Cardiac Ganglion Neurons Research Articles

Impairment of baroreflex control of heart rate and structural changes of cardiac ganglia in conscious streptozotocin (STZ)-induced diabetic mice

Baroreflex control of heart rate (HR) is impaired in human diabetes mellitus and in large experimental models. However, baroreflex impairment in diabetic mouse models and diabetes-induced remodeling of baroreflex circuitry are not well studied. We examined the impairment of baroreflex control of heart rate (HR) and assessed structural remodeling of cardiac ganglia in the streptozotocin (STZ)-induced diabetic mouse model. FVB mice were either injected with vehicle or STZ. Group 1: mice were anesthetized and the femoral artery and vein were catheterized at the 30th day after vehicle or STZ injection. On the second day after surgery, baroreflex-mediated HR responses to sodium nitroprusside (SNP) and phenylephrine (PE)-induced mean arterial blood pressure (MABP) changes were measured in conscious mice. Group 2: Fluoro-Gold was administered (i.p.) to label cardiac ganglia in each mouse at the 25th day after vehicle or STZ injection. After another five days, animals were perfused and cardiac ganglia were examined using confocal microscopy. Compared with control, we found in STZ mice: 1) the HR decreased, but MABP did not. 2) The PE-induced increases of MABP were decreased. 3) Baroreflex bradycardia was attenuated in the rapid MABP ascending phase but the steady-state ΔHR/ΔMABP was not different at all PE doses. 4) SNP-induced MABP decreases were not different. 5) Baroreflex tachycardia was attenuated. 6) The sizes of cardiac ganglia and ganglionic principal neurons were decreased. 7) The ratio of nucleus/cell body of cardiac ganglionic neurons was increased. We conclude that baroreflex control of HR is impaired in conscious STZ mice. In addition, diabetes may induce a significant structural remodeling of cardiac ganglia. Such an anatomical change of cardiac ganglia may provide new information for the understanding of diabetes-induced remodeling of the multiple components within the baroreflex circuitry.

Feedback From Peripheral Musculature to Central Pattern Generator in the Neurogenic Heart of the Crab Callinectes sapidus: Role of Mechanosensitive Dendrites

The neurogenic heart of decapod crustaceans is a very simple, self-contained, model central pattern generator (CPG)-effector system. The CPG, the nine-neuron cardiac ganglion (CG), is embedded in the myocardium itself; it generates bursts of spikes that are transmitted by the CG's five motor neurons to the periphery of the system, the myocardium, to produce its contractions. Considerable evidence suggests that a CPG-peripheral loop is completed by a return feedback pathway through which the contractions modify, in turn, the CG motor pattern. One likely pathway is provided by dendrites, presumably mechanosensitive, that the CG neurons project into the adjacent myocardial muscle. Here we have tested the role of this pathway in the heart of the blue crab, Callinectes sapidus. We performed "de-efferentation" experiments in which we cut the motor neuron axons to the myocardium and "de-afferentation" experiments in which we cut or ligated the dendrites. In the isolated CG, these manipulations had no effect on the CG motor pattern. When the CG remained embedded in the myocardium, however, these manipulations, interrupting either the efferent or afferent limb of the CPG-peripheral loop, decreased contraction amplitude, increased the frequency of the CG motor neuron spike bursts, and decreased the number of spikes per burst and burst duration. Finally, passive stretches of the myocardium likewise modulated the spike bursts, an effect that disappeared when the dendrites were cut. We conclude that feedback through the dendrites indeed operates in this system and suggest that it completes a loop through which the system self-regulates its activity.

