232

Brain Research, 525 (1990) 232-24l Elsevier

BRES 15776

Studies on the site and mechanism of the sympatholytic action of 8-OH DPAT Mark E. Clement and Robert B. McCall Cardiovascular Diseases Research, The Upjohn Company, Kalamazoo, MI 49001 (U.S.A.) (Accepted 20 February 1990) Key words: Serotonin; 8-OH DPAT; Sympathetic nerve activity; Blood pressure; Rostral ventrolateral medulla; Sympathetic preganglionic neurons

Studies in our laboratory indicate that the 5-HT1A agonist 8-OH DPAT acts in the central nervous system at postsynaptic receptor sites to inhibit sympathetic nerve activity and lower arterial blood pressure. The present study was designed to investigate possible postsynaptic sites on central sympathetic neurons where 8-OH DPAT might produce its sympatholytic action in anesthetized cats. The sympatholytic effect of 8-OH DPAT was compared in midcollicular transected and sham operated control animals. Administration of 8-OH DPAT (0.01-1.0 mg/kg, i.v.) inhibited sympathetic activity and decreased blood pressure in both the transected and sham animals to a similar degree. The effects of microiontophoretically applied 8-OH DPAT and 5-HT on antidromically identified sympathetic preganglionic neurons were determined. MicroiontophoreticaUy applied 5-HT consistently increased the firing rate of sympathetic preganglionic neurons. Iontophoretic 8-OH DPAT failed to affect the firing of sympathetic preganglionic neurons but blocked the excitatory effects of 5-HT. The effects of 8-OH DPAT and 5-HT on the firing of sympathoexcitatory neurons located in the rostral ventrolateral medulla were also determined. Sympathoexcitatory neurons were identified using spike triggered averaging techniques and by their response to baroreceptor activation. Intravenous administration of 8-OH DPAT inhibited the firing of sympathoexcitatory neurons in the rostral ventrolateral medulla. The inhibition of unit firing produced by 8-OH DPAT was exactly paralleled by the shutoff of inferior cardiac nerve activity. Microiontophoretic application of 8-OH DPAT and 5-HT onto sympathoexcitatory neurons in the rostral ventrolateral medulla failed to affect the firing rate of these neurons. These data suggest that 8-OH DPAT may act on central sympathetic neurons which lie antecedent to the ventrolateral sympathoexcitatory neurons. Alternatively, 8-OH DPAT may act on distal dendrites of the rostral ventrolateral sympathoexcitatory neurons. This implies that iontophoretically applied 8-OH DPAT may not gain access to these receptor sites. INTRODUCTION A substantial b o d y of evidence indicates the existence of multiple serotonin (5-HT) r e c e p t o r subtypes design a t e d 5-HT~, 5 - H T 2 and 5-HT 3 receptors. F u r t h e r m o r e , the 5-HT~ binding site is comprised of at least 4 subtypes designated 5-HT1A , 5-HT1B , 5 - H T l c and 5-HTiD (see ref. 30). The identification of selective ligands for some of these recognition sites has allowed for at least a partial u n d e r s t a n d i n g of the biological significance of these r e c e p t o r subtypes. For example, identification of the selective 5-HTIA agonist 8-hydroxy-2-(di-n-propylamino) tetralin ( 8 - O H D P A T ) has led to the recognition that 5-HT1A r e c e p t o r subtypes play an important role in the central nervous system regulation of sympathetic nerve discharge (SND). Thus, 8 - O H D P A T produces doserelated decreases in arterial blood pressure and heart rate in normotensive and spontaneously hypertensive rats 7' ~,30. The decrease in b l o o d pressure p r o d u c e d by 8 - O H D P A T is associated with an inhibition of spontaneous sympathetic activity r e c o r d e d from the inferior cardiac :2' 27, splanchnic27,2s and renal 12 sympathetic nerves. The

