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Central sites involved in the hypotensive effects of muscimol
Vol. 5, Suppl. 2, pp. 317-323. Printed in the U.S.A
Bruin ~c~.sporch Bulktin,
Central Sites Involved in the Hypotensive Effects of Muscimol DAVID W. SNYDER’ AND MICHAEL Squibb
Institute
for Medical
Research,
Department
J. ANTONACCIO
of Pharmacology,
P.O. Box 4000, Princeton,
NJ 08540
SNYDER, D. W. AND M. J. ANTONACCIO. Central sites invdwd in the hypotensive effeec?sof muscimol. BRAIN RES. 5: Suppl. 2, 317-323, 1980.-Areas within the brain mediating the hypotensive effects of muscimol, a GABA receptor agonist, were localized in chloralose anesthetized cats. Infusion of muscimol (0.003-0.03 &kg/min) into the lateral ventricle for 30 min resulted in a dose-related fall in blood pressure, heart rate and renal sympathetic nerve discharge when the perfusate was collected at the cistema magna (ICV). Under these conditions the largest dose produced decreases in blood pressure, heart rate and renal nerve discharge of 40%. 30% and 80% respectively. When bicuculline methiodide (0.5 &kg/min), a GABA receptor antagonist, was administered via the cerebral aqueduct simultaneously with the ICV infusion of muscimol, the cardiovascular effects of muscimol were antagonized. Infusion of bicuculline methiodide alone failed to alter blood pressure or heart rate. When muscimol was prevented from reaching the fourth ventricle by collecting the perfusate at the caudal end of the cerebral aqueduct, the largest dose produced a reduction in blood pressure and heart of i 15%. Administration of muscimol into the caudal region of the fourth ventricle failed to alter blood pressure, heart rate or renal nerve discharge. In contrast, administration of muscimol into tht anterior region of the fourth ventricle produced large reductions in blood pressure and heart rate. These results indicate that the major sites mediating the hypotensive actions of muscimol are located in the anterior region or anteroventral surface of the medulla with a small contribution stemming from supra-medullary sites. BULL.
Muscimol Central control of blood pressure GABA GABA receptors and blood pressure
acid (GABA), a putative neural transmitter, has been shown to lower blood pressure and heart rate in several species including man [5, 7, 17, 20, 29, 3 1, 321. These cardiovascular effects of GABA are mediated via a central reduction in sympathetic vasomotor outflow [5, 7, 321. Recently it was discovered that muscimol, a GABA mimetic which binds specifically to central GABA receptors [18,22], is 1000 times more potent than GABA in reducing blood pressure, heart rate and renal sympathetic nerve activity [5]. The cardiovascular effects of both GABA and muscimol can be antagonized by bicuculline, a GABA receptor antagonist [5]. The present study was designed to localize those areas of the brain which mediate the hypotensive effects of muscimol. GAMMA-aminobutyric
METHOD
Geneml
Adult mongrel cats were anesthetized with a-chloralose (60 mg/kg IV) following a brief induction period with ether. A cannula was placed in the trachea and the cat was artificially respired with a Harvard respirator. A femoral artery and vein were cannulated for the measurement of systemic blood pressure and the injection of drugs, respectively. Blood
Centrally-acting
antihypertensive
agents
pressure was measured with a Statham 23 Gb transducer and displayed on a Beckman Dynograph. Heart rate was monitored with a Beckman cardiotachometer triggered from the blood pressure pulse and also displayed on a Beckman Dynograph. Recortiing Sympathetic
Nerve
Acrivity
Cats used in the determination of the effects of muscimol on sympathetic nerve discharge were prepared as described above and immobilized with decamethonium bromide (0.2 mg/kg IV). Supplemental doses of decamethonium were administered as required during the experiment. A unilateral pneumothoracotomy was performed to reduce respiratory artifacts in the nerve recording. The left kidney was approached retroperitoneally and one of the renal nerves was ligated and severed. The proximal portion of the nerve was placed on a bipolar platinum electrode to record post-ganglionic action potentials monophasitally. Nerve potentials were amplified with a differential preamplifier with high and low pass filter settings at 1 and 1000 Hz, respectively. Integrated nerve activity was derived by feeding the signal into a Beckman resetting integrator (Model no. 9873B). The resetting integrator was shorted to 0 at a constant interval by a specially constructed external
‘Send reprint request to: David W. Snyder, Squibb Institute for Medical Research, Department of Pharmacology, NJ 08540
Copyright
0 1980 ANKHO
International
Inc.-0361-9230/80/080317-07$01.20/O
P.O. Box 4000, Princeton,
31x
SNYDER AND AN’I’ONAC?CIO
timer. Thus, the height of each epoch of integration was proportional to the amount of actual nerve activity emanating from the sympathetic nerve per unit of time. Neural data were expressed as percent of control (i.e., treated epoch height/control predrug epoch height x 100).
In 15 cats the entire ventricular system of the brain was perfused using the method described by Vollmer and Boccagno [33]. Briefly, a perfusion inflow cannula consisting ofa 22 ga hypodermic needle was lowered into the right lateral ventricle at the coordinates: anterior 15 mm, lateral 2.5 mm, and horizontal + 6.7 mm according to the atlas of Snider and Niemer 1271. An outflow cannula of PE 90 polyethelene tubing was advanced through the atlanto-occipital membrane into the cistema magna. Artificial cerebrospinal fluid (CSF) 1241 was infused continuously at a rate of 0.1 ml/mm using a Harvard infusion pump. Pl~~cement of Inflow Cu~~~la in the Cerebral A~uedact In 4 of the cats described above a second inflow cannula was placed in the cerebral aqueduct according to the procedure of Vollmer and Buckley [34] for administration of bicuculline methiodide (bicuculline), a GABA antagonist. The cannula, consisting of a 22 ga unbeveled hypodermic needle, was lowered through a hole in the skull and positioned in the cerebral aqueduct at coordinates: 1271anterior 1.O mm; lateral 0.0 mm and horizontal + 1.O mm. Placement of this cannula did not obstruct flow of CSF through the cerebral aqueduct. During the drug infusion period, bicuculline was administered via the cerebral aqueduct cannula while vehicle (CSF) or drug was simultaneously administered via the lateral ventricle cannula. As indicated by the distribution of methylene blue dye, this procedure prevented bicuculline from reaching the 3rd ventricle and directed the drug posteriorly toward the fourth ventricle. Perfusion of Supramedullary
Area
In 5 cats the supramedu~a~ region of the brain was perfused. An inflow cannda was placed in the lateral ventricle as described above and an outflow cannula (PE 160 “cuffed” with small piece of PE 205) was inserted into the cerebral aqueduct following retraction of the cerebellum to expose the caudal opening of the aqueduct. The diameter of the outflow cannula was such that it impeded flow to the fourth ventricle. Thus, only supramednllary structures were exposed to the drug. Proper placement of the cannulae was determined by injecting methylene blue dye into the inflow cannula at the end of each experiment. Absence of dye in the medulla was used as evidence for proper cannulation of the cerebral aqueduct. Perfusion of the Anterior Region of the Fourth Ventricle In 4 cats the anterior region of the fourth ventricle was perfused. In these cats an outflow cannula was placed in the cerebral aqueduct to prevent drugs from reaching supramedullary areas as described above. An inflow cannula (PE 50) was inserted beside the cerebral aqueduct outflow can&a so that the tip of the cannula was positioned 1.5 cm from the caudal tip of the cerebellum. With this procedure, the perfusate reaching the anterior region of the medulla was allowed to escape freely from the caudal region of the
brainstem. Dye (neutral red) was injected at the end of each experiment to determine the extent of the area perfused.
