Grant Number: 1R21NS058253-01
Project Title: Seizures & Amydala-Based Socioemotional
Dysfunction
PI Information: ASSOCIATE PROFESSOR LUDISE MALKOVA,
[email protected]
Abstract: DESCRIPTION (provided by applicant): A large proportion
of subjects with autism exhibit seizure disorders and epilepsy. This co
morbidity has been reported to be in the range of 30-40%. Epilepsy and
abnormal EEG patterns occur at a significantly higher rate in
individuals in the more impaired range of the autism spectrum. Seizures
in the autistic population often include complex partial seizures
involving the temporal lobe. The limbic network supporting these
seizures is composed of the amygdala, hippocampus, medio-dorsal
thalamus, piriform, rhinal, and orbitofrontal cortices. Recurrent and/or
prolonged complex partial seizures alter the functional connectivity of
this network in a manner that can impact both cognitive function and
socio-emotional regulation. Importantly, this is the same network
implicated in autistic pathophysiology and therefore these seizures may
pose the greatest risk for adverse psychiatric outcomes in this
population. Dysfunction (often in the absence of structural
abnormalities) in the network anchored in the amygdala, and
interconnected with the orbital frontal cortex, has been found in many
cases of autism. This dysfunction is likely to be exacerbated by the
aberrant plasticity induced in response to repeated seizure discharge.
The goal of this application is to determine if a history of repeated
seizure activity changes the responsiveness of this network and whether
it predisposes this network to amygdala-mediated behavioral
disturbances. Our recent findings showed that reversible manipulations
of the GABAA receptors within the basolateral amygdala (BLA) by focal
intracerebral infusions of GABAA receptor agonists or antagonists
resulted in profound changes in social interactions and reward
evaluation in nonhuman primates. The Specific Aims will determine
whether a history of complex partial seizures, focally-evoked from the
piriform cortex in one hemisphere, result in an increased vulnerability
to disinhibition within BLA in nonhuman primates. This shift in
sensitivity will be probed by evaluating specific behavioral responses
to focal manipulations of GABAA transmission within BLA: Social
interactions (Aim 1), reinforcer devaluation (Aim 2), and emotional
conditioning (Aim 3). The studies will address a recognized comorbidity,
not yet studied in pre-clinical animal models, for which clinical
studies cannot sort out the extent to which seizures may exacerbate the
autistic symptomatology. The combination of co-investigators provides a
unique blend of expertise in experimental epilepsy models, nonhuman
primate models of socio-emotional disturbances, and neural substrates of
human disorders of affect and psychopathy. The team is ideally suited to
approach the analysis of comorbidity of seizures and autistic-like
symptoms evoked from amygdala and to permit a translationally meaningful
analysis of the animal data.
Thesaurus Terms:
There are no thesaurus terms on file for this project.
Institution: GEORGETOWN UNIVERSITY
37TH AND O STS NW
WASHINGTON, DC 20057
Fiscal Year: 2006
Department: PHARMACOLOGY
Project Start: 18-SEP-2006
Project End: 31-AUG-2008
ICD: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
IRG: ZMH1
Behavioral/Systems/Cognitive
GABAA-Mediated Inhibition of Basolateral Amygdala Blocks Reward
Devaluation in Macaques
Laurie L. Wellman, Karen Gale, and Ludise Malkova
Interdisciplinary Program in Neuroscience and Department of
Pharmacology, Georgetown University, Washington, DC 20007
The Journal of Neuroscience, May 4, 2005, 25(18):4577-4586
Subjects
Six pigtail macaques (Macaca nemestrina), three females (OG, OH, JN) and
three males (OK, GR, ZC), were subjects in this study. They were 2-4
years of age and weighed 2.8-5.5 kg at the beginning of this study. All
subjects had been trained previously on a crossmodal auditory-visual
matching task. Two of them (OH and OK) also participated in a previous
experiment in which they were trained on visual delayed
nonmatching-to-sample tasks and received bilateral reversible
inactivations of perirhinal cortex by focal drug infusions (Malkova et
al., 2001 ). All monkeys were housed in pairs in a room with regulated
lighting (12 h light/dark cycle) and maintained on primate LabDiet
(number 5049; PMI Nutrition International, Brentwood, MD) supplemented
daily with fresh fruit. Water was available ad libitum in the home cage.
