Brain Research, 550 (1991) 353-357 © 1991 Elsevmr Science Pubhshers B V 0006-8993/91/$03 50 ADONIS 000689939124692V

353

BRES 24692

Effects of clonidine on sympathoexcitatory neurons in the rostral ventrolateral medulla Mark E Clement and Robert B. McCall Car&ovascular Disease Research, The UPlohn Company, Kalamazoo, M1 49001 (U S A ) (Accepted 5 March 1991) Key words Rostral ventrolateral medulla, Clomdme, Sympathetic nerve discharge, Norepmephnne, Cat

The effects of intravenous and lontophoreUc clomdme were determmed on the finng rates of sympathoexotatory neurons m the rostral ventrolateral medulla of the cat As previously reported m the rat, we found that sympathoexcltatory neurons could be dffferentmted based on their sensmwty m clomdme Approximately 50% of the neurons were inhibited by clomdme There was only a weak correlaUon between the mhlbmon of unit actwtty and whole sympathetic nerve actmty The discharge rates of the remaining neurons were either not altered o r were increased by clomdme Unhke the rat, these two groups of neurons could not be further differentiated on the barns of axonal conducUon velocity or discharge frequency These data are discussed and the effects of clomdme and 8-hydroxy-2-(dl-n-propylammo)tetrahn(8-OH-DPAT) on sympathoexotatory neurons are compared

T h e rostral v e n t r o l a t e r a l m e d u l l a ( R V L M ) ts a crlttcal structure m the central regulatton of v a s o m o t o r tone (see refs 4 and 11 for review) Electrophysiologtcal studies m several spectes indicate that neurons m this a r e a of the b r a l n s t e m p r o j e c t to the m t e r m e d t o l a t e r a l cell column ( I M L ) , are influenced by b a r o r e c e p t o r inputs, and exhtblt a firing p a t t e r n whtch is t e m p o r a l l y related to sympathettc nervous discharge (SND) and the cardiac cycle I Acttvatton of neuronal cell bodies m the R V L M ytelds d r a m a t i c increases in blood pressure and sympatheUc actw~ty suggestmg that these sympathetic-related neurons function m a s y m p a t h o e x o t a t o r y fashion 1° In addttton, a variety of mlcromjectlon studies uttltzlng inhibitory a m i n o acids and neurotoxins indicate that these neurons play a m a j o r role in the tontc regulatton of s y m p a t h e t i c acttvlty and artertal b l o o d pressure 2'7 In the rat, R V L M m e d u l l o s p m a l b a r o r e c e p t o r neurons can be subdivided mto two groups The first consists of n e u r o n s wtth axonal conducUon v e l o o t l e s of 3 5 - 8 0 m/s and discharge rates of 15-35 sptkes/s These neurons are not inhibited by the a2-receptor agonlst clontdme The second group of neurons are inhibited by clontdlne, have axonal conduction velocities of 0 4 - 0 8 m/s and have a slower discharge r a t e 11'15'16 In the cat, two groups of R V L M s y m p a t h o e x c t t a t o r y neurons have not been identified A x o n a l conduction velocities are a p p r o x i m a t e l y 2 5 m/s and discharge rates average 1-3 splkes/s 11 S e n s m w t y of clonldlne has not been e x a m i n e d

The R V L M has also b e e n t m p h c a t e d as the site at which centrally acting sympatholyttc c o m p o u n d s such as clonidine and 8 - h y d r o x y - 2 - ( d i - n - p r o p y l a m i n o ) t e t r a h n (8O H - D P A T ) exert their effects Mtcrolnlection of clomd m e or 8 - O H - D P A T m t o this a r e a results m a p r o f o u n d hypotenston 3'9 T h e electrophystological studies described a b o v e in the rat m a y p r o v i d e the basts by which microinlected clonidme lowers b l o o d pressure A n a t o m ically, the R V L M contains a substantial n u m b e r of epinephrme-synthestzing n e u r o n s which p r o j e c t to the m t e r m e d t o l a t e r a l cell column and receives a noradrenerglc m p u t from the A 5 a r e a of the b r a i n s t e m 8'14 T h e a f o r e m e n t i o n e d clonldme-insensittve n e u r o n s are apparently not adrenerglc neurons 16 D a t a from these experiments and others H-13 suggest that clontdine exerts its hypotenslve effects via a2-receptors l o c a t e d on sympathoexcttatory neurons in the R V L M In addttion, we recently d e m o n s t r a t e d a high d e g r e e of correlation b e t w e e n the sympatholytic effects of intravenous 8O H - D P A T and the mh~bitton of finng of sympathoexcitatory neurons in the cat 6 This suggests that Inhibition of R V L M s y m p a t h o e x o t a t o r y neurons ts critical in the central hypotenstve action of 8 - O H - D P A T In the present study we e x a m m e d the effects of clonidine and n o r e p m e p h r m e on the activity of sympathoexcltatory neurons located m the R V L M of the cat The p u r p o s e of the study was to (1) d e t e r m m e if, as m the rat, s y m p a t h o e x c t t a t o r y neurons can b e d~fferenttated

