THE MEDIAL PREFRONTAL CORTICAL CONTRIBUTION TO PATH
INTEGRATION: AN ANIMAL MODEL OF MEMORY AND BEHAVIORAL
DEFICITS FOUND IN NEURODEGENERATIVE DISEASES
A Thesis
Presented to the faculty of the Department of Psychology
California State University, Sacramento
Submitted in partial satisfaction of
the requirements for the degree of
MASTER OF ARTS
in
Psychology
by
Amanda L. Simmons
FALL
2012
© 2012
Amanda L. Simmons
ALL RIGHTS RESERVED
ii
THE MEDIAL PREFRONTAL CORTICAL CONTRIBUTION TO PATH
INTEGRATION: AN ANIMAL MODEL OF MEMORY AND BEHAVIORAL
DEFICITS FOUND IN NEURODEGENERATIVE DISEASES
A Thesis
by
Amanda L. Simmons
Approved by:
__________________________________, Committee Chair
Jeffrey Calton, Ph.D.
__________________________________, Second Reader
Kimberly Roberts, Ph.D.
__________________________________, Third Reader
Emily Wickelgren, Ph.D.
____________________________
Date
iii
Student: Amanda L. Simmons
I certify that this student has met the requirements for format contained in the University format
manual, and that this thesis is suitable for shelving in the Library and credit is to be awarded for
the thesis.
__________________________, Graduate Coordinator ___________________
Lisa Harrison, Ph.D
Date
Department of Psychology
iv
Abstract
of
THE MEDIAL PREFRONTAL CORTICAL CONTRIBUTION TO PATH
INTEGRATION: AN ANIMAL MODEL OF MEMORY AND BEHAVIORAL
DEFICITS FOUND IN NEURODEGENERATIVE DISEASES
by
Amanda L. Simmons
Theories of prefrontal cortical function in human and primate models include regulation
of cognitive processes such as working memory and executive functions, both of which
may be implicated in spatial navigation behavior. The role of working memory in path
integration navigation is not well understood. Lesions of the medial prefrontal cortex
administered to twenty rats assessed whether impairment in working memory associated
with the lesions produced navigational deficits similar to those found in humans with
neurodegenerative disorders. We hypothesized that medial prefrontal lesions would
produce impairment in navigation performance during a Whishaw table top path
integration task when compared with sham controls. We found no significant differences
between lesioned and sham animals on measures of path integration performance. These
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results are inconclusive in determining the possibility of functional similarities between
the rodent prefrontal cortex and the human manifestation of symptoms found in abnormal
aging affecting comparable brain regions.
_______________________, Committee Chair
Jeffrey Calton, Ph.D.
_______________________
Date
vi
TABLE OF CONTENTS
Page
Chapter
1. INTRODUCTION ……………. ………………………………………………….…….. 1
Navigation.................................................................................................................... 3
Working Memory……… ............................................................................................ 4
Prefrontal Cortical Anatomy ........................................................................................ 8
Anatomical Similarities Between Animals and Humans ................................ 9
The Prefrontal Cortex in Humans and Animals ......................................................... 10
WM and the PFC in Humans and Non-Human Primates:
Neuroimaging Studies................................................................................... 10
Single Cell Recordings in Non-Human Primates ......................................... 11
Human and Non-Human Primate Lesion Experiments................................. 12
Rodent Lesion Experiments .......................................................................... 12
Single Cell Recordings in Rodents ............................................................... 13
WM Debate .................................................................................................. 14
Navigation Requires WM .......................................................................................... 15
Path Integration and WM .............................................................................. 15
Lesions of the mPFC Impair Navigation ...................................................... 16
WM and Navigation Functions are Impaired by Lesions of the PFC ........... 16
The Connection Between WM, Executive Functions, and Navigation ..................... 17
Executive Functions Located in the PFC are Synonymous
with Navigational Deficits: Single Cell Recordings and fMRI Studies........ 19
PFC Involvement in Neurodegenerative Disease Found in Animals and Humans ... 20
The Presentation of Symptoms in Animals After the PFC Sustains….….....22
Damage is Similar to the Symptoms Found in Humans with
Neurodegenerative Disorders
Complex Diseases such as FTD and Dementia of the Alzheimer’s Type
Can Benefit From Research in Animal Models ............................................ 24
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2. METHOD ......................................................................................................................... 26
Animals ...................................................................................................................... 26
Apparatus .................................................................................................................. 26
Pre-Surgical Training ................................................................................................. 28
Surgical Procedure ..................................................................................................... 29
Post-Surgical Testing ................................................................................................. 30
Analysis ..................................................................................................................... 30
3. RESULTS ......................................................................................................................... 32
Histology.................................................................................................................... 34
4. DISCUSSION .................................................................................................................... 34
Conclusions and Future Study ................................................................................... 37
Appendix A. Subcortical Structures of the Rodent Brain .................................................... 39
Appendix B. An Infrared Picture of a Post-Surgical Rat ...................................................... 40
Appendix C. Pre-Operative Experimental Measures of Latency, Frequency
of Emergence, and Error in Lesioned and Sham Controls for
Five Trials, N = 16 .......................................................................................... 41
Appendix D. Post-Operative Experimental Measures Experimental Measures of Latency,
Frequency of Emergence, and Error in Lesioned and Sham Controls for
Five Trials, N = 16 .......................................................................................... 42
Appendix E. Post-Operative Latency Measures by Trials With Lesion and Sham Groups.. 43
Appendix F. Post-Operative Frequency Measures by Trials With Lesion and Sham
Groups .............................................................................................................. 44
Appendix G. Neurotoxic Lesion Extent................................................................................. 45
References ............................................................................................................................... 46
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1
Chapter 1
INTRODUCTION
The frontal cortex in humans is a complex region that regulates cognitive
functioning through various psychological processes including perception (Baddeley,
2000), which involves the scanning, selection and integration of information taken from
stimuli in the environment necessary for spatial and working memory (WM) processes
(Stuss & Knight, 2002; Chudasama & Robbins, 2006). Research also indicates that
frontal cortical functions contribute to WM and navigation in humans as well as rodents
(Chudasama & Robbins, 2006). While the extent of differences in human models of
disease include variability such as risk factors and demographics, animal models serve to
better differentiate the extent of impairment as a result of the degree of cortical tissue
damage in the absence of many of these confounding variables (Jones, 2002; Müller &
Knight, 2006). Path integration navigation is the process by which an individual uses the
distance and direction of travel from a previous known position to locate ones current
position. We hypothesize that path integration navigation is dependent on WM, and
because the medial prefrontal cortical region (mPFC) is thought to be necessary for WM
processes, we further hypothesize that lesions in this area will impair this form of
navigation in rodents.
While navigation tasks such as the radial arm maze are commonly utilized to
assess working memory performance in lesioned animals, a pure path integration task has
never been used to examine the effects of lesions expected to interfere with WM
2
performance. Lesions of the PFC in rodents have been found to result in profound
cognitive (Hoge & Kesner, 2007) and motor disturbances (Compton, Griffith, McDaniel,
Foster, & Davis, 1997; Heidbreder & Groenewegen, 2003; Granon & Poucet, 1995)
similar to neurodegenerative disorders in humans, such as Alzheimer’s disease,
(Chudasama & Robbins, 2006; Huntley & Howard, 2010), and Frontotemporal Dementia
(Boccardi, 2006; Hodges, 2001). Navigation such as path integration requires the use of a
“cognitive map” (Newman, Caplan, Kirschen, Korolev, Sekuler, & Kahana, 2006) which
is consistently updated in order to provide an “on-line” estimation of current location in
the environment, based on movement direction and distance from previously known
locations (Calton & Taube, 2009; Tolman, 1949). This use of information is similar to
models of WM where information is manipulated for temporary use (Baddeley, 2000).
