-Dox
food (40mg/kg). One month after Dox food was given, mice were placed in Context
"B" and had "Day 2" testing as described above, and then 24 hours later placed back
in Context "A" for "Day 3" testing. As a control for the month long gap between
Day 1 and 2 in the Dox off-on-on (Experiment 2) and Dox on-off-off (Experiment 3)
paradigm, a Dox-off-off-off (Experiment 1) experiment was conducted in which the
time between day 1 and 2 was one month, and the time between day 2 and 3 was 24
hours.
Three-Day 20 s Trace paradigms:
" MEC-III MEC-II-Cre+ (Control, CT (N=39)) and MEC-III-TeTX (Mutant,
MT (N=35).
" CA3 CA3-Cre+ mice (Control, CT (N=20)) and CA3-TeTx (Mutant, MT
(N=20)).
MEC-III 1-Month Induction Paradigms:
" Experiment 1 MEC-III-Cre+ (Control, CT (N=18)) and MEC-III-TeTX (Mutant, MT (N=14)).
* Experiment 2 MEC-III-Cre+ (Control, CT (N=26)) and MEC-III-TeTX (Mutant, MT (N=18)).
" Experiment 3 MEC-III-Cre+ (Control, CT (N=25)) and MEC-III-TeTX (Mutant, MT (N=19)).
40-second Trace Paradigm: This paradigm is the same as the 20-second trace
paradigm, except that the "trace" interval between the tone and the shock is 40
141
seconds. Day 2 and Day 3 testing are the same. MEC-III-Cre+ (Control, CT (N=8))
and MEC-III-TeTX (Mutant, MT (N=4)).
Backwards Trace Paradigm: This paradigm inverted the presentation order
of the stimuli in the 20 second trace paradigm. In this case, on Day 1, mice were
allowed to explore the Context "A" for 240 s and then received a 2 s, 0.75-mA
footshock, which was followed by a 20 s trace and then a 20 s, 75db, 2000Hz tone.
The shock-trace-tone bout was repeated 2 more times and the total time the mouse
spent in the conditioning chamber was 706 seconds. Day 2 and 3 remained the same
as that in the trace and delay paradigms. MEC-III-Cre+ (Control, CT (N=7)) and
MEC-III-TeTX (Mutant, MT (N=5)).
Un-Paired Acquisition Paradigm: In this paradigm, the tone and shock pairings were not temporally related, as they are in the trace and delay conditioning
paradigms. In this case, on Day 1, mice were allowed to explore Context "A" for 240
s and then received a 20 second tone presentation at 240 s, 402 s and 564 s, to match
the tone presentation schedule for the 20 s trace and delay paradigms. Three 2 s,
0.75mA shocks were delivered at 360 s, 502 s and 624 s, making the intervals between
the tone and shocks 100, 80 and 60 seconds, respectively. Day 2 and 3 remained the
same as that in the trace and delay paradigms. MEC-III Cre+ (Control, CT (N=9))
and MEC-III TeTX (Mutant, MT (N=7)).
Tone-Presentation only: In this paradigm, the tone as a stimulus was presented
alone, without any shock presentations. In this case, on Day 1, mice were allowed to
explore Context "A" for 240 s and then received a 20 s tone presentation at 240 s,
402 s and 564 s, to match the tone presentation schedule for the 20 s trace and delay
paradigms. Day 2 and 3 remained the same as that in the trace and delay paradigms.
This was done in Dox off-off-off mice over 3 days. MEC-III Cre+ (Control, CT (N=9))
and MEC-II TeTX (Mutant, MT (N=7)).
3-Shock Contextual Fear Conditioning: On Day 1, mice were placed in
Context "A" and allowed to explore for 240 s, at which point a 2 s, 0.75-mA footshock. This repeated two more times, starting at 402 s and 564 s. Mice remained in
the conditioning chamber for a total of 706 s. on Day 2, mice were placed back in
142
Context "A" for 5 minutes to assess their fear to the training context. MEC-II Cre+
(Control, CT) (N=13) and MEC-II TeTx (Mutant, MT) (N=11).
5-Shock Contextual Fear Conditioning: On Day 1, mice were placed in
Context "A" and allowed to explore for 240 s, at which point a 2 s, 0.75-mA footshock. This repeated 4 more times, starting at 322 s, 404 s, 486 s, and 568 s. Mice
remained in the conditioning chamber for a total of 706 s. on Day 2, mice were
placed back in Context "A" for 5 minutes to assess their fear to the training context.
MEC-III Cre+ (Control, CT) (N=7) and MEC-II TeTx (Mutant, MT) (N=5).
4.6.4
8-Arm Radial Maze Task
The 8-arm radial maze task was conducted as described (Miyakawa et al.
2001).
Prior to training mice were food-deprived until they reached 80-85% of their original
body weight. During food depravation, half of the food (by weight) given on a day
was regular food, and the other half was sucrose pellets (Bio-Serv (20mg, F0163)), to
adapt the mouse to the reward during the task. Mice were only then fed regular food
during the trials. Pre-training started when the co-hort of mice reached the desired
weight for each mouse. During pre-training, mice were placed in each arm of the
8-arm maze, one at a time, and allowed to eat scattered pellets on the arm. Pellets
were in the arm, as well as in the reward cup at the end of each arm. each mouse
spent 5 minutes in each arm. The following day, the actual maze acquisition started.
All eight arms were bated with one sugar pellet. Mice were placed in the central
platform and allowed to try and get all 8 pellets for a maximum of 15 minutes. A
trial was ended after 15 minutes had elapsed, or a mouse had consumed all 8 rewards.
For this task, a visit to an arm was counted when the animal traveled 5cm or more
into an arm. After an arm visit, when the mouse returned to the center, all doors
were closed and the mouse was confined to the center space for 5 seconds before all
of the doors opened again. Animals ran 1 trail per-day and this task was run for
18 days. During the task, revisiting errors, working memory errors, arm ommissions,
distance traveled, time-to-finish task, and the number of different arms chosen in the
first 8 arm choices was automatically recorded. Mouse genotype was unknown to the
143
experimenter. All mice were run off-Dox. MEC-III Cre+ (Control, CT) (N=27) and
MEC-II TeTx (Mutant, MT) (N=24).
4.6.5
Hot-Plate
Hot-Plate Pain Sensitivity test done on a plate heated to 55 degrees celsius.
15
Control (MEC-II Cre+) mice and 13 Mutant (MEC-III TeTx) mice 14-27 weeks of
age were used for the experiment. All animals were off-Dox for at least 4 weeks and
aged between 8 and 12 weeks old. Platform was heated to 550 Celsius and allowed
to equilibrate for 30 minutes. Mice were placed onto the hot plate and the timer was
started. Mice lifting paws indicates a reaction to the hot plate. Once both front paws
were lifted, clock was stopped. Mice were not allowed to stay on hot plate beyond
15 seconds to prevent burning. Average of "time-to-react" for each genotype were
calculated.
4.6.6
Elevated Plus Maze
The elevated plus-maze consisted of two open arms (25 x 5 cm) and two enclosed
arms of the same size, with 15-cm-high transparent walls.
The arms and central
square were made of white plastic plates and were elevated to a height of 50 cm
above the floor. To minimize the likelihood of animals falling from the apparatus,
1-cm-high Plexiglas ledges were provided for the open arms. Arms of the same type
were arranged at opposite sides to each other. Each mouse was placed in the central
square of the maze (5 x 5 cm), facing one of the open arms. Mouse behavior was
recorded during a 10 minute test period. The maze was cleaned with water after each
trial. The number of entries onto and the time spent on open and enclosed arms were
recorded. For data analysis, we used the following four measures: the percentage of
open arm entries, the percentage of time spent on the open arms, the total number of
arm entries, and total distance traveled (centimeters). Data acquisition and analysis
were performed automatically, using EthoVision XT Software version 4.1.106 (Noldus
Information Technology BV, Netherlands) . MEC-III Cre+ N=8 and MEC-III TeTx
144
N=8 mice that had been off-Dox for at least 4 weeks (14-27 weeks of age)
145
Chapter 5
The Role of Medial Entorhinal
Cortex in Spatial Learning,
Memory and Consolidation
This chapter addresses the necessity of MEC-II TeTx mice in learning spatial tasks
and addresses the specific claim that projections from EC are necessary for consolidation of spatial memories (Remondes and Schuman 2004). Testing the MEC-III TeTx
mice in various forms of the watermaze task, and the Barnes' maze, we find that the
MEC-II is not necessary for spatial learning tasks. Additionally, after learning the
hidden platform location in the watermaze, mice retain and thus consolidate memory of the platform location one month later, in the absence of MEC-III synaptic
transmission. This the MEC-III, contrary to proposals in the lesion literature, is not
necessary for consolidation of spatial memories.