In 13 cats, the caudal region of the fourth ventricle was perfused. A small inflow cannula (PE 50) was inserted through the dura covering the foramen magnum and advanced 8 mm into the caudal region of the fourth ventricle. The tip of the cannula rested on the dorsal surface of the brainstem immediately anterior to the obex. The outflow cannula was positioned in the cisterna magna, as described under “Perfusion of the Lateral Ventricle”. At the end of each experiment, methylene blue dye was added to the perfusion system to determine the region of the fourth ventricle which had received the drug. Compounds administered into the ventricular system of the brain were dissolved in artificial CSF prior to usage and infused with a Harvard infusion pump at a rate of 0. I mtimin. Compounds administered intravenously were dissolved in physiological saline and given as a bolus. Results are expressed as the mean + SE. Statistical significance was calculated using Student’s t-test or analysis of variance {ZS]. RESULTS
Intracerebroventricular
Infusion of Muscimol
Intracerebroventricular infusion of muscimol in chloralose anesthetized cats produced significant reductions in mean arterial pressure, heart rate and renal sympathetic nervous discharge (Fig. I). The cardiovascular effects of muscimol were dose related (Fig. 2). An infusion of 0.003 pg/kg/min produced a slight fall in blood pressure with virtually no change in heart rate. An infusion of 0.01 or 0.03 &kg/min produced significant decreases in both blood pressure and heart rate. Sympathetic nerve activity increased initially followed by a slight depression with 0.01 &kg/min of muscimol. Increasing the dose of muscimol produced significant inhibition of sympathetic nerve activity. ICV administration of bicuculline (I p&ikg) reversed the effects of muscimol on blood pressure and heart rate but failed to restore renaf nerve activity to its pre-muscimol level (Fig. 1). Administration oj’M~.~cimul to the Caudal Region of the Fourth Ventricle In 13 cats, muscimol was administered to the caudal region of the medulla since most of the centrally acting antihypertensive agents such as clonidine and L-DOPA produce their effects by stimulating neural components which lie in the lower brainstem. Infusion of 0.03 &kg/min of muscimol into the caudal region of the fourth ventricle failed to significantly alter blood pressure, heart rate or renal nerve activity (Figs. 3 and 4). In 8 of these cats the same dose of muscimol admi~st~red into the lateral ventricle produced marked reductions in blood pressure, heart rate and sympathetic nervous discharge (Figs. 3 and 5). Furthermore, in the 5 remaining cats, administration of clonidine (I.0 cLg!k@min for 10 min) to the caudal region of the medulla produced significant reductions in blood pressure and heart rate. Figure 6 summarizes the cardiovascular effects of muscimol and clonidine administered into the caudal region of the fourth ventricle. Administration
of Muscimol
to ~u~rameduila~
In 5 cats, an outflow cannula
Areas
was placed in the caudal
LOCATION
OF THE HYPOTENSIVE A Contrd
EFFECTS
B Muscii
OF MUSCIMOL TIME(min)
c Bic~ulline
pet-f usion period t 0 5 IO 15 I 1 I f
Heart Rate (bpm)
I 30 ,
45 I
60 b
1
1
Arterial Pressure (mmHg1
Mean
Arterial Pressure tmmHg)
-SO]
Renal Nerve Dischbrge
pM&ed
1 Dischaqe Nerve
t t Blc hi&mimol
O,O+g/kg/min
!d
be/M
+20+ IO-
g
o8_:;_ &
FIG. 1. A typical experiment demonstrating the effects of muscimol on blood pressure, heart rate and renal nerve discharge when administered into the lateral ventricle. A. Control record before the infusion of muscimol. Between A and B muscimol (0.03 FgjkJmin) was administered XV for 30 min. B. Record was taken after completion of the infusion. Between B and C bicu~ulline methi~ide (I pgikg ICV) was administered. C. Record was taken 30 see after the administration of bicuculline.
region of the cerebal aqueduct thus preventing the perfusate from reaching the fourth ventricle and bathing the lower brainstem. Restricting muscimol (0.03 pg/kg/min) to suprameduIlary areas resulted in a small (lO-15%) fall in blood pressure and heart rate (Figs. 7 and 8).
2 -3OE -4o;
I;;_.
d-70- eo-
-90-l Muscimol:
*p
Administration of Muscimnl Fourth Ventricle
I.C.V. I.C.V. I.C.V.
* *p
to the Anterior Region of the
In 4 cats an outflow cannula was placed in the cerebral aqueduct and an inflow cannula was placed in the anterior region of the fourth ventricle. Restricting muscimol (0.03 &kgimin) to the lower brainstem resulted in marked reductions in blood pressure and heart rate. The c~diovascular effects produced by muscimol administered to the anterior fourth ventricle were no different from those seen upon administration of muscimol to the entire ventricular system (Figs. 8 and 9). Bicaculline Antagonism
D 0.003 pg/kg/min, m O.OlO~g/kg/min, Cl O.O~~~k~rnin,
of’ Medullary Effects
of Muscimol
Attempts were made to antagonize the effects of muscimol by administe~ng low doses of bicuculline to the lower brainstem of the cat. In agreement with WiIl~ord et al., [35], bicuculline (0.5 ~~k~rnin) failed to alter blood pressure or heart rate when administered into the cerebral aqueduct. The perfusion system prevented bicuculline from reaching neural structures rostal to the cerebral aqueduct, but did allow the GABA antagonist to enter the 4th ventricle and bathe the lower brainstem. The simultaneous infusions of muscimol (0.03 pg/kgimin XV) and bicuculline (0.5 &kg/min cerebral aqueduct) produced a smail (IO-15%) reduction in blood
FIG. 2. Summary of the effects of muscimol administered ICV on blood pressure (top), heart rate (middle) and renal nerve discharge (bottom) of anesthetized cats. In all cases, the drug was administered for 30 min. Abscissa is time (min) after start of the infusion. Data are expressed as percent change from control. Number of animals is indicated in parenthesis. pressure and heart rate (Fig. 8). Thus bicuculline prevented most of the effects of muscimol. The hypotensive response that remained was comparable to that observed when muscimol was confined to the supramedullary region of the brain. DISCUSSION
These data indicate that the major cardiovascular action of muscimol is restricted to the anterior region of the medulla. This was demonstrated by the fact that infusion of muscimol into the anterior region of the 4th ventricle resulted in both hypotension and bradycardia, whereas infusion of muscimol solely into the posterior region of the fourth ventricle had no cardiovascular effect. The fall in blood pressure and heart rate seen with anterior medullary perfusion was comparable to that seen when muscimol was adminis-
SNYDER
AND ANTONACC’IO
TIME(min) perfusionperiod
(It=?) (n=6)
FIG. 3. A typical experiment demonstrating the lack of effect of muscimol on blood pressure, heart rate and renal nerve discharge when administered into the caudal region of the fourth ventricle. A. The intravenous ~rni~st~tion of a botus of norepineph~ne (1.0 &kg) produced a pressor response associated with a reflex fall in heart rate and reduction in renal nerve discharge which indicates the system is functioning properly. Control record was taken prior to administration of muscimol. Between A and B muscimol (0.03 ~~~rnin) was infused into the caudal region of the fourth venWicle for 30 min. B. Record was taken at the end of the infusion of muscimol. Between B and C muscimol(O.03 &kg/min) was infused into the lateral ventricle for 30 min. C. Record was taken at the end of the infusion of muscimol.