Anatomical references of the PFC in human and animal models have also
suggested several similarities between brain regions, indicating that rodents are capable
of complex processes necessary to support higher brain functioning (Jones, 2002; Stuss &
Knight, 2002). While the dorsolateral prefrontal cortex (dlPFC) in humans appears
largely responsible for WM, previous research has indicated that the rodent mPFC is
analogous to this area by sharing structural similarities and many of the same functions
(Jones, 2002; Uylings, Groenewegen, & Kolb, 2003). Brain areas responsible for
functional similarities in humans and animal models could indicate that mPFC lesions in
rodents are similar to brain insults found in humans with comparable symptoms, common
to neurodegeneration of the frontal lobe which manifest as memory loss and spatial
impairment (Stuss & Knight, 2002).
3
Navigation
Navigation of the spatial environment can be observed in rodents as well has
humans, and the basic processes of navigation seem to be somewhat similar among the
two species. Spatial navigation occurs when the organism is motivated and plans to
travel toward a goal in the environment, whether it is a building, car, or a reward in an
animal learning experiment. Normally navigation in an unfamiliar environment requires
the consistent update of spatial stimuli to process one’s orientation in the environment.
Two basic types of navigation, landmark navigation and path integration, have been
defined.
Landmark navigation (also known as place recognition or piloting) involves
locating ones position based on the location of familiar cues (landmarks) found in the
environment (Gallistel, 1990). As humans, we commonly use landmarks such as road
signs, buildings and geological features to make our way from one place to another. A
demonstration of landmark navigation in the laboratory can be observed in the Morris
water maze task when the rat uses landmarks in the surrounding room to return to a
hidden platform in a pool of water regardless of where it entered the pool.
A second form of navigation, path integration, relies not on the location of
familiar landmarks in the spatial environment, but on the knowledge of the distance and
direction moved since the start of the journey (Gallistel, 1990; Mittelstaedt &
Mittelstaedt, 1980; Touretzky & Redish, 1996). Path integration assumes a continual
process of integrating vestibular cues, efferent copy, and optic flow to locate one’s
position in space, and thereby allows accurate navigation even when familiar landmarks
4
are absent (Calton & Taube, 2008). An example of path integration would be an animal
taking a meandering route to find food in an unfamiliar environment and then using the
knowledge of the distance and direction traveled since beginning the journey in order to
return to its point of origin using a novel direct route.
These two forms of navigation are most likely complementary, in that landmarks
may be utilized when available, but when these external cues are absent, path integration
can be used to accurately navigate in an unfamiliar environment, (Calton & Taube, 2009;
Gallistel, 1990).
Working Memory
Traditionally, the term “working memory” (WM) is most often used in human
and nonhuman primate studies because researchers are reluctant to attribute higher
cognitive functioning to rodents. However, due to many anatomical and functional
similarities between primates and rodents, it has now become more acceptable to design
experiments studying WM in rodents in order to make comparisons with the human WM
model (Jones, 2002).
In the human literature, WM is defined as the system that uses temporary storage
and manipulation of information required for complex processes such as learning,
reasoning, language, and comprehension (Baddeley & Hitch, 1994; Baddeley, 2000). It
is the process whereby sensory material is actively held, making it easy to retrieve or
recall. This sensory material is temporarily held in an active state and becomes central to
processing, whereby additional sensory information cannot interfere with the active state
(Stuss & Knight, 2002). As one of the most specialized functions of the PFC, WM
5
includes attentional components which facilitate the on-line capabilities enabling
information processing (Stuss & Knight, 2002).
While this information processing can be analyzed through written and verbal
neuropsychological testing methods in humans, rodent models may incorporate
navigational tasks to identify these processing errors in WM functioning. In these animal
studies, errors in the temporary storage of various navigational trials, such as the
erroneous return to a previously visited (and no longer baited) arm of a radial arm maze,
may indicate the presence of WM dysfunction. The constituents of WM which facilitate
information processing consist of the “central executive,” the “visuospatial sketchpad,”
the “episodic buffer,” and the “phonological loop,” (Jones, 2002). While the
phonological loop pertains to sensory speech information in humans and is therefore not
likely to be relevant to rodent cognition, the central executive, visuospatial sketchpad,
and episodic buffer terms can still be applied to animal cognition.
The central executive refers to the regulation of decision-making, reasoning, and
the coordination of subsystems. Commonly referred to as the “supervisory attentional
system” (Stuss & Knight, 2002) the subsystems of the central executive are responsible
for the retention of visual and spatial information. This area is also responsible for
directing the attention systems which aid in the retention of stimuli from the visuospatial
sketchpad and phonological loop (Baddeley, 2000). With respect to rodent navigation, a
defect in the central executive would present as an inability to focus on stimuli
sufficiently enough to retain it in WM, or the failure to differentiate between correct and
incorrect navigational paths toward a task goal. These errors can be visualized using
6
common paradigms such as the spatial delayed-alternation task, or the delayed-response
task (Stuss & Knight, 2002).
The visuospatial sketchpad controls the processing of temporary visual and spatial
storage. In the human, the sketchpad activates the occipital lobe to process visual
patterns in the environment which has neural pathway connections to the frontal lobe.
The frontal lobes are also necessary for spatial processing, which is responsible for
control and coordination in the environment (Baddeley, 2002). Detection and processing
of visuospatial information by humans can be evaluated by neuropsychological testing
and imaging studies. The Wechsler Adult Intelligence Scale (WAIS) is used to measure
perceptual reasoning, visual processing, and spatial reasoning found in the Block Design,
Matrix Reasoning, and Coding subtests of the battery. Testing impairments would
indicate subsequent deficiencies in visuospatial processes. Evidence for human
impairment in these abilities is also observable in the activation of brain areas from fMRI
and PET scan studies, where individuals are required to remember letters and spatial
locations after a brief delay (Prabhakaran, Narayanan, Zhao, & Gabrieli, 2000).
Similar to the human, the rodent WM processes could be expected to hold visual
and spatial information temporarily for task completion. As an example, in the Delayed
Matching to Place T-maze task, the rat is first given a sample trial where it leaves the
start box and only one of the two goal boxes is available. In the following “match” trial,
both goal boxes are available but the rat is only rewarded if it chooses the goal box from
the sample trial. In subsequent pairs of trials, the reinforced goal box will change. In
7
essence, this task requires the animal to temporarily hold on-line the correct spatial
choice (Wenk, 2001).
The newly incorporated idea of the episodic buffer, controlled by the central
executive, has also been grouped in with the visuospatial sketchpad as a key component
of WM. The episodic buffer refers to the storage of information not organized within
WM, as well as the “rehearsal and coordination of temporally distinct but inter-dependent
information” (Jones, 2002), retrieved consciously. Capable of organizing, coding, and
storing information, this episodic buffer component is the intermediary between the
phonological loop and the visuospatial sketchpad as well as the long-term memory
(Baddeley, 2000).