The hippocampus is an essential processing center for spatial information. Neurons of each subregion within the hippocampal complex (DG, CA3, CA1) display
place cell properties. Individual neurons fire action potentials to specific spatial locations, and thus display a representation of that space. Additionally, physical lesions
of the hippocampus or molecular disruptions of CAl disrupt spatial learning. Interestingly, the molecular lesion of CA3 output does not disrupt spatial learning or
memory, implying that EC-III projections to CAl (mono-synaptic) may be neces146
sary (or sufficient) for these tasks. A physical lesion of the perforant pathway does
not disrupt spatial learning, but has been shown to disrupt consolidation of spatial
memories. Using the MEC-III-TeTx mouse as a model, we test the necessity and
sufficiency of the mono-synaptic input in spatial learning tasks, and test the role of
this input in consolidation of spatial memories.
5.1
Spatial Learning
Place cells.
The first discovery that the hippocampus represented spatial informa-
tion was by John O'Keefe (O'Keefe and Dostrovsky 1971), who demonstrated that
specific CA1 cells would fire action potentials to specific locations in space. These
cells are termed "place cells" as they represent the animal's specific location in any
given environment. It has been hypothesized that these place cells form the animal's
cognitive map of space (O'Keefe and Nadel 1978). CA3 pyramidal and DG granule
cells also encode spatial information.
Spatial learning and the hippocampus.
Lesions to the hippocampus disrupt
spatial learning (Logue et al. 1997) (Stoelzel et al. 2002), as assessed by the Morris
watermaze task (Morris 1984). Disruption of synaptic plasticity, by removal of the
NR1 subunit of the NMDA receptor specifically in CA1, impairs spatial information.
This is seen in the disruption of place cell spatial information (McHugh et al. 1996)
and in the retardation of spatial learning (Tsien et al. 1996).
Spatial learning and CA1.
Thus, CA1 seems to play an important role in spatial
memory tasks. CAl receives the majority of its inputs from CA3, which includes the
processed information from the tri-synaptic pathway, and additional input from LEC
and MEC
(Amaral and Witter 1995). Of the two entorhinal inputs, MEC seems
to process more spatial information, whereas LEC processes more object information
(Hafting et al. 2005) (Yoganarasimha et al. 2010) (Hargreaves et al. 2005). Thus,
the two major sources of spatial information into CA1 are from CA3 and MEC. While
plasticity in CAl is important for spatial learning, silencing transmission from CA3,
147
with the usage of a CA3-TeTx mouse, does not effect spatial learning (Nakashiba
et al.
2008).
This suggests that the spatial input from the MEC-III afferents is
sufficient, if not necessary for acquisition of spatial tasks.
5.2
Consolidation of Spatial Memories
Consolidation.
As originally noticed with H.M. (Milner 1972), patients with hip-
pocampal lesions cannot form new memories, but have intact episodic memories for
events that happened prior to the lesion. Episodic memory's temporal sensitivity
to disruption has been replicated in human patients treated with electroconvulsive
therapy (Squire et al. 1975) and mice similarly treated (Squire and Spanis 1984).
This implies that new episodic memories are formed in the hippocampus, but are
then moved over some time-course to some other brain structure, presumably the
cortex (Mishkin 1984) (Squire 1987) (Damasio 1989).
Perforant pathway and consolidation.
Remondes and Schuman have proposed
that it is the direct projections from MEC and LEC to CA1 that are necessary for
consolidation of spatial memories (Remondes and Schuman 2004). In this paper, the
authors used electrolytic ablation of the perforant pathway as it crosses through the
subiculum. Lesions prior to watermaze training did not affect learning of the task over
ten days, nor in a probe test 24 hours after the last day of training. Animals tested
in a probe test one month after the last day of training, however, showed a severe
impairment in target quadrant preference compared to controls. These results seem
to indicate that the short-term memory of the platform location that is assessed in
the day 11 probe cannot be consolidated properly and thus cannot be used for recall
one month later. As a control, un-lesioned rats learned the task for ten days and then
were given three weeks to form a long-term memory (presumably by consolidation)
before lesion of the perforant pathway. When tested one week after surgery, one
month after the final day of watermaze learning, both the lesioned and sham-lesioned
animals showed a preference for the target quadrant.
148
These results suggest that the perforant pathway in rats is necessary to allow
consolidation of spatial memories, and that three weeks is a sufficient amount of time
to consolidate these memories.
We used the MEC-II TeTX mouse to test the two hypotheses presented here. The
first hypothesis is that the MEC-II input into CA1 is necessary for learning spatial
memory tasks. The second hypothesis is that the TA pathway is necessary for longterm consolidation of spatial memories. The expression of tetanus toxin in essence
acts as a molecular lesion of the pathway, and we can be certain that we are lesioning
the MEC-II pathway specifically and not disrupting any other pathways/projections,
which is inevitable with any physical lesion.
We demonstrate here that MEC-II input to CA1 is not necessary for acquisiton
of spatial memory tasks, and that this input is not necessary for consolidation of
spatial memories.
5.3
5.3.1
Results
Spatial Learning in the Watermaze
To test whether MEC-III output is necessary for spatial learning we tested spatial
learning in 3 separate tasks. Firstly, we assessed these mutant mice in the standard
morris watermaze task, and then in the reversal version of that task, in which the
hidden platform was moved to the opposite quadrant after the first 11 days of learning.
Secondly, we tested these mice in a more difficult version of the watermaze task, in
which animals were given a single trial on each day for 18 days. Thirdly, we tested
spatial learning in the Barnes Maze task, which is a land-based spatial learning task.
We found that MEC-II TeTx mice performed as well as the control MEC-III Cre+
mice in all of these tasks.
MEC-III output is not necessary for spatial learning in the watermaze.
To test whether the molecular lesion of the MEC-II output is necessary for spatial
149
learning we tested these mice a standard version of the Morris watermaze. Morris
watermaze task run in mice off-Dox for at least 4 weeks. The average escape latencies
to the hidden platform location between the two genotypes were not significantly
different during learning of the first platform location over the first 11 days of the task
(Figure 5-1 A, page 5-1)(2-way ANOVA: Genotype x Day F(10,280)=0.64, P=0.7804;
Day F(10,280)=21.34, P < 0.0001; Genotype F(1,280)=0.72, P=0.4029).
MEC-III output is not necessary for reversal learning in the watermaze.
A typical challenge in the watermaze task is to move the platform after the mouse
has acquired the location of the first configuration. Given that there was no difference between the MEC-II TeTx and control mice in learning the location of the first
platform, we moved the location of the hidden platform to the opposite quadrant.
On the reversal task, the averaged latency curves over the 5 day task were also not
significantly different (see last five days in "A" from figure
5-1) (2-way ANOVA:
Genotype x Day F(4,112)=1.73, P=0.1477; Day F(4,112)=12.33, P < 0.0001; Genotype F(1,112)=0.52, P=0.4749).
MEC-III output is not necessary rapid learning on the first day of reversal in the watermaze.
Since there is no difference between the two genotypes on the standard or reversal
version of the watermaze, we took a closer look at the first 4 trials of the reversal, so
see if there were any early learning differences with the new challenge of the moved
platform. This analysis looked at the "savings" between trials on Day 12, the first day
of the reversal task. Savings are the defined as the improvement in task performance
as defined by a decrease in latency to find the hidden platform. Over and among the
four trials on the first day of the reversal task, there were no significant differences in
savings between the genotypes (Figure 5-2, page5-2)(TG, 12.34± 8.8, CT, 14.56 ±6.5;
p=0.8375 between trials 1 and 2; TG, 15.06 ± 8.5, CT, 26.1 ± 8.6; p=0.3718 between
trials 2 and 3; TG, 10.36 ± 10.28, CT, -2.19 ± 9.4; p=0.3748 between trials 3 and 4;
TG, 37.75 ± 7.9, CT, 38.48 ± 8.4; p=0.9508 between trials 1 and 4).
150
Quadrant Occupancy. Probe trials are inserted into learning tasks to get a
broader understanding of the mouse's representation of their goals during the task.
A probe trial requires the removal of the hidden platform, and is used to assess where
the mouse spends its time swimming. Mice that have acquired the location of the
hidden platform thus spend the majority of their time swimming in the quadrant that
normally contained the platform. (B) from Figure 5-1. Target quadrant occupancies
were not significantly different between the two genotypes (TG, 43.15 ± 3.9; CT,
46.47 ± 3.6; P=0.5355 on Day 6: TG, 51.29 ± 3.5; CT, 56.13 ± 4.3; P=0.3988 on
Day 11.) Quadrant occupancy after reversal (Day 16) for the New Target was not
significantly different between genotypes (TG, 35.89t4.9; CT,41.93±3.5; P=0.3165).
Similarly, there was no significant difference in occupancy of the old target quadrant
(TG, 19.71 ± 4.4; CT, 16.08 ± 3.2; P=0.4975). Occupancy percentages between the
Target (TA) and Opposite (OP) quadrants in the control mice were significantly
different (TA, 46.47 ± 3.6; OP, 11.26 ± 2.0; P < 0.0001 on Day 6: TA, 56.13 ± 4.3;
OP, 9.02 t 2.0; P < 0.0001 on Day 11).