sympatholytic effect of 8 - O H D P A T is a n t a g o n i z e d by the 5-HTaA antagonists s p i p e r o n e 27 and 8-methoxy2(N-2-chloroethyl-N-n-propyl)aminotetralin 8 but not by the a2-receptor antagonist idazoxan 28. These d a t a indicate that 8 - O H D P A T acts at 5-HTIA receptors in the central nervous system to inhibit s y m p a t h e t i c activity and therefore lower arterial b l o o d pressure. Recent studies suggest that the 5-HTaA r e c e p t o r may be identical to the 5 - H T a u t o r e c e p t o r located on serotonergic neurons in the central nervous system 16'33. Microiontophoresis of 8 - O H D P A T inhibits the firing 5-HT neurons located in the dorsal r a p h e nucleus 31. Similarly, we have found that iontophoretically applied 8-OH D P A T inhibits the firing of 5 - H T neurons located in the nucleus raphe obscurus and the nucleus raphe pallidus 22. Research in o u r l a b o r a t o r y indicates that these 5-HT neurons provide a tonic excitatory input to sympathetic preganglionic neurons located in the intermediolateral cell column ( I M L ) of the spinal cord 17'18"20'26. These observations suggest that 8 - O H D P A T produces its sympatholytic effect by inhibiting the activity of medullary 5-HT neurons. This would result in a disfacilitation

Correspondence: R.B. McCall, The Upjohn Company, 7243-209-3, Kalamazoo, M1 49001, U.S.A. 0006-8993/90/$03.50 © 1991) Elsevier Science Publishers B.V. (Biomedical Division)

233 of sympathetic nervous activity. Recent work, however, suggests that this is not the case. Destruction of the medullary raphe 5-HT cell bodies fails to alter the sympatholytic response to 8-OH D P A T 23. This suggests that 8-OH D P A T may act at a postsynaptic site to produce its cardiovascular effects. This hypothesis is supported by a lack of correlation between the inhibition of midline 5-HT neurons and the decrease in SND in response to systemic administration of 8-OH D P A T 23. Low doses of 8-OH D P A T completely inhibit the firing of medullary 5-HT neurons but have little effect on sympathetic activity. In addition, microinjection of 8-OH D P A T into the medullary raphe fails to produce a hypotensive response. Microinjection of 8-OH D P A T in the more lateral pressor area leads to decreases in blood pressure, heart rate and renal sympathetic activity 12. Taken together, this information indicates that 8-OH D P A T acts postsynaptically to produce its sympatholytic effects. The purpose of the present study was to further investigate the central sympatholytic mechanism of 8 - O H D P A T and to localize the post-synaptic site of action at which 8-OH D P A T exerts its cardiovascular effects. The 5-HT receptor which mediates 5-HT excitation of sympathetic preganglionic neurons appears pharmacologically similar to the 5-HT receptor on motoneurons ~7'2]. 8-OH D P A T acts as an antagonist to block the 5-HT-induced excitation of motoneurons ]°'29. Thus, the effect of 8-OH D P A T on sympathetic preganglionic firing was studied to determine if 8-OH D P A T antagonizes the spontaneous excitatory 5-HT input to these cells. In addition, the effects of 8-OH D P A T were determined on medullospinal sympathoexcitatory neurons located in the rostral ventrolateral medulla. These neurons receive a large number of inputs from the brainstem and spinal cord and project to the I M L 2"3. In

