Prenatal nicotine exposure enhances the trigeminocardiac reflex via serotonin receptor facilitation in brainstem pathways.

In this study we used a rat model for prenatal nicotine exposure to test whether clinically relevant concentrations of brain nicotine and cotinine are passed from dams exposed to nicotine to her pups, whether this changes the trigeminocardiac reflex (TCR), and whether serotonergic function in the TCR brainstem circuitry is altered. Pregnant Sprague-Dawley dams were exposed to 6 mg·kg(-1)·day(-1) of nicotine via osmotic minipumps for the duration of pregnancy. Following birth dams and pups were killed, blood was collected, and brain nicotine and cotinine levels were measured. A separate group of prenatal nicotine-exposed pups was used for electrophysiological recordings. A horizontal brainstem slice was obtained by carefully preserving the trigeminal nerve with fluorescent identification of cardiac vagal neurons (CVNs) in the nucleus ambiguus. Stimulation of the trigeminal nerve evoked excitatory postsynaptic current in CVNs. Our data demonstrate that prenatal nicotine exposure significantly exaggerates both the TCR-evoked changes in heart rate in conscious unrestrained pups, and the excitatory neurotransmission to CVNs upon trigeminal afferent nerve stimulation within this brainstem reflex circuit. Application of the 5-HT1A receptor antagonist WAY 100635 (100 µM) and 5-HT2A/C receptor antagonist ketanserin (10 µM)significantly decreased neurotransmission, indicating an increased facilitation of 5-HT function in prenatal nicotine-exposed animals. Prenatal nicotine exposure enhances activation of 5-HT receptors and exaggerates the trigeminocardiac reflex.

Activation of D2-like dopamine receptors inhibits GABA and glycinergic neurotransmission to pre-motor cardiac vagal neurons in the nucleus ambiguus.

The parasympathetic control of heart rate arises from premotor cardiac vagal neurons (CVNs) located in the nucleus ambiguus (NA). Previous microinjection studies in NA show that dopamine evokes a decrease in heart rate, but the underlying mechanisms responsible for these responses were not identified. This study tested whether dopamine modulates inhibitory GABAergic and glycinergic and/or excitatory glutamatergic neurotransmission to CVNs. Retrogradely labeled CVNs were identified in an in vitro rat brainstem slice preparation and synaptic events were recorded using whole cell voltage clamp techniques. Bath application of dopamine (100 µM) had no effect on excitatory synaptic events, but reversibly inhibited the frequency (but not amplitude) of GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs. Similarly, dopamine (10 µM and 100 µM) inhibited glycinergic IPSC frequency by ~50% and 70% respectively. The reduction in inhibitory neurotransmission to CVNs by dopamine was prevented by the sodium channel blocker TTX (1µM) indicating that the dopamine mediated effects were action potential dependent. Dopamine evoked responses were mimicked by the D2-like receptor agonist, Quinpirole but not D1-like receptor agonist, SKF 38393. In addition, the dopamine mediated depression of inhibitory synaptic responses were prevented by the D2-like receptor antagonist sulpiride, but not by D1-like or adrenergic or serotonergic receptor antagonists, suggesting that these responses were D2-like receptor mediated and not D1-like or adrenergic or 5-HT receptor mediated. These data suggest that dopamine acts via dis-inhibition, and diminishes inhibitory GABAergic and glycinergic neurotransmission to CVNs, which would be predicted to increase parasympathetic activity to the heart and evoke a bradycardia.

ß adrenergic receptor modulation of neurotransmission to cardiac vagal neurons in the nucleus ambiguus.