On the last day of reversal, occupancy
percentages between the New Target (NTA) and Old Target (OTA) in the control
mouse were significantly different (NTA 41.93
3.5; OTA, 16.08 ± 3.2; P < 0.0001 on
Day 16). Occupancy percentages between the target and opposite quadrants in the
mutant mice were significantly different (TA, 43.15± 3.9; OP, 14.18+2.5; P < 0.0001
on Day 6: TA, 51.29 ± 3.5; OP, 8.6
1.6; P < 0.0001 on Day 11). On the last day of
reversal, occupancy percentages between the New Target Area (NTA) and Old Target
Area (OTA) in the control mouse were significantly different (NTA 35.89 ± 4.9; OTA,
19.71 i 4.4; P = 0.0210 on Day 16).
Platform Crossings.
During the probe, a more specific assessment of the
mouse's memory of the platform location is crossings within the arena in which the
platform was previously. These are referred to as 'phantom' platform crossings, or
just platform crossings. (C) from Figure 5-1. Platform crossings during probe were
not significantly different between the two genotypes (TG, 3.64+0.71; CT, 4.56±0.75;
P=0.3851 on Day 6: TG, 7.14 ± 0.78; CT, 8.5 ± 1.16; P=0.3541 on Day 11.) Platform
crossings after reversal (Day 16) for the New Target were not significantly different
151
between genotypes (TG, 4.07 ± 0.85; CT,5.44 ± 0.77; P=0.2452). Similarly, there was
no significant difference in platform crossings of the Old Target platform area (TG,
1.57 t 0.53; CT, 1.63 ± 0.35; P=0.9321).
Platform crossings between Target (TA)
and Opposite (OP) in the control mice were significantly different (TA, 4.56 ± 0.75;
OP, 1.06 ± 0.21; P < 0.0001 on Day 6: TA, 8.5 ± 1.2; OP, 0.68 ± 0.40; P < 0.0001
on Day 11.) On the last day of reversal, occupancy percentages between the NTA
and OTA in the control mouse were significantly different (NTA 5.44 ± 0.77; OTA,
1.63 i 0.35; P < 0.0001).
Platform crossings between TA and OP in the mutant
mice were significantly different (TA, 3.6 ± 0.71; OP, 1.07 t 0.29; P = 0.0024 on
Day 6: TA, 7.1 ± 0.78; OP, 0.57 ± 0.23; P < 0.0001 on Day 11.) On the last day
of reversal, occupancy percentages between the NTA and OTA in the control mouse
were significantly different (NTA 4.07 t 0.85; OTA, 1.57 ± 0.53; P = .0198).
5.3.2
Morris Watermaze MWM: One-Trial Per Day Results
A more challenging version of the watermaze task is to give the mice 1 trial per day,
rather than the standard 4 trials. In this task, in order to improve over the length of
the task, mice must form a spatial representation of the platform location on a single
trial, and must recall that information for performance on the following day.
MEC-III output is not necessary for spatial learning with a one trial per
day task.
One-trial per day Morris water maze task run in mice off-Dox for at least 4 weeks
and mice were run for 18 days. The average escape latencies to the hidden platform
location between the two genotypes were not significantly different during learning
of the first platform location over the 18 days of the task (Figure 5-3 A, page 53)(2-way ANOVA: Genotype x Day F(17,408)=1.06, P=0.3896; Day F(17,408)=5.74,
P < 0.0001; Genotype F(1,408)=0.35, P=0.5582).
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5.3.3
Barnes Maze Result
The standard MWM, MWM-reversal and one-trial-per-day tasks described above are
all performed in the a rather stressful environment. Animals must find the platform
to stop swimming. A land-based version of this type of spatial learning is the Barnes
maze (Barnes 1979) (McLay et al. 1998). In this task, water-deprived mice are placed
on a raised platform in bright light and ambient noxious noise. There are 20 circular
holes around the circumference, one of which is an escape hole with water reward.
MEC-III output is not necessary for spatial learning in the land-based
Barnes maze task.
Escape Latency: 4 days Training, 4 days reversal. Barnes maze task,
and reversal run in mice off-Dox for at least 4 weeks. Mice were run for 8 days. For
training of first escape position, over first 4 days, the average escape latencies to the
hidden platform location between the two genotypes were not significantly different
(figure 5-4 A, page 168)(2-way ANOVA: Genotype x Day F(3,30)=1.13, P=0.3515;
Day F(3,30)=39.64, P < 0.0001; Genotype F(1,30)=0.15, P=0.7072). On the 4-day
reversal task from days 5 to 8, again, the average escape latencies to the new hidden
platform location between the two genotypes were not significantly different (2-way
ANOVA: Genotype x Day F(3,30)=0.31, P=0.8176; Day F(3,30)=24.03, P < 0.0001;
Genotype F(1,30)=0.01, P=0.9310).
Barnes Maze Probe. In a probe test on day five, prior to reversal training
these mice were run on a probe, in which there was no escape box. On the probe
trial on day 5, prior to reversal learning, there was a significant difference in the
percentage of time in the target (TA) quadrant (figure 5-4 B)(TG, 57.93% ± 3.030;
CT, 75.87% i 6.01); P=0.0237. There was no significant difference in the percent of
time spent in the opposite (OP) quadrant (TG, 5.075% ± 2.55; CT, 6.85% ± 2.74,
P=0.6455). While the mice did learn the task and had similar escape latencies, the
mutant mice did spend less time in the target quadrant during the probe.
In conclusion, the MEC-III functional output is not necessary for learning of
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spatial tasks.
5.3.4
Medial Entorhinal Cortex Layer III Output is Not Necessary for Consolidation of Spatial Learning
To specifically address the claim by Remondes et al (Remondes and Schuman 2004),
that the TA pathway is necessary for the consolidation of spatial memories, we trained
mice in the standard version of the watermaze task, and tested for the mouse's memory of the platform location 24 hours after training (short-term memory) and 28 days
after training (consolidated, long-term memory).
Morris watermaze task run in mice off-Dox for at least 4 weeks.
The aver-
age escape latencies to the hidden platform location between the two genotypes
were not significantly different (Figure 5-5 A, page 169)(2-way ANOVA: Genotype
x Day F(10,250)=0.93, P=0.5104; Day F(10,250)=26.99, P < 0.0001; Genotype
F(1,250)=0.00, P=0.9819).
MEC-III output is not necessary for consolidation of spatial memories.
In the probe trails, we found that MEC-III TeTx mice and control mice showed
a preference for the target quadrant both 24 hours after training and 28 days after training, demonstrating that the MEC-III functional output is not necessary for
consolidating spatial memories.
Quadrant Occupancy. Target quadrant occupancies were not significantly different between the two genotypes (Figure 5-5 B) (TG, 43.48 ± 4.3; CT, 44.69 i 3.5;
P=0.8268 on Day 6: TG, 46.72 ± 3.2; CT, 49.44 ± 3.2; P=0.5536 on Day 11: TG,
40.95 ± 3.9; CT, 43.81 i 3.2; P=0.5749 on Day 39). Occupancy percentages between the Target (TA) and Opposite (OP) quadrants in the control mice were significantly different (TA, 44.69±3.5; OP, 12.44± 1.9; P < 0.0001 on Day 6: TA, 49.44±3.2;
OP, 9.81+ 1.7; P < 0.0001 on Day 11: TA, 43.81±3.2; OP, 11.38± 4.0; P < 0.0001 on
Day 39). Occupancy percentages between the target and opposite quadrants in the
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mutant mice were significantly different (TA, 43.48 +4.3; OP, 14.34± 2.5; P < 0.0001
on Day 6: TA, 46.72 ± 3.2; OP, 10.91 ± 2.0; P < 0.0001 on Day 11: TA, 40.95 ± 3.9;
OP, 12.40 ± 2.3; P < 0.0001 on Day 39).
Platform Crossings. Mutant mice not only showed similar preferences for the
target quadrant, but also showed memory for the precise location of the hidden platform, both 24 hours and 28 days after training had finished.
Phantom platform
crossings during probe were not significantly different between the two genotypes
(Figure 5-5 C)(TG, 6.08 ±0.87; CT, 6.00
0.59; P=0.9417 on Day 6: TG, 7.3
0.68;
CT, 8.29 + 1.18; P=0.4882 on Day 11: TG, 5.77 ± 0.74; CT, 4.7 + 0.54; P=0.2533 on
Day 39). Platform crossings between phantom Target (TA) and Opposite (OP) in the
control mice were significantly different (TA, 6.00 +0.59; OP, 1.21 + 0.38; P < 0.0001
on Day 6: TA, 8.26
1.2; OP, 0.86±0.40; P < 0.0001 on Day 11: TA, 4.71±0.54; OP
1.00 ±0.43; P < 0.0001 on Day 39). Platform crossings between phantom Target (TA)
and Opposite (OP) in the mutant mice were significantly different (TA, 6.08 ± 0.87;
OP, 1.01 ± 0.26; P < 0.0001 on Day 6: TA, 7.31 ± 0.68; OP, 1.23 ± 0.41; P < 0.0001
on Day 11: TA, 5.77 ± 0.74; OP,0.69 ± 0.36; P < 0.0001 on Day 39).
5.3.5
Medial Entorhinal Cortex Layer III and Consolidation
of Contextual or Tone Memory
As an additional test for consolidation, we assayed the consolidation of a nonspatial memory with fear conditioning. Mice were trained in a specific context where
a tone and foot-shock co-terminated. MEC-III TeTx and control mice showed similar
short-term (24 hour) memory and similar long-term (3 week, consolidated) memory
for the training context.
