Adrenergic agonist induces rhythmic firing in quiescent cardiac preganglionic neurons in nucleus ambiguous via activation of intrinsic membrane excitability.

Cholinergic vagal nerves projecting from neurons in the brain stem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however, the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brain stem slices. Preganglionic NAm neurons were identified by ChAT-tdTomato fluorescence in young adult transgenic mice, and their cardiac projection was confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However, bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α1-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, because postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current and hyperpolarization-activated current are involved in this excitation pattern, although not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons and that persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone. NEW & NOTEWORTHY Cardiac preganglionic cholinergic neurons in the nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brain stem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

Long-term stimulation of cardiac vagal preganglionic neurons reduces blood pressure in the spontaneously hypertensive rat.

BACKGROUND: Arterial hypertension is associated with autonomic nervous system dysfunction. Different interventional strategies have been implemented in recent years for the reduction of sympathetic activity in patients with hypertension. However, the therapeutic benefit of increasing vagal tone in hypertensive patients remains largely unexplored. OBJECTIVE: Here, we describe the effects of long-term activation of vagal neural pathways on arterial pressure, heart rate arterial pressure variability and spontaneous baroreflex sensitivity in spontaneously hypertensive rats (SHR) and normotensive Wistar rats. METHODS: Brainstem vagal preganglionic neurons residing in the dorsal vagal motor nucleus (DVMN) were targeted with a lentiviral vector to induce the expression of an artificial G(s) protein-coupled receptor termed designer receptors exclusively activated by designer drugs (DREADD-Gs). The transduced neurons were activated daily by systemic administration of otherwise inert ligand clozapine-n-oxide. Arterial pressure measurements were recorded in conscious freely moving animals after 21 consecutive days of DVMN stimulation. RESULTS: Resting arterial pressure was significantly lower in SHRs expressing DREADD-Gs in the DVMN, compared with control SHRs expressing enhanced green fluorescent protein. No changes in arterial pressure were detected in Wistar rats expressing DREADD-Gs compared with rats expressing enhanced green fluorescent protein in the DVMN. Pharmacogenetic activation of DREADD-Gs-expressing DVMN neurons in SHRs was accompanied with increased baroreflex sensitivity and a paradoxical decrease in cardio-vagal components of heart rate and systolic arterial pressure variability in SHRs. CONCLUSION: These results suggest that long-term activation of vagal parasympathetic pathways is beneficial in restoring autonomic balance in an animal model of neurogenic hypertension and might be an effective therapeutic approach for the management of hypertension.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate.

Neuropharmacological lesion localization in idiopathic Horner's syndrome in Golden Retrievers and dogs of other breeds.

OBJECTIVE: To investigate whether idiopathic Horner's syndrome (HS) in Golden Retrievers is an exclusively preganglionic disorder based on denervation hypersensitivity pharmacological testing with phenylephrine. ANIMALS STUDIED: Medical records of dogs presented with HS between 2000 and 2012. Dogs presented with additional ocular or systemic signs were excluded. PROCEDURES: Clinical data examined included age, sex, duration of clinical signs, ancillary diagnostic test results, and time to mydriasis on topical ocular application of 1% phenylephrine. Lesions were diagnosed as postganglionic (mydriasis within 20 min) or preganglionic (mydriasis between 20 and 45 min). RESULTS: Medical records of 21 dogs of nine different breeds were included. An etiopathogenesis for Horner's syndrome was determined in five dogs, none of which were Golden Retrievers. All diagnoses correlated with pharmacological lesion localization. Ten Golden Retrievers were included (eight male and two female) with a mean age of 8.5 years (range: 4-13). Lesion localization was diagnosed as postganglionic in eight (mean: 10 min [range: 6-18]) and preganglionic in two Golden Retrievers (20 and 24 min). All cases were unilateral and had completely resolved within 15 weeks (range: 11-20). Recurrence was not reported in any of the patients. CONCLUSIONS: Idiopathic postganglionic HS was diagnosed in eight of 10 Golden Retrievers contradicting previous reports of a purely preganglionic localization. Etiopathogenesis of canine idiopathic HS remains to be determined; nevertheless, a vascular etiology cannot be excluded. Future studies using magnetic resonance angiography may aid in clarifying the pathogenesis.

Urotensin II promotes vagal-mediated bradycardia by activating cardiac-projecting parasympathetic neurons of nucleus ambiguus.

