Brain Research, 569 (1992) 221-228 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$03.50
221
BRES 17324
Plasma and organ catecholamine levels following stimulation of the rat insular cortex Stephen M. Oppenheimer 1'2'4, Tarek M. Saleh 1, John X. Wilson 3 and David F. Cechetto 1-3 ~Department of Stroke and Aging, Robarts Research Institute, London, Ont. (Canada), Departments of 2Clinical Neurological Sciences and 3Physiology, University of Western Ontario, London, Ont. (Canada) and 4Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD (U.S.A.) (Accepted 20 August 1991) Key words: Insular cortex; Phasic microstimulation; Heart rate; Plasma catecholamine; Organ catecholamine
The posterior insular cortex of the rat contains an area of cardiac chronotropic representation within which tachycardia sites occur rostrally to those producing bradycardia. In the current study using ketamine-anesthetized rats, the insular cortex was stimulated for 1 h using a phasic technique synchronized with the cardiac cycle. Taehycardia was associated with an increase in plasma norepinephrine concentration; epinephrine remained unchanged. This indicates a neural origin of the norepinephrine increment. The tachycardia response was completely blocked by atenolol. Plasma catecholamine levels remained unchanged during stimulation of insular bradycardia sites. Atenolol was without effect during stimulation-induced bradycardia which was completely blocked by atropine. Total cardiac norepinephrine concentration inversely correlated with change in heart rate during stimulation of tachycardia sites. No correlation between intracardiac catecholamines and heart rate variables was found for the bradycardia or control sites. These results indicate that in the ketamine-anesthetized rat, whereas insular stimulation-induced tachycardia is dependent on the sympathetic nervous system, bradycardia elicited by insular cortex stimulation is mediated by parasympathetic mechanisms. No correlation was identified between renal or skeletal muscle norepinephrine levels and any heart rate parameter. This implies that the sympathetic effects of phasic insular microstimulation may be exerted mainly on cardiac nerves, and less so in other visceral beds. INTRODUCTION The insular cortex receives a variety of visceral afferent projections and has efferent connectivity with subcortical sites involved in autonomic control 3'13'21. Recently, topographic organization for cardiac chronotropicity has been d e m o n s t r a t e d within the rat posterior insular cortex 16. B r a d y c a r d i a sites are r e p r e s e n t e d caudally to the m o r e rostrally situated tachycardia sites. Isolated cardiac chronotropic responses were elicited free of any concomitant blood pressure or respiratory changes (which accompany non-phasicalty induced heart rate alterations) using a new technique of phasic microstimulation linked to the R wave of the E C G . In chloraloseanesthetized rats, both tachycardia and bradycardia responses induced by insular cortical phasic stimulation were completely abolished by the fll antagonist atenolol suggesting that each was m e d i a t e d by the sympathetic nervous system ~6. A t r o p i n e was without effect on these heart rate changes, implying little or no parasympathetic involvement. More recently, phasic stimulation of the rat insular cortex linked to the T wave of the E C G was shown to
lead to increasing degrees of heart block, ventricular ectopy and asystolic death 17. A significant elevation of plasma n o r e p i n e p h r i n e but not epinephrine (implying a neural rather than adrenal origin 14) accompanied the earlier E C G changes which were also associated with evidence of structural damage to the heart. Similarly, elevations in plasma n o r e p i n e p h r i n e and in renal sympathetic nerve activity p r e c e d e d E C G changes and death of the experimental animal in the middle cerebral artery occlusion, rat stroke model, when the insular cortex was involved in the infarct 4. These observations suggest that the insular cortex may play an important role in the production of the E C G changes following stroke and in the occurrence of sudden unexpected death noted in that condition 15. The p h e n o m e n a may be m e d i a t e d by hyperactivity expressed by the cardiac sympathetic nerves. The current study was u n d e r t a k e n to investigate symp a t h o a d r e n a l activity in m o r e detail during insular stimulation: plasma, renal, cardiac and skeletal muscle catecholamine levels were d e t e r m i n e d following stimulation at bradycardia, tachycardia and control sites in ketamineanesthetized animals.