ß-adrenergic receptors are a class of G protein-coupled receptors that have essential roles in regulating heart rate, blood pressure, and other cardiorespiratory functions. Although the role of ß adrenergic receptors in the peripheral nervous system is well characterized, very little is known about their role in the central nervous system despite being localized in many brain regions involved in autonomic activity and regulation. Since parasympathetic activity to the heart is dominated by cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA), ß adrenergic receptors localized in the NA represent a potential target for modulating cardiac vagal activity and heart rate. This study tests the hypothesis that activation of ß adrenergic receptors alters the membrane properties and synaptic neurotransmission to CVNs. CVNs were identified in brainstem slices, and membrane properties and synaptic events were recorded using the whole-cell voltage-clamp technique. The nonselective ß agonist isoproterenol significantly decreased inhibitory GABAergic and glycinergic as well as excitatory glutamatergic neurotransmission to CVNs. In addition, the ß(1)-selective receptor agonist dobutamine, but not ß(2) or ß(3) receptor agonists, significantly decreased inhibitory GABAergic and glycinergic and excitatory glutamatergic neurotransmission to CVNs. These decreases in neurotransmission to CVNs persisted in the presence of tetrodotoxin (TTX). These results provide a mechanism by which activation of adrenergic receptors in the brainstem can alter parasympathetic activity to the heart. Likely physiological roles for this adrenergic receptor activation are coordination of parasympathetic-sympathetic activity and ß receptor-mediated increases in heart rate upon arousal.

Orexinergic modulation of GABAergic neurotransmission to cardiac vagal neurons in the brain stem nucleus ambiguus changes during development.

Cardiac vagal neurons (CVNs) in the nucleus ambiguus (NA) are the major determinant of parasympathetic activity to the heart. Spontaneous GABAergic neurotransmission to CVNs is modulated by hypothalamic neuropeptide orexin-A in postnatal days 2-5 (P5) rats; however, during early postnatal development, orexin expression changes, and the role of orexin-A in modulating CVN activity at other stages of development is unknown. In this study, we compared changes in GABAergic inhibitory postsynaptic currents (IPSCs) in CVNs evoked by orexin-A in P5, P16-20 (P20), and P27-30 (P30) rats using an in vitro brain stem slice preparation. Bath-applied orexin-A enhanced GABAergic IPSCs in all CVNs tested in P5 and P30 animals and in the majority of neurons tested in P20 pups. Focal application of orexin-A ejected from a pipette positioned within 30 µm of the patched CVN did not alter GABAergic signaling in P5 pups. In contrast, in both P20 and P30 rats, focal application of orexin-A inhibited GABAergic IPSCs, and this inhibition persisted in the presence of tetrodotoxin. These results indicate orexin-A facilitates GABAergic IPSCs likely by activating preceding GABAergic neurons that project to CVNs. Orexin-A also likely acts at GABAergic presynaptic terminals surrounding CVNs within the NA to inhibit GABA release. The latter mechanism is absent in P5 pups but occurs in P20 and P30 rats. In conclusion, this study elucidates an important maturation of the parasympathetic cardiac control system. Alterations in these developmental mechanisms may play a role in pathogenesis of disorders related to a specific stage of development maturation.

α1-adrenergic receptors facilitate inhibitory neurotransmission to cardiac vagal neurons in the nucleus ambiguus.

The cholinergic cardiac vagal neurons (CVNs), located in the nucleus ambiguus, are the origin of cardioinhibitory parasympathetic activity. Catecholaminergic neurons in nearby regions of the brainstem, including the C1 and C2 cell groups, are thought to play a key role in both arousing from sleep and maintaining wakefulness. Because norepinephrine (NE) could play an important role in influencing the activity of CVNs, particularly in response to sleeping/waking and arousal states, the present study investigated the contribution of α(1)-adrenergic receptor activation to augment inhibitory and/or blunt excitatory neurotransmission to CVNs. To test the effects of α(1)-adrenergic receptor activation, CVNs were labeled in rats by retrograde tracing and synaptic events were recorded by whole cell voltage clamp techniques in vitro. Prazosin, an inverse agonist of α(1)-adrenergic receptor, significantly decreased the frequency of both GABAergic and glycinergic neurotransmission to CVNs. Activation of α(1)-adrenergic receptors by the α(1)-adrenergic receptor agonists NE or phenylephrine (PE) both significantly increased GABAergic and glycinergic inhibitory event frequency. This effect was prevented by the sodium channel blocker tetrodotoxin (TTX). Activation of α(1)-adrenergic receptors did not alter glutamatergic neurotransmission to CVNs. This study indicates that α(1)-adrenergic receptor activation in the brainstem can facilitate inhibitory GABAergic and glycinergic neurotransmission so as to reduce CVN activity; this synaptic modulation may play a role in the tachycardia seen during NE-dependent behavioral arousal.