Urotensin II (U-II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U-II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U-II and its receptor at this level. We report here that U-II produces an increase in cytosolic Ca(2+) concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptor; and (ii) Ca(2+) influx through P/Q-type Ca(2+) channels. In addition, U-II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U-II into nucleus ambiguus elicits dose-dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U-II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

Cardioprotection evoked by remote ischaemic preconditioning is critically dependent on the activity of vagal pre-ganglionic neurones.

AIMS: Innate mechanisms of inter-organ protection underlie the phenomenon of remote ischaemic preconditioning (RPc) in which episode(s) of ischaemia and reperfusion in tissues remote from the heart reduce myocardial ischaemia/reperfusion injury. The uncertainty surrounding the mechanism(s) underlying RPc centres on whether humoral factor(s) produced during ischaemia/reperfusion of remote tissue and released into the systemic circulation mediate RPc, or whether a neural signal is required. While these two hypotheses may not be incompatible, one approach to clarify the potential role of a neural pathway requires targeted disruption or activation of discrete central nervous substrate(s). METHODS AND RESULTS: Using a rat model of myocardial ischaemia/reperfusion injury in combination with viral gene transfer, pharmaco-, and optogenetics, we tested the hypothesis that RPc cardioprotection depends on the activity of vagal pre-ganglionic neurones and consequently an intact parasympathetic drive. For cell-specific silencing or activation, neurones of the brainstem dorsal motor nucleus of the vagus nerve (DVMN) were targeted using viral vectors to express a Drosophila allatostatin receptor (AlstR) or light-sensitive fast channelrhodopsin variant (ChIEF), respectively. RPc cardioprotection, elicited by ischaemia/reperfusion of the limbs, was abolished when DVMN neurones transduced to express AlstR were silenced by selective ligand allatostatin or in conditions of systemic muscarinic receptor blockade with atropine. In the absence of remote ischaemia/reperfusion, optogenetic activation of DVMN neurones transduced to express ChIEF reduced infarct size, mimicking the effect of RPc. CONCLUSION: These data indicate a crucial dependence of RPc cardioprotection against ischaemia/reperfusion injury upon the activity of a distinct population of vagal pre-ganglionic neurones.

Agonist of 5-HT1A/7 receptors but not that of 5-HT2 receptors disinhibits tracheobronchial-projecting airway vagal preganglionic neurons of rats.

The vagus nerves supply the major cholinergic tone to airway smooth muscles physiologically and play critical roles in the genesis of airway hyperreactivity under some pathological conditions. Postganglionic airway cholinergic tone relies largely on the ongoing activity of medullary airway vagal preganglionic neurons (AVPNs), of which the tracheobronchial-projecting ones are primarily located in the external formation of the nucleus ambiguus (eNA). AVPNs are regulated by 5-HT, and 5-HT(1A/7) and 5-HT(2) receptors have been indicated to be involved. But the mechanisms at synaptic level are unknown. In the present study, tracheobronchial-projecting AVPNs (T-AVPNs) were retrogradely labeled from the trachea wall; fluorescently labeled T-AVPNs in the eNA were recorded with whole-cell voltage patch clamp; and the effects of 5-HT(1A/7) receptor agonist (±)-8-Hydroxy-2-(dipropylamino) tetralin hydrobromide (8-OH-DPAT) (1 µmol L(-1)) and 5-HT(2) receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (10 µmol L(-1)) on the synaptic inputs were examined. 8-OH-DPAT significantly inhibited the GABAergic and glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) of T-AVPNs in both the frequency and amplitude but had no effect on the GABAergic and glycinergic miniature inhibitory postsynaptic currents (mIPSCs). The 8-OH-DPAT inhibition of the GABAergic and glycinergic sIPSCs was prevented by 5-HT(1A/7) receptor antagonist N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl] ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY-100635) (1 µmol L(-1)). 8-OH-DPAT had no effect on the glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) and caused no alterations in the baseline current and input resistance of T-AVPNs. DOI had no effect on any types of the synaptic inputs of T-AVPNs. These results suggest that 5-HT(1A/7) receptor agonist causes "disinhibition" of T-AVPNs, which might, in part, account for the reflex increase of airway resistance.

Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties.

