Viral Delivery of Recombinant Growth Hormone to Rescue Effects of Chronic Stress on
Hippocampal Learning
by
Christopher M. Saenz
B.S. Biology
Hunter College, CUNY 2007
SUBMITTED TO THE DEPARTMENT OF THE BRAIN AND COGNITIVE SCIENCES IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN NEUROSCIENCE
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
February 2012
MASSACHUSE~T
NrrJr
ACUSETTS INSTriUTE
OF TECHNOLOGY
FEB 21 2012
© 2012 Christopher M. Saenz. All rights reserved.
The author hereby grants to MIT the permission to reproduce
and to distribute publicly paper and electronic
copies of this thesis document in whole or in part
in any medium noYw nown or hereafter created.
ARCHrES
Signature of Author:
Departme if6B in'and Cognitive Sciences
January 31, 2012
Al
Certified by:
JI),
Ki A. Goosens
Assistant Professor, Department of Brain
and Cognitive Sciences
'W
Accepted by:
L/
Earl K. Miller
Picower Professor of Neuroscience
Chairman, Committee for Graduate Education
THIS PAGE HAS INTENTIONALLY BEEN LEFT BLANK
Viral Delivery of Recombinant Growth Hormone to Rescue Effects of Chronic Stress on
Hippocampal Learning
by
Christopher M Saenz
Submitted to the Department of Brain and Cognitive Sciences
on January 31, 2012 in Partial Fulfillment of the
Requirements for the Degree of Master of Science in
Neuroscience
ABSTRACT
Chronic stress has been linked to variation in gene regulation in the hippocampus
(HIP) among other areas. These lead to cytoskeletal and volumetric rearrangements
in various nuclei of the central nervous system and are thought to contribute to
several stress-sensitive disorders. One such gene that has been shown to be downregulated in HIP in response to stress is somatotropin, colloquially known as growth
hormone (GH). These experiments were conducted to develop a novel assay for
examination of working memory in rats and explore the nature of stress-induced
impairment of hippocampal function and determine whether infusion of a modified
herpes simplex virus (HSV) carrying the recombinant rodent growth hormone (GH)
would be sufficient to restore normal hippocampal function. After 21 days of
chronic immobilization stress (CIS), animals received bilateral infusions into the
dorsal HIP of 2pl HSV carrying either GH with green florescent protein (GFP) or GFP
only. On the second day following the infusion, the animals received trace
conditioning, a HIP-dependent task, with five tone-shock pairings of a 16 second
tone followed by a 30 second trace interval terminating with a 1 second 0.85
milliamp footshock. An inter-trial interval of 3 minutes was used to separate the
tone-shock pairings. The following day the animals were tested for fear to the
context and for fear to the tone in a novel context, measured by amount of time the
animal spent freezing. Using this criterion, animals that had undergone stress that
received the control vector were less likely to freeze when presented with the tone,
indicating an impairment of hippocampal function. Viral-mediated overexpression
of GH in the dorsal HIP was able to reverse the CIS-related impairment in
hippocampal function. ELISA was used to verify the expression of GH from the
infused vector. These experiments may yield future directions of investigation for
stress-based disorders.
Thesis Supervisor: Ki Ann Goosens
Title: Assistant Professor
Acknowledgements
"there is a loneliness in this world so great
that you can see it in the slow movement of
the hands of a clock.
people so tired
mutilated
either by love or no love.
people just are not good to each other
one on one.
the rich are not good to the rich
the poor are not good to the poor.
we are afraid.
our educational system tells us
that we can all be
big-ass winners.
it hasn't told us
about the gutters
or the suicides.
or the terror of one person
aching in one place
alone
untouched
unspoken to
watering a plant."
-
Charles Bukowski
excerpt from "The Crunch"
from Love is a dog from hell
I would also like to take this time to acknowledge the lives of the animals used in
these experiments.
Table of Contents
Introduction
The hippocampus and trace conditioning
The hippocampus and context conditioning
The role of growth hormone in the hippocampus
6
7
10
11
Aims
12
Experiment 1: Exploration of trace footshock conditioning
Materials and Methods
Results
14
14
15
Experiment 2: Investigation of effects of chronic immobilization stress on trace and
17
context conditioning
17
Materials and Methods
19
Results
Experiment 3: Determination of level of functional output of the virus delivering
22
recombinant growth hormone to hippocampus
22
Materials and Methods
23
Results
Discussion
24
Figures
28
References
37
Introduction
Chronic stress leads to changes in the brain, including a region called the
hippocampus (HIP). These changes include atrophy of the dendritic arbors of HIP
neurons, reduction of HIP volume, and loss of plasticity (McEwen, 1994; 1999;
Sapolsky, 2000; Vyas et al., 2002; Donohue et al., 2006). Cognitive changes, seen in
humans as well as rodents, include deficits in HIP-dependent processes such as
working memory, which can be measured, in one way, by trace conditioning(Swaab
et al., 2005; Hales and Brewer, 2010; van Vugt et al., 2010). Although there are likely
many mechanisms leading to these changes, variation in expression of trophic
factors, including growth hormone (GH), has been shown to accompany chronic
stress (Donahue et al., 2006). Additionally, GH has been shown to be vital to longterm potentiation (LTP) in the HIP, a process of synaptic strengthening, (Zearfoss et
al., 2008), and leads to variation in hippocampal gene expression through the JAKSTAT and MEK-ERK pathways, two pathways shown to be important for learning
and memory (Rosenfeld and Hwa, 2009). Based on many studies linking stress to
mental disorders(Esch et al., 2002; Alfonso et al., 2005; Swaab et al., 2005; Bale,
2006), study of these phenomena may provide important novel insight into the
etiologies of several stress-linked disorders such as post-traumatic stress disorder
(PTSD), major depressive disorder (MDD) and anxiety disorders (AD) (Sapolsky,
2000; Gilbertson et al., 2002; Parker and Schatzberg, 2003; Alfonso et al., 2005;
Swaab et al., 2005; Bale, 2006; Spijker and van Rossum, 2009).