While the theory of the episodic buffer is referenced in humans, the idea of an
animal episodic buffer (let alone a rodent central executive), has not been thoroughly
investigated. However, research in single cell recordings in non-human primates have
suggested that PFC cells can have a combination of properties which contribute to the
WM process, and that these properties require the central executive episodic buffer (Stuss
& Knight, 2002). These properties may also include the initial cellular response in
conjunction with motor movement, the processing of stimuli, and the properties which
hold information on-line. One or more of these properties may be apparent in any given
cell at the time of stimuli exposure. During dlPFC single cell recordings of delayedresponse tasks, non-human primates were observed encoding information after briefly
observing visual stimuli on the computer screen. Specifically, WM encoding was
thought to have occurred when the cell showed peak activation or activity during the
8
delay periods when the animal was required to retain stimulus information in memory
(Stuss & Knight, 2002).
Episodic buffer impairment is presented in densely amnestic human patients that
fail to learn new material, or fail to recall a list of words after a delay (Stuss & Knight,
2002). This is a result of a failure in the central executive, (Baddeley, 1996) a structure
that when compromised, effects the integrated visuospatial sketchpad as well as the
episodic buffer subsystems, (Baddeley, 2002) structures known to effect WM.
Prefrontal Cortical Anatomy
The prefrontal cortex is important in maintaining a wide variety of processes
pertaining to executive functioning, memory, and navigation in the human (Stuss &
Knight, 2002), the non-human primate (Stuss & Knight, 2002), and the rat (Heidbreder &
Groenewegen, 2003; Hoover & Vertes, 2007). This section will be used to briefly
describe the anatomical regions which are suspected to be responsible for the processes
stated above (see Figure 1 in Appendix A), in order to provide justification for the
location of mPFC lesions in later sections.
The rodent PFC contains the orbital prefrontal cortex located dorsal to the
olfactory bulb, the rhinal sulcus, the agranular insular cortex located anterior to the rhinal
sulcus, and the mPFC which is located anterior and dorsal to the corpus callosum. The
mPFC can then be subdivided into the medial precentral, the anterior cingulate, the
prelimbic, and the infralimbic areas, which include the dorsal lateral (dl) and ventral
lateral (vl) PFC, (Heidbreder & Groenewegen, 2003). In primates, PFC structures have
been implicated by neuroimaging and single cell recordings to have connections with
9
brain regions such as the hippocampus (Müller & Knight, 2006). Afferent projections
using retrograde tracing techniques in rodents also discovered common neural pathways
from substructures of the mPFC to other brain areas. Connected areas include the midline
thalamus, amygdala, basal nuclei, subiculum of hippocampus, and the insular and
entorhinal cortices (Hoover & Vertes, 2007).
Anatomical Similarities Between Animals and Humans
Anatomical differences between the rat, non-human primate, and human models
are distinguished by cytoarchitecture, neurochemical, and anatomical regions in various
species. Identifying similarities in brain structures between species has been necessary in
order to determine the functional implications of damage to those structures. This in turn
can be used between species to apply and justify working theories of etiology for
neurodegenerative diseases in human models (Farovik, Dupont, Arce, and Eichenbaum,
2008; Jones, 2002).
Review of the animal model literature indicated that the PFC structure in rodents
was found to be homologous to several brain regions in non-human primates, (Kolb,
1990; Hoover & Vertes, 2007; Preuss, 1995; Uylings et al., 2003; Vertes, 2006). The
infralimbic cortex in the rodent was found to be functionally equivalent to the
orbitomedial PFC in non-human primates (Hoover & Vertes, 2007), as was the prelimbic
cortex of rats, and Brodmann’s area 32 in primates (Heidbreder & Groenewegen, 2003;
Ongur & Price, 2000), and the lateral and dlPFC in non-human primates and prelimbic
cortex of the rat (Hoover & Vertes, 2007).
The rodent mPFC has also been compared functionally to the human
10
ventromedial PFC (Hoover & Vertes, 2007) as well as the dorsomedial and dlPFC
regions in non-human primates (Heidbreder & Groenewegen, 2003). Additionally, rodent
PFC lesion studies have found comparable functional deficits to primates and humans
with dlPFC damage (Uylings, Groenewegen, & Kolb, 2003), which further describes the
prefrontal cortical reliance on both cortical and subcortical structures as described in the
neural pathway description above, (Chudasama and Robbins, 2006).
The Prefrontal Cortex and Working Memory in Humans and Animals
WM and the PFC in Humans and Non-Human Primates: Neuroimaging Studies
“The ability to hold information in the mind and work with it (manipulating,
monitoring, and transforming it),” (Stuss & Knight, 2002, pp 483) has been continuously
observed in neuroimaging studies. With the emergence of neuroimaging, the human PFC
has since been labeled as essential to WM, commonly referenced as the “neural substrate
of WM,” (Müller & Knight, 2006). Studies including functional imaging can reveal
distributed activity in involved cortical regions while a patient is completing a WM task
(Müller & Knight, 2006). Neuroimaging studies, such as those referenced by Stuss and
Knight (2002), describe activation of the dlPFC in patients completing the backward digit
span task, where the participants must repeat a series of numbers in the reverse order in
which they were given. This process of manipulating the series of numbers, monitoring it
in WM, and transforming it into a new backward number series, leads to activation of the
PFC region (Stuss and Knight, 2002). Mathematical applications such as multiplication
and addition, as well as randomization of numbers without repetitions also require the
essential faculties of the PFC (Stuss & Knight, 2002).
11
Single-Cell Recordings In Non-Human Primates
Lesion studies in humans that examine the role of the PFC in WM may be subject
to confounds that can make interpretation of function difficult (Müller & Knight, 2006).
For this reason, single cell recordings and lesion studies of this area in animals, have been
utilized to elucidate the cognitive and behavioral functioning deficits found in humans
with lesions of the PFC (Müller & Knight, 2006; Chudasama & Robbins, 2006). In
particular, single unit studies in non-human primates allow for functional assessment of
anatomical areas that at times cannot be identified with sufficient accuracy in human
imaging studies (Stuss & Knight, 2002). During single cell recordings with non-human
primates, the monkey dlPFC has been used to examine visual WM properties while the
animals complete spatial behavioral tasks. In these tasks, the animal is required to focus
on a visual stimulus for a brief moment, and then hold that image in WM during the
subsequent trials. WM performance is then tested by observing the eye movements
produced during each trial, where the monkey makes a saccade to the remembered
location of the previously viewed stimulus. Cellular recordings during this type of
delayed matching to sample task were able to explore the conglomeration of cells which
seem to indicate the WM process (Stuss & Knight, 2002). Neural PFC activity was also
reported in the research by Desimone (1996) suggesting the presence of WM processes in
this cortical area. During the task, the animal was shown one of two shapes. Then,
following a brief delay, the animal was rewarded upon correctly choosing the shape that
corresponds to the first shape. Neural activity in the PFC during the delay period
provided evidence for active WM processes.
12
Human & Non-Human Primate Lesion Experiments
Lesion studies located in the mPFC also suggest that the ventral and dorsal stream
neural pathways could help mediate WM functioning. Humans and non-human primate
studies involving the holding of information in memory have helped to identify neural
activity from the posterior cortex extending to the PFC region and neural connections
between ventral PFC and temporal cortex, and dorsal PFC to parietal regions (Müller &
Knight, 2006). Patients with lesions to both VM PFC and DL PFC areas displayed
impairments on a two-back test of WM. These impairments were not solely due to the
size of the lesions, but rather the area affected, suggesting that the PFC helps to sustain
WM, but is not the only region contributing to WM, (Müller & Knight, 2006).