MEC-III output is not necessary for consolidation of contextual fear
memories after 3 weeks.
This experiment looked at expression of fear memory, as assessed by freezing to
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the context in which the mice were conditioned with a 3-shock delay conditioning
paradigm. There was no significant difference in freezing between genotypes 24 hours
after conditioning (Figure 5-6 A, page 170 )(TG, 50.26% ± 6.7%; CT, 47.54% ±
7.2%; P=0.7980).
Additionally, when tested 3 weeks after conditioning, there was
no significant difference in freezing between the genotypes (TG, 36.33% ± 6.6%; CT,
41.98% ± 7.3%; P=0.5951).
MEC-III output is not necessary for consolidation of tone fear memories
after 3 weeks.
In a non-conditioned context, generalized fear was assessed 48 hours and 3 weeks
after conditioning. There was no significant difference in generalized fear 48 hours
(Figure 5-6 B)(TG, 9.3%
2.8%; CT, 14.64% ± 2.8%; P=0.2147) or 3 weeks (TG,
9.7% ± 2.1%; CT, 16.09% ± 3.4%; p=0.1758) after conditioning. In the same, nonconditioned context, the tone was played to assess fear associated with the conditioning tone. There was no significant difference in tone-conditioned fear 48 hours (TG,
62.20% ± 5.1%; CT, 55.88% ± 4.8%; P=0.3922) or 3 weeks (TG, 57.85% ± 6.9%; CT,
53.09% i 6.4%; P=0.6289) after conditioning.
In both the spatial and non-spatial tasks, MEC-II is not necessary for consolidation of these memories.
5.4
5.4.1
Discussion
Spatial Learning
Our results demonstrate that the mono-synaptic input into the hippocampus from
MEC-III is not necessary for spatial learning tasks. This is interesting because the
two major inputs into CA1 that demonstrate spatial representations, CA3 and MEC,
are individually not necessary for spatial learning, and yet CA1 is necessary for spatial
learning. The use of the molecular lesions of both regions show that both pathways
are sufficient for this task. The LEC mono-synaptic pathway was not disrupted in
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either molecular lesion, but is not known to demonstrate any spatial representation.
This has implications for the types of spatial information generated via the trisynaptic pathway and via the entorhinal cortex. For the purposes of spatial learning
tasks, these two pathways may generate very similar sets of input, or CA1 may not
distinguish between any differences in these inputs. This could be a built in redundancy in the hippocampal circuit. Given the importance of spatial navigation for mice
and other mammals, this redundancy could have evolved due to an increased survival
rate for animals that could use either pathway when the other was disrupted. Another
possible explanation is that these tasks simply do not allow us to test the differences
in the CA3 and MEC-II inputs. Either way, in the same task in which hippocampally
lesioned animals and CA1-plasticity-lesioned mice show a deficit, lesioning the CA3
or MEC-III inputs has no effect on performance in this task.
5.4.2
Spatial Memory Consolidation
Prior to creation of the MEC-III TeTX mouse, the prevailing hypothesis about the
role of the PP/TA pathway had been that this input was necessary for long-term
consolidation of spatial memories. With our more specific lesioning technique, we
have definitively rejected this hypothesis.
There are two possibilities as to why our data conflicts with that of the Remondes
et al study. First, this pathway may be necessary for spatial memory consolidation
in rats but not mice. This seems unlikely due to the overlapping neuro-anatomy
between the two species. Secondly, the physical lesion in the Remondes et al. study
caused collateral damage to structures, which when lesioned cause spatial memory
consolidation deficits. We believe the latter to be the case. Looking at the images
of the lesions they present, it is clear that they are lesioning the PP pathway, but
also lesioning the nucleus reuniens projections to the hippocampus, and parts of the
subiculum and hippocampal output. So, while they have identified that there is some
circuitry within their lesion area that is necessary for memory consolidation, it is
clear to us that it is not the PP/TA input to the hippocampus. Additionally, our
molecular lesion cut input for the MEC-III inputs traveling along both the PP and
157
alvear path (AP), making it a more complete disruption of this pathway.
What we cannot exclude is the possibility that the LEC-II inputs to CA1 are
necessary for spatial memory consolidation. This could warrant additional scrutiny,
but it is important to note that in the Remondes et al study, the lesions were made
to the PP in the dorsal sections of the hippocampus, which may not actually contain
the EC-II inputs at all, given that it has also been shown that in this pole of the
hippocampus, the majority of the inputs from EC-III to CA1 arrive via the AP.
We show that the MEC-III functional output is not necessary for spatial memory
consolidation.
158
5.5
5.5.1
Methods
Morris Watermaze MWM: Consolidation
The Morris watermaze (MWM) task was conducted with male mice between 14 and 22
weeks of age, with minor modifications of the method described previously (McHugh
et al. 2007). All the experiments were performed by operators who were blind to the
genotypes of the mice. Mice were all off-Dox for at least 4 weeks. Mice were kept in
a temperature-controlled room on a constant 12-hour light/dark cycle. Experiments
were conducted at approximately the same time of the day. The mice were transported
from the colony to a holding area where they sat undisturbed for 30 minutes prior
to the experiment. The facility was in a rectangular dim-lit room (340cm x 297 cm)
and consisted of a circular pool (160 cm diameter) filled with opaque water made
with color paints (White 5130, Berghause; Peach 2906, Pearl Tempera) at 19'C. Four
large illuminated objects were hung as extramaze, one cues on each wall. A hidden
circular platform (12 cm in diameter) was placed 1 cm below the water surface and
the mice were trained to find the platform in 4 trials per day for 11 days with an intertrial interval of approximately 60 minutes. During training, the mice were released
from 4 pseudorandomly assigned start locations (N, S, E, and W) and allowed to
swim for 90 s, and mice were manually guided to the platform and allowed to rest
on the platform for 15 s if they did not find the platform in 90 s. Probe trials were
conducted on day 6, day 11, and day 28, with the day 6 and day 11 probe trials
run prior to the 4 training trials. The mice were released at the "S" start location
of pool and were allowed to swim for 90 s in the absence of the platform.
Data
from the training session and probe trials were collected and analyzed with HVS
Image Water 2020 software. An escape latency to the hidden platform was measured
during training, and the quadrant occupancy as well as the number of crossing at the
phantom platform location were measured during probe trials. These data were then
averaged over mice of a particular genotype. Control (N=14) and Mutant (N=13).
159
5.5.2
Morris Watermaze (MWM): Reversal
The reversal version of the MWM was performed similarly to the MWM Consolidation
task (see above), with probes performed on day 6 and day 11. On day 12, however,
the hidden platform location was moved to the quadrant opposite to the original
platform location (180 degrees around the circumference of the pool), while the cues
remained in the same place. Mice were trained 4 trials/day for a further 5 days.
During training, the mice were released from four pseudorandomly assigned start
locations (N, S, E, and W) and allowed to swim for 90 s, and mice were manually
guided to the platform and allowed to rest on the platform for 15 s if they did not
find the platform in 90 s. A probe trial was run on day 16, prior to the trials for
that day to assess the animal's preference for the new "target" quadrant.
Data
from the training session and probe trials were collected and analyzed with HVS
Image Water 2020 software. An escape latency to the hidden platform was measured
during training, and the quadrant occupancy as well as the number of crossing at the
phantom platform location were measured during probe trials. These data were then
averaged over mice of a particular genotype. All mice off-Dox for at least 4 weeks.
Control (N=16) and Mutant (N=14).
5.5.3
Fear Conditioning Consolidation
3-Shock Delay Paradigm: Fear conditioning was performed with male mice between 14 and 27 weeks of age in the animal facility during the light cycle with minor
modifications of the method described previously (McHugh et al. 2007). Experiments
were performed by operators who were blind to the genotypes. There are two distinct
contexts for these behavioral assays which are in two distinct rooms. Context "A" is
a dimly lit room with red lights. In Context "A" The chambers had plexiglass fronts
and backs and aluminum side walls with a triangular plastic roof and measured 30
x 25 x 21 cm. The chamber floors consisted of 19 stainless steel rods spaced 16 mm
apart connected via a cable harness to a shock generator. Chambers were cleaned
prior to an introduction of an individual mouse into them with quatricide. A 1%
160
acetic acid solution was placed beneath the chamber floor during the experiment to
provide a dominant odor. Context "B" contains direct overhead fluorescent lighting
and distinct chambers, measuring 30 x 25 x 21 cm, with plexiglass front and back
walls, and aluminum side walls but with a flat roof. In addition, the floors of these
chambers were made up with white plastic and the odor was provided with 0.25% benzaldehyde (in 100% ethanol). These lighting, chamber materials and odor employed
on Day 3 provided a context quite distinct from Context "A". In each context, there
are 4 conditioning chambers. In each fear conditioning experiment, mice were transported from the colony room to a room adjacent to the conditioning behavioral suite
and were left undisturbed for 30 minutes prior to the start of the experiment. In all
of these experiments, the animals activity in the chamber was recorded using FreezeFrame software. Freezing behavior was assessed from the video image of the mouse
using FreezeView software, with a minimum bout time of 1 s. Freezing values were
then averaged over mice of a particular genotype for each session.