The HIP has been shown to be vital to many aspects of learning and memory
including spatial memory, short-term memory and consolidation from short to long-
term memory(Best et al., 2001; Hales and Brewer, 2010; van Vugt et al., 2010;
Epsztein et al., 2011; Fujisawa and Buzsaki, 2011; Vann and Albasser, 2011).
Another function of the HIP is recollection of the sequence in which events
occurred(Lee and Wilson, 2002). There are cells that fire in HIP in response to
locations in space, termed place cells(Best et al., 2001; Brun, 2002; McNaughton et
al., 2006; Moser et al., 2008; Epsztein et al., 2011); firing in individual cells is linked
to specific locations in space, which are stable over time. These locations are termed
the "place field" of that place cell. This phenomenon has been shown to be a
mechanism by which an organism can retain memory of a place(Best et al., 2001;
Frankland et al., 2004; Esclassan et al., 2009; Acheson et al., 2011). Chronic stress
leads to deterioration of this capability and contributing factors could be the
aforementioned HIP volumetric and cellular atrophy(Luine et al., 1994). In this
series of studies, I explored a behavioral protocol by which these changes can be
assayed and used them to assess a method of amelioration of such changes.
The hippocampus and trace conditioning
Pavlovian conditioning pairs a neutral conditional stimulus (CS) with an
unconditional stimulus (US) to elicit a conditional response (CR). For example,
some Pavlovian learning tasks involve pairing of a tone CS with a reward as the US;
the CR for this task is traditionally a measure of the animals prediction of reward
such as salivation or a nose poke into a reward port (Fanselow and Poulos, 2005;
Delamater, 2012). During a different type of Pavlovian learning, auditory fear
conditioning, an auditory CS is paired with an aversive US such as a footshock.
During this training, the animal learns to associate at least two stimuli with the
occurrence of the footshock. First, he learns that the context, the environment in
which the animal is conditioned, including the shape of the chamber, sights, and
smells, is a predictor of the aversive stimulus. Second, he learns that the auditory
cue predicts the occurrence of footshock(Otto and Poon, 2006). Several structures,
including the amygdala, prelimbic cortex and anterior cingulate cortex, form the
neural substrate for Pavlovian conditioning(Frankland et al., 2004; Dityatev and
Bolshakov, 2005; Bangasser, 2006; Krasne, 2011). Auditory delay conditioning, in
which the CS and US co-terminate, does not require the hippocampus, though trace
conditioning does(Bangasser, 2006; Esclassan et al., 2009). In this variation of fear
conditioning, the aversive US follows the termination of the CS after a temporal
delay. Unlike auditory delay conditioning, both context conditioning and auditory
trace conditioning require the HIP to establish the connection between the CS and
US(Bangasser, 2006; Esclassan et al., 2009; Pang et al., 2010; Moita, 2011).
Working memory, the ability to retain information in the short term for
mental manipulation, has been shown to be one of the fundamental roles for HIP. It
has long been speculated that the HIP plays a time-keeping role in memory in
several regards. In human fMRI studies of conditional fear, the HIP was shown to be
a predictor of the US with level of increase in HIP activity proportionate to accuracy
of prediction(Hales and Brewer, 2010). Human electrocorticograph studies
corroborate this with increase in the power of gamma oscillations in the
hippocampus during working memory tasks(van Vugt et al., 2010). In rats,
increases in power of gamma oscillations and coherence of gamma oscillations
between the CA1 and CA3 subfields of the HIP were shown to be fundamental to a Tmaze working memory task (Montgomery and Buzsaki, 2007). These oscillations
are the sum activity of local networks of neurons and are a mechanism by which the
actions of many neurons are coordinated. Coordinated activity between the HIP and
PFC has been shown to be a fundamental component of working memory, while
coordinated activity between HIP and the amygdala is a necessary component of
delay conditioning (Bauer et al., 2007; Hales and Brewer, 2010). Trace conditioning
studies in rats show a strong increase in neuronal excitability and an increase in
spine density in the CA1 subfield of the dorsal HIP(Moyer et al., 1996; Leuner et al.,
2003). In rabbits trace conditioning was found to have an effect on CA1 excitability
as well(Weible et al., 2006). Furthermore, after hippocampectomy, rabbits were
unable to learn CS-US pairings under a trace conditioning paradigm(Moyer et al.,
1990). Additionally, training during ripple events, periods of fast spiking activity in
relation to the oscillatory activity, in the hippocampus enhanced learning for this
task(Nokia et al., 2010). Stimulation of the hypothalamus, leading to release of GH,
following trace eye-blink conditioning training led to rapid training on the task and
coincided with increased hippocampal activity(Hyvarinen et al., 2006). This last
study is particularly of note to this study since GH secretion is regulated by the
hypothalamus and overexpression of GH in HIP may mimic this rapid training
effect(Muller and Locatelli, 1999).
