INTRODUCTION

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THE HYPOTHALAMUS and the amygdala appear to play important roles in cardiovascular regulation, particularly in the response to stress. Classic electrical stimulation studies suggest that activation of either the amygdala or the hypothalamus can elicit stresslike cardiovascular changes (2, 13, 15, 16, 19, 35). More recent studies employing the technique of microinjecting chemical stimulants have identified specific sites and receptor systems in the amygdala and the hypothalamus that are involved in producing these changes. DiMicco and colleagues (10, 11, 28, 29) have reported in a series of studies that GABA and excitatory amino acid (EAA) receptors in the dorsomedial hypothalamus (DMH) of rats regulate the activity of a population of neurons involved in mediating the cardiovascular and neuroendocrine responses to stress. Microinjection of the GABA_A receptor antagonist bicuculline methiodide (BMI) or EAA receptor agonists into the DMH produces increases in heart rate and arterial pressure and changes in vascular resistance and behavior that are characteristic of the defense reaction (10, 11, 25-29). Conversely, injection of the GABA_A receptor agonist muscimol or EAA receptor antagonists at this same site prevents stress-induced increases in heart rate, arterial pressure, and plasma levels of ACTH (30-32).

With regard to the amygdala, Shekhar and colleagues (22-27) have identified the basolateral nucleus of the amygdala (BLA) as a site where GABAergic mechanisms play a role in regulating cardiovascular function and behavior. Injection of GABA_A antagonists into the BLA produces increases in heart rate and arterial pressure along with anxiogenic-like behavior, changes similar to those seen on activation of the DMH (30-32). Taken together, these studies suggest that the DMH and the BLA play important and possibly integrative roles in regulating cardiovascular function as well as levels or the state of anxiety in rats.

The purpose of this study was to examine the interaction of the BLA and the DMH in regulating cardiovascular function. Based on known anatomic connections between the amygdala and hypothalamus (4, 17, 33) and given that activation of either nucleus produces stresslike cardiovascular changes and that EAA receptors in the DMH mediate stress-induced cardiovascular changes, we hypothesized that EAA receptors in the DMH mediate the cardiovascular changes resulting from activation of the amygdala. To test this hypothesis, the GABA_A antagonists BMI and picrotoxin were injected into the BLA before or after injection of the nonselective EAA receptor antagonist kynurenic acid into the DMH. The role of specific subtypes of EAA receptors in the DMH was examined using the NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and the DL--amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) at doses shown to be selective for their respective EAA receptor subtype. In addition, kynurenic acid was injected at sites outside the DMH before activation of the BLA to assess the anatomic specificity of this response.

	MATERIALS AND METHODS

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Animals. Male Sprague-Dawley rats (270-350 g, Harlan Sprague Dawley, Indianapolis, IN) were used in all experiments. Animals were housed individually under controlled temperature and light periodicity with free access to food and water. All procedures used were approved by Drake University Animal Care and Use Committee and followed guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Stereotaxic surgery. On day 1 of the surgical protocol, rats were anesthetized with ketamine (80 mg/kg ip) and xylazine (12 mg/kg ip) and positioned in a stereotaxic frame (Kopf Instruments) with the incisor bar positioned at 3.3 mm below the horizontal plane. Rectal temperature was monitored and maintained at 36-37°C with a heating pad. Once the overlying skin and connective tissue were cleared from the skull, holes were drilled into the skull to allow access to the brain. Chronic guide cannulas (26 gauge, 11 mm length; Plastics One, Roanoke, VA) were directed unilaterally at the BLA [anteroposterior (AP) 2.1, rostrolateral (RL) +4.9, height-depth (HD) 8.7, oriented rostrally at a 10° angle] and at the DMH (AP 3.1, RL 0.5, HD 8.9, oriented caudally at a 10° angle) and cemented in place with cranioplastic cement anchored to the skull with three jeweler's screws. The guide cannulas were fitted with injector cannulas (33 gauge, 12 mm length) during the implantation procedure to prevent fluids and tissue from accumulating in the guide cannulas. After the cranioplastic cement had set, the injector cannulas were removed and the guide cannulas were sealed with wire dummy cannulas. Animals were removed from the stereotaxic frame and allowed to recover in individual cages.