Rodent Lesion Experiments
Rodent studies using quinolinic lesions and lidocaine inactivations to the mPFC
resulted in a reduction in WM during various egocentric and navigational tasks
(Heidbreder & Groenewegen, 2003). WM deficits were also found in a study with rodent
PFC lesions using a conditional associative learning (CAL) procedure. For each CAL
trial, the rat was trained to select the retractable lever that was underneath one of four
lights on the apparatus wall that had previously been illuminated. Correct responses were
rewarded with a food pellet. Results indicated that rats with PFC lesions showed
response-selection deficits that could be attributed to impairments in WM (Winocur &
Eskes, 1998). Rodent studies have also determined that the PFC shares connections with
other areas thought to be important for WM. In a study by Jo et al. (2007), pattern
completion memory retrieval was tested on rodents with mPFC lesions.
During a water
13
maze navigational task, rodents were trained to search for a submerged platform which
varied in location dependent on the trial day. Between one and three cues were present to
represent the full or partial-cue in each trial. Results of this delayed matching-to-place
experiment indicated that spatial memory retrieval was inhibited regardless of full or
partial-cue conditions, suggesting a WM impairment and interaction between the
hippocampus and mPFC. Similarly, in a study by Izaki, Takita, and Akema (2008),
based on results from delayed radial arm maze tasks, rodent WM performance was found
to be affected by lesions to both the bilateral posterior dorsal hippocampus and the
bilateral PFC. This study concluded that the hippocampal-PFC anatomical pathways are
essential for efficient WM processes.
Single-Cell Recordings In Rodents
The presence of WM pathways in non-human primate data might also be used to
suggest similar PFC pathways in rodent physiology. In procedures designed to examine
WM during the delay period of spatial navigation tasks, Jones (2002) concludes that due
to known relevance of the hippocampus with WM, PFC neurons may also encode spatial
information. While single cell recording evidence for rodent WM processes in the PFC
are currently lacking, electrophysiological recordings suggest changes in the the level of
PFC activation during tasks designed to measure WM (Jones, 2002). Neurons located in
the deep PFC layer also exhibit short-term plasticity necessary in the encoding process of
WM; a complex process by which one could postulate that rodent PFC physiology
regulates more than simple spatial navigation processes (Jones, 2002).
14
WM Debate
Despite the recent evidence to the contrary, debate still exists regarding PFC
involvement in WM. Non-human primate lesion studies by Rushworth, Nixon, Eacott,
Passingham, (1997) have concluded that individuals with ventromedial lesions of PFC
preformed identical to normal controls in WM tasks. Similar observations were noted for
the dlPFC lesions (Müller & Knight, 2006), although many others have found spatial
WM (Ptito, Crane, Leonard, Amsel, & Caramanos, 1995; Bechara, Damasio, Tranel, &
Anderson, 1998; Shimamura, Janowsky, & Squire, 1990; Chudasama & Robbins, 2006;
Kesner, Hunt, Williams, & Long, 1996), as well as decision-making and WM to be
significantly impaired after lesions of this area (Chudasama & Robbins, 2006). WM in
particular, was found to have the greatest degree of impairment when both the
ventromedial and dorsolateral regions were lesioned (Müller & Knight, 2006), similar to
findings in human studies. Furthermore, in a task involving visual association with delay
periods, monkeys with lateral lesions of the inferior convexity maintained WM processes,
leading the authors to conclude that affected cortical regions were responsible for
attentional processes rather than directly responsible for WM impairments (Müller &
Knight, 2006, and Rushworth, Nixon, Eacott, Passingham, 1997). Similarly,
neuroimaging research also has suggested a non-mnemonic role of the PFC, where
processes that aid in memory are not mediated by the cortical region. Specifically, the
model of attention, monitoring, and integration of information from neuronal networks
was not theorized to come from the PFC (Jones, 2002; Müller & Knight, 2006).
15
Navigation Requires WM
Path Integration and WM
As discussed in the navigation section above, path integration relies on the
continual on-line use of movement related information in order to orient oneself in space.
One type of neuron that has been postulated to play a role in spatial orientation are place
cells. These cells, typically recorded in the CA region of the hippocampus, become
active when the animal is in a particular location of a known environment (O’Keefe &
Dostrosky, 1971). These cells have been shown to maintain an accurate navigational
signal for some time when landmarks are eliminated, suggesting that they are able to
maintain orientation using path integration (Touretzky & Redish, 1996).
The presumptive use of a “cognitive map” while navigating suggests some neural
scheme that organizes the locations of places in the world relative to each other, which
would allow for choosing optimal paths during navigation (Tolman, 1949). Since this
map is used to calculate the path between two points in the environment (Newman,
Caplan, Kirschen, Korolev, Sekuler, & Kahana, 2006), the “online” cognitive map could
suggest the presence of a WM component during path integration navigation when
landmarks are no longer present. Furthermore, since path integration employs the ability
to remember the environment when visual cues are absent, it can be deduced that this is a
function provided by the WM system. Consequently, if this system were to be impaired,
the continual integration of motor, vestibular, and visual cues during navigation would be
also be significantly compromised.
16
Lesions of the mPFC Impair Navigation
In support of the assumption that path integration navigation requires working
memory processes and the mPFC is necessary for working memory, a number of studies
have found navigational deficits following lesions of this area. Research by Grannon and
Poucet (1995) describe this impairment in trained animals with mPFC or sham lesions on
a water maze task. The task changed with increasing levels of difficulty, incorporating up
to four potential start positions and varying the occluded platform positions from trial to
trial. The authors found no apparent effects of mPFC lesions until all four start positions
were utilized. This indicated that mPFC lesioned animals were able to learn the spatial
position of the platform, but may have been impaired in WM functions involved in
forming a representation of the course of movements required to reach the platform.
WM and Navigation Functions are Impaired by Lesions of the PFC
Wolbers, Wiener, Mallot, and Büchel, (2007) utilized a virtual paradigm in
humans to examine the role of the mPFC in navigational behavior. To examine path
integration and relate its function to non-human primate and rodent models, a magnetic
resonance imaging virtual paradigm was presented to allow a first-person perspective
devoid of available landmarks. By navigating using a joystick, self-motion could be
inferred. For each task, the participant was required to path integrate to varying locations
within the virtual plane, then point to the start location. Activation of bilateral
hippocampal and mPFC regions were present during the path integration exercise and
correlated with higher response consistency in the hippocampus and mPFC. The mPFC
also corresponded to observed random error in navigation; results which effectively
17
associates the deficits from a complex spatial path integration task with higher order WM
processes in the PFC (Wolbers, Wiener, Mallot, and Büchel, 2007).