The hippocampus and context conditioning
A subset of cells in the hippocampus fire action potentials in response to
locations in space. These cells are known as place cells and their fields of activity
are known as place fields(Moser et al., 2008). These fields are typically stable for
each cell and remain stable day to day. Activity in these cells has been shown to be
shifted in response to context conditioning, namely, remapping of the place fields of
subsets of cells, presumably as a mechanism by which this structure can discern
between different states of an environment(Moita et al., 2004). Testing animals to
retention of the context in which they were fear conditioned is how this study
assayed HIP spatial memory. Animals that have been exposed to a context and
allowed to explore before fear conditioning have been shown to fear that
environment on subsequent exposure(Frankland et al., 2004). Electrolytic lesions
impair recall as assayed by freezing to fear conditioning context(McNish et al., 1997;
Gewirtz et al., 2000). Chemical lesioning studies have shown the HIP to be vital to
conditioning to a context and full hippocampectomy completely impairs contextual
conditioning(Maren et al., 1997; McEchron et al., 1998; Esclassan et al., 2009;
Acheson et al., 2011). Several immediate-early genes including NGF1-B, Arc, and cFos,genes known to be expressed as part of LTP, have also been shown to be
upregulated in the HIP of animals that recently experienced fear
conditioning(Hertzen and Giese, 2005; Huff et al., 2006; Lonergan et al., 2010;
Czerniawski et al., 2011). Scopolamine inactivation of muscarinic acetylcholine
receptors systemically administered disrupted context but not tone conditioning
suggesting a strong cholinergic component to context conditioning(Anagnostaras et
al., 1995). These effects are not due to an increase in motor activity but instead a
conditioning insufficiency(Maren et al., 1998). Functionality of the dorsal HIP
(dHIP) differs from the ventral HIP (vHIP). dHIP has been shown to be more
important to spatial memory and vHIP function seems to be more related to
emotional regulation. Lesions of vHIP affect fear expression and anxiety, without
effects on non-emotional spatial memory (Bannerman et al., 2003; Trivedi, 2004;
Weible et al., 2006).
The role of growth hormone in the hippocampus
As mentioned above, systemic GH secretion is regulated by the
hypothalamus. This release is partially regulated by HIP projections to the
hypothalamus(Dedovic et al., 2009). This occurs in a manner such that GH secretion
leads to subsequent release of somatostatin, the inhibitor of GH function, as a selfattenuating process(Muller and Locatelli, 1999). Hippocampal neurons in adult
animals but not juveniles have been shown to synthesize GH (Donahue et al., 2006).
Furthermore, GH levels in whole hippocampi (dorsal and ventral combined) have
been found to be elevated in response to an acute stressor(Donahue et al., 2006),
while other results suggest a decrease in GH expression in the dHIP after chronic
stress (Goosens lab, unpublished data). We are interested in GH because several
groups have found it to facilitate synaptic transmission (Mahmoud and Grover,
2006) and promote memory formation (Donahue et al., 2002; 2006; Zearfoss et al.,
2008). A role for GH in declarative memory was also found in humans, by exploiting
the negative feedback mechanism of release of GH to inhibit production of GH
during the trial; a study in which inhibition of GH release led to impairment of
ability to recall previously learned word-pairs, a measure of memory(Hallschmid et
al., 2011). The second experiment aims to further explore roles for GH in learning
and memory by examining effects of overexpression of GH in the stresscompromised brain.
Aims
This project is divided into three components: 1) exploring auditory trace
fear conditioning as a means to assay working and long-term memory; 2)
determining whether growth hormone overexpression can repair stress induced
deficits in these forms of memory as well as spatial memory; 3) assaying the
expression levels of transgenes from vectors developed for this set of experiments
using enzyme-linked immunosorbent assay (ELISA).
Trace conditioning has been studied in rats before but not extensively. The
studies that have been conducted typically consist of eye-blink conditioning during
which the CS is a tone or light, the US is a puff of air and the CR is the blinking
response. The time scale of the trace interval is approximately 500 ms. To optimize
the trace-conditioning paradigm used in the second set of experiments, several key
aspects of trace conditioning were investigated. The trace length was varied to
develop an understanding of the innate ability of rats to establish the association
across a time interval. Shock intensity and length were also varied to determine
which US was strong enough to induce a strong relationship with the CS but not so
strong that animals would freeze to any new environment on testing day, a
generalized fear to novel contexts. These experiments in and of themselves are
interesting in that the animals were able to develop the association between the CS
and US when the two stimuli were separated by a trace length of 50 s.
To explore the manner in which CIS alters hippocampal function, viralmediated gene transfer was used to overexpress rGH and a green florescent
reporter (GFP), or GFP only, in the dorsal hippocampus of rats following either CIS
or handling (no stress, NS). During this task, the animals were presented with
conditional stimuli (CS) separated by trace periods from the unconditional stimuli
(US) (Fig. 1). All animals were able to learn the task at the same rate, however,
animals that had undergone CIS were less likely to freeze to the tone the next day
during retesting, indicating either a deficit in consolidation or recall. Essentially, CIS
animals show deficit in ability to link the CS and US and, additionally, HSV-rGH
administration into the dorsal hippocampus reversed this effect.