P3.6 Metabolic inhibition increases intracellular calcium, alters the electrophysiological properties and ganglionic transmission in rat intrinsic cardiac ganglion neurones in vitro

P3.6 Metabolic inhibition increases intracellular calcium, alters the electrophysiological properties and ganglionic transmission in rat intrinsic cardiac ganglion neurones in vitro

ASIC2a and ASIC3 Heteromultimerize to Form pH-Sensitive Channels in Mouse Cardiac Dorsal Root Ganglia Neurons

Acid-sensing ion channels (ASICs) are Na+ channels that are activated by acidic pH. Their expression in cardiac afferents and remarkable sensitivity to small pH changes has made them leading candidates to sense cardiac ischemia. Four genes encode six different ASIC subunits, however it is not yet clear which of the ASIC subunits contribute to the composition of ASICs in cardiac afferents. Here, we labeled cardiac afferents using a retrograde tracer dye in mice, which allowed for patch-clamp studies of murine cardiac afferents. We found that a higher percentage of cardiac sensory neurons from the dorsal root ganglia respond to acidic pH and generated larger currents compared to those from the nodose ganglia. The ASIC-like current properties of the cardiac dorsal root ganglia neurons from wild-type mice most closely matched the properties of ASIC2a/3 heteromeric channels. This was supported by studies in ASIC-null mice: acid-evoked currents from ASIC3(-/-) cardiac afferents matched the properties of ASIC2a channels, and currents from ASIC2(-/-) cardiac afferents matched the properties of ASIC3 channels. We conclude that ASIC2a and -3 are the major ASIC subunits in cardiac dorsal root ganglia neurons and provide potential molecular targets to attenuate chest pain and deleterious reflexes associated with cardiac disease.

Cardiac defibrillator neurones

The cardiac vagal motor system exerts a myriad of actions upon the beating mammalian heart and its nutrient vascular supply, but one of its most fascinating actions is to quell potentially lethal arrhythmias. It has been recognised for a long time that the amount of nervous vagal tone to a heart has a direct bearing on the outcome of potentially fatal cardiac arrhythmias following a heart attack (La Rovere et al. 1998). Life and death in a coronary care unit is sometimes governed by cardiac vagal tone. In 2007, Brack et al. provided evidence that nitric oxide mediates the protective effect of the vagi on ventricular fibrillation. In this issue of The Journal of Physiology this group has succeeded in measuring nitric oxide release in the beating left ventricle in response to vagal stimulation (Brack et al. 2009). One interpretation of this work is that there is a population of cardiac nitrergic postganglionic neurones which project to the left ventricle and protect it from fibrillation. This is the conception that I am drawn to, being richer in possibilities than other hypotheses. For almost 10 years this group of physiologists and cardiologists has employed the century old method of Oscar Langendorff but with a twist; they leave the cardiac autonomic nerves attached. Like countless investigators before them, they have shown that there is no vagal inotropic effect on the left ventricle, provided the heart rate is maintained constant. Interestingly they have reported that the vagus can affect dispersion of ventricular repolarisation (Mantravadi et al. 2007). This was followed by experiments that showed vagal stimulation could also raise the ventricular threshold to fibrillation. Pharmacological experimentation provided evidence that nitric oxide (NO) mediates this vagal protection via effects on action potential duration restitution (Brack et al. 2007). The next logical step is described in this issue of The Journal of Physiology: to detect an increase of NO in the wall of the left ventricle during vagal stimulation (Brack et al. 2009). The question arises: what is the source of this NO? Is it nitrergic nerves or nitrergic myocytes? The burst of NO is probably not due to constriction and ischaemia causing increased NO production due to disturbed energy metabolism (because vagus nerve stimulation did not increase perfusion pressure under constant flow conditions i.e. did not increase coronary resistance). One caveat is that the researchers measured pressure a little remotely from the ostia of the coronary arteries. Of note, the NO increase also occurred in response to acetylcholine infusion, but this was not blocked by an antagonist of the neuronal form of NO synthase. In contrast, the vagally released NO was sensitive to this antagonist. If the NO is from nitrergic nerves then the heart should contain these NO-producing postganglionic neurones. There is no difficulty in finding supportive evidence for this proposal in many species e.g. 37% of human cardiac ganglion neurones contain NO synthase (Singh et al. 2009). The work of Brack et al. (2009) should act as a stimulus to trace the trajectory of these NOS-positive neurones within atrial ganglionated plexuses. The question of vagal innervation of mammalian ventricles may have become embroiled in controversy in the past, partly because there has been too much emphasis on cholinergic markers. The paper is a testament to the ingenuity of its authors and an enduring method of Carl Ludwig who in 1866, together with Elias Cyon created the first isolated perfused frog heart.