The study was conducted under a protocol approved by the Georgetown
University Animal Care and Use Committee and in accordance with the
Guide for Care and Use of Laboratory Animals adopted by the National
Institutes of Health.
The monkeys were implanted with stereotaxically positioned
chronic-infusion platforms, which allowed a removable injector, fitted
with an infusion cannula of adjustable length, to be inserted into
predetermined sites in the brain through the guiding channels of the
platform.
Infusion platform
The design and construction of the platform assembly are a modification
of the method of Dooley et al. (1981 ). A custom mold was fabricated
(Elmeco Engineering, Rockville, MD) with a total of 12 rows by 14
columns spaced 2 mm apart to maximize the number of sites for
injections. The holes were arranged into two groups of seven columns,
and the two groups were separated at the midline by a 5.5-mm-wide
section in which two threaded holes were located for placement on a
stereotaxic holder (Fig. 1 A). Stainless-steel pins were placed in the
holes and then liquid acrylic (Kooliner; Patterson Dental Supply, St.
Paul, MN) was poured into the mold to form the platform. Once the
platform had cured, the pins were removed, and any irregularities were
sanded until smooth.
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Figure 1.
A, The infusion platform. A top view of
the platform, which contains holes 2 mm apart in 12
rows by 16 columns. The holes are separated at the
midline by 5.5 mm, where two threaded holes were
located for placement on a stereotaxic holder.
B, The injector apparatus. The left
picture shows the separation of the three parts, and
the right picture shows the three disks flush
together as they appear during infusion. |
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Injector
A custom-built telescoping infusion apparatus (Elmeco Engineering) was
designed to fit snugly into the infusion platform and allow an easy
adjustment of infusion cannula length. The apparatus consists of a
series of three lengths of stainless-steel tubing, each positioned in a
solid stainless-steel disc (Small Parts, Miami Lakes, FL). The discs are
serially interlocked, with the central tubes acting as a guide. The
three lengths of tubing are of successively larger diameters, allowing
for concentric placement (Fig. 1 B). The 27 ga tube of the topmost disc
serves as the infusion cannula and is connected via polyethylene tubing
(BioLab, Decatur, GA) to a 50 �l Hamilton syringe. To perform the
infusion, the telescoping apparatus is slowly inserted through the
chronic platform until the discs are flush with each other and the
platform and the drug is infused (see below, Intracerebral drug
infusions) using an injection pump (model 341; Sage Instruments; Orion
Research, Cambridge, MA).
Magnetic resonance imaging
Approximately 2 weeks before the surgery, each monkey received a
T1-weighted magnetic resonance imaging (MRI) structural brain scan to
calculate stereotaxic coordinates for the platform implantation.
