Autoactivity of A5 neurons: role of subthreshold oscillations and persistent Na+ current.

A5 noradrenergic neurons play a key role in autonomic regulation, nociception, and respiration. The purpose of the present experiments was to characterize some of the intrinsic properties of A5 cells in vitro. Whole cell recordings were obtained from 85 spinally projecting neurons of the ventrolateral pons of neonate rats. Immunohistochemistry showed that 60% of the ventrolateral pontine cells were noradrenergic. Eighty percent of A5 neurons were spontaneously active (0.1-5.5 spikes/s). Their discharge rate was unchanged by a mixture of synaptic blockers that eliminated postsynaptic potentials (PSPs). The nonnoradrenergic cells could not be distinguished from A5 cells on the basis of discharge rate, action potential duration, inward rectification, input resistance, or accommodation. A5 cells displayed subthreshold irregular oscillations of the membrane potential (main frequency component 0.5-2 Hz). These oscillations were unchanged in the presence of low external Ca(2+)-high Mg2+ and were very reduced by hyperpolarizing the cells below -65 mV. The oscillations were partially attenuated by 1 microM tetrodotoxin (TTX) and were eliminated by reducing external Na+ (27 mM). Stepping the membrane potential from -65 to -50 mV for 200 ms revealed the presence of a transient and a persistent inward current that were both blocked by 0.1 microM TTX or by extracellular Na+ reduction. In conclusion, most A5 neurons are spontaneously active in vitro. They display irregular subthreshold membrane potential oscillations generated by voltage-activated conductances that include a persistent TTX-sensitive Na+ current. Most of the activity of A5 cells appears due to intrinsic properties rather than to synaptic inputs.

Alpha 2-adrenergic autoreceptors in A5 and A6 neurons of neonate rats.

A5 noradrenergic neurons control sympathetic outflow, nociception, and respiration. The presence of alpha 2-adrenergic receptors (alpha 2-ARs) in A5 cells has been suggested by immunohistochemistry. In the present experiments, we analyze the response of spinally projecting A5 cells to alpha 2-AR agonists, and we compare it with that of locus ceruleus (A6) neurons. Whole cell recordings were obtained from 52 spinally projecting neurons in the ventrolateral pons of neonate rats. Immunohistochemistry showed that 60% of the recorded cells were A5 cells. In A5 cells clamped at -55 mV, norepinephrine (NE) in the presence of the alpha 1-AR antagonist prazosin produced a Ba(2+)-sensitive outward current (20.4 +/- 2.6 pA; n = 28). The alpha 2-AR-induced current reversed at the K+ equilibrium potential (EK) at three different extracellular K+ concentrations. Replacement of 82% of the extracellular Na concentration with N-methyl-D-glucamine did not change the reversal potential. The 19 presumably noncatecholaminergic neurons responded weakly or not at all to NE (2.5 +/- 0.6 pA outward current). Pontospinal A6 neurons (n = 11) were also recorded. Six A6 cells displayed large tetrodotoxin (TTX)-resistant membrane oscillations. In these cells, the current induced by alpha 2-AR stimulation did not reverse over the voltages tested (-50 to -130 mV) or reversed at potentials more negative than EK (less than -114 mV). In A6 neurons that did not display large oscillations (n = 5), the alpha 2-AR-induced current reversed at or close to the EK (-90 +/- 1.6 mV). In conclusion, A5 cells, like locus ceruleus neurons, have alpha 2-ARs that may function as autoreceptors. In both cases, alpha 2-AR activation increases an inwardly rectifying K+ conductance. In A5 cells, we found no evidence that alpha 2-AR activation decreases a resting Na+ conductance. The inhibition of A5 cells by clonidine and other agents with alpha 2-AR agonist activity is likely to contribute to the ability of these drugs to decrease sympathetic tone and arterial pressure.

Effect of cholinergic agonists on bulbospinal C1 neurons in rats.