Cardiovascular instrumentation. Five to seven days after implantation of the guide cannulas, animals were reanesthetized with ketamine-xylazine. The right femoral artery was cannulated with a 3.5-cm length of Microrenathane tubing (0.01 in. ID; Braintree Scientific, Braintree, MA) attached to a length of Tygon tubing (0.02 in. ID) filled with heparinized saline (100 U/ml), and the end was sealed with a stylet. The tubing was then routed subcutaneously to the nape of the neck, exteriorized, and secured to skin and underlying muscle with sutures. The animals were allowed to recover in their individual cages for 24-48 h before testing. By this time, the animals had resumed their regular eating, drinking, and grooming habits and exhibited no signs of pain or stress.

Microinjection procedure. Animals were tested while freely moving or resting in their home cage between 1000 and 1600. Arterial pressure was measured and recorded by connecting the arterial line in series to a pressure transducer, a MacLab/4 data acquisition system, and a Macintosh LCIII computer. Heart rate was derived from the arterial pulse pressure and measured and recorded on a separate channel. Once a steady baseline of cardiovascular parameters was attained, the injector cannulas containing drug solution or vehicle were inserted into the guide cannulas. All injections were unilateral (250 nl into the BLA, 100 nl into the DMH, infused over 30 s) and were made using 5-µl Hamilton syringes mounted in a Harvard infusion pump. Injection cannulas remained in place for 1 min after the end of the infusion and then were removed. The order of injections for dose-response curves and antagonist pretreatments was given in a staggered or varied sequence to control for order effects. Animals received no more than two injections per day and no more than five injections at one site.

Histology. Microinjection sites were marked at the completion of each study. After induction of anesthesia (ketamine-xylazine), 100 nl of a 5% solution of Alcian blue dye was infused at each site. The rat was perfused transcardially with 60 ml of heparinized saline followed by 150 ml of buffered 10% Formalin. The brain was removed and stored in buffered 10% Formalin. Coronal sections (60 µm) were cut on a microtome/cryostat, mounted on gelatin-coated slides, and stained with 1% neutral red solution. The locations of the sites of injection were determined according to the atlas of Paxinos and Watson (20).

Chemicals. Drugs used in these experiments included BMI, CPP, NBQX, N-methyl-D-aspartate (NMDA), and AMPA from Research Biochemicals International (Natick, MA) and picrotoxin, kynurenic acid, and xanthurenic acid from Sigma Chemical (St. Louis, MO). All drugs were dissolved in saline, and the final pH was adjusted to 6-8. The anesthetic (ketamine-xylazine) was obtained in a ready-to-use formulation from Research Biochemicals International.

Statistics. Results are expressed as means ± SE. The data were analyzed using one-way ANOVA (with repeated measures where appropriate) and Scheffé's post hoc test to determine differences between groups. In those experiments in which the data are expressed as peak changes in heart rate or arterial pressure, the peak was defined as the highest value sustained for 1 min or longer. In those experiments in which the data are expressed as a time course, the data were analyzed after calculation of the area under the curve (AUC). The AUC was determined for each experiment by plotting the data (change from baseline) over a grid (grid block = 5 min × mmHg or beats/min) and calculating the area using the trapezoidal method. All grids for a given experiment were summed (including both positive and negative areas, relative to baseline) to determine the total AUC (12). The 5% limits of probability were accepted as significant.

