Intracardiac Ganglia Research Articles

Reduced N-Type Ca2+ Channels in Atrioventricular Ganglion Neurons Are Involved in Ventricular Arrhythmogenesis.

BackgroundAttenuated cardiac vagal activity is associated with ventricular arrhythmogenesis and related mortality in patients with chronic heart failure. Our recent study has shown that expression of N‐type Ca2+ channel α‐subunits (Cav2.2‐α) and N‐type Ca2+ currents are reduced in intracardiac ganglion neurons from rats with chronic heart failure. Rat intracardiac ganglia are divided into the atrioventricular ganglion (AVG) and sinoatrial ganglion. Ventricular myocardium receives projection of neuronal terminals only from the AVG. In this study we tested whether a decrease in N‐type Ca2+ channels in AVG neurons contributes to ventricular arrhythmogenesis.Methods and ResultsLentiviral Cav2.2‐α shRNA (2 μL, 2×107 pfu/mL) or scrambled shRNA was in vivo transfected into rat AVG neurons. Nontransfected sham rats served as controls. Using real‐time single‐cell polymerase chain reaction and reverse‐phase protein array, we found that in vivo transfection of Cav2.2‐α shRNA decreased expression of Cav2.2‐α mRNA and protein in rat AVG neurons. Whole‐cell patch‐clamp data showed that Cav2.2‐α shRNA reduced N‐type Ca2+ currents and cell excitability in AVG neurons. The data from telemetry electrocardiographic recording demonstrated that 83% (5 out of 6) of conscious rats with Cav2.2‐α shRNA transfection had premature ventricular contractions (P<0.05 versus 0% of nontransfected sham rats or scrambled shRNA‐transfected rats). Additionally, an index of susceptibility to ventricular arrhythmias, inducibility of ventricular arrhythmias evoked by programmed electrical stimulation, was higher in rats with Cav2.2‐α shRNA transfection compared with nontransfected sham rats and scrambled shRNA‐transfected rats.ConclusionsA decrease in N‐type Ca2+ channels in AVG neurons attenuates vagal control of ventricular myocardium, thereby initiating ventricular arrhythmias.

Ganglion cardiacum or juxtaductal body of human fetuses.

The ganglion cardiacum or juxtaductal body is situated along the left recurrent laryngeal nerve in the aortic window and is an extremely large component of the cardiac nerve plexus. This study was performed to describe the morphologies of the ganglion cardiacum or juxtaductal body in human fetuses and to compare characteristics with intracardiac ganglion. Ganglia were immunostained in specimens from five fetuses of gestational age 12–16 weeks and seven fetuses of gestational age 28–34 weeks. Many ganglion cells in the ganglia were positive for tyrosine hydroxylase (TH; sympathetic nerve marker) and chromogranin A, while a few neurons were positive for neuronal nitric oxide synthase (NOS; parasympathetic nerve marker) or calretinin. Another ganglion at the base of the ascending aorta carried almost the same neuronal populations, whereas a ganglion along the left common cardinal vein contained neurons positive for chromogranin A and NOS but no or few TH-positive neurons, suggesting a site-dependent difference in composite neurons. Mixtures of sympathetic and parasympathetic neurons within a single ganglion are consistent with the morphology of the cranial base and pelvic ganglia. Most of the intracardiac neurons are likely to have a non-adrenergic non-cholinergic phenotype, whereas fewer neurons have a dual cholinergic/noradrenergic phenotype. However, there was no evidence showing that chromogranin A- and/or calretinin-positive cardiac neurons corresponded to these specific phenotypes. The present study suggested that the ganglion cardiacum was composed of a mixture of sympathetic and parasympathetic neurons, which were characterized the site-dependent differences in and near the heart.

Bradykinin excites intracardiac ganglion neurons by activating non-selective cation channels and inhibiting M-currents

Bradykinin excites intracardiac ganglion neurons by activating non-selective cation channels and inhibiting M-currents

GAREM1 regulates the PR interval on electrocardiograms.