addition, these neurons are critical to the maintenance of spontaneous sympathetic activity 3. Microinjection of 8-OH D P A T into the rostral ventrolateral medulla inhibits SND and lowers arterial blood pressure 9'12. Thus, it is possible that 8 - O H D P A T produces its sympatholytic effect by inhibiting the firing of these sympathoexcitatory neurons. MATERIALS AND METHODS General procedures Twenty seven cats (2.5-4.0 kg) were anesthetized by an intravenous injection of diallybarbiturate sodium (60 mg/kg), urethane (240 mg/kg), and monoethylurea (240 mg/kg). The animals were placed in a David Kopf Instruments stereotaxic apparatus and spinal investigation unit, and a glass tracheal tube was inserted. A femoral artery and vein were cannulated to measure arterial blood pressure and to permit intravenous drug administration, respectively. An arterial embolectomy catheter (American Edwards Laboratories) was inserted in the opposite femoral artery to permit occlusion of the descending aorta. Heart rate was recorded continuously with a Grass 7P4 tachograph triggered by the electrocardiogram. Rectal temperature was maintained between 36 and 38 °C with a heating pad and/or lamp. The dorsal aspect of the medulla was exposed by removal of portions of the overlying occipital bone and cerebellum. The thoracic spinal cord was exposed by removal of the first to the third thoracic vertebrae. Dorsal roots were sectioned after removal of the dura and pia to allow for visualization of the dorsolateral sulcus. Following surgery, animals were immobilized with gallamine triethiodide (4 mg/kg, i.v.) and artificially respired. Bilateral pneumothoracotomy was performed to minimize movements associated with artificial respiration. Supplemental doses of gallamine were administered as required during the experiment. In addition, 5 animals underwent midcollicular transection. Neural recordings The dorsal lateral sulcus was used as a reference for electrode placement in the spinal cord studies. Neurons were identified by antidromic activation in an area in which we have previously found sympathetic preganglionic neurons (i.e., within 200 ~m of the sulcus and 1.0-2.0 mm below the surface of the cord) ]7. In the medullary studies the obex was used as a surface landmark for placement of the recording electrode. Neurons were recorded in the rostral

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8-OH DPAT (ug/Kg) Fig, 2. Dose-response curves illustrating the effects of i.v. 8-OH DPAT on mean arterial pressure (MAP) and SND in midcollicular transected and control animals. Open squares: SND, transected. Filled squares: SND, control. Open circles: MAP, transected. Filled circles: MAP, control (n = 5). S.E.M. bars are illustrated. ventrolateral area previously described 2'19'2° (i.e., 4.5-6.0 mm anterior to the obex, 3.5-3.7 mm lateral to the midline, and 0.3-2.0 mm from the ventral surface of the medulla). A platinum reference electrode was placed on the frontal bone. Single-unit action potentials were amplified using capacity-coupled preamplification (low and high half-amplitude responses at 0.3 and 3.0 kHz, respectively) and displayed on an oscilloscope, Unitary discharges were typically recorded using extracellular techniques with a tungsten microelectrode (1/~m tip diameter, 2-4 Mr2 impedance). Microiontophoretic experiments utilized 5-barrel glass microelectrodes (tip 4-8 /~m) containing monofilament glass fibers to

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Fig. 3. Oscillograph traces triggered by stimulus applied to the cervical sympathetic nerve illustrating antidromic activation of a sympathetic preganglionic neuron. Traces 1, 3 and 4 show constant onset of activation. Trace 2 illustrates collision between spontaneous and antidromic potentials. Vertical calibration is 20 pV. Horizontal calibration is 5 ms.

simultaneously record the extracellular potentials from single neurons and to apply drugs at the recording site. The center barrel was filled with 2 M NaC1 and was used to record extracellular unit activity. The impedance of the recording barrel typically ranged from 1 to 4 M~2. One side barrel was filled with 4 M NaCI and used for automatic current balancing. The remaining barrels were filled with serotonin HC1 (Research Biochemicals Inc., 0.04 M, pH 4.2), 8-OH DPAT HBr (Research Biochemicals Inc., 0.02 M, pH 4.0), the monosodium salt of L-glutamate (Sigma, 0.1 M, pH 8.0) or an appropriate control solution. Solutions were prepared before each experiment. A retaining current of 15 nA was applied between ejecting periods. The brainstem or the spinal cord was removed and fixed in 10% buffered Formalin at the end of each experiment. Frontal sections of 30 pm thickness were cut with a cryostat microtome and stained with Cresyl violet. Stimulating sites were reconstructed from electrode tracks located in these sections. The constancy of the action potential contour and amplitude were assessed using a storage oscilloscope to confirm the unitary nature of the recordings. Recordings were likely made from the region of the neuronal cell body rather than axons because (1) an inflexion on the rising phase of the action potential that reflects invasion of the initial segment and somatodendritic regions was often observed, (2) the duration of the action potential was longer than that for axonal action potentials and (3) the firing rates of the neurons increased during microiontophoresis of L-glutamate (L-glutamate excites cell