Cholinergic inputs to the rostral ventrolateral medulla (RVLM) may contribute to sympathetic tone generation. The present study analyzes the response of RVLM neurons to cholinergic agonists. In chloralose-anesthetized rats iontophoresis of carbachol excited RVLM sympathoexcitatory neurons (+69% from resting level of 11.9 +/- 2 spikes/s; n = 28). This effect was reduced 85% by iontophoresis of methylatropine and abolished by intravenous scopolamine. Iontophoresis of nicotine or hexamethonium was ineffective. In contrast, most RVLM respiratory units were inhibited by carbachol. Whole cell recordings of bulbospinal RVLM neurons were made in neonatal rat brain slices (54 cells, 24 C1 adrenergic neurons). In current-clamp recordings (without tetrodotoxin) carbachol produced depolarization, increased postsynaptic potential frequency, and decreased input resistance. In voltage-clamp recording (-50 to -60 mV; 1 microM tetrodotoxin) carbachol produced inward current [50% effective concentration (EC50): 10 +/- 1 microM; 12.6 +/- 2 pA at 30 microM; n = 16] that persisted in low Ca2+/high Mg2+ (n = 6). Muscarine (30 microM) caused smaller inward currents (2.6 +/- 0.6 pA; n = 16). The carbachol-induced current was reduced 46% by 5 microM methylatropine (n = 15) and 84% by 200 microM hexamethonium (n = 9). The current was linear as a function of the holding potential (extrapolated reversal potential: -22 +/- 2 mV). In conclusion, carbachol exerts both pre- and postsynaptic effects on C1 and other putative sympathoexcitatory RVLM neurons. In vitro the postsynaptic effect of carbachol has a mixed nicotinic and muscarinic pharmacology. In vivo, iontophoretically applied carbachol produces muscarinic excitation of barosensitive RVLM neurons.

Mechanism of the hypotensive action of anandamide in anesthetized rats.

We studied the effects of the endogenous cannabinoid ligand anandamide on blood pressure, single unit activity of barosensitive neurons in the rostral ventrolateral medulla, and postganglionic splanchnic sympathetic nerve discharge in urethane-anesthetized rats. In rats with an intact baroreflex, an intravenous bolus of 4 mg/kg anandamide caused a triphasic blood pressure response: transient hypotension, followed by a brief pressor and more prolonged depressor phase. Anandamide evoked a "primary" increase in neuronal firing coincident with its pressor effect and a "secondary," baroreflex-mediated rise coincident with its depressor effect at both sites. Pretreatment of rats with phentolamine or trimethaphan did not inhibit either the pressor response or the primary increase in splanchnic nerve discharge elicited by anandamide. In barodenervated rats, electrical stimulation of the rostral ventrolateral medulla increased blood pressure and splanchnic nerve discharge. Anandamide treatment blunted the rise in blood pressure without affecting the increase in splanchnic nerve discharge. Anandamide did not affect the rise in blood pressure in response to an intravenous bolus dose of phenylephrine. The results indicate that (1) the brief pressor response to anandamide is not sympathetically mediated, and (2) the prolonged hypotensive response to anandamide is not initiated in the central nervous system, in ganglia, or at postsynaptic adrenergic receptors but is due to a presynaptic action that inhibits norepinephrine release from sympathetic nerve terminals in the heart and vasculature.

Sympatholytic effect of tricyclic antidepressants: site and mechanism of action in anesthetized rats.

Intravenous desipramine (DMI) and amitriptyline, but not fluoxetine, dose dependently inhibited splanchnic sympathetic nerve discharge (sSND; -64 +/- 3% after 4 mg/kg iv DMI, 172 ng/ml plasma) in urethan-anesthetized debuffered rats. Inhibition was reversed or prevented by microinjection of the alpha 2-adrenergic receptor (alpha 2-AR) antagonist 2-methoxyidazoxan (MOI) into the rostral ventrolateral medulla (RVLM, 1 nmol/side). sSND inhibition (-58 +/- 12%) by baclofen (8 mg/kg iv, a gamma-aminobutyric acid-B receptor agonist) was unaffected by MOI. MOI alone raised sSND 46 +/- 9%. Microinjection of 6-hydroxydopamine into the RVLM (2 micrograms/side, 10-13 days, to destroy noradrenergic terminals) did not change the effect of intravenous DMI or MOI on sSND. Slow-firing presympathetic neurons of the RVLM were activated by iontophoretic MOI (26 +/- 4%) and inhibited by 4 mg/kg iv DMI (-44 +/- 12%, effect reversed by alpha 2-AR antagonists iv). We interpret these findings as follows: 1) alpha 2-ARs in the RVLM are activated at rest, probably by catecholamines released by C1 adrenergic cells, 2) this activation reduces sSND, 3) DMI and amitriptyline reduce sSND by increasing alpha 2-AR activation in the RVLM, and 4) these effects are due to neither serotonin uptake inhibition nor blockade of norepinephrine uptake by noradrenergic fibers.

Epibatidine, an alkaloid from the poison frog Epipedobates tricolor, is a powerful ganglionic depolarizing agent.

