Grant Number: 2R01MH041479-19A1
Project Title: Anatomy of the Primate Amygdaloid Complex
PI Information: PROFESSOR DAVID G. AMARAL,
Abstract: DESCRIPTION (provided by applicant):
The primate amygdala is a complex brain region comprised of 13 nuclei
and cortical regions in the rostral portion of the medial temporal lobe.
This grant has funded studies with the overarching goal of defining the
cytoarchitectonic organization and intrinsic and extrinsic connections
of the macaque monkey amygdaloid complex.
We have also been investigating neuropathology in the autistic
amygdala. We reported that the amygdala in typically developing boys
undergoes a 40% increase in volume between 7 and 18 years of age.This
expansion occurs at a time when the cerebral volume decreases by about
10%. In boys with autism, the amygdala reaches its adult size by 7 years
and does not increase thereafter.
Given the association of the amygdala with a variety of psychiatric
disorders including anxiety, depression, autism and schizophrenia, many
of which are first manifest during the peripubertal period, it would be
valuable to determine the morphological features of the amygdala's
It is not feasible, however, to carry out this type of analysis in
postmortem human brains. Thus, with this renewal we will begin studies
to investigate the postnatal development of the macaque amygdala.
First, we will carry out a longitudinal magnetic resonance imaging
study of the brains of developing male and female rhesus monkeys to
determine whether the dramatic and disproportionate growth of the
amygdala in boys is also a feature of macaque development.
Second, we will quantitatively analyze the emergence of species
typical behaviors in the imaged animals.
Third, we will implement modern design-based stereological techniques
to measure the volume, count neuron number, and characterize neuron
morphology in the amygdala throughout postnatal development.
Fourth, we will use histochemical and immunohistochemical techniques
to characterize the postnatal development of four major neurotransmitter
systems (i.e., glutamatergic, GABAergic, cholinergic and serotonergic)
in the amygdala. In addition, we will investigate the development of
myelination and the expression of non-phosphorylated neurofilaments and
synaptic markers (synaptophysin).
Finally, we will use the Golgi technique and intracellular labeling
of neurons in the in vitro slice preparation to quantify the maturation
of dendrites in the major nuclei of the amygdaloid complex.
This work provides essential normative data to study influences like
circulating hormones or social experience on amygdala maturation.
amygdala, animal developmental psychology, autism, brain mapping, brain
morphology, developmental neurobiology, neurochemistry, neurogenesis,
neurotransmitter dendrite, gamma aminobutyrate, gender difference,
myelin, psychopathology, serotonin, synaptophysin Macaca mulatta,
behavioral /social science research tag, clinical research,
immunocytochemistry, magnetic resonance imaging, neuroimaging,
Institution: UNIVERSITY OF CALIFORNIA DAVIS
OFFICE OF RESEARCH - SPONSORED PROGRAMS
DAVIS, CA 95618
Fiscal Year: 2006
Department: PSYCHIATRY & BEHAVIORAL SCIENCES
Project Start: 01-SEP-1986
Project End: 31-JUL-2011
ICD: NATIONAL INSTITUTE OF MENTAL HEALTH
The Journal of Neuroscience, July 11, 2007, 27(28):7386-7396;
in Fear-Potentiated Startle: Effects of Chronic
Lesions in the Rhesus Monkey
Elena A. Antoniadis,1,2 James T.
Winslow,4 Michael Davis,5,6
and David G. Amaral1,2,3
1Department of Psychiatry and
Behavioral Sciences, 2California
National Primate Research Center, and 3Medical
Investigation of Neurodevelopmental Disorders Institute, University of
California, Davis, Davis, California 95616, 4National
Institute of Mental Health, Bethesda, Maryland 20842, and
5Yerkes National Primate Research Center and
6Department of Psychiatry and
Behavioral Science and Center for Behavioral Neuroscience, Emory
University, Atlanta, Georgia 30320
Subjects and living arrangements
The 18 adult male rhesus monkeys (Macaca mulatta) used in this study
were born and mother reared at the California National Primate Research
Center (CNPRC) in outdoor half-acre enclosures and lived among a group
of conspecifics in troops ranging from 70 to 120 monkeys.
The subjects were all relocated from outdoor cages to indoor CNPRC
housing at the same time and were habituated to the new conditions.
Monkeys were housed individually in cages (28 x 22 x 46 inches). The
rooms were automatically regulated on a 12 h light/dark cycle with
lights on at 6:00 A.M. and off at 6:00 P.M., and room temperature
maintained at 75–85°F. The subjects were fed a diet of monkey chow
(Ralston Purina, St. Louis, MO) supplemented with fruit and vegetables
and ad libitum water.
