Methods

Animals

Experiments were conducted on male Wistar rats (Harlan Laboratories, Indianapolis, IN, 275–300 g). They were individually housed in a temperature controlled room (72°F) on a 12-hr day/night cycle and were given food and water ad libitum.

Surgical Techniques

Arterial catheterization.

Animals were given atropine (1 mg/kg) and anesthetized with pentobarbitol (50 mg/kg). Arterial catheters were made up of 5 cm of 0.01 in. Tygon tubing (Fisher Scientific, Pittsburgh) inside 30 cm of 0.02 inches using cyclohexanone to fuse the tubing together. The 0.01-inch tubing was inserted into the artery while the 0.02-inch tubing was routed s.c. to the dorsal aspect of the neck where it was secured with a leather jacket as previously described (Sanders and Shekhar, 1991). To improve patency, catheters were soaked in heparinized saline (2.5 U/ml) prior to insertion and filled with the same heparinized saline after placement.

Implantation of chronic injection cannulae into the BLA.

Immediately after catheterization, animals were placed into a stereotaxic instrument (Kopf Instruments, Tujunga, CA) with the incisor bar set at -3.3 mm. Two stainless steel guide cannulae (26 gauge, 10 mm length) were fixed onto the stereotaxic arms and then lowered into position of the BLA using the coordinates (A, -2.0; L, +5.0; V, -8.0) according to the atlas of Paxinos and Watson (1986). The guide cannulae were secured in place using three 2.4-mm stainless steel screws anchored to the skull and cranioplastic cement. The guide cannulae were sealed with dummy cannulae (Plastic Products, Roanoke, VA). Animals were removed from the stereotaxic instrument and allowed 72 hr to recover.

Intracranial Drug Infusions

Acute microinjections of drugs into the BLA, utilizing injection cannulae (33 gauge) that fitted into and extended 1 mm beyond the guide cannulae, were delivered bilaterally in 100 nl of a-CSF. A 10-μl Hamilton syringe placed on an infusion pump (Sage Instruments, Boston, MA, model 355) was connected to the injection cannulae via polyethylene (PE-50) tubing (Fisher Scientific, Pittsburgh, PA). The pump was subsequently turned on for 30 sec during which time the 100 nl of solution per site was delivered. The injection cannulae remained in place for an additional minute before being removed. Smooth flow of the solutions via the tip of the injection cannulae was ascertained before and after each injection to ensure drug delivery. The drugs used in i.c. injections (all obtained from RBI, Natick, MA) were the GABA_A antagonist BMI, the competitive NMDA receptor antagonist AP5 (± 2-amino-5-phosphopentanoic acid), the noncompetitive antagonist dizocilpine (+ MK-801), the competitive AMPA(α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)/kainate antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and the noncompetitive AMPA antagonist GYKI 52466.

Behavioral Measurement (Social Interaction)

Experimental anxiety was measured by the SI test, a fully validated test of anxiety (File, 1980) which has been previously used in our laboratory (Shekhar and Katner, 1995a, b). The apparatus used was a solid wood box 36“ L × 36” W × 12“ H with an open roof. A video camera was fixed above the SI box and all behavioral tests were recorded. During the test session, the ”experimental“ rat was placed into the social interaction box with a ”partner“ rat for a total of five minutes. The ”partner“ rat was another male Wistar rat that was housed individually and had never previously encountered the experimental rat. The amount of time the ”experimental“ rat spent interacting, i.e., making physical contact (grooming, sniffing, crawling upon, etc.) with the ”partner“ rat was recorded. Sessions were scored at a later time by two raters of whom at least one was blind to any drug treatment. Inter-observer reliability for the time of social interaction has been 0.9 to 0.97 in our laboratory. A decrease in interaction time was taken as an increase in ”anxiety“ and vice versa.

