Mark E. Clement

Identification of serotonergic and sympathetic neurons in medullary raphe nuclei

The purpose of the present study was to identify midline medullary serotonin (5-HT) neurons and to determine if these neurons were distinct from previously identified sympathoinhibitory and sympathoexcitatory neurons. Identification of medullary 5-HT neurons was based on electrophysiological and pharmacological similarities to dorsal raphe 5-HT neurons. Sympathoinhibitory and sympathoexcitatory neurons were characterized by an irregular discharge pattern which was temporally related to inferior cardiac sympathetic nerve discharge (SND) and to the cardiac cycle. Sympathoinhibitory neurons increased their discharge rate and the discharge of sympathoexcitatory neurons decreased during baroreceptor reflex activation. A third type of neuron fired in an extremely regular fashion (as judged by interspike interval analysis), fired at a rate of 1.1 spikes/s and had spike durations of approximately 2 ms. The discharges of regularly firing neurons were not temporally related to SND and were not affected during baroreceptor reflex activation. Regularly firing neurons and sympathoinhibitory neurons could be antidromically activated by electrical stimulation of the intermediolateral cell column of the spinal cord. Axonal conduction velocity of sympathoinhibitory neurons (2.4 m/s) was significantly greater than that for regularly firing neurons (1.3 m/s). Regularly firing neurons were completely inhibited by low doses of the 5-HT1A agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) (i.e. 2 micrograms/kg, i.v.) while much higher doses of the drug failed to affect the discharges of sympathoinhibitory and sympathoexcitatory neurons. Microiontophoretic application of 5-HT and 8-OH-DPAT profoundly depressed the firing of regularly discharging neurons. Based on the striking similarities between regularly firing medullary neurons and dorsal raphe 5-HT neurons it is concluded that the regularly firing neurons were 5-HT-containing neurons. Furthermore, these medullary 5-HT neurons are distinct from sympathoinhibitory and sympathoexcitatory neurons.

Studies on the site and mechanism of the sympatholytic action of 8-OH DPAT

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. Microiontophoretically 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of the sympatholytic effect of 8-OH DPAT: lack of correlation between inhibition of serotonin neuronal firing and sympathetic activity

Previous studies indicate that the selective 5-HT1A agonist, 8-OH DPAT, acts in the central nervous system to inhibit sympathetic nerve activity. Based on the observations that: (1) 8-OH DPAT acts at serotonin (5-HT) autoreceptors to inhibit 5-HT neuronal firing; and (2) medullary 5-HT neurons provide a tonic excitatory input to sympathetic preganglionic neurons, we have hypothesized that 8-OH DPAT produces its sympatholytic effects by inhibiting medullary 5-HT neuronal firing and thereby removing an excitatory input to sympathetic preganglionic neurons. The present study was designed to critically test this hypothesis. The sympatholytic effects of 8-OH DPAT were compared in intact animals and in animals which received large electrolytic lesions in the midline area of the lower brainstem. These lesions extended from the obex rostral through the level of the facial motor nucleus and encompassed the brain stem from the dorsal to the ventral surface. The sympatholytic effect of 8-OH DPAT was identical in intact animals and in animals receiving the lesion. The inhibitory effects of 8-OH DPAT on activity recorded simultaneously from the inferior cardiac sympathetic nerve and from medullospinal 5-HT neurons were determined. Medullary 5-HT neurons were identified using criteria modeled after the electrophysiological and pharmacological characteristics previously described for dorsal raphe 5-HT neurons. Medullary 5-HT neuronal activity was more sensitive to the inhibitory effects of 8-OH DPAT than was sympathetic activity. Indeed, low doses of 8-OH DPAT completely suppressed the firing of medullary 5-HT neurons but had little effect on sympathetic nerve activity. These data fail to support the hypothesis that inhibition of 5-HT neuronal firing is responsible for the central sympatholytic effects of 8-OH DPAT. Rather, the data suggest that 8-OH DPAT acts postsynaptically on 5-HT1A receptors located on central sympathetic neurons to inhibit sympathetic nerve activity.

Studies on the site and mechanisms of the sympathoexcitatory action of 5-HT2 agonists

Intravenous administration of the selective 5-HT2 agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), produced an increase in sympathetic activity recorded from the inferior cardiac nerve in chloralose-anesthetized cats. Microiontophoretically applied 5-HT increased the firing rate of antidromically identified sympathetic preganglionic neurons. Microiontophoretic DOI failed to affect the firing of sympathetic preganglionic neurons. The effect of DOI was also studied on medullospinal sympathoexcitatory neurons located in the rostral ventrolateral medulla. Intravenous DOI increased the firing of sympathoexcitatory neurons and sympathetic nerve discharge to a similar extent. Microiontophoretic application of DOI failed to affect the firing of sympathoexcitatory neurons. The data are discussed in relation to the site and mechanism of the sympathoexcitatory action of 5-HT2 agonists.

