Abstract
Blockade of γ-aminobutyric acid (GABAA) receptors in the anterior basolateral amygdala (BLA) with bicuculline methiodide results in an increase in heart rate, blood pressure and “anxiety” in rats. Glutamate receptors in the BLA are also reported to be involved in eliciting anxiety responses. The purpose of this study was to investigate the interaction between GABAergic inhibition and glutamatergic excitation in the BLA. Male Wistar rats were implanted with femoral arterial catheters and bilateral chronic microinjection cannulae into the BLA. Each animal was injected with either artificial cerebrospinal fluid (100 nl), bicuculline methiodide (20 pmol/100 nl) or bicuculline methiodide + one dose of an antagonist of either the N-methyl-d-aspartate receptor [AP5 (20 and 100 pmol) and dizocilpine (25 and 125 pmol)] or the non-N-methyl-d-aspartate ionotropic receptor [CNQX (10 and 50 pmol) and GYKI 52466 (50 and 250 pmol)]. Increases in heart rate, blood pressure and “anxiety” (as measured in the social interaction test) observed in rats after bicuculline methiodide injections into the BLA were blocked in a dose dependent manner with the concurrent injections of either N-methyl-d-aspartate or non-N-methyl-d-aspartate antagonists, suggesting that activation of both subtypes of glutamate ionotropic receptors may be necessary for the responses elicited by GABAA receptor blockade in the anterior basolateral amygdala.
Anxiety and stress are mental states that are characterized by emotional responses of fear and apprehension, along with physiological responses such as increases inHR, BP, respiration and gastrointestinal motility. Both animal studies (Hilton and Zbrozyna, 1963; Kapp et al., 1982) and human studies (Feindel and Penfield, 1954) have shown that stimulation of the amygdala results in behavioral and physiological responses associated with anxiety. In contrast, lesions of this area are associated with a decrease in fear and anxiety (Weiskrantz, 1956;Narabayashi et al., 1963). The basolateral nucleus in particular has been implicated in regulating “anxiety” in rats. Inhibiting the activity of the BLA with glutamate antagonists or lesions will block both the acquisition and expression of conditioned fear in rats (Miserendino et al., 1990; Kim and McGaugh, 1992; Campeau and Davis, 1995). Thus, it has been suggested that the BLA acts as an integration center for sensory and memory information in the anxiety response and that glutamate neurotransmission is critical in regulating these responses (LeDoux et al., 1990; Campeau and Davis, 1995).
Blockade of GABAA receptors in the anterior portion of the BLA leads to increases HR, BP as well as experimental “anxiety” as assessed in the SI and conflict tests (Sanders and Shekhar 1991; 1995a, b). In addition, this area of the amygdala contains the highest concentration of benzodiazepine receptors (Niehoff and Kuhar, 1983). The anxiolytic effects produced by systemic benzodiazepines can be reversed by direct injections of the benzodiazepine receptor antagonist flumazenil into the BLA (Sanders and Shekhar, 1995a). It appears that the GABA-benzodiazepine receptor complex in the BLA also plays a critical role in regulating anxiety responses.
Within the BLA, stimulation of the afferent projections will result in both a fast-excitatory postsynaptic potential (EPSP) that appears to be mediated via a non-NMDA ionotropic receptor of the AMPA/kainate type and a slow-EPSP mediated via the NMDA receptor (Rainnie et al., 1991a, b). In addition, there is also a GABAA-mediated IPSP which overlaps the NMDA-regulated slow-EPSP. The GABAergic inhibition appears to be critical in determining the excitability of the BLA neurons and the IPSPs can be blocked by glutamate antagonists and reduced in amplitude by an NMDA antagonist, suggesting the presence of a close interaction between GABA and glutamate neurotransmission in the BLA neurons. Based on such a circuitry, it appears that a balance between the EAA-mediated EPSPs and the GABAA receptor mediated IPSP ultimately determines the primary state of excitability of the BLA neurons.