On Day 1, mice were placed in Context "A" and allowed to explore for 240 s, at
which point the first of 3 tone-shock pairings started. The pairing consisted of a 20
s tone, 75 db, 2000Hz which co-terminated in a 2 s, .75-mA foot-shock. The second
tone-shock pair initiated at 380 s into the experiment and the third pair initiated
at 520 s. Mice remained in the conditioning chamber for a total of 706 s at which
point they were removed back into their transport (home-cage) and to the adjacent
room. 24 hours later, on day 2, mice were placed back in Context "A" and allowed
to explore for 300 s. 24 hours later, on day 3, mice were placed in a novel Context
"B" for 3 minutes to assess their generalized "Pre-Tone" fear and their freezing to
the conditioning "Tone." On day 21, mice were placed back into Context "A" to
assess long-term memory of the conditioning context and freezing was assessed. On
day 22, mice were placed in Context "B" and freezing was assessed under the same
paradigm as day 3. These experiments were carried out in off-Dox animals. Control
mice (N=12), Mutant mice (N=8).
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5.5.4
One-Trial Per Day
The one-trial per day version of the MWM task was conducted with male mice between 14 and 22 weeks of age. All mice were off-Dox for at least 4 weeks (MEC-III
transmission inhibited). All the experiments were performed by operators who were
blind to the genotypes of the mice. Mice were kept in a temperature-controlled room
on a constant 12-hour light/dark cycle.
Experiments were conducted at approxi-
mately the same time of the day. The mice were transported from the colony to
a holding area where they sat undisturbed for 30 minutes prior to the experiment.
The facility was in a rectangular dim-lit room (340cm x 297 cm) and consisted of
a circular pool (160 cm diameter) filled with opaque water made with color paints
(White 5130, Berghause; Peach 2906, Pearl Tempera) at 19 C. Four large illuminated
objects were hung as extramaze cues on each wall. A hidden circular platform (12
cm in diameter) was placed 1 cm below the water surface and the mice were trained
to find the platform with 1 trial per day for 18 days. For each day during training,
the mice were released from pseudorandomly assigned start locations (N, S, E, and
W) and allowed to swim for a maximum of 90 s. Mice were manually guided to the
platform and allowed to rest on the platform for 30 s if they did not find the platform
within 90 s. Data from the training session was collected and analyzed with HVS
Image Water 2020 software. An escape latency to the hidden platform was measured
during training. These data were then averaged over mice of a particular genotype.
Control (N=13) and Mutant (N=13).
5.5.5
Barnes Maze
Room conditions: Lights on maximum setting for the room white noise machine set
at 85 dB during all trials to incentivize mouse to find dark escape box. Distinct cues
were hung on each of the room's 4 walls. Mice were held in the dark anteroom when
not being tested The two rooms were separated by a translucent curtain.
Falcon
tubes filled with Hydrogel were taped in four locations on the base of the maze so
that the mild odor of the Hydrogel could not be used as a cue.
162
Maze: White plastic circle (92 cm in diameter and 2 cm thick) with 20 equally
spaced, 2 cm wide holes. An dark escape tunnel (5 cm deep, 5 cm wide and 11.5
cm long) is placed underneath one of the holes. The escape tunnel contains a capful
(from a 50 mL Falcon tube) of Hydrogel reward. The mouse is placed in an opaque
plastic cylinder at the start of each trial. After 15 s, the cylinder is lifted. Mice
are tracked with EthoVision software. The maze was thoroughly cleaned with 70%
ethanol between trials.
Water restriction/handling/habituation: The animals were water restricted for 4
days prior to habituation. To habituate the mice to Hydrogel and the experimenter:
For 2 days, mice were handled 2 minutes each and a bag of Hydrogel was placed in
the cage. The water bottle was also left in the cage. On the third day the water
bottle was removed from cage in the afternoon and mice were weighed and handled
for 2 minutes/each. For the next three days, and for duration of experiment mice
were given 10 minutes access to water bottle per day. To habituate the mice to the
maze, mice were placed in the anteroom for 15 minutes prior to maze exposure. In
the maze room, the lights were on and the Hydrogel reward was in place but the
aversive noise was not turned on. One by one, the mice were placed in the opaque
cylinder, located in the center of the maze. After 15 s, the cylinder was lifted and the
mouse was allowed to explore the maze for 120 s, after which the experimenter led
the mouse to the escape box. The mouse remained in the escape box for 1 minute.
Each mouse received one habituation trial.
Training: Mice received 4 training trials a day for 4 days. Each mouse was placed
in the opaque cylinder. The white noise was turned on, and the cylinder was lifted
after a 15 s delay. Once the mouse escaped into the escape tunnel, the aversive noise
was turned off and the animal was left in the escape tunnel for 1 minute to consume
the reward. If the mouse did not escape before 120 seconds elapsed, the mouse was
led to the escape tunnel, at which point the noise was turned off and the animal was
left in the tunnel for 1 minute to consume the reward. The ITI between trials is 15-20
minutes.
Probe: Each animal received one 24-hour probe trial after the first 4 days of
163
training. The escape tunnel was removed before each probe trial. Each mouse was
placed in the opaque cylinder and the aversive noise was turned on. After 15 s, the
cylinder was lifted. After 60 s, the escape tunnel was returned and the animal was
led to the tunnel, where it was left for 1 minute to consume the reward.
Reversal Training: The mice received 4 reversal training trials a day for 4 days,
using the same procedure as above except the new target hole was placed in the
opposite hole from the original escape hole. The average ITI was 15 minutes.
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........
..............................
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Watermaze Learning 11 Days
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Day 6 Probe
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60'
50,
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10,
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Left
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Figure 5-1: Watermaze Learning and Reversal Training. Quadrant Occupancy and Platform Crossings on Probe Trials. Blue is Control Mouse. Red is
MEC-Ill TeTx mouse. (A) Escape Latency for mice learning first platform position
for 11 days and then escape latency for mice learning new platform location from day
12-16. Reversal platform was placed in the opposite quadrant from the original platform. (B) Probe trials on day 6, 11, and 16. Probes were conducted before training
on day 6, 11, and 16 with a 90 s trial in which the escape platform was removed.
Percent time in quadrant each of the four quadrants was assessed. Grey line indicates
what behavior would be if mice performed at chance. (C) Platform Crossings on
probe trials. During probes on day 6, 11 and 16, crossings of the phantom platform
were counted.
165
.....
...........
Trial-To-Trial Lateny Savings
Day 1 Reversal
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30
20
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1 to 2
2 to 3
3 to 4
Control
Mutant
1 to 4
0
'5
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lo-
-20
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Trial to Trial
Figure 5-2: Trial-to-Trial Savings on Day 1 of Watermaze Reversal Latency
improvements, "Savings" between trials on first day of reversal learning. Blue is
Control Mouse. Red is MEC-II TeTx mouse. Savings are assessed between Trial 1
and Trial 2, between Trials 2 and 3, between Trials 3 and 4, and between Trials 1 and
4.
166
* Control
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B
A
One Trial/Day
2 Day Blocks
One Trial Per Day: 17 days
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Figure 5-3: Watermaze: One Trial per Day. Blue circles represent Control (MECIII Cre+) and Red circles represent Mutant (MEC-III TeTx) mice. (A) Averaged
escape latency (seconds) per genotype over the 18-day training period. (B) Averaged
escape latency (seconds) per genotype represented in nine 2-day blocks.
167
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...................................
.....
.........................
Day Averages Both
Training and Reversal
A
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Figure 5-4: Spatial Learning: Barnes Maze. Blue circles/bars represent Control
(MEC-III Cre+) and Red circles/bars represent Mutant (MEC-III TeTx) mice. (A)
Averaged escape latency (seconds) for 4 days of learning, and 4 days of the reversal
task. (B) Day 5 Probe. Percentage of time spent in quadrants during the probe:
Target refers to the quadrant of the maze platform that housed the escape box during
training.
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0
Right
Opposite
Lef
Platform
Target
Figure 5-5: Spatial Memory Consolidation. Blue Circles and bars represent
Control (MEC-II Cre+) and Red Circles and Bars represent Mutant (MEC-II TeTx)
mice. (A) Water-maze escape latency learning curve for hidden platform over 11 days.
(B) Quadrant occupancy during probe trials on days 6, 11 and 28. Grey bar represents
chance if animal explored all quadrants equally and had no quadrant preference. (C)
Platform Crossings during probe trials on days 6, 11, and 28. Crossings represent
number of crossing of area where platform would have been.
169
Control
Mutant
A
B
TI T
T
24 Hour
Context Test
PkM
Pre-tone
3 Week
48 Hours
Tone
3 Weeks
Figure 5-6: Fear Conditioning: Consolidation Blue is control (MEC-III Cre+)
and red is mutant (MEC III-TeTx) mouse. (A) Freezing to conditioning context
24 hours after conditioning and 3 weeks after conditioning. (B) Freezing to different
context "Pre-Tone" and to tone from conditioning "Tone" 48 hours after conditioning
and 3 weeks after conditioning.
