Celiac Neurons Research Articles

Immunohistochemical analysis of the mouse celiac ganglion: An integrative relay station of the peripheral nervous system

Celiac ganglia are important sites of signal integration and transduction. Their complex neurochemical anatomy has been studied extensively in guinea pigs but not in mice. The goal of this study was to provide detailed neurochemical characterization of mouse celiac ganglia and noradrenergic nerves in two target tissues, spleen and stomach. A vast majority of mouse celiac neurons express a noradrenergic phenotype, which includes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, and the norepinephrine transporter. Over 80% of these neuron also express neuropeptide Y (NPY), and this coexpression is maintained by dissociated neurons in culture. Likewise, TH and NPY were colocalized in noradrenergic nerves throughout the spleen and in stomach blood vessels. Somatostatin was not detected in principal neurons but did occur in small, TH-negative cells presumed to be interneurons and in a few varicose nerve fibers. Cholinergic nerves provided the most abundant input to the ganglia, and small percentages of these also contained nitric oxide synthase or vasoactive intestinal polypeptide. A low-to-moderate density of nerves also stained separately for the latter markers. Additionally, nerve bundles and varicose nerve fibers containing the sensory neuropeptides, calcitonin gene-related polypeptide, and substance P, occurred at variable density throughout the ganglia. Collectively, these findings demonstrate that principal neurons of mouse celiac ganglia have less neurochemical diversity than reported for guinea pig and other species but receive input from nerves expressing an array of neurochemical markers. This profile suggests celiac neurons integrate input from many sources to influence target tissues by releasing primarily norepinephrine and NPY.

Functional interactions between nicotinic and P2X receptors in celiac ganglia neurons

Here we characterized the cross-inhibitory interactions between nicotinic and P2X receptors of celiac neurons from the guinea pig by recording whole-cell currents induced by 1 mM ACh ( I ACh), 1 mM ATP ( I ATP) and by the simultaneous application of both agonists ( I ACh + ATP ). I ACh and I ATP were inhibited by hexamethonium (nicotinic channel blocker) and PPADS (P2X receptor antagonist), respectively. The amplitude of I ACh + ATP was equal to the current induced by the most effective agonist, indicating a current occlusion. Various observations indicate that I ACh + ATP is carried out through both nicotinic (nACh) and P2X channels: i) I ACh + ATP desensitisation kinetics were in between that of I ACh and I ATP; ii) application of ATP + ACh, decreased I ACh and I ATP, whereas no cross-desensitisation was observed between nACh and P2X receptors; iii) ATP did not affect I ACh in the presence of PPADS or after P2X receptor desensitisation; and iv) ACh did not affect I ATP when nACh channels were blocked with hexamethonium or after nACh receptor desensitisation. Current occlusion is not mediated by activation of metabotropic receptors as it is: i) voltage dependent (was not observed at + 5 mV); ii) present at low temperature (10 °C) and after inhibition of protein kinase activity (with staurosporine); and iii) absent at 30 µM ATP and 30 µM ACh (concentrations that should activate metabotropic receptors). In conclusion, current occlusion described here is similar to the previously reported myenteric neurons. This occlusion is likely the result of allosteric interactions between these receptors.

Effects of hydrogen peroxide on neuronal firing in cultured rat celiac neurons

Elevated reactive oxygen species (ROS) and altered patterns of firing associated with reduced potassium currents were found in celiac ganglion neurons from hypertensive rats. In this study, we investigated whether these changes could be mimicked by application of H2O2. Using whole‐cell patch clamp, dissociated celiac neurons from 3‐week old SD rats were divided into three groups based on their action potential (AP) firing patterns in response to a suprathreshold depolarizing current step (100pA; 5sec). Phasic neurons fired several APs and adapted quickly; tonic‐phasic neurons fired several APs and adapted slowly; tonic neurons fired continuously. After a 1 min exposure to 3mM H2O2, 20% of tonic neurons were converted to phasic neurons; 80% of tonic neurons continued to fire tonically but at a reduced frequency. These effects were reversible in 77% of neurons. Tonic‐phasic and phasic neurons showed decreased firing frequency. The amplitude of the sustained potassium current measured at 50mV from a holding potential of ‐70 mV was significantly increased by 21% in these neurons. When catalase (100 kU/ml) was applied intracellularly in the patch electrode, subsequent H2O2 application had no effect. These findings demonstrate that H2O2 acts intracellularly in celiac neurons to reduce neuronal firing frequency and alter firing patterns. These changes in firing properties are associated with increased amplitude of steady‐state potassium currents.