	RESULTS

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Microinjection of the GABA_A receptor antagonist BMI (100 pmol) into the BLA of conscious rats produced marked increases in heart rate and modest elevations in arterial pressure. When the nonselective EAA receptor antagonist kynurenic acid (0.1-10 nmol) was injected into the DMH 5-7 min before injection of the same dose of BMI into the BLA, the tachycardic and pressor responses to BMI were blocked in a dose-dependent manner (Figs. 1 and 2). In contrast, pretreatment in the DMH with xanthurenic acid (10 nmol), an analog of kynurenic acid that has no activity at EAA receptors, did not alter the response to injection of BMI into the BLA. The heart rate (330 ± 6 beats/min) and mean arterial pressure (108 ± 2 mmHg) were not significantly different among the groups before injection of BMI.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Tracings of heart rate (HR; beats/min) and arterial pressure (AP; mmHg) depicting the effects of bicuculline methiodide (BMI; 100 pmol/250 nl) injected into the basolateral amygdala (BLA) after injection of saline or various doses of kynurenic acid (Kyn) into the dorsomedial hypothalamus (DMH) of a single conscious rat. BPM, beats/min.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Maximal changes in HR (A) and mean AP (MAP; B ) after injection of 100 nl saline or various doses of kynurenic acid into the DMH followed by injection of 100 pmol BMI into the BLA of conscious rats. * Significant differences from BMI+Saline and BMI+xanthurenic (XAN). HR: F(4,22) = 26.90, P < 0.0001. MAP: F(4,22) = 6.15, P = 0.002; n = 5 or 6 for each group.

In another series of experiments, we used a different microinjection protocol and a different GABA antagonist injected into the BLA to provide further evidence that EAA receptors in the DMH mediate the cardiovascular changes resulting from activation of the amygdala. Microinjection of the noncompetitive, postsynaptic GABA antagonist picrotoxin (100 pmol) into the BLA produced significant increases in heart rate and arterial pressure similar to those seen with local injection of BMI (Fig. 3). Microinjection of kynurenic acid (10 nmol) into the DMH 10 min after intra-amygdalar injection of picrotoxin reversed the picrotoxin-induced tachycardic and pressor responses. With the use of this same protocol, hypothalamic injection of 10 nmol of the inactive analog xanthurenic acid did not significantly alter the magnitude or time course of cardiovascular changes resulting from injection of picrotoxin into the BLA. The resting heart rate (334 ± 9 beats/min) and mean arterial pressure (110 ± 3 mmHg) were not significantly different among the groups before injection of picrotoxin.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Time course of changes in HR (A ) and MAP (B ) after injection of 100 pmol picrotoxin (; n = 4) into the BLA alone or followed by injection of 10 nmol kynurenic acid (; n = 6) or 10 nmol xanthurenic acid (; n = 4) into the DMH. * Significant differences from picrotoxin alone and picrotoxin + xanthurenic acid. HR: F(2,11) = 17.22, P = 0.0004. MAP: F(2,11) = 29.65, P < 0.0001. Values for area under the curve: HR (beats/min): picrotoxin, 1,727 ± 193; picrotoxin + xanthurenic acid, 1,705 ± 133; picrotoxin + kynurenic acid, 739 ± 114. MAP (mmHg): picrotoxin, 528 ± 17; picrotoxin + xanthurenic acid, 487 ± 17; picrotoxin + kynurenic acid, 347 ± 18.

