Download Enlargement of Axo-Somatic Contacts Formed by

Cerebral Cortex, June 2016;26: 2574–2589
doi: 10.1093/cercor/bhv087
Advance Access Publication Date: 15 May 2015
Original Article
ORIGINAL ARTICLE
Enlargement of Axo-Somatic Contacts Formed by
GAD-Immunoreactive Axon Terminals onto Layer V
Pyramidal Neurons in the Medial Prefrontal Cortex of
Adolescent Female Mice Is Associated with Suppression
of Food Restriction-Evoked Hyperactivity and Resilience
to Activity-Based Anorexia
Yi-Wen Chen, Gauri Satish Wable, Tara Gunkali Chowdhury, and Chiye Aoki
Center for Neural Science, New York University, New York, NY 10003, USA
Address correspondence to Chiye Aoki, Center for Neural Science, New York University, 4 Washington Place, New York, NY 10003, USA. Email: [email protected]
Abstract
Many, but not all, adolescent female mice that are exposed to a running wheel while food restricted (FR) become excessive
wheel runners, choosing to run even during the hours of food availability, to the point of death. This phenomenon is called
activity-based anorexia (ABA). We used electron microscopic immunocytochemistry to ask whether individual differences in ABA
resilience may correlate with the lengths of axo-somatic contacts made by GABAergic axon terminals onto layer 5 pyramidal
neurons (L5P) in the prefrontal cortex. Contact lengths were, on average, 40% greater for the ABA-induced mice, relative to controls.
Correspondingly, the proportion of L5P perikaryal plasma membrane contacted by GABAergic terminals was 45% greater for the
ABA mice. Contact lengths in the anterior cingulate cortex correlated negatively and strongly with the overall wheel activity after
FR (R = −0.87, P < 0.01), whereas those in the prelimbic cortex correlated negatively with wheel running specifically during the
hours of food availability of the FR days (R = −0.84, P < 0.05). These negative correlations support the idea that increases in the
glutamic acid decarboxylase (GAD) terminal contact lengths onto L5P contribute toward ABA resilience through suppression of
wheel running, a behavior that is intrinsically rewarding and helpful for foraging but maladaptive within a cage.
Key words: anxiety, cingulate cortex, electron microscopic immunocytochemistry, exercise, prelimbic cortex
Introduction
Adolescence is a period of peak physical health and mental
capacity, though brain maturation is still incomplete. Brain maturation during adolescence is susceptible to environmental influences (Andersen et al. 2000; Chowdhury et al. 2014) and
many mental illnesses emerge for the first time during adolescence (Spear 2000; Romeo 2010). One mental illness that is
particularly prevalent among adolescent females is anorexia
nervosa (AN), features of which include refusal to maintain normal body weight, fear of weight gain, and dysmorphia (American
Psychiatric Association 2013). Once succumbed to the condition
of AN, relapse is prevalent (30–50%) (Birmingham et al. 2005)
and mortality is extremely high (10–20%) (Sullivan 1995; Zipfel
et al. 2000; Birmingham et al. 2005; Bulik et al. 2007), even
surpassing depression. Yet, there is at present no accepted pharmacological treatment for this condition. Nearly all adolescent
© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: [email protected]
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Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
females experiment with dieting, while only 1% of them succumb
to the condition of AN (Hudson et al. 2007). What is the basis for
the individual differences in vulnerability to AN? This was the
question of our study strived to answer through the use of a
mouse model of AN, namely activity-based anorexia (ABA).
ABA is a condition whereby severe voluntary hyperactivity
and weight loss is induced simply by restricting food access
while providing ad libitum access to a running wheel (Gutierrez
2013). ABA induction captures 3 hallmarks of AN (American
Psychiatric Association 2013): Severe weight loss (Gutierrez
2013), over-exercise (Epling et al. 1983; Davis et al. 1997, 1999;
Hebebrand and Bulik 2011; Zunker et al. 2011; Gutierrez 2013),
and anxiety (Kaye et al. 2004; Perdereau et al. 2008; Dellava
et al. 2010; Thornton et al. 2011; Wable et al. 2015). Importantly,
although ABA is induced by the initial imposition of food restriction (FR), once animals have begun to lose body weight, their voluntary wheel activity becomes so excessive that animals choose
wheel running over eating, even during the limited hours of food
access. In this way, ABA-induced animals exhibit one additional
striking hallmark of the human condition of AN—namely, voluntary FR. When applied to adolescent C57BL6 female mice, this
animal model also reveals individual differences in vulnerability,
with approximately half of the animals exhibiting severe hyperactivity and weight loss that would be lethal unless removed
from the ABA-inducing environment, while approximately a
quarter of the population exhibits resilience, due to minimal
hyperactivity and weight loss (Chowdhury et al. 2013). Having
identified a cohort with variable degrees of vulnerability to ABA
induction, our next goal has been to identify the cellular bases
for the individual differences in ABA vulnerability.
It has recently been demonstrated convincingly that wheel
running is not an abnormal behavior induced by rearing in captivity but is highly rewarding, even to mice in wilderness (Meijer
and Robbers 2014). A number of ideas have been proposed to explain why restricted food access evokes heightened activity on
the wheel. One idea is that wheel running is in response to hypothermia, since FR-evoked hyperactivity can be reversed through
high ambient temperature (Gutierrez et al. 2006). Another is
that wheel running is anxiolytic (Hare et al. 2012), thus being perpetuated by individuals that are experiencing anxiety due to the
stress of FR. Wheel running may also be an attempt at food foraging [discussed by Gutierrez (2013)]. However, when occurring
within a closed environment, wheel running is detrimental, because it exacerbates weight loss (Chowdhury et al. 2013) without
any opportunity of increasing food access. Individual differences
in ABA vulnerability could arise, at least in part, from differences
in the animals’ neural circuitry underlying the decision to suppress wheel running, after undergoing an experience where
wheel running is not suitable for survival. This idea is based on
our observation that the resilient animals are those that reduce
wheel running, thereby minimizing body weight loss and extending survival, while those animals that exhibit the greatest ABA
vulnerability, based on the severity of weight loss, are the ones
that continue to respond to FR with excessive running on the
wheel (Chowdhury et al. 2013). In search for differences in neural
circuitry underlying the decision to suppress wheel running, we
have turned our attention to the prefrontal cortex (PFC).
Our rationale for analyzing the PFC stems from human data
(Kaye et al. 2009). Among individuals with AN, presentations of
visual stimuli depicting physical activity result in an exaggerated
response of the PFC: The authors suggest that visual stimuli in
the form of physical activity may be more rewarding, thereby placing increased demand on the inhibitory control system of AN
patients when challenged with a Go/NoGo task (Kullmann et al.
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2014). Another study reported that AN patients’ performance of
a PFC cognitive task, the Wisconsin Card Sorting Test, was significantly poorer than that of healthy controls (Sato et al. 2013).
There is also an intriguing case study, indicating that symptoms
of AN were relieved in a male after glioma in the frontal lobe was
removed surgically (Goddard et al. 2013). Not only is activation of
the PFC of individuals with AN consistently altered, but also in the
rat model of ABA, the PFC has been shown to be hypometabolic by
micro-positron emission tomography (PET; van Kuyck et al. 2007).
These studies suggest that altered activity of the PFC may contribute to the behavioral phenotype of AN among humans and of ABA
among rodents, thereby ultimately affecting individuals’ decisions
to exercise or to eat.
Excitability of the PFC is determined, in part, by the intracortical circuitry comprised of excitatory pyramidal neurons and inhibitory interneurons. Of them, the layer V pyramidal neurons
contribute strongly to the excitatory outflow from the PFC to descending cortical and subcortical regions mediating motor output
(Berendse et al. 1992; Gabbott et al. 2005; Riga et al. 2014). Furthermore, while excitability of the layer V pyramidal neurons in
the PFC is controlled by the balance of their excitatory and inhibitory inputs, individual gamma-aminobutyric acid (GABA)ergic
axo-somatic inhibitory synapses are more influential than the
GABAergic axo-dendritic synapses or the numerous but more
distally located excitatory axo-spinous synapses. Moreover, parvalbumin (PV) neurons in the PFC, which exert the axo-somatic
inhibition, have been shown to fire as animals make decisions to
leave a reward (Kvitsiani et al. 2013). This indicates that GABAergic
axo-somatic inhibition of pyramidal neurons within the PFC could
play a particularly central role in an animal’s choice-related behaviors, such as to eat food or to run on the wheel. Thus, among
the many types of synapses that exist within the PFC, we chose
to assess the extent of axo-somatic inhibitory contacts formed
by GABAergic axon terminals onto layer V pyramidal neurons.
We chose to take an electron microscopic approach for 2 reasons: First, we wished to be able to study a well-defined single
class of neurons targeted by GABAergic innervation, in case environmentally and experientially evoked changes in GABAergic
contacts differed depending on the postsynaptic neuronal type
( pyramidal vs. interneuronal; layer II/III vs. layer V vs. layer VI
pyramidal cells). Electron microscopy (EM) allows for differentiation of cell bodies belonging to pyramidal cells versus interneurons (Peters and Jones 1984; White and Keller 1989). Moreover, we
wished to be able to use the resolving capability of EM for differentiating those contacts that are direct versus others that are indirect, due to the interposition of thin (<30 nm, below the limit of
resolution of light microscopes) astrocytic processes in between
GABAergic axon terminals and perikaryal plasma membranes
that can reduce the activation of GABAergic receptors [review in
Zhou and Danbolt (2013)].
The source of PFC tissue for this analysis was derived, in part,
from an earlier published study that examined GABAergic innervation of pyramidal cells in the hippocampus of adolescent
female C57BL6 mice exhibiting variable degrees of hyperactivity
following 2 ABA inductions during adolescence, spaced 1 week
apart (Chowdhury et al. 2013).
Materials and Methods
Animals and ABA Induction
Fourteen female mice, 7 ABA animals, and 7 controls, from 4 litters, were used in this study. All animals used in this study were
bred in New York University’s animal facility. All but one of the
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PFC tissue used for this study came from brains used for another
anatomical study on the hippocampus (Chowdhury et al. 2013).