Decreased sustained potassium current contributes to different firing patterns of celiac neurons in DOCA‐salt hypertensive and normotensive Rats

It is not known if changes in the electrophysiological properties of sympathetic ganglia neurons contribute to increased sympathetic activity in hypertension. Using whole‐cell patch clamp dissociated celiac neurons from hypertensive and normotensive rats were divided into three groups based on their action potential (AP) firing patterns to a constant suprathreshold depolarizing current (100pA; 400 ms). Phasic neurons fired several APs and adapted quickly; tonic‐phasic neurons fired several APs and adapted slowly; tonic neurons fired continuously. The distribution of these three groups was significantly different in neurons from hypertensive rats. There were more phasic neurons, less tonic‐phasic neurons and similar amount of tonic neurons in hypertensive rats. The firing frequency in tonic neurons was higher in hypertensive rats compared to normotensive rats. Potassium channel currents were tested to explore the reason of these neuronal firing changes in hypertension. Sustained potassium current was significantly lower in celiac neurons from hypertensive rats compared to those from normotensive rats. The M‐type and A‐type potassium currents were not different between hypertensive and normotensive rats. In normotensive rats, 23.44mM tetraethylammonium (TEA) reduced the amplitude of sustained potassium currents by 50%. TEA also decreased the firing frequency in tonic neurons and converted tonic‐phasic neurons to phasic neurons. The different celiac neuronal firing patterns contributed by decreased sustained potassium current may play a compensatory role in elevated sympathetic activity in hypertension.Support HL70687

Different Firing Patterns of Celiac Neurons in DOCA‐salt hypertensive and Normotensive Rats

It is not known if changes in the electrophysiological properties of sympathetic ganglia neurons contribute to increased activity in hypertension. Using whole-cell patch clamp of dissociated neurons the resting membrane potential, membrane resistance and membrane capacitance of neurons from hypertensive rats were not different in neurons from normotensive rats. Neurons were divided into five groups based on their action potential (AP) firing patterns to increasing amounts of constant suprathreshold depolarizing current (20–100pA; 400 ms). Group 1 fired only 1 AP; Group 2 fired several APs and adapted quickly; Group 3fired several APs and adapted slowly; Group 4 fired continuously during low level current injection, but fired only a shout burst during higher level current injection; Group 5 fired continuously in response to all levels of depolarizing current. The distribution of these five groups was significantly different in neurons from hypertensive rats (n=68) using X2 test. There were more Group 4 neurons and less Group 3 neurons in hypertensive rats; although the average instantaneous firing frequency in each group was the same in hypertensive rats compared to normotensive rats. These differences indicate that in hypertensive rats a larger subset of celiac neurons firing more APs by depolarization compared to normotensive rats. Support HL70687

Calcium currents and their modulation by strong depolarization in cultured hypertensive and normotensive rat celiac neurons