Correspondence: D.E Cechetto, The Department of Stroke and Aging, The John P. Robarts Research Institute, 100 Perth Drive, London, Ont. N6A 5K8, Canada.
222 MATERIALS AND METHODS Eighteen male Wistar rats (mean weight 241 g) were divided into 3 groups each of 6 animals according to their response to insular stimulation (bradycardia, tachycardia or control). A reactive site was so designated if initial stimulation for 30-60 s produced a change in heart rate of 20 bpm or more unaccompanied by significant (greater than 5 mm Hg) change in blood pressure. The animals were initially anesthetized with ketamine-xylazine 0.1 ml/100 g i.m. (ketamine 100 mg/ml, xylazine 20 mg/ml), and the femoral artery and vein cannulated. The arterial cannula was connected to a Statham P23D pressure transducer for measurement of the blood pressure. Heart rate was obtained from the pulse pressure, using a Grass 7P44 tachograph. An endotracheal tube was inserted into each animal, which inspired 100% oxygen. Respiration was measured with a Fleisch pneumotachograph. Blood pressure, heart rate, and respiration were recorded by a Grass RPS 7C8 polygraph. The animals' core temperatures were maintained at 37°C with a rectal probe heating pad and a YSI temperature controller. The venous cannula was connected to a compact infusion pump model 975 (Harvard Apparatus Co.) and ketamine infused at a rate of 0.001 ml/min. This rate had been determined to maintain an adequate and constant depth of anesthesia. The animals were placed in a stereotactic frame, and the parietal bone drilled. A glass microelectrode filled with 3 M saline, of internal tip diameter 10-20/~m, was inserted into the left insular cortex using co-ordinates previously determined from stimulation experiments in this region 16. The ECG was monitored in the lead II configuration, and relayed to a signal discriminator which differentiated the R wave from other parts of the ECG waveform. The method of cortical phasic microstimulation which produces cardiac chronotropic responses without accompanying blood pressure or respiratory effects if applied for 1 min, or large blood pressure if applied for 1 h, has been detailed elsewhere 16. In brief, paired stimuli separated by 20 ms were delivered to the insular cortex during every second or third ECG cycle coincident with the R wave and approximately 80-100 ms before the P wave. Allowing for central conduction delay 2, this was anticipated to generate activity in cardiac nerves during the period when the sinoatrial node would be most receptive to chronotropic stimuli. The stimulus parameters were: 2 ms pulse duration, 500 ~A current. Responses were ascertained at progressive 200-/~m steps of microelectrode advancement and the experimental site chosen was that giving maximum responsiveness. Once identified, the site was stimulated for 1 h. One ml of blood was obtained before the onset of stimulation for baseline catecholamine determination. Another sample was obtained after 1 h of stimulation, at which time the heart, right kidney and a portion of the left gastrocnemius muscle were rapidly removed, weighed and then frozen in liquid nitrogen. Plasma and tissue specimens were stored at -80°C prior to catecholamine extraction and analysis. Organ cateeholamine results are expressed as pg per gm of organ wet weight. At the end of each experiment, the animal was perfused using an intra-aortic catheter. The perfusate comprised a solution of 0.9% N saline, followed by 10% formalin. The brain was removed and stored in 10% formalin for at least 2 days, before being cut in the coronal plane on a microtome. The sections were then stained with Thionin and examined using a Leitz Diaplan microscope. Camera lucida tracings of the stimulation sites were thus obtained. Plasma catecholamines were assayed as previously described ~. The organs and muscle were homogenized as follows: the tissue was minced with scissors at 4°C. To this, a volume of ice-cold 0.4 N perchloric acid-GSH solution equivalent to 10 times the wet weight of tissue was added. The mixture was homogenized using a Brinkman Polytron homogenizer fitted with a PT-10 head, and then centrifuged at 2700 rpm (2000 g) for 30 min at 4°C to obtain a protein-free supernatant ~'23. The samples were stored at -80°C until subsequently assayed. Good catecholamine stability is reported under these conditions8. In 6 additional rats which had undergone the general surgical
'so t
MAP
.................
