honors thesis final
TRANSCRIPT
THE FLORIDA STATE UNIVERSITY
COLLEGE OF ARTS AND SCIENCES
USE OF ZINC SUPPLEMENTATION TO IMPROVE BEHAVIORAL
RESILIENCY IN TRUAMATIC BRAIN INJURY
By
DANIEL PIERCE
A thesis submitted to the Department of Psychology
in partial fulfillment of the requirements for graduation withHonors in the Major
Degree Awarded:Spring 2016
The members of the Defense Committee approve the thesis of Daniel Pierce defended on April 23, 2015.
Dr. Cathy LevensonThesis Director
Dr. Orenda JohnsonOutside Committee Member
Dr. Heather FlynnCommittee Member
Abstract
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Some of the most prominent outcomes associated with traumatic brain injury (TBI) are
deficits in learning and memory, anxiety, and depression. Previous work in a pre-clinical model
showed that chronic zinc supplementation can provide resilience to these poor outcomes. The
connection that exists between memory and the hippocampus, as well as the role of the
hippocampus in regulatory mood, led us to hypothesize that zinc supplementation acts via
hippocampal mechanisms. To test this hypothesis we used the Porsolt swim test, spontaneous
alternation, open field behavior, and novel object recognition test in a TBI model with zinc
supplementation and hippocampal irradiation. Prior to surgical procedures for TBI, rats were fed
zinc adequate (30 ppm) or zinc supplemented (180 ppm) diets for 4 weeks. We found that the
Porsolt swim test is not an appropriate measure of depression-like behavior in TBI models.
Traumatic brain injury had no significant effect on locomotor behavior in spontaneous
alternation or open field behavior tests. However, TBI did decrease the time spent grooming and
amount of rearing, but zinc supplementation did not show any improvements. Irradiation of the
hippocampus increases anxiety and reduces locomotor activity, which is also uncorrected by zinc
supplementation. Traumatic brain injury impaired novel object recognition performance. Zinc
supplementation did not have any improvements on these impairments. Traumatic brain injury
combined with irradiation of the hippocampus did not cause any further deficits. Therefore,
given that zinc supplementation has been shown to improve hippocampus dependent spatial
learning and memory, we conclude that the action of zinc supplementation in traumatic brain
injury on learning and memory is primarily in the hippocampus. These data further suggest that
zinc supplementation is less effective in improving cortical mechanisms of learning and memory.
Introduction
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Zinc and the Brain
The essential trace element zinc is most highly concentrated in the hippocampus in
glutaminergic neuron rich areas. Zinc is transported to the hippocampus through the blood brain
barrier. However, soma staining that targeted vesicular zinc was not observed following TBI,
which lends evidence that vesicular zinc plays no role in neuronal damage following TBI
(Doering et al, 2007). Furthermore, clinical studies have shown that following traumatic brain
injuries, patients have a much higher risk to develop zinc deficiency with a majority of zinc
being lost in the urine (McClain et al, 1986). This provides reason to look into dietary options
and outcomes. Dietary zinc deprivation may influence zinc homeostasis in the brain, resulting in
brain dysfunction such as learning impairment (Takeda, 2000). Dietary zinc deprivation rarely
causes any decrease of zinc concentration in the brain, unlike in the peripheral tissues. However,
the brain functions are affected by zinc deprivation (Takeda, 2000). It was previously unknown if
dietary zinc levels had any link to the major depressive symptoms so closely related to TBI, but a
study was conducted that found when rats were fed a zinc deficient diet (1 ppm) they displayed
signs of depression such as anorexia, anhedonia, and increased anxiety (Tassabehji et al, 2008).
When zinc supplementation was added to the diet of a sample of rats, a 90% increase in zinc
present in the hippocampus was observed; further linking zinc, hippocampus, and memory
(Sowa-Kucma et al, 2011). Alterations induced by zinc administration in the hippocampus may
be related to specific zinc mechanisms (Sowa-Kucma et al, 2011).