PR interval is the period from the onset of P wave to the start of the QRS complex on electrocardiograms. A recent genomewide association study (GWAS) suggested that GAREM1 was linked to the PR interval on electrocardiograms. This study was designed to validate this correlation using additional subjects and examined the function of Garem1 in a mouse model. We analyzed the association of rs17744182, a variant in the GAREM1 locus, with the PR interval in 5646 subjects who were recruited from 2 Korean replication sets, Yangpyeong (n = 2471) and Yonsei (n = 3175), and noted a significant genomewide association by meta-analysis (P = 2.39 × 10-8). To confirm the function of Garem1 in mice, Garem1 siRNA was injected into mouse tail veins to reduce the expression of Garem1. Garem1 transcript levels declined by 53% in the atrium of the heart (P = 0.029), and Garem1-siRNA injected mice experienced a significant decrease in PR interval (43.27 ms vs. 44.89 ms in control, P = 0.007). We analyzed the expression pattern of Garem1 in the heart by immunohistology and observed specific expression of Garem1 in intracardiac ganglia. Garem1 was expressed in most neurons of the ganglion, including cholinergic and adrenergic cells. We have provided evidence that GAREM1 is involved in the PR interval of ECGs. These findings increase our understanding of the regulatory signals of heart rhythm through intracardiac ganglia of the autonomic nervous system and can be used to guide the development of a therapeutic target for heart conditions, such as atrial fibrillation.

Correlation of Ventricular Arrhythmogenesis with Neuronal Remodeling of Cardiac Postganglionic Parasympathetic Neurons in the Late Stage of Heart Failure after Myocardial Infarction.

Introduction: Ventricular arrhythmia is a major cause of sudden cardiac death in patients with chronic heart failure (CHF). Our recent study demonstrates that N-type Ca2+ currents in intracardiac ganglionic neurons are reduced in the late stage of CHF rats. Rat intracardiac ganglia are divided into the atrioventricular ganglion (AVG) and sinoatrial ganglion. Only AVG nerve terminals innervate the ventricular myocardium. In this study, we tested the correlation of electrical remodeling in AVG neurons with ventricular arrhythmogenesis in CHF rats.Methods and Results: CHF was induced in male Sprague-Dawley rats by surgical ligation of the left coronary artery. The data from 24-h continuous radiotelemetry ECG recording in conscious rats showed that ventricular tachycardia/fibrillation (VT/VF) occurred in 3 and 14-week CHF rats but not 8-week CHF rats. Additionally, as an index for vagal control of ventricular function, changes of left ventricular systolic pressure (LVSP) and the maximum rate of left ventricular pressure rise (LV dP/dtmax) in response to vagal efferent nerve stimulation were blunted in 14-week CHF rats but not 3 or 8-week CHF rats. Results from whole-cell patch clamp recording demonstrated that N-type Ca2+ currents in AVG neurons began to decrease in 8-week CHF rats, and that there was also a significant decrease in 14-week CHF rats. Correlation analysis revealed that N-type Ca2+ currents in AVG neurons negatively correlated with the cumulative duration of VT/VF in 14-week CHF rats, whereas there was no correlation between N-type Ca2+ currents in AVG neurons and the cumulative duration of VT/VF in 3-week CHF.Conclusion: Malignant ventricular arrhythmias mainly occur in the early and late stages of CHF. Electrical remodeling of AVG neurons highly correlates with the occurrence of ventricular arrhythmias in the late stage of CHF.