Therefore, our study was conducted in order to test the relationship between the excitatory (i.e., glutamate) and the inhibitory (i.e., GABA) neurotransmission in the BLA in the regulation of “anxiety” in the rat. It was hypothesized that GABAA receptor blockade in the BLA would result in activation of one or more glutamate receptor subtypes leading to an anxiety response. In order to test this hypothesis, microinjections of the GABAA antagonist BMI alone were compared to injections of BMI with an excitatory amino acid antagonist (either NMDA or non-NMDA ionotropic types) given bilaterally into the BLA. Changes in physiological parameters of HR and BP along with behavioral measures assessed in the SI test were monitored.
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 GABAA 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.
Results
NMDA Antagonists
Competitive antagonist AP5.
Figure1A shows the changes in HR seen after intracranial injection of AP5 (20 or 100 pmol), a-CSF and BMI. Injections of the GABAA antagonist BMI alone as well as BMI with AP5 (20 pmol) showed a significant increase in HR as compared to a-CSF, although the changes in HR of animals administered BMI and AP5 (100 pmol) were significantly less than that of BMI alone. A mixed ANOVA with dose of AP5 as a nested factor revealed a significant difference among the treatments [F(2,33) = 39.67; P = .0001], as well as between groups [F(1,10) = 9.6632; P = .0107]. In addition, there was a significant interaction for group × treatment [F(2,19) = 4.7995; P = .0206]. In contrast to HR, changes in BP (fig. 1B) were not significantly different between the two groups nor was there a significant group treatment interaction. However, the changes in BP seen with AP5 (100 pmol), although not significant, was decreased as compared to BMI.
Changes in (A) heart rate (HR, beats/min), (B) blood pressure (BP, mm of HG), and (C) social interaction time (SI, % of a-CSF time in sec) elicited by bilateral injection of either a-CSF (100 nl), BMI (20 pmol/100 nl), BMI (20 pmol/100 nl) + AP5 (20 pmol/100 nl), or BMI (20 pmol/100 nl) + AP5 (100 pmol/100 nl) into the BLA of rats. Significantly different from *a-CSF and #BMI by a mixed ANOVA model with a nested design (using the antagonist dose as the nested factor) along with a least squares means test post hoc test, P < .05 for HR and BP analysis and by a one-way ANOVA with a Newman-Keuls post hoctest, P < .05 for SI analysis.
During this set of experiments a-CSF was used as the baseline for SI time, and as such, the percent increase in SI time after injections of AP5 (20 pmol only) and BMI (fig. 1C) showed a significant difference among the three treatment conditions using a one-way ANOVA [F (2,14) = 19.701; P = .0001]. Follow-up Newman-Keuls tests revealed that the 20 pmol dose of AP5 significantly increase SI time compared to both BMI and a-CSF conditions with a P < .05.
The effects of coadministering the different isomers of AP5 with BMI into the BLA are shown in Figure 2. As before, injecting BMI (20 pmol) bilaterally into the BLA elicited increases in HR (fig. 2A) and BP (fig. 2B) as well as decreases in SI time (fig. 2C) compared to a-CSF. Coadministering the active isomer of AP5 (R-AP5) completely blocked the BMI induced HR [F(3,12) = 19.6; P = .0001], BP [F(3,12) = 7.1; P = .007] and SI changes [F(3,12) = 8.3; P = .003], although L-AP5 (inactive isomer) had no significant effect, suggesting a stereo-specific receptor-mediated effect of AP5 in blocking the BMI effects.
Changes in (A) heart rate (HR, beats/min), (B) blood pressure (BP, mm of Hg), and (C) social interaction time (SI, % of a-CSF time in sec) elicited by bilateral injection of either a-CSF (100 nl), BMI (20 pmol/100 nl), BMI (20 pmol/100 nl) + L-AP5 (100 pmol/100 nl) or BMI (20 pmol/100 nl) + R-AP5 (100 pmol/100 nl) into the BLA of rats. Significantly different from *a-CSF and #BMI by one-way ANOVA with repeated measures and Newman-Keuls post hoctest, P < .05.
Noncompetitive antagonist dizocilpine.