TBI-Effects on Learning and Memory
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An estimated 1.7 million people suffer from a traumatic brain injury (TBI) each year, and
of them 52,000 die and 275,000 are hospitalized. This makes TBI a contributing factor to a third
of all injury related deaths in the United States (CDC, 2015). Patients with severe brain injury
typically present with significant cognitive impairment, especially in the domains of attention
and concentration, psychomotor speed, memory, and executive function, in addition to fatigue
and problems with motivation (Fleminger, 2010). Most of these impairments would fall under
the class of working or short term memory, which is crucial not only to making seemingly basic
decisions like determining if a corridor had been entered before, but also in conversion of
experiences into long term memory such as the ability to identify novel objects in a familiar
setting after a period of time. The capacity of this working memory load was evaluated between
mild traumatic brain injury (MTBI) patients and healthy patients. MTBI patients showed
disproportionately increased activation of working memory circuitry during the moderate
processing load condition, but very little increase in activation associated with the highest
memory processing load condition (McAllister et al, 2001). Task performance did not differ
significantly between groups on any task condition, but MTBI patients showed a different pattern
of allocation of processing resources associated with a high processing load condition compared
to healthy controls, despite similar task performance (McAllister et al, 2001). In a longitudinal
study there was progressive normalization of the working memory activation pattern after diffuse
axonal injury in severe TBI, coinciding with an improvement in performance on this function
(Sanchez-Carrion et al, 2008). This suggests that injury-related changes in ability to activate or
modulate working memory processing resources might underlie some of the memory complaints
after MTBI (McAllister et al, 2001). This work made a good case for explaining what type of
memory is usually affected following a TBI, but did little to explore potential treatments.
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It is likely that the changes in the hippocampus are crucial for predicting the severity of
memory deficits following a TBI. Hippocampal lesions produced a severe and selective
impairment in the capacity of rats to remember the sequential ordering of a series of odors,
despite an intact capacity to recognize odors that recently occurred (Fortin et al, 2002). These
findings support the hypothesis that hippocampal networks mediate associations between
sequential events that constitute elements of an episodic memory (Fortin et al, 2002). Previously,
it had been shown that rats experiencing a mild traumatic brain injury (MTBI) showed little
preference towards a novel object when placed among familiar objects (Munyon et al, 2014). It
was concluded that memory deficits after MTBI are associated with decreased intrinsic burst
activity in cells and impaired context-specific firing patterns in the hippocampus during object
exploration (Munyon et al, 2014).
Most importantly, a recent study looked at the effects of dietary zinc on learning and
memory. Morris Water Maze (MWM) has been correlated to improvements in learning and
memory. According to the study, zinc supplementation prior to injury significantly improved
MWM performance after a frontal cortex TBI and enabled zinc supplemented animals to perform
as well as uninjured sham-operated controls (Cope et al, 2011).
TBI-Effects on Mood
In addition to memory deficits, major depression is a consequence of TBI that affects
nearly 40% of all patients suffering from brain injuries (Jorge and Starkstein, 2005). A variety of
pre-clinical and clinical reports have shown that supplemented zinc has antidepressant activity.
For example, a preliminary clinical report suggested augmentation of antidepressant therapy by
zinc in patients treated for depression (Siwek et al, 2009). Zinc supplementation also
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significantly reduced depression scores and facilitated the treatment outcome in antidepressant
treatment resistant patients (Siwek et al, 2009). Additionally, pre-clinical models show that zinc
increases resilience to TBI-associated depression, making it potentially useful in populations at
risk for injury (Cope et al, 2011). These populations include, but are not limited to, the elderly,
athletes, and military personnel. All of which whom are naturally exposed to high levels of stress
making them even more susceptible to major depression due to traumatic brain injury.
Experimental Questions
1. Previous work showed that zinc supplementation prevented TBI-associated depression
measured by the 2-bottle saccharine preference test (Cope et al, 2011). Therefore, this
work sought to determine if zinc supplementation would also reduce immobility in the
Porsolt swim test after traumatic brain injury.