170
Chapter 6
The Role of Medial Entorhinal
Cortex Pattern Completion
In this chapter, using the MEC-III TeTx mouse as a model, we test the role of MECIII synaptic output in a fear conditioning version of a pattern completion task. In
this pre-exposure task, we show that MEC-II TeTX mice are impaired in rapidly
recalling contextual representations prior to a foot-shock, and thus are impaired in
forming contextual fear memories.
6.1
Pattern Completion
Pattern completion as a phenomenon describes the process by which an entire experience of memory can be retrieved with a smaller subset of cues (Marr 1971) (Rudy
and O'Reilly 1999). Larry Squire proposed that pattern completion is essential for
the declarative memory system (Squire 1992), as it allows for a single consciously
recalled memory to evoke a larger subset of consciously recallable memories. Within
the hippocampus, the nature of the recurrent collateral projections in CA3 make CA3
a potential locus for pattern completion (Marr 1971). Recurrent collaterals are projections from CA3 neurons that synapse on neighboring CA3 neurons, and allow for
a small subset of activated CA3 neurons to activate other CA3 neurons.
In support of this theory, knocking out NMDA-receptor mediated plasticity specif171
ically in the recurrent collaterals disrupts pattern completion in a watermaze task
(Nakazawa et al. 2002). In this experiment, mice learned the location of a hidden
platform in the MWM task based on the platform's relationship to extra-maze cues.
In a probe test with full extra-maze cues, both the mutant and control mice searched
the target quadrant more than the other quadrants, demonstrating knowledge of
where the platform should be. However, with three of the four cues removed, the
control mice still explored the target quadrant, but the mutants explored all quadrants equally. The control mice used the single cue to recall a spatial map of where the
hidden platform was, but the mutants could not. Thus, plasticity in CA3 is necessary
for pattern completion, as proposed.
Pattern completion is also testable in a pre-exposure fear conditioning paradigm
(Fanselow 1990) (Rudy and O'Reilly 1999). Mice brought into a context and 'immediately' shocked, meaning that the shock occurred in under thirty seconds after
placement in the context, display very little freezing to the context (Fanselow 1990).
The rational for why they do not associate the contextual CS with the foot-shock
US suggests that a contextual representation is not fully formed in the short period
before the shock, and thus cannot associate with it.
However, altering the 'immediate-shock' paradigm by inserting a pre-exposure
day before the shock day significantly changes the performance. In the pre-exposure
paradigm, mice explore the context for ten minutes on the day before the immediate
shock occurs. Mice with pre-exposure on day one, and immediate shock on day two
form a better contextual memory than animals just immediately shocked on day one,
even though the same amount of time in the context is given before the shock on
the shock day (Fanselow 1990). This suggests that during the time prior to shock
in the pre-exposure paradigm, animals are recalling the familiar environment, and
this process occurs more quickly than novel contextual formation. Limited exposure
during exploration prior to shock in the pre-exposure paradigm allows the animals
pattern complete a full contextual representation.
The CA3-TeTx mice are disrupted in this pre-exposure contextual fear conditioning (PECFC) paradigm, when the time-to-shock on day two was ten seconds, further
172
supporting the notion that CA3 and the hippocampus itself, is important in pattern
completion (Nakashiba et al. 2008).
We tested the MEC-III TeTx mice in the PECFC paradigm to assess whether
other circuits in the MTL are also necessary in pattern-completion. MEC-II TeTx
mice show a pattern completion deficit.
6.2
6.2.1
Results
Pre-exposure Paradigm
The pre-exposure contextual fear conditioning (PECFC) task was run in mice off-Dox
for at least 4 weeks. For the pre-exposure, all mice were exposed to the context for 10
minutes on Day 1 without any tone of foot-shock (Figure 6-1 A, page 174). On day
2, mice were placed back into the same context, and given different lengths of time
prior to shock: 0, 10, 20 or 60 seconds. Contextual fear memory was then assessed
on the third day and measured by freezing levels over a three minute period in the
context.
MEC-III TeTX mice not impaired when time-to-shock is 0 seconds.
There was not a statistically significant difference in freezing to the conditioning
context as assessed on day three, when the animals time-to-shock on Day 2 was 0 s
(CT, 4.37% ± 2.39%, TG, 11.40% ± 5.38%, P=0.1941). Mice in this experiment had
no time to recall the familiar context, and thus this serves as a control for no-recall
in the pre-exposure paradigm.
ME C-III TeTX mice are impaired when time-to-shock is 10 seconds.
There was a statistically significant difference in freezing to context on Day 3,
however, when the time-to-shock on Day 2 was 10 s, with mutants freezing less than
controls (CT, 38.52% ± 3.96%, TG, 25.25% ± 3.54%, P=0.0187).
Over the three
minute period, the curves followed very different profiles, with the controls freezing
173
. ........
.....
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...
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..
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..
* n~s.
n.s.
7060
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B
A
50
n.s.
40
30 |L.
30-
20-
a
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20
10
0
10
20
60
Seconds to Shock (Day 2)
60
120
180
Seconds
Figure 6-1: MEC-III TeTx mice impaired with 10s recall in Pre-Exposure
Paradigm. Control (MEC-III Cre+ mice) are represented by blue and Mutant
(MEC-IlI TeTx) are represented by red. (A) Day 3 freezing-to-context percentages
averaged over first 3 minutes of context exposure with relation to time-to-shock on
day 2. On day 2, mice were either shocked immediately, 10, 20 or 60 s after placement
in context. (B) Day 3 freezing profile for mice shocked 10 s after placement on day 2.
significantly more (Figure 6-1 B).
ME C-III TeTX mice are not impaired when time-to-shock is 20 or 60
seconds.
Freezing to context was not statistically different when the time-to-shock on Day
2 was 20 s ((CT(N=17), 37.60% ± 4.96%, TG(N=12), 27.65% ±4.02%, P=0.1556) or
when the time-to-shock was 60 s (CT(N=14), 56.17% i 4.60%, TG(N=14), 56.35%i
7.09%, P=0.9834).
There was a trend for the MEC-Ill TeTx mice to freeze less when shocked 20 s
after placement, but this was not significant. These results suggest that in short intervals, control mice can pattern complete the contextual representation, but synaptic
transmission from MEC-IlI is required for this pattern completion.
174
6.2.2
Immediate Shock Paradigm
As a control for pattern completion, mice were run in an 'immediate' shock paradigm
to show that pre-exposure can enhance contextual learning, in MEC-III TeTx mice.
MEC-III TeTX mice are not impaired when in immediate shock paradigm.
Immediate shock contextual fear conditioning (ISCFC) task was run in mice offDox for at least 4 weeks. This experiment removed the context exposure pre-exposure
day in order to assess the effect of the pre-exposure on subsequent memory recall.
Mice that were shocked 10 s after placement into a novel chamber, and subsequently
tested for freezing behavior to that context on the following day showed no statistically
significant difference between the two genotypes (Figure 6-2, page 176) (CT, 13.72% ±
5.60%, TG, 10.00% ± 3.46%, P=0.6218). More importantly, the freezing levels, as
compared to the Day 3 freezing levels in the PECFC are notably lower, suggesting
that pre-exposure alone aids rapid contextual memory recall that can be bound with
the foot-shock after 10 s. Mice shocked 60 s after placement into a novel chamber,
and subsequently tested for freezing behavior the following day showed no statistically
significant difference between the two genotypes (CT, 28.06% ±5.13%, TG, 32.08%
9.92%, P=0.7036).
6.3
Discussion
In this chapter we demonstrate a pattern-completion deficit in the MEC-III TeTx
mice in the pre-exposure contextual fear conditioning task. Intact synaptic function
in MEC-III is thus necessary for this task.
6.3.1
Pattern Completion
These results indicate that brain structures other than CA3 compute or participate
in the process of pattern completion. A local intra-MEC circuit could serve as a
175
Control
A
-Mutant
70'
n.s.
60a
50-
40IL
n.s.
30
~20
1
0
0
60
10
Seconds to Shock (Day 1)
Figure 6-2: MEC-III TeTx mice Similar to Controls in Immediate-shock
Paradigm. Control (MEC-II Cre+ mice) are represented by blue cirlces and Mutant
(MEC-III TeTx) are represented by red circles. (A) Averaged freezing time over the
first 3 minutes of day 2 context exposure with relation to time-to-shock on day 1,
without pre-exposure.
176
recurrent network.
We see staining spread in layer III EC with the injection of
the AAV8-CHR2-EYFP virus (Figure 3-1 C, page 80).
Whether these are axons
or dendrites has not been elucidated, but this result indicates that there is some
intra-MEC synaptic processing. Alternatively, a loop formed within the entorhinal
and hippocampal complex, the MECIII-CA1-MECV-MECIII loop, could support a
pattern completion-like process.
Our results show a significant difference in freezing to the context in the MEC-II
TeTx mice when the prior day's shock was given 10 seconds after placement into the
chamber. Additionally there was a trend for less freezing when the shock was given
20 seconds after placement into the chamber. This timeline fits well with our data in
the 20 second trace conditioning paradigm that produces a deficit in contextual fear
association (Figure 4-2 A, page 4-2), which we hypothesize to be a pattern completion
deficit as well. For discussion on that, please read 4.5.2 on page 130.