Two breeding pairs were wild-type C57BL/6J mice obtained
from Jackson Laboratories. Two other breeding pairs were also
obtained from Jackson Laboratories (Bar Harbor, ME, USA) on a
C57BL6/J background but crossed with BALB/c strains: CB6-Tg
(Gad1-EGFP)G42Zjh/J (G42), which express green fluorescent protein (GFP) in PV-containing interneurons (Chattopadhyaya et al.
2004). GFP label was not utilized as a marker for the electron
microscopic study described here. Details of these animals with
GFP expression are described in a previous publication from this
laboratory: in short, these mice exhibited behavioral and glutamic acid decarboxylase (GAD) expression phenotypes that were no
different from wild types (Chowdhury et al. 2013).
After weaning at postnatal day 25 (P25), the animals were
group-housed 2–4 per cage with their littermates, separated
from males. Starting on P36, the animals were assigned into 2
groups: ABA animals and controls. Control (CON) animals were
singly housed in standard home cages with ad libitum access
to food and no running wheel access for the duration of this
study. ABA animals were housed individually with unrestricted
food and water access. Starting on P36, ABA animals were given
24-h/day access to a running wheel (Med Associates Low-Profile
Wireless Running Wheel for Mouse Product: ENV-044), and
their wheel-running activity was recorded continuously on a
PC, using Med Associates’ “Wheel Manager” software (Product:
SOF-860). In addition, wheel running during the hours 7 PM to
9 PM and the 24-h running record (from 7 PM to 7 PM) was manually recorded, together with the animals’ weight and food intake.
The cages were kept under a 12-h light/dark cycle (lights on at 7 AM;
lights off at 7 PM). The daily measurements of animals’ body
weight, food consumption, and running wheel activity for 3 days
served as baseline data under the singly housed condition, to be
compared against the additional impact of FR that began on P40.
On P40, food was removed at 12 PM. ABA mice were only allowed
free access to food for the first 2 h of the dark cycle (7 PM–9 PM)
for 3 days (first ABA). After 3 days of exposure to ABA, starting at
12 PM, ad libitum food access was restored and the running
wheel was removed from the cage. The animals’ body weights returned to baseline levels within 48 h. After 7 days of 24-h/day access
to food, and without wheel access, animals were placed in a cage
with a running wheel, so as to reassess the running wheel activity
in the absence of FR. After reacclimation to the running wheel
for 4 days, ABA animals were returned to the ABA environment
(second ABA), whereby they were given restricted food access for
2 h, at the beginning of the dark cycle, in the presence of a running
wheel. The second ABA continued for 4 days.
The animals’ estrous cycles were not monitored, because
cycles are already known to become disrupted by FR (Nelson
1985; Dixon et al. 2003) and because pubertal animals are too immature to be cycling regularly (Hodes and Shors 2005). We also
reasoned that vaginal smears, needed for assessing the estrous
cycle, is a stressful procedure that could exacerbate or mask the
effect of stress associated with FR.
All procedures relating to the use of animals were according to
the NIH Guide for the Care and Use of Laboratory Animals and
also approved by the Institutional Animal Care and Use Committee of New York University (A3317-01).
Preparation of Brains
ABA animals were euthanized at the end of the second ABA, at
ages P59 or P60, by anesthetizing them with urethane (i.p. 34%;
0.15 mL/20 g body weight) during the hours of 12 PM to 4 PM,
then transcardially perfusing them with a buffer containing 4%
paraformaldehyde. Glutaraldehyde fixation was withheld until
after immunocytochemistry, so as to optimize antigen retention.
Brains were also collected from CON mice at ages P59 or P60.
The GAD Immunocytochemistry Procedure
Details of the GAD immunocytochemical procedure are similar to
the steps described in our previous publication (Chowdhury et al.
2013). Tissues from all animals were processed for immunocytochemistry and EM synchronously, so as to minimize variabilities
arising from differences in reagent qualities, incubation time,
ambient temperature, etc. The rabbit anti-GAD antibody used
in this study (Lot 2069924 of Millipore, Bellerica, MA, USA; catalog
number AB11; hereafter referred to as anti-GAD) recognizes both
the GAD65 and GAD67 isoforms. Multiple vibratome sections
from each animal’s brain, prepared in the coronal plane at a
thickness of 40 μm and containing 3 regions [cingulate cortex,
area 1 (Cg1), prelimbic cortex (PrL), and medial orbital cortex
(MO)] of the PFC (Paxinos and Franklin 2001; Fig. 1), were incubated overnight, at room temperature, under constant agitation,
in a buffer consisting of 0.01 M phosphate buffer/0.9% sodium
chloride (PBS), adjusted to pH 7.4, together with 1% bovine
serum albumin (BSA, w/v, from Sigma Chem., St Louis, MO,
USA), 0.05% sodium azide (w/v, from Sigma Chem.), and the
GAD antibody at a dilution of 1 : 400. At the end of the incubation
period, sections were rinsed in PBS over a 30-min period, and
then incubated for 1 h at room temperature under constant agitation in the PBS/BSA/azide buffer containing biotinylated goat
anti-rabbit IgG (Vector, Inc., Burlingam, CA, USA) at a dilution of
1 : 200. At the end of the incubation period, the sections were
rinsed in PBS over a 30-min period and then incubated in PBS containing the avidin-biotinylated horseradish peroxidase complex
(HRP) (ABC, from Vector’s ABC Elite kit) for 30 min at room temperature, under constant agitation. The sections were then rinsed
in PBS and reacted with the HRP substrate, consisting of 3,3′diaminobenzidine hydrochloride (DAB, 10 mg tablets from
Sigma Chem.) per 44 mL of PBS buffer and hydrogen peroxide
(4 μL of 30% hydrogen peroxide per 44 mL of DAB solution). The
HRP reaction was begun by adding hydrogen peroxidase, and terminated at the end of 11 min by rinsing repeatedly in PBS. Vibratome sections underwent post-fixation using 2% glutaraldehyde
for 16 min (EM Sciences, Martifield, PA, USA) in PBS.
Tissue Processing for EM
Following the immunocytochemical procedure, the vibratome
sections were processed by a conventional electron microscopic
procedure, consisting of post-fixation by immersing in 1%
osmium tetroxide/0.1 M phosphate buffer for 1 h, then dehydrated
using graded concentrations of alcohol up to 70%, and then postfixed overnight using 1% uranyl acetate (Terzakis 1968; Lozsa 1974)
dissolved in 70% ethanol. On the following day, the sections were
further dehydrated using 90% ethanol, followed by 100% ethanol,
then were rinsed in acetone, and infiltrated in EPON 812 (EM
Sciences), which was cured by heating the tissue at 60 °C, while
sandwiched between 2 sheets of Aclar plastic, with lead weights
placed on top of the Aclar sheets, so as to ensure flatness of the
EPON-embedded sections. These flat-embedded vibratome sections were re-embedded in BEEM® capsules (EM Sciences) filled
with EPON 812, then ultrathin-sectioned at a plane tangential to
the vibratome sections. The ultrathin sections were collected
onto formvar-coated, 400 mesh thin-bar nickel grids (EM
Sciences). Ultrathin sections were counterstained with Reynolds’
lead citrate before viewing.
Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
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Figure 1. Description of the sampling procedure for quantification of GAD terminal contact sizes onto layer V pyramidal neurons of the PFC. EM was used to verify direct
contact between GAD axon terminals and pyramidal cell bodies. (A) Mouse brain atlas highlighting the 3 regions of interest in the PFC, consisting of the cingulate cortex
area 1 (Cg1), prelimbic cortex (PrL), and medial orbital cortex (MO). Anterior–posterior position (from bregma) of a schematic of a mouse brain’s coronal section is indicated
below the atlas, but the actual sampling ranged from 2.34 to 1.98 mm. Each trapezoid represents the position of the ultrathin section prepared for electron microscopic
analysis. The atlas was modified from figures of Paxinos and Franklin’s atlas (Paxinos and Franklin 2001). Calibration bars on the left = 1 mm. (B) An illustration of the
procedure we followed to quantify the lengths of direct contacts made by GAD-immunoreactive axons along the perikaryal plasma membrane. The blue lines
represent the total membrane length of a profile identified to be belonging to a pyramidal neuron. The purple lines represent the contact lengths of GAD axon
terminals. The red polygons represent the cross-sectional area of GAD axon terminals forming contacts. (C) This example shows 6 GAD axon terminals forming direct
contacts onto a cell body, taken from MO of the PFC of a CON animal. The smooth contour of the nuclear membrane and absence of asymmetric excitatory synapses
indicates that this is a cell body of a pyramidal neuron. The white asterisks (to the right of contact 6; below contact 6; and below contact 2) are GAD axon profiles that
are near but not directly contacting the cell body plasma membrane. The 2 gray arrows below contact 2 are examples of axon terminals with levels of HRP-DAB
reactivity that are indistinguishable from background labeling. To maintain consistency, only those terminals with HRP-DAB labeling that were detectably more
electron dense than those of mitochondria were considered immunolabeled, and included in the quantitative analysis. Calibration bar = 2 μm. (D) Contact 3 is shown
at a higher magnification of 60 000× to reveal synaptic specialization (synaptic junctions, between the 2 arrows), indicated by the clustering of vesicles and parallel
alignment of plasma membranes of the axon terminal and of the cell body. The arrowheads indicate the lateral extents of the contact, which includes the flanking
regions that lack clearly parallel alignment of the 2 plasma membranes. Ast, astrocytic profile in (D), which often flanks synaptic junctions. Calibration bar = 500 nm.
The Sampling Procedure
Two steps were taken to maintain random, unbiased sampling.
One, the electron microscopist sampling the ultrathin section
was blinded to the animal’s ante mortem behavioral characteristics and treatments. Two, for each cortical area of each animal,
somatic profiles were analyzed, strictly in the order that they
were encountered from the surface-most portions of vibratome
sections spanning layer V. Cell bodies of pyramidal neurons
were identified as being positioned in layer V, if they were located
midway between pial surface and the white matter. Care was
taken to never re-sample an area, so as to avoid analysis of overlapping sectors of perikaryal plasma membrane.