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bodies but not axons of neurons). Peripheral sympathetic nerve activity was recorded from the central end of the sectioned left postganglionic inferior cardiac nerve. The nerve was located distal to the stellate ganglion and isolated outside the pleural cavity after removal of the vertebral portion of the first rib. Potentials were recorded monophasically under mineral oil with a bipolar platinum electrode after capacitycoupled preamplification. A band pass of 1-1000 Hz was used to display the synchronized discharges of the whole sympathetic nerve in the form of slow waves.

Neural stimulation The left preganglionic cervical sympathetic nerve was isolated at the level of the third cervical vertebrae and attached to a shielded bipolar electrode. Electrode placement and nerve isolation were determined to be correct if mydriasis of the ipsilateral pupil could be elicited by high frequency (50 Hz) stimulation. This electrode was used to antidromically activate sympathetic preganglionic neurons. Neurons were considered to be antidromically activated if (1) the neuron responded to the stimulus with a constant onset latency, (2) the unitary discharge followed high frequencies of stimulation and (3) collisions between antidromic and orthodromic spikes could be observed. Electrical pulses were applied to the cervical sympathetic nerve by a WPI 302T square-wave stimulator, the output of which was passed through a stimulus isolation unit.

Data analysis Inferior cardiac SND, unitary discharges, blood pressure, and pulses derived from the R wave of the electrocardiogram and stimuli applied to the cervical sympathetic nerve were recorded on magnetic tape. The data on tape were analyzed using a Nicolet 1170 signal averager or an RC Electronics Computerscope. Unit discharges were subjected to window discrimination before presentation to the computer. In addition, action potentials were counted during 8 s intervals and recorded as an integrated rate histogram. The memory content of the computers were displayed in analog form on an oscilloscope and on an X - Y recorder or plotter. Methods of analysis included (1) post R-wave time interval histogram analysis of unit activity, (2) post R-wave average of inferior cardiac SND and arterial blood pressure, (3) unitary discharge triggered average of SND, (4) random pulse (i.e. generated by the stimulator) triggered average of SND, and (5) spike-triggered post event time interval histogram analysis. Student's t-test for unpaired data was used to evaluate statistical significance (P < 0.05).

RESULTS

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236 blood pressure, heart rate and inferior cardiac sympathetic nerve activity were determined in 5 midcollicular transected animals. The effects of 8-OH DPAT in transected animals were compared to those observed in sham operated control animals to determine if the forebrain mediated the sympatholytic action of 8-OH DPAT. An example of the sympatholytic action of 8-OH D P A T in a midcollicular transected animal is illustrated in Fig. 1. Fig. 2 illustrates dose-response curves for the effects of 8-OH DPAT on SND and mean arterial blood pressure (MAP) in control and midcollicular (n = 5) transected animals. In transected animals, the lowest i.v. dose test (10/~g/kg) produced reductions in sympathetic activity and blood pressure of 20%, with maximal reductions at 300/tg/kg. These results are similar to those obtained in control preparations, where 10 yg/kg produced a 32% reduction in SND and 300 #g/kg resulted in complete inhibition. Decreases in MAP were nearly identical in both groups. Similarly, 8-OH DPAT produced a dose-related bradycardia (10-25% of control