In the next series of experiments, we used the NMDA receptor antagonist CPP and the AMPA receptor antagonist NBQX to examine the role of EAA receptor subtypes in the DMH in mediating the cardiovascular changes resulting from intra-amygdalar injection of BMI. We previously showed that injection of EAA receptor agonists into the DMH of conscious or anesthetized rats produces increases in heart rate and arterial pressure (27, 29). In the present study, we repeated these experiments as part of an effort to define doses of CPP and NBQX that selectively block their respective EAA receptor subtypes in the DMH. In these experiments, either saline (100 nl), CPP (10 pmol), or NBQX (50 pmol) was injected into the DMH 8-10 min before injection of the EAA receptor agonists NMDA or AMPA at the same site. With saline as a pretreatment, both NMDA (10 pmol) and AMPA (4 pmol) produced significant increases in heart rate and arterial pressure (Fig. 4). Local pretreatment with the NMDA receptor antagonist CPP (10 pmol) blocked NMDA-induced cardiovascular changes but did not significantly affect the response to a similar injection of AMPA. Conversely, local pretreatment with the AMPA receptor antagonist NBQX (50 pmol) blocked the response to injection of AMPA at this site but did not significantly affect the response to local injection of NMDA. Thus these experiments define doses of CPP and NBQX that are selective for their respective EAA receptor subtypes in the DMH. In the next set of experiments, we used these selective doses as a pretreatment in the DMH 4-5 min before injection of BMI into the BLA. Hypothalamic injection of either CPP (10 pmol) or NBQX (50 pmol) significantly attenuated the tachycardic and pressor responses resulting from injection of BMI (50 pmol) into the BLA (Fig. 5).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Maximal changes in HR (A ) and MAP (B ) after bilateral injection into the DMH of either N-methyl-D-aspartate (NMDA; left; n = 8) or DL--amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA; right; n = 8) alone or after local injection of either 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10 pmol; n = 4) or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX; 50 pmol, n = 4). * Significant differences from the agonist alone. HR: F(5,26) = 10.68, P < 0.0001. MAP: F(5,26) = 11.40, P < 0.0001.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Maximal changes in HR (A ) and MAP (B ) after injection of 100 nl saline, 10 pmol CPP, or 50 pmol NBQX into the DMH followed by injection of 50 pmol BMI into the BLA of conscious rats. * Significant differences from BMI+Saline. HR: F(2,8) = 36.42, P = 0.0001. MAP: F(2,8) = 24.42, P = 0.0004. n = 5 for each group (repeated measures).

In all experiments described above, subsequent histology indicated that the amygdalar injections were localized to the anterior region of the BLA and that the hypothalamic injections were localized to an area in or immediately adjacent to the DMH (Fig. 6). To determine if the site of action of the EAA antagonists was limited to the area of the DMH, we injected kynurenic acid (10 nmol) or saline (100 nl) at sites dorsal or lateral to the DMH before injection of BMI (100 pmol) into the BLA. With saline injected at sites lateral, dorsal, or into the DMH as a pretreatment, intra-amygdalar injection of BMI (100 pmol) produced significant increases in heart rate and arterial pressure (Fig. 7). Injection of kynurenic acid at sites 1.0 mm dorsal or lateral to the DMH did not significantly alter the changes in heart rate and arterial pressure resulting from injection of BMI into the BLA. Injection of kynurenic acid at sites 0.3-0.5 mm lateral to the DMH (i.e., perifornical area) significantly reduced but did not fully suppress the BMI-induced tachycardic responses. The pressor response to injection of BMI into the BLA was not significantly affected by pretreatment with kynurenic acid into the perifornical area. In a repeat of experiments shown in Fig. 2, injection of kynurenic acid into the DMH eliminated the BMI-induced tachycardic and pressor responses.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Schematic representation of injection sites in coronal sections of the rat brain through portions of the amygdala and hypothalamus. Sites of injection are identified for those experiments summarized in Figs. 2, 3, and 5. Filled symbols on the templates on the left depict injections into the amygdala. Matching open symbols on the templates on the right depict the corresponding injections into the hypothalamus. Squares represent the injection sites from data summarized in Fig. 2 in which 100 pmol BMI was injected into BLA and 1-10 nmol kynurenic acid into DMH. Triangles represent the injection sites from data summarized in Fig. 3 in which 100 pmol picrotoxin was injected into BLA and 10 nmol kynurenic acid into DMH. Circles represent the injection sites from data summarized in Fig. 5 in which 50 pmol BMI was injected into BLA and CPP and NBQX into DMH. Stars represent the injection sites from data summarized in Fig. 7 in which 100 pmol BMI was injected into BLA and 10 nmol kynurenic acid at sites designated as lateral or dorsal to DMH. Diamonds represent the injection sites from data summarized in Fig. 7 in which 100 pmol BMI was injected into the BLA and 10 nmol kynurenic acid was injected at sites designated as the perifornical area. ACo, anterior cortical amygdala; BLP, posterior basolateral amygdala; BLV, ventral basolateral amygdala; BM, basomedial amygdala; CNA, central amygdala; DEn, dorsal endopiriform nucleus; DMH, dorsomedial hypothalamus; f, fornix; LaDL, dorsolateral lateral amygdala; LaV, ventrolateral amygdala; mt, mammillothalamic tract; OT, optic tract; Pir, piriform cortex; PVN, paraventricular hypothalamus; PLCo, posterolateral cortical amygdala; VMH, ventromedial hypothalamus; 3V, third ventricle. Adapted from the atlas of Paxinos and Watson (20).