Postoperatively, each monkey received at least one T1-weighted scan to
verify the coordinates for the position of the platform. For each scan,
the monkey was anesthetized with a 4:1 (v/v) mixture of ketamine
(ketamine hydrochloride, 10-20 mg/kg, i.m.) and xylazine (0.2-0.4 mg/kg,
i.m.) and placed in a nonferrous stereotaxic frame (David Kopf
Instruments, Tujunga, CA). MRI was performed in a 1.5 T Vision scanner
(Siemens Magnetom Vision, Munich, Germany) using a human head coil. MR
images were obtained using a three-dimensional FLASH (fast low-angle
shot) pulse sequence (repetition time, 25 ms; echo time, 5 ms;
field-of-view, 20 cm; slice thickness, 1 mm). The frame and the monkey
were aligned in the MR unit using a landmark laser-alignment system and
bubble levels to ensure that the planes of the MR unit were parallel to
those of the stereotaxic instrument. The MRI scans were used to obtain
the coordinates of the basolateral amygdala relative to both the
interaural plane (marked by the ear bars) and the midline, which were
visible on the scan (Saunders et al., 1990 ). Based on these
measurements, coordinates for positioning the infusion platform and
coordinates for the infusion sites were calculated. Postoperatively, the
position of the predetermined infusion sites was validated by another
MRI scan using either 19 ga plastic tubes filled with vitamin E or
tungsten microelectrodes (FHC, Bowdoinham, ME), which were visible on
the scan. One of the tubes or electrodes was inserted, via the guiding
channels and predrilled holes in the skull, into a specified location in
each hemisphere at least 15 mm above the intended infusion site. The
final coordinates for the drug infusions were determined with respect to
the position of the tubes (or electrodes) and the ear bars.
Surgery for implantation of the infusion platform
After first sedating the monkey with ketamine, a surgical level of
anesthesia was established and maintained with isoflurane gas (1-2%, to
effect). Atropine (0.1 ml/kg, s.c.) was administered to counteract the
decrease in heart rate caused by the anesthetics and maintain cardiac
output close to normal. Throughout the aseptic procedure, the monkey
received an intravenous drip solution of isotonic fluids; heart rate,
respiration rate, blood pressure, expired CO2, and body temperature were
monitored.
For the surgery, the monkey was placed in a stereotaxic headholder, and
a sterile field was established. The skull was exposed over the area of
the cranium, where the placement of the platform was determined based on
the preoperative MRI. The platform (with stainless-steel pins placed
into the guide tubes to prevent their closure) was positioned
stereotaxically, four plastic anchor bolts were inserted under the
skull, and an oval wall was created with Palacos R bone cement (BioMet
Orthopedics, Warsaw, IN). Kooliner acrylic was poured around and under
the prefabricated platform. Once the acrylic cured, the pins were
removed from the platform, and a removable acrylic cap was secured with
screws to cover the platform and protect the guide channels when not in
use. All monkeys received postoperative analgesics as determined in
consultation with the facility veterinarian. Approximately 1 week after
the surgery, the channels intended for drug infusions were opened by
hand-held drill under anesthesia and aseptic conditions.
Intracerebral drug infusions
The GABAA agonist muscimol was used to transiently suppress synaptic
activity in the vicinity of the focal infusion site. Previous work
(Martin, 1991 ; Martin and Ghez, 1999 ) showed that a 1 �l infusion of
MUS affected a sphere of tissue 2 mm in diameter surrounding the site of
infusion. In other studies in our own laboratory, MUS in the same volume
and concentration as used in the present studies, when placed into sites
2 mm apart, induced distinct changes in motor function (Dybdal et al.,
1997 ) or social behavior (Lower et al., 2002 ; Wellman et al., 2004 ).
The infusions in these studies were aimed at the BLA (Fig. 2 A), which
includes basal, accessory basal, and lateral nuclei (Amaral et al., 1992
), bilaterally. MUS (9 nmol in 1 �l; Sigma, St. Louis, MO) or sterile
saline (0.9% NaCl solution) of the same volume was infused at a rate of
0.2 �l/min. The drug infusions were performed using an aseptic technique
while the monkey was seated in a primate chair (Crist Instrument
Company, Hagerstown, MD) with minimal restraint. The entire infusion
procedure lasted 15 min.
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Figure 2.
A, Coronal sections showing the intended
infusion site (shaded) in both the left and right
basolateral amygdala. Numerals indicate the distance
in millimeters from the interaural plane. B,
Photomicrograph of Nissl-stained coronal sections
through the basolateral amygdala of the animal OH,
which was used for histological evaluation (see
Materials and Methods), at approximately the same
level as that in A showing the
infusion cannula tracts bilaterally. The additional
cannula tracts visible laterally on each side are
from drug infusions into perirhinal cortex done in a
previous experiment (Malkova et al., 2001). |
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Injection site verification
We performed a detailed histological analysis on one monkey (OH), to
validate the accuracy of the MRI-based calculation of injection sites.