Figure3A shows the effects of injecting dizocilpine into the BLA on HR of rats. Similar to the effects of the antagonist AP5, dizocilpine also showed a significant difference among the three treatments (a-CSF, BMI and BMI +dizocilpine) [F(2,33) = 36.37; P = .0001], as well as a main interaction effect between group and treatment [F(2,20) = 6.6867, P = .006]. Further analysis revealed significant increases in HR with microinjections of both BMI and the lowest dose of the antagonist dizocilpine as compared to the a-CSF injection. A significant difference among treatment groups was also seen as well for changes in BP [fig. 3B; F (2,33) = 4.50; P = .02]. Animals receiving injections with BMI had significant increases in BP when compared to the a-CSF condition, although these same rats given dizocilpine (25 pmol and 125 pmol) showed a significant reversal of the BMI effects as seen in the difference among treatments. Although both doses of dizocilpine decreased BP compared to the BMI condition, the 25 pmol dose reversed the BP back to changes equal to that of a-CSF, and 125 pmol elicited a decrease below that of baseline.
Changes in (A) heart rate (HR, beats/min), (B) blood pressure (BP, mm of Hg), and (C) social interaction time (SI, % of a-CSF time in sec) elicited by bilateral injection of either a-CSF (100 nl), BMI (20 pmol/100 nl), BMI (20 pmol/100 nl) + dizocilpine (25 pmol/100 nl) or BMI (20 pmol/100 nl) + dizocilpine (125 pmol/100 nl) into the BLA of rats. Significantly different from *a-CSF and #BMI by a nested design mixed ANOVA with a least squares means post hoc test, P < .05
Animals administered dizocilpine and tested in SI showed a significant difference in their interaction time among treatment groups [fig. 3C; F (2,33) = 6.07; P = .0087]. Similar to BP, the BMI treatment was significantly decreased compared to a-CSF, although the antagonist showed an incremental increase in SI time.
Histology.
Figure 4 shows the schematic representation of the injection cannulae implantation sites in the BLA of rats used in the AP5 and dizocilpine study. An actual histological section (inset) is also shown. All of these were sites where microinjections of BMI elicited significant increases in HR and BP.
Schematic representation of the sites of cannula implantation within the BLA of rats used in the AP5 (open circles) and MK-801 (closed circles) microinjection experiments as verified by histological examination. Abbreviations: BLA, anterior basolateral amygdala; BLP, posterior basolateral amygdala; BLV, ventral basolateral amygdala; BMA, anterior basomedial amygdala; BMP, posterior basomedial amygdala; Ce, central nucleus of the amygdala; La, lateral nucleus of the amygdala; LaDL, lateral nucleus of amygdala, dorsolateral division; LaVL, lateral nucleus of amygdala, ventrolateral division.
Non-NMDA (AMPA/Kainate) Antagonists
Competitive antagonist CNQX.
The HR changes seen in the animals of the CNQX group are given in figure5A. Statistical analysis showed significant differences among the a-CSF, BMI and BMI + CNQX treatments [F(2,33) = 27.81; P = .0001], between the groups [F(1,10) = 27.2247; P = .0004] and an interaction effect between group and treatment [F(2,20) = 6.5824; P = .0064]. Post hoctesting further revealed a significant increase in HR when BMI and CNQX (10 pmol) were administered to the animals as compared to a-CSF; however, rats receiving the highest dose of CNQX showed significant inhibition of HR increases due to BMI. Similarly, there were significant differences in the changes in BP (fig. 5B) among the three treatment conditions [F (2,33) = 13.21; P = .0002]. Again, the BMI condition elicited a significantly greater increase in BP as compared to a-CSF, although the CNQX (10 and 50 pmol) treatments were able to significantly reverse this effect. The lowest dose of CNQX was only able to partially reverse the BMI effect although the highest dose completely blocked the HR increase. SI (fig. 5C) also showed a significant difference in interaction time among the three treatment conditions [F (2,10) = 11.66; P = .0024] with the Newman-Keulspost hoc showing both BMI and CNQX (10 pmol) being significantly less than that of a-CSF.