This study investigates the firing properties of the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons located in the external formation of the nucleus ambiguus. The results showed that inspiratory-activated and inspiratory-inhibited neurons are distributed with different density and site preference in this area. Inspiratory-inhibited neurons exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential under current clamp. The afterhyperpolarization in inspiratory-activated neurons was blocked by apamin, a blocker of the small-conductance Ca(2+)-activated K(+) channels; and that in inspiratory-inhibited neurons by charybdotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) channels. Under voltage clamp, depolarizing voltage steps evoked tetrodotoxin-sensitive rapid inward sodium currents, 4-aminopyridine-sensitive outward potassium transients and lasting outward potassium currents. 4-Aminopyridine partially blocked the lasting outward potassium currents of inspiratory-activated neurons but was ineffective on those of inspiratory-inhibited neurons. These findings suggest that inspiratory-activated and inspiratory-inhibited neurons are differentially organized and express different types of voltage-gated ion channels.

Thyrotropin-releasing hormone causes a tonic excitatory postsynaptic current and inhibits the phasic inspiratory inhibitory inputs in inspiratory-inhibited airway vagal preganglionic neurons.

The airway vagal preganglionic neurons (AVPNs) in the external formation of the nucleus ambiguus (eNA), which include the inspiratory-activated AVPNs (IA-AVPNs) and inspiratory-inhibited AVPNs (II-AVPNs), predominate in the control of the trachea and bronchia. The AVPNs receive particularly dense inputs from terminals containing thyrotropin-releasing hormone (TRH). TRH microinjection into the nucleus ambiguus (NA) caused constriction of the tracheal smooth muscles. However, it is unknown whether TRH affects all subtypes of the AVPNs in the eNA, and as a result affects the control of all types of target tissues in the airway (smooth muscles, submucosal glands, and blood vessels). It is also unknown how TRH affects the AVPNs at neuronal and synaptic levels. In this study, the AVPNs in the eNA were retrogradely labeled from the extrathoracic trachea, the II-AVPNs were identified in rhythmically firing brainstem slices, and the effects of TRH were examined using patch-clamp. TRH (100 nmol L(-1)) enhanced both the rhythm and the intensity of the hypoglossal bursts, and caused a tonic excitatory inward current in the II-AVPNs at a holding voltage of -80 mV. The frequency of the spontaneous excitatory postsynaptic currents (EPSCs) in the II-AVPNs, which showed no respiratory-related change in a respiratory cycle, was not significantly changed by TRH. At a holding voltage of -50 mV, the II-AVPNs showed both spontaneous and phasic inspiratory (outward) inhibitory postsynaptic currents (IPSCs). TRH had no effect on the spontaneous IPSCs but significantly attenuated the phasic inspiratory outward currents, in both the amplitude and area. After focal application of strychnine, an antagonist of glycine receptors, to the II-AVPNs, the spontaneous IPSCs were extremely scarce and the phasic inspiratory inhibitory currents were abolished; and further application of TRH had no effect on these currents. Under current clamp configuration, TRH caused a depolarization and increased the firing rate of the II-AVPNs during inspiratory intervals. These results demonstrate that TRH affects the II-AVPNs both postsynaptically via a direct excitatory current and presynaptically via attenuation of the phasic glycinergic synaptic inputs.

Differential activation of adrenal, renal, and lumbar sympathetic nerves following stimulation of the rostral ventrolateral medulla of the rat.

Under acute and chronic conditions, the sympathetic nervous system can be activated in a differential and even selective manner. Activation of the rostral ventrolateral medulla (RVLM) has been implicated in differential control of sympathetic outputs based on evidence primarily in the cat. Although several studies indicate that differential control of sympathetic outflow occurs in other species, only a few studies have addressed whether the RVLM is capable of producing varying patterns of sympathetic activation in the rat. Therefore, the purpose of the present study was to determine whether activation of the RVLM results in simultaneous and differential increases in preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activities. In urethane-chloralose anesthetized rats, pre-ASNA, RSNA, and LSNA were recorded simultaneously in all animals. Microinjections of selected concentrations and volumes of glutamate increased pre-ASNA, RSNA, and LSNA concurrently and differentially. Pre-ASNA and RSNA (in most cases) exhibited greater increases compared with LSNA on a percentage basis. By varying the volume or location of the glutamate microinjections, we also identified individual examples of differential and selective activation of these nerves. Decreases in arterial pressure or bilateral blockade of RVLM GABA(A) receptors also revealed differential activation, with the latter having a 3- to 4-fold greater effect on sympathetic activity. Our data provide evidence that activation of the rat RVLM increases renal, lumbar, and preganglionic adrenal sympathetic nerve activities concurrently, differentially, and, in some cases, selectively.

