Jijiang Wang

Synaptic and Neurotransmitter Activation of Cardiac Vagal Neurons in the Nucleus Ambiguus

Abstract: Cardiac vagal neurons play a critical role in the control of heart rate and cardiac function. These neurons, which are primarily located in the nucleus ambiguus (NA) and the dorsal motor nucleus of the vagus (DMNX), dominate the neural control of heart rate under normal conditions. Cardiac vagal activity is diminished and unresponsive in many disease states, while restoration of parasympathetic activity to the heart lessens ischemia and arrhythmias and decreases the risk of sudden death. Recent work has demonstrated that cardiac vagal neurons are intrinsically silent and therefore rely on synaptic input to control their firing. To date, three major synaptic inputs to cardiac vagal neurons have been identified. Stimulation of the nucleus tractus solitarius evokes a glutamatergic pathway that activates both NMDA and non‐NMDA glutamatergic postsynaptic currents in cardiac vagal neurons. Acetylcholine excites cardiac vagal neurons via three mechanisms, activating a direct ligand‐gated postsynaptic nicotinic receptor, enhancing postsynaptic non‐NMDA currents, and presynaptically by facilitating transmitter release. This enhancement by nicotine is dependent upon activation of pre‐ and postsynaptic P‐type voltage‐gated calcium channels. Additionally, there is a GABAergic innervation of cardiac vagal neurons. The transsynaptic pseudorabies virus that expresses GFP (PRV‐GFP) has been used to identify, for subsequent electrophysiologic study, neurons that project to cardiac vagal neurons. Bartha PRV‐GFP‐labeled neurons retain their normal electrophysiological properties, and the labeled baroreflex pathways that control heart rate are unaltered by the virus.

Respiratory Sinus Arrhythmia: Endogenous Activation of Nicotinic Receptors Mediates Respiratory Modulation of Brainstem Cardioinhibitory Parasympathetic Neurons

Abstract— The heart rate increases during inspiration and decreases during expiration. This respiratory sinus arrhythmia (RSA) occurs by modulation of premotor cardioinhibitory parasympathetic neuron (CPN) activity. However, RSA has not been fully characterized in rats, and despite the critical role of CPNs in the generation of RSA, little is known about the mechanisms that mediate this cardiorespiratory interaction. This study demonstrates that RSA in conscious rats is similar to that in other species. The mechanism of RSA was then examined in vitro. Rhythmic inspiratory-related activity was recorded from the hypoglossal rootlet of 700- to 800-&mgr;m medullary sections. CPNs were identified by retrograde fluorescent labeling, and neurotransmission to CPNs was examined using patch-clamp electrophysiological techniques. During inspiratory bursts, the frequency of both spontaneous &ggr;-aminobutyric acidergic (GABAergic) and spontaneous glycinergic synaptic events in CPNs was significantly increased. Focal application of the nicotinic antagonist dihydro-&bgr;-erythroidine in an &agr;4&bgr;2-selective concentration (3 &mgr;mol/L) abolished the respiratory-evoked increase in GABAergic frequency. In contrast, the increase in glycinergic frequency during inspiration was not altered by nicotinic antagonists. Prenatal nicotine exposure exaggerated the increase in GABAergic frequency during inspiration and enhanced GABAergic synaptic amplitude both between and during inspiratory events. Glycinergic synaptic frequency and amplitude were unchanged by prenatal nicotine exposure. This study establishes a neurochemical link between neurons essential for respiration and CPNs, reveals a functional role for endogenous acetylcholine release and the activation of nicotinic receptors in the generation of RSA, and demonstrates that this cardiorespiratory interaction is exaggerated in rats prenatally exposed to nicotine.

Synaptic activation of hypoglossal respiratory motorneurons during inspiration in rats

Recent work has suggested glutamatergic and cholinergic synapses, and electric coupling may be involved in the activation of hypoglossal motorneurons during inspiration, however their relative importance is unknown. In this study we examined the excitatory inputs to hypoglossal motorneurons in a brainstem slice preparation. Focal application of D-2-amino-5-phosphonovalerate significantly inhibited a long lasting inward current evoked during inspiration. 6-Cyano-7-nitroquinoxaline-2,3-dione completely blocked the post-synaptic currents that increased in frequency and amplitude during inspiration and also reduced the long lasting inward current. Nicotinic receptors and gap junctional communication, blocked by D-tubocurare and carbenoxolone, respectively, contributed significant but smaller inputs to hypoglossal motorneurons during inspiration. In summary, non-N-methyl-D-aspartate (NMDA) receptors constitute the largest excitatory drive to hypoglossal neurons during inspiration, while NMDA, nicotinic receptors and gap junctions are also actively involved.