Dorsal spinal cord stimulation obtunds the capacity of intrathoracic extracardiac neurons to transduce myocardial ischemia

Populations of intrathoracic extracardiac neurons transduce myocardial ischemia, thereby contributing to sympathetic control of regional cardiac indices during such pathology. Our objective was to determine whether electrical neuromodulation using spinal cord stimulation (SCS) modulates such local reflex control.MethodsIn 10 anesthetized canines, middle cervical ganglion neurons were identified that transduce the ventricular milieu. Their capacity to transduce a global (rapid ventricular pacing) versus regional (transient regional ischemia) ventricular stress was tested before and after SCS (50 Hz, 0.2 msec duration at 90% motor threshold) applied to the dorsal, cranial thoracic spinal cord. Rapid ventricular pacing and transient myocardial ischemia both activated cardiac related middle cervical ganglion neurons. SCS obtunded their capacity to reflexly respond to the regional ventricular ischemia, but not rapid ventricular pacing.ConclusionsSpinal cord inputs to the intrathoracic extracardiac nervous system obtund the latter's capacity to transduce regional ventricular ischemia, but not global cardiac stress. Given the substantial body of literature indicating the adverse consequences of excessive adrenergic neuronal excitation on cardiac function, these data delineate the intrathoracic extracardiac nervous system as a potential target for neuromodulation therapy in minimizing such effects. (Supported by HL71830)

Intracellular recording from cardiac ganglion neurons receiving functional vagal inputs

Cardiac vagal tone is an important indicator of cardiovascular health, and its loss is an independent risk factor for arrhythmias and cardiac mortality. Thus, it is important to elucidate the factors that modulate cardiac vagal ganglionic transmission. Using the working heart‐ brainstem preparation (Paton 1996, J.Neurosci Meth. 65, 63‐), we report the first intracellular recordings from functionally connected cardiac vagal ganglion cells in situ. The atria were dissected from the ventricles, pinned out, stabilized with a nylon mesh foot, and ganglion cells (n=33) were impaled with sharp microelectrodes (0.5M KCl; 80‐120 MOhm). Spontaneous EPSPs and action potentials (AP) occurred most commonly in post‐inspiration. Activation of bradycardic reflexes (baro‐, chemo‐, nasotrigeminal) increased dramatically EPSP frequency producing AP and atrial rhythm slowing. Spontaneous and reflexly‐evoked AP were mostly triggered by suprathreshold unitary EPSPs rather than by summation of subthreshold EPSPs. Stimulation of the right cervical vagus evoked EPSPs with a latency of 30‐40 ms. Thus, it is possible to record intracellularly from functionally intact cardiac vagal ganglion cells, which exhibit appropriate patterns of ongoing and reflex excitatory synaptic drives. Future studies will elucidate how cardiac vagal transmission may be modified in health and disease.RMcA holds an NHMRC Principal Research Fellowship; JFRP holds a Royal Society Wolfson Research Merit Award; AEP is a Wellcome Trust Advanced Clinical Fellow

Nitric oxide and carbon monoxide synthesizing enzymes and soluble guanylyl cyclase within neurons of adult human cardiac ganglia

Nitric oxide and carbon monoxide are diffusible gas messengers, synthesized by nitric oxide synthase or heme oxygenase 2, respectively, that can activate soluble guanylyl cyclase in adjacent cells. Nitric oxide and carbon monoxide neuromodulation in cardiac ganglia has been demonstrated. However, identification of nitric oxide or carbon monoxide in human cardiac ganglia needs to be confirmed as suggested from animal model studies. Immunohistochemistry was used to demonstrate neuronal nitric oxide synthase, heme oxygenase 2, and soluble guanylyl cyclase immunoreactivity within neurons of adult human cardiac ganglia. Nitric oxide synthase immunoreactivity was present in 37% of neurons within cardiac ganglia, heme oxygenase 2 immunoreactivity in 79%, and soluble guanylyl cyclase in 53%. Our findings support the hypothesis that nitric oxide and carbon monoxide are modulators of neurotransmission in cardiac ganglia and in neural control of the adult human heart.

