Brain Research, 576 (1991) 101-110 © 1991 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/91/$03.50
101
BRES 17247
Blockade of G A B A A receptors in the region of the anterior basolateral amygdala of rats elicits increases in heart rate and blood pressure* S.K. Sanders and A. Shekhar Department of Psychiatry, Pharmacology and Toxicology, and Program in Medical Neurobiology, Indiana University School of Medicine, Indianapolis, IN 46202 (U.S.A.) (Accepted 30 July 1991) Key words: 7-Aminobutyric acid; Amygdala; Bicuculline methiodide; Anxiety; Locomotor activity
Stimulation of the amygdala in rats is known to elicit increases in heart rate (HR) and blood pressure (BP) as well as locomotor activity associated with emotional arousal. The present study was conducted to localize and characterize the role of the GABA system of the amygdala in regulating these cardiovascular responses. Male Sprague-Dawley rats with arterial catheters placed for physiological measurements were implanted with chronic microinjection cannulae in the anterior basolateral (BLA) and central (Ce) amygdaloid nuclei under pentobarbital anesthesia. After recovering, rats were microinjected bilaterally with saline (250 nl) and bicuculline methiodide (BMI, 5-25 ng/250 nl), a selective GABAA antagonist. Microinjection of BMI in the BLA caused significant increases in HR and BP as well as locomotor stimulation while saline had no effect. The cardiovascular response to BMI was blocked by pentobarbital anesthesia. Microinjection of equimolar concentrations of (+)-baclofen HCI (GABAB agonist), phaclofen (GABAn antagonist), or strychnine (glycine antagonist) into the BLA or BMI into the Ce had no significant cardiovascular effects. The cardiovascular effects of BMI injection in the BLA does not appear to be secondary to generalized seizure activity. These results suggest that endogenous GABA, acting on GABA A receptors in the region of the BLA, may be involved in cardiovascular regulation.
INTRODUCTION Since the early suggestions of Papez 24, the amygdaloid complex is thought to play a significant role in emotional arousal. Electrical stimulation of the amygdaloid nucleus has been shown to elicit behavioral and cardiovascular changes associated with the defense reaction 11'~4't6. The typical cardiovascular changes characteristic of a defense reaction include increases in heart rate (HR) and blood pressure (BP), muscle vasodilation, and mesenteric, renal, and cutaneous vasoconstriction Las. More recently, chemical stimulation of the amygdala has been shown to produce cardiovascular and behavioral changes. Microinjecting D,L-homocysteic acid ( D L H ) into the basolateral (BL) nucleus of the amygdala also activates the defense reaction 2°, but altered blood pressure in markedly fewer rats than did electrical stimulation 9. This suggests that cell bodies in the B L as well as fibers of passage may be involved in cardiovascular control. The amygdala has also been implicated in regulation
of anxiety states. The amygdala has a high concentration of benzodiazepine receptors, the highest density being localized to the anterior basolateral (BLA) amygdaloid nucleus 21, and may be one of the important sites of action of these anxiolytic drugs. Lesions of the B L A have been shown to produce anxiolytic effects in the conflict test 33, an animal model of anxiety. In addition, a dosedependent anticonflict action was produced by intraamygdalar administration of benzodiazepines and barbiturates 29'33. Microinjection of the F-aminobutydc acid ( G A B A ) agonist, muscimol, into the amygdala also has anticonflict effects 2s. In man, electrical stimulation of the amygdaloid region in patients under local anesthesia evokes feelings of fear and confusional states 7. Based on these studies, it appears that the G A B A - b e n z o d i a z e p i n e receptor complex in the B L A may play a significant role in the regulation of anxiety. Preliminary studies in this laboratory suggest that blockade of G A B A in the region of the B L A of the amygdala in conscious rats brings about increases in H R
* Preliminary results of this study were presented at the 1990 annual meeting of the Society for Neuroscience at St. Louis, MO. Correspondence: A. Shekhar, Department of Psychiatry, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202, U.S.A.
102 and B P a n a l o g o u s to w h a t is s e e n in a d e f e n s e r e a c t i o n 28. M i c r o i n j e c t i o n of the G A B A antagonist, bicuculline m e t h i o d i d e ( B M I ) , into the B L A b r o u g h t a b o u t i m m e d i a t e c a r d i o v a s c u l a r stimulation. A l t h o u g h a g r e a t d e a l of att e n t i o n has b e e n
given to the r o l e of t h e G A B A -
b e n z o d i a z e p i n e system in the B L A in regulating anxiety, relatively little is k n o w n a b o u t the role o f this system in r e g u l a t i n g the physiological c o n c o m i t a n t s of anxiety such as changes in H R , BP, etc. T h e o b j e c t i v e of the p r e s e n t study is to c h a r a c t e r i z e the role of the G A B A system o f the B L A in r e g u l a t i n g the c a r d i o v a s c u l a r responses.