While the freezing deficit in the pre-exposure paradigm strongly suggests a recall
problem in the MEC-II TeTX mice, this has not been conclusively shown. With the
Dox-on/Dox-off system, this can be specifically addressed. In this paradigm, mice
would experience the pre-exposure with synaptic transmission on (Dox-on) to ensure
that context formation is normal. Prior to day 2, Dox removal from the animal's
diet would ensure inhibition of synaptic transmission in the MEC-II pathway. Mice
receiving shock after 10 seconds of context exposure in this paradigm should be impaired in contextual memory on the next day's context test, and should freeze less.
Such a result would confirm a recall deficit and further support the idea that these
mice are deficient in pattern completion.
MEC-III TeTx mice with the pre-exposure day off-Dox then conditioned and
tested a month later should show the opposite results. MEC-III TeTx mice in this
experiment should freeze similarly to controls in the context memory test.
6.3.2
Other Assessments of Pattern Completion
Watermaze.
Similarly to Nakazawa et al. (Nakazawa et al.
2002), these mice
should display a pattern-completion deficit in the pattern completion version of the
177
Morris watermaze. As shown in "A" in figure 5-1 on page 165 , the MEC-III TeTx
mice an acquire the location of the hidden platform as well as their control litter
mates, and under full cue conditions, search the target quadrant as much as controls
in the probes.
MEC-III TeTx mice, when run in partial cue conditions during a probe trial may
display a deficit as compared to controls, if the MEC-II projection is necessary for
spatial memory pattern completion. It may be the case that different types of memories are pattern completed by different brain circuitry, or that pattern completion is
a process that utilizes many systems together.
Active Place Avoidance.
The active place avoidance task provides another way
to assess spatial pattern completion. Active place avoidance is more similar to fear
conditioning, but includes a spatial component and requires spatial memory in the
mouse for proper performance (Cimadevilla et al. 2000). This task uses a rotating
grid-floor and spatial cues. During training, the animal is only shocked when it enters
a zone as defined by the visual cues surrounding the maze. In this task, in order to
avoid the shock zone, the mice must constantly move away from the shock zone, and
can not simply sit in the opposite quadrant, as the rotating maze eventually carries
them back to the shock zone. Mice must develop a representation of the shock zone
and constantly use that map to avoid the foot-shock.
Using this paradigm, it would be interesting to see if MEC-Ill TeTx mice can
learn this task. We assume that they can, because our spatial memory acquisition
assays show no deficit. If the MEC-IlI TeTx mice can use cues to develop a spatial
representation we would then probe these mice with full and partial cues, in a manner
similar to the probes in the pattern completion version of the watermaze. We expect
that these mice will demonstrate knowledge of the shock-zone during the full cue
probe, but may not fully pattern complete in the partial cue probe, and thus will
enter the shock zone more than control mice.
178
6.4
6.4.1
Methods
Pre-exposure and Immediate Shock
Fear conditioning was performed with male mice between 14 and 27 weeks of age
in the animal facility during the light cycle with minor modifications of the method
described previously (McHugh et al. 2007). Experiments were performed by operators
who were blind to the genotypes.
Context "A" is a dimly lit room with red lights. In context "A" the chambers
had plexiglass fronts and backs and aluminum side walls with a triangular plastic
roof and measured 30 x 25 x 21 cm. The chamber floors consisted of 19 stainless
steel rods spaced 16 mm apart connected via a cable harness to a shock generator.
Chambers were cleaned prior to an introduction of an individual mouse into them with
quatricide. A 1% acetic acid solution was placed beneath the chamber floor during
the experiment to provide a dominant odor. There are 4 conditioning chambers.
In each fear conditioning experiment, mice were transported from the colony room
to a room adjacent to the conditioning behavioral suite and were left undisturbed for
30 minutes prior to the start of the experiment.
In all of these experiments, the animals activity in the chamber was recorded using
FreezeFrame software. Freezing behavior was assessed from the video image of the
mouse using FreezeView software (Coulbourn Instruments, Whitehall, PA), with a
minimum bout time of 1 second. Freezing values were then averaged over mice of a
particular genotype for each session.
Pre-Exposure On day 1, mice were brought into the conditioning chamber in
context "A", and allowed to explore freely for 10 minutes, and then transported
back to their home cages. On day 2, the mice were transported individually to the
conditioning chamber in context "A" and received a single 1.25 mA foot-shock (2 s
duration) 0, 10, 20 or 60 s after being placed in the chamber. The mice remained
in the chamber for 30 s after the shock, then were transported back to their home
cages. On day 3, contextual fear was assessed by placing the mice in the conditioning
chamber (context "A") for 5 minutes. For pre-exposure mice, time-to-shock on Day 2
179
0 s: Control (N=11) and Mutant (N=7), for time-to-shock 10 s: Control (N=17) and
Mutant (N=16), for time-to-shock 20 s: Control (N=17) and Mutant (N=12) and for
time-to-shock 60 s: Control (N=14) and Mutant (N=14).
Immediate shock In this paradigm, the pre-exposure day was removed. Mice
were shocked either 10 seconds or 60 seconds after being placed into novel chamber
in context "A". On day 2, mice were placed back in context "A" and freezing was
assessed over a 5 minute testing session. For immediate-shock mice, time-to-shock
10s, Control (N=7) Mutant (N=5) and for time-to-shock 60s, Control (N=7) and
Mutant (N=5).
180
Appendix A : 1-shock Delay Fear Conditioning
.1
CA3-TeTx mice are impaired in contextual, but not tone fear conditioning in the
1-shock delay paradigm (Nakashiba et al. 2008). As MEC-III provides one of the
other major inputs into CA1, we tested the MEC-III TeTx mice in this same task.
We find that these mice are impaired in conditioning to both the context and to the
tone.
1-Shock Delay Conditioning
.2
.2.1
1-shock Delay Task
MEC-III TeTx mice are impaired in tone and contextual fear conditioning in a 1-shock delay taslk
One-shock delay conditioning task was run in mice off-Dox for at least 4 weeks.
There was a statistically significant difference in freezing levels during the conditioning, with the mutant mice freezing less than controls ("A" from -3)(2-way ANOVA:
Genotype x Time F(20,880)=4.47, P < 0.0001; Time F(20,880)=15.93, P < 0.0001;
Genotype F(1,880)=11.57, P=0.0014).
On the second day, testing of freezing to the conditioning context also showed a
significant difference between the genotypes, again with mutants freezing less during
the session ("B" -3)(2-way ANOVA: Genotype x Time F(29,1276)=1.31, P=0.1242;
Time F(29,1276)=2.72, P < 0.0001; Genotype F(1,1276)=13.89, P=0.0005), this is
also represented in "E" over the 5 minute period (CT, 34.80% ± 3.37%, TG, 17.05%
3.35%, P=0.0005).
In a novel context, there was a significant difference in pre-tone freezing between
the genotypes, indicating possibly more generalized fear in the control mouse (CT,
5.97% ± 1.04%, TG, 2.80% + 0.77%, P=0.02), although it must be noted that the
freezing levels for both genotypes are almost at expected baseline.
Additionally, in the novel context, there was a significant difference in the freezing
percentages during the tone-presentation, with the mutant mice freezing less ("C" and
181
"D" in -3)(CT, 49.11% i 3.59%, TG, 28.10% ± 3.50%, P=0.0001).
.2.2
Methods
On Day 1, mice were placed into a chamber in Context "A" and allowed to freely explore for 180 seconds. A 75db, 2000Hz tone started at 150 seconds and co-terminated
with a a single, 2 second, 1.25-mA footshock. Following the shock delivery, the mice
remained in the chamber for 30 seconds, and then were returned to the home cages
and transported back to the holding room. On Day 2, the mice were returned to the
conditioning chambers under the conditions identical (Context "A") to those on Day
1 for a five minutes context test. On Day 3, the mice were transferred to Context "B."
The mice were placed in this chamber for 3 minutes during which freezing responses
were measured. This response compared to the response to the conditioning chamber on Day 2 gives a measure of the context specificity of contextual conditioning.
The mice were then given the same tone as the one given on Day 1, but this time
for 2 minutes and freezing responses were monitored. Control (N=24) and Mutant
(N=22).
182
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B
40
Control
60
050T
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Io0a
0
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dif
o
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360
2010
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contraMutant
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ToneIFoBt-Shock
Seconds
Figure -3: MEC-IlI TeTx mice are impaired in 1-Shock Delay Conditioning.
Control (MEC-Ill Cre+ mice) are represented by blue cirlees and Mutant (MECTeTx) are represented by red circles. (A) Freezing profile over 210 s conditioning
paradigm. Green block - tone, red line = shock. (B) Day 2 Freezing profiles in
same context as conditioning chamber. (C) Freezing Profile on day 3 in Context "B"
without tone and with tone (Green block). (D) Bar graphs representing averaged
freezing for 3 minute pre-tone in Context "B" and averaged freezing over 2 minute
tone presentation. (B) Bar graph represeting averaged freezing percentage over total
5 minutes of Day 2 context exposure.