2. We further hypothesized that zinc supplementation would improve open field behaviors
after traumatic brain injury including thigmotaxic behavior, grooming, and rearing.
3. The hippocampus is enriched in neuronal precursor cells. This work was designed to
begin to test the hypothesis that these cells play a role in the behaviors we test after
traumatic brain injury. This hypothesis was tested after irradiation of the hippocampus to
prevent neuronal precursor proliferation.
4. Previous data show that zinc supplementation prevents deficits in spatial learning and
memory associated with traumatic brain injury. Because we know that spatial learning
and memory is governed by the hippocampus, we designed an experiment to test the
degree to which zinc supplementation would improve learning and memory not directed
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by the hippocampus. To accomplish this, we tested the effect of zinc supplementation on
TBI-associated deficits in a novel object recognition test.
Materials and Methods
Animals
The FSU Animal Care and Use Committee (ACUC) approved all animal experiments.
Young adult male rats (6 weeks of age) were divided into four groups (n=8): zinc adequate
sham-operated controls, zinc adequate TBI rats, zinc supplemented TBI rats, and zinc
supplemented TBI rats with hippocampal irradiation. Prior to surgical procedures for TBI or
irradiation, rats were fed zinc adequate (30 ppm) or zinc supplemented (180 ppm) diets for 4
weeks. During this time, animals were handled a minimum of 3 times/week.
Surgical Procedures
All surgical procedures were conducted aseptically under isofluorane anesthesia,
followed by a 1 cm scalp incision and 6 mm diameter craniotomy. All rats (except the sham
group) then received a TBI administered by a controlled cortical impact as previously described
(Cope et al, 2011). Post-operatively, rats were weighed and monitored for 10 days.
Irradiation
The group of zinc supplemented TBI rats that received hippocampal irradiation were
anesthetized for the duration of exposure to the radiation using 2% isoflurane in oxygen at a flow
rate of 2 liters per minute. The rats were then placed into a stereotaxic frame (David Kopf
Instruments, Tujunga, CA, USA), which is fitted with a custom-built and adjustable lead shield.
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We used coordinates obtained from the rat brain atlas (Paxinos, Watson, 2013) to position the
shield with a 6mm exposure window over the hippocampus. A secondary lead shield was used to
protect the body and tail from collateral x-ray exposure. At this point, each rat was subjected to a
single 10Gy dose of X-ray radiation using an X-Rad 320 self-contained precision x-ray generator
(PXi precision x-ray, North Bradford, CT, USA) that has been previously shown in our lab to
eliminate stem cell proliferation.
Novel Object Recognition
The novel object recognition test consisted of two phases. Each rat first spent a five-
minute period in an enclosed area with two identical objects to allow them to become familiar
with the objects. After a one-hour hiatus, each rat was then returned to the enclosed space, but
one of the original objects was replaced with a novel object. Interactions with the novel and
familiar object were video recorded for 5 minutes. The videos were scored for the amount of
time spent interacting with the familiar object and the amount of time spent interacting with the
novel object.
Spontaneous Alteration
Next, each rat went to the spontaneous alteration test, which was administered in an
eight-arm radial maze. Each rat was placed in the center of the maze and given one minute to
acclimate to the environment, but restricted from entering any arms. Rats were then allowed to
enter all eight arms of the maze at will. Alterations between the 8 arms were video recorded for 5
minutes and later analyzed for patterns in arm selection. The pattern was defined by 4 arm
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segments, and considered repeated if any one arm was present more than once in any block of
four arms given in non-overlapping succession.
Open Field Test
Each rat was placed in the center of a three-foot by five-foot open space that was marked
with a four by six grid pattern. Every rat had five minutes to freely explore the space. The
following recordings were taken: total number of lines crossed, number of lines crossed through
the middle grids, number of lines crossed along the outside edge, number of rearings, and
amount of time spent grooming. Percentages were measured of percent of crosses in the middle
vs. percent of crosses along the outside edge out of total line crosses.