Changes in (A) heart rate (HR, beats/min), (B) blood pressure (BP, mm of Hg), and (C) social interaction time (SI, % of a-CSF time in sec) elicited by bilateral injection of either a-CSF (100 nl), BMI (20 pmol/100 nl), BMI (20 pmol/100 nl) + CNQX (10 pmol/100 nl) or BMI (20 pmol/100 nl) + CNQX (50 pmol/100 nl) into the BLA of rats. Significantly different from *a-CSF and #BMI by a nested design mixed ANOVA with a least squares means test post hoc test, P < .05 for HR and BP analysis and a one-way ANOVA with a Newman-Keuls post hoc test, P < .05 for SI analysis.
Injecting CNQX alone in another group of rats placed in the SI test revealed that CNQX by itself had an “anxiolytic” effect (a-CSF = 100%; CNQ × 50 pmol = 257%) without significant changes in HR or BP. When CNQX (50 pmol) was coadministered with either AMPA (500 pmol/100 nl) or NMDA (500 pmol/100 nl) in the same animals, only AMPA was able to completely prevent the CNQX-mediated increases in SI time (decreased to 68% compared to a-CSF), although NMDA had no effect (300% compared to a-CSF).
Noncompetitive antagonist GYKI 52466.
Figure6A represents the changes seen in the HR of animals administered the antagonist GYKI. These animals showed a significant difference among treatments [F (2,33) = 41.76; P = .0001]. In addition a significant difference was observed between the groups [[F(1,10) = 7.9380; P = .0182] as well as a main effect [F(2,20) = 18.7884; P = .0001]. Similar to the competitive antagonist animals, GYKI at the lowest dose was similar to BMI in that both treatments yielded significantly greater increases in HR as compared to a-CSF, although the highest dose of the compound was able to significantly prevent the BMI increases and was no different than a-CSF-treated rats. Significant changes in BP (fig. 6B) were seen between the two groups [F(1,10) = 8.7262; P = .0144]. Furthermore, there was a significant interaction effect between group x treatment [F(2,20) = 6.8066; P = .0056]. Figure 6C shows that when SI changes were measured animals administered the highest dose of GYKI showed a significant increase in SI time as compared to BMI. In addition, both BMI and GYKI (50 pmol) treatments elicited significant decreases in the rats’ interaction time as compared to baseline,i.e., a-CSF treatment. Statistical analysis showed both a significant treatment effect [F(2,20) = 6.7123; P = .0059] and an interaction effect between group and treatment [F(2,20) = 13.0231; P = .0002] for the non-NMDA antagonist as well.
Changes in (A) heart rate (HR, beats/min), (B) blood pressure (BP, mm of Hg), and (C) social interaction time (SI, % of a-CSF time in sec) elicited by bilateral injection of either a-CSF (100 nl), BMI (20 pmol/100 nl), BMI (20 pmol/100 nl) + GYKI 52466 (50 pmol/100 nl) or BMI (20 pmol/100 nl) + GYKI 52466 (250 pmol/100 nl) into the BLA of rats. Significantly different from *a-CSF and #BMI by a nested design mixed ANOVA with a least squares means post hoc test, P < .05.
Histology.
Figure 7 represents the areas of injection within the BLA for rats used in this the CNQX and the GYKI 52466 group, respectively. All of these cannulae placements were in the region of the BLA where microinjections of BMI elicited significant increases in HR and BP. Histological data from the nonreactive rats is eliminated for convenience.
Schematic representation of the sites of cannula implantation within the BLA of rats used in the CNQX (open circles) and GYKI 52466 (closed circles) microinjection experiments as verified by histological examination. Abbreviations: BLA, anterior basolateral amygdala; BLP, posterior basolateral amygdala; BLV, ventral basolateral amygdala; BMA, anterior basomedial amygdala; BMP, posterior basomedial amygdala; Ce, central nucleus of the amygdala; La, lateral nucleus of the amygdala; LaDL, lateral nucleus of amygdala, dorsolateral division; LaVL, lateral nucleus of amygdala, ventrolateral division.