Modulation of rat parasympathetic cardiac ganglion phenotype and NGF synthesis by adrenergic nerves

Cardiac function is regulated by interactions among intrinsic and extrinsic autonomic neurons, and the mechanisms responsible for organizing these circuits are poorly understood. Parasympathetic neurons elsewhere synthesize the neurotrophin NGF, which may promote postganglionic axonal associations where parasympathetic axons inhibit sympathetic transmitter release. Previous studies have shown that parasympathetic NGF content and neurochemical phenotype are regulated by sympathetic innervation. In this study we assessed contributions of sympathetic input on cardiac ganglion neuronal phenotype and NGF expression. Because cardiac ganglia are reported to contain putative noradrenergic neurons, we eliminated sympathetic input both surgically (extrinsic) and chemically (extrinsic plus intrinsic). In controls, most cardiac ganglion neurons expressed vesicular acetylcholine transporter, frequently colocalized with vesicular monoamine transporter, but lacked catecholamine histofluorescence. Most cardiac ganglion neurons expressed NGF transcripts, and 40% contained mature and 47% proNGF immunoreactivity. Guanethidine treatment for 7 days decreased numbers of neurons expressing vesicular acetylcholine transporter, NGF transcripts and NGF immunoreactivity, but did not affect proNGF or vesicular monoamine transporter immunoreactivity. Stellate ganglionectomy had comparable effects on neurochemical phenotype and mature NGF immunoreactivity, but proNGF expression was additionally reduced. These findings show that individual cardiac ganglion neurons display markers of both cholinergic and noradrenergic transmission. Sympathetic noradrenergic innervation maintains levels of cholinergic but not noradrenergic marker protein. Sympathetic innervation also promotes cardiac ganglion neuronal NGF synthesis. Because chemical blockade of all noradrenergic transmission is no more effective than extrinsic sympathectomy, local intrinsic noradrenergic transmission is not a factor in regulating ganglion neuron phenotype.

Cholinergic neurons of mouse intrinsic cardiac ganglia contain noradrenergic enzymes, norepinephrine transporters, and the neurotrophin receptors tropomyosin-related kinase A and p75

Half of the cholinergic neurons of human and primate intrinsic cardiac ganglia (ICG) have a dual cholinergic/noradrenergic phenotype. Likewise, a large subpopulation of cholinergic neurons of the mouse heart expresses enzymes needed for synthesis of norepinephrine (NE), but they lack the vesicular monoamine transporter type 2 (VMAT2) required for catecholamine storage. In the present study, we determined the full scope of noradrenergic properties (i.e. synthetic enzymes and transporters) expressed by cholinergic neurons of mouse ICG, estimated the relative abundance of neurons expressing different elements of the noradrenergic phenotype, and evaluated the colocalization of cholinergic and noradrenergic markers in atrial nerve fibers. Stellate ganglia were used as a positive control for noradrenergic markers. Using fluorescence immunohistochemistry and confocal microscopy, we found that about 30% of cholinergic cell bodies contained tyrosine hydroxylase (TH), including the activated form that is phosphorylated at Ser-40 (pSer40 TH). Dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) were present in all cholinergic somata, indicating a wider capability for dopamine metabolism and catecholamine uptake. Yet, cholinergic somata lacked VMAT2, precluding the potential for NE storage and vesicular release. In contrast to cholinergic somata, cardiac nerve fibers rarely showed colocalization of cholinergic and noradrenergic markers. Instead, these labels were closely apposed but clearly distinct from each other. Since cholinergic somata expressed several noradrenergic proteins, we questioned whether these neurons might also contain trophic factor receptors typical of noradrenergic neurons. Indeed, we found that all cholinergic cell bodies of mouse ICG, like noradrenergic cell bodies of the stellate ganglia, contained both tropomyosin-related kinase A (TrkA) and p75 neurotrophin receptors. Collectively, these findings demonstrate that mouse intrinsic cardiac neurons (ICNs), like those of humans, have a complex neurochemical phenotype that goes beyond the classical view of cardiac parasympathetic neurons. They also suggest that neurotrophins and local NE synthesis might have important effects on neurons of the mouse ICG.