Porsolt Swim Test
The final test was the Porsolt swim test and it consisted of two phases. The first phase
was a ten-minute period spent in a clear cylindrical water tank (3 feet tall and 1 foot in diameter),
which was designed to allow the rats an opportunity to learn how to swim and realize that there
was no escape. After twenty-four hours, each rat was returned to the water tank for five minutes.
Video recordings of the 5-minute test phase were evaluated for the depression-like activity of
immobility. Immobility is defined by minimal efforts of swimming that produce just enough to
keep the nose above water, which is usually accomplished by kicks from the back feet only. The
amount of time spent immobile was recorded and compared to the amount of time spent in the
tank.
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Statistical Analysis
All data were expressed as the mean ± SEM and statistical significance was set at p<0.05
with 95% confidence (Prism; GraphPad, San Diego, CA). All behavioral data was analyzed by
one-way ANOVA with a Newman-Keuls Multiple Comparison Test.
Results
Novel Object Recognition Test
The injured animals spent a majority of their 5-minute period interacting with neither the
familiar or novel object. Injury reduced the amount of time spent with the novel object by over
50% (p<0.05). Zinc supplementation prior to TBI did not increase the amount of time spent with
the novel object (Fig. 1). This behavior was not impaired by irradiation of the hippocampus.
Fig. 1 Effect of zinc supplementation on learning and memory following traumatic brain injury.
Rats were fed a zinc adequate (ZA) or zinc supplemented (ZS) for 4 weeks followed by either a
sham surgery, injury, or injury and irradiation (IRR) of proliferating cells. Sham-operated
controls were fed the ZA control diet. Rats that received irradiation to the hippocampal region
were fed a ZS diet. Novel Object (NO) interactions were measured for 5 min. Bars represent
mean ± SEM. *Significantly different from sham operated rats at p<0.05.
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Memory – Spontaneous Alternation
Following TBI, there were no significant differences in working memory based on the
spontaneous alternation test. ZS rats entered approximately the same number of total arms (Fig.
2A) as the ZA diet rats, which was only slightly more than the sham-operated controls. However,
there was a significant difference when comparing the sham and ZS TBI IRR groups’ alternation
scores. Rats that had newly proliferating cells in the hippocampus eliminated following injury
were unable to methodically enter each arm without repeating a significant number of arms
(p<0.001, Fig. 2B).
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Fig. 2 Effect of zinc supplementation on working memory following traumatic brain injury. Rats
were fed a zinc adequate (ZA) or zinc supplemented (ZS) for 4 weeks followed by either a sham
surgery, injury, or injury and irradiation (IRR) of proliferating cells. Sham-operated controls
were fed the ZA control diet. Rats that received irradiation to the hippocampal region were fed a
ZS diet. Working memory was measured for 5 minutes using an eight-arm radial maze and
quantified by (A) the total number of arms entered and (B) alternation score, which was
equivalent to the number of consecutive novel 4 arm patterns over the total number of arms. Bars
represent mean ± SEM. *Significantly different from sham operated rats at p<0.05.
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Open Field Test
TBI did not significantly alter the total number of lines crossed in the open field test (Fig.
3A). Neither zinc supplementation or irradiation of the hippocampus altered locomotor activity
in this test. Fig. 3B shows that irradiation of the hippocampus caused a 26% decrease in time
spent in the center of the open field as compared to the sham-operated controls, which spent 33%
of their time in the center (p<0.05, Fig. 3B). Open-field testing revealed that zinc
supplementation did not alter the amount of time spent grooming. When comparing the sham-
operated group to the ZS TBI and ZS TBI IRR groups, the injured animals allocated no time to
grooming (p<0.05, Fig. 4). Injury significantly decreased time spent grooming, regardless of ZS,
(p<0.05, Fig. 4) by more than 50% as seen between the sham control and ZA TBI groups. Injury
also caused a significant decrease in number of rearings (p<0.05, Fig. 5).
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Fig. 3 Effect of zinc supplementation on anxiety and depression following traumatic brain injury.