50 "J
~,5o ~- 50
500
. . . . .
300
HR m
S
............
500 ~300
m
1 Nlin
A. Fig. 1. Demonstration of cardiac chronotropic responses to phasic cortical microstimulation for 60 s at insular tachycardia (A) and bradycardia (B) sites. MAP mean arterial pressure (ram Hg): FIR. heart rate (bpm); S. stimulus time marker: R, integrated respiration trace (ml); bpm, beats per minute.
procedures described above, sympathetic or parasympathetic involvement in the responses to insular cortical stimulation under ketamine anesthesia were compared with those previously obtained for chloralose-anesthetized animals TM. A cardiac chronotropic site in the insular cortex was stimulated for 30-60 s before and twice after the injection of atenolol 5 mg/kg ].v. or atropine 0.3 mg/kg i.v. Stimulation occurred within 1 rain of the injection, and approximately 5 rain later. Experiments were performed at tachycardia sites (3 rats) and at bradycardia sites (3 rats).
Statistical analysis The results were analyzed using t-test for data. Relationships between organ and plasma cateeholamines ,or heart rate were evaluated using regression analysis (method of least squares fit) and the t-value calculated from the square root of the coeffieienl of determination (R). 22 The level of statistical significance was set at P < 0.05. RESULTS T h e a v e r a g e b a s e l i n e h e a r t r a t e a n d b l o o d p r e s s u r e for all g r o u p s ( w h i c h w e r e n o t significantly d i f f e r e n t f r o m e a c h o t h e r ) w e r e 347 b p m a n d 92 m m H g , r e s p e c t i v e l y , Bradycardia the
1 min
and tachycardia responses elicited during of insular
cortex
phasic
microstimulation
( w h i c h w e r e u s e d t o d e f i n e t h e site p r i o r t o p r o l o n g e d s t i m u l a t i o n ) a r e i l l u s t r a t e d in Fig. 1. C h a n g e s in p u l s e r a t e o f s i m i l a r m a g n i t u d e b u t o f o p p o s i t e sign o c c u r r e d during the 1 h of stimulation at bradycardia and tachyc a r d i a sites a n d w e r e m a i n t a i n e d f o r this p e r i o d ( T a b l e I). A s m a l l b u t s i g n i f i c a n t d e p r e s s o r e f f e c t w a s s e e n d u r ing 1 h o f s t i m u l a t i o n o f b r a d y c a r d i a sites ( P < 0.05), w h e r e a s i n s u l a r s t i m u l a t i o n a t t a c h y c a r d i a sites d i d n o t affect b l o o d p r e s s u r e . D i a g r a m s o f t h e d i s t r i b u t i o n o f i n s u l a r s t i m u l a t i o n sites
223
,4
4.0-5
-I-0-0
Fig. 2. Diagram of the distribution of cardiac chronotropic sites responsive to stimulation within the insular cortex, and control (unresponsive) sites. The numbers refer to the distance of each section in millimeters from the bregma in the plane of Paxinos and Watson ~9. Filled triangles, bradycardia sites; filled circles, tachycardia sites; open circles, control sites. Sites used in the neuropharmacological experiments are included in this depiction. AC, anterior commissure; AI, agranular insular cortex; CI, claustrum; CPu, caudate-putamen; DI, dysgranular insular cortex; EC, external capsule; En, endopyriform nucleus; F, fornix; GI, granular insular cortex; LV, lateral ventricle; Pir, piriform cortex; 3V, third ventricle.