tered to the entire ventricular system. These cardiovascular responses were mediated through activation of GABA receptors since both hypotension and bradycardia were attenuated by the GABA receptor antagonist bicuculline. In this study, administration of bicuculline alone to the hindbrain, failed to alter blood pressure or heart rate confirming the findings of Williford et ul., [35]. The caudal region of the medulla can be excluded as a site of action since administration of muscimol solely to this region failed to alter blood pressure or heart rate. In contrast, ICV administration of muscimol did produce the expected cardiovascular response, indicating that the animals were not refractory to the drug. In addition, administration of clonidine, a centrally acting antihypertensive agent, onto the caudal region of the medulla did lower blood pressure and heart rate, suggesting that the caudal region of the medulla was responsive. This difference between the sites of action of clonidine and muscimof indicates that the hypotensive actions of these two drugs are mediated through activation of different receptors. Clonidine is believed to produce its centrally mediated cardiovascular effects by stimulating a-adrenergic receptors [23,25] while muscimol is a GABA mimetic. Compared to most centrally acting antihypertensive agents, the lack of effect of muscimol in the caudal region of the medulla is unique. Other reports have also suggested that more anterior regions of the medulla are important for the cardiovascular effects of muscimol. Guertzenstein [2Ol demonstrated that GABA applied topically to the anteroventral surface of the medulla of anesthetized cats elicited a fall in blood pressure. Bousquet et al., f91 demonstrated that microinjections of muscimol into the ventrolateral medulla between the trapezoid bodies and the root of the
8 +40-
2 +30% +20$ + IO4 5
- IO OI - 20-30wsada_
* O,O&&kg/min, 4#hw~trkle 0 O.~~,~ wltt?kltt
FIG. 4. Lack of effect on blood pressure (top), heart rate (middle) and renal nerve discharge (bottom) of muscimol infused into the caudal region of the fourth ventricle. Data are expressed as percent change from controLAbscissa is time (min) from start of the perfusion period which lasted 30 min. Number of animals is indicated in parenthesis.
hypoglossal nerve reduced blood pressure and heart rate in urethane-anesthetized cats. However the dose of muscimol used by Bousquet to evoke the response in a localized area of brain was high compared to the dose needed in the present study to produce a similar change in blood pressure when administered ICV. Neurons mediating sympathetic activity in the ventrolateral medulla which were exposed to the drug in these previous studies probably comprise part of the lateral reticular nucleus (211. In the present study the caudal extent of this nucleus was bathed with muscimol during perfusion of the caudal medulla, yet neither blood pressure nor heart rate was altered. Thus, muscimol may be acting at the more rostra1 extent of this brainstem nucleus as suggested by Guertzenstein and Silver [21]. It is possible that the region of periaqueductal gray comprising the caudal extent of the nucleus raphe dorsalis is cont~buting to the cardiovascular effects of musci~ol. This nucleus is composed largely of serotonergic neurons USI. Serotonergic neurons are distributed throughout the lower brainstem [ 151and pharmacological evidence indicates that they can raise or lower blood pressure depending on their Iocation [ 11,30]. Recently Beiin et al., [6] localized GABAergic neurons in nucleus raphe dorsalis in rats. Electrical stimulation of this nucleus increases blood pressure with lit-
LOCATION
OF THE HYPOTENSIVE
EFFECTS
TIME(minf perfusion period t I 0 5 IO I5 30 45 t
x
/
321
OF MUSCIMOL TIME (min) perfusion
period
60 (n=7)
r
x
,:<:
p
(n-8)
@ Muscimol 0 Muscimol a Clonidine
+20+ IOO- IO-2o-3o-4o-5o-6O-?O-90 -
ventricle
,4th ventricle
I .O ~g/kg/min~ 4th ventricle f IO min perfusion)
FIG. 6. Comparison of the effects on blood pressure (top) and heart rate (bottom) of muscimol and clonidine administered into the caudal region of the fourth ventricle of anesthetized cats. Muscimol was infused for 30 min. Clonidine (1 .O pg/kg/min) was infused for 10 min. Data are expressed as percent change from control. Abscissa is time (min) from start of perfusion period. Number of animals is indicated in parenthesis. A
Muscimot: 0 0.03 ~g/k~rnin, 0 O.O3~g/kg/min, *p
4th ventricle I.C.V.
**p
FIG. 5. Comparison of the effects on blood pressure (top), heart rate (middle) and renal nerve discharge (bottom) of muscimol infused ICV and into the caudal region of the fourth ventricle of anesthetized cats. Muscimol (0.03 @kg/min) was infused for 30 min. Data are expressed as percent change from control. Abscissa is time (min) from start of perfusion period. Statistical significance was measured between the cardiovascular changes induced by the two routes of administration of muscimol. Number of animals is indicated in parenthesis.
in heart rate [26]. Possibly, the cardiovascular responses to electrical activation of the nucleus raphe dorsalis is due to activation of serotonergic neurons. In addition it has been shown that local administration of GABA to the nucleus inhibits the firing of serotonergic neurons; an action which is blocked by picrotoxin, a GABA antagonist [I]. Thus, GABA-ergic neurons may in part influence cardiovascular function by modulation of serotonergic neurons in this nucleus. Since, in this study, muscimol did have access to this area of the brainstem during perfusion of the anterior medulla, one cannot rule out the possibility that part of the hypotensive effect of muscimol may be mediated by these GABA-ergic neurons. The small forebrain effect of muscimol was demonstrated in experiments in which muscimol was restricted to the lat-
KZ (bpmf
180
B
CONTROL
240
tie change
0.01 pg/kg/min,4th
0.03 pg/kg/min
__~__
SUPRAMEDULLARY AREA .-_ .^
Arterial Preaaure (mmtrZlf 01
Mean Arterial +s.ere (~~)
180 S0 I----
d
t
Muscimol
t---i
1 min
0.03~gkgfmin
FIG. 7. Typical experiment demonstrating the SupramedulIary actions of muscimol on heart rate and arterial pressure. A. Control record was taken prior to the infusion of muscimol. Between A and B muscimol (0.03 pglkgimin) was infused into the lateral ventricle and perfusate was collected at the caudal end of the cerebral aqueduct. B. Record was taken at the end of the 30 min infusion of muscimol. eral and third ventricles.