Epibatidine, a newly discovered alkaloid from the skin of Dendrobatidae frogs, has structural similarities to nicotine. We examined the effects of epibatidine on cardiorespiratory function and ganglionic synaptic transmission. Superior cervical or splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded along with arterial pressure (AP) in urethane-anesthetized, paralyzed and artificially ventilated rats. Epibatidine administered i.v. at low doses (0.5-2 micrograms/kg) produced a transient increase in AP and sSND, followed by a decrease and return to baseline; this low dose of epibatidine also produced a dose-dependent increase in PND. At high doses (cumulative dose of 8-16 micrograms/kg), epibatidine produced bradycardia, a profound depression in sSND and a transient elimination of PND. After i.v. administration of the ganglionic blocker chlorisondamine (5 mg/kg), AP was still increased by 1 microgram/kg epibatidine (+39 +/- 11 mm Hg). This pressor effect was not altered by pretreatment with the alpha-1 adrenergic antagonist phentolamine (+40 +/- 10 mm Hg); however, it was blocked by additional pretreatment with the vasopressin antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropiony1, O-ET-Tyr2,Val4,Arg8]vasopressin (50 micrograms/kg i.v.; +2 +/- 0.4 mm Hg). Low doses of epibatidine (0.5-2 micrograms/kg) produced firing of postganglionic neurons in a decentralized ganglion preparation and potentiated synaptic transmission; at high doses (cumulative dose of 8-16 micrograms/kg), the alkaloid blocked ganglionic synaptic transmission. These results suggest that epibatidine is a potent agonist of ganglionic nicotinic receptors and that the alkaloid elicits cardiorespiratory effects similar to those of nicotine.

Role of serotonin and catecholamines in sympathetic responses evoked by stimulation of rostral medulla.

In halothane-anesthetized, paralyzed rats, single-pulse stimulation of nucleus raphe pallidus/obscurus (NR) evoked an inhibition followed by a single wave of excitation in 1) the mass discharge of lumbar and splanchnic sympathetic nerves (modal onset latencies: LSND 210 +/- 14 ms, SSND 161 +/- 5 ms) and 2) the unit activity of lumbar sympathetic vasoconstrictor neurons (LSVNs). Stimulation of the rostral ventrolateral medulla (RVL) produced two excitatory responses in LSND, SSND, and LSVN units (modal onset latencies: RVL peak I 98 +/- 8 ms for LSND and 62 +/- 2 ms for SSND; RVL peak II 210 +/- 15 ms for LSND and 160 +/- 6 ms for SSND). Most LSVNs received excitatory inputs from both raphe and RVL. NR peak was selectively attenuated (76%) by 5,7-dihydroxytryptamine [intracisternally (ic), 2-wk] and acute intrathecal (it) methiothepine (10 micrograms, 69%) or methysergide (40 micrograms, 72%). 6-Hydroxydopamine (2-wk ic) produced no effect. Phentolamine (20 micrograms it) or prazosin (20 micrograms it) reduced RVL peak II by > 94% and attenuated NR peak (phentolamine 67%, prazosin 46%). Intrathecal kynurenic acid produced proportionately larger reduction of RVL peak I than raphe peak or RVL peak II. Our interpretations are 1) LSVNs receive convergent excitatory inputs from midline raphe, parphyramidal area, and RVL, 2) bulbospinal serotonergic neurons mediate most of the sympathoexcitation evoked from NR and a portion of RVL peak II, 3) a catecholamine probably released by C1 cells mediates most of RVL peak II and a portion of the NR response, and 4) RVL peak I is predominantly mediated by an excitatory amino acid.

Central respiratory control of A5 and A6 pontine noradrenergic neurons.

Sympathetic nerve discharge (SND), phrenic nerve discharge (PND), and unit activity of locus ceruleus (LC) and of putative A5 noradrenergic cells were recorded in vagotomized rats anesthetized with urethan. SND was activated by stimulation of carotid chemoreceptors with hypoxia (N2 inhalation, 5-15 s or 12% O2 inhalation, 2-5 min) and displayed a prominent central respiratory modulation during the hypoxic challenge (postinspiratory pattern). LC cells were also activated by peripheral chemoreceptor stimulation. The discharge of most LC units (28 of 31) exhibited central respiratory modulation. 15 LC units had a postinspiratory pattern and 11 had an inspiratory one. Putative A5 cells were also excited by hypoxia and also displayed a clear central respiratory modulation (mostly postinspiratory pattern). These experiments indicate that 1) the firing rate of most pontine noradrenergic cells is increased by peripheral chemoreceptor stimulation, and 2) pontine noradrenergic neurons receive afferent information of a respiratory nature, possibly from their ventrolateral medullary inputs.