For histological processing, the monkey was given an overdose of
barbiturate (sodium pentobarbital, 100 mg/kg, i.p.) and perfused through
the heart with normal saline, followed by aldehyde fixatives. The brain
was removed from the skull, and the tissue was sectioned in the coronal
plane at 50 �m on a freezing stage microtome. Every section through the
amygdala was mounted, defatted, stained with thionin, and coverslipped.
A photomicrograph of a representative section showing cannula placement
is shown in Figure 2 B.
The placement of the cannula documented in the histological sections was
coaligned with the placement of the microelectrode registered on the MRI
scan. After adjustments were made for the tissue shrinkage associated
with fixation, the target site as determined on the MRI and the cannula
artifact visualized on tissue sections coincided with an error of less
than �0.5 mm. (The remaining five animals are still undergoing
additional testing and were not available for histological processing.)
Apparatus and materials
The monkeys were trained in a Wisconsin General Testing Apparatus
located in a darkened sound-shielded room. The test compartment was
illuminated with a 15 W fluorescent bulb (Philips F15T8-CW; Philips
Electronics, New York, NY), but the monkey's compartment was always
unlit. The test tray, which was located at the level of the floor of the
monkey transport cage, contained three food wells spaced 18 cm apart,
center to center on the midline of the tray. The wells were 25 mm in
diameter and 5 mm deep. For the present task, only the two outer wells
were used. The stimuli were 120 junk objects that differed widely in
shape, size, color, and texture. There were two different food rewards.
One was one-half of a "fruit snack" (food 1), a chewy candy made from
fruit juice, 10 mm in size (Safeway, Pleasanton, CA), and the other was
one-half of a peanut (food 2). Based on previous studies (Malkova et
al., 1997 ), these two food rewards were found to be equally palatable.
In addition, before initiation of the behavioral training, we tested
that the monkeys were readily taking both rewards and replaced any
reward if it appeared undesirable. For one monkey, the fruit snack was
replaced by a piece of dried pineapple of the same size (Brown's Fruit
Bites, Sinking Spring, PA) and for two monkeys, the peanut was replaced
by a single Cheerio (Cheerios; General Mills, Minneapolis, MN).
Behavioral testing procedure
Overview. Postoperatively, all monkeys were trained on a task described
previously by Malkova et al. (1997 ). The behavioral procedure has three
phases. In phase I, the animals are trained to form an association
between objects and food rewards. One food is presented with a set of 30
objects that are always baited with that specific food and are each
simultaneously presented with one of a set of 30 nonrewarded objects;
this is done for two separate foods, each with its own set of 30 object
pairs. The animals are trained to criterion on the two different sets of
food-object associations, intermixed within a session. Once they have
been trained to criterion on the object discrimination, their object
preference under baseline conditions is evaluated in a "probe session"
in which the animals are allowed to chose between two objects, each of
which is derived from one of the two sets (e.g., yellow block covering
peanut vs blue bowl covering fruit snack). By repeating this choice with
30 different rewarded object pairs, it is then possible to generate a
preference score for each food-object set without repeating any objects.