The 3 PFC regions of interest that were sampled and analyzed
separately were Cg1, PrL, and MO. The anatomical landmarks
of white matter, sulci, overall shape of the coronal section, cell
density, and all other features shown in Figure 1A were used to
determine the AP levels and boundaries of the subregions of
the PFC. We sampled the neuropil surrounding cell bodies of
pyramidal cells located in layer V of the PFC.
Statistical analysis did not reveal any difference in the proportion of perikaryal plasma membrane contacted by GAD terminals
or of the individual lengths of contact formed by GAD terminals
onto cell bodies of layer V pyramidal neurons across the AP levels.
There is also no behavioral literature pointing to differences in function across the AP levels. Therefore, data from all AP levels were
combined for each subregion of the PFC (Fig. 1A) of each animal.
Sampling of the axon terminals contacting cell bodies of
layer V pyramidal neurons in prefrontal areas was designed to
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optimize immunodetection. To achieve this, for each region of
interest, multiple ultrathin sections revealing the surface-most
portions of vibratome sections for that region were chosen. Five
to 10 somata were sampled from each subregion of the PFC of
each animal. Since there were 3 subregions of the PFC analyzed
for each animal, the total number of somata sampled per animal
was 15–30. The number of ultrathin sections collected from any
one animal’s brain section depended on the natural curvature
that the vibratome section took on, even while cured under
lead weights. Usually, 5–10 ultrathin sections needed to be collected from each vibratome section, so as to reach the target of
sampling from the 3 subregions of the PFC.
Quantification of Axo-Somatic Contact Sizes Made by GAD Axon
Terminals and the Proportion of Perikaryal Plasma Membranes
Contacted by GAD Axon Terminals
Somatic profiles were identified using standard morphological criteria (Peters et al. 1991). Somatic profiles were further identified to
be of pyramidal cells and not GABAergic, based on the absence of
deep indentations along the nuclear membranes, homogeneous
distribution of chromatin within the nucleoplasm, and the absence of GAD-immunoreactivity in the cytoplasm (Peters and
Jones 1984; White and Keller 1989). Contacts formed by GADimmunoreactive axon terminals (heretofore referred to as “GAD
terminal contacts,” for short) were considered to be synaptic
when they exhibited >5 vesicles within the profile, a parallel alignment between the plasma membrane of the axon terminal and of
the soma and thin or complete absence of postsynaptic density.
Essentially, these are the features associated with symmetric
synapses (Peters and Jones 1984). Typically, symmetric synaptic
junctions were flanked by plasma membranes lacking the preto-postsynaptic parallel alignment but with the 2 plasma membranes still juxtaposed directly against one another. These nonjunctional flanking regions were included in the quantification
of GAD terminal contact lengths (see below for further details).
Digital electron microscopic images were captured from ultrathin sections by using a 1.2-megapixel Hamamatsu CCD camera
from AMT (Boston, MA, USA) from the JEOL 1200 XL electron
microscope (JEOL Ltd, Tokyo, Japan). The extent of contact
formed by GAD terminals upon layer V pyramidal cells of the
Cg1, PrL, and MO regions of the medial PFC (Fig. 1A) was quantified using the segmented line tool of NIH’s software, Image J
(version 1.46r), to measure plasma membrane lengths of the
neuronal profiles within digital images of the ultrathin sections.
After determining the total perikaryal plasma membrane length
of a profile identified to be belonging to a pyramidal neuron, the
proportion (in units of percent) of the plasma membrane
contacted by GAD terminals was determined by retracing those
portions of the plasma membrane directly opposite to the HRPDAB-labeled axon terminals. Portions of GAD-immunoreactive
axon terminals lacking the parallel alignment to the plasma
membrane of the cell bodies but still directly juxtaposed were included in our measurements of GAD terminal contacts (Figs 1–3).
So as to optimize consistency in discriminating immunolabeled
from unlabeled or background labeling, axon terminals containing faint HRP-DAB-like density in the cytoplasm that was less
than the electron density of mitochondria were categorized as
below threshold for definitively positive immunoreactivity and
not included in the quantification (Fig. 1C). Conversely, some of
the profiles contacting cell bodies were so densely filled with
HRP reaction products that vesicles could not be visualized easily.
However, inspection of the same profiles recurring in adjacent ultrathin sections more removed from the surface of vibratome
section surfaces verified that these did contain vesicles (Fig. 3),
thereby indicating that these intensely darkened profiles were
GAD-immunoreactive axon terminals that could be included in
the quantification.
The size of GAD terminals forming contacts with layer V pyramidal neurons’ cell bodies was assessed in 2 ways: The length of
contact, using the measurement described above, and cross-sectional areas of GAD terminal cytoplasm at the site forming direct
contacts with the cell bodies (Fig. 1B). GAD terminal area was
measured using Image J’s polygon tool.
The group mean values of the proportion of layer V pyramidal
cells’ perikaryal plasma membrane contacted by GAD terminals,
plasma membrane lengths contacted by GAD terminals, and the
cytoplasmic areas of the GAD terminals at the site of axo-somatic
contact were compared across the ABA versus CON groups. The
group mean values were compared for each of the 3 PFC subregions separately and after pooling across the areas.
Statistical Analyses
Statistical analysis was done by SPSS statistical software 22.0
(IBM Corp., Armonk, NY, USA) and GraphPad Prism version 6.0
(GraphPad Software, San Diego, CA, USA). Data were verified to
be of normal distribution by using the Kolmogorov–Smirnov,
the Lilliefors, and Shapiro–Wilk’s W tests. All variables were
found to be normally distributed. For comparison of 2 groups,
Student’s t-test was used to determine significance of the difference. For comparison of 3 groups, an one-way repeated-measures ANOVA was conducted, followed by Bonferroni’s post hoc
analysis. Pearson correlation was conducted between behavioral
activities and GAD-immunoreactive measurements. P-values of
<0.05 were considered statistically significant.
Results
Variability in Wheel Activity during the First
and Second ABA
Seven female C57BL6J mice, derived from 3 litters, were subjected
to 2 sessions of ABA inductions. The ABA inductions were separated by a week of recovery, consisting of ad libitum food access
for 24 h and no wheel access. Both ABA inductions occurred during adolescence (P36–P60).
All 7 mice exhibited hyperactivity within 24 h after the
onset of FR, relative to the pre-FR baseline activity, and all but
one were hyperactive within the first 6 h of FR. The average of
the 7 animals’ wheel activity (in km/day) during the 3 days of
FR of the first ABA induction increased by 68%, relative to the
baseline activity that preceded FR and this increase was significant (18.7 ± 1.68 km/day during first ABA; 11.14 ± 1.55 km/day
during baseline; F2,12 = 5.08, P < 0.05, by one-way repeated-measures ANOVA; P < 0.05, Bonferroni’s post hoc test, comparing
first ABA with baseline).
Notably, although all animals exhibited FR-evoked hyperactivity, their degrees of hyperactivity were variable. Two of the
7 animals exhibited only a minimal increase in activity following
the first ABA (<3.5 km/day increase, Animals 3 and 4), whereas
the remaining animals exhibited an increase in activity by at
least 7 km/day (Animals 5–8 and 11; Fig. 4A). The time that animals dwelled on the wheel correlated strongly with the distance
that animals ran on the wheel (R = 0.9926, P < 0.0001; Fig. 4B), indicating that individual differences in wheel-running distance
arose mostly from the individual differences in the “preference”
of using the running wheel, rather than the speed with which
they ran on the wheel.
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Figure 2. GAD axon terminals forming direct contacts onto a cell body of the PrL of the PFC of an ABA animal are shown. Same criteria used for the CON cell bodies were
applied to verify that this cell body belongs to a pyramidal neuron (E). Five white asterisks (near contact 3, in between contacts 4 and 5, to the right of contact 1, and 2 in
between contacts 7 and 8) are GAD axons that are near but not directly contacting the perikaryal plasma membrane. The 8 direct GAD terminal contacts onto the cell body,
indicated by arrows, are shown in higher magnification in (A–D) and (F–I). Arrowheads indicate the lateral borders used to measure GAD terminal contact lengths. Arrows
(A and D) indicate the lateral borders identifiable as synaptic junctions, based on the parallel alignment of the plasma membranes of the axon terminal and of the cell
body. The contacts showing only one or the other of the salient features of synaptic junctions could be verified to be synaptic by viewing adjacent ultrathin sections,
examples of which are shown in Figure 3. Sometimes, an endoplasmic reticulum saccule abutted the intracellular surface of the plasma membrane of the contact site
(C). ER, endoplasmic reticulum; Ast, astrocytic profile. Calibration bar = 2 μm in (E); 500 nm in (I), and applies to all other panels as well, except for E.
Animals restored their body weight within the first 48 h of the
recovery period, as was reported earlier (Chowdhury et al. 2013).
Greater variability in wheel activity was observed following exposure to the second ABA induction. One of the animals (Animal 3) exhibited strong resistance to the second ABA exposure, in that its
activity level after FR was less than the level measured during
the initial acclimation to the wheel. Two animals exhibited only
a minimal increase in activity following the second ABA (≤5 km/
day increase, relative to the running during the baseline; Animals
4, 6, and 7). The remaining animals exhibited an increase in activity by at least 7 km/day (Animals 5, 8, and 11; Fig. 4A). These individual differences were apparent within the first 12 h of the
second ABA. Owing to this individual variability in the FR-evoked
hyperactivity, there was no statistically significant difference in
the group mean average of wheel running during the second exposure to ABA, relative to the pre-FR baseline activity (17.81 ± 2.49
km/day during second ABA; 11.14 ± 1.55 km/day during baseline;
P = 0.296).