Rats were fed a zinc adequate (ZA) or zinc supplemented (ZS) for 4 weeks followed by either a
sham surgery, injury, or injury and irradiation (IRR) of proliferating cells. Sham-operated
controls were fed the ZA control diet. Rats that received irradiation to the hippocampal region
were fed a ZS diet. Anxiety and depression style behaviors were measured for 5 minutes using
an open field box and quantified by (A) the total number of line crosses and (B) percent of time
spent in center. Bars represent mean ± SEM. *Significantly different from sham operated rats at
p<0.05.
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Fig. 4 Effect of zinc supplementation on anxiety and depression following traumatic brain injury.
Rats were fed a zinc adequate (ZA) or zinc supplemented (ZS) for 4 weeks followed by either a
sham surgery, injury, or injury and irradiation (IRR) of proliferating cells. Sham-operated
controls were fed the ZA control diet. Rats that received irradiation to the hippocampal region
were fed a ZS diet. Anxiety and depression style behaviors were measured for 5 minutes using
an open field box and quantified by the amount of time spent grooming. Bars represent mean ±
SEM. *Significantly different from sham operated rats at p<0.05.
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Fig. 5 Effect of zinc supplementation on anxiety and depression following traumatic brain injury.
Rats were fed a zinc adequate (ZA) or zinc supplemented (ZS) for 4 weeks followed by either a
sham surgery, injury, or injury and irradiation (IRR) of proliferating cells. Sham-operated
controls were fed the ZA control diet. Rats that received irradiation to the hippocampal region
were fed a ZS diet. Anxiety and depression style behaviors were measured for 5 minutes using
an open field box and quantified by the total number of rearings. Bars represent mean ± SEM.
*Significantly different from sham operated rats at p<0.05.
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Porsolt Swim Test
Rats receiving a TBI spent 34%-43% less time immobile (p<0.001, Fig. 6) than sham
controls. Irradiation completely eliminated immobility (Fig. 6). There was no significant
difference between injured animals on ZA or ZS diets.
Fig. 6 Effect of zinc supplementation on anxiety and depression following traumatic brain injury.
Rats were fed a zinc adequate (ZA) or zinc supplemented (ZS) for 4 weeks followed by either a
sham surgery, injury, or injury and irradiation (IRR) of proliferating cells. Sham-operated
controls were fed the ZA control diet. Rats that received irradiation to the hippocampal region
were fed a ZS diet. Anxiety and depression style behaviors were measured for 5 minutes using
the Porsolt Swim Test and quantified by the percent of time spent immobile versus total time
spent in tank. Bars represent mean ± SEM. *Significantly different from sham operated rats at
p<0.05.
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Discussion
TBI-induced depression.
Previous work used a rat model of TBI to show that injury to the frontal cortex results in
the depression-like behavior anhedonia (Cope et al, 2011). This test uses a two-bottle test to
measure the relative intake of saccharin-sweetened water compared to deionized water.
Anhedonia, identified as decreases in saccharin consumption is a depression-like behavior. This
finding is consistent with clinical work showing that depression is the single most common
problem in patients with TBI (Jorge and Starkstein, 2005).
While TBI clearly induces depression in both clinical and pre-clinical studies, 4 weeks of
dietary zinc supplementation prior to the induction of cortical injury in rats completely prevented
the development of TBI-associated anhedonia. The current work sought to determine the extent
to which other measures could be used to evaluate the efficacy of zinc in a rat model. We thus,
chose to use the well-characterized Prosolt swim test. This test, which is employed over a two
day period measures the reduction in immobility as a measure of treatment efficacy, such that
rats that have successfully undergone treatment will reduce the amount of time they spend
immobile in the swim tank over a 5 minute period. Curiously, we found that TBI completely
abolished immobility in the Porsolt swim test. In fact, all groups receiving a TBI (regardless of
treatment) had significant reductions in immobility when compared to the sham-operated group.
These data led to the hypothesis that TBI induces hyperactivity that significantly increases
swimming behavior. Regardless of the mechanism, it is clear from these results that the Porsolt
swim test is not appropriate in this model of TBI, and cannot thus be used to evaluate the
efficacy of zinc supplementation in cortical injury.