NGF expression within parasympathetic cardiac ganglion neurons is reduced in an experimental model of heart failure

NGF expression within parasympathetic cardiac ganglion neurons is reduced in an experimental model of heart failure

Degeneration of vagal efferent axons and terminals in cardiac ganglia of aged rats

Baroreflex control of the heart rate is significantly reduced during aging. However, neural mechanisms that underlie such a functional reduction are not fully understood. We injected the tracer DiI into the left nucleus ambiguus (NA), then used confocal microscopy and a Neurolucida Digitization System to examine qualitatively and quantitatively vagal efferent projections to cardiac ganglia of young adult (5-6 months) and aged (24-25 months) rats (Sprague Dawley). Fluoro-Gold was injected intraperitoneally to counterstain cardiac ganglionic principal neurons (PNs). In aged, as in young rats, NA axons projected to all cardiac ganglia and formed numerous basket endings around PNs in the hearts. However, significant structural changes were found in aged rats compared with young rats. Vagal efferent axons contained abnormally swollen axonal segments and exhibited reduced or even absent synaptic-like terminals around PNs, such that the numbers of vagal fibers and basket endings around PNs were substantially reduced (P < 0.01). Furthermore, synaptic-like varicose contacts of vagal cardiac axons with PNs were significantly reduced by approximately 50% (P < 0.01). These findings suggest that vagal efferents continue to maintain homeostatic control over the heart during aging. However, the marked morphological reorganization of vagal efferent axons and terminals in cardiac ganglia may represent the structural substrate for reduced vagal control of the heart rate and attenuated baroreflex function during aging.

Role of TRPV1 and intracellular Ca2+ in excitation of cardiac sensory neurons by bradykinin

Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.

Correspondence

We found the most interesting aspect of the article by Oh et al 1 Oh S. Zhang Y. Bibevski S. Marrouche N.F. Natale A. Mazgalev T.N. Vagal denervation and atrial fibrillation inducibility: epicardial fat pad ablation does not have long-term effects. Heart Rhythm. 2006; 3: 701-708 Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar in the June 2006 issue of Heart Rhythm was the restoration of sinus node slowing and AV conduction block by right vagal stimulation 4 weeks after acute denervation of two clusters of ganglionated plexi (GP) on the heart. However, several major problems were evident throughout the article, starting with the article title, “Vagal denervation and atrial fibrillation inducibility: Epicardial fat pad ablation does not have long-term effects.” Previous studies have clearly shown that both parasympathetic and sympathetic elements reside in the GP on the heart 2 Lazzara R. Scherlag B.J. Robinson M.J. Samet P. Selective in situ parasympathetic control of the canine sinoatrial and atrioventricular nodes. Circ Res. 1973; 32: 393-401 Crossref PubMed Scopus (123) Google Scholar , 3 Ardell J.L. Structure and function of mammalian intrinsic cardiac neurons. in: Armour J.A. Ardell J.L. Neurocardiology. Oxford University Press, New York1994: 95-114 Google Scholar , 4 Singh S. Johnson P.I. Javed A. Gray T.S. Lonohyna V.A. Wurster R.D. Monomine and histamine synthesizing enzymes and neurotransmitters within neurons of adult human cardiac ganglia. Circulation. 1999; 99: 411-419 Crossref PubMed Scopus (75) Google Scholar ; therefore, the implication that only vagal denervation was induced is misleading. The authors must recognize that their epicardial fat pad ablation procedure was only partial because they left intact the GPs at the antra of the left superior and left inferior pulmonary veins, as well as other GP found along the posterior left atrium. Furthermore, use of right cervical vagal trunk stimulation may be inappropriate because the form of atrial fibrillation (AF) induced probably is due to macroreentry, which is short-lived (≤1 minute) and not due to sustained focal ectopy arising from pulmonary veins (PVs). 5 Po S.S. Scherlag B.J. Yamanashi W.S. Edwards J. Zhou J. Wu R. Geng N. Lazzara R. Jackman W.M. Experimental model for paroxysmal atrial fibrillation arising at the pulmonary vein–atrial junction. Heart Rhythm. 2006; 3: 201-208 Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar The latter has been shown to occur by acetylcholine injection into fat pads containing GP adjacent to a right or left PV. The focal AF induced arises from the PV and sustains for periods longer that 20 minutes. To the Editors—ResponseHeart RhythmVol. 3Issue 10PreviewThe letter by Dr. Scherlag and associates commenting on our article published in Heart Rhythm1 provides a welcome opinion from one of the strong proponents of autonomic neural denervation as a potential novel therapeutic strategy for patients with atrial fibrillation (AF). Full-Text PDF