Hypocretin-1 (orexin A) prevents the effects of hypoxia/hypercapnia and enhances the GABAergic pathway from the lateral paragigantocellular nucleus to cardiac vagal neurons in the nucleus ambiguus.

Hypocretins (orexins) are hypothalamic neuropeptides that play a crucial role in regulating sleep/wake states and autonomic functions including parasympathetic cardiac activity. We have recently demonstrated stimulation of the lateral paragigantocellular nucleus (LPGi), the nucleus which is thought to play a role in rapid eye movement (REM) sleep control, activates an inhibitory pathway to preganglionic cardiac vagal neurons in the nucleus ambiguus (NA). In this study we test the hypothesis that hypocretin-1 modulates the inhibitory neurotransmission to cardiac vagal neurons evoked by stimulation of the LPGi using whole-cell patch-clamp recordings in an in vitro brain slice preparation from rats. Activation of hypocretin-1 receptors produced a dose-dependent and long-term facilitation of GABAergic postsynaptic currents evoked by electrical stimulation of the LPGi. Hypoxia/hypercapnia diminished LPGi-evoked GABAergic current in cardiac vagal neurons and this inhibition by hypoxia/hypercapnia was prevented by pre-application of hypocretin-1. The action of hypocretin-1 was blocked by the hypocretin-1 receptor antagonist SB-334867. Facilitation of LPGi-evoked GABAergic current in cardiac vagal neurons under both normal condition and during hypoxia/hypercapnia could be the mechanism by which hypocretin-1 affects parasympathetic cardiac function and heart rate during REM sleep. Furthermore, our findings indicate a new potential mechanism that might be involved in the cardiac arrhythmias, bradycardia, and sudden cardiac death that can occur during sleep.

Endogenous inhibition of the trigeminally evoked neurotransmission to cardiac vagal neurons by muscarinic acetylcholine receptors.

Stimulation of the nasal mucosa by airborne irritants or water evokes a pronounced bradycardia accompanied by peripheral vasoconstriction and apnea. The dive response, which includes the trigeminocardiac reflex, is among the most powerful autonomic responses. These responses slow the heart rate and reduce myocardial oxygen consumption. Although normally cardioprotective, exaggeration of this reflex can be detrimental and has been implicated in cardiorespiratory diseases, including sudden infant death syndrome (SIDS). An essential component of the diving response and trigeminocardiac reflex is activation of the parasympathetic cardiac vagal neurons (CVNs) in the nucleus ambiguus that control heart rate. This study examined the involvement of cholinergic receptors in trigeminally evoked excitatory postsynaptic currents in CVNs in an in vitro preparation from rats. CVNs were identified using a retrograde tracer injected into the fat pads at the base of the heart. Application of the acetylcholinesterase inhibitor neostigmine significantly decreased the amplitude of glutamatergic neurotransmission to CVNs on stimulation of trigeminal fibers. Whereas nicotine did not have any effect on the glutamatergic responses, the muscarinic acetylcholine receptor (mAChR) agonist bethanechol significantly decreased the excitatory neurotransmission. Atropine, an mAChR antagonist, facilitated these responses indicating this trigeminally evoked brain stem pathway in vitro is endogenously inhibited by mAChRs. Tropicamide, an m4 mAChR antagonist, prevented the inhibitory action of the muscarinic agonist bethanechol. These results indicate that the glutamatergic synaptic neurotransmission in the trigeminally evoked pathway to CVNs is endogenously inhibited in vitro by m4 mAChRs.

5-HT2 receptors modulate excitatory neurotransmission to cardiac vagal neurons within the nucleus ambiguus evoked during and after hypoxia.