Thus, wheel analysis confirmed that all 7 adolescent female
mice exhibited FR-evoked hyperactivity but that the extent of
this hyperactivity was variable, especially during the second
ABA induction. Each animal’s average hyperactivity evoked during the first and second ABA inductions relative to the baseline
activity was ranked and shown in Figure 4A. Using this measure
to rank ABA resilience, Animals 3, 4, and 6 were the most resilient
among the 7 mice.
As was reported earlier by others (Gelegen et al. 2008; Gutierrez 2013) and us (Chowdhury et al. 2013), a more detailed analysis
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Figure 3. Ultrastructure of synaptic profiles re-captured in an adjacent ultrathin section. Contacts 3 (Panel A) and 1 (Panel B), captured from an ultrathin section adjacent to
the one shown in Figure 2, verified that these contacts are associated with synaptic specializations, based on the clearer visualization of synaptic vesicles, due to the
lightened DAB reaction product intensity (contact 3), and of the more clearly parallel alignment of the plasma membranes of the axon terminals and of cell body
(arrows, contacts 3 and 1). The portions of the plasma membrane with synaptic specializations contrast the longer contact lengths (arrowheads at the lateral-most
borders) that include plasma membranes lacking the definitively parallel alignments (arrowheads). Calibration bar = 500 nm applies to both panels.
of the animals’ wheel running revealed that once food access becomes limited to certain hours of the day, animals begin to exhibit particularly high wheel running during the 6-h bin that
immediately precedes feeding, referred to as the food anticipatory activity (FAA). The remaining 18 h also contribute significantly, albeit less, to the overall increase in wheel running that
is evoked by FR (Chowdhury et al. 2013). Analysis of this cohort’s
wheel running indicated that these animals also exhibited FAA
during the 6-h preceding feeding (Fig. 4C). The 4 animals exhibiting the greatest vulnerability (7, 5, 8, and 11, ranked in the order of
ABA severity) exhibited strong correlation between FAA running
and their 24 h activity during the first ABA induction (R = 0.9524;
P < 0.05) as well as a marginal correlation with their total wheel
activity, spanning from the baseline days to the end of the second
ABA (R = 0.8771; P = 0.12).
To determine whether animals would reduce the running behavior to gain more food during the period of limited food access
(7 PM to 9 PM) under FR, the wheel running activity during 7 PM to
9 PM in baseline, first ABA, re-acclimation, and second ABA were
measured. No difference in the ABA group mean values of wheel
running was found in first ABA and second ABA, compared with
baseline (Fig. 4D). This indicates that animals voluntarily chose to
run on the wheel, rather than eat, even during the hours of food
availability, after they had become entrained to the restrictive
feeding schedule and had lost, on average, 18% of their body
weight.
If the emergence of increased wheel activity during the 6-h
preceding feeding is a reflection of animals’ entrainment to the
limited hours of food access, then those animals that exhibit
heightened FAA might also be the ones to maximize feeding by
suppressing wheel running during the hours of 7 PM to 9 PM,
when food is available. However, no correlation was found
between individual animals’ FAA and wheel activity during
food access (R = 0.22; P = 0.63).
GAD Terminal Contacts onto Cell Bodies of Layer V
Pyramidal Neurons in the PFC of Animals Following ABA
We assessed the extent of axo-somatic contacts formed by GAD
terminals onto layer V pyramidal neurons by quantifying the proportion of the perikaryal plasma membrane contacted directly by
axon terminals immunoreactive for GAD, the rate-limiting enzyme for the synthesis of GABA (Fig. 1B). These GAD-immunoreactive axon terminals formed only symmetric synaptic
junctions, characterized by the parallel alignment of the pre- to
postsynaptic plasma membranes and vesicles on the presynaptic
side (Figs 1–3). With the use of EM, these contacts were verified to
be direct, that is, not interposed by astrocytic or other axonal processes that can be as fine as 30 nm in diameter (Figs 1 and 2).
Symmetric synaptic specializations (synaptic junctions) were
typically flanked by plasma membranes that lacked the clearly
parallel pre-to-postsynaptic alignment, but nevertheless maintained direct contact. Measurements of the GAD terminal contacts included these non-parallel flanking portions, so long as
the 2 plasma membranes were not interposed by astrocytic processes (Figs 1–3).
EM revealed that in all 3 regions of the PFC of CON animals,
GAD terminals contacted 11.9 ± 0.95% of the cell body plasma
membrane lengths. In contrast, the extent of contacts formed
by GAD terminals onto cell bodies of the ABA animals was 17.3 ±
0.64%, reflecting a 45% increase, relative to the CON values [t (12) =
4.762, P < 0.05]. This difference was significant (P < 0.05), as were
differences when comparing each PFC area separately (Fig. 5A,
all P < 0.05). This difference, in percentage, could have arisen
due to changes in the presynaptic GAD terminals or the postsynaptic perikaryal plasma membranes. The mean lengths of
the ultrastructurally analyzed perikaryal plasma membranes
were 26710.4 ± 1080.45 nm for the ABA group and this was not
significantly different from the values of the CON groups (27466.8
± 1544.15 nm), whether pooled across the 3 groups or analyzed
separately for each PFC area (Fig. 5B, P > 0.05 for all), thereby
pointing to the change being presynaptic.
Differences in the proportion of plasma membrane covered by
GAD terminals across the groups could have arisen due to differences in GAD terminal contact sizes and/or the frequency of GAD
terminals contacting the somata. The mean number of GAD
terminals encountered along the perikaryal plasma membrane
was 4.07 ± 0.18 for the PFC of the ABA group and this was not significantly different from the values for the CON group (3.47 ±
0.22), whether analyzed across the 3 regions together (P > 0.05)
or separately (P > 0.05; Fig. 4). The possibility that percentage
differences in the lengths of contact formed by GAD terminals
Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
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Figure 4. Individual differences in vulnerability to ABA. (A) Mice varied in their baseline wheel activity, but all exhibited increased activity in response to the first ABA. The
y-axis depicts the average wheel activity in km per day across 2 days, while each group of 3 bars depicts the wheel activity exhibited by an individual animal. The bar graphs
are presented in an order of increasing vulnerability to ABA, from left to right. The average of increases wheel running during the first and second ABA, relative to baseline,
is indicated as the white star on each gray bar. The degree of change in activity following FR, compared with baseline, fell into 2 groups, with some exhibiting an increase
that was >7 km per day on average between the first ABA and baseline (animals 7, 5, 8, and 11), and the remaining animals exhibiting an increase that was modest (<4 km
per day). (B) The total time that animals dwelled on the wheel was positively correlated with the total distance that animals ran on the wheel. Correlation reveals
significant effect at 0.0001 level (two-tailed). (C) The wheel running during FAA (1 to 7 PM) and during the hours other than the FAA was increased significantly after
FR. Lines connect the same animal before and after FR. Bar graphs show mean values. *P < 0.05. (D) To determine whether wheel running was changed during the
hours of food access (7–9 PM) after FR, the wheel running activity during food access of baseline, first ABA, recovery, and second ABA were measured. No difference
was found after FR. Lines connect the same animal before and after FR.
onto cell bodies reflected differences in GAD terminal contact
sizes was tested by analyzing the mean lengths and cross-sectional areas of the boutons formed by GAD terminals directly
contacting the somatic plasma membrane of layer V pyramidal
cells. The mean value of individual GAD terminal contact profile
lengths was 4381.8 ± 154.9 nm for the ABA group, representing a
40% enlargement compared with the value from the CON tissue
(3125.6 ± 216.9 nm). Each PFC area also exhibited significant difference between ABA and CON groups (Fig. 5C, all P < 0.05). Correspondingly, the mean cross-sectional area of GAD boutons
forming contacts onto cell bodies of pyramidal cells was 1 816
382 ± 70886.55 nm2 for the ABA group, representing a >60% enlargement compared with the value from the CON tissue (1 137
852 ± 128575.4 nm2). The group difference was also significant
for each of the 3 PFC regions that were analyzed separately
(Fig. 5D).
This difference in GAD contact terminal lengths and areas at
sites of contact with cell bodies of layer V pyramidal neurons
indicates that enlargement of GAD terminals is likely to have
contributed most significantly to the differences in the proportion of the perikaryal plasma membrane receiving GAD terminal
contact.
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Figure 5. Quantification of GAD-immunoreactive axon terminals contacting cell bodies of layer V pyramidal neurons in PFC areas following ABA. (A) Quantification of the
proportion of somatic plasma membrane contacted by GAD-immunoreactive axon terminals in 3 different regions of prefrontal areas—Cg1 (cingulate cortex, area 1), PrL
( prelimbic cortex), and MO (medial orbital cortex). Comparisons of the somatic profiles revealed that the plasma membrane from the ABA animals were more extensively
covered by GAD-immunoreactive axon terminals than were the profiles from brains of CON animals across all 3 regions (n = 5–7 for each group, representing the number of
animals). (B) The mean lengths of the ultrastructurally analyzed perikaryal plasma membranes in 3 regions. No difference was found between the values of the CON
groups and ABA animals. (C) GAD terminal lengths at sites of contact with the somatic plasma membrane of layer V pyramidal neurons. The same profiles analyzed
for (A) were re-analyzed to determine the mean lengths of GAD terminals forming contact with the somatic plasma membrane of pyramidal cells in prefrontal areas.
Statistically significant enlargement of GAD terminal contacts onto somatic membrane of the ABA, relative to the CON tissue, was detected in all 3 regions of
prefrontal areas. Graph represents mean + SEM values. *P < 0.05. (D) GAD terminal areas at sites of contact with the somatic plasma membrane of layer V pyramidal
neurons. Statistically significant enlargement of GAD terminal area of the ABA, relative to the CON tissue, was detected in all 3 regions of prefrontal areas. Graph
represents mean + SEM values. *P < 0.05.
Correlation between the Running Wheel Activity
and GAD Terminals’ Axo-Somatic Contact
Lengths on Layer V Pyramidal Neurons
Previous studies indicated that the anterior cingulate cortex (ACg)
(Cg1), located at the dorsal-most region of the PFC, is linked to
motor behaviors (Heidbreder and Groenewegen 2003; Vertes
2006). We investigated whether the inter-animal differences in
behavioral responses to the FR might correlate with differences
in the inhibitory input onto layer V pyramidal neurons in Cg1.