E x p e r i m e n t s w e r e con-
d u c t e d to localize the a m y g d a l a r regions m o s t sensitive to eliciting a physiological r e s p o n s e of i n c r e a s e d H R and B P u p o n i n t r a c e r e b r a l a d m i n i s t r a t i o n o f G A B A antagonists. T h e b l o c k a d e o f G A B A was a c h i e v e d by injecting B M I t h r o u g h m i c r o i n j e c t i o n c a n n u l a e , chronically imp l a n t e d in the a m y g d a l a . A n a t o m i c a l specificity of the effects o f m i c r o i n j e c t i n g B M I into specific a m y g d a l a r areas was e x a m i n e d by i m p l a n t i n g m i c r o i n j e c t i o n c a n n u l a e in areas s u r r o u n d i n g the active sites, including t h e central a m y g d a l o i d ( C e ) nucleus. P h a r m a c o l o g i c a l specificity was
examined
by
microinjecting
(+)-baclofen
HCI,
p h a c l o f e n , and strychnine into the a m y g d a l a r sites previously d e t e r m i n e d r e a c t i v e to B M I injection.
MATERIALS AND METHODS Male Sprague-Dawley rats (obtained from Harlan, Indianapolis), aged 12-15 weeks and weighing about 300 g were used. Free access to food and water was allowed during the study. Animals were maintained on a 12-12-h light-dark schedule (light off at 19.00 h) at a temperature of 72 °F. All animals underwent femoral artery catheterization under sodium pentobarbital (50 mg/kg i.p.) anesthesia. The catheters were made of 5 cm of 0.01 in. "l~gon tubing (Fisher Scientific) inside 30 cm of 0.02 in. Tygon tubing, using eyclohexanone to fuse the tubing together. The 0.01-in. tubing was inserted into the right femoral artery and the 0.02-in. tubing was routed subdermally to the dorsal aspect of the neck where it was stabilized using a leather jacket. The catheters were kept patent using heparinized saline (2.5 units/ml) and were later used for HR and BP measurements. The catheter was connected to a Beckman R511 Dynograph by a pulse pressure transducer to determine BP (mean arterial pressure) and HR was obtained via the pulse pressure signal coupled to a cardiotachometer. Following a 1-2-day recovery period in individual plastic boxes, the animals were again anesthetized with sodium pentobarbital (50 mg/kg i.p.) and fixed into a stereotaxic apparatus with the incisor bar at -3.3 mm. Two stainless steel guide cannulae (26 gauge) were mounted on both arms of the stereotaxic apparatus with injection cannulae (33 gauge) inside them. The injection cannulae were linked to 1-ml syringes by PE-50 polyethylene tubing and the system was filled with saline. The injection cannulae, as well as the guide cannulae were lowered into the BLA. The target coordinates with respect to bregma were A, -2.0 ram; L, +5.0 mm; V, 7.0 mm according to the atlas of Paxinos and Watson (1986). Cannulae were also placed in areas surrounding the B L A : L, +3.8 mm; L, +6.0 mm; V, 8.5 mm; V, 5.8 mm. When both cannulae were in place, the guide cannulae were fixed in position with three 2.4-mm stainless steel screws anchored to the skull and cranioplastic cement
(Plastics One, Roanoke, VA). After the cranioplastic cement solidified, the injection cannulae were removed and replaced with steel wire dummy cannulae. The rats were removed from the stereotaxic apparatus and allowed a 4-5-day recovery period in individual plastic cages and given ad libitum food and water. Before recovering from anesthesia, 4 rats were microinjected with 25 ng/ 250 nl BMI. In order to accomplish this, the 1-ml syringes were replaced with 10-gl Hamilton microinjection syringes and the system was filled with a 0.1 mg/ml solution of BMI in saline. The microinjection syringes were placed on an infusion pump (Sage Instruments, model 355) set to deliver 500 nl/min. With the injection cannulae inside the guide cannulae, 250 nl (25 ng BMI) of fluid was delivered to the BLA in a 30-s period. The injection cannulae were left in place for one additional minute. Controls were also accomplished by injecting 250 nl of saline in a 30-s period. Any HR and/or BP changes were recorded using the arterial catheters and the Beckman Dynograph. The remaining animals did not receive BMI injections under anesthesia. Following the 4-5-day recovery