The Connection Between WM, Executive Functions, and Navigation
The process of navigation, in humans as well as animals, requires coordinated
movements based on independent task cues integrated together to form a goal or purpose
(Chudsama & Robbins, 2006; Müller & Knight, 2006; Heidbreder & Groenewegen,
2003). As described in previous sections, the functions of the PFC includes elements of
perception, sensory and motor processes in navigation, executive functioning, learning,
and memory components (Miller & Cohen, 2001; Jones, 2002). The organization of
spatial information is an executive process, whereby perceptual, sensory and motor
stimuli from the navigating individual are integrated and manipulated, then passed
through various neural networks in the PFC, thereby producing “executive behavior,”
(Jones, 2002). An example of executive behavior would be the “cognitive map” used in a
path integration experiment, where the rat would produce a visual image of the
immediate environment using WM in order to effectively navigate the surroundings from
various locations within that environment. As described in the WM section, the central
executive is responsible for executive processes and is essential to functions such as WM
and navigation (de Saint Blanquat, Hok, Alvernhe, Save, & Poucet, 2010). The central
executive as well as WM processes could be thought of as components of spatial
learning, where it is theorized that these components require various demands on
different structures and processes. If one of these structures such as the PFC is
compromised by a lesion or other brain insult, the result is the loss of one or more
18
components necessary for navigation (Compton, Griffith, McDaniel, Foster, & Davis,
1997).
Jacobsen (1935) was among the first to reference a connection with bilateral PFC
lesions and memory dysfunction through observed impairment in delayed response tasks
in non-human primates. After presenting two objects, the task required the ability to
locate and choose the object that had been rewarded on the previous trial. Subsequent
studies noted in a review by Chudasama & Robbins (2006), found that PFC lesioned
animals had significant spatial navigation and WM deficits when completing a test of
spatial short-term memory. The resulting impairments from those studies were related to
the inability to hold information on-line, and the way in which the PFC neural networks
manipulate information on new stimuli to produce a behavior (Chudasama & Robbins,
2006) in, for example, a path integration task.
Müller & Knight, (2006) also noted that the human ventrolateral PFC supports
object information and the dlPFC the maintenance of spatial information, with executive
functions distributed along the aforementioned brain regions. When both regions are
lesioned in humans, simple spatial tasks were impaired. Results from the one-back test
required the participant to inhibit previous responses to stimuli in order to prevent false
alarms when selecting the stimulus in the environment during future trials (Müller &
Knight, 2006). Several other studies also report similar findings of patients with frontal
lobe lesions (see review by Baldo and Shimamura, 2002).
WM and spatial navigation have also been tested with rodent PFC lesion studies
using the T-maze, radial arm, and water mazes which were effective in identify the
19
resulting level of impairment in WM and associated navigation processes (Jones, 2002;
Grannon & Poucet, 1995; Heidbreder & Groenewegen, 2003; Hoge & Kesner, 2007).
These mazes employ the use of available cues in testing spatial navigation through
delayed response tasks in which the frequency of error and inability to navigate in a maze
environment suggest both WM and navigation dysfunction. Since these functions
employ the use of PFC networks to operate successfully, PFC impairment would produce
a negative effect on WM and spatial navigation processes (Kolb, Buhrmann, McDonald,
& Sutherland, 1994; Jones, 2002). Research by Kolb, Buhrmann, McDonald, &
Sutherland (1994), also concluded that spatial navigation was impaired in rodents with
mPFC lesions when completing a water maze WM paradigm. During this task, prior to
the lesion, the rat was trained to navigate to search for a visual platform. After surgery,
rodents with mPFC lesions presented with a higher frequency of directional “heading
error,” increased latency in learning and task completion, as well as perseveration or the
repetition of a fixed navigation pattern in the water maze. These findings support the link
between spatial orientation and navigation in the PFC, but also suggest that impairment
could result from failure to organize spatial information required to produce cognitive
maps of the immediate environment necessary for effective navigation and WM function
(Chudasama & Robbins, 2006; Kolb et al., 1994; Newman et al., 2006; Tolman, 1949).
Executive Functions Located in the PFC are Synonymous with Navigational
Deficits: Single Cell Recordings and fMRI Studies
As referenced by Curtis and Lee (2010), persistent neuronal activity in the PFC
from a transient stimulus and resulting behavioral response also provides evidence for
20
WM processes by way of the central executive. Rodent “executive control” can be
exhibited during the training of navigational tasks which require previous trial
information to be integrated within the context of current task demands. To summarize,
previous task experiences are used to make predictions about future tasks, in which
behaviors are adjusted to produce the most desired outcome in the spatial navigation task
(Curtis and Lee, 2010; de Saint Blanquat et al., 2010). Additionally, the rodent mPFC
region has been implicated in the management of executive processes when completing a
navigation task (de Saint Blanquat et al., 2010). In this experiment the ability to learn the
location of the four rewarded radial arms in an eight arm radial maze was measured
during single cell recordings of the PFC. Neural correlates of the PFC were shown to
reflect navigation to various baited and non-baited radial arms (de Saint Blanquat et al.,
2010). These results represent the neural correlates associated with the identification of
previously baited arms and the executive control of decision making during navigation of
the radial arm maze.
PFC involvement in neurodegenerative disease found in animals and humans
Pathological aging in humans is represented by neurodegenerative disorders such
as Alzheimer’s disease and fronto-temporal lobe dementia. Alzheimer’s disease is the
most common neurodegenerative disease of the elderly, categorized by the presence of
neurofibullary plaques and tangles found in the hippocampus and temporal cortical
tissues (Kendel, Schwartz, & Kendle, 2000). Amyloid plaques build up between the
neurons in the brain tissues in conjunction with proteins called Beta Amyloid,
accumulating to produce insoluble, hard bundles around neurons. Tangles or stringy
21
fibers clumped together inside the neurons of the brain are formed out of Tau proteins,
which help carry nutrients to the neurons. The accumulation of these tangles inhibit
neural connections causing cell death and resulting atrophy (Kendel, Schwartz, &
Kendle, 2000. Older adult populations with dementia of the Alzheimer’s type often
sustain severe hippocampal, PFC, and temporal lobe atrophy (Sweeny & Ranstack, 2009)
concurrent with cognitive impairment.
Cognitive impairment in the disease presentation is most often characterized by
an amnestic type, affecting memory processes. However, several cognitive domains such
as executive functioning, language, and visuospatial processes have also been implicated
in contributing to chronic disease progression (Tomaszewski Farias et al., 2011). Several
fMRI studies, (Olichney et al., 2010; Nordahl et al. 2006; Nordahl et al., 2005), have
also validated the presence of multiple cognitive domains regulated by frontal cortical
systems (Marshuetz, Reuter-Lorenz, Smith, Jonides, & Noll, 2006), specifically memory
and executive functions which regulate verbal encoding and the episodic memory of
autobiographical events.
Frontotemporal Dementia (FTD) is also affected by similar structural
neurodegeneration as Alzheimer’s disease with acute frontal and temporal lobe atrophy.
The manifestations of disease present as behavioral abnormalities, often mistaken for
psychiatric disorders, in which dramatic changes to personality, social or emotional
inhibitions, and the ability to use and understand language (Stuss & Knight, 2002).
Previously defined as Pick’s Disease, this form of dementia is characterized by pick-like
bodies which occur in the affected frontal lobe and surrounding regions. Pick bodies
22
contain abnormal spherical Tau proteins which accumulate within neurons, damaging
neural connections. In addition to perfuse gray and white matter atrophy of the frontotemporal region (Hodges, 2001), the amygdala, striatum, and hippocampal regions can
also sustain considerable brain insult (Boccardi, 2006). Damage to the PFC can cause
difficulty encoding and recalling contextual information, in which the ability to learn is
impaired (Müller & Knight, 2006). In addition to these deficits, individuals can be prone
to distraction, have long-term memory retrieval problems, and display deficient spatial
WM during navigation (Hoge & Kesner, 2007; Kesner, 1998).