μ-opioid receptors are located postsynaptically and endomorphin-1 inhibits voltage-gated calcium currents in premotor cardiac parasympathetic neurons in the rat nucleus ambiguus

Activation of opioid receptors in the CNS evokes a dramatic decrease in heart rate which is mediated by increases in inhibitory parasympathetic activity to the heart. Injection of opiates into the nucleus ambiguus, where premotor cardiac parasympathetic nucleus ambiguus neurons are located elicits an increase in parasympathetic cardiac activity and bradycardia. However, the mechanisms responsible for altering the activity of premotor cardiac parasympathetic nucleus ambiguus neurons is unknown. This study examined at the electron microscopic level whether premotor cardiac parasympathetic nucleus ambiguus neurons possess postsynaptic opioid receptors and whether mu-opioid receptor agonists alter voltage-gated calcium currents in these neurons. Premotor cardiac parasympathetic nucleus ambiguus neurons were identified in the rat using retrograde fluorescent tracers. One series of experiments utilized dual-labeling immunocytochemical methods combined with electron microscopic analysis to determine if premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. In a second series of experiments whole cell patch clamp methodologies were used to determine whether activation of postsynaptic opioid receptors altered voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons in brainstem slices. The perikarya and 78% of the dendrites of premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. Voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons were comprised nearly entirely of omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents. Activation of mu-opioid receptors inhibited these voltage-gated calcium currents and this inhibition was blocked by pretreatment with pertusis toxin. The mu-opioid receptor agonist endomorphin-1, but not the mu-opioid receptor agonist endomorphin-2, inhibited the calcium currents. In summary, mu-opioid receptors are located postsynaptically on premotor cardiac parasympathetic nucleus ambiguus neurons. The mu-opioid receptor agonist endomorphin1 inhibited the omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents in premotor cardiac vagal nucleus ambiguus neurons. This inhibition is mediated via a G-protein mediated pathway which was blocked by pretreatment with pertusis toxin. It is possible that the inhibition of calcium currents may act to indirectly facilitate the activity of premotor cardiac parasympathetic nucleus ambiguus neurons by disinhibition, such as by a reduction in inhibitory calcium activated potassium currents.

Pentobarbital enhances GABAergic neurotransmission to cardiac parasympathetic neurons, which is prevented by expression of GABA(A) epsilon subunit.

BACKGROUND Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation. METHODS The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission. RESULTS Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents. CONCLUSIONS Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

Ketamine inhibits presynaptic and postsynaptic nicotinic excitation of identified cardiac parasympathetic neurons in nucleus ambiguus.

Background Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated possible central nervous system actions of ketamine to inhibit cardiac parasympathetic neurons in the brainstem by inhibiting multiple nicotinic excitatory mechanisms. Methods The authors used a novel in vitro approach to study the effect of ketamine on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording. The effects of ketamine were tested on nicotine-evoked ligand-gated currents and spontaneous glutamatergic miniature synaptic currents (mini) in cardiac parasympathetic preganglionic neurons. Results Ketamine (10 &mgr;m) inhibited (1) the nicotine (1 &mgr;m)-evoked presynaptic facilitation of glutamate release (mini frequency, 18 ± 7% of control; n = 9), and (2) the direct postsynaptic ligand-gated current (27 ± 8% of control; n = 9), but ketamine did not alter the amplitude of postsynaptic miniature non–N-methyl-d-aspartate currents. &agr; Bungarotoxin, an antagonist of &agr;7 containing nicotinic presynaptic receptors, blocked ketamine actions on mini frequency (n = 10) but not mini amplitude. Conclusions Ketamine inhibits the presynaptic nicotinic receptors responsible for facilitating neurotransmitter release, as well as the direct ligand-gated inward current, but does not alter the nicotinic augmentation of non–N-methyl-d-aspartate currents in brainstem parasympathetic cardiac neurons. Such actions may mediate the decrease in parasympathetic cardiac activity and increase in heart rate that occurs with ketamine.

Arginine vasopressin enhances GABAergic inhibition of cardiac parasympathetic neurons in the nucleus ambiguus.

Previous studies have shown that arginine vasopressin is an important neuropeptide that can modulate the reflex control of blood pressure and heart rate. The nucleus ambiguus, where cardiac parasympathetic neurons are located, receives dense arginine vasopressin projections. However the mechanisms by which arginine vasopressin alters cardiac parasympathetic activity are unknown. We tested the hypothesis that arginine vasopressin can alter the activity of cardiac parasympathetic neurons by altering the spontaneous GABAergic input to these neurons. Experiments were conducted using whole cell patch clamp recordings of cardiac parasympathetic neurons in an in vitro slice preparation in rats. The results of this study demonstrate that arginine vasopressin increases the frequency and amplitude of GABAergic inhibitory post-synaptic currents in cardiac parasympathetic neurons. Arginine vasopressin did not alter the GABAergic currents evoked by exogenous application of GABA. Similarly, in the presence of tetrodotoxin, arginine vasopressin did not alter the frequency, amplitude or decay time of GABAergic miniature synaptic events evoked by high osmolarity. These results indicate that arginine vasopressin likely acts on neurons precedent to cardiac parasympathetic neurons and that arginine vasopressin likely acts not at the synaptic terminal but at the soma or dendrites of the precedent neuron. Oxytocin and agonists for the V(2)-arginine vasopressin and V(1b)-arginine vasopressin receptors had no effect. By contrast, the arginine vasopressin-evoked responses were completely abolished by a selective V(1a)-arginine vasopressin receptor antagonist indicating arginine vasopressin responses are mediated by V(1a)-arginine vasopressin receptors. We conclude that the V(1a)-arginine vasopressin receptor-mediated increase in frequency and amplitude of inhibitory GABAergic activity to cardiac parasympathetic neurons may be at least one mechanism by which central arginine vasopressin may increase heart rate and inhibit reflex bradycardia.