Calcium channels play an important role in decreased neuron spike accommodation in superior cervical ganglia of spontaneously hypertensive rats. We investigated calcium currents in dissociated celiac neurons from normotensive rats and from rats made hypertensive by treatment with Deoxycorticosterone acetate and high salt diet. Both normotensive and hypertensive celiac neurons showed a high-voltage activated calcium current with peak current at −10mv and reversal potential at +45mv. The currents were totally blocked by 200nM cadmium and 30% blocked by 10μM nifedipine. There were no differences in the amplitude and kinetic properties of these calcium currents between normotensive and hypertensive celiac neurons. G-protein inhibitory modulation of calcium channels is critical in the regulation of calcium currents in rat sympathetic neurons. A brief strong depolarization temporarily reverses G-protein inhibition in superior cervical ganglion. We used a +80mv, 200ms depolarization prior to a 0mv voltage step as the strong depolarization. The strong depolarization did not change the amplitude of calcium currents in neurons from either normotensive or hypertensive rats. However, beginning 2s after the application of the strong depolarization, the peak amplitude of the calcium current was decreased by 20% in neurons from hypertensive rats but was not changed in neurons from normotensive rats. This reduction in current amplitude persisted up to 1 minute. These findings show that strong depolarization induces a long-lasting inhibition of calcium current in sympathetic neurons from hypertensive rats.

Different types of potassium channels underlie the long afterhyperpolarization in guinea-pig sympathetic and enteric neurons

Ca 2+-activated K + channels play an important role in the control of neuronal excitability via the generation of the afterhyperpolarization. While both small and large conductance Ca 2+-activated K + channels underlie afterhyperpolarizations in different neuron types, the role of intermediate conductance Ca 2+-activated K + channels (IK Ca) in the generation of afterhyperpolarizations remains unclear. The effects of blockade of IK Ca on guinea pig coeliac and ileal myenteric neurons were studied using single microelectrode current and voltage clamp. In coeliac neurons, TRAM-39, a selective blocker of IK Ca, depressed the amplitude of the prolonged conductance underlying the slow afterhyperpolarization, (gKCa2) by 57%. In contrast, the conductance underlying the prolonged afterhyperpolarization in AH-type myenteric neurons was unaffected by TRAM-39, although it has been suggested that this AHP is mediated by IK Ca. In both types of neurons, TRAM-39 did not alter the resting cell properties or the properties of the action potential. TRAM-39 had no effect on the amplitude of the fast component of the afterhyperpolarization present in sympathetic LAH neurons. The results of this study suggest that in sympathetic LAH neurons, activation of IK Ca underlies at least part of the prolonged afterhyperpolarization while the nature of the channel underlying the AHP in enteric neurons remains unclear.

Effects of Testosterone on the Electrical Properties and Nicotinic Transmission of the Major Pelvic and Coeliac Ganglion Neurones

The effects of testosterone on the electrical properties and nicotinic activation of prevertebral ganglion neurones were investigated in vitro on the male rat major pelvic ganglion and rabbit coeliac ganglion. The electrical activity of the neurones was recorded using intracellular recording techniques. Nicotinic activation was triggered for neurones of the major pelvic ganglion by stimulating the hypogastric, pelvic and cavernous nerves and for coeliac neurones by stimulating the splanchnic nerves. Testosterone modified the resting membrane potential of neurones in the major pelvic ganglion by triggering a slow depolarization, and was without significant effect on the resting membrane potential of coeliac ganglion neurones. In neurones of the major pelvic and coeliac ganglia, testosterone had no significant effect on the firing pattern, on the characteristics of the action potential (firing threshold, duration, overshoot) and on the after-hyperpolarization (amplitude and duration). Testosterone affected, in opposite ways, the nicotinic activation of neurones of the two prevertebral ganglia. In the major pelvic ganglion, testosterone triggered an increase in the amplitude of excitatory postsynaptic potentials induced by stimulation of the hypogastric, pelvic and cavernous nerves with a single pulse, revealing a facilitation of nicotinic activation. On coeliac ganglion neurones, testosterone elicited a decrease in the amplitude of excitatory postsynaptic potentials induced by stimulation of the splanchnic nerves, indicating an inhibition of nicotinic activation. Our study shows that testosterone acts differently on neurones of prevertebral ganglia involved in the nervous control of different functions, its facilitatory action being exerted on neurones of the major pelvic ganglion which is particularly involved in the control of the urogenital tract. Our study reinforces the concept, derived from neuroanatomical and pharmacological studies, of the major pelvic ganglion as a major peripheral target for testosterone.