ELISA is a popular method to determine the concentration of a substance in a
solution. A vital question after the development of the HSV-rGH vector was to
determine if the transgene was sufficient to induce over-expression of growth
hormone in the HIP. By using this method, it was determined that in HIP infected
with this vector, growth hormone was elevated compared to the unstressed
controls.
I hypothesize there is an optimum trace and shock duration for trace
conditioning in rats, CIS impairs ability to pair the CS to the US as assayed by
freezing during the trace interval and the rGH-HSV vector will prove efficacious as
both a delivery mechanism and restorative to the HIP after CIS.
Experiment 1: Exploration of trace footshock conditioning
Materials and Methods
Animal subjects
This set of experiments used two cohorts of animals, each consisting of male
rats (250-275g;Taconic). Animals were single-housed in under a 12-hour on, 12hour off light cycle and given ad libitum access to both standard rodent chow and
water. The first cohort consisted of 16 rats divided into four groups of four animals
that underwent conditioning using 2 different trace intervals and 2 different shock
durations. These animals were handled for 2 days prior to the behavior assays
described below. The second cohort of animals consisted of 18 animals divided into
3 groups of 6 to investigate three different shock intensities. These animals were
also handled for 2 days prior to the behavior experiments.
BehaviorAssays.
Animals in the first experiment, exploring trace and shock duration, were
divided into four groups: 35 s trace with a 1 s shock, 35 s trace with a 2 s shock, 50 s
trace with a 1 s shock, 50 s trace with a 2 s shock. These animals underwent a
training protocol that consisted of 3 minutes habituation to the chamber and 4 tone
shock pairings of a 16s 2kHz tone (CS) followed by the trace interval corresponding
to the subject grouping and followed by a 0.5SmA scrambled footshock. 24 hours
later animals were presented with a series of 15 tones at 2 10s intervals to test for
tone-elicited freezing during the tone, trace interval, and interstimulus interval (ISI)
periods in a novel context. All freezing was calculated by VideoFreeze (Med
Associates, Inc. St Albans, VT). Statistics were calculated in Prism 4.0 (GraphPad, Inc.
La Jolla, CA).
The protocol used for the second experiment consisted of 5 tone shock
pairings with 16s 2kHz tone CS, a 20s trace interval and a US of 0.40, 0.60, or 0.85
mA scrambled footshocks. These animals were also tested 24 hours later for
retention of the training by presenting the 16s CS tone 15 times at 180s intervals in
a novel context. A shorter ISI was used in this experiment because the shorter trace
represented a smaller part of the ISI compared to the 50s trace for the previous
cohort. Analysis was conducted as above.
Results
Shock duration was found to affect conditioning though trace interval was
not (during TRACE: repeated measures ANOVA; effect of trace interval length, F(1,5)
= 1.076, p = 0.3824; effect of shock duration, F(1,5) = 2.385, p = 0.0487; interaction
of trace length x shock duration, F(1,5) = 0.466, p = 0.7998) (during TONE: repeated
measures ANOVA; effect of trace interval length, F(1,5) = 0.595, p = 0.7037; effect of
shock duration, F(1,5) = 1.221, p = 0.3104; interaction of trace length x shock
duration, F(1,5) = 1.113, p = 0.3631) (during ISI: repeated measures ANOVA; effect
of trace interval length, F(1,5) = 2.359, p = 0.0508; effect of shock duration, F(1,5)
1.954, p = 0.0987; interaction of trace length x shock duration, F(1,5) = 1.358, p =
0.2530) (Fig. 2). Freezing for all groups except the 35 s trace + 1 s tone group
reached maximum freezing following the first tone shock pairing during both the
tone and trace epochs.
=
During testing, all animals froze to the CS after the first tone presentation and
no significant differences were seen in the freezing to the tone (p=ns), during the
trace (p=ns) or during the ISI (p=ns) by repeated measures ANOVA (during TRACE:
repeated measures ANOVA; effect of trace interval length, F(1,15) = 1.340 p =
01837; effect of shock duration, F(1,15) = 0.987, p = 0.4711; interaction of trace
length x shock duration, F(1,15) = 2.674, p = 0.6900) (Fig. 3, top; only trace interval
data shown). However, during the period prior to onset of the first tone, 3 of the
groups were freezing at levels above 25%, a sign of generalized fear. The exception
to this was a trend of the 35s trace + is shock group which froze approximately
20% of the time during the 3 minutes prior to onset of the first tone (ANOVA; effect
of trace interval length, F(1,1) = 0.018 p = 0.8950; effect of shock duration, F(1,1)
5.712, p = 0.0341; interaction of trace length x shock duration, F(1,1) = 1.305, p
=
=
2.755) (Fig. 3, bottom).