Electrophysiological, pharmacological and molecular profile of the transient outward rectifying conductance in rat sympathetic preganglionic neurons in vitro.

Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K+ channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1-10 µM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Kα (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Kα and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 µM). Single-cell RT-PCR revealed mRNA expression for the α-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory ß-subunits was detected for Kvß2 in all SPN with differential expression of mRNA for KChIP1, Kvß1 and Kvß3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the ß-subunit Kvß2. Differential expression of the accessory ß subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons.

Erythromycin inhibited glycinergic inputs to gastric vagal motoneurons in brainstem slices of newborn rats.

BACKGROUND: Motilin has been known to stimulate the motility of digestive organs peripherally via activation of motilin receptors located at gastrointestinal (GI) cholinergic nerve endings and/or smooth muscle cells. Recent studies have indicated that motilin may also promote GI motility via actions in the central nervous system; however the sites of action and the mechanisms are not clear yet. The present study aimed to test the hypothesis that motilin receptor agonist erythromycin alters the synaptic inputs of preganglionic gastric vagal motoneurons (GVMs) located in the dorsal motor nucleus of the vagus (DMV). METHODS: Gastric vagal motoneurons were retrogradely labeled by fluorescent tracer from the stomach wall of newborn rats. Fluorescently labeled GVMs in DMV were recorded using whole-cell patch-clamp in brainstem slices and the effects of motilin receptor agonist erythromycin on the synaptic inputs were examined. KEY RESULTS: Erythromycin (100 nmol L(-1), 1 µmol L(-1), 10 µmol L(-1)) significantly inhibited the frequency of glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) of GVMs and significantly inhibited the amplitude at the concentration of 10 µmol L(-1). These responses were prevented by GM-109, a selective motilin receptor antagonist. In the pre-existence of tetradotoxin (TTX, 1 µmol L(-1)), erythromycin (10 µmol L(-1)) caused significant decreases of the glycinergic miniature inhibitory postsynaptic currents (mIPSCs), in both the frequency and the amplitude. However, erythromycin (10 µmol L(-1)) didn't cause significant changes of the GABAergic sIPSCs. CONCLUSIONS & INFERENCES: Erythromycin selectively inhibits the glycinergic inputs of GVMs.

Spontaneous rhythmogenic capabilities of sympathetic neuronal assemblies in the rat spinal cord slice.

Neuronal networks generating rhythmic activity as an emergent property are common throughout the nervous system. Some are responsible for rhythmic behaviours, as is the case for the spinal cord locomotor networks; however, for others the function is more subtle and usually involves information processing and/or transfer. An example of the latter is sympathetic nerve activity, which is synchronized into rhythmic bursts in vivo. This arrangement is postulated to offer improved control of target organ responses compared to tonic nerve activity. Traditionally, oscillogenic circuits in the brainstem are credited with generating these rhythms, despite evidence for the persistence of some frequencies in spinalized preparations. Here, we show that rhythmic population activity can be recorded from the intermediolateral cell column (IML) of thoracic spinal cord slices. Recorded in slices from 10- to 12-day-old rats, this activity was manifest as 8-22 Hz oscillations in the field potential and was spatially restricted to the IML. Oscillations often occurred spontaneously, but could also be induced by application of 5-HT, α-methyl 5-HT or MK212. These agents also significantly increased the strength of spontaneous oscillations. Rhythmic activity was abolished by TTX and attenuated by application of gap junction blockers or by antagonists of GABA(A) receptors. Together these data indicate that this rhythm is an emergent feature of a population of spinal neurons coupled by gap junctions. This work questions the assumption that sympathetic rhythms are dependent on supraspinal pacemaker circuits, by highlighting a surprisingly strong rhythmogenic capability of the reduced sympathetic networks of the spinal cord slice.

Dorsal vagal preganglionic neurons: differential responses to CCK1 and 5-HT3 receptor stimulation.