Photosensitive neurogenic heart of the isopod crustacean Ligia exotica.

The heart of animals is regulated through the central nervous system in response to external sensory stimuli. We found, however, that the adult neurogenic heart of the isopod crustacean Ligia exotica has photosensitivity. The beat frequency of the isolated heart decreased in response to a light stimulus. Magnitude of the response was stimulus intensity dependent and the heartbeat frequency decreased to less than 80% of the dark value during illumination of the white light with an intensity of 6.0 mW cm-2. The spectral sensitivity curve of the heart photoresponse peaked at a wavelength around 520 nm. In response to 530 nm monochromatic light, the relationship between light intensity and response magnitude was linear and the threshold intensity was 7.26 x 1012 quanta cm-2 s-1. Bursting activity of the cardiac ganglion, which is located in the heart and acts as the cardiac pacemaker deceased in frequency in response to illumination by white light. This fact suggests that the heart photoresponse of L. exotica results from the photosensitivity of the cardiac ganglion neurons. The photoresponse of the heart therefore contributes to regulation of cardiac output in addition to other regulatory systems.

Modulation of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) Expression in Explant‐Cultured Guinea Pig Cardiac Neurons

Pituitary adenylate cyclase-activating polypeptide (PACAP) expression was quantified in explant-cultured guinea pig cardiac ganglia neurons. In explant culture, both the percentage of PACAP-immunoreactive neurons and pro-PACAP transcript levels increased significantly. Treatment with neurturin or glial-derived neurotrophic factor significantly suppressed the percentage of PACAP-IR neurons, but not pro-PACAP transcript levels.

Developmental Changes in Electrophysiological Properties and Synaptic Transmission in Rat Intracardiac Ganglion Neurons

We charted postnatal changes in the intrinsic electrophysiological properties and synaptic responses of rat intrinsic cardiac ganglion (ICG) neurons. We developed a whole-mount ganglion preparation of the excised right atrial ganglion plexus. Using intracellular recordings and nerve stimulation we tested the hypothesis that substantial transformations in the intrinsic electrical characteristics and synaptic transmission accompany postnatal development. Membrane potential (E(m)) did not change but time constant (tau) and cell capacitance increased with postnatal development. Accordingly, input resistance (R(in)) decreased but specific membrane resistance (R(m)) increased postnatally. Comparison of the somatic active membrane properties revealed significant changes in electrical phenotype. All neonatal neurons had somatic action potentials (APs) with small overshoots and small afterhyperpolarizations (AHPs). Adult neurons had somatic APs with large overshoots and large AHP amplitudes. The range of AHP duration was larger in adults than in neonates. The AP characteristics of juvenile neurons resembled those of adults, with the exception of AHP duration, which fell midway between neonate and adult values. Phasic, multiply adapting, and tonic evoked discharge activities were recorded from ICG neurons. Most neurons displayed phasic discharge at each developmental stage. All neurons received excitatory synaptic inputs from the vagus or interganglionic nerve trunk(s), the strength of which did not change significantly with postnatal age. The changes in the electrophysiological properties of the postganglionic neuron suggest that increased complexity of parasympathetic regulation of cardiac function accompanies postnatal development.