Ketamine inhibits sodium currents in identified cardiac parasympathetic neurons in nucleus ambiguus.

Background Ketamine increases both blood pressure and heart rate, effects commonly thought of as sympathoexcitatory. The authors investigated the possibility that ketamine increases heart rate by inhibiting the central cardiac parasympathetic mechanisms. Methods We used a novel in vitro approach to study the effect of ketamine on the identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with the fluorescent tracer by placing rhodamine into the pericardial sac. Dye-labeled neurons were visually identified for patch clamp recording, and ketamine effects on isolated potassium (K+) and sodium (Na+) currents were studied. Results Cardiac nucleus ambiguus neurons (n = 14) were inherently silent, but depolarization evoked sustained action potential trains with little delay or adaptation. Ketamine (10 &mgr;m) reduced this response but had no effect on the voltage threshold for action potentials (n = 14;P > 0.05). The current–voltage relations for the transient K+ current and the delayed rectified K+ current (n = 5) were unaltered by ketamine (10 &mgr;m–1 mm). Ketamine depressed the total Na+ current dose-dependently (10 &mgr;m–1 mm). In addition, ketamine shifted the Na+ current inactivation curves to more negative potentials, thus suggesting the enhancement of the Na+ channel inactivation (P < 0.05; n = 7). In the presence of Cd2+, ketamine (10 &mgr;m) continued to inhibit voltage-gated Na+ currents, which recovered completely within 10 min. Conclusions Ketamine inhibits Na+ but not K+ channel function in brainstem parasympathetic cardiac neurons, and such actions may mediate the decrease in parasympathetic cardiac activity and increase in heart rate that occurs with ketamine.

Endomorphin-2 inhibits GABAergic inputs to cardiac parasympathetic neurons in the nucleus ambiguus

The nucleus ambiguus is an area containing cardiac vagal neurons, from which originates most of the parasympathetic control regulating heart rate and cardiac function. GABAergic pathways to these neurons have recently been described, yet modulation of this GABAergic input and its impact upon cardiac vagal neurons is unknown. The nucleus ambiguus has been shown to contain mu-opioid receptors and endomorphin-1 and endomorphin-2, the endogenous peptide ligands for the mu-receptor, whilst microinjections of opioids in the ambiguus area evoke bradycardia. The present study therefore examined the effects of endomorphin-1, endomorphin-2 and DAMGO (a synthetic, mu-selective agonist) on spontaneous GABAergic IPSCs in cardiac parasympathetic neurons. Only endomorphin-2 (100 microM) produced a significant inhibition, of both the frequency (-22.8%) and the amplitude (-30.5%) of the spontaneous IPSCs in cardiac vagal neurons. The inhibitory effects of endomorphin-2 were blocked by naloxonazine (10 microM), a selective mu(1) receptor antagonist. Naloxonazine alone (10 microM) had a potentiating effect on the frequency of the GABAergic IPSCs (+161.43%) but not on the amplitude, indicating that GABA release to cardiac vagal neurons may be under tonic control of opioids acting at the mu(1) receptor. Endomorphin-2 did not reduce the responses evoked by exogenous application of GABA. These results indicate that endomorphin-2 acts on mu(1) receptors located on precedent neurons to decrease GABAergic input to cardiac vagal neurons located in the nucleus ambiguus. The subsequent increase in parasympathetic outflow to the heart may be one mechanism by which mu-selective opioids act to induce bradycardia.

Firing properties of identified superior laryngeal neurons in the nucleus ambiguus in the rat.

Superior laryngeal motoneurons control muscles in the larynx and recent work has shown they also have axon collaterals that project to cardiac vagal neurons in the nucleus ambiguus. The present study was undertaken to identify and examine the firing properties of superior laryngeal neurons (SLNs) in the rat. SLNs typically fired spontaneously and repetitively at a rate of 4-7 Hz. The firing was continuous and showed little bursting activity. Firing evoked afterhyperpolarizations were insensitive to apamin but blocked by charybdotoxin. The voltage-gated currents in SLNs consist of a TTX-sensitive Na current and a 4-aminopyridine sensitive K current. It is likely that the activity of these neurons not only control respiratory laryngeal muscles, but may also provide an interaction between the respiratory system and the control of the heart rate.