The relationship between lengths of GAD terminal contact with
the somatic plasma membrane of Cg1 layer V pyramidal cells and
running wheel activity in the ABA paradigm was examined using
the Pearson correlation analysis. There was no correlation between
running activity prior to FR and GAD terminal contact lengths
(Fig. 6A). In contrast, GAD terminal contact lengths were negatively
correlated with the wheel-running activity during the first ABA
(Table 1 and Fig. 6B), meaning that the animals exhibiting the greatest increase in wheel activity were the ones that exhibited the lowest increase of GAD terminal contact lengths at layer V cell bodies.
The negative correlation was even stronger during the second ABA
exposure (Table 1 and Fig. 6C). The negative correlation between
GAD terminal contact length and wheel running was also strong
for the total running activity (sum of running from baseline
through the first as well as the second ABA; Table 1 and Fig. 6D).
GAD terminal size analysis further confirmed that the cytoplasmic area of GAD terminals at the site of contact with cell bodies was also negatively correlated with the total wheel-running
activity (Table 1).
In contrast to Cg1, which is linked to motor behaviors, the
ventral regions of the PFC areas PrL and Mo are associated with
diverse emotional, cognitive, and mnemonic processes and less
to motor behaviors (Heidbreder and Groenewegen 2003; Vertes
2006). The strong correlations observed between the contact
by GAD terminals onto cell bodies of layer V pyramidal neurons
and running activity were specific for the Cg1, in that the PrL
(Fig. 6E) and MO (Fig. 6F) did not exhibit these correlations
(Table 1).
Correlation between the FAA, Running during Food
Access, and GAD Terminal Contact Lengths with Somatic
Plasma Membranes of Layer V Pyramidal Neurons
Although axo-somatic contact lengths formed by GAD terminals
in the PrL and MO did not exhibit correlation with wheel running
during the first or second ABA, a possibility remained that some
other subcomponent of the wheel activity might. Indeed, correlation was also found between GAD terminal contacts onto somata in the PrL and the running during the hours of food access,
from 7 PM to 9 PM, of the second ABA (Table 1 and Fig. 7B). The
Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
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Figure 6. Correlation between the running wheel activity and GAD terminal lengths at sites of contact with the somatic plasma membrane of layer V pyramidal neurons.
(A) No correlation was found between GAD terminal length in Cg1 and the average wheel activity in 3 days baseline. Here and in other panels, GAD terminal lengths of CON
animals are shown for comparison with the values of ABA animals. (B) There is a negative correlation between GAD terminal length in Cg1 and the average wheel activity
during the 3 days of the first ABA. Those animals that exhibited the greatest mean value of GAD axon terminal length ran the least. By the Pearson correlation analysis,
correlation is significant at 0.0489 level (two-tailed). (C) There is a negative correlation between GAD terminal length in Cg1 and the average wheel activity during the 3 days
of exposure to the second ABA. Correlation reveals significant effect at 0.009 level (two-tailed). (D) A negative correlation was found between GAD terminal length in Cg1
and total running activity at 0.0267 significant level (two-tailed). Total running activity was measured as the sum of baseline, during the first ABA induction, and during the
second ABA induction. (E) No correlation was found between GAD terminal length in PrL and total running activity (P = 0.94). (F) No correlation was found between GAD
terminal length in MO and total running activity (P = 0.21).
correlation was negative and significant, meaning that animals
exhibiting the least wheel activity during the food access period
were the ones that exhibited the greatest increase of GAD terminal contact lengths. GAD terminal size analysis further confirmed
that the area of GAD terminals at axo-somatic contact sites in the
PrL was also negatively correlated with the running during the
hours of food access (Table 1). In contrast, GAD terminal contact
lengths onto cell bodies in the Cg1 and MO did not correlate significantly with wheel running during food access in the second
ABA (Fig. 7A for Cg1; Fig. 7C for MO; and Table 1). None of the 3
PFC regions exhibited correlation between GAD terminal contact
lengths onto cell bodies of layer V pyramidal cells and running
during the hours of food access of the first ABA (Table 1).
Interestingly, correlation was also found between GAD terminal contact lengths in the PrL and FAA, spanning the 6 h that
preceded feeding during the first ABA (Fig. 7E and Table 1). The
correlation was “positive,” meaning that animals exhibiting the
greatest wheel activity during the FAA period were the ones
that exhibited the greatest increase of GAD terminal contact
lengths at cell bodies. MO, the most ventral part of PFC, also exhibited marginally positive correlation between GAD terminal
contact lengths at cell bodies and FAA of the first ABA (Fig. 7F
and Table 1). In contrast, GAD terminal contact lengths onto pyramidal cells in layer V of Cg1 did not correlate significantly with
FAA during the first ABA (Fig. 7D).
Discussion
The present study is consistent with the idea that induction
of FR-evoked hyperactivity on the wheel (i.e., ABA) caused enlargement of GAD axon terminals targeting layer V pyramidal
cell bodies in the PFC. However, contrary to our expectation, this
circuitry change is not likely to have supported the FR-evoked
hyperactivity, since the 2 parameters correlated negatively and
strongly (R = −0.87, P < 0.01): Individuals with the most enlarged
GAD terminal contacts onto somata exhibited the greatest suppression of FR-evoked hyperactivity, whereas those animals
with terminal contact sizes no different from controls’ exhibited
Pearson
r = −0.03
P = 0.95
Pearson
r = −0.28
P = 0.55
Pearson
r = −0.17
P = 0.72
Pearson
r = 0.02
P = 0.97
Bold values indicate statistically significant, P <.05.
Contact size:
area (nm2)
ABA, activity-based anorexia; Cg1, cingulate cortex, area 1; PrL, prelimbic cortex; MO, medial orbital cortex; FAA, food anticipatory activity.
Pearson
r = 0.09
P = 0.86
Pearson
r = 0.22
P = 0.64
Pearson
r = 0.56
P = 0.19
Pearson
r = 0.59
P = 0.16
Pearson
r = −0.51
P = 0.24
Pearson
r = −0.69
P = 0.09
Pearson
r = 0.75
P = 0.05
Pearson
r = 0.48
P = 0.27
Pearson
r = 0.69
P = 0.08
Pearson
r = 0.69
P = 0.08
Pearson
r = −0.68
P = 0.09
Pearson
r = −0.66
P = 0.11
Pearson
r = −0.25
P = 0.58
Pearson
r = −0.71
P = 0.07
PrL
Cg1
PrL
MO
MO
Pearson
r = −0.84
P = 0.01
Pearson
r = −0.79
P = 0.03
Cg1
Cg1
PrL
Contact size:
length (nm)
Contact size:
area (nm2)
MO
Running during food access in the first ABA Running during food access in the second ABA
FAA during the first ABA
Pearson
r = −0.81
P = 0.02
Pearson
r = −0.78
P = 0.04
Pearson
r = −0.13
P = 0.78
Pearson
r = 0.34
P = 0.46
Pearson
r = −0.87
P = 0.01
Pearson
r = −0.37
P = 0.41
Pearson
r = 0.71
P = 0.074
Pearson
r = 0.72
P = 0.07
Pearson
r = 0.36
P = 0.43
Pearson
r = −0.1
P = 0.84
Pearson
r = 0.33
P = 0.48
Pearson
r = 0.13
P = 0.78
Pearson
r = 0.11
P = 0.82
Pearson
r = 0.13
P = 0.79
Contact size:
length (nm)
PrL
Pearson
r = −0.76
P = 0.04
Pearson
r = −0.69
P = 0.08
Pearson
r = 0.1
P = 0.84
Pearson
r = −0.37
P = 0.41
Pearson
r = −0.08
P = 0.86
Pearson
r = −0.19
P = 0.68
PrL
Cg1
Total running activity
MO
PrL
Running during the second ABA
Cg1
MO
Cg1
PrL
Running during the first ABA
Cg1
MO
Running during baseline
Brain region
Table 1 Summary of correlation coefficients and P-values between running activities and contact sizes
Pearson
r = −0.53
P = 0.21
Pearson
r = −0.28
P = 0.54
| Cerebral Cortex, 2016, Vol. 26, No. 6
MO
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the greatest hyperactivity. FR-evoked hyperactivity can lead to
death (Chowdhury et al. 2013; Gutierrez 2013). Therefore, suppression of FR-evoked hyperactivity is clearly adaptive. We propose that the enhancement of axo-somatic inhibition of layer V
pyramidal neurons in the PFC may contribute to this adaptive behavior. In support of this idea, it has been shown by others that
GABAergic synapses exhibit activity-dependent structural plasticity, including enlargement of synapse sizes [review by Flores
and Mendez (2014)], and that this enlargement can be accompanied by increased expression of GABAergic receptors at synapses
(Nusser et al. 1998). Unfortunately, our attempt to quantify α1
and β2/3 subunits of GABAA receptors at axo-somatic synapses
has not been successful, so we have yet to determine whether
ABA induction increases GABAA receptor density at these contact
sites.
Another unexpected finding was that although individual differences in GAD terminal contact sizes at cell bodies correlated
most strongly with those in hyperactivity during the second
ABA induction, this correlation was already detectable 10 days
prior, during the first ABA induction. Although identification of
the molecular cascades underlying this change was beyond the
scope of this study, an idea compatible with the current finding
is that some yet-to-be-identified individual difference supporting
activity-dependent changes in GABAergic synapses existed prior
to ABA induction. A number of previously published findings
point to individual differences in the activity-dependent brain
derived neurotrophic factor (BDNF) expression as a possible
mechanism linking the FR-evoked stress or heightened exercise
to the upregulation of GABA receptors and expansion of contact
sites formed by GAD terminals onto somata (Neeper et al. 1996;
Gomez-Pinilla et al. 2002; Lee et al. 2002; Duman 2005; Stranahan
et al. 2007, 2009; Jiao et al. 2011).