The primary motor cortex located in the precentral gyrus, and the motor
association cortex found rostral to the primary motor region in humans, is responsible for
the movement of joints and motor preparation necessary for navigation (Kendel,
Schwartz, & Kendle, 2000). Damage to these regions found in the frontal lobe can
negatively affect spatial navigation functions often found in virtual reality paradigms
where an inability to navigate using efficient routes from cues in the environment is
important for orientation (Newman et al., 2007), and arguably the cognitive map.
The presentation of symptoms in animals after the PFC sustains damage is similar
to the symptoms found in humans with neurodegenerative disorders
In rodent studies, perseveration has been observed after the PFC was lesioned,
suggesting a deficit in the ability to hold information in WM sufficiently in order to
develop a behavioral navigation strategy to reach a new stimulus (Gemmell & O’Mara,
1999). Perseveration occurs when attention is fixed to one task, and that task is
consistently repeated over a period of time. When perseveration is present during
23
navigational tasks, it may represent an impairment in “response shifting” to a new
stimulus allowing methodical navigation toward it in the relative environment (Gemmell
& O’Mara, 1999). As discussed in earlier sections, this behavior is also a function of
executive control and requires attention necessary for planning and organization of
actions (Chudasama & Robbins, 2006), a function which is likely controlled by the PFC.
This perseveration is also found in humans with dementia disorders, such as Alzheimer’s
disease.
Damage to the frontal lobe can also inhibit the ability to organize and plan
behaviors in humans with FTD. Dopaminergic neural connections in both humans and
animals are comprised in the PFC region (Chudasama & Robbins, 2006; Stuss & Knight,
2002; Kendel, Schwartz, & Kendle, 2000). In a study designed to test the effects of
dopamine on behavior, dopamine was depleted from that area in monkeys using an
injection of 6-hydroxydopamine, destroying post-synaptic dopamine terminals in
neurons. Following this, monkeys were unable to remember the stimuli after a short
delay, exhibiting WM dysfunction similar to humans with lesions of the same area
(Kendel, Schwartz, & Kendle, 2000). Human neuropsychological testing involving the
Wisconsin Card Sorting Task is also designed to test frontal lobe functioning using set
shifting rules with cards designed with colored shapes and symbols. Impairment on this
test would also present with impairment in response-shifting commonly exhibited by
perseveration (Stuss & Knight, 2002). Tests of visual discrimination in monkeys apply
these same response-shift concepts to test the ability of “reversal learning” involved in
remembering the changing rules of the task in order to accomplish a goal, also known as
24
set shifting (Stuss & Knight, 2002). The serial reaction time task (5CSRTT) for animals
is modeled after the human continuous performance test (CPT), used to measure
attentional processes to monitor infrequent and random pictures of letters; a process
dependent on neural networks of the PFC (Chudasama & Robbins, 2006). The animal
paradigm involves food reinforcement and an operant chamber with apertures required to
monitor rodent attention and response accuracy to attend to the correct stimulus. These
procedures are used to examine the impulsivity of responses or inhibition, as well as
compulsions to perseverate toward one stimulus verses others and the level of motivation
to complete the task (Chudasama & Robbins, 2006). These are common behavioral
deficits exhibited by individuals with Alzheimer’s disease and frontotemporal lobe
dementias.
Complex Diseases such as FTD and Dementia of the Alzheimer’s Type can Benefit
from Research in Animal Models
Animal research has been critical in understanding new models of disease, in
particular the understanding of the PFC structures and function (Stuss & Knight, 2002).
Animal models of cognition provide advantages with minimal cohort differences by
eliminating variation in age, education, and intelligence; thereby determining a more
precise contribution between various brain structures and corresponding cognitive
behavior (Chudasama & Robbins, 2006). Studies involving lesions also have similar
benefits with the ability to resect a region and duplicate the procedure to determine neural
pathways responsible for behavior and cognition during particular cognitive tasks. Even
imaging and single cell recordings in animals have advanced our understanding of the
25
functions dependent on the PFC. Research on the learning abilities and plasticity of PFC
neurons in particular have challenged the idea that brain areas are only responsible for
certain processes (Stuss & Knight, 2002). In this respect, the contribution of animal
research regarding PFC systems and functions has proved to be indispensable in
advancing our understanding of complex disease processes in humans.
26
Chapter 2
METHOD
It is hypothesized that rodents with mPFC lesions will show indications of
impaired navigation when compared with controls during a path integration task. This
hypothesis has been constructed from the assumptions introduced above, namely that
rodents with lesions of mPFC will possess deficits in WM and our hypothesis that
navigation through the use of path integration relies on WM processes. Since evidence
suggests an analogous relationship between the PFC of rats and humans, it can also be
suggested that any observed WM or navigational deficit would be similar to those found
in humans. Deficits in navigation and WM are also commonly found in many
neurodegenerative diseases such as: Alzheimer’s disease and frontotemporal lobe
dementia. The similarities between animal and human deficits found in navigation and
WM would also bring further insight into the process of human neurodegeneration.
Animals
Twenty adult female Long–Evans rats, weighing 250–300 gm, were
housed in wire metal cages located in the Department of Psychology animal vivarium on
the campus of California State University, Sacramento. They were maintained on a 16/8
hour light/dark cycle in a room with a consistent temperature of 20–21°C. A food
restricted diet was used to establish motivation during navigational tasks. Water was
freely available. All cages were located next to one another to ensure social contact.
27
Apparatus
The Whishaw Tabletop Homing Task (Whishaw & Maaswinkel, 1998) was
utilized to assess the effects of mPFC lesions on path integration navigation and WM.
Figure 2 presents a picture of the apparatus used in the task. A circular wooden table 204
cm in diameter and elevated 64 cm off the floor served as the primary apparatus. A
bearing located in the middle of the table allowed rotation during the individual task
trials. The table was painted white to eliminate landmark cues and contained eight holes,
11.5 cm each, spaced at equal distances from each other and located 13.5 cm from edge
of the table. Below one of these holes was the refuge ( home cage), where the rat was
able to enter and exit the table with the food pellet for each trial. The top of the table was
marked with 17 pseudo-randomly placed black dots located relatively equal distance
from one another. These black dots were made with black permanent marker rather than
raised black markers or cups to decrease landmark cues during trials. During each trial, a
sugar pellet (Bioserve; Frenchtown, NJ) was randomly placed on one of the 17 black
dots. The apparatus was located in a classroom with many cues such as, tables, chairs,
computers, cupboards, and a sink. However, these cues were available only during the
initial phase of pretraining described below. During other phases the lights were
extinguished and curtains were drawn around the table to eliminate the use of external
visual cues from the testing room. An infrared camera was positioned over the table to
document performance and navigation behavior. The signal from the camera was
viewable live in an adjacent room away from the training and was also saved to videotape
for later analysis.
28
Pre-Surgical Training
Pre-surgical training was necessary to habituate the rats to the path integration
task prior to commencing the experimental procedure. Pre-training involved placing the
rat in the refuge cage, rewarding the rat for climbing out of the refuge onto the table,
navigating across the surface of the table to the sugar pellet, and returning to the refuge to
eat the sugar pellet. Pre-surgical trials began by placing one sugar pellet near the
entrance/exit of the home cage with a small cardboard box surrounding the region
occluding all visual cues. Over time, the rat began to search for the pellet on the table.