period, awake and freely moving rats were injected with 250 nl of saline and 25 ng/250 nl BMI using the procedure described above. Some of the rats showing a response to BMI (an increase in HR and BP, n = 16) were also microinjected with 5 ng/250 nl (n = 6) or 10 ng/250 nl (n = 6) BMI. The results were tabulated as mean change in HR or BP from baseline -+ S.E.M. Statistical analysis was performed by the t-test or analysis of variance coupled with the Newman-Keuis multiple range test. In order to determine if the increases in HR and BP were caused by generalized seizure-like activity beginning in the amygdala and spreading to other parts of the brain, one rat was subjected to EEG recordings. The rat used for monitoring EEG activity was anesthetized with sodium pentobarbital (50 mg/kg, i.p.), implanted with arterial and venous catheters according to the procedure described earlier, and fixed into a stereotaxic apparatus. Bipolar electrodes were placed in the right temporal cortex (Tel) and left dorsal hippocampus with coordinates from bregma being (in ram) A, -3.8; L, 6.0; V, -4.2; and A, -4.0; L, +2.3; V, -3.0, respectively. A stainless steel screw (ground) electrode was placed rostral to the other electrodes. Bilateral guide cannulae were also placed into the BLA using the procedure described earlier. The electrode leads were inserted into an Amphenol plug and the entire assembly was fixed to the skull with cranioplastic cement (Plastics One, Roanoke, VA). The rat was allowed to recover for 2 days in a plastic box with ad libitum food and water. Before the EEG experiment began, the rat (awake and freely moving) was first injected intracerebrally with saline (250 nl) and BMI (25 ng/250 ni) while HR and BP were recorded on a Beckman Dynograph. This was done to determine proper placement of the cannulae since HR and BP could not be monitored during the experiment as they were in a sound-attenuated chamber. The site (BLA) of implantation was confirmed (/>50 beats/rain increase in HR) following the BMI injection and the EEG experiment followed. During the EEG experiment, the rat was housed in a glass cylinder in a sound-attenuated environmental chamber equipped with a ventilation fan and internal lighting. The Amphenol plug was connected with Microdot cable to a slip ring contact assembly (Airflyte Electronics Co.). A fine insulated wire loosely attached to the cable indicated the rat's movement. Signals from the EEG electrodes and movement detector were recorded on a Grass Model 78 polygraph. The rat was allowed 30 min to become accustomed to the environment while baseline recordings were made. Sequentially, 30-rain recordings were made after injecting saline (250 nl) and BMI (25 ng/250 nl) intracerebrally, and pentylenetetrazole (PTZ, 24 mg) i.v. The PTZ was used to induce a seizure in the rat in order to compare definitive EEG seizure activity with the EEG activity following the BMI injection. In addition, HR and BP were monitored following a 24 mg i.p. injection of PTZ to assess the similarities and/or differences in the physiological responses of the two treatments. To further test the receptor type involved in this cardiovascular
103 RESULTS
response, a GABAB agonist, (+)-badofen HCI (12 and 50 ng/250 nl; RBI), and a GABAB antagonist, phaciofen (12 and 50 ng/250 nl; RBI), were microinjected into the BLA of a rat implanted with bilateral microinjeetion cannulae. Furthermore, strychnine sulfate (38 ng/250 hi; Sigma), a glycine antagonist and a CNS convulsant, was microinjeeted into the BLA to address the possibility that the observed cardiovascular response seen with BMI administration is only a non-specific convulsant effect. At the end of the experiment, the rats were anesthetized and the site of injection was marked with 250 nl of a 50% solution of India ink. In addition, the sites of the bipolar electrode implants in the rat used for the EEG were marked by electrolytic lesions using a DC anodal current of 7-10 mA for 3 s. Brains were removed and stored in a 10% neutral buffered formalin solution (Sigma) for at least 48 h. Microtome sections (50/~m) were stained with Neutral red (Sigma) and the precise location and extent of India ink diffusion was determined.