Discussion
The results of this study show that when animals are administered BMI (20 pmol) directly into the BLA, an increase in HR, BP and experimental anxiety are observed, as seen in previous studies (Sanders and Shekhar, 1995a, b). Further, the increases seen in HR are reversed by concomitant injections of either NMDA and non-NMDA ionotropic antagonists in a dose dependent fashion. In addition to increases in HR and BP, the amount of time the rat spent interacting with the partner rat in the SI test was significantly decreased with the injection of BMI as compared to a-CSF indicating an increase in “anxiety.” Concomitant injections of AP5 significantly reversed the BMI induced anxiogenic effect even at the lowest dose used in this study (fig 1C). The noncompetitive NMDA antagonist dizocilpine also significantly blocked the BMI response at the lowest dose and the high dose (125 pmol) elicited a significant anxiolytic effect compared to the a-CSF baseline (fig 3C). The lower doses of the non-NMDA antagonists CNQX and GYKI 52466 had no effect on SI. However, the highest dose of the noncompetitive non-NMDA antagonist GYKI 52466 was able to significantly reverse the anxiogenic behavior of BMI (fig 6C). Overall, these results reveal that both the physiological effects as well as the behavior effects observed with GABAA blockade in the BLA can be significantly blocked by coadministration of either NMDA or AMPA/kainate antagonists.
One of the concerns with coadministration of the EAA antagonists with BMI would be the possibility of nonspecific effects of the glutamate antagonists, such as changes in osmolarity, pH, etc., rather than a receptor mechanism attenuating the BMI effects. Comparing the effects of injecting the active isomers of a drug with those of an inactive isomer is one way to separate the receptor-mediated responses from such nonspecific effects of a drug. Such an experiment was conducted with the NMDA antagonist AP5, using the active isomer R-AP5 and the inactive L-AP5 (fig. 2). Coadministration of the active R-AP5 and not the inactive isomer was able to block the BMI induced HR, BP and SI changes, suggesting that the effects of the NMDA antagonist is indeed mediated by its receptor action rather than nonspecific effects.
Although the EAA antagonists used in this study are relatively selective to the NMDA vs. the non-NMDA ionotropic receptors, the specificity of the drugs to the putative receptors under these experimental conditions also need to be clarified. In a previous study, we have shown that at the highest doses used here, the NMDA antagonist AP5 and the non-NMDA antagonist CNQX did not have any HR or BP effects when injected alone into the BLA of rats (Sajdyk and Shekhar, 1997, in press). However, injection of these drugs into the BLA did increase the SI time suggesting an anxiolytic effect. To test the specificity of CNQX doses used in this study to the non-NMDA receptor without significant NMDA antagonism, we tried to reverse the anxiolytic effects of injecting CNQX alone into the BLA by concomitant injection of either NMDA (500 pmol/100 nl) or AMPA (500 pmol/100 nl). The anxiolytic effect of CNQX injected into the BLA was blocked by AMPA and not NMDA injections. This suggests that under these experimental conditions, the non-NMDA antagonists may be selective to the AMPA/kainate receptors and have no significant effects mediated via the NMDA receptors. This finding further supports the involvement of both the NMDA and non-NMDA receptors in the responses elicited by BMI injections into the BLA of rats.
The above results also suggest that within the BLA, the basal firing rates of the projection neurons involved in the regulation of the physiological and behavioral expression of “anxiety” is under the influence of both tonic GABA inhibition and glutamate excitation. Characterization of this interaction between inhibition and excitation in BLA neurons has also been done with single cell recordings (Rainnieet al., 1991 a, b). Within the BLA, the GABAA receptor effectively prevents the neurons from firing beyond a brief period even when a strong excitatory stimulus is presented to the cell. If this chronic GABA inhibition is blocked (as in injection of BMI), the BLA neurons demonstrate a burst firing pattern suggesting that the tonic inhibition in this nucleus is a major regulating factor in the state of excitability of the BLA (Rainnie et al., 1991a, b). Further, the IPSP produced by GABA in the BLA itself can be blocked or reduced in amplitude by both NMDA and AMPA/kainate antagonists, thus suggesting that glutamate excitation may activate the GABA neurons in the form of a feed-forward inhibition.