Effects of Testosterone on the Electrical Properties and Nicotinic Transmission of the Major Pelvic and Coeliac Ganglion Neurones

AbstractThe effects of testosterone on the electrical properties and nicotinic activation of prevertebral ganglion neurones were investigated in vitro on the male rat major pelvic ganglion and rabbit coeliac ganglion. The electrical activity of the neurones was recorded using intracellular recording techniques. Nicotinic activation was triggered for neurones of the major pelvic ganglion by stimulating the hypogastric, pelvic and cavernous nerves and for coeliac neurones by stimulating the splanchnic nerves. Testosterone modified the resting membrane potential of neurones in the major pelvic ganglion by triggering a slow depolarization, and was without significant effect on the resting membrane potential of coeliac ganglion neurones. In neurones of the major pelvic and coeliac ganglia, testosterone had no significant effect on the firing pattern, on the characteristics of the action potential (firing threshold, duration, overshoot) and on the after‐hyperpolarization (amplitude and duration). Testosterone affected, in opposite ways, the nicotinic activation of neurones of the two prevertebral ganglia. In the major pelvic ganglion, testosterone triggered an increase in the amplitude of excitatory postsynaptic potentials induced by stimulation of the hypogastric, pelvic and cavernous nerves with a single pulse, revealing a facilitation of nicotinic activation. On coeliac ganglion neurones, testosterone elicited a decrease in the amplitude of excitatory postsynaptic potentials induced by stimulation of the splanchnic nerves, indicating an inhibition of nicotinic activation. Our study shows that testosterone acts differently on neurones of prevertebral ganglia involved in the nervous control of different functions, its facilitatory action being exerted on neurones of the major pelvic ganglion which is particularly involved in the control of the urogenital tract. Our study reinforces the concept, derived from neuroanatomical and pharmacological studies, of the major pelvic ganglion as a major peripheral target for testosterone.

Synaptically activated low-threshold muscarinic inward current sustains tonic firing in rabbit prevertebral sympathetic neurons.

Whole-cell patch-clamp experiments were performed on non-dissociated rabbit coeliac sympathetic neurons in the presence of nicotinic blockers. Coeliac neurons were classified as either silent or spontaneously active (pacemaker) cells. Under voltage-clamp conditions, pacemaker cells exhibited a steady-state N-shaped current-voltage relationship due to the presence of a persistent voltage-dependent inward current in the potential range of -100 to approximately -20 mV. This inward current sustained the regular firing activity of pacemaker cells and was absent from quiescent neurons. It disappeared in the presence of tetrodotoxin and in low Ca(2+)-high Mg2+ external solutions and was enhanced by eserine. Splanchnic nerve stimulation induced slow regenerative depolarizations and firing discharges in silent neurons by activating a low-threshold voltage-sensitive inward current. The synaptic current had a U-shaped voltage-dependence from -96 to approximately -20 mV and exhibited the dynamic properties of the muscarinic voltage-dependent inward current INa,M. It gave the current-voltage relationship an N shape similar to that observed in spontaneously active cells. The muscarinic antagonists atropine and pirenzepine abolished the inward current present in pacemaker cells and that induced by nerve stimulation in silent neurons. These data provide evidence that both spontaneous firing activity and nerve-evoked depolarizing responses in coeliac neurons are sustained by the activation of the muscarinic Na,M current. The tonic activation of INa,M in spontaneously firing cells results from a sustained Ca(2+)-dependent tetrodotoxin-sensitive release of acetylcholine. This study provides evidence that the role of the muscarinic receptors is not purely a neuromodulatory one, but that these receptors are directly involved in ganglionic neurotransmission.

Electrophysiological properties of P2X‐purinoceptors in rat superior cervical, nodose and guinea‐pig coeliac neurones.