183
..
..
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- - -- - - _-
.3
Appendix B: Control Behaviors
.4
Rota-Rod
A0...
....... .t
.
MEC-III TeTx mice normal in Rota-Rod Behavior.
To assess potential motor deficits, MEC-III TeTx were run on the rota-rod test,
a standard test for motor function, balance and coordination (Jones and Roberts
1968). MEC-1II TeTx mice showed a trend towards falling off the apparatus sooner,
but this difference was not statistically different. (2-way ANOVA: Genotype x Time
F(2.52)=1.91, P=0.1577; Time F(2,52)=14.26, P < 0.0001; Genotype F(1,52)=0.2,
P=0.6596). See -4.
Rota-Rod Learning
Control
Mutant
300= 250200*@ 150C
100co
500-
2
Trial
3
Figure -4: Rota-Rod Learning. Blue Bars represent Control (MEC-III Cre+) and
Red Bars represent Mutant (MEC-III TeTx) mice. Averaged seconds to fall off RotaRod spinning wheels during each of three trials during the day.
184
- -
4
lzzz
.5
Open Field
Open-field behavioral assessment tests anxiety in mouse lines (Carola et al. 2002).
We saw no differences in center distance or center crossings, but did MEC-II TeTx
mice did move more during the first three days, and displayed more rearing activity
during the first two days. These results may indicate an inability to process this
context as familiar over the first two to three days, as rearing activity is induced by
novelty (Crusio et al. 1989).
MEC-III TeTx move more distances on days 1 to 3. On day 1 ("A" -5,
mutant mice moved significantly more over the 10 minute session (2-way ANOVA:
Genotype x Time F(9,252) =0.8, P=0.6161; Time F(9,252) =11.93, P < 0.0001;
Genotype F(1,252) =11.88, P =0.0018) and this genotype effect was the same through
day 3. "B" Day 2 (2-way ANOVA: Genotype x Time F(9.252) =0.81, P=0.6053; Time
F(9,252) =8.86, P < 0.0001; Genotype F(1,252) =10.97, P =0.0026). "C" Day 3 (2way ANOVA: Genotype x Time F(9,252) =0.40, P=0.9353; Time F(9,252) =1.82,
P=0.0658; Genotype F(1,252) =8.95, P =0.0057).
MEC-III TeTx do not move more than controls on days 4 to 7. On
days 4 to 7 "D-G" there was no genotype effect on the total distance moved during
the 10 minute session. "D" Day 4 (2-way ANOVA: Genotype x Time F(9,252) =1.23,
P=0.2786; Time F(9,252) =1.77, P=0.0752; Genotype F(1,252) =3.72, P =0.0639).
"E" Day 5 (2-way ANOVA: Genotype x Time F(9,252) =1.11, P=0.3547; Time
F(9,252) =2.38, P=0.0136; Genotype F(1,252) =3.37, P =0.0770).
"F" Day 6 (2-
way ANOVA: Genotype x Time F(9,252) =0.96, P=0.4701; Time F(9,252) =1.96,
P=0.0441; Genotype F(1,252) =1.82, P =0.1881). "G" Day 7 (2-way ANOVA: Genotype x Time F(9,252) =1.10, P=0.3640; Time F(9,252) =3.55, P=0.0004; Genotype
F(1,252) =1.18, P =0.2869).
"H" Total distance in the first minute of each day.
There was no significant difference between genotypes in the first minute on day 1
(TG, 224.2cm ± 20; CT, 205.5cm ± 25.85; P=0.5718), but there was a significant
difference on day 2 (TG, 235.1cm
21; CT, 155.4cm± 15.85; P=0.0047), day 3 (TG,
162.1cm ± 19, 3; CT, 98.07cm ± 220.96; P=0.0326), and day 4 (TG, 146.2cm ± 18.33;
185
I
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B 30
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A300'
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123456789
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$
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Day 7 Total Distance
300-
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Over 7 Days
300-
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20
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3
4
5
Mintues
6
7
8
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i
i
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4
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Day
Figure -5: Open-Field Total Distance. Blue circles represent Control (MEC-III
Cre+) and Red circles represent Mutant (MEC-III TeTx) mice. (A-G) Averaged
total distance (cm) per minute of the ten minute daily session for Day 1-7. (H)
Averaged total distance (cm) during first minute of each of the seven days.
186
....
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Day 1 Rearing
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Day 5 Rearing
Day 6 Rearing
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7"
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00
" Control
* Mutant
0
10
Mintues
Average Number of Rearing Events
Per Day: 7 days
Day 7RearingH
91
8'
7'
f
6,{i
I
1 2
u3es 6'
Mintues
9 10
0
1
2
3 D7 4
Day
5
6
Figure -6: Open-Field Rearing. Blue circles represent Control (MEC-IJI Cre+)
and Red circles represent Mutant (MEC-II TeTx) mice. (A-G) Averaged total
number of rearing events per minute of the ten minute daily session over Days 1-7.
(H) Averaged number of rearing events per day, over the 7 days.
187
CT, 84.27cm ± 20; P=0.0302).
There was no significant difference between geno-
types in the first minute on day 5 (TG, 101.4cm ± 19.29; CT, 63.27cm ± 19.98;
P=0.1805), day 6 (TG, 111.4cm ± 23.41; CT, 77.80cm ± 21.89; P=0.3035) and day 7
(TG, 94.33cm t 28.91; CT, 78.4cm ± 24.89; P=0.6793).
MEC-III TeTx mice rear more on days 1 and 2. On day 1 (-6(A), mutant
mice reared significantly more over the 10 minute session (2-way ANOVA: Genotype x
Time F(9,252) =1.38, P=0.1990; Time F(9,252) =1.91, P=0.0514; Genotype F(1,252)
=7.15, P =0.0124) and this genotype effect was the same through day 2. "B" Day 2
(2-way ANOVA: Genotype x Time F(9,252) =1.26, P=0.2574; Time F(9,252) =1.96,
P=0.045; Genotype F(1,252) =6.34, P =0.0178).
MEC-III TeTx mice rear similarly to controls on days 3 to 7. On days
3 to 7 (C-G) there was no genotype effect on the rearing events during the 10 minute
session. "C" Day 3 (2-way ANOVA: Genotype x Time F(9,252) =0.48, P=0.8857;
Time F(9,252) =1.56, P=0.1277; Genotype F(1,252) =3.92, P =0.0577). "D" Day 4
(2-way ANOVA: Genotype x Time F(9,252) =0.39, P=0.9382; Time F(9,252) =2.22,
P=0.0212; Genotype F(1,252) =1.28, P =0.2666).
"E" Day 5 (2-way ANOVA:
Genotype x Time F(9,252) =1.35, P=0.2126; Time F(9,252) =4.99, PiO.0001; Genotype F(1,252) =0.52, P =0.4778).
"F" Day 6 (2-way ANOVA: Genotype x Time
F(9,252) =0.65, P=0.7521; Time F(9,252) =5.50, Pi0.0001; Genotype F(1,252) =0.10,
P =0.7504). "G" Day 7 (2-way ANOVA: Genotype x Time F(9,252) =0.95, P=0.4791;
Time F(9,252) =6.59, PiO.0001; Genotype F(1,252) =0.11, P =0.7422).
"H" Aver-
aged rearing events per day, over 7 day trials. There was a significant difference
between genotypes in the averaged number of rearing events on day 1 (unpaired t
test P=0.0124), and Day 2 (P=0.0178).
On days 3-7, however there was no sig-
nificant difference between genotypes: day 3 (P=0.0577), day 4 (P=0.2666), day 5
(P=0.4778), day 6 (P=0.7504) and day 7 (P=0.7422).
188
.6
.6.1
Methods
Rota-Rod
Rota-Rod experiment done using Ugo Basile Rota-rod, model 7650-Acceler Rota-Rod
for mice. All mice were off-Dox for at least 4 weeks and aged between 8 and 12 weeks
old. Mice were tested for three trials over one day. Mice were placed onto the black
rollers and machine was turned on. The roller moved with a constantly accelerating
pace and for each mouse, the time before falling from the rod was recorded. Mice
were given a maximum time of 300 s if they did not fall off. Once all mice in a cohort
were tested, trial two began. Control (N=23) and Mutant (N=21).
.6.2
Open-Field
Locomotor activity, exploration and anxiety were assessed using an open field test.
All mice were off-Dox for at least 4 weeks and experimenter was blind to the genotypes. Each subject was placed in the center of the open field apparatus (40 x 40 x
30.5 cm; Omnitech Electronics, Columbus, OH) and the experiment was started. Apparatus consisted of clear Plexiglas cages with infrared monitors ever 2.54 cm along
the perimeter (16 on each side) and 4.5 cm above the floor. Data was collected with
the VersaMax analyzer from AccuScan Instruments, Inc, and analyzed with VersaDat
software V3.20-127E (www.accusan-usa.com). The maze was cleaned with quatricide
after each trial. Horizontal activity (in centimeters), vertical activity (rearing measured by counting the number of photobeam interruptions), and time spent in the
center were measured. Mice were given one 10 minute trial per day for 7 days. Control
(N=15) Mutant (N=15).
189
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