Ventrolateral medulla and sympathetic chemoreflex in the rat.

Splanchnic sympathetic nerve discharge (SND), phrenic nerve activity (PND) and putative sympathetic premotor neurons of the rostral ventrolateral medulla (RVL) were recorded in urethane-anesthetized vagotomized rats without aortic baroreceptor afferents. Carotid chemoreceptor stimulation with brief N2 inhalation increased SND by 101 +/- 7%, raised mean arterial pressure (MAP) and increased the discharge rate of RVL premotor neurons by 46 +/- 12% (N = 32). During chemoreceptor activation. SND and most RVL neurons displayed pronounced central respiratory rhythmicity with maximal firing probability immediately after cessation of the PND (postinspiratory phase) and lowest probability during PND (inspiratory phase). Bilateral microinjection of the breed spectrum glutamate receptor antagonist kynurenic acid (Kyn, 5 nmol in 100 nl) into RVL blocked the sympathetic chemoreflex but left the sympathetic baroreflex intact. In contrast, bilateral microinjection of the same dose of Kyn into the caudal ventrolateral medulla (at obex level CVL) blocked the baroreflex but left the sympathetic chemoreflex intact. Bilateral microinjection of the GABAA agonist muscimol (87.5 pmol in 50 nl) into CVL produced effects identical to those of Kyn. These results confirm that the caudal ventrolateral medulla contains an essential relay of the sympathetic baroreflex and demonstrate that the same area plays no role in the sympathetic chemoreflex. The data suggests that these two reflexes could have a largely independent course through the medulla oblongata and that integration between the baroreceptor and chemoreceptor information used for sympathetic vasomotor control may occur as late as the premotor neuronal stage in RVL.

Central respiratory modulation of facial motoneurons in rats.

Facial motoneurons (FMN) were recorded intracellularly in Sprague-Dawley rats anesthetized with halothane. The animals were vagotomized, paralyzed, and artificially ventilated. The average membrane potential of the cells was 62.6 +/- 1.9 mV and their input impedance ranged from 5 to 30 M omega (9.8 +/- 1.1 M omega, n = 38). The membrane potential of most FMNs varied throughout the central respiratory cycle and four distinct patterns were detected. Type I (post-inspiratory) cells (21/44) showed a two-phase Cl(-)-mediated hyperpolarization during the respiratory cycle, one during central inspiration and the second during late expiration. Type II cells (early inspiratory, n = 10) showed early inspiratory depolarization. Type III (n = 2, stage-2 expiratory) cells displayed late expiratory depolarization and one cell (type IV or throughout inspiratory) exhibited expiratory Cl(-)-mediated hyperpolarization. The remaining 10 cells showed no detectable respiratory modulation. The results reflect the heterogeneity of the central respiratory modulation of FMNs and suggest that these cells receive both excitatory and inhibitory inputs from elements of the central respiratory pattern generating network.

Rostral ventrolateral medullary neurons projecting to locus coeruleus have cardiorespiratory inputs.

This study was designed to characterize some of the properties of the rostral ventrolateral medullary (RVLM) cells with axonal projection to the locus coeruleus (LC) in urethane anesthetized, vagotomized, paralyzed and artificially respirated rats. The vast majority of RVLM units antidromically (AD) activated from LC (RVLM-LC units) were silent and unresponsive to peripheral chemoreceptor stimulation or nociceptive stimulation. Twenty seven spontaneously active RVLM-LC neurons, AD activated from LC with currents below 30 microA (17 +/- 2 microA) were analyzed. AD mapping (n = 18) indicated that the lowest threshold for AD activation occurred within the LC itself. Axonal branching within or close to LC was suggested by the presence of sudden jumps in AD latency. Maximal AD latencies ranged from 7 to 37 ms. Most spontaneously active RVLM-LC neurons displayed marked central respiratory modulation characterized by either a post-inspiratory or an inspiratory pattern. The majority of the tested neurons were affected (excited or inhibited) by brief peripheral chemoreceptor stimulation (N2 inhalation). Most cells were inhibited by raising arterial pressure but none exhibited any detectable pulse synchrony. Reticulospinal sympathetic premotor neurons of RVLM were not found to project to LC (sample of 9) and very few RVLM cells with on-off respiratory discharges appeared to project to LC (2 out of 110). This study suggests that much of the information conveyed by the RVLM to LC could be of a mixed cardiorespiratory nature.