They were randomly assigned to either receive bilateral ibotenic acid
lesions of the amygdala (amygdala group, n = 6) or of the hippocampus
(hippocampus group, n = 6) or to serve as the operated control group (n
= 6). The hippocampus lesion group was included as a medial temporal
lobe lesion control group.
Before the fear-potentiated startle experiment, these monkey cohorts
had been tested on a set of socio-emotional tasks including emotional
responsiveness, dyadic social interaction (Mason et al., 2006 ), and
human intruder (Emery et al., 2001 ). After the fear-potentiated startle
experiment, animals were tested on a spatial learning task (Banta-Lavenex
et al., 2006 ).
Magnetic resonance imaging. Animals were anesthetized
individually with ketamine hydrochloride (10 mg/kg, i.m.) and
medetomidine (25–50 µg/kg, i.m.) and were then placed in a magnetic
resonance imaging (MRI)-compatible stereotaxic apparatus (Crist
Instrument, Hagerstown, MD).
After scan completion, the medetomidine was reversed with atipamazole
(0.15 mg/kg, i.m.). MRI scans served as brain atlases and were used to
generate individualized injection coordinate matrices. T1 images were
exported to Photoshop (version 5; Adobe Systems, San Jose, CA) and then
Canvas (version 5; Deneba System, Miami, FL), to superimpose a
calibrated grid that was used to calculate injection coordinates.
Lesion surgery: ibotenic acid injections. Anesthesia was
induced with ketamine hydrochloride (10 mg/kg, i.m.), after which the
animals were maintained on isoflurane (1.2–2%). After reaching a
surgical anesthesia level, the animal was placed in the stereotaxic
apparatus. Fentanyl (7–10 mg/kg/min, i.v.) was administered in
combination with isoflurane to provide a stable level of anesthesia
throughout the surgical procedure.
Using sterile procedures, the skull was exposed, and openings were
made dorsal to the amygdala or to the hippocampal formation.
The dorsoventral location of the amygdala or the hippocampus was
verified electrophysiologically by lowering a tungsten microlectrode
into the locations calculated initially by the MRI analysis. Adjustments
were made according to salient electrophysiological features of the
spontaneous neuronal activity of the amygdala and hippocampus.
Two identical 10 µl (26 gauge beveled needle) Hamilton syringes were
used to simultaneously infuse ibotenic acid (10 mg/ml in 0.1 M PBS;
Biosearch Technologies, Novato, CA) into each amygdala or each
A unilateral amygdala lesion required three to four rostrocaudal
injection planes, each with one to four mediolateral levels and one to
three dorsoventral injection sites. A unilateral hippocampal lesion
required seven to eight rostrocaudal injection planes, each with one to
two mediolateral levels and one to two dorsoventral injection sites.
One microliter was infused into each injection site at 0.2 µl/min,
for a total of 13–25 µl per amygdala or 10–16 µl per hippocampus. For
all operated animals, the ibotenic acid injections were followed by (1)
suturing of the dura, (2) filling the craniotomy with GelFoam (Amersham
Biosciences, Peapack, NJ), and (3) suturing of the facia and skin in
The six sham-operated control animals had the same presurgical
preparations, midline incision, and skull exposure. They were
anesthetized for the average lesion surgery duration and had facia and
skin suturing in two layers. Postsurgical care for all experimental
groups included vital sign monitoring as well as administration of
antibiotics and analgesics when deemed necessary by veterinary staff.
Postoperative T2-weighted scans: lesion verification. Ibotenic
acid-induced edema appears as a hyperintense signal in T2-weighted MR
images and is used as a general indication of the injection locus
(Saunders et al., 1990 ; Malkova et al., 2001 ). After a 10–14 d
recovery period, animals underwent a second MRI procedure, and
T2-weighted signals for each of the 12 lesion subjects were evaluated to
confirm the location of the lesion.
General experimental procedure
At the time of the experiment ( 4.5 years after the lesions had been
made), the mean age was 11.4 ± 0.4 years in the control group, 11 ± 0.6
years in the amygdala group, and 11.7 ± 0.6 years in the hippocampus
group. The mean weight was 13.1 ± 0.6 kg in the control group, 12.8 ±
0.9 kg in the amygdala group, and 12.2 ± 0.4 kg in the hippocampus
Each monkey was provided a primate collar (Primate Products, Miami,
FL) and underwent daily pole and collar training for 60 d to permit
habituation to the primate restraint chair. Aluminum transport cages
(0.5 x 0.03 x 0.04 m) were used for transferring subjects from the
colony home cage to the experimental room.