In phase II, the animals are given unlimited access to one of the two
foods in their home cage until they refuse additional offers of that
food. This procedure in monkeys and in humans is called "selective
satiation" (Malkova et al., 1997 ; Thornton et al., 1998 ; Baxter et
al., 2000 ; Gottfried et al., 2003 ; Izquierdo and Murray, 2004a ,b ;
Izquierdo et al., 2004 ) [a similar procedure in rats has been called
"specific satiety treatment" (Balleine and Dickinson, 2000 )], an
operational term defined as the process of allowing the monkeys to eat
ad libitum only one of the two food rewards. This procedure results in
the loss of interest in whichever food was presented during selective
satiation; this is referred to as "devaluation." The devaluation of a
food transfers from the home cage to the testing apparatus: when the
monkeys are presented with 30 pairs of food rewards (food 1 vs food 2),
they avoid the devalued food rewards in favor of the nondevalued reward
(Izquierdo et al., 2004 ). In phase III, the animals are evaluated in a
probe session for object preference. Under control conditions, many of
the objects associated with the sated (devalued) food are rejected in
favor of the objects associated with the nonsated food, resulting in a
clear shift in the preference score. This shift occurs regardless of
which food is used for selective satiation. The rejection of an object
associated with the sated food indicates that the devaluation of the
food has successfully been transferred to the object. In our
experimental design, we inactivated BLA either just before the satiation
session (i.e., during phase II) or immediately after satiation and
before the testing of preference in the probe session (i.e., during
phase III). We reasoned that, if the BLA is necessary for the
registration or encoding of the devaluation in a manner that can be
subsequently transferred to the objects, then inactivation during
satiation in phase II will impair the shift in object preference
measured in phase III. Furthermore, if the BLA is necessary for either
the memory of the devaluation or the transfer of the devaluation to the
objects, then inactivation during phase III will impair the shift in
object preference. Conversely, if BLA inactivation during phase III does
not impair the shift in object preference, then it will indicate that
BLA is not required for either the memory of the devaluation or its
association with the objects.
Phase 1: object discrimination training. Monkeys were first trained to
discriminate 60 pairs of objects. In each pair, one object was
designated as positive (i.e., baited with a food reward) and the other
was designated as neutral (i.e., unbaited), with one-half of the
positive objects (30) baited with food 1 and the other half baited with
food 2. The monkeys were trained at a rate of one session per day, 5
d/week, until they reached criterion, which was set at a mean of 90%
correct responses over 5 consecutive days (i.e., 270 correct responses
of 300).
Phase II: reinforcer devaluation by selective satiation. Approximately
24 h after the last feeding, a food box attached to the monkey's home
cage was filled with one of the food rewards (either food 1 or food 2)
of measured quantity while the monkey was in its home cage. The monkey
was allowed to eat the food without being directly observed for 25 min.
The experimenter then entered the room and checked the amount of food
eaten. In all cases, there was food still remaining in the food box. The
experimenter then observed the monkey from outside of the housing room
until the monkey did not take any more food for a 5 min period. At that
time, the food box was removed, and the amount of remaining food was
measured. In all cases, 30 min was a sufficient time to complete this
procedure. Each monkey received each of the following treatments before
phase II: (1) MUS, (2) saline, and (3) "no infusion." MUS was infused
with both food 1 and food 2 sessions; saline was infused once with one
of the foods, food 1 in OH, OK, OG, and ZC and food 2 in JN and GR. As
indicated in Figure 3, each of the two foods was used for satiation on
three occasions. Within the randomized schedule, selective satiation
with food 1 and food 2 alternated between weeks so that at least 2 weeks
intervened between repeated satiation with the same food.
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Figure 3.
Behavioral testing schedule. Days 1-7 represent a
sequence of behavioral testing for each of the six
infusions (a-f). |
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Phase III: experimental probe sessions. All phase II treatment
conditions are tested in phase III. Each monkey received each of the
following treatments before phase III: (1) MUS, (2) saline, and (3) no
infusion. For the no-infusion treatment, the probe sessions (phase III)
followed immediately after the satiation. For MUS and saline, the
intracerebral infusions were administered just before the probe
sessions. MUS was infused with both food 1 and food 2 for each animal,
and saline was infused with food 2 in OH, OK, OG, and ZC and food 1 in
JN and GR.
The timing schedule for the two different infusion probe sessions is
presented in Figure 4, A and B. As indicated, the selective satiation
procedure lasted 30 min; the time required for transport and infusion
was 25 min, of which 15 min was infusion time.