Functional Significance of the Increased Lengths of
Contacts Formed by GABAergic Axon Terminals onto
Layer V Pyramidal Neurons in the PFC
PFC is a site of integration of multiple sensory modalities in addition to signals associated with visceral states, rewards, social
conflicts, stress, attention, and “affective qualities” [reviewed by
Vertes (2006)]. The PFC converts these signals into efferent signals that contribute toward behavior that are in accordance
with internal goals. The PFC has been described as contributing
to executive functioning which, for rodents, includes attentional
selection, resistance to interference, reduction in impulsive behavior, and ultimately generating behavioral flexibility for planning and decision-making (Dalley et al. 2004; Erlich et al. 2011).
Because layer V pyramidal neurons are the major efferent neurons of the PFC to motor centers (Berendse et al. 1992; Gabbott
et al. 2005; Riga et al. 2014), increased GABAergic terminals’ contact lengths onto layer V pyramidal neurons may be the cellular
mechanism that facilitates response selection through inhibition
of impulsive behavior. For rodents, an impulsive initial response
to starvation is to enhance foraging behavior. It has been hypothesized that the increased wheel activity that was elicited
by all 7 of our experimental animals during the initial ABA induction may be a substitute for the foraging behavior elicited by
hungry rodents in captivity (Gutierrez 2013). Conversely, the
dampened wheel activity that was observed by some of the
mice during the second ABA induction may reflect a learned
behavior of suppressing what turned out to be an unproductive
behavior during the first ABA induction, mediated through enhanced GABAergic inhibition of excitatory outflow from the PFC
to the motor centers. As for the mechanism supporting enhanced
Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
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Figure 7. Correlation between the FAA, running during food access, and GAD terminal lengths at sites of contact with the somatic plasma membrane of layer V pyramidal
neurons. (A) No correlation was found between GAD terminal contact length in Cg1 and running during food access in the second ABA (P = 0.86). Here and in other panels,
GAD terminal contact lengths of CON animals are shown for comparison with the values of ABA animals. (B) There was a negative correlation between GAD terminal
contact length in PrL and running during food access in the second ABA. Pearson correlation was significant at P = 0.016 level (two-tailed). (C) No correlation was found
between GAD terminal contact length in MO and running during food access in the second ABA (P = 0.71). (D) No correlation was found between GAD terminal contact
length in Cg1 and FAA during the first ABA (P = 0.24). (E) There was a positive correlation between GAD terminal contact length in PrL and FAA during the first ABA.
Pearson correlation was significant at P = 0.05 level (two-tailed). (F) A marginal positive correlation was found between GAD terminal contact length in MO and FAA
during the first ABA, at P = 0.08 level (two-tailed).
GABAergic inhibition, elevated levels of BDNF in the PFC have
been hypothesized for another learned behavior—the extinction
of fear memory (Bredy et al. 2007), and may be at work for the
ABA-induced mice as well.
Functional Significance of the Regional Differences in the
Correlation between GAD Terminals’ Axo-Somatic
Contact Lengths and Wheel Activity
Cg1, PrL, and MO are all parts of the medial PFC, but receive different afferents (Hoover and Vertes 2007). Specifically, the dorsally
located Cg1 receives primarily sensorimotor cortical input,
whereas the more ventrally located PrL and MO receive primarily
limbic inputs that are both cortical and subcortical, including
the amygdala, hippocampus, basal forebrain area, the monoaminergic nuclei, and the midline thalamus. All 3 of these medial
PFC regions exhibited increased GAD terminal sizes in response
to ABA induction. However, we also noted individual differences
in GAD terminal contact sizes among the ABA-induced mice and
discovered that GAD terminal sizes within specific subregions
correlate with distinct components of the FR-evoked hyperactivity. Specifically, within the area PrL, GAD terminal sizes correlated negatively and strongly with wheel activity during the 2 h
of food availability of the food-restricted days. In other words, animals with the largest GAD terminals in the PrL were the ones
that were able to suppress wheel activity the most during the
2 h of food availability. Although the PrL region is part of the medial PFC, as is the Cg1, highly localized lesion studies within the
medial PFC have revealed that there are behavioral tasks linked
more to the PrL than to the Cg1. These include the delayed
non-matching-to-sample response (Delatour and GisquetVerrier 1999), extra-dimensional and cross-modal set-shifting,
and shifts between new strategies or rules (Dalley et al. 2004).
As was described earlier, cage-housed mice that are resilient
and survive ABA induction are those that can learn a new rule,
namely that wheel running does not yield a new source of food
and is detrimental within the confines of a cage. The enlargement of GAD terminal contact sizes at the PrL cell bodies may
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have contributed toward the animal’s learned behavior and/or
shifting to this new rule, even if running is intrinsically rewarding (Meijer and Robbers 2014) and helpful for foraging.
GAD terminal sizes in Cg1 also correlated with individuals’
wheel activity. However, in contrast to the PrL, GAD terminal
sizes in the Cg1 correlated most strongly with the animals’ total
wheel activity during the second ABA, and not specifically to the
2 h of food availability. Although weaker, Cg1 GAD terminal contact sizes also correlated significantly with the 24-h activity during the first ABA, even though these preceded euthanasia by
more than 10 days. Within the medial PFC, the most dorsally located Cg1 (corresponding to ACg in rats; Paxinos and Watson
1998; Ongur and Price 2000) projects most prominently to the dorsal striatum (Gabbott et al. 2005). Based on this trajectory, it is not
surprising that inhibition of these excitatory outflows from the
PFC would also suppress locomotor activity.
Among the 3 animals—3, 4, and 6—that exhibited the
greatest resilience to ABA induction by reducing wheel activity,
it was Animal 4 that exhibited the largest GAD terminal contact
lengths and bouton areas in PrL and the least wheel activity
during the hours of food availability. The other 2 animals
(3 and 6) exhibited the largest GAD terminal contact lengths
and areas in Cg1 and minimal overall wheel activity during
the FR days. These observations indicate that the cellular response for attaining resilience to ABA induction varied across
animals—enlargement of GAD terminal contacts in PrL or in
Cg1. For both the Cg1 and PrL, the correlation between GAD
terminal contact lengths and wheel activity suppression was
more robust for the second ABA than the first. Perhaps, the
GAD terminal enlargement was cumulative through the first
ABA, the recovery phase, and the second ABA.
It is reasonable to consider that those animals exhibiting the
most heightened FAA are the ones most strongly entrained to the
restricted feeding schedule and have undergone the most robust
alteration in PFC circuitry. This idea is supported by our observation that a positive correlation exists between FAA and GAD terminal sizes—those individuals with largest GAD axo-somatic
contact lengths in the PrL and MO are the ones that exhibited
the highest FAA. Interestingly, there was no correlation between
FAA and suppression of activity during the non-FAA period, including the 2 h of food access. This indicates that entrainment
to the feeding schedule (as expressed by the heightened FAA)
was not sufficient to elicit behavior according to the new rule—
to suppress the intrinsically rewarding wheel activity during
the hours of food availability, when food access is limited. The
lack of correlation between FAA and wheel running suppression
during the non-FAA hours is explained by the diversity among
the mice in terms of attaining resilience. As described above,
one attained resilience by reducing wheel activity during the
2 h of food availability, while 2 attained resilience by reducing
overall activity during the FR days. The remaining 4 did not attain
resilience because they did not reduce activity during the nonFAA hours, even though their FAAs were high. These 4 that failed
to elicit behavior according to the new rule were the ones with
smaller GAD terminal sizes, no different from controls’.
Prior to this study, we showed that ABA induction during adolescence evokes a change in the GABAergic system within 2 other
brain regions—the hippocampal CA1 and the basolateral amygdala. The change evoked in the hippocampal CA1 was to increase
GABAergic inhibition through increased contact lengths by
GABAergic axon terminals onto cell bodies and dendrites of pyramidal cells (Chowdhury et al. 2013). After just one exposure to
ABA, we also observed increased plasmalemmal expression of
non-synaptic GABAergic receptors at spines in the hippocampus
(Aoki et al. 2012, 2014). Since upregulation of GABAergic terminal
contact sizes onto cell bodies and of non-synaptic GABAergic receptors at spines was greater among animals exhibiting suppression of the FR-evoked hyperactivity (Chowdhury et al. 2013; Aoki
et al. 2014), these changes to the hippocampal GABAergic system
may have contributed, together with the medial PFC, toward reducing the FR-evoked hyperactivity. Whether other cortical
areas also undergo changes in the GABAergic system and may
contribute toward suppression of hyperactivity or whether the
changes evoked in the hippocampus and/or PFC are sufficient
to alter behavior remains to be determined.
Basolateral amygdala, a hub of emotion circuits in the brain
(LeDoux 2000), was analyzed within brains of rats, immediately
after they underwent single ABA induction (Wable et al. 2014).
Here, the change evoked by ABA induction was an increase in
the expression of non-synaptic GABA receptor expression by inhibitory interneurons, which would be expected to increase the
excitatory outflow and possibly contribute toward increased anxiety and the initial FR-evoked hyperactivity (Wable et al. 2015). In
the current study, we observed a modest positive correlation between GABAergic axon terminal contact sizes (lengths and areas)
impinging upon layer V pyramidal neurons of the MO and FAA
wheel activity. MO is a visceromotor limbic cortex with projections to the dorsal motor nucleus of vagus, nucleus of the solitary
tract and amygdala, through which it is proposed to modulate
autonomic activity and emotion (Fisk and Wyss 2000; Vogt
et al. 2004). Assuming the murine basolateral amygdala undergoes similar GABAergic reorganization as observed in rats, the remodeling of the GABAergic system in MO may be a compensatory
mechanism to dampen the heightened anxiety brought about by
the increased excitatory outflow from the basolateral amygdala.