TABLE I Change in heart rate, arterial blood pressure and mean norepinephrine concentrations (NE) following 1 h of phasic microstimulation in the insular cortex at bradycardia, tachycardia and control sites
Values are expressed as the mean -+ S.E.M. for 6 experiments in each group. Bradycardia
Mean heart rate change (Bpm) Mean arterial pressure change (ram Hg) Mean plasma NE change ( p g / m l )
Tachycardia
Control
-119+24 *** 128-+24*** -13-+4'
-6-+6
0-+6
- 4 6 6 - + 2 7 8 783-+185"*
0+3 -131-+106
Statistically significant differences from control values: * P < 0.05; ** P < 0.0h ***P < 0.001.
from which cardiac chronotropic or control responses were elicited comprise Fig. 2. Sites from which tachycardia responses were obtained were generally situated medially or rostrally to those producing bradycardia. Responses were elicitable from all 3 areas of the insular cortex (granular, dysgranular and agranular zones). Control sites, from which no change in heart rate was obtained during phasic microstimulation, were located either outside, or at the periphery of, the insular cortex. The basal norepinephrine and epinephrine values for all 18 animals were 560 -+ 117 pg/ml and 109 _+ 26 pg/ml, respectively. The initial catecholamine levels did not differ significantly between the 3 groups. At tachycardia sites, stimulation elicited a statistically significant increase both in the mean plasma norepinephfine concentration when compared to the effect of stimulation at control sites (P < 0.05; Fig. 3A), and in the mean change in plasma norepinephrine concentration (P < 0.01; Table I). No significant change was observed
224
B
T~lcm~lia 1600
1200
~
1200
~. 8oo
800
4OO
_A
1600
o
~
Control
t200
-
800
• •
stim. stim.
4OO
~ C Norepimq~hrine f:pinaph~ ne
Fig. 3. Plasma norepinephrine and epinephrine concentrations before, and after 1 h of stimulation at tachycardia, bradycardia and comrol sites within the insular cortex. The asterisk indicates a statistically significant result (P < 0.05).
following stimulation at bradycardia or control sites (Fig. 3B,C). Plasma epinephrine concentrations were unchanged by insular stimulation at any site. A highly significant correlation was demonstrated between the change in plasma norepinephrine concentration and the changes in heart rate following 1 h of insular cortex stimulation when the 18 animals from all 3 groups were considered together (Fig. 4A; R = 0. 79; P < 0.001). A similar relationship was determined for the plasma norepinephrine concentration and the heart rate change after 1 h of stimulation (Fig. 4B; R = 0.69; P < 0.01). No such relationships were observed for plasma epinephrine (Fig, 4C,D). The mean organ catecholamine levels after 1 h of insular cortex stimulation are shown in Table II. Following stimulation at bradycardia sites, a statistically significant (P < 0,05) increase in mean renal norepinephrine concentration over control levels was demonstrated; this was not noted for any of the other organs. No change in the mean organ catecholamine concentration was detectable when the insular cortex was stimulated at tachycardia sites. Organ epinephrine levels were generally undetectable excepting one control and one bradycardia muscle sample.
In the animals stimulated at tachycardia sites, an inverse relationship between cardiac norepinephrine and change in heart rate following 1 h of insular stimulation was demonstrated (Fig. 5A: R = 0.90: P < 0.02). No relationship was detectable between these parameters in rats stimulated at bradycardia sites (Fig. 5B). No significant relationships were observed between renal or skeletal muscle catecholamine levels and any plasma or heart rate variable when animals in each group were individually analyzed after 1 h of stimulation of cardiac or control sites within the insular cortex. The intravenous administration of atenolol prior to insular stimulation decreased the baseline heart rate by 64 --- 19 bpm. In animals stimulated at tachycardia sites atenoloi completely abolished the chronotropie response (Fig. 6). On the other hand, atenolol was without any effect in rats stimulated at bradycardia sites (Fig. 6), Atropine injection raised the heart rate by 12 ~ 9 bpm prior to stimulation and completely abolished the bradycardia response evoked by insular cortex stimulation (Fig. 6). Stimulation-induced tachycardia was unaffected by atropine (Fig. 6).