To examine the role of 5-HT2 receptors in the central cardiorespiratory network, and in particular the respiratory modulation of parasympathetic activity to the heart, we used an in vitro medullary slice that allowed simultaneous examination of rhythmic inspiratory-related activity recorded from hypoglossal rootlet and excitatory inspiratory-related neurotransmission to cardioinhibitory vagal neurons (CVNs) within the nucleus ambiguus (NA). Focal application of ketanserin, a 5-HT2 receptor antagonist, did not significantly alter the frequency of spontaneous excitatory postsynaptic excitatory currents (EPSCs) in CVNs in control conditions. However, ketanserin diminished spontaneous excitatory neurotransmission to CVNs during hypoxia. The inhibitory action of ketanserin was on 5-HT3 mediated EPSCs during hypoxia since these responses were blocked by the 5-HT3 receptor antagonist ondansetron. In addition, a robust inspiratory-related excitatory neurotransmission was recruited during recovery from hypoxia. Focal application of ketanserin during this posthypoxia period evoked a significant augmentation of the frequency of inspiratory-related, but not spontaneous EPSCs in CVNs. This excitatory effect of ketanserin was prevented by application of the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These results demonstrate 5-HT2 receptors differentially modulate excitatory neurotransmission to CVNs during and after hypoxia. Activation of 5-HT2 receptors acts to maintain excitatory neurotransmission to CVNs during hypoxia, likely via presynaptic facilitation of 5-HT3 receptor-mediated neurotransmission to CVNs. However, activation of 5HT2 receptors diminishes the subsequent inspiratory-related excitatory neurotransmission to CVNs that is recruited during the recovery from hypoxia likely exerting an inhibitory action on inspiratory-related purinergic signaling.

Serotonergic modulation of the trigeminocardiac reflex neurotransmission to cardiac vagal neurons in the nucleus ambiguus.

Stimulation of the trigeminal nerve evokes a dramatic decrease in heart rate and blood pressure, and this reflex has generally been termed the trigeminocardiac reflex. A subset of the trigeminocardiac reflex is the diving reflex in which the nasal mucosa is stimulated with water or air-borne chemical irritants. Activation of the diving reflex evokes a pronounced bradycardia, mediated by increased parasympathetic cardiac activity, and is the most powerful autonomic reflex. However, exaggeration of this protective response could be detrimental and has been implicated in Sudden Infant Death Syndrome (SIDS). Despite the importance and strength of the trigeminocardiac reflex, there is little information about the cellular mechanisms and brain stem pathways that constitute this reflex. To address these issues, stimulation of trigeminal afferent fibers and the evoked excitatory postsynaptic currents were recorded in cardiac vagal neurons (CVNs) in an in vitro brain stem slice preparation. This synaptic pathway is robust and activation of the trigeminal pathway often evoked action potentials in CVNs. Application of the serotonin (5-HT) reuptake inhibitor citalopram significantly enhanced these responses. Consistent with the hypothesis this pathway is endogenously modulated by 5-HT receptors the 5-HT1A receptor antagonist, WAY 100635 inhibited, whereas the 5-HT2A/C receptor antagonist, ketanserin facilitated the excitatory neurotransmission to CVNs. The 5-HT1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin hydrobromide increased, whereas the 5-HT2 receptor agonist, alpha-methylserotonin maleate salt inhibited this reflex pathway. These results indicate stimulation of trigeminal fibers evokes a powerful excitatory and polysynaptic pathway to CVNs, and this pathway is endogenously modulated and differentially enhanced and depressed, by 5-HT1A and 5-HT2 receptors, respectively.

Mapping and identification of GABAergic neurons in transgenic mice projecting to cardiac vagal neurons in the nucleus ambiguus using photo-uncaging.

The neural control of heart rate is determined primarily by the activity of preganglionic parasympathetic cardiac vagal neurons (CVNs) originating in the nucleus ambiguus (NA) in the brain stem. GABAergic inputs to CVNs play an essential role in determining the activity of these neurons including a robust inhibition during each inspiratory burst. The origin of GABAergic innervation has yet to be determined however. A transgenic mouse line expressing green florescent protein (GFP) in GABAergic cells was used in conjunction with caged glutamate to identify both clusters and individual GABAergic neurons that evoke inhibitory GABAergic synaptic responses in CVNs. Transverse slices were taken with CVNs patch-clamped in the whole cell configuration. Sections containing both the pre-Botzinger complex as well as the calamus scriptorius were divided into approximately 90 quadrants, each 200 x 200 microm and were sequentially photostimulated. Inhibitory post synaptic currents (IPSCs) were recorded in CVNs after a 5-ms photostimulation of 50 microM caged glutamate. The four areas that contained GABAergic cells projecting to CVNs were 200 microm medial, 400 microm medial, 200 microm ventral, and 1,200 microm dorsal and 1,000 microm medial to patched CVNs. Once foci of GABAergic cells projecting to CVNs were determined, photostimulation of individual GABAergic neurons was conducted. The results from this study suggest that GABAergic cells located in four specific areas project to CVNs, and that these cells can be individually identified and stimulated using photouncaging to recruit GABAergic neurotransmission to CVNs.