Correspondence R B McCall, The Upjohn Company, 7243-209-3, Kalamazoo, MI 49001, U S A

354 on the basis of clonldme sensitivity and (2) determine d clomdme and 8 - O H - D P A T have slmdar sites of sympatholytlc action m the R V L M

a d m m l s t r a u o n , respecUvely A n arteNal embolectomy catheter (American Edwards Laboratories) was inserted in the opposite femoral artery to permit occlusion ot the

Twenty-seven cats (2 5 - 4 0 kg) were anesthetized by

descending aorta A m m a l s were Immobilized with gallamine trlethlodlde (4 mg/kg, I v ) and artlhoallv respired

an intravenous rejection of dlallylbarblturate sodmm (60 mg/kg), urethan (240 mg/kg), and m o n o e t h y l u r e a (240 kg/mg) The ammals were placed in a David Kopt

The dorsal aspect of the medulla was exposed by removal of portions of the overlying occipital bone and cerebel-

Instruments stereotaxlc apparatus and spinal investigation umt, and a glass tracheal tube was inserted A

lum The obex was used as a surface landmark tor placement of the recording electrode Neurons were

femoral artery and veto were cannulated to measure arterial blood pressure and to permit intravenous drug

recorded in the rostral ventrolateral area previously described 6 U m t a r y discharges were recorded using exL

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Fig 1 Effects of 1 v clomdme on blood pressure (BP), sympathetic nerve discharge (SND), and umt actlvtty (Counts) Panel A represents a clomdme-sensltwe neuron, while the neuron m panel B was msensmve to l v clomdme Vertical cahbratlon for SND is 100/zV BP umts are expressed as mmHg

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Fig 2 Dose-response curves dlustratmg the effects of i v clomdme on clomdme-sensmve (filled circles) and clomdme-lnsensltwe (open ctrcles) neurons of the RVLM Triangles sympathetic nerve &scharge Filled squares mean artenal pressure (n -- 12) Standard error bars are illustrated

tracellular techniques with a tungsten microelectrode (1 /~m tip dmmeter, 2-4 Mg2 impedance) or a 5-barrel glass mlcroelectrode (tip 10-15/~m) containing monofilament glass fibers to simultaneously record the extracellular potentials from single neurons and to apply drugs at the recording site The center barrel was filled with 2 M NaCI and was used to record extracellular unit activity (impedance, 1-4 Mg2) One side barrel was filled with 4 M NaCI and used for automatic current balancing The remaining barrels were filled with clonIdine HCI (Research BIochemicals I n c , 0 1 M, pH 4 0), arterenol (Research Biochemicals I n c , 0 1 M, pH 4 0), the monosodium salt of L-glutamate (Sigma, 0 1 M, pH 8 0) or an appropriate control solutmn Solutions were prepared fresh before each expenment A retaining current of 15 nA was apphed between ejecting periods The constancy of the action potentml contour and amplitude were assessed using a storage oscilloscope to confirm the unitary nature of the recordings and to monitor for possible anesthetic effects of the drugs The first and second thoraoc spinal segments were exposed by lammectomy and a tungsten stimulating electrode was placed m the IML The electrode was determined to be in the vlcImty of the IML if single electrical pulses evoked a potential recorded from the inferior cardiac nerve with an onset latency of about 15 ms Peripheral sympathetic nerve activity was recorded from the central end of the sectioned left postganghonic inferior cardiac nerve Potentials were recorded monophasically under mineral oil with a bipolar platinum electrode A band pass of 1-1000 Hz was used to display the synchronized discharges of the whole sympatheuc nerve in the form of slow waves Inferior cardiac SND, unitary discharges, blood pressure, and pulses derwed from the R wave of the electrocar&ogram and stimuli were recorded on magnetic tape and