225
I• E ~
2000 •
1000
2250
R: 0-79; P
221
BRES 17324
Plasma and organ catecholamine levels following stimulation of the rat insular cortex Stephen M. Oppenheimer 1'2'4, Tarek M. Saleh 1, John X. Wilson 3 and David F. Cechetto 1-3 ~Department of Stroke and Aging, Robarts Research Institute, London, Ont. (Canada), Departments of 2Clinical Neurological Sciences and 3Physiology, University of Western Ontario, London, Ont. (Canada) and 4Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD (U.S.A.) (Accepted 20 August 1991) Key words: Insular cortex; Phasic microstimulation; Heart rate; Plasma catecholamine; Organ catecholamine
The posterior insular cortex of the rat contains an area of cardiac chronotropic representation within which tachycardia sites occur rostrally to those producing bradycardia. In the current study using ketamine-anesthetized rats, the insular cortex was stimulated for 1 h using a phasic technique synchronized with the cardiac cycle. Taehycardia was associated with an increase in plasma norepinephrine concentration; epinephrine remained unchanged. This indicates a neural origin of the norepinephrine increment. The tachycardia response was completely blocked by atenolol. Plasma catecholamine levels remained unchanged during stimulation of insular bradycardia sites. Atenolol was without effect during stimulation-induced bradycardia which was completely blocked by atropine. Total cardiac norepinephrine concentration inversely correlated with change in heart rate during stimulation of tachycardia sites. No correlation between intracardiac catecholamines and heart rate variables was found for the bradycardia or control sites. These results indicate that in the ketamine-anesthetized rat, whereas insular stimulation-induced tachycardia is dependent on the sympathetic nervous system, bradycardia elicited by insular cortex stimulation is mediated by parasympathetic mechanisms. No correlation was identified between renal or skeletal muscle norepinephrine levels and any heart rate parameter. This implies that the sympathetic effects of phasic insular microstimulation may be exerted mainly on cardiac nerves, and less so in other visceral beds. INTRODUCTION The insular cortex receives a variety of visceral afferent projections and has efferent connectivity with subcortical sites involved in autonomic control 3'13'21. Recently, topographic organization for cardiac chronotropicity has been d e m o n s t r a t e d within the rat posterior insular cortex 16. B r a d y c a r d i a sites are r e p r e s e n t e d caudally to the m o r e rostrally situated tachycardia sites. Isolated cardiac chronotropic responses were elicited free of any concomitant blood pressure or respiratory changes (which accompany non-phasicalty induced heart rate alterations) using a new technique of phasic microstimulation linked to the R wave of the E C G . In chloraloseanesthetized rats, both tachycardia and bradycardia responses induced by insular cortical phasic stimulation were completely abolished by the fll antagonist atenolol suggesting that each was m e d i a t e d by the sympathetic nervous system ~6. A t r o p i n e was without effect on these heart rate changes, implying little or no parasympathetic involvement. More recently, phasic stimulation of the rat insular cortex linked to the T wave of the E C G was shown to
lead to increasing degrees of heart block, ventricular ectopy and asystolic death 17. A significant elevation of plasma n o r e p i n e p h r i n e but not epinephrine (implying a neural rather than adrenal origin 14) accompanied the earlier E C G changes which were also associated with evidence of structural damage to the heart. Similarly, elevations in plasma n o r e p i n e p h r i n e and in renal sympathetic nerve activity p r e c e d e d E C G changes and death of the experimental animal in the middle cerebral artery occlusion, rat stroke model, when the insular cortex was involved in the infarct 4. These observations suggest that the insular cortex may play an important role in the production of the E C G changes following stroke and in the occurrence of sudden unexpected death noted in that condition 15. The p h e n o m e n a may be m e d i a t e d by hyperactivity expressed by the cardiac sympathetic nerves. The current study was u n d e r t a k e n to investigate symp a t h o a d r e n a l activity in m o r e detail during insular stimulation: plasma, renal, cardiac and skeletal muscle catecholamine levels were d e t e r m i n e d following stimulation at bradycardia, tachycardia and control sites in ketamineanesthetized animals.