Abolishment of serotonergic neurotransmission to cardiac vagal neurons during and after hypoxia and hypercapnia with prenatal nicotine exposure.

Cardioinhibitory cardiac vagal neurons (CVNs) do not receive inspiratory-related excitatory inputs under normal conditions. However, excitatory purinergic and serotonergic pathways are recruited during inspiratory activity after episodes of hypoxia and hypercapnia (H/H). Prenatal nicotine (PNN) exposure is known to dramatically change cardiorespiratory responses and decrease the ability to resuscitate from H/H. This study tested whether PNN exposure alters excitatory neurotransmission to CVNs in the nucleus ambiguus during and after H/H. Spontaneous and inspiratory evoked excitatory postsynaptic currents were recorded in CVNs from rats that were exposed to nicotine (6 mg x kg(-1) x d(-1)) throughout the prenatal period. In contrast to unexposed animals, in PNN animals H/H recruited excitatory neurotransmission to CVNs during inspiratory-related activity that was blocked by the alpha3beta4 nicotinic acetylcholine receptor (nAChR) blocker alpha-conotoxin AuIB (alpha-CTX AuIB, 100 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 50 microM) and d(-)-2-amino-5-phosphonopentanoic acid (AP5, 50 microM), selective AMPA/kainate and N-methyl-d-aspartate receptor blockers, respectively. Following H/H, there was a significant increase in inspiratory-related excitatory postsynaptic currents that were unaltered by alpha-CTX AuIB or ondansetron, a 5-HT3 receptor blocker, but were subsequently inhibited by pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (100 microM), a purinergic receptor blocker and CNQX and AP5. The results from this study demonstrate that with PNN exposure, an excitatory neurotransmission to CVNs is recruited during H/H that is glutamatergic and dependent on activation of alpha3beta4-containing nAChRs. Furthermore, exposure to PNN abolishes a serotonergic long-lasting inspiratory-related excitation of CVNs that is replaced by recruitment of a glutamatergic pathway to CVNs post H/H.

Recruitment of excitatory serotonergic neurotransmission to cardiac vagal neurons in the nucleus ambiguus post hypoxia and hypercapnia.

Inhibitory GABAergic and glycinergic neurotransmission to cardioinhibitory cardiac vagal neurons (CVNs) increase during inspiratory activity and likely mediate respiratory sinus arrhythmia, while the frequency of excitatory postsynaptic currents (EPSCs) in CVNs are unaltered during the different phases of respiration. However, following hypoxia and hypercapnia (H/H), the parasympathetic activity to the heart increases and thus far, identification of the pathways and neurotransmitters that are responsible for exciting CVNs post H/H are unclear. This study identifies different excitatory pathways to CVNs recruited post H/H. Spontaneous and inspiratory-related EPSCs were recorded in CVNs before, during, and after 10 min of H/H in an in vitro slice preparation that retains rhythmic respiratory activity. Before and during H/H, EPSCs in CVNs were completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5), selective AMPA/kainate and N-methyl-d-apartate (NMDA) receptor blockers, respectively. However, after H/H, there was a significant increase in EPSCs during each inspiratory burst. While some of the inspiratory-related EPSCs were blocked by the broad purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and the specific P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate monolithium trisodium salt (TNP-ATP) a P2X receptor blocker, most of the recruited excitatory neurotransmission to CVNs is serotonergic because odansetron, a selective 5-HT3 antagonist, abolished the majority of the spontaneous and inspiratory-related EPSCs evoked during recovery from H/H. The results from this study suggest that following episodes of H/H, two nonglutamatergic excitatory pathways, purinergic and serotonergic, activating P2X and 5-HT3 receptors, respectively, are recruited to excite CVNs in the post H/H recovery period.

5-HT(2) receptor subtypes mediate different long-term changes in GABAergic activity to parasympathetic cardiac vagal neurons in the nucleus ambiguus.