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Maximal changes in HR (A ) and MAP (B ) after injection of saline (100 nl) or kynurenic acid (10 nmol) at sites in the perifornical area (PFA; n = 5) or at sites dorsal (n = 4), lateral (n = 5), or directly into the DMH (n = 4) followed by injection of BMI 100 pmol into the BLA of conscious rats. * Significant difference from all other kynurenic-treated groups. ** Significant difference from all other kynurenic-treated groups. HR: F(7,28) = 14.89, P < 0.0001. MAP: F(7,28) = 6.96, P < 0.0001.

	DISCUSSION

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Data presented in this study provide evidence that blockade of EAA receptors in the area of the DMH prevents the cardiovascular changes resulting from removal of GABAergic inhibition in the BLA of conscious rats. This study expands on a growing body of evidence that indicates the DMH and the BLA play important roles in forebrain mechanisms of cardiovascular regulation.

In the present study, microinjection of GABA receptor antagonists into the BLA was used as a method of activating the amygdala. Previous reports have demonstrated that intra-amygdalar injection of these agents produces both cardiovascular and behavioral changes (22-24) that are consistent with anxiety-like or defenselike reactions in this species (15, 16). In addition, these previous reports indicate that the site of action for the GABA receptor antagonists is localized to the anterior portion of the BLA. In the present study, we used similar volumes of injection and doses of GABA receptor antagonists and verified histologically that the site of injection was directed at the anterior region of the BLA. Therefore, the method of pharmacological activation of the amygdala used in this study is consistent with an established model for eliciting both behavioral and cardiovascular defenselike reactions in rats.

The cardiovascular changes resulting from activation of the amygdala were blocked or reversed by injection of the nonselective EAA receptor antagonist kynurenic acid into the DMH. When kynurenic acid was injected into the DMH before intra-amygdalar injection of BMI, the EAA receptor antagonist attenuated in a dose-dependent manner the BMI-induced increases in heart rate and arterial pressure. Similarly, injection of kynurenic acid into the DMH 10 min after injection of the GABA receptor antagonist picrotoxin into the BLA reversed the picrotoxin-induced tachycardic and pressor responses. Conversely, xanthurenic acid, an inactive analog of kynurenic acid (14), failed to alter the response when injected into the DMH before intra-amygdalar injection of BMI or 10 min after intra-amygdalar injection of picrotoxin. Therefore, when the staggered order of treatments within the series of experiments, the negative control using xanthurenic acid, and the different injection protocols (pretreatment blockade vs. posttreatment reversal) are taken together, it is likely that the reduced response seen in the presence of kynurenic acid can be attributed to blockade of EAA receptors and not to changes in the responsiveness of the preparation or to nonspecific actions of kynurenic acid.

Given that nonselective blockade of EAA receptors in the DMH prevents the cardiovascular changes resulting from withdrawal of GABAergic inhibition in the BLA, we next examined the role of specific EAA receptor subtypes in the DMH in mediating these effects. The NMDA receptor antagonist CPP and the AMPA receptor antagonist NBQX are considered selective for their respective EAA receptor subtypes. However, because the DMH appears to be exquisitely sensitive to EAA receptor agonists (10, 28) and the doses of CPP and NBQX cited in other microinjection studies vary widely (1, 3, 6, 8), we performed experiments to define selective doses of these two agents for the current experimental conditions. Injection of CPP (10 pmol) into the DMH blocked the increases in heart rate and arterial pressure caused by local injection of NMDA. However, CPP at this dose did not significantly affect comparable cardiovascular changes caused by AMPA. Conversely, injection of NBQX (50 pmol) into the DMH attenuated the tachycardic and pressor responses to local injection of AMPA but, at this same dose, did not affect NMDA-induced changes. In these experiments, the antagonists were injected into the DMH 8-10 min before local injection of the EAA receptor agonists. This protocol was chosen to determine the selectivity and effectiveness of the antagonists at a time point that, in later experiments, represents the peak effect of BMI when injected into the BLA 4-5 min after pretreatment with CPP or NBQX in the DMH. In these subsequent experiments, injection of either CPP or NBQX into the DMH attenuated the tachycardic and pressor responses resulting from injection of BMI into the BLA. These results suggest that the cardiovascular response to activation of the BLA is mediated through the DMH by two distinct components. One component relies on activity at NMDA receptors, whereas the other is mediated through AMPA receptors.