Under these conditions, muscimol lowered blood pressure and heart rate IO-W%. Evidence for a supramedullary action of muscimol also was obtained in experiments in which the medullary effects of muscimol were blocked by bicuculline. Since bicuculline did not reach the third or lateral ventricles, the forebrain mediated hypotensive and bradycardic effect of muscimol were expressed.
SNYDER Time (mid
perfusionperiod b L
5
II
10
15
1
3b
A 45
I
CONTROL
60
I
0
Heart Rate fbpm)
e!
I
240
t!J20 -
1
160
Arterial
180
Pressure @)mW
w 0 380
2 "
B ANTERKM 4th VENTRICLE
120 -10 -
&I 3
H:
AND ANTONACCIO
.30 -
Mean Arterial
.40 -
90 0
ae
3 -- t
H 1
min
Muacimol
en
O.~~g~g/min 0-
f!
-lO-
P .5
.20 -
FIG. 9. Typical experiment demonstrating the effects on blood pressure and heart rate of muscimol administered to the anterior region of the fourth ventricle. A. Control record prior to the infusion of muscimol. Between A and B muscimol(O.03 pg/kg/min) was infused into the anterior region of the fourth ventricle. B. Record was taken after the 30 min infusion of muscimol.
! z
-30 -
40-
D
Muccimol, 0.03pp/kg/min,i.e.v. (n-81
FIG. 8. Effects of the simultaneous infusions of bicuculline methiodide via the cerebral aqueduct and muscimol ICV on blood pressure (top) and heart rate (bottom). These effects are compared to those produced by muscimol alone (ICV) and when muscimol was restricted to supramedullary areas. Data are expressed as percent change from control. Abscissa is time (min) from start of drug. Number of animals is indicated in parenthesis.
There was no evidence of a forebrain effect of muscimol if muscimol was first infused into the anterior region of the fourth ventricle. This would suggest that muscimol was acting at two different sites along the same neural pathway. If this is the case then one may question the significance of a forebrain site of action. However, forebrain GABA receptors which influence blood pressure and heart rate may be important in expressing the cardiovascular action of benzodiazepines. Diazepam has been demonstmted to reduce resting blood pressure and heart rate [8, 10, 161. It is believed that diazepam has a supramedullary site of action since it reduced supramedullary evoked pressor responses without altering medullary evoked pressor responses [2]. Muscimol also inhibited the pressor response evoked by electric%l stimulation of the diencephalon [3]. Moreover, ICV but not intracisternal administration of diazepam was effective in lowering blood pressure, an action that was attenuated by
intraperitoneal injection of picrotoxin 181. The cardiovascular effect of diazepam is probably not mediated by central a-adrenergic receptors, since the a-adrenergic receptor blocking agent piperoxane could not antagonize the reduction in arteriai pressure caused by diazepam [ 191. Biochemical evidence suggests that at least some actions of diazepam and other benzodiazepines may be mediated via increases in GABA receptor activity [12, 13, 14, 191. Thus, the forebrain cardiovascular effects of muscimol demonstrate the existence of a neural substrate which may be mediating the cardiovascular action of benzodiazepines. In the present study, a 30 min i&&ion of muscimol into the entire ventricular system of the cat lowered blood pressure, heart rate and renal sympathetic nerve activity. Similar observations have been reported by others when muscimol was infused ICV [29] or when administered as a bolus into the third ventricle 13, 4, 51. Infusion of muscimol over a 30 min period did not produce dose related reductions in renal sympathetic nerve discharge. At low doses renal sympathetic nerve activity increased initially. This increase in sympathetic activity was associated with a decrease in blood pressure and thus was probably related to a com~nsato~ action of the baroreceptor reflex. The increase in renal nerve activity was usually followed by a decrease. Similar results have been reported by others in which muscimol was administered as a bolus into the third ventricle. The inhibition of renal nerve activity seen with the high dose of muscimol paralleled the f&l in blood pressure and heart rate and was likely due to a cent& action of the drug. The data in the present study indicate that the primary cardiovascular effects of muscimol are mediated through the activation of GABA receptors in the anterior or anteroventral region of the medulla. A small but significant component is mediated by forebrain structures probably lying in close proximity to the lateral and third ventricle and cerebral aqueduct.
LOCATION
OF THE HYPOTENSIVE
EFFECTS OF MUSCIMOL
323
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19. Fuxe, K., L. F. Agnati, P. Bolme. T. Hokfelt, P. Ledbrink, A. Ljungdahl, M. Perez de la Mora and S. 0. Ogren. The possible involvement of GABA mechanisms in the action of benzodiazepines on central catecholamine neurons. In: Mrchnnism of Acfiorl
of Brn;oditrzrpirrP.s.
Adt~ar~cc.c in Biochpmiccrl
Psy-
c:ilophurmrr;,o/t,a?‘. Vol. ‘14. edited by E. Costa and P. Greengard. New York: Raven Press, 1976, pp. 45-61. 20 Guertzenstein, P. G. Blood pressure effects obtained by drugs applied to the ventral surface of the brainstem. J. fh.v.sio/.. Lend. 229: 395408, 1973. 21. Guertzenstein. P. G. and A. Silver. Fall in blood pressure produced from discrete regions of the ventral surface of the medulla by glycine and lesions. J. Physiof., Lontl, 242: 489-503, 1974.
22. Johnston, G. A. R. Physiologic pharmacology of GABA and its antagonists in the vertebrate nervous system. In: GABA in Ncn~cms Sy.~rcv~ Function, edited by E. Roberts, T. N. Chase and D. B. Tower. New York: Raven Press, 1976, pp. 395-411. W. and L. Pichler. Evidence for direct 23. Kohinger, u-adrenoceptor stimulation of effector neurons in cardiovascular centers by clonidine. Errr. J. ~~7~ir~?~~~~,. 27: 151-154, 1974. 24. Pappenheimer, J. R., S. R. Heisey. E. F. Jordan and J. De C. Downer. Perfusion of the cerebral ventricular system in unanesthetized goats. An7. J. Ph.v.sio/. 203: 763-774, 1962. 25. Schmitt, H., H. Schmitt and S. Fenard. Evidence for an tr-sympathomimetic component in the effects of catapresan on vasomotor centers: antagonism by piperoxane. Eltr. J. f’har~?~~~c’. 14: 98-100, 1971. 26. Smlts, J. F. M., H. van Essen and H. A. J. Struyker-Boudier. Serotonin-mediated cardiovascular responses to electrical stinlulation of the raphe nuclei in the rat. L$i> SC!. 23: 173-178, 1978. 27 Snider, R. S. and W. T. Niemer. A Sfert,ottr.ric Adtrs c$thc Cot Brnir~. Chicago: University of Chicago Press, 1961. 28. Steel, R. G. D. and J. H. Torrie. Principles um/ Procdrrres of S~urisfics. New York: McGraw-Hill Book Company, l%O. 29. Sweet. C. S.. H. C. Wenger and D. M. Gross. Central antihypertensive properties of muscimol and related y-aminobutyric acid agonists and the interaction of muscimol with baroreceptor reflexes. Cnir. .I. Ph.v$ioi. Phtrnrtcrc~. 57: 600605,
1979.
