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Stop Animal Exploitation NOW!
S. A. E. N.
"Exposing the truth to wipe out animal experimentation"

Government Grants Promoting Cruelty to Animals

University of California, Davis CA

DAVID G. AMARAL - Primate Testing - 2006

Grant Number: 2R01MH041479-19A1
Project Title: Anatomy of the Primate Amygdaloid Complex

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 postnatal development.

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.

Thesaurus Terms:
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, stereotaxic technique

DAVIS, CA 95618
Fiscal Year: 2006
Project Start: 01-SEP-1986
Project End: 31-JUL-2011

The Journal of Neuroscience, July 11, 2007, 27(28):7386-7396; doi:10.1523/JNEUROSCI.5643-06.2007

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 ).

Surgical procedures
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 hippocampus.

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 three layers.

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 group.

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 experimental group.

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 two-panel platform.

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.

Please email:  DAVID G. AMARAL,  to protest the inhumane use of animals in this experiment. We would also love to know about your efforts with this cause:

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Rats, mice, birds, amphibians and other animals have been excluded from coverage by the Animal Welfare Act. Therefore research facility reports do not include these animals. As a result of this situation, a blank report, or one with few animals listed, does not mean that a facility has not performed experiments on non-reportable animals. A blank form does mean that the facility in question has not used covered animals (primates, dogs, cats, rabbits, guinea pigs, hamsters, pigs, sheep, goats, etc.). Rats and mice alone are believed to comprise over 90% of the animals used in experimentation. Therefore the majority of animals used at research facilities are not even counted.

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