'
\
7E
Injection sites were most p r o m i n e n t from - 1 , 8 to -2.3 m m posterior to Bregma (Fig. 1). The largest increases in H R (>100 beats/min) and BP ( > 3 0 m m Hg) occurred in areas within, ventral, and lateral to the B L A while no increase in H R or BP was observed in areas medial, anterior, or posterior to the BLA. The central amygdaloid (Ce) nucleus appears to be void of any significant activity resulting from the B M I injection. The areas marked in Fig. 1 represent 11 rats with implantations in reactive areas and 5 rats with implantations in non-reactive areas. O n e of the rats is only represented unilater-
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Fig. 1. Results of histology showing the location of the sites in the amygdala where increases in HR and BP were elicited with microinjection of the GABA antagonist BMI. Note that most of the sites are in and around the anterior basolateral region of the amygdala. Also note that no response was elicited in the central amygdaloid nucleus with BMI microinjection. Brain sections are represented according to the atlas of Paxinos and Watson. Numbers on the right indicate distance (ram) posterior from bregma. The scale on the left represents distance (ram) ventral to bregraa. ACo, anterior cortical amygdaloid nucleus; BLA, anterior basolateral amygdaloid nucleus; BLP, posterior basolateral amygdaloid nucleus; BLV, ventral basolateral amygdaloid nucleus; BMA, anterior basornedial amygdaloid nucleus; Ce, central amygdaloid nucleus; CeL, lateral central amygdaloid nucleus; CPu, striatum; DEn, dorsal endopiriform nucleus; I, intercalated amygdaloid nuclei; IM, main intercalated araygdaloid nucleus; La, lateral amygdaloid nucleus; LaDL, dorsolaterai lateral amygdaloid nucleus; Pir, piriform cortex; PLCo, posterolateral cortical amygdaloid nucleus; VEn, ventral endopiriform nucleus; 3V, 3rd ventricle.
104 India ink was found to be 0.68 --- 0.06 mm diameter in an anterior-posterior direction and 0.63 --- 0.07 mm diameter in a mediolateral orientation. The India ink, being particulate, does not diffuse to the same extent as the drug solution. Although diffusion of the India ink may differ from the extent of diffusion of the drug solution, it may give a crude estimate of the area of injection.
TABLE I
Physiological responses of animals with nonreactive (NR) and reactive (R) sites NR sites include areas medial, anterior, and posterior to the BLA. R sites include the BLA as well as areas ventral and lateral to it. Note that the BMI injections are not significantly different from saline injections for the NR sites, while the BMI injections are significantly different from saline injections for the R sites. Data are presented as changes in HR or BP (mean -+ S.E.M.) from baseline following the corresponding i.c. injection.
Treatment
Site
Change in heart rate (bpm)
Change in blood pressure (rnmng)
Saline (n = 9) B M I 2 5 n g ( n = 9) Saline (n = 16) B M I 2 5 n g ( n = 16)
NR NR R R
3 -+ 7 7--- 8 3 -+ 6 91 --- 8*
-2 - 2 4-+ 3 2 -+ 1 27--- 4*
Physiological effects Heart rate and BP were monitored on all animals using a Beckman Dynograph. When BMI was microinjected into the reactive sites in the BLA, there was a significant and dose-dependent increase in H R and BP compared to saline injections (see Table I). Fig. 2 shows a typical physiograph recording obtained after a single injection of 25 ng/250 nl BMI into the region of the BLA. In this particular recording, H R increased 90 beats/min (bpm) and BP increased 40 mm Hg from baseline. From the time of injection, the physiological effects reached a maximum in 11.5 min, and lasted 23 min. In addition, a somewhat delayed locomotor response, quantified as crossings, was also observed (Fig. 2). It should be noted that crossings (movement of the rat from one side of the box to the other across an imaginary line through the center of the box) began only after the response reached a maximum and that movement of the rat did not cause or enhance the physiological response. To understand the relationship between changes in H R and BP in the animals, a time course plot was utilized. Upon injecting BMI (25 ng/250 nl) into the BLA,
* Significantly different from all other groups by ANOVA coupled with Newman-Keuls test, P < 0.001.
ally as the second site did not fall in the anterior-posterior range of Fig. 1. Five brains having reactive implants were not included in this figure because 4 were not processed due to technical difficulties and one brain had inadequate India ink marks so that the site of injection could not be clearly determined. Four brains having non-reactive implants were not included in the figure because all extended beyond the anterior-posterior limits of Fig. 1. India ink was used not only to mark the sites of injection, but to approximate the extent of diffusion of the drug solutions. At the injection site, tissue stained with A c t u a l Record
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Fig. 2. Representative physiological recording showing increases in heart rate, blood pressure, and locomotor activity in an awake and freely moving rat following an intracerebral injection of BMI (25 ng/250 nl) into the region of the anterior basolateral amygdala. HR is represented as bpm and blood pressure in mm Hg. Each down stroke of the crossings line represents a single crossing.