30. Tadepatli, A. S.. E. Mills and S. M. Schanberg. Central depression of carotid baroreceptor pressor response, arterial pressure and heart rate by 5-hydroxytryptophan: influence of supracollicular areas of the brain. .I. Phcrrmclc,. c.rp. Thor. 202: 310-319, 1977. 31. Takahashi, H., M. Tiba, M. Ino and T. Takayasu. The effect of y-aminobutyric acid on blood pressure. .!(lp. .I. Physiol. 5: 334339, 1955. 32. Takahashi, H., M. Tiba, T. Yamazaki and F. Noguchi. On the site of action of ~-aminobuty~c acid on blood pressure. Jop. f. Pl7~siol. s: 378-379, 1958. 33. Vollmer, R. R. and J. A. Boccagno. Central cardiovascular effects of SQ 14.225, an angiotensin-converting enzyme inhibitor in chloralose-anesthetized cats. Eur. .I. Phtrm~. 45: 117-125. 1977. 34. Vollmer, R. R. and J. P. Buckley. Central cardiovascular effects of phentolamine in chloralose-anesthetized cats. Eur. J. Phclrnr(r(,.43: 17-25. 1977. 35. Williford, D. J., B. L. Hamilton, J. Dias Souza, T. P. Williams, J. A. Di Micco and R. A. Gillis. Arterial pressure and heart rate changes produced by activation of y-aminobutyric acid (GABA) receptors in the brainstem of cats. F&I P~oc.. 38: 2366, 1979.
Bruin ~c~.sporch Bulktin,
Central Sites Involved in the Hypotensive Effects of Muscimol DAVID W. SNYDER’ AND MICHAEL Squibb
Institute
for Medical
Research,
Department
J. ANTONACCIO
of Pharmacology,
P.O. Box 4000, Princeton,
NJ 08540
SNYDER, D. W. AND M. J. ANTONACCIO. Central sites invdwd in the hypotensive effeec?sof muscimol. BRAIN RES. 5: Suppl. 2, 317-323, 1980.-Areas within the brain mediating the hypotensive effects of muscimol, a GABA receptor agonist, were localized in chloralose anesthetized cats. Infusion of muscimol (0.003-0.03 &kg/min) into the lateral ventricle for 30 min resulted in a dose-related fall in blood pressure, heart rate and renal sympathetic nerve discharge when the perfusate was collected at the cistema magna (ICV). Under these conditions the largest dose produced decreases in blood pressure, heart rate and renal nerve discharge of 40%. 30% and 80% respectively. When bicuculline methiodide (0.5 &kg/min), a GABA receptor antagonist, was administered via the cerebral aqueduct simultaneously with the ICV infusion of muscimol, the cardiovascular effects of muscimol were antagonized. Infusion of bicuculline methiodide alone failed to alter blood pressure or heart rate. When muscimol was prevented from reaching the fourth ventricle by collecting the perfusate at the caudal end of the cerebral aqueduct, the largest dose produced a reduction in blood pressure and heart of i 15%. Administration of muscimol into the caudal region of the fourth ventricle failed to alter blood pressure, heart rate or renal nerve discharge. In contrast, administration of muscimol into tht anterior region of the fourth ventricle produced large reductions in blood pressure and heart rate. These results indicate that the major sites mediating the hypotensive actions of muscimol are located in the anterior region or anteroventral surface of the medulla with a small contribution stemming from supra-medullary sites. BULL.
Muscimol Central control of blood pressure GABA GABA receptors and blood pressure
acid (GABA), a putative neural transmitter, has been shown to lower blood pressure and heart rate in several species including man [5, 7, 17, 20, 29, 3 1, 321. These cardiovascular effects of GABA are mediated via a central reduction in sympathetic vasomotor outflow [5, 7, 321. Recently it was discovered that muscimol, a GABA mimetic which binds specifically to central GABA receptors [18,22], is 1000 times more potent than GABA in reducing blood pressure, heart rate and renal sympathetic nerve activity [5]. The cardiovascular effects of both GABA and muscimol can be antagonized by bicuculline, a GABA receptor antagonist [5]. The present study was designed to localize those areas of the brain which mediate the hypotensive effects of muscimol. GAMMA-aminobutyric
METHOD
Geneml
Adult mongrel cats were anesthetized with a-chloralose (60 mg/kg IV) following a brief induction period with ether. A cannula was placed in the trachea and the cat was artificially respired with a Harvard respirator. A femoral artery and vein were cannulated for the measurement of systemic blood pressure and the injection of drugs, respectively. Blood
Centrally-acting
antihypertensive
agents
pressure was measured with a Statham 23 Gb transducer and displayed on a Beckman Dynograph. Heart rate was monitored with a Beckman cardiotachometer triggered from the blood pressure pulse and also displayed on a Beckman Dynograph. Recortiing Sympathetic
Nerve
Acrivity
Cats used in the determination of the effects of muscimol on sympathetic nerve discharge were prepared as described above and immobilized with decamethonium bromide (0.2 mg/kg IV). Supplemental doses of decamethonium were administered as required during the experiment. A unilateral pneumothoracotomy was performed to reduce respiratory artifacts in the nerve recording. The left kidney was approached retroperitoneally and one of the renal nerves was ligated and severed. The proximal portion of the nerve was placed on a bipolar platinum electrode to record post-ganglionic action potentials monophasitally. Nerve potentials were amplified with a differential preamplifier with high and low pass filter settings at 1 and 1000 Hz, respectively. Integrated nerve activity was derived by feeding the signal into a Beckman resetting integrator (Model no. 9873B). The resetting integrator was shorted to 0 at a constant interval by a specially constructed external
‘Send reprint request to: David W. Snyder, Squibb Institute for Medical Research, Department of Pharmacology, NJ 08540
Copyright
0 1980 ANKHO
International
Inc.-0361-9230/80/080317-07$01.20/O
P.O. Box 4000, Princeton,
31x
SNYDER AND AN’I’ONAC?CIO
timer. Thus, the height of each epoch of integration was proportional to the amount of actual nerve activity emanating from the sympathetic nerve per unit of time. Neural data were expressed as percent of control (i.e., treated epoch height/control predrug epoch height x 100).