Abstract 11882: Reduced N-type Calcium Channels in Ventricular Vagal Neurons and Ventricular Arrhythmogenesis

Attenuated cardiac vagal activity is linked to ventricular arrhythmogenesis, and is associated with mortality in patients with chronic heart failure (CHF). However, the sites and mechanisms responsible for decreased cardiac vagal activity are still poorly understood. Our previous studies showed that expression of N-type Ca ++ channel α-subunits (Ca v 2.2-α) and N-type Ca ++ currents were reduced in intracardiac ganglion neurons from coronary artery ligation-induced CHF rats. Rat intracardiac ganglia are divided into the atrioventricular ganglion (AVG) and sinoatrial ganglion. Ventricular myocardium only receives projection of neuronal terminals from the AVG. Here, we tested the hypothesis that reducing N-type Ca ++ channels in AVG neurons contributes to ventricular arrhythmogenesis. Lentiviral Ca v 2.2-α shRNA (2 μl, 1x10 6 pfu/ml) or scrambled shRNA was in-vivo transfected into rat AVG neurons. Non-transfected sham rats served as control. Using real-time single-cell PCR and reverse-phase protein array, we found that in-vivo transfection of Ca v 2.2-α shRNA decreased expression of Ca v 2.2-α mRNA and protein in rat AVG neurons. The results from whole-cell patch clamp recording showed that Ca v 2.2-α shRNA reduced N-type Ca ++ currents and cell excitability in AVG neurons. The data obtained from telemetry ECG recording demonstrated that 83% (5 out of 6) of conscious rats with Ca v 2.2-α shRNA transfection had premature ventricular contraction (p&lt;0.05 vs. 0% of non-transfected sham rats or scrambled shRNA transfected rats). Additionally, an index of susceptibility to ventricular arrhythmias, inducibility of ventricular arrhythmias evoked by programmed electrical stimulation is higher in rats with Ca v 2.2-α shRNA transfection (1.8 ± 0.3 ventricular arrhythmia score), compared to non-transfected sham rats and scrambled shRNA transfected rats (0 ventricular arrhythmia score, p&lt;0.05 vs. these two groups). From these results, we conclude that reduced N-type Ca ++ channels in AVG neurons cause vagal neuronal dysfunction, which is a trigger to initiate ventricular arrhythmias. These results suggest that reduced N-type Ca ++ channels in AVG neurons might be involved in ventricular arrhythmogenesis and sudden cardiac death in the CHF state.

Zebrafish heart as a model to study the integrative autonomic control of pacemaker function

The cardiac pacemaker sets the heart's primary rate, with pacemaker discharge controlled by the autonomic nervous system through intracardiac ganglia. A fundamental issue in understanding the relationship between neural activity and cardiac chronotropy is the identification of neuronal populations that control pacemaker cells. To date, most studies of neurocardiac control have been done in mammalian species, where neurons are embedded in and distributed throughout the heart, so they are largely inaccessible for whole-organ, integrative studies. Here, we establish the isolated, innervated zebrafish heart as a novel alternative model for studies of autonomic control of heart rate. Stimulation of individual cardiac vagosympathetic nerve trunks evoked bradycardia (parasympathetic activation) and tachycardia (sympathetic activation). Simultaneous stimulation of both vagosympathetic nerve trunks evoked a summative effect. Effects of nerve stimulation were mimicked by direct application of cholinergic and adrenergic agents. Optical mapping of electrical activity confirmed the sinoatrial region as the site of origin of normal pacemaker activity and identified a secondary pacemaker in the atrioventricular region. Strong vagosympathetic nerve stimulation resulted in a shift in the origin of initial excitation from the sinoatrial pacemaker to the atrioventricular pacemaker. Putative pacemaker cells in the sinoatrial and atrioventricular regions expressed adrenergic β2 and cholinergic muscarinic type 2 receptors. Collectively, we have demonstrated that the zebrafish heart contains the accepted hallmarks of vertebrate cardiac control, establishing this preparation as a viable model for studies of integrative physiological control of cardiac function by intracardiac neurons.

Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons

Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of −60 mV. Removal of extracellular Ca2+ markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca2+ was fully inhibited by removal of extracellular Na+, indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration–inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca2+/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca2+ from intracellular Ca2+ stores and calmodulin.The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

Intracardiac Ganglion Neuron Dysfunction in Type 2 Diabetes

Reduced cardiac parasympathetic activity is involved in sudden cardiac death and is responsible for high mortality in patients with type 2 diabetes mellitus (T2DM). Nicotinic acetylcholine receptor (nAChR) mediates synaptic transmission in intracardiac ganglion (ICG) and regulates the excitability of postganglionic neurons. We hypothesize that changes in the responsiveness of ICG neuronal excitability and postsynaptic nAChR to nicotine (a nAChR agonist) might be involved in attenuated cardiac vagal tone in type 2 diabetic rats. Type 2 diabetic rats were induced by a combination of both high‐fat diet and low‐dose streptozotocin (30 mg/kg i.p.) injection. As an index of cardiac vagal tone, changes of heart rate in response to vagal efferent nerve stimulation were blunted in T2DM rats, compared to sham rats. In Langendorff‐perfused hearts, there was no significant different on sensitivity of the heart to acetylcholine in sham and T2DM rats. Whole‐cell patch‐clamp data showed that nicotine increases action potential frequency and Ca2+ currents in ICG neurons from sham and T2DM rats, which was completely abolished by hexamethonium (a nAChR antagonist). T2DM reduced the responsiveness of ICG neruonal excitability and Ca2+ channel to nicotine. Additionally, T2DM decreased nAChR currents and sensitivity of the nAChR channel to nicotine in ICG neurons. Data from immunofluorescence staining studies showed that T2DM did not alter the protein expression of nAChR subunits in ICG neurons. These results suggest that the low sensitivity of ICG neurons to nicotine might contribute to impairment of the cardiac vagal activity in T2DM.

Nicotinic Acetylcholine Receptors and Cardiac Vagal activity in Rats with Type 2 Diabetes

Reduced cardiac parasympathetic activity is involved in sudden cardiac death and is responsible for high mortality in patients with type 2 diabetes mellitus (T2DM). Nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in the intracardiac ganglia (ICG) and regulate the excitability of postganglionic parasympathetic neurons. We hypothesized that changes in the functional responsiveness of postsynaptic nAChRs to nicotine (a nAChR agonist) might be involved in attenuated cardiac parasympathetic (vagal) activity in type 2 diabetic rats. A rat model of T2DM was induced by a combination of both high-fat diet and injection of low-dose streptozotocin (30 mg/kg, i.p.). As an index of cardiac vagal function, changes of heart rate in response to graded vagal efferent nerve stimulation were blunted in T2DM rats, compared to sham rats. In isolated Langendorff-perfused hearts, there was no significant difference on sensitivity of the heart to acetylcholine in sham and T2DM rats. Whole-cell patch-clamp data showed that T2DM decreased nAChR currents and sensitivity of the nAChR channel to nicotine in ICG neurons. However, the data from immunofluorescence staining showed that there is no significant difference in the protein expression of ?3 and ?4 nAChR subunits in ICG neurons of sham and T2DM rats. These results suggest that the low sensitivity of nAChR channels to nicotine might contribute to impairment of the cardiac vagal activity in T2DM.

微細加工を用いた副交感神経細胞-心筋細胞共培養系の構築

The sympathetic neurons (SN) and the parasympathetic neurons (PN) have important roles in regulation of the cardiac activity. Imbalance of the SN and the PN activities results in abnormal cardiac electrophysiology and can cause several diseases such as atrial fibrillation. While activities of SN and cardiomyocytes have been studied using microfabrication technique, relationship between activities of PN and cardiomyocytes remains unclear. In this study, we developed a device for co-culture of intracardiac ganglion (ICG) neurons which are parasympathetic neurons and atrial myocytes (AMs) using microfabrication technique. Morphological connections between ICG neurons and AMs were observed by immunostaining. Inter-beat-intervals (IBI) were modulated after electrical stimulation of ICG neurons. This modulation completely blocked after applying atropine, a muscarinic receptor antagonist. These results suggest that the co-culture system can be utilized in the research of cell-cell interactions between parasympathetic neurons and cardiomyocytes.