Blocking GABAA receptors in the BLA not only elicits an increase in HR, BP and behavioral anxiety as assessed in the SI test and the conflict test (Sanders and Shekhar, 1995a, b), repeated daily administration of doses of BMI that are subthreshold to elicit increases in HR (6 pmol) directly into the BLA will also lead to full increases in HR, BP and SI times by day 4 or 5 (Sanders et al., 1995). Development of threshold responses with subthreshold injections of BMI into the BLA suggest that synaptic plasticity may be involved in the “priming” of anxiety responses. Preliminary studies suggest that this “priming” of physiological and behavioral effects with BMI can be blocked by pretreatment with NMDA antagonists (Sajdyk and Shekhar, unpublished data). Similarly, when the NMDA antagonist AP5 was injected bilaterally into the BLA, a dose-dependent attenuation of the acquisition of fear-potentiated startle, another test of anxiety, was observed (Miserendino et al., 1990). However, once fear conditioning has been established, infusion of AP5 was not able to block the expression of fear, although CNQX was able to dose dependently block the expression (Kim et al., 1993). All these behavioral studies taken together suggest that the modulatory influences of GABA and glutamate in the BLA is capable of regulating both the learning, (i.e., acquisition) and expression of emotions such as anxiety. The cellular mechanisms of such an interaction between inhibitory and excitatory inputs in the induction and maintenance of anxiety responses in the BLA still need to be elucidated.
The anatomical circuitry of the amygdala is also such that it enables this region to integrate emotional information. It has direct connections with the neocortex, hypothalamic neuroendocrine sites and the preganglionic autonomic areas of the brainstem, providing somatomotor, visceromotor and neuroendocrine information (Gray, 1991). The BLA possesses connections with the cortical areas (Tigges et al., 1982, 1983; Amaral and Price, 1984) that are involved in relaying multimodal sensory information for perception (Downer, 1961). In addition, the BLA also projects to the insular cortex, lateral orbital cortex, medial orbital cortex and the medial wall of the frontal lobe (Amaral and Price, 1984; Mufson et al., 1981;Friedman et al., 1986; Carmichael and Price, 1989; Barbas and DeOlmos, 1990). The BLA has a dense projection to the central nucleus of the amygdala (McDonald, 1992). The central nucleus sends fibers caudally to many areas associated with autonomic control, such as the lateral hypothalamus, the periaqueductal gray, the parabrachial nucleus, the dorsal vagal nuclei, the reticular formation, the solitary tract and the locus coeruleus (Fallon et al., 1978; Norita and Kawamura, 1980; Price and Amaral, 1981). Direct projections to the striatum from the BLA are also seen (Krettek and Price, 1978; Parentet al., 1983; Russchen et al., 1985). These particular anatomical connections within the BLA support the idea that this nucleus is possibly acting as an integration center for sensory and memory information necessary to elicit an anxiety response.
In conclusion, blockade of GABAA receptors leads to both physiological and behavioral changes similar to those seen in an anxiety response. These changes can be completely reversed with the coadministration of either an NMDA or a non-NMDA (AMPA/kainate) antagonist. Therefore, there appears to be a balance between GABAA receptor-mediated inhibition and EAA-mediated excitation that regulates behavioral and physiological responses associated with anxiety. The nature of the GABA-EAA balance in the BLA may be critical in learning and expression of emotions such as anxiety by regulating synaptic plasticity through mechanisms such as priming and LTP.
Acknowledgments
The authors thank Kuolung Hu for his assistance in statistical analysis.
Footnotes
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Send reprint requests to: Dr. Anantha Shekhar, Dept. Of Psychiatry, Indiana University Medical Center, 791 Union Drive, Indianapolis, IN 46202.
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↵1 This study was supported by Public Health Service Grant MH 52691, PDP grant from the IUPUI biomedical Grant Program and a grant from the Association for the Advancement of Mental Health Research and Education Inc.
- Abbreviations:
- a-CSF
- artificial cerebrospinal fluid
- BLA
- basolateral amygdala
- BMI
- bicuculline methiodide
- BP
- blood pressure
- EAA
- excitatory amino acid
- EPSP
- excitatory postsynaptic potential
- GABA
- γ-aminobutyric acid
- HR
- heart rate
- i.c.
- intracranial
- IPSP
- inhibitory postsynaptic potential
- NMDA
- N-methyl-d aspartate
- SI
- social interaction
- ANOVA
- analysis of variance
- Received December 23, 1996.
- Accepted July 23, 1997.
- The American Society for Pharmacology and Experimental Therapeutics