Splanchnic nerve response to A5 area stimulation in rats.

Microinjection of the excitatory amino acid N-methyl-D-aspartate (NMDA, 0.5 nmol) into ventrolateral pons (the A5 area) of halothane-anesthetized, paralyzed rats increased splanchnic (sSND) and renal sympathetic nerve discharges (approximately 45%) and usually decreased lumbar SND. These effects were accompanied by 1) regionally specific changes in vascular resistances, 2) an increase in the gain of the sympathetic baroreflex, and 3) modest reductions in blood pressure and heart rate. sSND activation was greatest when NMDA injections were made in the vicinity of A5 noradrenergic (NE) cells. Injection of 6-hydroxydopamine (6-OH-DA) into A5 area (after 15 days) destroyed 83% of NE neurons and reduced NMDA activation of sSND by 76%. Stimulation of sSND by NMDA in A5 area was reduced 1) 1-2 h after bilateral intraspinal injection of 6-OHDA, but not vehicle or 5,7-dihydroxytryptamine, and 2) by administration of prazosin [alpha 1-NE receptor antagonist, 1 mg/kg iv or 10-20 micrograms intrathecal (it)], but not by idazoxan (alpha 2-NE receptor antagonist, 10-20 micrograms it) or propranolol (0.2 mg/kg iv). We conclude that A5 NE cells have a visceral vasomotor sympathoexcitatory function mediated in large part by their spinal projection.

A5 noradrenergic unit activity and sympathetic nerve discharge in rats.

Unit recording experiments were designed to determine whether A5 noradrenergic neurons contribute to the generation of the splanchnic sympathetic nerve discharge (SSND) of halothane-anesthetized rats. Neurons (presumed A5 cells) were selected on the following bases: location in the ventrolateral tegmentum rostrolateral to facial nucleus (FN), antidromic (AD) activation from thoracic spinal cord, and complete inhibition by clonidine (10-15 micrograms/kg iv). These cells (n = 59) had low rates of spontaneous firing (1.4 +/- 0.2 spikes/s) and slow conduction velocities (2.6 +/- 0.2 m/s). The AD activation of seven of eight neurons was abolished within 1 h after intraspinal microinjection of 6-hydroxydopamine (4 micrograms), but the drug failed to affect the AD responses of eight sympathoexcitatory cells located caudal to the FN (control cells). The terminal fields of 16 A5 area neurons were found in the intermediolateral cell column of the spinal cord. Most neurons (63%, 37/59) were inhibited by raising arterial pressure and by train stimulation of the aortic depressor nerve (ADN, 47%, 9/20). A few cells responded to ADN stimulation but not to arterial pressure elevation or vice versa. The discharge of the cells was correlated to the SSND and preceded a peak of SSND by 69 +/- 6 ms (12/29 in intact and 3/9 in debuffered rats). We conclude that 40% of A5 cells may have a visceral vasomotor sympathoexcitatory function.

Sympatholytic response to stimulation of superior laryngeal nerve in rats.

The central pathway mediating a sympatholytic response to stimulation of the superior laryngeal nerve (SLN) was studied in halothane-anesthetized, paralyzed rats. Single-pulse stimulation of SLN inhibited lumbar sympathetic nerve discharge (LSND) with onset latency of 113 +/- 1.7 ms. LSND inhibition was markedly attenuated by bilateral microinjection of kynurenic acid (Kyn, glutamate receptor antagonist, 4.5 nmol/side) into the caudal ventrolateral medulla (CVL) or by bilateral administration of bicuculline methiodide (Bic; gamma-aminobutyric acid-receptor antagonist, 225 pmol/side) into the rostral ventrolateral medulla (RVL). In 13 of 14 cases, the baroreceptor reflex was also severely reduced. Injections of Bic or Kyn elsewhere in the medullary reticular formation were ineffective. Single-pulse stimulation of SLN inhibited 19 of 26 RVL reticulospinal barosensitive cells (onset latency 46 +/- 1.4 ms). This inhibition was attenuated (from 92 +/- 6 to 14 +/- 12%) by iontophoretic application of Bic (n = 7), which also reduced the cells' inhibitory response to aortic coarctation. The remaining seven barosensitive neurons were unaffected by SLN stimulation. In conclusion, the sympathetic baroreflex and the sympathoinhibitory response to SLN stimulation appear to be mediated by similar medullary pathways.