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Fig 3 Rate histogram dlustratmg effects of mlcromntophoretlcally apphed clomdme and norepmephrme on &scharge rate of a

clomdme-sensmve neuron Bars represent mntophoreUcperiods of 60-nA ejecUon currents Umt could subsequently be shut off by a total dose of 30/~g/kg i v clomdme Vertical axis is number of neuronal &scharges Vertical cahbratlon for SND is 50/~V BP umts are expressed as mmHg

analyzed using a RC Electronics Computerscope Unit discharges were subjected to window dlscnmlnatton before presentation to the computer Individual neurons were initially Identified by a onesecond spike triggered mid-signal average of sympathetic activity Units were determined to be sympathoexcitatory if a transient increase in blood pressure elicited by occlusion of the descending aorta produced an immediate and complete shut-off of the unit and sympathetic activity Twenty-six neurons were tdentified which had activity temporally related to the 2-6 Hz slow wave pattern of sympathetic nervous &scharge, were inhibited during baroreceptor actwation and could be antidromIcally activated from the mtermedxolateral cell column Indwidual firing rates ranged from 0 75 to 4 1 Hz (mean = 1 67) The onset latencles of antidromlcally acUvated neurons ranged from 38 to 76 ms, corresponding to conduction velocltwS of 1 4 to 3 9 m/s Latencles of activation were normally distributed over the range, with the mean onset latency equal to 50 8 +_ 4 7 ms and the conduction velooty equal to 2 2 m/s Clonidine was given intravenously in incremental doses to 18 of these sympathoexotatory RVLM neurons Nine neurons exhibited an inhibitory response to intravenous clomdine while the remaining half showed either no response or an increase in activity Panel A of Fig 1 illustrates a sympathoexcitatory neuron which was sensitive to intravenous clomdme Complete mhIbltmn of clonidme-sensltive units could usually be achieved by a cumulative dose of 30/~g/kg clonidlne Unit inhibition was roughly correlated to SND lnhlbmon, although complete shut-off of SND was generally achieved before complete inhibition of unit discharge Panel B of Fig 1

356 represents a s y m p a t h o e x c l t a t o r y R V L M umt which was insensitive to intravenous clonldlne Units oÀ this type could not be inhibited by cumulative doses totahng 100 /zg/kg of c l o m d m e despite complete inhibition ot sympathetic activity These neurons did, however, show a rapid and c o m p l e t e shut-off in response to mtravenous 8O H - D P A T (Fig l b ) as we have previously d e m o n strated Fig 2 ts a d o s e - r e s p o n s e curve s u m m a n z m g the effects of mcreaslng doses of intravenous clonldlne on sensitive and insensitive sympathoexcttatory neurons m the R V L M Clomdlne-senslttve and -msensttwe neurons could not be differentiated on the basis ol axonal conduction velocity or flrmg rate In o r d e r to m o r e d~rectly examine the effects of az-agonists on sympathoexcttatory R V L M neurons we mtcrolontophorettcally applied clomdme and noreptn e p h r m e on these units MlCrolontophoretic ejection currents ranged from 10 to 120 n A and ejection periods ranged from 30 to 120 s In no case did we observe an Inhibitory response to mlcro~ontophoretlc clontdme or n o r e p m e p h r l n e by a sympathoexcltatory neuron, although several units could be subsequently shut off by intravenous clonldlne Fig 3 is an example of a clomdlnesensitive n e u r o n which was unresponsive to 60-hA ejection currents of clonldlne and n o r e p m e p h r m e Clom d m e - l n s e n s l t w e neurons hkew~se showed no response to direct application of c l o m d m e or n o r e p m e p h r l n e In some instances, a slight excitatory response to mtcrolont o p h o r e t l c n o r e p m e p h r i n e was observed (not shown) The p u r p o s e of the present study was to investigate s y m p a t h o e x c l t a t o r y R V L M neurons m the cat and to d e t e r m i n e their role, if any, m the hypotenswe effect of az-agonlsts We also sought to d e t e r m m e whether these umts could be differentiated based on sensitivity to a2-agonlsts in a s~mllar m a n n e r as has been done in the rat To this end we discovered that a subpopulation of s y m p a t h o e x c l t a t o r y neurons m the R V L M were inhibited in a d o s e - d e p e n d e n t m a n n e r in response to intravenous c i o m d m e whde the r e m a m l n g were unaffected or even excited The Increased activity of clomdlne-msensltlve umts was likely due to a b a r o r e c e p t o r influence on these neurons (1 e dlslnhlbttton during hypotenswe effect of clontdlne) In the rat, clomdlne-senstttve and clonldmeinsensitive s y m p a t h o e x c l t a t o r y neurons have been further dlstlngmshed based on differences in their conduction velocities and firmg rates Clonidme-sensmve neurons are slow-conducting, exhibiting mean conduction velocities of 0 6 m/s while the clomdme-lnsensittve neurons are fast-conducting (3 3 m/s) and have a higher