In the above experiments different doses of BMI were used to activate the BLA in the presence of kynurenic acid and in the presence of CPP and NBQX. In the presence of kynurenic acid, the response to the higher dose of BMI (100 pmol) was completely blocked, suggesting that the cardiovascular changes were mediated entirely by EAA receptors. In the second series of experiments, a lower dose of BMI (50 pmol) was used for two reasons. First, if the BLA was activated to its maximal level with the higher dose of BMI, it may be difficult to demonstrate a significant attenuation with the relatively small but selective doses of CPP and NBQX. Secondly, if one of the selective antagonists produced a significant blockade and the other failed under conditions of "low-dose stimulation" with BMI, subsequent experiments using the higher dose of BMI would have been carried out to determine if the second receptor subtype would begin to contribute under conditions of "high-dose stimulation." This second scenario is based on reports of experiments using hippocampal slices in which low-frequency stimulation of afferent pathways produced excitatory postsynaptic potentials (EPSPs) in hippocampal neurons that were mediated exclusively by AMPA receptors, whereas high-frequency stimulation of the same pathway produced EPSPs with components mediated by both NMDA and AMPA receptors (5, 9). In the current study, such a phenomenon was not evident and, therefore, subsequent experiments with the higher dose of BMI were not performed.

Histological analysis from this study indicates that the DMH is the most likely site at which the EAA receptor antagonists are acting to block the cardiovascular changes that result from activation of the amygdala. The present study used appropriate pharmacological methods, a fairly restrictive volume of injection (100 nl) for studies involving conscious animals, and a series of control experiments to attempt to establish the boundaries of the reactive area by injecting at sites 0.3-0.5 mm and 1.0 mm away from the proposed site of action. Areas dorsal to the DMH were examined based on the cannula tract (located dorsally), which represented the path of least resistance for drug diffusion and, therefore, a likely site of action. Sites lateral to the DMH were examined because this area includes the lateral hypothalamus, a site described as mediating the autonomic effects resulting from stimulation of the amygdala and the insular cortex (7, 18). We also examined sites in the perifornical area located immediately lateral to the DMH based on anatomic evidence that suggests this area receives a direct connection from nuclei in the amygdala (21). In the present study, injection of kynurenic acid at sites 1.0 mm dorsal or 1.0 mm lateral to the DMH did not significantly alter the cardiovascular response resulting from injection of BMI into the BLA. Injection of kynurenic acid at sites 0.3-0.5 mm lateral to the DMH (perifornical area) significantly attenuated the BMI-induced tachycardic response but not to the extent seen with injection of this same dose of kynurenic acid directly into the DMH. Therefore, it is possible that EAA receptors in the perifornical area play a role in mediating the tachycardic responses that result from activation of BLA. However, it is likely that this attenuated response is a result of the drug diffusing from the site of injection in the perifornical area to the DMH. Therefore, the present study indicates that the DMH is the site of an obligatory EAA receptor-mediated synapse involved in mediating the cardiovascular changes that result from removal of GABAergic inhibition in the BLA. The present study did not systematically test sites throughout the anterior and posterior limits of the lateral hypothalamus, and, therefore, we cannot totally rule out involvement of the lateral hypothalamus nor can we rule out the possibility that the sites tested do play a role but involve other neurotransmitter systems.

Perspectives