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Government Grants Promoting Cruelty to Animals

Georgetown University, Washington, DC

Ludise Malkova - Primate Testing - 2006

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:
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Fiscal Year: 2006
Project Start: 18-SEP-2006
Project End: 31-AUG-2008

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

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.

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.


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 Ál 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 Ál 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 Ál; Sigma, St. Louis, MO) or sterile saline (0.9% NaCl solution) of the same volume was infused at a rate of 0.2 Ál/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.

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).


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 Ám 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.

Figure 3. Behavioral testing schedule. Days 1-7 represent a sequence of behavioral testing for each of the six infusions (a-f).


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.  

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.


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. 

Please email:  LUDISE MALKOVA, [email protected]  to protest the inhumane use of animals in this experiment. We would also love to know about your efforts with this cause: [email protected]

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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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