Heart failure-induced changes of voltage-gated Ca2+ channels and cell excitability in rat cardiac postganglionic neurons

Chronic heart failure (CHF) is characterized by decreased cardiac parasympathetic and increased cardiac sympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in patients with CHF. We hypothesized that the molecular and cellular alterations of cardiac postganglionic parasympathetic (CPP) neurons located in the intracardiac ganglia and sympathetic (CPS) neurons located in the stellate ganglia (SG) possibly link to the cardiac autonomic imbalance in CHF. Rat CHF was induced by left coronary artery ligation. Single-cell real-time PCR and immunofluorescent data showed that L (Ca(v)1.2 and Ca(v)1.3), P/Q (Ca(v)2.1), N (Ca(v)2.2), and R (Ca(v)2.3) types of Ca2+ channels were expressed in CPP and CPS neurons, but CHF decreased the mRNA and protein expression of only the N-type Ca2+ channels in CPP neurons, and it did not affect mRNA and protein expression of all Ca2+ channel subtypes in the CPS neurons. Patch-clamp recording confirmed that CHF reduced N-type Ca2+ currents and cell excitability in the CPP neurons and enhanced N-type Ca2+ currents and cell excitability in the CPS neurons. N-type Ca2+ channel blocker (1 μM ω-conotoxin GVIA) lowered Ca2+ currents and cell excitability in the CPP and CPS neurons from sham-operated and CHF rats. These results suggest that CHF reduces the N-type Ca2+ channel currents and cell excitability in the CPP neurons and enhances the N-type Ca2+ currents and cell excitability in the CPS neurons, which may contribute to the cardiac autonomic imbalance in CHF.

Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia

Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the β1 and β4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular β subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory β subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.

Alterations of calcium channels and cell excitability in intracardiac and stellate ganglion neurons from chronic heart failure rats

Chronic heart failure (CHF) is characterized by increased cardiac sympathetic and decreased cardiac parasympathetic nerve activity. This autonomic imbalance increases the risk of arrhythmias and sudden death in CHF patients. As the final pathways for the autonomic control of the cardiac function, sympathetic postganglionic neurons from the stellate ganglia (SG) release norepinephrine and parasympathetic postganglionic neurons of the intracardiac ganglia (ICG) release acetylcholine upon neuronal excitation. In the present study, we measured the protein expression and current density of Ca2+ channels in the SG and ICG neurons and investigated the role of Ca2+ channels in neuronal excitability and also recorded the cardiac sympathetic nerve activity in sham and coronary artery ligation‐induced CHF rats. Immunofluorescent data showed that L, P/Q, N, and R types of Ca2+ channels were expressed in the SG and ICG neurons, but only protein expression of N‐type Ca2+ channels was decreased in the ICG neurons from CHF rats. Using whole‐cell patch clamp technique, we found that CHF significantly enhanced the Ca2+ currents and cell excitability in SG neurons and reduced the Ca2+ currents and cell excitability in ICG neurons. N‐type Ca2+ channel blocker (1 μM ω‐Conotoxin GVIA) lowered the Ca2+ currents and cell excitability to the same level in SG and ICG neurons from sham and CHF rats. In addition, the cardiac sympathetic nerve activity was augmented in CHF rats. Our results indicate that CHF caused changes of Ca2+ channels and cell excitability in SG and ICG neurons may contribute to the cardiac autonomic imbalance in CHF.

Electrophysiological and pharmacological evaluation of the nicotinic cholinergic system in chagasic rats