Serotonin (5-HT), and in particular 5-HT(2) receptors, play an important role in cardiorespiratory function within the brainstem. In addition, abnormalities in the 5-HT system have been implicated in many cardiorespiratory disorders, including sudden infant death syndrome. However, little is known about the mechanisms of action of 5-HT(2) receptors in altering the activity of parasympathetic cardiac neurons in the brainstem. In this study we examined the effects of activation of different subtypes of 5-HT(2) receptors on spontaneous and respiratory-evoked GABAergic neurotransmission to cardioinhibitory vagal neurons within the nucleus ambiguus as well as rhythmic fictive inspiratory-related activity in rats. A single application of alpha-Me-5-hydroxytryptamine maleate (alpha-Me-5-HT), a 5-HT(2) receptor agonist, did not significantly alter the frequency of spontaneous or respiratory-evoked GABAergic inhibitory postsynaptic currents (IPSCs) in cardiac vagal neurons. However, repetitive successive applications of alpha-Me-5-HT elicited a long-lasting (>/=1 h) decrease in the frequency of spontaneous as well as inspiratory-related GABAergic IPSCs to cardiac vagal neurons. This study demonstrates multiple, but not single applications of the 5-HT(2) receptor agonist alpha-Me-5-HT caused a long-lasting inhibition of both spontaneous and fictive inspiratory-related GABAergic neurotransmission to CVNs, which can be prevented by the 5-HT(2B) receptor antagonist SB204741, but persisted with the 5-HT(2A/2C) receptor antagonist ketanserin. The 5-HT(2) receptor agonist alpha-Me-5-HT also reversibly and transiently excited central fictive inspiratory activity, which was abolished by ketanserin, but was unaffected by the 5-HT(2B) receptor antagonist SB204741.

Nicotinic receptor activation occludes purinergic control of central cardiorespiratory network responses to hypoxia/hypercapnia.

Prenatal nicotine exposure alters the cardiorespiratory network responses to hypoxia/hypercapnia; however the mechanism(s) responsible for these cardiorespiratory network responses and their alteration by prenatal nicotine exposure are unknown. We used an in vitro medullary slice that allows simultaneous examination of rhythmic respiratory-related activity and excitatory synaptic neurotransmission to cardioinhibitory vagal neurons (CVNs). Respiratory related increases in glutamatergic neurotransmission only occurred on recovery from hypoxia/hypercapnia in unexposed animals. These responses were not altered by nicotinic antagonists but were mediated in part by activation of P2 purinergic receptors. Prenatal nicotine exposure transformed central cardiorespiratory responses to hypoxia/hypercapnia; CVNs received a respiratory related glutamatergic neurotransmission during periods of hypoxia and hypercapnia, whereas increases in glutamatergic neurotransmission during recovery were absent. The excitatory neurotransmission to CVNs during hypoxia/hypercapnia in prenatal nicotine-exposed animals were wholly dependent on nicotinic receptor activation. In the presence of nicotinic antagonists, the responses in prenatal nicotine animals reverted to the pattern of responses in unexposed animals in which an increase in glutamatergic neurotransmission occurred not during but only on recovery from hypoxia/hypercapnia, and this recruited excitatory pathway was blocked by P2 receptor antagonists. These data identify a new functional role for purinergic receptors in the cardiorespiratory responses to hypoxia/hypercapnia and their role in occluding nicotinic receptor activation with prenatal nicotine exposure.

Hypoxia recruits a respiratory-related excitatory pathway to brainstem premotor cardiac vagal neurons in animals exposed to prenatal nicotine.