The cardboard box was then removed, and the sugar pellet was placed randomly on one
of the black dots marked on the table. Once the rat entered the table it was given five
minutes to find the sugar pellet and return with it to the refuge. If this time limit was
reached or the rat located the sugar pellet, the trial would end and the rat would have 30
seconds to reach the refuge, after which it would be removed from the table by the
experimenter. Each rat was rewarded with one additional sugar pellet if the task was
completed successfully. If the sugar pellet was consumed on the table or the task was not
completed, a reward was not given.
Each rat was rotated in order though the pre-training trails. For each trial the
locations of the pellet and home cage would change through random assignment. After a
rat completed 20 consecutive trials with at least a 95% success rate, the procedure was
repeated (without the initial carboard box phase) with the lights turned off and the
curtains drawn to eliminate visual cues. After the animal performed the task successfully
29
95% of the time over a 30 day period the animal was moved to the surgical phase of the
experiment.
Surgical procedure
A surgical procedure approved by the CSUS Institutional Animal Care and Use
Committee was used. After the pre-training phase ten of the rats received surgical
neurotoxic lesions of the mPFC and the ten remaining rats underwent a sham operation.
Prior to surgery, the rodents were anesthetized with an intra-muscular injection
containing ketamine (30 mg/kg), xylazine (6 mg/kg), and acepromazine (1 mg/kg). The
head was then shaved and disinfected using Betadine. After being placed in the
stereotaxic device, an incision was made to expose the skull. In the case of the rats
receiving neurotoxic ibotenic acid lesions (Jarrard, 2002), burr holes were drilled in the
skull and injections were made at two sites by slow infusion of ibotenic acid (0.06 M in
sterile phosphate buffer saline solution; 0.3 μl) over 3 minutes using a 0.5 μl Hamilton
syringe with a 30-gauge beveled needle (Tait et al., 2009). Injections were administered
with the bevel of the needle facing medially using the following coordinates relative to
Bregma provided by Paxinos & Watson (1996): (1) AP = +2.5 mm; ML = +0.6 mm; DV
= -5.0 mm, and (2) AP = +3.5 mm; ML = +0.6 mm; DV = -5.2 mm, (Tait et al., 2009).
For each injection, the needle was left in place for two minutes to allow for diffusion.
Once the lesion procedure had been completed, the incision was sutured closed and a
topical Neosporin antibiotic was placed on the site. The rats receiving sham surgeries
experienced the same procedures except that no burr holes were drilled and no neurotoxin
was injected. Analgesics were given for one to two days following the surgical
30
procedure. Prior to post-surgical testing, the rodents were allowed to recover for an
average of 25 days.
Post-Surgical Testing
Following the recovery period, each animal identity was recoded by non-research
personnel to ensure that the testing was performed blindly relative to the identity of the
animals. The rats were then giving testing for 5 trials each using the same testing method
as the pre-surgical training phase. During the testing period, rats in both the control and
lesioned group were subjected to up to four path integration trials per day. Infrared video
recordings were made of these trials for later scoring purposes.
Analysis
After post-surgical testing, all video recordings for lesioned and control groups
were individually analyzed to document navigational performance. Performance was
analyzed by counting the number of incorrect refuge choices on each trial. A refuge
choice was indicated if the snout of the rat was within 3 cm of the hole. Latency of
emergence, the time the pellet was grabbed, and the time necessary to find the correct
refuge entrance was also assessed, as well as the frequency of emergence onto the table.
The total time was calculated in seconds after the body of the rat entered onto the table in
search of the food pellet. Once the food pellet was retrieved, time was then calculated
from the pellet to the correct refuge choice (Tait, Marston, Shahid, & Brown, 2009;
Whishaw & Maaswinkel, 1998). Navigational routes were documented and “wandering”
or perseverative behaviors were noted if applicable.
31
To determine if lesioned and control animals differed in these measures, an
analysis of variance (ANOVA) tests were conducted.
32
Chapter 3
RESULTS
The total sample included 191 trials, with pre-operative (111 trials) and postoperative trial observations (80 trials) for 16 rats. Out of the 20 original rats, three rats
died of surgical complications and one developed health complications that required its
removal from the study.
Differences between lesion and sham groups in pre-surgical training performance
were assessed using each measure of latency, the frequency of emergence onto the table,
and number of errors. Results of the seven one-way between subjects ANOVAs
determined that the emergence time [F(1, 14) = 0.0002, p = 0.99)], the time to take the
pellet [F(1, 14) = 1.27, p = 0.28)], the time to return to the cage [F(1, 14) = 0.81, p =
0.38)], the difference between the emergence time and the time the pellet was grabbed
[F(1, 14) = 2.44, p = 0.14)], the difference between the time the pellet was grabbed and
the time to return to the cage [F(1, 14) = 3.32, p = 0.09)], the number of times on the
table [F(1, 14) = 1.80, p = 0.20)], and the frequency of error [F(1, 14) = 0.62, p = 0.44)]
were not statistically significant (see Table 1 in Appendix C). The fact that the groups
did not show differences in the pre-surgical training phase is important, as it demonstrates
that the groups were equivalent prior to the surgical procedure.
The average performance for each group across the five post-training test trials
are presented in Table 2 of Appendix D, and Figures 3 and 4 present these measures
calculated for each individual test trial. To determine a possible main effect of the lesion
33
condition on post-surgical performance, seven 2 x 5 (Groups x Trials) ANOVAs were
performed on the post-surgical test data on each of the dependent measures. For the
dependent measure of emergence time, the analyses showed no significant main effect for
group [F(1, 36) = 3.08, p = 0.09], trials [F(4, 36) = 0.64, p = 0.09] or the interaction
between these factors [F(4, 36) = 0.42, p = 0.79]. For the measure of the time the pellet
was grabbed, the main effect of group was not significant [F(1, 36) = 0.18, p = 0.67], nor
was the main effect of trials [F(4, 36) = 0.02, p = 1.0], or the interaction between the two
factors [F(4, 36) = 1.0, p = 0.42]. On the variable of the time the rodent returned to the
home cage the main effect of group was not significant [F(1, 36) = 0.14, p = 0.72], nor
was the main effect of trials [F(4, 36) = 0.01, p = 1.0], or the interaction [F(4, 36) = 1.0, p
= 0.42]. Regarding the difference between the time of emergence and the time the pellet
was grabbed, there was no main effect of group [F(4, 36) = 0.02, p = 0.96], trials [F(4,
36) = 0.10, p = 0.98], or interaction between these factors [F(4, 36) = 1.11, p = 0.37].
Similarly, regarding the difference between the time the pellet was grabbed and the time
returned to the home cage, there was no main effect of group [F(1, 36) = 01.08, p = 0.31],
trials [F(4, 36) = 1.51, p = 0.22], or interaction between the two [F(4, 36) = 0.23, p =
0.92] (see Figure 3 in Appendix E). Measures of frequency also failed to show significant
differences, including the frequency of emergence main effect of group [F(1, 36) = 0.69,
p = 0.41], trials [F(3, 36) = 1.47, p = 0.23], and interaction [F(1, 4) = 0.24, p = 0.91] and
number of errors main effect of group [F(1, 36) = 0.06, p = 0.81], trials [F(4, 36) = 1.54,
p = 0.21], and the interaction between these factors [F(4, 36) = 0.19, p = 0.94] were also
34
not significant (see Figure 4, in Appendix F). To summarize, there were no significant
differences in post-operative performance between lesioned and sham animals.