For testing order, all subjects were randomly assigned to one of
three, six-animal cohorts. Each cohort was composed of two subjects from
the amygdala group, two from the hippocampus group, and two from the
control group. Cohort 1 was tested on day 1, cohort 2 was tested on day
2, and cohort 3 was tested on day 3. Testing order was fixed across
experimental phases, with each animal tested at the same time each day.
Time of day was counterbalanced among the groups so that every
experimental time slot was occupied by at least one animal from each
The rodent fear-potentiated startle apparatus (Cassella and Davis, 1986
) modified for primate research is detailed and depicted in the study by
Winslow et al. (2002) .
Briefly, a custom-built primate restraint chair within which the
monkey was comfortably positioned for startle response recordings was
enclosed within a ventilated, light- and sound-attenuated wooden
chamber. The restraint chair was secured on the upper panel of a
Startle amplitude was measured with an accelerometer (model 7201-50;
Endevco Corporation, San Juan Capistrano, CA) that was center mounted
underneath the upper panel (60 x 40 x 1.91 cm). The two panels were
bolted together and separated by heavy compression springs that
maintained an interpanel distance of 10 cm. A rubber stopper (6.57 cm
diameter) was mounted on a 5.08 cm plastic block resting on the lower
panel, located directly underneath the accelerometer. When the bolts
connecting the panels were tightened, the accelerometer was pressed
against the stopper, resulting in a highly dampened interface.
Movement of the restraint box, resulting from a whole-body startle
response, displaced the accelerometer and produced a signal that was
integrated by the Endevco amplifier (model 104). The resulting voltage
signal was proportional to the displacement velocity of the chair (Cassella
and Davis, 1986 ). This signal was digitized and fed to a Macintosh
computer and analyzed using custom software (Experimenter 3.0; Glass
Beads, Newtown, CT).
Startle response measurement was defined as the maximal peak
accelerometer output during the first 600 ms after the startle-eliciting
noise onset. Baseline activity was the maximal peak accelerometer output
during a similar 600 ms time window but 30 s after the startle-eliciting
noise offset (i.e., in the absence of any startle-eliciting noise).
The startle stimulus was a computer-generated burst of white noise
delivered through a wall-mounted speaker located 12 cm behind the
animal's head. The conditioned stimulus (CS) consisted of light
presentation for 4.2 s through four halogen lights (400 lux each) corner
mounted to the ceiling. The noxious unconditioned stimulus (US) was the
presentation of a 1.2 s, 100 pound per square inch compressed air burst
with the nozzle located 26 cm from the animal's face and neck.
Specific behavioral procedures
Phase I: baseline startle amplitude assessment. The animal was
transferred to the experimental chair and placed in the test chamber.
For the first 10 min of the baseline testing session, there were no
startle stimuli to let the animal adjust to the darkness, isolation, and
ambient noise (Cassella and Davis, 1986 ). At the end of the 10 min
adjustment period, a 50 min test session began. During this 50 min
period, blocks of startle stimuli consisting of white noise bursts (5–20
kHz) were presented at each of the following intensities: 80, 90, 100,
105, 110, 115, and 120 dB. There were seven blocks of the seven startle
stimuli, so the animal was exposed to 49 randomly presented noise bursts
at a 60 s intertrial interval (ITI).
Phase II: light test to measure unconditioned effects of the light
on startle amplitude. The animal was placed in the test chamber and for
the first 10 min was acclimated as described above. At the end of the 10
min period, a 20 min test session began. The 20 min session consisted of
20 startle stimuli at a 60 s ITI: 10 110 dB white noise bursts delivered
alone (noise-alone trials), intermixed with 10 110 dB white noise bursts
delivered 3.5 s after onset of a 4.2 s light. This test session was used
to evaluate whether the light would have any unconditioned facilitatory
or inhibitory effect on startle amplitude before its being paired with
the aversive air blast.
Phase III: fear-potentiated startle training and testing. The
animal was placed in the test chamber and for the first 10 min was
acclimated as described above.
After this, a 16 min session began that consisted of four training
trials randomly intermixed with 12 testing trials each separated by a 60
s ITI. A training trial was used to produce the association between the
light (CS) and the noxious air puff (US).
Each training trial was initiated by light onset and followed by an
air puff at one of the following delays: 1.5, 2.0, 2.5, or 2.7 s after
the onset of the light. US onset time was varied in an effort to make
the entire CS duration aversive (Davis et al., 1989 ).
Testing trials were of two types. Either a startle stimulus was
delivered alone or the startle stimulus was delivered 1.5 s after light
onset. When training trials are intermixed with testing trials,
relatively stable levels of fear-potentiated startle can be maintained
across repeated training–testing sessions (Winslow et al., 2007 ).
When the conditioned light came on, the animal did not know whether
it would be followed by a startle stimulus, to measure fear, or an
aversive air blast, to condition fear to the light.