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Spinal cord studies Fifteen sympathetic preganglionic neurons within the IML were identified following stimulation of the cervical sympathetic nerve. In general, the action potentials of the neurons exhibited a biphasic negative-positive wave complex. The onset latency of antidromically elicited action potentials ranged from 12 to 47 ms. This range approximates the total duration of the compound action potential elicited in the cervical sympathetic nerve by stimulation of thoracic ventral roots 32. An example of an antidromically activated sympathetic preganglionic neuron is shown in Fig. 3. The neuron responded with a constant onset latency to the stimulus applied to the cervical sympathetic nerve (traces 1, 3 and 4). An

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example of collision between an orthodromic and antidromic elicited spike is illustrated in trace 2 of Fig. 3. Neurons were often quiescent or fired in an irregular manner. The discharge rate of the spontaneously active units ranged from 0.2 to 1.3 spikes/s, with a mean rate of 0.6 spikes/s.

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Microiontophoresis of 5-HT (10-120 nA) increased the firing rate of all 15 sympathetic preganglionic neurons studied. Examples of three of these units are illustrated in Fig. 4. The excitatory response could be elicited by currents as low as 10 nA and averaged 45 nA. Cells responded to higher ejecting currents with an increased excitatory response. The amplitude of the action potentials remained unaltered by 5-HT, even at higher ejecting currents. 8-OH D P A T consistently failed to elicit a response from sympathetic preganglionic neurons (n = 11), even at elevated (115 hA) ejecting currents (Fig. 4A) or for extended periods (Fig. 4C). While 8-OH DPAT alone produced neither an excitatory or an inhibitory response, iontophoresis of 8-OH D P A T invariably led to a blockade of the usual excitatory response to 5-HT (n = 11). The 5-HT antagonist effect was typically observed after a 1 min period of iontophoresed 8-OH DPAT. The antagonist effect of 8-OH D P A T was at times complete (Fig. 4B), although at other times the excitatory response was only partially attenuated. Occasionally a latent excitation was observed after co-administration of these compounds was discontinued (Fig. 4B, n = 4). More often the antagonistic effects continued for some time (15

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min) after the 8-OH DPAT ejection was terminated (Fig. 4C, n --- 7). Partial recovery of the 5-HT response was observed 7-30 min after discontinuation of 8-OH DPAT (Fig. 4A,B). Despite the blockade of excitation to 5-HT during and after 8-OH DPAT iontophoresis, the sympathetic preganglionic neurons still responded to glutamate (Fig. 4C). Furthermore, the excitation produced by iontophoretic glutamate was not altered by 8-OH DPAT at a time when the 5-HT excitation was totally inhibited (n = 2). Importantly, microiontophoretic application of 8-OH DPAT for periods up to 10 min failed to inhibit the spontaneous discharge rate of sympathetic preganglionic neurons. Rostral ventrolateral medulla studies Sympathoexcitatory neurons in the rostral ventrolateral medulla were identified using previously described criteria 2"19"2°. Briefly, these units were characterized by (1) an inhibition of firing during an increase in blood pressure produced by occlusion of the descending aorta (Fig. 5A); (2) a temporal relation between the unit firing and the inferior cardiac sympathetic nervous discharge (Fig. 5B); (3) a slow (2 Hz) irregular spontaneous discharge rate and (4) a temporal relation between unit firing and the cardiac cycle (Fig. 5C). To determine whether the sympatholytic action of 8-OH DPAT resulted from inhibition of sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM), activity from the inferior cardiac nerve and sympathoexcitatory neurons was simultaneously recorded (n = 9). Fig. 6 illustrates the relationship between sympathoexcitatory unit activity and SND in response to increasing i.v. doses of 8-OH DPAT. The lowest dose (3