105
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Effects of Microinjecting Different Doses of BMI Into The Amygdala On Heart Rate --"
*$ +80
100
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101
BRES 17247
Blockade of G A B A A receptors in the region of the anterior basolateral amygdala of rats elicits increases in heart rate and blood pressure* S.K. Sanders and A. Shekhar Department of Psychiatry, Pharmacology and Toxicology, and Program in Medical Neurobiology, Indiana University School of Medicine, Indianapolis, IN 46202 (U.S.A.) (Accepted 30 July 1991) Key words: 7-Aminobutyric acid; Amygdala; Bicuculline methiodide; Anxiety; Locomotor activity
Stimulation of the amygdala in rats is known to elicit increases in heart rate (HR) and blood pressure (BP) as well as locomotor activity associated with emotional arousal. The present study was conducted to localize and characterize the role of the GABA system of the amygdala in regulating these cardiovascular responses. Male Sprague-Dawley rats with arterial catheters placed for physiological measurements were implanted with chronic microinjection cannulae in the anterior basolateral (BLA) and central (Ce) amygdaloid nuclei under pentobarbital anesthesia. After recovering, rats were microinjected bilaterally with saline (250 nl) and bicuculline methiodide (BMI, 5-25 ng/250 nl), a selective GABAA antagonist. Microinjection of BMI in the BLA caused significant increases in HR and BP as well as locomotor stimulation while saline had no effect. The cardiovascular response to BMI was blocked by pentobarbital anesthesia. Microinjection of equimolar concentrations of (+)-baclofen HCI (GABAB agonist), phaclofen (GABAn antagonist), or strychnine (glycine antagonist) into the BLA or BMI into the Ce had no significant cardiovascular effects. The cardiovascular effects of BMI injection in the BLA does not appear to be secondary to generalized seizure activity. These results suggest that endogenous GABA, acting on GABA A receptors in the region of the BLA, may be involved in cardiovascular regulation.
INTRODUCTION Since the early suggestions of Papez 24, the amygdaloid complex is thought to play a significant role in emotional arousal. Electrical stimulation of the amygdaloid nucleus has been shown to elicit behavioral and cardiovascular changes associated with the defense reaction 11'~4't6. The typical cardiovascular changes characteristic of a defense reaction include increases in heart rate (HR) and blood pressure (BP), muscle vasodilation, and mesenteric, renal, and cutaneous vasoconstriction Las. More recently, chemical stimulation of the amygdala has been shown to produce cardiovascular and behavioral changes. Microinjecting D,L-homocysteic acid ( D L H ) into the basolateral (BL) nucleus of the amygdala also activates the defense reaction 2°, but altered blood pressure in markedly fewer rats than did electrical stimulation 9. This suggests that cell bodies in the B L as well as fibers of passage may be involved in cardiovascular control. The amygdala has also been implicated in regulation
of anxiety states. The amygdala has a high concentration of benzodiazepine receptors, the highest density being localized to the anterior basolateral (BLA) amygdaloid nucleus 21, and may be one of the important sites of action of these anxiolytic drugs. Lesions of the B L A have been shown to produce anxiolytic effects in the conflict test 33, an animal model of anxiety. In addition, a dosedependent anticonflict action was produced by intraamygdalar administration of benzodiazepines and barbiturates 29'33. Microinjection of the F-aminobutydc acid ( G A B A ) agonist, muscimol, into the amygdala also has anticonflict effects 2s. In man, electrical stimulation of the amygdaloid region in patients under local anesthesia evokes feelings of fear and confusional states 7. Based on these studies, it appears that the G A B A - b e n z o d i a z e p i n e receptor complex in the B L A may play a significant role in the regulation of anxiety. Preliminary studies in this laboratory suggest that blockade of G A B A in the region of the B L A of the amygdala in conscious rats brings about increases in H R
* Preliminary results of this study were presented at the 1990 annual meeting of the Society for Neuroscience at St. Louis, MO. Correspondence: A. Shekhar, Department of Psychiatry, Indiana University School of Medicine, 791 Union Drive, Indianapolis, IN 46202, U.S.A.
102 and B P a n a l o g o u s to w h a t is s e e n in a d e f e n s e r e a c t i o n 28. M i c r o i n j e c t i o n of the G A B A antagonist, bicuculline m e t h i o d i d e ( B M I ) , into the B L A b r o u g h t a b o u t i m m e d i a t e c a r d i o v a s c u l a r stimulation. A l t h o u g h a g r e a t d e a l of att e n t i o n has b e e n
given to the r o l e of t h e G A B A -
b e n z o d i a z e p i n e system in the B L A in regulating anxiety, relatively little is k n o w n a b o u t the role o f this system in r e g u l a t i n g the physiological c o n c o m i t a n t s of anxiety such as changes in H R , BP, etc. T h e o b j e c t i v e of the p r e s e n t study is to c h a r a c t e r i z e the role of the G A B A system o f the B L A in r e g u l a t i n g the c a r d i o v a s c u l a r responses.