The most ubiquitous form of arrhythmia is respiratory sinus arrhythmia in which the heart beat slows during expiration and heart rate increases during inspiration. Whereas respiratory sinus arrhythmia benefits pulmonary gas exchange respiratory dysfunction presents a major challenge to the cardiorespiratory system. Hypoxia evokes a pronounced bradycardia mediated by increases in parasympathetic cardiac activity. It has been hypothesized that the fatal events in sudden infant death syndrome (SIDS) are exaggerated cardiorespiratory responses to hypoxia. This study tests whether premotor cardiac vagal neurons receive rhythmic respiratory-related excitatory synaptic inputs during normoxia and hypoxia, and if animals exposed to nicotine in the prenatal period have exaggerated responses to hypoxia. Premotor cardiac vagal neurons in the nucleus ambiguus were identified in rats by the presence of a fluorescent tracer in medullary slices that generate rhythmic inspiratory-related motor discharge. Respiratory activity was recorded from the hypoglossal nerve and excitatory synaptic events in cardiac vagal neurons were isolated using patch clamp techniques. Adult female rats were implanted with osmotic minipumps that delivered nicotine at a level approximately equivalent to those that occur in moderate to heavy smokers. During normal eupneic respiration, as well as during hypoxia, premotor cardiac vagal neurons from control animals did not receive any rhythmic respiratory-related excitatory inputs. However in animals exposed to nicotine throughout the prenatal period respiratory bursts during hypoxia dramatically increased the frequency of excitatory synaptic events in cardiac vagal neurons. In summary, in animals exposed to nicotine throughout the prenatal period, but not in unexposed animals, respiratory bursts that occur during hypoxia dramatically increase the frequency of excitatory synaptic events in cardiac vagal neurons. This study establishes a likely neurochemical mechanism for the heart rate responses to hypoxia and a link between prenatal nicotine exposure and exaggerated bradycardia responses during hypoxia that may contribute to sudden infant death syndrome.

Action of kappa and Delta opioid agonists on premotor cardiac vagal neurons in the nucleus ambiguus.

Both enkephalin and dynorphin containing fibers are in close proximity to neurons in the nucleus ambiguus, including cardiac vagal neurons. Microinjection of Delta and kappa agonists into the nucleus ambiguus have been shown to evoke decreases in heart rate. Yet little is known about the mechanisms by which Delta and kappa opioid receptors alter the activity of cardiac vagal neurons. This study tests whether kappa and Delta opioid agonists can alter the activity of cardiac vagal neurons by modulating likely opioid targets including voltage gated calcium currents, and both glycinergic and GABA) neurotransmission to cardiac vagal neurons. Cardiac vagal neurons were identified in vitro by a fluorescent tracer and studied using patch clamp techniques. Neither the kappa agonist spiradoline or the Delta agonist [D-Pen(2), D-Pen(5)]enkephalin (DPDPE) modulated the voltage gated calcium currents in cardiac vagal neurons. DPDPE also did not alter either glycinergic or GABAergic synaptic neurotransmission. Spiradoline did not change GABAergic synaptic inputs, but did significantly inhibit glycinergic synaptic inputs to cardiac vagal neurons. At a concentration of 1 microM, spiradoline inhibited the amplitude of glycinergic events, and at a concentration of 5 microM, spiradoline inhibited both glycinergic amplitude and frequency. Spiradoline also inhibited both the amplitude and frequency of glycinergic miniature inhibitory post-synaptic currents, indicating kappa agonists likely act at both presynaptic and postsynaptic sites to inhibit glycinergic neurotransmission to cardiac vagal neurons.

Mu-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.

Distribution of mu-opioid receptors in rat visceral premotor neurons.

Agonists of the mu-opioid receptor (MOR) can modulate the activity of visceral premotor neurons, including cardiac premotor neurons. Neurons in brainstem regions containing these premotor neurons also contain dense concentrations of the MOR1. This study examined the distribution of MOR1 within two populations of visceral premotor neurons: one located in the dorsal motor nucleus of the vagus and the other in the nucleus ambiguus. Visceral premotor neurons contained the retrograde tracer Fluoro-Gold following injections of the tracer into the pericardiac region of the thoracic cavity. MOR1 was localized using immunogold detection of an anti-peptide antibody. Visceral premotor neurons in both regions contained MOR1 at somatic and dendritic sites, although smaller dendrites were less likely to contain the receptor than larger dendrites, suggesting there may be selective trafficking of MOR1 within these neurons. MOR1 labeling in nucleus ambiguus neurons was more likely to be localized to plasma membrane sites, suggesting that ambiguus neurons may be more responsive to opioid ligands than neurons in the dorsal motor nucleus of the vagus. In addition, many of the dendrites of visceral premotor neurons were in direct apposition to other dendrites. MOR1 was often detected at these dendro-dendritic appositions that may be gap junctions. Together these findings indicate that the activity of individual visceral premotor neurons, as well as the coupling between neurons, may be regulated by ligands of the MOR.

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.

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.