/~g/kg) has little effect on unit firing rate, SND or MAP. Increasing the dose to 10 ag/kg resulted in a 40% reduction in SND and unit activity and a 20% reduction in MAP. 8-OH DPAT (30 btg/kg, i.v.) caused further reductions in unit activity, SND and MAP, while a cumulative dose to 100 ag/kg produced very nearly complete inhibition of sympathoexcitatory neuronal firing and SND. The inhibition of unit activity and SND by 8-OH DPAT was reversible utilizing the 5-HTIA antagonist spiperone (n = 3). Spiperone (1 mg/kg, i.v.) resulted in complete recovery of both neuronal and sympathetic activity (Fig. 7). In addition, spiperone returned arterial blood pressure to the level observed prior to administration of 8-OH DPAT. Fig. 8 summarizes the results of 9 experiments in which the inhibitory effects of 8-OH DPAT on RVLM sympathoexcitatory neuronal activity and SND were simultaneously determined. The dose of 8-OH DPAT required to produce a 50% inhibition of unit and sympathetic activity was 15 /tg/kg. Doses of 100 ag/kg produced almost complete (3 min). Although iontophoretic 8-OH DPAT failed affect the unit illustrated in Fig. 9A, the unit was inhibited following i.v. administration of 8-OH DPAT (Fig. 9B). DISCUSSION Previous studies from our laboratory and others indicate that the selective 5-HTIA agonist 8-OH DPAT acts in the central nervous system to reduce sympathetic nerve activity and thereby lower arterial blood pressure 79,12,22,23,27,28 8-OH DPAT might produce its sympatholytic effect by inhibiting medullary 5-HT neurons which provide a tonic excitatory input to sympathetic preganglionic neurons located in the IML of the spinal cord 17' 22,27. In this regard, 5-HT1A receptors are located on medullary 5-HT neurons (i.e. autoreceptors) and microiontophoresis of 8-OH DPAT inhibits the firing of these neurons 22. However, recent experiments in our laboratory revealed that medullary 5-HT neuronal activity was more sensitive to the inhibitory effect of 8-OH DPAT than was sympathetic activity23. In addition, electrolytic lesions encompassing the majority of the midline area of the lower brainstem failed to affect the sympatholytic action of 8-OH DPAT 23. These data provide strong evidence to indicate that direct inhibition of medullospinal 5-HT neuronal firing is not sufficient to explain the central sympatholytic effects of 8-OH DPAT. Rather, the data indicate that 8-OH DPAT acts on postsynaptic 5-HTIA receptors to produce its cardiovascular actions. The purpose of the present investigation was to study the postsynaptic effects of 8-OH DPAT by determining the effects of 8-OH DPAT on two types of neurons known to play an important role in the generation of sympathetic nerve discharge: sympathetic preganglionic neurons and medullospinal sympathoexcitatory neurons located in the rostral ventrolateral medulla. The effect of 8-OH DPAT on sympathetic preganglionic neurons was determined since these cells receive a dense 5-HT innervation 4,13,~4. In agreement with previous studies 6A7, we found that microiontophoretically applied 5-HT consistently excited sympathetic preganglionic neurons (Fig. 4). In contrast, iontophoretic appli-