E x p e r i m e n t s w e r e con-
d u c t e d to localize the a m y g d a l a r regions m o s t sensitive to eliciting a physiological r e s p o n s e of i n c r e a s e d H R and B P u p o n i n t r a c e r e b r a l a d m i n i s t r a t i o n o f G A B A antagonists. T h e b l o c k a d e o f G A B A was a c h i e v e d by injecting B M I t h r o u g h m i c r o i n j e c t i o n c a n n u l a e , chronically imp l a n t e d in the a m y g d a l a . A n a t o m i c a l specificity of the effects o f m i c r o i n j e c t i n g B M I into specific a m y g d a l a r areas was e x a m i n e d by i m p l a n t i n g m i c r o i n j e c t i o n c a n n u l a e in areas s u r r o u n d i n g the active sites, including t h e central a m y g d a l o i d ( C e ) nucleus. P h a r m a c o l o g i c a l specificity was
examined
by
microinjecting
(+)-baclofen
HCI,
p h a c l o f e n , and strychnine into the a m y g d a l a r sites previously d e t e r m i n e d r e a c t i v e to B M I injection.
MATERIALS AND METHODS Male Sprague-Dawley rats (obtained from Harlan, Indianapolis), aged 12-15 weeks and weighing about 300 g were used. Free access to food and water was allowed during the study. Animals were maintained on a 12-12-h light-dark schedule (light off at 19.00 h) at a temperature of 72 °F. All animals underwent femoral artery catheterization under sodium pentobarbital (50 mg/kg i.p.) anesthesia. The catheters were made of 5 cm of 0.01 in. "l~gon tubing (Fisher Scientific) inside 30 cm of 0.02 in. Tygon tubing, using eyclohexanone to fuse the tubing together. The 0.01-in. tubing was inserted into the right femoral artery and the 0.02-in. tubing was routed subdermally to the dorsal aspect of the neck where it was stabilized using a leather jacket. The catheters were kept patent using heparinized saline (2.5 units/ml) and were later used for HR and BP measurements. The catheter was connected to a Beckman R511 Dynograph by a pulse pressure transducer to determine BP (mean arterial pressure) and HR was obtained via the pulse pressure signal coupled to a cardiotachometer. Following a 1-2-day recovery period in individual plastic boxes, the animals were again anesthetized with sodium pentobarbital (50 mg/kg i.p.) and fixed into a stereotaxic apparatus with the incisor bar at -3.3 mm. Two stainless steel guide cannulae (26 gauge) were mounted on both arms of the stereotaxic apparatus with injection cannulae (33 gauge) inside them. The injection cannulae were linked to 1-ml syringes by PE-50 polyethylene tubing and the system was filled with saline. The injection cannulae, as well as the guide cannulae were lowered into the BLA. The target coordinates with respect to bregma were A, -2.0 ram; L, +5.0 mm; V, 7.0 mm according to the atlas of Paxinos and Watson (1986). Cannulae were also placed in areas surrounding the B L A : L, +3.8 mm; L, +6.0 mm; V, 8.5 mm; V, 5.8 mm. When both cannulae were in place, the guide cannulae were fixed in position with three 2.4-mm stainless steel screws anchored to the skull and cranioplastic cement
(Plastics One, Roanoke, VA). After the cranioplastic cement solidified, the injection cannulae were removed and replaced with steel wire dummy cannulae. The rats were removed from the stereotaxic apparatus and allowed a 4-5-day recovery period in individual plastic cages and given ad libitum food and water. Before recovering from anesthesia, 4 rats were microinjected with 25 ng/ 250 nl BMI. In order to accomplish this, the 1-ml syringes were replaced with 10-gl Hamilton microinjection syringes and the system was filled with a 0.1 mg/ml solution of BMI in saline. The microinjection syringes were placed on an infusion pump (Sage Instruments, model 355) set to deliver 500 nl/min. With the injection cannulae inside the guide cannulae, 250 nl (25 ng BMI) of fluid was delivered to the BLA in a 30-s period. The injection cannulae were left in place for one additional minute. Controls were also accomplished by injecting 250 nl of saline in a 30-s period. Any HR and/or BP changes were recorded using the arterial catheters and the Beckman Dynograph. The remaining animals did not receive BMI injections under anesthesia. Following the 4-5-day recovery