Analysis of the different shock intensities showed that there is a difference in
freezing during the trace interval though not in response to the tone but post-hoc
TMP showed no differences (during TONE: repeated measures ANOVA; effect of
shock intensity, F(2,8) = 11.54, p = 0.7563; interaction of shock intensity x time, F(2,
8) = 3.383, p = 0.0862) (during TRACE: repeated measures ANOVA; effect of shock
intensity, F(2,8) = 4.563, p = 0.0283; interaction of shock intensity x time, F(2,8)
=
3.061, p = 0.0065), when the animals were presented with the tone the animals in
the 0.85 mA shock group froze more than the other groups (during TONE: repeated
measures ANOVA; effect of shock intensity, F(2,15) = 4.426, p = 0.02 19; interaction
of shock intensity x time, F(2,15) = 7.454, p = 0.0104)(Fig. 4). Additionally, during
the trace interval, a better predictor of the animal's ability to associate the CS and
US, the 0.85 mA group froze more as well (during TRACE: repeated measures
ANOVA; effect of shock intensity, F(2,15) = 14.65, p = 0.0 003; interaction of shock
intensity x time, F(2,15) = 2.405, p = 0.0155).
Experiment 2: Investigation of the effects of CIS on trace and context
conditioning
Materials and Methods
Virus generation
The gene for somatotropin was cloned into pHSVgfp, a backbone plasmid, to
become the HSV-rGH; the control virus was generated using solely the backbone to
express GFP, to become the HSV-GFP. Viral plasmids were transfected into 2-2 cells
via Lipofectamine (Invitrogen, Inc, Grand Island, NY), which were then
superinfected with Sdl1.2 replication deficient HSV1 virus. The 2-2 cells contain the
portion of the IE2 gene deleted from the 5d1.2 that allows the packaging of the
virus backbone material into functional vectors. After three rounds of amplification,
the viruses were purified by centrifugation across a series of 10%, 30% and 60%
sucrose gradients. Final concentrations of the viruses were 9 x 109 particle forming
units for the rGH-HSV and 4 x 108 for the GFP-HSV. Helper virus concentrations
were 1.7 x 1010 and 1.2 x 109 respectively(Lim and Neve, 2001).
Animal Subjects
Animals were 20 male Long-Evans rats acquired at 250-275 grams from
Taconic for the first cohort of 12 animals. The second cohort consisted of 14
animals from Charles River Laboratories. Animals were single-housed in under a 12hour on, 12-hour off light cycle. The CIS group underwent daily immobilization
stress in decapitation cones 4 hours/day for 21 days prior to surgery while the NS
group was handled the 2 days prior to surgery.
Infusion
On the day following the last bout of CIS, all animals were anesthetized with
Nembutal (0.65mg/kg, intraperitoneally) or isoflurane at a concentration of 3-5% in
oxygen and bilaterally infused to the coordinates of AP -3.30mm, ML ± 2.00mm DV 3.20mm relative to bregma which targeted the dorsal hippocampus (see Fig 5).
Animals were administered ketoprofen (1mg, s.c.) for swelling and pain at the time
of surgery and 24 hours following surgery. These experiments used 24 animals
divided into four groups: non-stressed with the control GFP vector (NS + GFP, n=7),
non-stressed with the GH vector (NS + rGH, n=4), chronic immobilization stressed
with the control GFP vector (CIS + GFP, n=7), and chronic immobilization stressed
with the growth hormone vector (CIS + rGH, n=6). Following the last day of stress,
the animals were anesthetized with isoflurane and 2 pd of at least 108 infectious
units/mL of either rGH-HSV or control GFP-HSV was infused over the course of 20
minutes to maximize penetrance through the extracullular matrix.
Behavioral analysis
After 72 hours the animals were trace conditioned with 5 tone shock
pairings. A CS of 15 seconds of 2 kHz tone, a trace of 35 seconds and US consisting of
a 1 second foot shock at 0.85 mA were the parameters for training. The animals
were tested the next day in the same context in the absence of the CS to assay
contextual fear and, four hours later tested in a novel context, consisting of white
plastic inserts in the testing chamber and a different scent cue, for retention of
freezing to the tone, a conditioned response (CR). Video was taken during trace
conditioning and the tone recall for analysis by VideoFreeze software (Med
Associates; Georgia, VT).
Results
During conditioning, no clear differences were seen between the treatment
groups. All animals were able to associate the US with the CS even across the 35s
trace interval. No differences were seen in freezing during the trace, tone or interstimulus intervals (during TRACE: repeated measures ANOVA; effect of stress F(1,4)
= 0.442, p = 0.775; effect of vector, F(1,4) = 0.675, p = 0.6150; interaction of stress x
vector, F(1,4) = 0.838, p = 0.5125) (during TONE repeated measures ANOVA; effect
of stress F(1,4) = 1.413, p = 0.2555; effect of vector, F(1,4) = 1.164, p = 0.3477;
interaction of stress x vector, F(1,4) = 1.135, p = 0.3604) (during ISI: repeated
measures ANOVA; effect of stress F(1,4) = 0.808, p = 0.5305; effect of vector, F(1,4)
= 0.859, p = 0.5005; interaction of stress x vector, F(1,4) = 0.775, p = 0.5508)) (Fig.
6). This is in direct contrast to what was originally hypothesized and will be
addressed in the discussion section.