In 15 cats the entire ventricular system of the brain was perfused using the method described by Vollmer and Boccagno [33]. Briefly, a perfusion inflow cannula consisting ofa 22 ga hypodermic needle was lowered into the right lateral ventricle at the coordinates: anterior 15 mm, lateral 2.5 mm, and horizontal + 6.7 mm according to the atlas of Snider and Niemer 1271. An outflow cannula of PE 90 polyethelene tubing was advanced through the atlanto-occipital membrane into the cistema magna. Artificial cerebrospinal fluid (CSF) 1241 was infused continuously at a rate of 0.1 ml/mm using a Harvard infusion pump. Pl~~cement of Inflow Cu~~~la in the Cerebral A~uedact In 4 of the cats described above a second inflow cannula was placed in the cerebral aqueduct according to the procedure of Vollmer and Buckley [34] for administration of bicuculline methiodide (bicuculline), a GABA antagonist. The cannula, consisting of a 22 ga unbeveled hypodermic needle, was lowered through a hole in the skull and positioned in the cerebral aqueduct at coordinates: 1271anterior 1.O mm; lateral 0.0 mm and horizontal + 1.O mm. Placement of this cannula did not obstruct flow of CSF through the cerebral aqueduct. During the drug infusion period, bicuculline was administered via the cerebral aqueduct cannula while vehicle (CSF) or drug was simultaneously administered via the lateral ventricle cannula. As indicated by the distribution of methylene blue dye, this procedure prevented bicuculline from reaching the 3rd ventricle and directed the drug posteriorly toward the fourth ventricle. Perfusion of Supramedullary
Area
In 5 cats the supramedu~a~ region of the brain was perfused. An inflow cannda was placed in the lateral ventricle as described above and an outflow cannula (PE 160 “cuffed” with small piece of PE 205) was inserted into the cerebral aqueduct following retraction of the cerebellum to expose the caudal opening of the aqueduct. The diameter of the outflow cannula was such that it impeded flow to the fourth ventricle. Thus, only supramednllary structures were exposed to the drug. Proper placement of the cannulae was determined by injecting methylene blue dye into the inflow cannula at the end of each experiment. Absence of dye in the medulla was used as evidence for proper cannulation of the cerebral aqueduct. Perfusion of the Anterior Region of the Fourth Ventricle In 4 cats the anterior region of the fourth ventricle was perfused. In these cats an outflow cannula was placed in the cerebral aqueduct to prevent drugs from reaching supramedullary areas as described above. An inflow cannula (PE 50) was inserted beside the cerebral aqueduct outflow can&a so that the tip of the cannula was positioned 1.5 cm from the caudal tip of the cerebellum. With this procedure, the perfusate reaching the anterior region of the medulla was allowed to escape freely from the caudal region of the
brainstem. Dye (neutral red) was injected at the end of each experiment to determine the extent of the area perfused.
In 13 cats, the caudal region of the fourth ventricle was perfused. A small inflow cannula (PE 50) was inserted through the dura covering the foramen magnum and advanced 8 mm into the caudal region of the fourth ventricle. The tip of the cannula rested on the dorsal surface of the brainstem immediately anterior to the obex. The outflow cannula was positioned in the cisterna magna, as described under “Perfusion of the Lateral Ventricle”. At the end of each experiment, methylene blue dye was added to the perfusion system to determine the region of the fourth ventricle which had received the drug. Compounds administered into the ventricular system of the brain were dissolved in artificial CSF prior to usage and infused with a Harvard infusion pump at a rate of 0. I mtimin. Compounds administered intravenously were dissolved in physiological saline and given as a bolus. Results are expressed as the mean + SE. Statistical significance was calculated using Student’s t-test or analysis of variance {ZS]. RESULTS
Intracerebroventricular
Infusion of Muscimol
Intracerebroventricular infusion of muscimol in chloralose anesthetized cats produced significant reductions in mean arterial pressure, heart rate and renal sympathetic nervous discharge (Fig. I). The cardiovascular effects of muscimol were dose related (Fig. 2). An infusion of 0.003 pg/kg/min produced a slight fall in blood pressure with virtually no change in heart rate. An infusion of 0.01 or 0.03 &kg/min produced significant decreases in both blood pressure and heart rate. Sympathetic nerve activity increased initially followed by a slight depression with 0.01 &kg/min of muscimol. Increasing the dose of muscimol produced significant inhibition of sympathetic nerve activity. ICV administration of bicuculline (I p&ikg) reversed the effects of muscimol on blood pressure and heart rate but failed to restore renaf nerve activity to its pre-muscimol level (Fig. 1). Administration oj’M~.~cimul to the Caudal Region of the Fourth Ventricle In 13 cats, muscimol was administered to the caudal region of the medulla since most of the centrally acting antihypertensive agents such as clonidine and L-DOPA produce their effects by stimulating neural components which lie in the lower brainstem. Infusion of 0.03 &kg/min of muscimol into the caudal region of the fourth ventricle failed to significantly alter blood pressure, heart rate or renal nerve activity (Figs. 3 and 4). In 8 of these cats the same dose of muscimol admi~st~red into the lateral ventricle produced marked reductions in blood pressure, heart rate and sympathetic nervous discharge (Figs. 3 and 5). Furthermore, in the 5 remaining cats, administration of clonidine (I.0 cLg!k@min for 10 min) to the caudal region of the medulla produced significant reductions in blood pressure and heart rate. Figure 6 summarizes the cardiovascular effects of muscimol and clonidine administered into the caudal region of the fourth ventricle. Administration
of Muscimol
to ~u~rameduila~
In 5 cats, an outflow cannula
Areas
was placed in the caudal
LOCATION
OF THE HYPOTENSIVE A Contrd
EFFECTS
B Muscii
OF MUSCIMOL TIME(min)
c Bic~ulline
pet-f usion period t 0 5 IO 15 I 1 I f
Heart Rate (bpm)
I 30 ,
45 I
60 b
1
1
Arterial Pressure (mmHg1
Mean
Arterial Pressure tmmHg)
-SO]
Renal Nerve Dischbrge
pM&ed
1 Dischaqe Nerve
t t Blc hi&mimol
O,O+g/kg/min
!d
be/M
+20+ IO-
g
o8_:;_ &
FIG. 1. A typical experiment demonstrating the effects of muscimol on blood pressure, heart rate and renal nerve discharge when administered into the lateral ventricle. A. Control record before the infusion of muscimol. Between A and B muscimol (0.03 FgjkJmin) was administered XV for 30 min. B. Record was taken after completion of the infusion. Between B and C bicu~ulline methi~ide (I pgikg ICV) was administered. C. Record was taken 30 see after the administration of bicuculline.
region of the cerebal aqueduct thus preventing the perfusate from reaching the fourth ventricle and bathing the lower brainstem. Restricting muscimol (0.03 pg/kg/min) to suprameduIlary areas resulted in a small (lO-15%) fall in blood pressure and heart rate (Figs. 7 and 8).