period, awake and freely moving rats were injected with 250 nl of saline and 25 ng/250 nl BMI using the procedure described above. Some of the rats showing a response to BMI (an increase in HR and BP, n = 16) were also microinjected with 5 ng/250 nl (n = 6) or 10 ng/250 nl (n = 6) BMI. The results were tabulated as mean change in HR or BP from baseline -+ S.E.M. Statistical analysis was performed by the t-test or analysis of variance coupled with the Newman-Keuis multiple range test. In order to determine if the increases in HR and BP were caused by generalized seizure-like activity beginning in the amygdala and spreading to other parts of the brain, one rat was subjected to EEG recordings. The rat used for monitoring EEG activity was anesthetized with sodium pentobarbital (50 mg/kg, i.p.), implanted with arterial and venous catheters according to the procedure described earlier, and fixed into a stereotaxic apparatus. Bipolar electrodes were placed in the right temporal cortex (Tel) and left dorsal hippocampus with coordinates from bregma being (in ram) A, -3.8; L, 6.0; V, -4.2; and A, -4.0; L, +2.3; V, -3.0, respectively. A stainless steel screw (ground) electrode was placed rostral to the other electrodes. Bilateral guide cannulae were also placed into the BLA using the procedure described earlier. The electrode leads were inserted into an Amphenol plug and the entire assembly was fixed to the skull with cranioplastic cement (Plastics One, Roanoke, VA). The rat was allowed to recover for 2 days in a plastic box with ad libitum food and water. Before the EEG experiment began, the rat (awake and freely moving) was first injected intracerebrally with saline (250 nl) and BMI (25 ng/250 ni) while HR and BP were recorded on a Beckman Dynograph. This was done to determine proper placement of the cannulae since HR and BP could not be monitored during the experiment as they were in a sound-attenuated chamber. The site (BLA) of implantation was confirmed (/>50 beats/rain increase in HR) following the BMI injection and the EEG experiment followed. During the EEG experiment, the rat was housed in a glass cylinder in a sound-attenuated environmental chamber equipped with a ventilation fan and internal lighting. The Amphenol plug was connected with Microdot cable to a slip ring contact assembly (Airflyte Electronics Co.). A fine insulated wire loosely attached to the cable indicated the rat's movement. Signals from the EEG electrodes and movement detector were recorded on a Grass Model 78 polygraph. The rat was allowed 30 min to become accustomed to the environment while baseline recordings were made. Sequentially, 30-rain recordings were made after injecting saline (250 nl) and BMI (25 ng/250 nl) intracerebrally, and pentylenetetrazole (PTZ, 24 mg) i.v. The PTZ was used to induce a seizure in the rat in order to compare definitive EEG seizure activity with the EEG activity following the BMI injection. In addition, HR and BP were monitored following a 24 mg i.p. injection of PTZ to assess the similarities and/or differences in the physiological responses of the two treatments. To further test the receptor type involved in this cardiovascular
103 RESULTS
response, a GABAB agonist, (+)-badofen HCI (12 and 50 ng/250 nl; RBI), and a GABAB antagonist, phaciofen (12 and 50 ng/250 nl; RBI), were microinjected into the BLA of a rat implanted with bilateral microinjeetion cannulae. Furthermore, strychnine sulfate (38 ng/250 hi; Sigma), a glycine antagonist and a CNS convulsant, was microinjeeted into the BLA to address the possibility that the observed cardiovascular response seen with BMI administration is only a non-specific convulsant effect. At the end of the experiment, the rats were anesthetized and the site of injection was marked with 250 nl of a 50% solution of India ink. In addition, the sites of the bipolar electrode implants in the rat used for the EEG were marked by electrolytic lesions using a DC anodal current of 7-10 mA for 3 s. Brains were removed and stored in a 10% neutral buffered formalin solution (Sigma) for at least 48 h. Microtome sections (50/~m) were stained with Neutral red (Sigma) and the precise location and extent of India ink diffusion was determined.