On testing day, CIS + GFP animals were impaired in this form of learning
(during TRACE: repeated measures ANOVA; effect of stress F(1,9) = 2.365, p =
0.0153; effect of vector, F(1,9) = 1.681, p = 0.0153; interaction of stress x vector,
F(1,9) = 1.153, p = 0.3286) and froze less during the trace period and this effect was
not rescued by rGH (Fig. 7, Top). This effect was verging on significance though did
not make the cutoff for freezing during the trace period though the Tukey's multiple
comparison that was conducted did show a difference between CIS + GFP and NS
+GFP. Additionally, the animals in the NS + rGH group also were impaired in this
form of learning since the repeated measures ANOVA showed this group was not
significantly different from any other group. Interestingly, the fact that the animals
in the NS + rGH group also seem to show deficit during testing is suggestive of an
optimum rGH level in the hippocampus above which connectivity suffers. Strangely,
the animals in the NS + rGH group do freeze during the tone epoch(during TONE
repeated measures ANOVA; effect of stress F(1,9) = 2.169, p = 0.0264; effect of
vector, F(1,9) = 0.716, p = 0.6938; interaction of stress x vector, F(1,9) = 0.726, p =
0.6848) however it seems that once the tone ceases, they do not recall the trace
interval indicating an element of generalization (Fig. 7, Bottom).
During the first three minutes of the first 4 5-minute ISI differences in
freezing were seen in response to the CS (during ISI: repeated measures ANOVA;
effect of stress F(1,11) = 1.011, p = 0.4376; effect of vector, F(1,11) = 1.036, p =
0.4158; interaction of stress x vector, F(1,11) = 1.020, p = 0.4296). CIS did impair
learning of this task; freezing in the CIS + GFP group froze less than NS + GFP
(Tukey's multiple comparison (TMP); p 0.001). CIS + rGH was found to be elevated
compared to CIS + GFP (TMP; ps 0.05) though was still found to be different from
NS + GFP (TMP; p9 0.05), thus GH was able to partially restore this decrease in
learning for this task. The NS + rGH group was found to be different from the NS +
GFP (TMP; ps p.001) and not significantly different from CIS + rGH or CIS + GFP
(TMP; p=n.s.). This is an interesting effect indicating that aberrant GH signaling in
the NS brain may actually impair learning of this task.
Figure 8 shows the data for the first eight minutes of context testing. CIS
impaired performance for memory to the context; the CIS + GFP group froze less
than the NS + GFP group (repeated measures ANOVA; effect of stress F(1,15) =
1.323, p = 0.1872; effect of vector, F(1,15) = 0.599, p = 0.8748; interaction of stress x
vector, F(1,15) = 0.462, p = 0.9577; TMP, ps0.001). GH was unable to reverse this
effect; the CIS + rGH group displayed the same amount of CR to the context as CIS +
GFP (TMP, p=n.s.). Similar to the tone testing, GH administration to healthy animals
impaired memory; the NS + CIS group responded less than the NS + GFP group (TMP
p 0.001) though this impairment was not as robust as CIS impairment. The NS +
rGH group responded more robustly than the CIS + GFP group (TMP 0.001).
When the freezing across the eight minutes was averaged, the visualization
became clearer and subsequent analysis confirmed the previously seen statistics (
ANOVA; effect of stress F(1,1) = 52.826, p < 0.001; effect of vector, F(1,1) = 14.622, p
= 0.0.002; interaction of stress x vector, F(1,1) = 4.787, p = 0.0.0293). Post-hoc
analysis via TMP re-iterated the effect of CIS on context retention; CIS + GFP froze
less than NS + GFP (TMP, ps 0.001). GH was not able to reverse this effect; CIS +
rGH was not found to be different from CIS + GFP (TMP, p=n.s.). Again, GH
administration in HIP impeded learning in the healthy animal; NS + rGH froze less
than NS + GFP (TMP, ps 0.05) and this impairment was found to be as severe as
CIS(TMP vs CIS + GFP, p= n.s.).
These data indicate an ability for GH to restore function for some aspects of
trace conditioning but not context conditioning.
Experiment 3: Determination of level of functional output of the virus
delivering recombinant growth hormone gene to hippocampus
Materials and Methods
Animal subjects
Animals were 8 male Long-Evans rats acquired at 250-275 grams from
Taconic. Animals were single-housed in under a 12-hour on, 12-hour off light cycle.
The CIS group underwent daily immobilization stress in Decapicones 4 hours/day
for 21 days prior to surgery while the US group was handled the 2 days prior to
surgery.
Infusion.
On the day following the last bout of CIS animals were anesthetized by Nembutal
(0.65mg/kg, intraperitoneally) and bilaterally infused to the coordinates of AP -33.0,
ML ± 20.0 DV -32.0. Animals were administered ketoprofen (1mg, s.c.) for swelling
and pain at the time of surgery and 24 hours following surgery. These experiments
used 8 animals divided into four groups: NS + GFP (n=2), NS + rGH (n=2), CIS + GFP
(n=2), and CIS + rGH (n=2). Chronic immobilization stress consisted of 4 hours of
immobilization every day for 21 days. Following the last day of stress, the animals
were anesthetized with isoflurane and 2 d of at least 108 infectious units/mL of
either rGH-HSV or control GFP-HSV were infused over the course of 20 minutes to
maximize penetrance through the extracellular matrix.