2 -3OE -4o;
I;;_.
d-70- eo-
-90-l Muscimol:
*p
Administration of Muscimnl Fourth Ventricle
I.C.V. I.C.V. I.C.V.
* *p
to the Anterior Region of the
In 4 cats an outflow cannula was placed in the cerebral aqueduct and an inflow cannula was placed in the anterior region of the fourth ventricle. Restricting muscimol (0.03 &kgimin) to the lower brainstem resulted in marked reductions in blood pressure and heart rate. The c~diovascular effects produced by muscimol administered to the anterior fourth ventricle were no different from those seen upon administration of muscimol to the entire ventricular system (Figs. 8 and 9). Bicaculline Antagonism
D 0.003 pg/kg/min, m O.OlO~g/kg/min, Cl O.O~~~k~rnin,
of’ Medullary Effects
of Muscimol
Attempts were made to antagonize the effects of muscimol by administe~ng low doses of bicuculline to the lower brainstem of the cat. In agreement with WiIl~ord et al., [35], bicuculline (0.5 ~~k~rnin) failed to alter blood pressure or heart rate when administered into the cerebral aqueduct. The perfusion system prevented bicuculline from reaching neural structures rostal to the cerebral aqueduct, but did allow the GABA antagonist to enter the 4th ventricle and bathe the lower brainstem. The simultaneous infusions of muscimol (0.03 pg/kgimin XV) and bicuculline (0.5 &kg/min cerebral aqueduct) produced a smail (IO-15%) reduction in blood
FIG. 2. Summary of the effects of muscimol administered ICV on blood pressure (top), heart rate (middle) and renal nerve discharge (bottom) of anesthetized cats. In all cases, the drug was administered for 30 min. Abscissa is time (min) after start of the infusion. Data are expressed as percent change from control. Number of animals is indicated in parenthesis. pressure and heart rate (Fig. 8). Thus bicuculline prevented most of the effects of muscimol. The hypotensive response that remained was comparable to that observed when muscimol was confined to the supramedullary region of the brain. DISCUSSION
These data indicate that the major cardiovascular action of muscimol is restricted to the anterior region of the medulla. This was demonstrated by the fact that infusion of muscimol into the anterior region of the 4th ventricle resulted in both hypotension and bradycardia, whereas infusion of muscimol solely into the posterior region of the fourth ventricle had no cardiovascular effect. The fall in blood pressure and heart rate seen with anterior medullary perfusion was comparable to that seen when muscimol was adminis-
SNYDER
AND ANTONACC’IO
TIME(min) perfusionperiod
(It=?) (n=6)
FIG. 3. A typical experiment demonstrating the lack of effect of muscimol on blood pressure, heart rate and renal nerve discharge when administered into the caudal region of the fourth ventricle. A. The intravenous ~rni~st~tion of a botus of norepineph~ne (1.0 &kg) produced a pressor response associated with a reflex fall in heart rate and reduction in renal nerve discharge which indicates the system is functioning properly. Control record was taken prior to administration of muscimol. Between A and B muscimol (0.03 ~~~rnin) was infused into the caudal region of the fourth venWicle for 30 min. B. Record was taken at the end of the infusion of muscimol. Between B and C muscimol(O.03 &kg/min) was infused into the lateral ventricle for 30 min. C. Record was taken at the end of the infusion of muscimol.
tered to the entire ventricular system. These cardiovascular responses were mediated through activation of GABA receptors since both hypotension and bradycardia were attenuated by the GABA receptor antagonist bicuculline. In this study, administration of bicuculline alone to the hindbrain, failed to alter blood pressure or heart rate confirming the findings of Williford et ul., [35]. The caudal region of the medulla can be excluded as a site of action since administration of muscimol solely to this region failed to alter blood pressure or heart rate. In contrast, ICV administration of muscimol did produce the expected cardiovascular response, indicating that the animals were not refractory to the drug. In addition, administration of clonidine, a centrally acting antihypertensive agent, onto the caudal region of the medulla did lower blood pressure and heart rate, suggesting that the caudal region of the medulla was responsive. This difference between the sites of action of clonidine and muscimof indicates that the hypotensive actions of these two drugs are mediated through activation of different receptors. Clonidine is believed to produce its centrally mediated cardiovascular effects by stimulating a-adrenergic receptors [23,25] while muscimol is a GABA mimetic. Compared to most centrally acting antihypertensive agents, the lack of effect of muscimol in the caudal region of the medulla is unique. Other reports have also suggested that more anterior regions of the medulla are important for the cardiovascular effects of muscimol. Guertzenstein [2Ol demonstrated that GABA applied topically to the anteroventral surface of the medulla of anesthetized cats elicited a fall in blood pressure. Bousquet et al., f91 demonstrated that microinjections of muscimol into the ventrolateral medulla between the trapezoid bodies and the root of the
8 +40-
2 +30% +20$ + IO4 5
- IO OI - 20-30wsada_
* O,O&&kg/min, 4#hw~trkle 0 O.~~,~ wltt?kltt
FIG. 4. Lack of effect on blood pressure (top), heart rate (middle) and renal nerve discharge (bottom) of muscimol infused into the caudal region of the fourth ventricle. Data are expressed as percent change from controLAbscissa is time (min) from start of the perfusion period which lasted 30 min. Number of animals is indicated in parenthesis.
hypoglossal nerve reduced blood pressure and heart rate in urethane-anesthetized cats. However the dose of muscimol used by Bousquet to evoke the response in a localized area of brain was high compared to the dose needed in the present study to produce a similar change in blood pressure when administered ICV. Neurons mediating sympathetic activity in the ventrolateral medulla which were exposed to the drug in these previous studies probably comprise part of the lateral reticular nucleus (211. In the present study the caudal extent of this nucleus was bathed with muscimol during perfusion of the caudal medulla, yet neither blood pressure nor heart rate was altered. Thus, muscimol may be acting at the more rostra1 extent of this brainstem nucleus as suggested by Guertzenstein and Silver [21]. It is possible that the region of periaqueductal gray comprising the caudal extent of the nucleus raphe dorsalis is cont~buting to the cardiovascular effects of musci~ol. This nucleus is composed largely of serotonergic neurons USI. Serotonergic neurons are distributed throughout the lower brainstem [ 151and pharmacological evidence indicates that they can raise or lower blood pressure depending on their Iocation [ 11,30]. Recently Beiin et al., [6] localized GABAergic neurons in nucleus raphe dorsalis in rats. Electrical stimulation of this nucleus increases blood pressure with lit-
LOCATION
OF THE HYPOTENSIVE
EFFECTS
TIME(minf perfusion period t I 0 5 IO I5 30 45 t
x
/
321
OF MUSCIMOL TIME (min) perfusion
period
60 (n=7)
r
x
,:<:
p
(n-8)
@ Muscimol 0 Muscimol a Clonidine
+20+ IOO- IO-2o-3o-4o-5o-6O-?O-90 -
ventricle
,4th ventricle
I .O ~g/kg/min~ 4th ventricle f IO min perfusion)
FIG. 6. Comparison of the effects on blood pressure (top) and heart rate (bottom) of muscimol and clonidine administered into the caudal region of the fourth ventricle of anesthetized cats. Muscimol was infused for 30 min. Clonidine (1 .O pg/kg/min) was infused for 10 min. Data are expressed as percent change from control. Abscissa is time (min) from start of perfusion period. Number of animals is indicated in parenthesis. A
Muscimot: 0 0.03 ~g/k~rnin, 0 O.O3~g/kg/min, *p
4th ventricle I.C.V.