Experimental Protocol

First a total of 70 rats were implanted with bilateral injection cannulae in the BLA. From this group, 48 animals were successfully implanted with bilateral cannulae into the BLA and were used in the final data collection. Seventy-two hours after recovery, rats were divided into eight groups (n = 6 each). Each group was then assigned to one dose of an antagonist. On experimental day 1, the arterial catheter of the rat was connected to a Beckman R511 Dynograph in order to monitor HR and BP and the injection cannulae were placed into the guide cannulae of the rat. All animals were given sufficient time for a steady baseline of their HR and BP to be reached before any injections were made into the BLA. Each rat was then injected bilaterally with either a-CSF 100 nl/site, 20 pmol BMI in 100 nl of a-CSF/site, or 20 pmol BMI plus the assigned dose of one of the antagonists in 100 nl a-CSF/site. This procedure was repeated on experimental days 3 and 5 to complete all three injections. The order of the three injections was randomized among animals using a latin square design for each group. The doses of EAA antagonists used were AP5: 20 and 100 pmol; dizocilpine: 25 and 125 pmol; CNQX: 10 and 50 pmol; and GYKI 52466: 50 and 250 pmol. HR and BP were monitored for 15 min immediately after each injection and the maximum change during this interval was noted. Rats were allowed to move freely in their home cages during this period. After the physiological data were collected, the animals were immediately placed in the SI test for 5 min of behavioral assessment. In rats receiving the highest doses of AP5 (100 pmol) and CNQX (50 pmol), only HR and BP were measured and social interaction were not obtainable due to technical difficulty.

Another additional group of eight rats were also implanted with bilateral BLA cannulae as well as arterial and venous catheters. After recovery, one group (n = 4) received either a-CSF 100 nl/site, 20 pmol of BMI in 100 nl of a-CSF, 20 pmol of BMI + 100 pmol of R-AP5 (active isomer) or 20 pmol of BMI + 100 pmol L-AP5 (inactive isomer). This was done to test if the AP5 response is stereospecific and not due to nonspecific factors such as osmolarity, pH or other mechanical effects. In the second group (n = 4), rats were injected bilaterally into the BLA with either a-CSF, CNQX (50 pmol) alone, CNQX (50 pmol) + AMPA (500 pmol) in 100 nl of a-CSF/site or CNQX (50 pmol) + NMDA (500 pmol) in 100 nl of a-CSF/site and the response of the animal in the SI test was measured. This experiment was conducted to determine if the effects of the highest dose of CNQX used in this study would still be specific to blocking the AMPA receptors as opposed to the NMDA receptors.

Histology

On completion of the experiment, animals were anesthetized with halothane and 100 nl of 50% solution of India ink was injected via the amygdalar cannulae. They were then immediately sacrificed using a guillotine. The brains were quickly removed and placed in 0.9% saline. They were then immediately frozen in a cryostat, sectioned (40 μm) and mounted on slides. Slides were subsequently stained with Neutral Red to determine the location of the i.c. injection by comparing them with the atlas of Paxinos and Watson (1986). Only data from the rats that had successful implants into the BLA were used in the analyses.

Statistical Analysis

The results of the HR and BP data were recorded as the maximal change from baseline that occurred during the 15 min of monitoring. Social interaction data were converted to percent change in social interaction time as compared to a-CSF (which was represented as 100% for each rat). All data are presented as mean ± S.E.M. A mixed ANOVA with dose of antagonist (high or low) as a nested factor and treatment (a-CSF, BMI or antagonist) as the within factor was conducted. A least square mean post hoc test was calculated on the significant interactions or main effects. In the case of SI measurements after AP5,CNQX and CNQX + AMPA or NMDA treatment data as well as all data analysis of the AP5 inactive isomer group, a repeated measure ANOVA with a Newman-Keuls post hoc test was conducted. All calculated F values and subsequent post hocanalyses met an α = 0.05 criteria. Computerized statistical programs (SAS 6.12) were used and a statistician was available for consultation.