firing frequency (15-35 spikes/s) It should be noted that these researchers did observe at least one fast-conducting neuron which was sensitive to clontdme In the cat however we found no such distinction between clomdme-sensttive and -msensttlve neurons Antldromically activated s y m p a t h o e x c i t a t o r y R V L M units are unitorm m that they displayed a normal distribution with regard to conduction velocity and discharge frequency Thus, s y m p a t h o e x c l t a t o r y neurons in the cdt are simdar to those m the rat in that they can be divided into two groups based on c l o m d m e sensitivity H o w e v e r , unlike the rat, there are no differences in axonal conduction velocity or firmg rates between these groups of neurons in the cat This observation may be e x p l a m e d by lnterspecles differences, although since a btmodai distribution of conduction velocities of R V L M neurons is found in both the rat and the rabbit 17, it may also be that the small population of neurons sampled in the present study simply did not include a clomdme-sensltwe tastconducting neuron Neurons which were inhibited by intravenous clonldlne and those which were not were also similar in their unresponsiveness to direct application of clontdlne and n o r e p l n e p h r m e The fact that m l c r o l o n t o p h o r e t i c clonidine had no effect on clomdlne-sensltlVe neurons may mdlcate that the az-receptors mediating the inhibitory effects of clontdme are located on dendrites r e m o v e d far enough from the cell bodies b e m g r e c o r d e d that clonldlne could not diffuse to these sites A l t e r n a t i v e l y clomdlne may act on m t e r n e u r o n s located in the R V L M which m o d u l a t e the activity of the identified sympathoexcltatory neurons Mtcrolnjectlon of either clontdine or the 5-HTIA agomst 8 - O H - D P A T into the R V L M results in an inhibition of sympathetic actwlty and a decrease m blood pressure 39 Previously we found an absolute correlation between the lnhib~tton of R V L M sympathoexcltatory neurons and the shut-off of sympathetic actlwty p r o d u c e d by 8 - O H - D P A T 6 All s y m p a t h o e x c l t a t o r y neurons were sensitive to the inhibitory effects of 8 - O H - D P A T In contrast, only about 50% of these neurons are inhibited by ciomdme U n h k e 8 - O H - D P A T , the correlation between inhibition of unit and whole nerve activity followlng clontdine was weak Typically, whole sympathetic nerve activity was completely inhibited by clomdlne before the unit was inhibited This suggests that inhibition of R V L M s y m p a t h o e x c l t a t o r y neurons is critical to the hypotenslve effect of 8 - O H - D P A T , while clomdlne has multiple sites of action 6 1~

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autonomic response, Bram Research, 238 (1982) 65-76 11 McCall, R B , Role of neurotransm~tters m the central regulauon of the cardiovascular system, Prog Drug Res, 35 (1990) 25-84 12 McCall, R B , Schuette, M R , Humphrey, S J , Lahtl, R A and Barsuhn, C , Evidence for a central sympathoexcatatory action of alpha-2 adrenerglc antagomsts, J Pharmacol Exp Ther, 224 (1983) 501-507 13 Pnvltera, P J , Granata, A R , Underwood, M D , Gaffney, T E and Rels, D J , C1 area of the rostral ventrolateral medulla as a site for the central hypotenswe action of propranolol, J Pharmacol Exp Ther, 246 (1988) 529-533 14 Ross, C A , Rugglero, D A , Joh, T H , Park, D H and Rels, D J , Rostral ventrolateral medulla selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons, J Comp Neurol, 228 (1984) 168-185 15 Sun, M -K and Guyenet, P G , Effect of clomdlne and G A B A on the discharges of medullosplnal sympathoexotatory neurons m the rat, Bram Research, 368 (1986) 1-19 16 Sun, M -K and Guyenet, P G , Rostral ventrolateral medullary neurons w~th intrinsic pacemaker properties are not catechoiammerglc, Brain Research, 451 (1988) 345-349 17 Terul, N , Saeko, Y and Kumada, M , Barosensory neurons m the ventrolateral medulla in rabbits and their response to various afferent inputs from peripheral and central sources, Jpn J Phystol, 36 (1986) 1141-1164