The dorsal motor nucleus of the vagus (DMV) is the main source of the vagal innervation of the pancreas. Several studies in vitro have demonstrated that the DMV consists of a heterogeneous population of preganglionic neurons but little is known about their electrophysiological characteristics in vivo. The aims of this study were to (i) identify DMV preganglionic neurons in vivo with axons in the pancreatic vagus and (ii) characterize their responses to stimulation of cholecystokinin (CCK(1)) and serotonin (5-HT(3)) receptors which are major regulators of pancreatic secretion. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O(2)) were used throughout. Dorsal vagal preganglionic neurons were identified by antidromic activation in response to stimulation of the pancreatic vagus. Dorsal vagal preganglionic neurons had axonal conduction velocities in the C-fibre range (0.7+/-0.03 m/s). Forty-four neurons were identified within the rostral, intermediate and caudal DMV and thirty-eight were tested for responsiveness to CCK-8S (CCK(1) agonist) and phenylbiguanide (PBG; 5-HT(3) receptor agonist). CCK-8S and PBG (0.1-10 microg/kg, i.v.) produced three types of response: (i) preganglionic neurons in the intermediate DMV were inhibited by CCK-8S (n=18) and PBG (n=10), (ii) neurons in the caudal DMV were activated by CCK (n=5) and PBG (n=2) and (iii) CCK-8S (n=9) and PBG (n=7) had no effect on preganglionic neurons in the rostral DMV. CCK-8S and PBG have complex actions on preganglionic neurons in the DMV that may be related to their effects on pancreatic secretion.

Targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced sustained ventricular tachycardia in conscious rats.

The Cardiac Arrhythmia Suppression Trial demonstrated that antiarrhythmic drugs not only fail to prevent sudden cardiac death, but actually increase overall mortality. These findings have been confirmed in additional trials. The "proarrhythmic" effects of most currently available antiarrhythmic drugs makes it essential that we investigate novel strategies for the prevention of sudden cardiac death. Targeted ablation of cardiac sympathetic neurons may become a therapeutic option by reducing sympathetic activity. Thus cholera toxin B subunit (CTB) conjugated to saporin (a ribosomal inactivating protein that binds to and inactivates ribosomes; CTB-SAP) was injected into both stellate ganglia to test the hypothesis that targeted ablation of cardiac sympathetic neurons reduces the susceptibility to ischemia-induced, sustained ventricular tachycardia in conscious rats. Rats were randomly divided into three groups: 1) control (no injection); 2) bilateral stellate ganglia injection of CTB; and 3) bilateral stellate ganglia injection of CTB-SAP. CTB-SAP rats had a reduced susceptibility to ischemia-induced, sustained ventricular tachycardia. Associated with the reduced susceptibility to ventricular arrhythmias were a reduced number of stained neurons in the stellate ganglia and spinal cord (segments T(1)-T(4)), as well as a reduced left ventricular norepinephrine content and sympathetic innervation density. Thus CTB-SAP retrogradely transported from the stellate ganglia is effective at ablating cardiac sympathetic neurons and reducing the susceptibility to ventricular arrhythmias.

Electrophysiological responses of sympathetic preganglionic neurons to ANG II and aldosterone.

The intermediolateral cell column (IML) of the spinal cord is an important area where sympathetic impulses propagate to peripheral sympathetic organs. ANG II and aldosterone are important components of the renin-angiotensin-aldosterone system (RAAS), which activate the sympathetic nervous system. Each is partly synthesized in the brain and plays a paracrine role in the regulation of blood pressure independently of RAAS in the periphery. Our purpose in the present study was to clarify the contributions of sympathetic preganglionic neurons in the IML (IML neurons) and the effects of ANG II and aldosterone on the sympathetic nervous system. To examine responses to ANG II and aldosterone, we intracellularly recorded 104 IML neurons using a whole cell patch-clamp technique in spinal cord slice preparations. IML neurons were classified into two types: silent and firing. Both neuron types were significantly depolarized by ANG II, and candesartan inhibited this depolarization. After pretreatment with TTX, firing neurons (but not silent neurons) were significantly depolarized by ANG II. Aldosterone significantly increased the number of excitatory postsynaptic potentials (EPSPs) in both neuron types, but this response disappeared after pretreatment with TTX. ANG II and aldosterone had no synergistic effects on the IML neurons. The silent neurons had large cell soma, and many more dendrites than the firing neurons. These results suggest that ANG II acts presynaptically and postsynaptically in IML neurons, while aldosterone acts mainly presynaptically. Thus, the physiological effects of these substances are likely to be transmitted via specific membrane receptors of IML and/or presynaptic neurons.

Effects of thoracic epidural anaesthesia on cardiac repolarization.