Evidence that the lateral tegmental field plays an important role in the central sympathoinhibitory action of 8-OH-DPAT

We examined the effects of 8-OH-DPAT and 5-HT on three types of sympathetic-related neurons identified in the lateral tegmental field of anesthetized cats using spike-triggered averaging techniques. Based on their response to baroreceptor activation, these neurons could be classified as sympathoexcitatory, sympathoinhibitory, or baroreceptor activation unresponsive. 8-OH-DPAT administered intravenously was found to inhibit sympathoexcitatory neurons with a high degree of correlation to inhibition of sympathetic activity, and to excite sympathoinhibitory neurons in a dose-dependent manner. Iontophoretic application of 8-OH-DPAT and 5-HT to the majority of sympathoexcitatory neurons caused inhibition of spontaneous activity while iontophoretic application of 8-OH-DPAT to sympathoinhibitory neurons had variable effects although 5-HT consistently caused excitation. Baroreceptor unresponsive neurons were insensitive to iontophoretic 8-OH-DPAT and showed only limited response to 5-HT. It is concluded that the lateral tegmental field plays an important role in the sympathoinhibitory action of 8-OH-DPAT.

EFFECTS OF CLONIDINE ON SYMPATHOEXCITATORY NEURONS IN THE ROSTRAL VENTROLATERAL MEDULLA

The effects of intravenous and iontophoretic clonidine were determined on the firing rates of sympathoexcitatory neurons in the rostral ventrolateral medulla of the cat. As previously reported in the rat, we found that sympathoexcitatory neurons could be differentiated based on their sensitivity in clonidine. Approximately 50% of the neurons were inhibited by clonidine. There was only a weak correlation between the inhibition of unit activity and whole sympathetic nerve activity. The discharge rates of the remaining neurons were either not altered or were increased by clonidine. Unlike the rat, these two groups of neurons could not be further differentiated on the basis of axonal conduction velocity or discharge frequency. These data are discussed and the effects of clonidine and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on sympathoexcitatory neurons are compared.

Impairment of baroreceptor reflexes following kainic acid lesions of the lateral tegmental field.

We examined the effects of kainic acid lesions of the lateral tegmental field on baroreceptor function in the anesthetized cat. Kainic acid lesions prevented the reflex inhibition of inferior cardiac sympathetic nerve activity observed during an increase in blood pressure. The temporal locking of sympathetic slow waves to the cardiac cycle was also abolished following tegmental field lesions. Finally, the periodicity of sympathetic nerve discharge shifted to a higher frequency range following kainic acid lesions. These observations are consistent with the conclusion that lesions of the lateral tegmental field impair baroreceptor reflexes.

Lateral tegmental field involvement in the central sympathoinhibitory action of 8-OHDPAT

This study examined the effects of kainic acid and NMDA microinjections into the lateral tegmental field on the sympatholytic effect of the 5-HT1A agonist 8-OH-DPAT. Kainic acid has been reported to destroy cell bodies while leaving fibers of passage intact while NMDA excites the cell bodies but not the axons of neurons. Microinjection of kainic acid was found to block the usual sympatholytic effect of 8-OH-DPAT but not the sympathoinhibition produced by the alpha 2 agonist clonidine. Microinjection of NMDA elicited profound pressor responses related to an increase in sympathetic activity. Sympatholytic effects of 8-OH-DPAT and clonidine were transiently overridden by microinjections of NMDA, but not glutamate. A role for the lateral tegmental field in the generation of sympathetic tone and in the sympatholytic mechanism of 8-OH-DPAT is supported by the chemical lesion and stimulation studies.

Pharmacological characterization of medullary serotonin neurons

In the present study we investigated the characteristics of medullary raphe serotonergic neurons. Specifically, we sought to examine further the similarities between medullospinal 5-HT neurons and the more extensively studied neurons of the dorsal raphe. Intravenous administration of 5-methoxy-dimethyltryptamine (5-MeODMT) produced a dose-related inhibition of the firing of midline medullary 5-HT neurons. Microiontophoretically applied 5-MeODMT also inhibited medullary 5-HT neurons. The inhibitory potency of 5-MeODMT was nearly identical to that observed for dorsal raphe 5-HT neurons. Microiontophoretic or intravenous administration of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) did not alter the firing rate of medullary 5-HT neurons. Intravenous administration of the alpha 1-receptor antagonist prazosin resulted in an inhibition of the medullary 5-HT neuronal firing. The discharge of medullary 5-HT neurons increased during iontophoresis of norepinephrine. These data are discussed in relation to the identification and characterization of medullary 5-HT neurons. In addition, the data suggest that the firing rate of medullary 5-HT neurons is regulated in part by a tonic excitatory noradrenergic input.

8-OH-DPAT-induced inhibition of renal sympathetic nerve activity and serotonin neuronal firing

The effects of the 5-HT1A receptor 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) administered i.v. on medullary 5-HT neuronal firing and renal sympathetic nerve activity were determined in spontaneously breathing anaesthetized cats. Low doses of 8-OH-DPAT (1-3 micrograms/kg) caused a similar reduction in 5-HT neuronal firing and renal nerve activity while high doses (10-30 micrograms/kg) completely inhibited neuronal firing but caused only 80% inhibition of renal nerve activity. These data are discussed in relationship to the mechanism of sympatholytic action of 8-OH-DPAT and the serotonergic regulation of sympathetic nerve activity.