**p
FIG. 5. Comparison of the effects on blood pressure (top), heart rate (middle) and renal nerve discharge (bottom) of muscimol infused ICV and into the caudal region of the fourth ventricle of anesthetized cats. Muscimol (0.03 @kg/min) was infused for 30 min. Data are expressed as percent change from control. Abscissa is time (min) from start of perfusion period. Statistical significance was measured between the cardiovascular changes induced by the two routes of administration of muscimol. Number of animals is indicated in parenthesis.
in heart rate [26]. Possibly, the cardiovascular responses to electrical activation of the nucleus raphe dorsalis is due to activation of serotonergic neurons. In addition it has been shown that local administration of GABA to the nucleus inhibits the firing of serotonergic neurons; an action which is blocked by picrotoxin, a GABA antagonist [I]. Thus, GABA-ergic neurons may in part influence cardiovascular function by modulation of serotonergic neurons in this nucleus. Since, in this study, muscimol did have access to this area of the brainstem during perfusion of the anterior medulla, one cannot rule out the possibility that part of the hypotensive effect of muscimol may be mediated by these GABA-ergic neurons. The small forebrain effect of muscimol was demonstrated in experiments in which muscimol was restricted to the lat-
KZ (bpmf
180
B
CONTROL
240
tie change
0.01 pg/kg/min,4th
0.03 pg/kg/min
__~__
SUPRAMEDULLARY AREA .-_ .^
Arterial Preaaure (mmtrZlf 01
Mean Arterial +s.ere (~~)
180 S0 I----
d
t
Muscimol
t---i
1 min
0.03~gkgfmin
FIG. 7. Typical experiment demonstrating the SupramedulIary actions of muscimol on heart rate and arterial pressure. A. Control record was taken prior to the infusion of muscimol. Between A and B muscimol (0.03 pglkgimin) was infused into the lateral ventricle and perfusate was collected at the caudal end of the cerebral aqueduct. B. Record was taken at the end of the 30 min infusion of muscimol. eral and third ventricles.
Under these conditions, muscimol lowered blood pressure and heart rate IO-W%. Evidence for a supramedullary action of muscimol also was obtained in experiments in which the medullary effects of muscimol were blocked by bicuculline. Since bicuculline did not reach the third or lateral ventricles, the forebrain mediated hypotensive and bradycardic effect of muscimol were expressed.
SNYDER Time (mid
perfusionperiod b L
5
II
10
15
1
3b
A 45
I
CONTROL
60
I
0
Heart Rate fbpm)
e!
I
240
t!J20 -
1
160
Arterial
180
Pressure @)mW
w 0 380
2 "
B ANTERKM 4th VENTRICLE
120 -10 -
&I 3
H:
AND ANTONACCIO
.30 -
Mean Arterial
.40 -
90 0
ae
3 -- t
H 1
min
Muacimol
en
O.~~g~g/min 0-
f!
-lO-
P .5
.20 -
FIG. 9. Typical experiment demonstrating the effects on blood pressure and heart rate of muscimol administered to the anterior region of the fourth ventricle. A. Control record prior to the infusion of muscimol. Between A and B muscimol(O.03 pg/kg/min) was infused into the anterior region of the fourth ventricle. B. Record was taken after the 30 min infusion of muscimol.
! z
-30 -
40-
D
Muccimol, 0.03pp/kg/min,i.e.v. (n-81
FIG. 8. Effects of the simultaneous infusions of bicuculline methiodide via the cerebral aqueduct and muscimol ICV on blood pressure (top) and heart rate (bottom). These effects are compared to those produced by muscimol alone (ICV) and when muscimol was restricted to supramedullary areas. Data are expressed as percent change from control. Abscissa is time (min) from start of drug. Number of animals is indicated in parenthesis.
There was no evidence of a forebrain effect of muscimol if muscimol was first infused into the anterior region of the fourth ventricle. This would suggest that muscimol was acting at two different sites along the same neural pathway. If this is the case then one may question the significance of a forebrain site of action. However, forebrain GABA receptors which influence blood pressure and heart rate may be important in expressing the cardiovascular action of benzodiazepines. Diazepam has been demonstmted to reduce resting blood pressure and heart rate [8, 10, 161. It is believed that diazepam has a supramedullary site of action since it reduced supramedullary evoked pressor responses without altering medullary evoked pressor responses [2]. Muscimol also inhibited the pressor response evoked by electric%l stimulation of the diencephalon [3]. Moreover, ICV but not intracisternal administration of diazepam was effective in lowering blood pressure, an action that was attenuated by
intraperitoneal injection of picrotoxin 181. The cardiovascular effect of diazepam is probably not mediated by central a-adrenergic receptors, since the a-adrenergic receptor blocking agent piperoxane could not antagonize the reduction in arteriai pressure caused by diazepam [ 191. Biochemical evidence suggests that at least some actions of diazepam and other benzodiazepines may be mediated via increases in GABA receptor activity [12, 13, 14, 191. Thus, the forebrain cardiovascular effects of muscimol demonstrate the existence of a neural substrate which may be mediating the cardiovascular action of benzodiazepines. In the present study, a 30 min i&&ion of muscimol into the entire ventricular system of the cat lowered blood pressure, heart rate and renal sympathetic nerve activity. Similar observations have been reported by others when muscimol was infused ICV [29] or when administered as a bolus into the third ventricle 13, 4, 51. Infusion of muscimol over a 30 min period did not produce dose related reductions in renal sympathetic nerve discharge. At low doses renal sympathetic nerve activity increased initially. This increase in sympathetic activity was associated with a decrease in blood pressure and thus was probably related to a com~nsato~ action of the baroreceptor reflex. The increase in renal nerve activity was usually followed by a decrease. Similar results have been reported by others in which muscimol was administered as a bolus into the third ventricle. The inhibition of renal nerve activity seen with the high dose of muscimol paralleled the f&l in blood pressure and heart rate and was likely due to a cent& action of the drug. The data in the present study indicate that the primary cardiovascular effects of muscimol are mediated through the activation of GABA receptors in the anterior or anteroventral region of the medulla. A small but significant component is mediated by forebrain structures probably lying in close proximity to the lateral and third ventricle and cerebral aqueduct.
LOCATION
OF THE HYPOTENSIVE
EFFECTS OF MUSCIMOL
323
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