Presence and co-localization of vasoactive intestinal polypeptide with neuronal nitric oxide synthase in cells and nerve fibers within guinea pig intrinsic cardiac ganglia and cardiac tissue

The presence of vasoactive intestinal polypeptide (VIP) has been analyzed in fibers and neurons within the guinea pig intrinsic cardiac ganglia and in fibers innervating cardiac tissues. In whole-mount preparations, VIP-immunoreactive (IR) fibers were present in about 70% of the cardiac ganglia. VIP was co-localized with neuronal nitric oxide synthase (nNOS) in fibers innervating the intrinsic ganglia but was not present in fibers immunoreactive for pituitary adenylate cyclase-activating polypeptide, choline acetyltransferase (ChAT), tyrosine hydroxylase, or substance P. A small number of the intrinsic ChAT-IR cardiac ganglia neurons (approximately 3%) exhibited VIP immunoreactivity. These few VIP-IR cardiac neurons also exhibited nNOS immunoreactivity. After explant culture for 72 h, the intraganglionic VIP-IR fibers degenerated, indicating that they were axons of neurons located outside the heart. In cardiac tissue sections, VIP-IR fibers were present primarily in the atria and in perivascular connective tissue, with the overall abundance being low. VIP-IR fibers were notably sparse in the sinus node and conducting system and generally absent in the ventricular myocardium. Virtually all VIP-IR fibers in tissue sections exhibited immunoreactivity to nNOS. A few VIP-IR fibers, primarily those located within the atrial myocardium, were immunoreactive for both nNOS and ChAT indicating they were derived from intrinsic cardiac neurons. We suggest that, in the guinea pig, the majority of intraganglionic and cardiac tissue VIP-IR fibers originate outside of the heart. These extrinsic VIP-IR fibers are also immunoreactive for nNOS and therefore most likely are a component of the afferent fibers derived from the vagal sensory ganglia.

Selective Modulation of Neuronal Nicotinic Acetylcholine Receptor Channel Subunits by Go-Protein Subunits

G-protein modulation of neuronal nicotinic acetylcholine receptor (nAChR) channels in rat intrinsic cardiac ganglia was examined using dialyzed whole-cell and excised membrane patch-recording configurations. Cell dialysis with GTPgammaS increased the agonist affinity of nAChRs, resulting in a potentiation of nicotine-evoked whole-cell currents at low concentrations. ACh- and nicotine-evoked current amplitudes were increased approximately twofold in the presence of GTPgammaS. In inside-out membrane patches, the open probability (NP(o)) of nAChR-mediated unitary currents was reversibly increased fourfold after bath application of 0.2 mm GTPgammaS relative to control but was unchanged in the presence of GDPbetaS. The modulation of nAChR-mediated whole-cell currents was agonist specific; currents evoked by the cholinergic agonists ACh, nicotine, and 1,1-dimethyl-4-phenylpiperazinium iodide, but not cytisine or choline, were potentiated in the presence of GTPgammaS. The direct interaction between G-protein subunits and nAChRs was examined by bath application of either G(o)alpha or Gbetagamma subunits to inside-out membrane patches and in glutathione S-transferase pull-down and coimmunoprecipitation experiments. Bath application of 50 nm Gbetagamma increased the open probability of ACh-activated single-channel currents fivefold, whereas G(o)alpha (50 nm) produced no significant increase in NP(o). Neuronal nAChR subunits alpha3-alpha5 and beta2 exhibited a positive interaction with G(o)alpha and Gbetagamma, whereas beta4 and alpha7 failed to interact with either of the G-protein subunits. These results provide evidence for a direct interaction between nAChR and G-protein subunits, underlying the increased open probability of ACh-activated single-channel currents and potentiation of nAChR-mediated whole-cell currents in parasympathetic neurons of rat intrinsic cardiac ganglia.

Developmental changes in expression of GABAA receptor‐channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was approximately 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.