ELISA
On the third day after surgery, animals were sacrificed. Whole hippocampi
were dissected after sacrifice under deep isoflurane anesthesia. Tissue was
homogenized in ice cold homogenization buffer at 10 times the volume of the tissue
using a VWR rotating tissue homogenizer. Homogenization buffer consisted of PBS
containing one tablet each of PhosSTOP Phosphotase Inhibitor (Roche; Basel,
Switzerland) and cOmplete Mini, EDTA-free Protease Inhibitor Cocktail (Roche;
Basel, Switzerland) per 10mL of solution as indicated. Following homogenization,
the homogenate was centrifuged in a tabletop centrifuge for 30 minutes at 14000
rpm and the supernatant was decanted. The assay was conducted using a Millipore
rodent growth hormone ELISA kit on the decanted supernatant, pooled by group
and imaged on a Bio-Rad iMark microplate absorbance reader (Bio-Rad Hercules,
CA).
Results
The ELISA was conducted in duplicate, pooled by treatment group (Fig 6). However,
in the absence of treatment group replicates, only trends can be discussed. The NS+
GFP group showed discernible expression of rGH. The CIS + rGH seemed to show a
distinct increase over CIS + GFP as to be expected but surprisingly, also a higher
level than NS + rGH.
Discussion
The data for the analysis of trace and shock length in Experiment 1 were not
found to be statistically significantly conclusive, however, certain trends stand out.
Firstly, though animals are able to make the association between the CS and US for
all of the trace and shock lengths, all groups except the 35s trace + is shock group
tend to generalize their responses and, when presented with the novel testing
context, freeze aberrantly. Using the shorter 35s trace with the largest shock
intensity, produced robust learning of the task while minimizing the risk of having
the animals generalize and present with the CR during the preliminary epoch of the
testing session. Additionally, though the animals in the 0.85mA group froze when
presented with the CS, they did not reach a ceiling effect in which all the animals
froze all the time as a response. Such an effect would be an indicator of overtraining, leaving little room for meaningful interpretation of data in later
experiments.
Though the rGH was sufficient to recover some aspects of working memory,
the recovery was not complete. CIS does seem to negatively impact retention of the
CS-US pairing as initially hypothesized and rGH is able to lead to recovery of the CR
during ISIs during tone testing. The hippocampus is a large structure; perhaps
further restoration of this structure could lead to reduction in trace conditioning
deficit seen after CIS. That the rGH-HSV administration was able to restore some
ability to associate during tone testing but not context testing is at odds with current
literature which states that both are the domain of the dorsal hippocampus (dHIP)
(Esclassan et al., 2009). However, the ventral HIP (vHIP) has been shown to be
important for regulating the HPA axis(Radley and Sawchenko, 2011) and maybe
restoration of the dorsal dHIP without restoration of the vHIP attenuates overall
mitigation of deleterious stress effects. Tough the dorsal HIP is partially restored,
control of further release of stress hormones by ventral HIP is not(HERMAN et al.,
2005). Additionally, the amygdala (AMY) forms the basis for associative memory
and it has been shown that trace conditioning does not require the hippocampus
during the tone test but is a vital part of association to the context(Raybuck and
Lattal, 2011).
Further exploration with GH may further restore trace-conditioning learning
in a dose dependent manner though. Two injection sites per hemisphere would
cover the bulk of the dorsal hippocampus and may further ameliorate effects of CIS
on dHIP-dependent tasks. Alternatively, a smaller virus particle capable of more
fully penetrating the extracellular matrix such as AAV could be used to further
distribute the transgene (Davidson and Breakefield, 2003).
One of the more interesting aspects of this second experiment was the
deterioration of the ability of the NS + rGH group to pair the tone or context with the
US. The NS + rGH group froze more less than the NS + GFP group but more than
either of the CIS groups. Since GH is upregulated in response to memory formation
and has been found to be present in levels low enough that they cannot be detected
in the HIP of a rat that has not undergone conditioning, it may be that excess rGH
signaling in the dorsal HIP may lead to an occlusion of plasticity(Donahue et al.,
2002). Occlusion has been shown to occur by a group that tested mice with a
constitutively active CAMKII gene product, an initiator of LTP, on a spatial memory
task(Bejar et al., 2002). Mice with the mutant CAMKII had higher escape latencies
than their wild type litter mates indicating a necessity of control over induction of
LTP. Growth hormone has been shown to be involved in long term
plasticity(Zearfoss et al., 2008); over-expression in HIP that is not compromised by
CIS may block this time sensitive rGH function.
The ELISA data does show that rGH-HSV administration is able to lead to
overexpression of rGH. The antibody adhered to the ELISA plate was able to bind
the polypeptide thus the epitope the antibody is attracted to is present in the gene
product. The CIS + rGH group showed a robust increase in growth hormone level
compared to the NS + GFP and CIS + GFP groups. This may be due to a synergistic
effect of the transgene and endogenous restorative mechanisms. As stated above,
GH is upregulated in response to HIP-dependent learning, these animals underwent
no experiment that would induct GH expression and that may explain the lack of
signal in the NS + GFP group(Donahue et al., 2002).
In sum, rats are able to establish meaningful connections between the CS and
US over a 35s trace interval though extending beyond that may lead to generalized
expression of fear. Additionally, long shock durations seem to lead to an increase in
freezing. Saliency of the US is important to develop a strong link to the CS. CIS has
been shown to alter freezing in response to the training context though, under these
criteria, did not alter freezing to the tone or during the trace. CIS did decrease
freezing during the ISI but that may be due to an increase in anxiety though not fear.