Correspondence: D.E Cechetto, The Department of Stroke and Aging, The John P. Robarts Research Institute, 100 Perth Drive, London, Ont. N6A 5K8, Canada.
222 MATERIALS AND METHODS Eighteen male Wistar rats (mean weight 241 g) were divided into 3 groups each of 6 animals according to their response to insular stimulation (bradycardia, tachycardia or control). A reactive site was so designated if initial stimulation for 30-60 s produced a change in heart rate of 20 bpm or more unaccompanied by significant (greater than 5 mm Hg) change in blood pressure. The animals were initially anesthetized with ketamine-xylazine 0.1 ml/100 g i.m. (ketamine 100 mg/ml, xylazine 20 mg/ml), and the femoral artery and vein cannulated. The arterial cannula was connected to a Statham P23D pressure transducer for measurement of the blood pressure. Heart rate was obtained from the pulse pressure, using a Grass 7P44 tachograph. An endotracheal tube was inserted into each animal, which inspired 100% oxygen. Respiration was measured with a Fleisch pneumotachograph. Blood pressure, heart rate, and respiration were recorded by a Grass RPS 7C8 polygraph. The animals' core temperatures were maintained at 37°C with a rectal probe heating pad and a YSI temperature controller. The venous cannula was connected to a compact infusion pump model 975 (Harvard Apparatus Co.) and ketamine infused at a rate of 0.001 ml/min. This rate had been determined to maintain an adequate and constant depth of anesthesia. The animals were placed in a stereotactic frame, and the parietal bone drilled. A glass microelectrode filled with 3 M saline, of internal tip diameter 10-20/~m, was inserted into the left insular cortex using co-ordinates previously determined from stimulation experiments in this region 16. The ECG was monitored in the lead II configuration, and relayed to a signal discriminator which differentiated the R wave from other parts of the ECG waveform. The method of cortical phasic microstimulation which produces cardiac chronotropic responses without accompanying blood pressure or respiratory effects if applied for 1 min, or large blood pressure if applied for 1 h, has been detailed elsewhere 16. In brief, paired stimuli separated by 20 ms were delivered to the insular cortex during every second or third ECG cycle coincident with the R wave and approximately 80-100 ms before the P wave. Allowing for central conduction delay 2, this was anticipated to generate activity in cardiac nerves during the period when the sinoatrial node would be most receptive to chronotropic stimuli. The stimulus parameters were: 2 ms pulse duration, 500 ~A current. Responses were ascertained at progressive 200-/~m steps of microelectrode advancement and the experimental site chosen was that giving maximum responsiveness. Once identified, the site was stimulated for 1 h. One ml of blood was obtained before the onset of stimulation for baseline catecholamine determination. Another sample was obtained after 1 h of stimulation, at which time the heart, right kidney and a portion of the left gastrocnemius muscle were rapidly removed, weighed and then frozen in liquid nitrogen. Plasma and tissue specimens were stored at -80°C prior to catecholamine extraction and analysis. Organ cateeholamine results are expressed as pg per gm of organ wet weight. At the end of each experiment, the animal was perfused using an intra-aortic catheter. The perfusate comprised a solution of 0.9% N saline, followed by 10% formalin. The brain was removed and stored in 10% formalin for at least 2 days, before being cut in the coronal plane on a microtome. The sections were then stained with Thionin and examined using a Leitz Diaplan microscope. Camera lucida tracings of the stimulation sites were thus obtained. Plasma catecholamines were assayed as previously described ~. The organs and muscle were homogenized as follows: the tissue was minced with scissors at 4°C. To this, a volume of ice-cold 0.4 N perchloric acid-GSH solution equivalent to 10 times the wet weight of tissue was added. The mixture was homogenized using a Brinkman Polytron homogenizer fitted with a PT-10 head, and then centrifuged at 2700 rpm (2000 g) for 30 min at 4°C to obtain a protein-free supernatant ~'23. The samples were stored at -80°C until subsequently assayed. Good catecholamine stability is reported under these conditions8. In 6 additional rats which had undergone the general surgical
'so t
MAP
.................