Histology
Following the completion of the study, the histological study of the brains of lesioned rats
was conducted to determine the extent of the neurotoxic lesions. During this process, the
rats were anesthetized deeply and perfused with saline and then a 10% formalin solution.
After two formalin rinses over a 48 hour period, the brains were cut into 40μm slices in
the coronal plane, and stained with cresyl violet. Lesion extent was determined by
microscopic examination of obvious cell death and identified by the largest and smallest
affected area at selected coordinates from Paxinos and Watson (1996). All lesions were
consistent with complete mPFC ablation (see Figure 3 in Appendix G).
35
Chapter 4
DISCUSSION
The present study tested the hypothesis that rodents with neurotoxic lesions of the mPFC
are impaired relative to sham controls in a path integration task designed to assess
navigation and possibly WM. Results failed to find differences between rodents trained
in the task prior to surgery verses those that received the sham or lesion surgery and
completed post-surgical testing.
Measures of mean latency suggested that post-surgical (both sham and control)
animals took longer than their pre-surgical counterparts to emerge from the home cage
and once the pellet was grabbed, path integrate back to the home cage. Whereas the
lesion group took longer than the sham control group to emerge and locate the pellet,
grab the pellet and return to the home cage. The difference between the time of
emergence and the time the pellet was retrieved was also found to be longer than the
sham control group. However, since the differences were slight, and most likely due to
the effects of surgery, none of the interactions were statistically significant.
Additional measures found that the post-surgical lesioned group committed
more errors on average than sham controls and that the sham group had a higher
frequency for the number of times on the table in order to find the food pellet, but again
these differences were not found to be significant. Potential differences in the frequency
of emergence from the refuge was first noted as a behavioral observation. Rats searching
for the pellet appeared to be monitoring the location of the home cage by creating various
36
short trajectories in order to cover different areas on the apparatus. During the test
procedure it was suspected that this navigation technique was used to compensate for
possible WM impairment resulting from the inability to locate the home cage once the
pellet was retrieved.
Altogether, however, the hypothesis that mPFC lesions would impair path
integration navigation was not supported. The failure to uncover deficits in the path
integration task following mPFC lesions could be due to several possibilities. First, path
integration navigation may not depend on WM processes as originally hypothesized. Our
view that path integration relies on working memory is based on the fact that previous
movement events are continually integrated in order to provide a “real time” estimate of
position in space. While this process likely contains some demands on the WM system,
the demand on the system may not be as strong as hypothesized. As an example, suppose
the animal travels to position A, then B, then C, before finally arriving at point D. A path
integration navigation strategy that would impose a high demand on the WM system
might require the animal to retain all of the previous movements leading to the arrival at
position D. On the other hand, a navigation strategy that would impose a lower demand
on the WM system would involve the animal only retaining in memory the most recent
position and the movements since leaving that position. Diffuse neural networks
unaffected by the lesion could also explain the plasticity of WM and navigation
processes. Structures such as the cerebellum, responsible for motor memory during
navigation, could potentially regulate WM functional impairments to a greater degree
37
than anticipated. It is possible that our lesions only marginally impaired WM and hence
did not produce a measurable effect on path integration.
On a related note, it is possible that the mPFC lesions utilized were somehow
ineffective at impairing WM processes as expected. This could have occurred if our
lesions were not inclusive or if other areas of the brain besides mPFC are involved in
working memory, such as the hippocampus, amygdala, or striatum. This possibility could
have been assessed by inclusion of a task that is already recognized as a valid
measurement of working memory performance, such as the working memory version of
the radial arm maze where the rats must remember the previously visited arms to avoid
entering an unbaited arm (Izaki, Takita, and Akema, 2008; Jones, 2002).
Finally, it is possible that the present methods were insensitive at detecting
impaired path integration performance in our animals. For instance, the small sample
size (especially in the lesion group due to postsurgical mortality) likely resulted in a
decreased ability to detect lesion effects. A sample of 20 or more in each group would
have been optimal. In addition, an extended postsurgical testing period may have
allowed group differences to emerge. Finally, no direct controls were utilized to ensure
that the animals were truly performing the task via path integration rather than using an
undetected landmark.
Conclusions and Future Study
This study failed to provide evidence that mPFC lesions in rats results in a deficit
in path integration processes. Additional research is needed to assess the theory that
mPFC lesions contribute to functional behavioral impairment similar to humans with
38
neurodegenerative diseases. However, results can be compared to the research in
aforementioned sections which describe the plasticity of the PFC which produces
complex behaviors (see review by Stuss & Knight, 2002) such as path integration.
Although results were negative regarding impairment in this task following mPFC
lesions, one could argue that successful completion of the path integration task, even
prior to PFC ablation, is evidence of higher cortical functioning and the presence of intact
WM processes.
39
APPENDIX A
Subcortical Structures of the Rodent Brain
Figure 1. Figure adapted from Paxinos, G. & Watson, C. (1996)
40
APPENDIX B
An Infrared Picture of a Post-Surgical Rat
Figure 2. An Infrared Picture of a Post-Surgical Rat Completing the Path Integration
Task Using the Whishaw Table Top Apparatus
41
APPENDIX C
Table 1
Pre-Operative Experimental Measures of Latency, Frequency of Emergence, and Error
in Lesioned and Sham Controls for Five Trials, N =16.
Measures
Procedure
Sham
Lesion
Emergence Time Seconds
18.48 (19.53)
10.53 (7.79)
Time Grabbed
67.55 (67.86)
79.42 (68.58)
Time Returned to Cage/Total Time
96.0 (78.08)
90.04 (68.14)
1.0 (0.0)
1.27 (0.64)
0.44 (0.71)
0.27 (0.52)
Difference Emergence-Grabbed Pellet
49.07 (65.62)
68.88 (68.72)
Difference Grabbed Pellet-Cage
28.45 (42.10)
10.62 (7.93)
Times on Table
Errors
Note. Numbers in parentheses are standard deviations.
42
APPENDIX D
Table 2
Post-Operative Experimental Measures of Latency, Frequency of Emergence, and Error
in Lesioned and Sham Controls for Five Trials, N =16.
Measures
Procedure
Sham
Lesion
Emergence Time Seconds
20.73 (22.16)
37.56 (44.27)
Time Grabbed
123.14 (89.05)
187.86 (157.89)
Time Returned to Cage/Total Time
135.41 (88.45)
196.97 (157.66)
Times on Table
1.59 (1.01)
1.38 (0.82)
Errors
0.59 (0.91)
0.5 (0.93)
Difference Emergence-Grabbed Pellet
102.41 (79.75)
150.30 (159.66)
Difference Grabbed Pellet-Cage
12.27 (10.37)
9.13 (4.89)
Note. Numbers in parentheses are standard deviations.
43
APPENDIX E
Figure 3. Error and Frequency of Emergence by Condition and Procedure
44
APPENDIX F
Figure 4. Measures of Latency by Condition in Lesion & Sham Groups
45
APPENDIX G
mPFC Neurotoxic Lesion Extent
Figure 5. Black and gray regions indicate maximum and minimum ablation.
Figure adapted from Paxinos, G. & Watson, C. (1996)
46
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