BackgroundTwo theories attempt to explain the changes observed in the nicotinic acetylcholine receptors (nAChRs) in chagasic cardiomyopathy. The neurogenic theory proposes that receptor changes are due to loss of intracardiac ganglia parasympathetic neurons. The immunogenic theory proposes that the nAChRs changes are the result of autoantibodies against these receptors. Both theories agreed that nAChRs functional expression could be impaired in Chagas disease.MethodsWe evaluated nAChRs functional integrity in 54 Sprague Dawley rats, divided in two groups: healthy and chronic chagasic rats. Rats were subjected to electrocardiographic studies in the whole animal under pentobarbital anesthesia, by isolation and stimulation of vagus nerves and in isolated beating hearts (Langendorff’s preparation).ResultsNicotine, 10 μM, induced a significant bradycardia in both groups. However, rats that had previously received reserpine did not respond to nicotine stimulation. β-adrenergic stimulation, followed by nicotine treatment, induced tachycardia in chagasic rats; while inducing bradycardia in healthy rats. Bilateral vagus nerve stimulation induced a significantly higher level of bradycardia in healthy rats, compared to chagasic rats; physostigmine potentiated the bradycardic response to vagal stimulation in both experimental groups. Electric stimulation (e.g., ≥ 2 Hz), in the presence of physostigmine, produced a comparable vagal response in both groups. In isolated beating-heart preparations 1 μM nicotine induced sustained bradycardia in healthy hearts while inducing tachycardia in chagasic hearts. Higher nicotine doses (e.g.,10 – 100 uM) promoted the characteristic biphasic response (i.e., bradycardia followed by tachycardia) in both groups. 10 nM DHβE antagonized the effect of 10 μM nicotine, unmasking the cholinergic bradycardic effect in healthy rats only. 1 nM α-BGT alone induced bradycardia in healthy hearts but antagonized the 10 μM nicotine-induced tachycardia in chagasic rats. In healthy but not in chagasic hearts, 10 μM nicotine shortened PQ and PR interval, an effect counteracted by MA, DHβE and αBGTConclusionOur results suggest that cholinergic function is impaired in chronic Chagas disease in rats, a phenomena that could be related to alteration on the nAChR expression.

Remodelling of the intracardiac ganglia in diabetic Goto-Kakizaki rats: an anatomical study

BackgroundAlthough cardiac autonomic neuropathy is one of major complications of diabetes mellitus (DM), anatomical data on cardiac innervation of diabetic animal models is scant and controversial. We performed this study to check whether long-term diabetic state impacts the anatomy of intracardiac ganglia in Goto-Kakizaki (GK) rats, a genetic model of type 2 DM.MethodsTwelve GK rats (276 ± 17 days of age; mean ± standard error) and 13 metabolically healthy Wistar rats (262 ± 5 days of age) as controls were used for this study. Blood glucose was determined using test strips, plasma insulin by radioimmunoassay. Intrinsic ganglia and nerves were visualized by acetylcholinesterase histochemistry on whole hearts. Ganglion area was measured, and the neuronal number was assessed according to ganglion area.ResultsThe GK rats had significantly elevated blood glucose level compared to controls (11.0 ± 0.6 vs. 5.9 ± 0.1 mmol/l, p < 0.001), but concentration of plasma insulin did not differ significantly between the two groups (84.0 ± 9.8 vs. 67.4 ± 10.9 pmol/l, p = 0.17). The GK rats contained significantly fewer intracardiac ganglia, decreased total area of intracardiac ganglia (1.4 ± 0.1 vs. 2.2 ± 0.1 mm2, p < 0.001) and smaller somata of ganglionic neurons. Mean total number of intracardiac neurons in GK rats was 1461 ± 62, while this number in control rats was higher by 39% and reached 2395 ± 110 (p < 0.001).ConclusionsResults of our study demonstrate the decreased number of intracardiac neurons in GK rats compared to metabolically healthy Wistar rats of similar age. It is likely that the observed structural remodelling of intracardiac ganglia in GK rats is caused by a long-term diabetic state.

Complexity of the frog intracardiac neurons. Intracellular injection study

The goal of this study was to determine the structure of intracardiac neurons in the frog Rana temporaria . Fifty-six intracardiac neurons from 8 animals were labelled ionophoretically by the intracellular markers AlexaFluor 586 and Lucifer Yellow CH. Among the labelled neurons, we found the cells of unipolar, bipolar, multipolar and pseudounipolar types. Multiple neuronal processes originated from the soma, the axon hillock and the initial segment of axon. With respect to the soma, the neuron contained (Mean ± SE) 3.5 ± 0.3 long and 5.5 ± 0.6 short processes. Most neurons had the spine, the bubble or the flake like extensions on their soma surface and were classified as Golgi I typeneurons. Few Golgi II type neurons, the presumptive interneurons, were also found. Our findings contradict to a general view that the frog intracardiac ganglia contain only the adendritic neurons of the unipolar type. Our findings demonstrate that the frog intracardiac neurons are structurally complex and diverse. This diversity may account for the complicated integrative functions of the frog intrinsic cardiac ganglia.