50 "J
~,5o ~- 50
500
. . . . .
300
HR m
S
............
500 ~300
m
1 Nlin
A. Fig. 1. Demonstration of cardiac chronotropic responses to phasic cortical microstimulation for 60 s at insular tachycardia (A) and bradycardia (B) sites. MAP mean arterial pressure (ram Hg): FIR. heart rate (bpm); S. stimulus time marker: R, integrated respiration trace (ml); bpm, beats per minute.
procedures described above, sympathetic or parasympathetic involvement in the responses to insular cortical stimulation under ketamine anesthesia were compared with those previously obtained for chloralose-anesthetized animals TM. A cardiac chronotropic site in the insular cortex was stimulated for 30-60 s before and twice after the injection of atenolol 5 mg/kg ].v. or atropine 0.3 mg/kg i.v. Stimulation occurred within 1 rain of the injection, and approximately 5 rain later. Experiments were performed at tachycardia sites (3 rats) and at bradycardia sites (3 rats).
Statistical analysis The results were analyzed using t-test for data. Relationships between organ and plasma cateeholamines ,or heart rate were evaluated using regression analysis (method of least squares fit) and the t-value calculated from the square root of the coeffieienl of determination (R). 22 The level of statistical significance was set at P < 0.05. RESULTS T h e a v e r a g e b a s e l i n e h e a r t r a t e a n d b l o o d p r e s s u r e for all g r o u p s ( w h i c h w e r e n o t significantly d i f f e r e n t f r o m e a c h o t h e r ) w e r e 347 b p m a n d 92 m m H g , r e s p e c t i v e l y , Bradycardia the
1 min
and tachycardia responses elicited during of insular
cortex
phasic
microstimulation
( w h i c h w e r e u s e d t o d e f i n e t h e site p r i o r t o p r o l o n g e d s t i m u l a t i o n ) a r e i l l u s t r a t e d in Fig. 1. C h a n g e s in p u l s e r a t e o f s i m i l a r m a g n i t u d e b u t o f o p p o s i t e sign o c c u r r e d during the 1 h of stimulation at bradycardia and tachyc a r d i a sites a n d w e r e m a i n t a i n e d f o r this p e r i o d ( T a b l e I). A s m a l l b u t s i g n i f i c a n t d e p r e s s o r e f f e c t w a s s e e n d u r ing 1 h o f s t i m u l a t i o n o f b r a d y c a r d i a sites ( P < 0.05), w h e r e a s i n s u l a r s t i m u l a t i o n a t t a c h y c a r d i a sites d i d n o t affect b l o o d p r e s s u r e . D i a g r a m s o f t h e d i s t r i b u t i o n o f i n s u l a r s t i m u l a t i o n sites
223
,4
4.0-5
-I-0-0
Fig. 2. Diagram of the distribution of cardiac chronotropic sites responsive to stimulation within the insular cortex, and control (unresponsive) sites. The numbers refer to the distance of each section in millimeters from the bregma in the plane of Paxinos and Watson ~9. Filled triangles, bradycardia sites; filled circles, tachycardia sites; open circles, control sites. Sites used in the neuropharmacological experiments are included in this depiction. AC, anterior commissure; AI, agranular insular cortex; CI, claustrum; CPu, caudate-putamen; DI, dysgranular insular cortex; EC, external capsule; En, endopyriform nucleus; F, fornix; GI, granular insular cortex; LV, lateral ventricle; Pir, piriform cortex; 3V, third ventricle.