Conclusions
It is well established that food deprivation evokes robust and reliable increases in voluntary wheel running by rodents (Sherwin
1998). Among the many ideas about why food deprivation evokes
wheel running in rodents, one prevalent idea is that this reflects
the adaptive behavior of foraging within the wild by hungry animals, but could also be related to the maladaptive, over-exercise
response elicited by a large proportion of patients diagnosed with
AN (Epling et al. 1983; Davis et al. 1997, 1999; Hebebrand and Bulik
2011; Zunker et al. 2011; Gutierrez 2013). This idea is the basis for
using ABA as an animal model for understanding and developing
treatments for AN (Gutierrez 2013). AN is a complex psychiatric
illness with biological bases. Twin studies indicate that there is
a genetic basis for vulnerability to AN (Bulik et al. 2007), and animal models have pointed to a link between genes modulating
anxiety and ABA vulnerability (Gelegen et al. 2006, 2007, 2008,
2010). However, unlike other mental and neurological illnesses
such as schizophrenia, autism, epilepsy, or depression, the largest scale search for a genetic link to AN has yet to identify candidate genes (Boraska et al. 2014). In our opinion, one important
contribution we have made to the field of ABA is the utility of correlating individual differences in vulnerability to ABA with cellular and molecular markers.
Our findings indicate that ABA induction among adolescent
female mice has an impact on PFC circuitry and could be contributing toward prevention of weight loss caused by FR-evoked
hyperactivity The individual differences in response to ABA induction and AN vulnerability may arise due to GAD gene polymorphism, which exists (Addington et al. 2005; Hettema et al.
2006; Zhao et al. 2007), or epigenetic regulations of the GAD
gene promoter (Kundakovic et al. 2007; Satta et al. 2008; Zhang
Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
et al. 2010) in the medial PFC. More studies are needed to identify
the molecular mechanisms underlying the differential responses
of GAD terminals in the PFC and whether it is mediated through
the activity-dependent release of BDNF or other neurotrophic
factors, such as NGF1-A (Zhang et al. 2010). What is also needed
is a direct test of whether increased GABAergic inhibition of layer
V pyramidal neurons in the PFC will enable individuals to exert
cognitive control over the impulsive desire to exercise, rather
than to eat.
Funding
This study was supported by the National Institutes of Health
[NEI Core grant EY13079, 1 R21MH091445-01, 1 R21MH10584601, R01NS066019-01A1, and R01NS047557-07A1 to C.A., T32
MH019524 to G.S.W., and the UL1 TR000038 from the National
Center for the Advancement of Translational Science (NCATS)
to T.G.C.]; The Fulbright Graduate Study Grant (Fulbright Taiwan,
to Y.-W.C.); and NYU’s Research Challenge Fund to C.A.
Notes
We thank Kei Tateyama for her help with electron microscopy.
Conflict of Interest: None declared.
References
Addington AM, Gornick M, Duckworth J, Sporn A, Gogtay N,
Bobb A, Greenstein D, Lenane M, Gochman P, Baker N, et al.
2005. GAD1 (2q31.1), which encodes glutamic acid decarboxylase (GAD67), is associated with childhood-onset schizophrenia and cortical gray matter volume loss. Mol Psychiatry.
10:581–588.
American Psychiatric Association. 2013. Diagnostic and
Statistical Manual of Mental Disorders: DSM-5. Washington,
DC: American Psychiatric Publishing.
Andersen SL, Thompson AT, Rutstein M, Hostetter JC,
Teicher MH. 2000. Dopamine receptor pruning in prefrontal
cortex during the periadolescent period in rats. Synapse.
37:167–169.
Aoki C, Sabaliauskas N, Chowdhury T, Min JY, Colacino AR,
Laurino K, Barbarich-Marsteller NC. 2012. Adolescent female
rats exhibiting activity-based anorexia express elevated levels
of GABA(A) receptor alpha4 and delta subunits at the plasma
membrane of hippocampal CA1 spines. Synapse. 66:391–407.
Aoki C, Wable G, Chowdhury TG, Sabaliauskas NA, Laurino K,
Barbarich-Marsteller NC. 2014. alpha4betadelta-GABAARs in
the hippocampal CA1 as a biomarker for resilience to activity-based anorexia. Neuroscience. 265:108–123.
Berendse HW, Galis-de Graaf Y, Groenewegen HJ. 1992. Topographical organization and relationship with ventral striatal
compartments of prefrontal corticostriatal projections in the
rat. J Comp Neurol. 316:314–347.
Birmingham CL, Su J, Hlynsky JA, Goldner EM, Gao M. 2005. The
mortality rate from anorexia nervosa. Int J Eat Disord.
38:143–146.
Boraska V, Franklin CS, Floyd JA, Thornton LM, Huckins LM,
Southam L, Rayner NW, Tachmazidou I, Klump KL,
Treasure J, et al. 2014. A genome-wide association study of
anorexia nervosa. Mol Psychiatry. doi:10.1038/mp.2013.187.
Bredy TW, Wu H, Crego C, Zellhoefer J, Sun YE, Barad M. 2007. Histone modifications around individual BDNF gene promoters
in prefrontal cortex are associated with extinction of conditioned fear. Learn Mem. 14:268–276.
Chen et al.
| 2587
Bulik CM, Slof-Op’t Landt MC, van Furth EF, Sullivan PF. 2007. The
genetics of anorexia nervosa. Annu Rev Nutr. 27:263–275.
Chattopadhyaya B, Di Cristo G, Higashiyama H, Knott GW,
Kuhlman SJ, Welker E, Huang ZJ. 2004. Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical
period. J Neurosci. 24:9598–9611.
Chowdhury TG, Rios MB, Chan TE, Cassataro DS, BarbarichMarsteller NC, Aoki C. 2014. Activity-based anorexia during
adolescence disrupts normal development of the CA1
pyramidal cells in the ventral hippocampus of female rats.
Hippocampus. doi:10.1002/hipo.22320.
Chowdhury TG, Wable GS, Sabaliauskas NA, Aoki C. 2013. Adolescent female C57BL/6 mice with vulnerability to activity-based
anorexia exhibit weak inhibitory input onto hippocampal CA1
pyramidal cells. Neuroscience. 241:250–267.
Dalley JW, Cardinal RN, Robbins TW. 2004. Prefrontal executive
and cognitive functions in rodents: neural and neurochemical
substrates. Neurosci Biobehav Rev. 28:771–784.
Davis C, Katzman DK, Kaptein S, Kirsh C, Brewer H, Kalmbach K,
Olmsted MP, Woodside DB, Kaplan AS. 1997. The prevalence
of high-level exercise in the eating disorders: etiological implications. Compr Psychiatry. 38:321–326.
Davis C, Katzman DK, Kirsh C. 1999. Compulsive physical activity
in adolescents with anorexia nervosa: a psychobehavioral
spiral of pathology. J Nerv Ment Dis. 187:336–342.
Delatour B, Gisquet-Verrier P. 1999. Lesions of the prelimbicinfralimbic cortices in rats do not disrupt response selection
processes but induce delay-dependent deficits: evidence for
a role in working memory? Behav Neurosci. 113:941–955.
Dellava JE, Thornton LM, Hamer RM, Strober M, Plotnicov K,
Klump KL, Brandt H, Crawford S, Fichter MM, Halmi KA,
et al. 2010. Childhood anxiety associated with low BMI in
women with anorexia nervosa. Behav Res Ther. 48:60–67.
Dixon DP, Ackert AM, Eckel LA. 2003. Development of, and recovery from, activity-based anorexia in female rats. Physiol
Behav. 80:273–279.
Duman RS. 2005. Neurotrophic factors and regulation of mood:
role of exercise, diet and metabolism. Neurobiol Aging. 26
(Suppl 1):88–93.
Epling WF, Pierce WD, Stefan L. 1983. A theory of activity-based
anorexia. Int J Eat Disord. 3:27–46.
Erlich JC, Bialek M, Brody CD. 2011. A cortical substrate for memory-guided orienting in the rat. Neuron. 72:330–343.
Fisk GD, Wyss JM. 2000. Descending projections of infralimbic
cortex that mediate stimulation-evoked changes in arterial
pressure. Brain Res. 859:83–95.
Flores CE, Mendez P. 2014. Shaping inhibition: activity dependent
structural plasticity of GABAergic synapses. Front Cell
Neurosci. 8:327.
Gabbott PL, Warner TA, Jays PR, Salway P, Busby SJ. 2005. Prefrontal cortex in the rat: projections to subcortical autonomic, motor, and limbic centers. J Comp Neurol. 492:
145–177.
Gelegen C, Collier DA, Campbell IC, Oppelaar H, Kas MJ. 2006.
Behavioral, physiological, and molecular differences in response to dietary restriction in three inbred mouse strains.
Am J Physiol Endocrinol Metab. 291:E574–E581.
Gelegen C, Collier DA, Campbell IC, Oppelaar H, van den Heuvel J,
Adan RA, Kas MJ. 2007. Difference in susceptibility to
activity-based anorexia in two inbred strains of mice. Eur
Neuropsychopharmacol. 17:199–205.
Gelegen C, Pjetri E, Campbell IC, Collier DA, Oppelaar H, Kas MJ.
2010. Chromosomal mapping of excessive physical activity
2588
| Cerebral Cortex, 2016, Vol. 26, No. 6
in mice in response to a restricted feeding schedule.
Eur Neuropsychopharmacol. 20:317–326.
Gelegen C, van den Heuvel J, Collier DA, Campbell IC, Oppelaar H,
Hessel E, Kas MJ. 2008. Dopaminergic and brain-derived
neurotrophic factor signalling in inbred mice exposed to a restricted feeding schedule. Genes Brain Behav. 7:552–559.
Goddard E, Ashkan K, Farrimond S, Bunnage M, Treasure J. 2013.
Right frontal lobe glioma presenting as anorexia nervosa: further evidence implicating dorsal anterior cingulate as an area
of dysfunction. Int J Eat Disord. 46:189–192.
Gomez-Pinilla F, Ying Z, Roy RR, Molteni R, Edgerton VR. 2002.
Voluntary exercise induces a BDNF-mediated mechanism
that promotes neuroplasticity. J Neurophysiol. 88:2187–2195.
Gutierrez E. 2013. A rat in the labyrinth of anorexia nervosa:
contributions of the activity-based anorexia rodent model to
the understanding of anorexia nervosa. Int J Eat Disord.