Large-conductance calcium-activated potassium current modulates excitability in isolated canine intracardiac neurons

We studied principal neurons from canine intracardiac (IC) ganglia to determine whether large-conductance calcium-activated potassium (BK) channels play a role in their excitability. We performed whole cell recordings in voltage- and current-clamp modes to measure ion currents and changes in membrane potential from isolated canine IC neurons. Whole cell currents from these neurons showed fast- and slow-activated outward components. Both current components decreased in the absence of calcium and following 1-2 mM tetraethylammonium (TEA) or paxilline. These results suggest that BK channels underlie these current components. Single-channel analysis showed that BK channels from IC neurons do not inactivate in a time-dependent manner, suggesting that the dynamic of the decay of the fast current component is akin to that of intracellular calcium. Immunohistochemical studies showed that BK channels and type 2 ryanodine receptors are coexpressed in IC principal neurons. We tested whether BK current activation in these neurons occurred via a calcium-induced calcium release mechanism. We found that the outward currents of these neurons were not affected by the calcium depletion of intracellular stores with 10 mM caffeine and 10 μM cyclopiazonic acid. Thus, in canine intracardiac neurons, BK currents are directly activated by calcium influx. Membrane potential changes elicited by long (400 ms) current injections showed a tonic firing response that was decreased by TEA or paxilline. These data strongly suggest that the BK current present in canine intracardiac neurons regulates action potential activity and could increase these neurons excitability.

Functional Na V 1.8 Channels in Intracardiac Neurons

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform Na V 1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for Na V 1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense Na V 1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest Na V 1.8 expression within the myocardium. Immunocytochemistry further revealed substantial Na V 1.8 staining in isolated neurons from murine intracardiac ganglia but no Na V 1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the Na V 1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (I Na ) density or I Na gating kinetics in isolated myocytes but significantly reduced I Na density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of Na V 1.8-based I Na . In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for Na V 1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /Na V 1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.

Alterations of calcium channels and cell excitability in intracardiac ganglion neurons from type 2 diabetic rats

Clinical study has demonstrated that patients with type 2 diabetes with attenuated arterial baroreflex have higher mortality rate compared with those without arterial baroreflex dysfunction. As a final pathway for the neural control of the cardiac function, functional changes of intracardiac ganglion (ICG) neurons might be involved in the attenuated arterial baroreflex in the type 2 diabetes mellitus (T2DM). Therefore, we measured the ICG neuron excitability and Ca(2+) channels in the sham and T2DM rats. T2DM was induced by a combination of both high-fat diet and low-dose streptozotocin (STZ, 30 mg/kg ip) injection. After 12-14 wk of the above treatment, the T2DM rats presented hyperglycemia, hyperlipidemia, and insulin resistance but no hyperinsulinemia, which closely mimicked the clinical features of the patients with T2DM. Data from immunofluorescence staining showed that L, N, P/Q, and R types of Ca(2+) channels were expressed in the ICG neurons, but only protein expression of N-type Ca(2+) channels was decreased in the ICG neurons from T2DM rats. Using whole cell patch-clamp technique, we found that T2DM significantly reduced the Ca(2+) currents and cell excitability in the ICG neurons. ω-Conotoxin GVIA (a specific N-type Ca(2+) channel blocker, 1 μM) lowered the Ca(2+) currents and cell excitability toward the same level in sham and T2DM rats. These results indicate that the decreased N-type Ca(2+) channels contribute to the suppressed ICG neuron excitability in T2DM rats. From this study, we think high-fat diet/STZ injection-induced T2DM might be an appropriate animal model to test the cellular and molecular mechanisms of cardiovascular autonomic dysfunction.