1. Prolongation of the QT interval is associated with a risk of life-threatening cardiac arrhythmias. In the present study, we examined whether reversible blockade of preganglionic sympathetic fibres of the heart, achieved by thoracic epidural anaesthesia, affects cardiac repolarization and thus confers an anti-arrhythmic effect. 2. Fifty-two men, aged between 28 and 65 years, were included in the study: 28 were patients scheduled for thoracic epidural anaesthesia (Group T) and 24 were patients scheduled for lumbar epidural anaesthesia (Group L). Epidural blockade was achieved with 0.5% isobaric bupivacaine solution. Measurements were taken from electrocardiogram fragments obtained before epidural anaesthesia and after detection of blockade (T1 or T8 segment sensory block in Groups T and L, respectively). Correction of the QT interval was calculated using Bazett's formula (QTcb), Fridericia's correction (QTcf) and the Framingham formula (QTcF). Transmural dispersion of repolarization (TDR) was determined using the Tpeak--Tend time, where Tpeak is the peak of the T wave and Tend is the end of the T wave. 3. Significant shortening of the QTcb interval and TDR was detected after blockade in Group T. However, there were no changes in the ECG parameters in Group L. 4. In conclusion, reversible blockade of preganglionic sympathetic fibres to the heart, achieved by thoracic epidural anaesthesia, results in a reduction in QTcb, as well as the TDR. These changes may explain the anti-arrhythmic action seen with central blockade.

omega-Conotoxin-GVIA-sensitive calcium channels on preganglionic nerve terminals in mouse pelvic and celiac ganglia.

Release of acetylcholine (ACh) from preganglionic nerve terminals requires calcium entry through voltage-gated calcium channels. The calcium channel subtype required for ACh release varies depending on the particular ganglionic synapse. I have investigated the functional role of calcium channels in transmitter release from parasympathetic and sympathetic preganglionic terminals in pelvic and celiac ganglia of female mice. Single electrode voltage clamp was used to measure EPSC amplitude in the absence and presence of selective calcium channel antagonists. In pelvic ganglia omega- conotoxin GVIA, a selective N-type calcium channel antagonist, reduced the amplitude of EPSCs evoked by pelvic nerve stimulation by 46+/-5% (n=8, P=0.015). In contrast, in the celiac ganglion, omega- conotoxin GVIA had no effect on the amplitude of EPSCs evoked by splanchnic nerve stimulation (P=0.09, n=7). EPSCs in both pelvic and celiac ganglia were resistant to the P-type calcium channel antagonist agatoxin (50 nM, n=5 for both ganglia) and the R-type calcium channel antagonist SNX482 (100 nM, n=4 for both ganglia). These results indicate that in female mice, release of ACh in sympathetic pathways to prevertebral ganglia does not require calcium entry from N-type calcium channels. However, release of ACh from sacral parasympathetic preganglionic neurons requires calcium entry from both N-type and toxin-resistant calcium channels.

Long-term depression in the superior cervical ganglion of the rat.

Long-term depression (LTD) is a use-dependent decrease in synaptic efficacy widely recognized as a form of synaptic plasticity related to cognitive function in the central nervous system. Such response has previously not been demonstrated in autonomic ganglia. In the isolated superior cervical ganglion (SCG) of the rat (superfused with Locke solution containing 100 microM choline), low-frequency stimulation (LFS, 3-5 Hz/15 min) of the preganglionic nerve produced a long-lasting (up to 3 h ), significant (20-40%) decrease in the amplitude of the extracellularly recorded postganglionic compound action potential. Pretreatment of ganglia with the 5-HT(3) receptor antagonist tropisetron (0.5 microM) completely prevented the induction of ganglionic LTD (gLTD). Treatment of ganglia with the 5-HT(3) receptor antagonist MDL 72222 (0.5 microM) during the maintenance phase of established gLTD (1 h after LFS) antagonized the LFS-induced depression. Inhibition of nitric oxide (NO) synthase with l-NOARG (20-50 microM), applied before or after LFS, failed to affect the expression of gLTD. Additionally, pretreatment with the protein synthesis inhibitor emetine (1 microM) totally prevented the expression of gLTD. However, inhibition of protein phosphatase with cantharidin (30 microM) did not interfere with the expression of gLTD. These results indicate the presence of LTD in the rat SCG and suggest that expression of gLTD involves activation of 5-HT(3) receptor.