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Injection sites were most p r o m i n e n t from - 1 , 8 to -2.3 m m posterior to Bregma (Fig. 1). The largest increases in H R (>100 beats/min) and BP ( > 3 0 m m Hg) occurred in areas within, ventral, and lateral to the B L A while no increase in H R or BP was observed in areas medial, anterior, or posterior to the BLA. The central amygdaloid (Ce) nucleus appears to be void of any significant activity resulting from the B M I injection. The areas marked in Fig. 1 represent 11 rats with implantations in reactive areas and 5 rats with implantations in non-reactive areas. O n e of the rats is only represented unilater-
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Fig. 1. Results of histology showing the location of the sites in the amygdala where increases in HR and BP were elicited with microinjection of the GABA antagonist BMI. Note that most of the sites are in and around the anterior basolateral region of the amygdala. Also note that no response was elicited in the central amygdaloid nucleus with BMI microinjection. Brain sections are represented according to the atlas of Paxinos and Watson. Numbers on the right indicate distance (ram) posterior from bregma. The scale on the left represents distance (ram) ventral to bregraa. ACo, anterior cortical amygdaloid nucleus; BLA, anterior basolateral amygdaloid nucleus; BLP, posterior basolateral amygdaloid nucleus; BLV, ventral basolateral amygdaloid nucleus; BMA, anterior basornedial amygdaloid nucleus; Ce, central amygdaloid nucleus; CeL, lateral central amygdaloid nucleus; CPu, striatum; DEn, dorsal endopiriform nucleus; I, intercalated amygdaloid nuclei; IM, main intercalated araygdaloid nucleus; La, lateral amygdaloid nucleus; LaDL, dorsolaterai lateral amygdaloid nucleus; Pir, piriform cortex; PLCo, posterolateral cortical amygdaloid nucleus; VEn, ventral endopiriform nucleus; 3V, 3rd ventricle.
104 India ink was found to be 0.68 --- 0.06 mm diameter in an anterior-posterior direction and 0.63 --- 0.07 mm diameter in a mediolateral orientation. The India ink, being particulate, does not diffuse to the same extent as the drug solution. Although diffusion of the India ink may differ from the extent of diffusion of the drug solution, it may give a crude estimate of the area of injection.
TABLE I
Physiological responses of animals with nonreactive (NR) and reactive (R) sites NR sites include areas medial, anterior, and posterior to the BLA. R sites include the BLA as well as areas ventral and lateral to it. Note that the BMI injections are not significantly different from saline injections for the NR sites, while the BMI injections are significantly different from saline injections for the R sites. Data are presented as changes in HR or BP (mean -+ S.E.M.) from baseline following the corresponding i.c. injection.
Treatment
Site
Change in heart rate (bpm)
Change in blood pressure (rnmng)
Saline (n = 9) B M I 2 5 n g ( n = 9) Saline (n = 16) B M I 2 5 n g ( n = 16)
NR NR R R
3 -+ 7 7--- 8 3 -+ 6 91 --- 8*
-2 - 2 4-+ 3 2 -+ 1 27--- 4*
Physiological effects Heart rate and BP were monitored on all animals using a Beckman Dynograph. When BMI was microinjected into the reactive sites in the BLA, there was a significant and dose-dependent increase in H R and BP compared to saline injections (see Table I). Fig. 2 shows a typical physiograph recording obtained after a single injection of 25 ng/250 nl BMI into the region of the BLA. In this particular recording, H R increased 90 beats/min (bpm) and BP increased 40 mm Hg from baseline. From the time of injection, the physiological effects reached a maximum in 11.5 min, and lasted 23 min. In addition, a somewhat delayed locomotor response, quantified as crossings, was also observed (Fig. 2). It should be noted that crossings (movement of the rat from one side of the box to the other across an imaginary line through the center of the box) began only after the response reached a maximum and that movement of the rat did not cause or enhance the physiological response. To understand the relationship between changes in H R and BP in the animals, a time course plot was utilized. Upon injecting BMI (25 ng/250 nl) into the BLA,
* Significantly different from all other groups by ANOVA coupled with Newman-Keuls test, P < 0.001.
ally as the second site did not fall in the anterior-posterior range of Fig. 1. Five brains having reactive implants were not included in this figure because 4 were not processed due to technical difficulties and one brain had inadequate India ink marks so that the site of injection could not be clearly determined. Four brains having non-reactive implants were not included in the figure because all extended beyond the anterior-posterior limits of Fig. 1. India ink was used not only to mark the sites of injection, but to approximate the extent of diffusion of the drug solutions. At the injection site, tissue stained with A c t u a l Record
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Fig. 2. Representative physiological recording showing increases in heart rate, blood pressure, and locomotor activity in an awake and freely moving rat following an intracerebral injection of BMI (25 ng/250 nl) into the region of the anterior basolateral amygdala. HR is represented as bpm and blood pressure in mm Hg. Each down stroke of the crossings line represents a single crossing.
105
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