TABLE I Change in heart rate, arterial blood pressure and mean norepinephrine concentrations (NE) following 1 h of phasic microstimulation in the insular cortex at bradycardia, tachycardia and control sites
Values are expressed as the mean -+ S.E.M. for 6 experiments in each group. Bradycardia
Mean heart rate change (Bpm) Mean arterial pressure change (ram Hg) Mean plasma NE change ( p g / m l )
Tachycardia
Control
-119+24 *** 128-+24*** -13-+4'
-6-+6
0-+6
- 4 6 6 - + 2 7 8 783-+185"*
0+3 -131-+106
Statistically significant differences from control values: * P < 0.05; ** P < 0.0h ***P < 0.001.
from which cardiac chronotropic or control responses were elicited comprise Fig. 2. Sites from which tachycardia responses were obtained were generally situated medially or rostrally to those producing bradycardia. Responses were elicitable from all 3 areas of the insular cortex (granular, dysgranular and agranular zones). Control sites, from which no change in heart rate was obtained during phasic microstimulation, were located either outside, or at the periphery of, the insular cortex. The basal norepinephrine and epinephrine values for all 18 animals were 560 -+ 117 pg/ml and 109 _+ 26 pg/ml, respectively. The initial catecholamine levels did not differ significantly between the 3 groups. At tachycardia sites, stimulation elicited a statistically significant increase both in the mean plasma norepinephfine concentration when compared to the effect of stimulation at control sites (P < 0.05; Fig. 3A), and in the mean change in plasma norepinephrine concentration (P < 0.01; Table I). No significant change was observed
224
B
T~lcm~lia 1600
1200
~
1200
~. 8oo
800
4OO
_A
1600
o
~
Control
t200
-
800
• •
stim. stim.
4OO
~ C Norepimq~hrine f:pinaph~ ne
Fig. 3. Plasma norepinephrine and epinephrine concentrations before, and after 1 h of stimulation at tachycardia, bradycardia and comrol sites within the insular cortex. The asterisk indicates a statistically significant result (P < 0.05).
following stimulation at bradycardia or control sites (Fig. 3B,C). Plasma epinephrine concentrations were unchanged by insular stimulation at any site. A highly significant correlation was demonstrated between the change in plasma norepinephrine concentration and the changes in heart rate following 1 h of insular cortex stimulation when the 18 animals from all 3 groups were considered together (Fig. 4A; R = 0. 79; P < 0.001). A similar relationship was determined for the plasma norepinephrine concentration and the heart rate change after 1 h of stimulation (Fig. 4B; R = 0.69; P < 0.01). No such relationships were observed for plasma epinephrine (Fig, 4C,D). The mean organ catecholamine levels after 1 h of insular cortex stimulation are shown in Table II. Following stimulation at bradycardia sites, a statistically significant (P < 0,05) increase in mean renal norepinephrine concentration over control levels was demonstrated; this was not noted for any of the other organs. No change in the mean organ catecholamine concentration was detectable when the insular cortex was stimulated at tachycardia sites. Organ epinephrine levels were generally undetectable excepting one control and one bradycardia muscle sample.
In the animals stimulated at tachycardia sites, an inverse relationship between cardiac norepinephrine and change in heart rate following 1 h of insular stimulation was demonstrated (Fig. 5A: R = 0.90: P < 0.02). No relationship was detectable between these parameters in rats stimulated at bradycardia sites (Fig. 5B). No significant relationships were observed between renal or skeletal muscle catecholamine levels and any plasma or heart rate variable when animals in each group were individually analyzed after 1 h of stimulation of cardiac or control sites within the insular cortex. The intravenous administration of atenolol prior to insular stimulation decreased the baseline heart rate by 64 --- 19 bpm. In animals stimulated at tachycardia sites atenoloi completely abolished the chronotropie response (Fig. 6). On the other hand, atenolol was without any effect in rats stimulated at bradycardia sites (Fig. 6), Atropine injection raised the heart rate by 12 ~ 9 bpm prior to stimulation and completely abolished the bradycardia response evoked by insular cortex stimulation (Fig. 6). Stimulation-induced tachycardia was unaffected by atropine (Fig. 6).
225
I• E ~
2000 •
1000
2250
R: 0-79; P