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Locomotor Activity.
To test the hypothesis that this model of TBI induces hyperactivity we tested all animals
in an open field. Measurements of line crossing showed that, contrary to our hypothesis, that TBI
did not increase locomotor activity and did not indicate hyperactivity. In fact, less time was spent
by TBI animals in grooming and rearing. Zinc supplementation did not correct either of these
behaviors, suggesting that the effects of zinc on depression-like behaviors may be specific and
include exploratory or motor behaviors. Additionally, the conclusion that this model of TBI does
not induce hyperactivity or increased locomotor activity was confirmed by the finding the total
number of arms explored in the spontaneous alternation test was not changed by TBI. Finally, in
the open field test, animals face a conflict between avoiding an open area and staying to the safer
areas such as by walls and corners in a novel environment (Ahn et al, 2013). There was no
significant difference between the injured and uninjured animals in time spent in the center.
Learning and Memory.
Traumatic brain injury has repeatedly been shown to result in cognitive impairment in
both humans and animal models. For example, it has been previously shown that hippocampal
lesions produced a severe and selective impairment in the capacity of rats to remember the
sequential ordering of a series of odors, despite an intact capacity to recognize odors that recently
occurred (Fortin et al, 2002). Additionally, spatial learning and memory, as assessed by the
Morris Water Maze is impaired by our model of TBI (Cope et al, 2011; Cope et al, 2012).
Zinc supplementation prior to frontal cortex injury improves spatial learning and memory
as demonstrated by Morris Water Maze performance (Cope et al, 2011). Because spatial learning
and memory is dependent on the hippocampus, we hypothesized that chronic zinc
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supplementation prevented memory deficits via hippocampal mechanisms. We tested this
hypothesis with a novel object recognition test that is primarily mediated by the cortex (Wilson
et al, 2013). We show that chronic dietary zinc supplementation before TBI produced marginal
differences between the ZA and ZS TBI experimental groups. In light of this, if spatial learning
and memory is indeed regulated through hippocampal mechanisms, and it is known that novel
object recognition works through the cortex, then the failure of zinc supplementation to improve
novel object recognition provides evidence that zinc supplementation is improving learning and
memory via hippocampal mechanisms. To further test this, we irradiated the hippocampus of all
newly proliferated stem cells. As expected, the irradiated groups did not perform significantly
worse in the novel object recognition task. These data support the hypothesis that novel object
recognition is a test of cortical memory and not hippocampal. If it were hippocampal, then we
would have seen significantly worse if not a completely elimination of performance in novel
object recognition.
Together these data support the conclusion that the beneficial effects of zinc
supplementation in TBI are provided by actions in the hippocampus. Given the large
concentration of zinc in this important region of the brain, future work will be needed to
understand the cellular and molecular mechanisms responsible for the protective effects of zinc
supplementation in cortical injury. Our lab is currently exploring the role of zinc
supplementation in the regulation of hippocampal stem cells after TBI as well as the role of
synaptic zinc after TBI.
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Conclusions
1. Porsolt swim test is not an appropriate measure of depression-like behavior in traumatic
brain injury.
2. Traumatic brain injury does not significantly impair locomotor behavior as measure in
the spontaneous alternation and open field test.
3. Traumatic brain injury decreases time spent grooming and amount of rearing, however,
zinc supplementation did not correct these behaviors.
4. Irradiation of the hippocampus increases anxiety as measured by time spent in
thigmotaxic behavior, and reduces locomotor behavior in spontaneous alternation.
5. Traumatic brain injury decreases novel object recognition, which was measuring
cortically derived learning and memory. Zinc supplementation did not improve these
measures. Traumatic brain injury combined with irradiation of the hippocampus did not
cause any further deficits. Therefore, given that zinc supplementation has been shown to
improve hippocampus dependent spatial learning and memory, we conclude that the
action of zinc supplementation in traumatic brain injury on learning and memory is
primarily in the hippocampus. These data further suggest that zinc supplementation is
less effective in improving cortical mechanisms of learning and memory.
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