cation of 8-OH DPAT failed to affect the spontaneous firing of sympathetic preganglionic neurons. Similarly, intrathecal administration of 8-OH DPAT fails to lower arterial blood pressure 34. However, 8-OH DPAT concomitantly iontophoresed with 5-HT blocked the excitatory effect of the monoamine on preganglionic firing (Fig. 4). The blockade of the 5-HT-induced excitation did not result from a local anesthetic action of iontophoretic 8-OH DPAT as evidenced by the consistency of action potential amplitude and configuration. In addition, the unit continued to respond to the excitatory effects of iontophoretic glutamate. Previous studies from our laboratory 22'23 suggest that the amount of 8-OH DPAT microiontophoresed has an effect similar to that of a small i.v. dose of 8-OH DPAT (e.g. 1-10 /~g/kg). However, a possible 5-HT antagonist action of i.v. 8-OH DPAT was not studied since i.v. 8-OH DPAT inhibits the firing of sympathetic preganglionic neurons 27. Recent studies indicate that 8-OH DPAT also blocks the excitatory effect of iontophoretic 5-HT on spinal motoneurons and on facial motoneurons 1°'29. Thus, 8-OH DPAT has antagonist activity on 5-HT receptors located on sympathetic preganglionic neurons and on spinal and facial motoneurons. The receptor subtype mediating the effect of 8-OH DPAT on sympathetic preganglionic neurons was not determined in this study. A number of studies from our laboratory suggest that medullospinal 5-HT neurons provide a tonic excitatory input to sympathetic preganglionic neurons. Microiontophoresis of the 5-HT antagonists methysergide or metergoline block the excitatory effects of microiontophoretic 5-HT and decrease the spontaneous discharge rate of sympathetic preganglionic neurons 17. In contrast, 5-HT antagonists fail to affect the spontaneous discharge rate of sympathetic preganglionic neurons in spinal animals 17. In addition, 5-HT antagonists fail to inhibit sympathetic activity in animals depleted of 5-HT 26 or in which the descending serotonergic neurons have been destroyed 17' 24. Electrical stimulation of midline medulla elicits sympathoexcitatory as well as sympathoinhibitory responses 18'25. The sympathoexcitatory responses are blocked by 5-HT antagonists and potentiated by 5-HT uptake inhibitors TM. The sympathoinhibitory response results from a GABA-mediated inhibition of sympathoexcitatory medullospinal neurons located in the rostral ventrolateral medulla2°,25. Collectively, these data provide strong evidence to indicate that midline medullary 5-HT neurons provide a tonic excitatory input to sympathetic preganglionic neurons. In the present study we found that 8-OH DPAT blocked the excitatory effect of iontophoretic 5-HT but failed to alter the firing rate of preganglionic sympathetic neurons. The fact that 8-OH DPAT antagonized the excitatory effect of 5-HT but

24(I failed to reduce the spontaneous firing rate of sympathetic preganglionic neurons is inconsistent with our previous studies. The possibility exists that excitatory medullospinal 5-HT neurons terminate on either distal dendrites of the preganglionic neurons or on closely adjacent interneurons. In this case, iontophoretic 8-OH DPAT may not gain access to these more distant excitatory 5-HT receptors but could block the effects of iontophoretic 5-HT which presumably acts on or near the soma. However, it should be noted that 5-HT neurons have been shown to terminate on the soma of adrenal sympathetic preganglionic neurons I. Alternatively, multiple receptors may mediate the excitatory effect of 5-HT on sympathetic preganglionic neurons. In this case, 8-OH DPAT may not act as an antagonist at the receptors receiving the endogenous 5-HT input. This explanation is supported by the observation that microiontophoresis of the non-specific 5-HT antagonists methysergide and metergoline reduce the firing rate of sympathetic preganglionic neurons 17. In any case, the fact that iontophoretic 8-OH DPAT failed to affect the firing of preganglionic neurons suggests that 8-OH DPAT acts elsewhere in the central nervous system to produce its sympatholytic action. Recent studies indicate that microinjection or local application of 8-OH DPAT in the area of the rostral ventrolaterai medulla results in a decrease in arterial blood pressure 9'12. This is a significant finding since the rostral ventrolateral medulla contains neurons which represent a major source of excitatory sympathetic input to sympathetic preganglionic neurons 3. Therefore, we investigated the effects of both i.v. and iontophoretic 8-OH DPAT on the firing of medullospinal sympathoexcitatory neurons located in the rostral ventrolateral medulla. Neurons were identified as previously described 2'19'2° and were considered to subserve a sympathoexcitatory function based on the observations that the neuronal discharge was temporally related to sympathetic activity and the cardiac cycle and was inhibited during baroreceptor reflex activation (Fig. 5). We found that i.v. administration of 8-OH DPAT markedly inhibited the firing of sympathoexcitatory neurons in the rostral ventrolateral medulla (Fig. 6). The inhibition of neuronal firing produced by 8-OH DPAT was reversed by the 5-HT1A antagonist spiperone (Fig. 7). Although spiperone has affinity at other receptors (e.g. 5-HT2 and D2) it is likely that spiperone acted as a 5-HT1A antagonist since 8-OH DPAT is highly selective for the 5-HTIA receptor subtype. In addition, spiperone blocks the 5-HTIA effects of 8-OH DPAT on dorsal raphe neurons and on sympathetic nerve activity ~5'26. These data strongly suggest that the inhibition of neuronal firing produced by 8-OH DPAT was associated with the