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Figure 4. Time
schedule for infusion sessions. A,
B, Timing of the infusion sessions when
the muscimol or saline was infused before (A)
and after (B) selective satiation.
C, D, Timing of the
follow-up control experiments. |
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Experimental testing schedule. As presented in Figure 3, on day 1, the
monkey was given a practice session on the food-object association task
with the original 60 pairs of objects. On day 2, the monkey was given a
baseline probe session with the rewarded objects (each choice was
between a food 1 rewarded object and a food 2 rewarded object) to
determine the baseline number of choices of food 1 and food 2 objects.
On day 3, the monkey was again given a practice session on the
food-object association task with 60 pairs of objects. On day 4,
according to a randomized schedule, the monkey was infused with MUS or
saline either in phase II or in phase III and subsequently given an
infusion probe session to determine the number of choices of food 1 and
food 2 objects. The monkey was then given at least 2 d of rest without
any testing before this testing schedule began again. Each monkey
completed six of these testing schedules (Fig. 3, a-f), typically over a
period of 6 weeks.
The practice sessions (days 1 and 3) with the original 60 pairs of
objects were administered to ensure that the effects of the satiation
procedure did not carry over to the subsequent probe sessions.
Furthermore, it was important to be sure that the repeated devaluations
of each food did not alter the monkey's choices of objects during the
baseline probe sessions. Therefore, each infusion probe session (day 4)
was compared with its preceding baseline probe session (day 2). In
addition, to avoid the possibility that the repeated devaluation might
affect the monkeys' choices and also their willingness to eat a large
amount of the same food repeatedly, the saline infusions were limited to
one infusion in phase II with one of the foods and one infusion in phase
III with the other food.
"Difference score" for measuring devaluation. Previous studies have
found that monkeys often consistently prefer one type of food reward
versus the other when the same task is used [e.g., mean ratio of choices
for control and operated monkeys, respectively: 22:8 and 19:11 in
Malkova et al. (1997 ); 23:7 and 25:5 in Thornton et al. (1998 ); and
21:9 and 25:5 in Baxter et al. (2000 )]. Because the number of possible
choices in a probe session is constant (i.e., 30), the number of chosen
food 1 objects limits the choice of food 2 objects. Given an
asymmetrical baseline, satiation with one ("preferred") food can result
in a stronger devaluation effect. To overcome the asymmetry, ensure
maximum statistical power, and conform to the previous literature
(Malkova et al., 1997 ; Baxter et al., 2000 ; Izquierdo and Murray,
2004a ,b ; Izquierdo et al., 2004 ), we combined the difference scores
obtained from each devaluation to generate cumulative difference scores.
We recognize that one food may contribute significantly more to the
overall effect than the other food. However, if, under control
conditions, the devaluation effect shows the same trend for each of the
foods, then the cumulative effect will be a valid representation of the
response to satiation. In view of this fact, we planned our analysis to
take the possible asymmetry into account.
Thus, in each probe session, the number of food 1 and food 2 objects
chosen was recorded for each monkey. The number of objects rewarded with
the devalued (sated) food chosen in each drug-treatment probe session
was then subtracted from the number of objects rewarded with that same
food chosen in the preceding baseline probe session, resulting in a
difference score (expressed as a negative number to reflect the
direction of the change). A greater devaluation effect is reflected in a
larger difference score. For MUS infusion in phase II, the difference
score was assessed for each satiation (food 1 and food 2) separately,
and the two values were then added to obtain a cumulative difference
score. Similarly, the cumulative difference score was obtained for MUS
infusion in phase III. For the purpose of data analysis, all saline
infusion results for each animal (both phase II and phase III) were
pooled to generate a cumulative difference score; this ensured that both
foods were represented in the control condition. The differences between
the MUS infusion in phase II, phase III, and saline were analyzed by
within-subject ANOVA with repeated measures. All statistical comparisons
were within subjects.
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