Growth hormone over-expression was able to attenuate though not completely
reverse that CIS-induced decrease in freezing during the ISI. ELISA confirmed that
over-expression produced functional hormone. In the future, GH and its related
pathways are a promising avenue of exploration for treatment of psychiatric
diseases that coincide with chronic stress and its corresponding alteration of the
hippocampus.
Figures
Delay Conditioning
cs
m M
I
Trace Conditioning
us~~
u
Figure 1 During delay conditioning, the cessation
of the CS and US coincide. For the trace
conditioning paradigm the US follows CS
termination by a trace period.
Freezing during ISIs
-4- 35s Trace
-e- 35s Trace
- 50s Trace
S50s Trace
Is Shock
29 Shock
Ia Shock
2s Shock
4
q 4'..
Freezing during tones
-a- 35s Trace Ia Shock
- 3S Trace 2s Shock
50 e-Trace 1a Shock
- 50s Trace 2s Shock
Freoaing during trace intervals
36s Trace ia Shock
-.-
36s Trace 2s Shock
-_-50s Trace is Shock
-
-+-
5s Trace 2s Shock
Figure 2 Freezing during 3 different time periods during fear
conditioning for analysis of trace length and shock length. Top:
Freezing during the 3 min ISI. Middle: Freezing during the 16s CS
presentation.
Freezing during trace interval
100-35s
Trace is Shock
-7--
35s Trace 2s Shock
50s Trace 2s Shock
50s Trace 2s Shock
Freezing prior to first testing tone
~25100
Figure 3 Top: Freezing during the trace intervals for each group for
analysis of effects of trace and shock duration on conditioning..
Bottom: Freezing during the initial introduction to the novel context
in which the CS is presented for testing.
Freezing to ton* duding conditioning
-6-0.85 mA
mA
mA
74-.-0.60
I-6-0.40
LP
Freezing to tonm during testing
--
0.85 mA
-*- 0.60 mA
--
0.40 mA
-a- 0.85 mA
--
0.85 mA
-e-0.60 mA
-*-0.60 mA
-'--0.40 mA
--
so
es.
2a
26
400
Freezing to trace during conditioning
Free Ing to trace during testing
0.40 mA
Figure 4 For analysis of three different shock intensities, during conditioning no significant difference
was seen in response to the tone however during the trace interval a significant difference was seen
between the groups. For testing each block represents responses for three traces or tones. There was
no sign of elevated freezing during the initial exposure to the novel context in which testing occurred.
Upon presentation with the CS, the 0.85 mA shock group displayed the greatest level of the CR which was
sustained almost to the end of the 15 tones. During the trace interval this group also displayed
significantly more freezing as well.
Figure 5 A representative Infection In dHIP at SX
magnification 4 days after Infusion with 2 pl of the
control HSV-GFP. The cell bodies of pyramidal cells
In the CA1 layer are visible.
Freaing during te Inter-stimulus Intervals during conditioning
-.- NS + S0109
-- NS + rGH
St + Solos
-+- St + rGH
Freezing to the tone during condl~onlng
-- NS + S0 109
NS + rGH
-9-St + S0109
-e-
+ St + rGH
/
~/~b/
Freezing during the trace Intrval during conditioning
10- NSo+- 109
--
NS +
rGH
-*-CIS + 50109
-CIS+
rGH
Figure 6 No differences were seen during conditioning between groups during
exploration of interaction between CIS and rGH. For all three epochs assayed
during fear conditioning, there was no significant difference in the rates of
learning for these four groups.
Freezing during the trace period
-a- CIS + GFP
-.- CIS + rGH
-M-NS + GFP
- NS + rGH
Freezing to the tone
-w- CIS + GFP
-,-CIS+ rGH
- -NS + GFP
NS + rGH
10 I
10%
ri
% Freezing by mbtmt during first four Me~
-
-*-CIS+GFP
--- CIS+ rGH
nI:
90
-.-
NS + GFP
NS + rGH
300
20
.b
5
Figure 7 After conditioning the animals in Experiment 2 were tested
for freezing to the CS in a novel environment Neither freezing to the
tone nor the trace was found to be significantly different. However,
when examining the ISI by minute, the CIS+GFP group froze
significantly less than other groups.
Context Testing
-w- CIS + GFP
-*-CIS + rGH
-- NS + GFP
-+-NS + rGH
Freezing to the Context on Testing Day
s9,
CIS+ GFPCI;rGH NS +GFP NS+ rGH
Figure 8 Top: Freezing when rats were re-exposed to the context in
which they were conditioned, in 30s bins for 8 minutes. Via repeated
measures ANOVA the groups were found to be different (and by
Tukey's multiple comparison test, all groups were shown to be
different from one another save the two CIS groups. Bottom: The
average freezing across that 8 minutes was also found to be
significantly different for all groups except the two CIS groups.
Pooled Growth Hormone Extracted from dHIP
U
250
CIS + GFP
2CIS + rGH
NS + GFP
1NS + rGH
CI8 + OFPCIB + rH NI + QFP NO6 r0H
Figure 9. A comparison of the GH levels In the hippocampi of
each group. t = No bar Is shown for the NS + GFP group
because no growth hormone was detectable in this sample.
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