5-HT~A agonist properties of the drug. Simultaneous determination of the affect of 8-OH DPAT on sympathoexcitatory neurons in the rostral ventrolateral medulla and on sympathetic nerve discharge revealed an extremely close relationship between unit and sympathetic inhibition (Fig. 8). This observation indicates that inhibition of medullospinal sympathoexcitatory neurons in the rostral ventrolateral medulla is critical for the expression of the sympatholytic action of 8-OH DPAT. The remarkable correlation between the inhibition of sympathoexcitatory neuronal firing and the inhibition of peripheral sympathetic nerve discharge suggests that 8-OH DPAT acts directly on rostral ventrolateral sympathoexcitatory neurons to produce its sympatholytic action. This was tested directly by determining the effects of microiontophoretically applied 8-OH DPAT on the firing rate of these neurons. Surprisingly, microiontophoretically applied 8-OH DPAT failed to affect the firing of the sympathoexcitatory neurons in the rostral ventrolateral medulla (Fig. 9). Similarly, iontophoretic 5-HT had no obvious affect on these neurons. These observations suggest that 8-OH DPAT may inhibit the firing of sympathoexcitatory neurons by acting on distal dendrites of these neurons. In this regard, iontophoretically applied 8-OH DPAT might not gain access to 5-HT1A receptors located on dendrites removed a great distance from the cell body of these neurons. Alternatively, 8-OH DPAT may produce its sympatholytic effect by acting on sympathetic neurons which lie antecendent to the sympathoexcitatory neurons located in the rostral ventrolateral medulla. It is not likely that sympathetic neurons in the midline area of the medulla play a role in the sympatholytic action of 8-OH DPAT since we have previously demonstrated that neither iontophoretically applied or i.v. administered 8-OH DPAT affects the firing of midline sympathetic neurons 22. To eliminate the forebrain as a possible sight for the sympatholytic action of 8-OH DPAT, the effects of 8-OH DPAT in control and mid-collicular transected animals were compared (Figs. 1,2). We found that 8-OH DPAT inhibited sympathetic activity and lowered arterial blood pressure in the transected animal. Indeed, the doseresponse curves illustrating the sympatholytic and hypotensive effect of 8-OH DPAT in control and transected animals were nearly identical. This observation indicates that forebrain structures do not play a critical role in the sympatholytic action of 8-OH DPAT. In summary, the present paper characterizes the effects of 8-OH DPAT on sympathetic preganglionic neurons located in the spinal cord and on sympathoexcitatory neurons located in the rostral ventrolateral medulla. We found that iontophoretically applied 8-OH DPAT failed to alter the spontaneous firing rate of

241 s y m p a t h e t i c preganglionic neurons but acted as an antagonist to block the excitatory effect of i o n t o p h o r e tically a p p l i e d 5-HT. In addition, we d e m o n s t r a t e d a r e m a r k a b l e correlation b e t w e e n the inhibition of firing of s y m p a t h o e x c i t a t o r y neurons in the rostral ventrolateral m e d u l l a and p e r i p h e r a l sympathetic activity. This indicates that inhibition of these neurons is critical in the

expression of the sympatholytic action of 8 - O H DPAT. A direct action of 8 - O H D P A T is q u e s t i o n e d since iontophoretic application of the drug failed to affect the firing of s y m p a t h o e x c i t a t o r y neurons. Similarly, i o n t o p h o r e t i c 5-HT had no affect on these neurons. Finally, forebrain structures do not play an i m p o r t a n t role in the sympatholytic action of 8 - O H DPAT.

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