46:289–301.
Gutierrez E, Baysari MT, Carrera O, Whitford TJ, Boakes RA. 2006.
High ambient temperature reduces rate of body-weight
loss produced by wheel running. Q J Exp Psychol (Hove).
59:1196–1211.
Hare BD, D’Onfro KC, Hammack SE, Falls WA. 2012. Prior stress interferes with the anxiolytic effect of exercise in C57BL/6J mice.
Behav Neurosci. 126:850–856.
Hebebrand J, Bulik CM. 2011. Critical appraisal of the provisional
DSM-5 criteria for anorexia nervosa and an alternative proposal. Int J Eat Disord. 44:665–678.
Heidbreder CA, Groenewegen HJ. 2003. The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based
upon functional and anatomical characteristics. Neurosci
Biobehav Rev. 27:555–579.
Hettema JM, An SS, Neale MC, Bukszar J, van den Oord EJ,
Kendler KS, Chen X. 2006. Association between glutamic
acid decarboxylase genes and anxiety disorders, major depression, and neuroticism. Mol Psychiatry. 11:752–762.
Hodes GE, Shors TJ. 2005. Distinctive stress effects on learning
during puberty. Horm Behav. 48:163–171.
Hoover WB, Vertes RP. 2007. Anatomical analysis of afferent projections to the medial prefrontal cortex in the rat. Brain Struct
Funct. 212:149–179.
Hudson JI, Hiripi E, Pope HG Jr, Kessler RC. 2007. The prevalence
and correlates of eating disorders in the National Comorbidity
Survey Replication. Biol Psychiatry. 61:348–358.
Jiao Y, Zhang Z, Zhang C, Wang X, Sakata K, Lu B, Sun QQ. 2011.
A key mechanism underlying sensory experience-dependent
maturation of neocortical GABAergic circuits in vivo. Proc Natl
Acad Sci USA. 108:12131–12136.
Kaye WH, Bulik CM, Thornton L, Barbarich N, Masters K. 2004. Comorbidity of anxiety disorders with anorexia and bulimia nervosa. Am J Psychiatry. 161:2215–2221.
Kaye WH, Fudge JL, Paulus M. 2009. New insights into symptoms
and neurocircuit function of anorexia nervosa. Nat Rev
Neurosci. 10:573–584.
Kullmann S, Giel KE, Hu X, Bischoff SC, Teufel M, Thiel A, Zipfel S,
Preissl H. 2014. Impaired inhibitory control in anorexia
nervosa elicited by physical activity stimuli. Soc Cogn Affect
Neurosci. 9:917–923.
Kundakovic M, Chen Y, Costa E, Grayson DR. 2007. DNA methyltransferase inhibitors coordinately induce expression of the
human reelin and glutamic acid decarboxylase 67 genes.
Mol Pharmacol. 71:644–653.
Kvitsiani D, Ranade S, Hangya B, Taniguchi H, Huang JZ, Kepecs A.
2013. Distinct behavioural and network correlates of two interneuron types in prefrontal cortex. Nature. 498:363–366.
LeDoux JE. 2000. Emotion circuits in the brain. Annu Rev Neurosci.
23:155–184.
Lee J, Duan W, Mattson MP. 2002. Evidence that brain-derived
neurotrophic factor is required for basal neurogenesis and
mediates, in part, the enhancement of neurogenesis by dietary
restriction in the hippocampus of adult mice. J Neurochem.
82:1367–1375.
Lozsa A. 1974. Uranyl acetate as an excellent fixative for lipoproteins
after electrophoresis on agarose-gel. Clin Chim Acta. 53:43–49.
Meijer JH, Robbers Y. 2014. Wheel running in the wild. Proc Biol
Sci. 281. doi:10.1098/rspb.2014.0210.
Neeper SA, Gomez-Pinilla F, Choi J, Cotman CW. 1996. Physical
activity increases mRNA for brain-derived neurotrophic
factor and nerve growth factor in rat brain. Brain Res.
726:49–56.
Nelson M. 1985. Level of living and fertility among a rural population of the Philippines. Stud Comp Int Dev. 20:31–46.
Nusser Z, Hajos N, Somogyi P, Mody I. 1998. Increased number of
synaptic GABA(A) receptors underlies potentiation at hippocampal inhibitory synapses. Nature. 395:172–177.
Ongur D, Price JL. 2000. The organization of networks within
the orbital and medial prefrontal cortex of rats, monkeys
and humans. Cereb Cortex. 10:206–219.
Paxinos G, Franklin KBJ. 2001. The mouse brain in stereotaxic
coordinates. 2nd ed. San Diego: Academic Press.
Paxinos G, Watson C. 1998. The rat brain in stereotaxic
coordinates. 4th ed. San Diego: Academic Press.
Perdereau F, Faucher S, Wallier J, Vibert S, Godart N. 2008. Family
history of anxiety and mood disorders in anorexia nervosa: review of the literature. Eat Weight Disord. 13:1–13.
Peters A, Jones EG. 1984. Cellular components of the cerebral
cortex. New York (NY): Plenum Press.
Peters A, Palay SL, Webster HF. 1991. The fine structure of
the nervous system: neurons and their supporting cells.
New York (NY): Oxford University Press.
Riga D, Matos MR, Glas A, Smit AB, Spijker S, Van den Oever MC.
2014. Optogenetic dissection of medial prefrontal cortex circuitry. Front Syst Neurosci. 8:230.
Romeo RD. 2010. Adolescence: a central event in shaping stress
reactivity. Dev Psychobiol. 52:244–253.
Sato Y, Saito N, Utsumi A, Aizawa E, Shoji T, Izumiyama M,
Mushiake H, Hongo M, Fukudo S, et al. 2013. Neural basis of
impaired cognitive flexibility in patients with anorexia nervosa. PLoS ONE. 8:e61108.
Satta R, Maloku E, Zhubi A, Pibiri F, Hajos M, Costa E, Guidotti A.
2008. Nicotine decreases DNA methyltransferase 1 expression
and glutamic acid decarboxylase 67 promoter methylation
in GABAergic interneurons. Proc Natl Acad Sci USA. 105:
16356–16361.
Sherwin CM. 1998. Voluntary wheel running: a review and novel
interpretation. Anim Behav. 56:11–27.
Spear LP. 2000. The adolescent brain and age-related behavioral
manifestations. Neurosci Biobehav Rev. 24:417–463.
Stranahan AM, Khalil D, Gould E. 2007. Running induces
widespread structural alterations in the hippocampus and
entorhinal cortex. Hippocampus. 17:1017–1022.
Stranahan AM, Lee K, Martin B, Maudsley S, Golden E, Cutler RG,
Mattson MP. 2009. Voluntary exercise and caloric restriction
enhance hippocampal dendritic spine density and BDNF
levels in diabetic mice. Hippocampus. 19:951–961.
Sullivan PF. 1995. Mortality in anorexia nervosa. Am J Psychiatry.
152:1073–1074.
Terzakis JA. 1968. Uranyl acetate, a stain and a fixative.
J Ultrastruct Res. 22:168–184.
Enlarged GABA Synapses in the Medial Prefrontal Cortex Correlates with ABA Resilience
Thornton LM, Dellava JE, Root TL, Lichtenstein P, Bulik CM. 2011.
Anorexia nervosa and generalized anxiety disorder: further
explorations of the relation between anxiety and body mass
index. J Anxiety Disord. 25:727–730.
van Kuyck K, Casteels C, Vermaelen P, Bormans G, Nuttin B, Van
Laere K. 2007. Motor- and food-related metabolic cerebral
changes in the activity-based rat model for anorexia nervosa:
a voxel-based microPET study. Neuroimage. 35:214–221.
Vertes RP. 2006. Interactions among the medial prefrontal cortex,
hippocampus and midline thalamus in emotional and cognitive processing in the rat. Neuroscience. 142:1–20.
Vogt BA, Vogt L, Farber NB. 2004. Cingulate Cortex and Disease
Models. In: Paxinos G, editor. The Rat Nervous System.
3rd ed. San Diego (CA): Elsevier. p. 705–727.
Wable GS, Barbarich-Marsteller NC, Chowdhury TG,
Sabaliauskas NA, Farb CR, Aoki C. 2014. Excitatory synapses
on dendritic shafts of the caudal basal amygdala exhibit elevated levels of GABAA receptor alpha4 subunits following
the induction of activity-based anorexia. Synapse. 68:1–15.
Wable GS, Min JY, Chen Y-W, Aoki C. Forthcoming 2015. Anxiety is
correlated with running in adolescent female mice undergoing
activity-based anorexia. Behav Neurosci. 129(2):170–182.
Chen et al.
| 2589
White EL, Keller A. 1989. Cortical circuits: synaptic organization
of the cerebral cortex—structure, function, and theory.
Boston: Birkhä user.
Zhang TY, Hellstrom IC, Bagot RC, Wen X, Diorio J, Meaney MJ.
2010. Maternal care and DNA methylation of a glutamic acid
decarboxylase 1 promoter in rat hippocampus. J Neurosci.
30:13130–13137.
Zhao X, Qin S, Shi Y, Zhang A, Zhang J, Bian L, Wan C, Feng G,
Gu N, Zhang G, et al. 2007. Systematic study of association
of four GABAergic genes: glutamic acid decarboxylase 1
gene, glutamic acid decarboxylase 2 gene, GABA(B) receptor
1 gene and GABA(A) receptor subunit beta2 gene, with schizophrenia using a universal DNA microarray. Schizophr Res. 93:
374–384.
Zhou Y, Danbolt NC. 2013. GABA and glutamate transporters in
brain. Front Endocrinol (Lausanne). 4:165.
Zipfel S, Lowe B, Reas DL, Deter HC, Herzog W. 2000. Long-term
prognosis in anorexia nervosa: lessons from a 21-year follow-up study. Lancet. 355:721–722.
Zunker C, Mitchell JE, Wonderlich SA. 2011. Exercise interventions for women with anorexia nervosa: a review of the literature. Int J Eat Disord. 44:579–584.