1. Whole-cell recordings were made from guinea-pig coeliac, rat nodose and rat superior cervical (SCG) neurones in culture, and currents in response to fast-flow (concentration clamp) application of ATP and other ATP analogues were measured. 2. At a holding potential of -70 mV, ATP evoked inward currents in all neurons. ATP-induced currents reversed at approximately 0 mV and showed strong inward rectification. Halving the external sodium concentration shifted the reversal potential by -15 to -17 mV, while increasing external potassium from 2 to 20 mM produced a 6-10 mV shift in reversal potential. Latency to onset of ATP current was < 1 ms; rise time was concentration dependent with maximum time to peak of 5-20 ms in nodose and coeliac neurones but 80 ms in superior cervical neurones. 3. Threshold concentrations of ATP were 0.1 microM for nodose and coeliac neurones but 10 microM for superior cervical neurones; EC50 values were approximately 3 microM for both nodose and coeliac neurones and 43 microM for superior cervical ganglia. Hill slopes for ATP concentration-response curves were not significantly different from unity in nodose and coeliac neurones whereas the Hill slope in superior cervical neurones was two. 4. 2-MethylthioATP (2-MeSATP) acted as a full agonist in all three neuronal preparations; EC50 values were 0.4, 2.8 and 46 microM for nodose, coeliac and superior cervical neurones, respectively. alpha,beta-Methylene ATP (alpha,beta-MeATP) was a full agonist in nodose and coeliac neurones with EC50 values of 9 and 13 microM, respectively. 5. In superior cervical neurones alpha,beta-MeATP had little or no agonist action but produced a concentration-dependent attenuation of the ATP current. Thus, alpha, beta-MeATP appears to behave as a partial agonist at P2X-purinoceptors in superior cervical neurones. 6. The non-selective purinoceptor antagonists suramin (1-100 microM) and pyridoxal-5'-phosphate (30 microM), as well as the putative P2Y-selective antagonist, Cibacron Blue (30 microM), inhibited all agonist-evoked responses to a similar degree in all three neuronal populations. 7. This study demonstrates that an agonist potency profile of 2-MeSATP > or = ATP > or = alpha,beta-MeATP is characteristic of ligand-gated P2X-purinoceptors in isolated peripheral neurones. We also suggest that the P2X-receptor in superior cervical neurones may represent a distinct subtype of P2X-purinoceptor from that present in nodose and coeliac neurones.

The muscarinic receptor agonist oxotremorine methiodide evokes a nicotinic response in mammalian sympathetic neurons

Oxotremorine methiodide, a congener of oxotremorine, is used as a muscarinic receptor agonist. Responses to oxotremorine methiodide and nicotinic receptor agonists were examined in cultured guinea-pig celiac neurons using whole-cell voltage clamp techniques. At holding potentials between −30 and −60 mV, a brief application of oxotremorine methiodide produced fast and slow inward current transients, depending upon the concentration applied. Slowly developing inward current transients, characteristic of muscarinic responses, were produced by lower concentrations (EC 50: 0.3 μM) and were blocked by atropine. Rapid inward current transients, characteristic of nicotine responses, were produced by higher concentrations of oxotremorine methiodide (EC 50: 168 μM) and were blocked by d-tubocurarine. Thus oxotremorine methiodide, at concentrations of 10 μM and greater, produced an initial nicotinic fast inward current transient followed by a slow muscarinic inward transient. The fast inward transients were similar to responses evoked by the nicotinic receptor agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenyl-piperazinium iodide and were not antagonized by atropine. We conclude that oxotremorine methiodide acts as a nicotinic and muscarinic receptor agonist in celiac sympathetic ganglion neurons.

Muscarinic inhibition of two potassium currents in guinea-pig prevertebral neurons: Differentiation by extracellular cesium

Muscarinic responses were studied in dissociated guinea-pig celiac ganglion neurons using the whole-cell voltage-clamp technique. Muscarine (0.025–1 mM; ec 50 = 95 μM) administered to cells for 1.5 s evoked inward shifts in holding current in 53 of 74 cells. The amplitude of the inward current transients decreased with hyperpolarization and the null potential averaged −71 ± 3.4 mV ( n = 11). The currents that underlie the responses to muscarine were examined with hyperpolarizing voltage stepping protocols to − 100 mV from a holding potential of − 30 mV. Eighty-one per cent of cells displayed voltage-dependent current relaxations characteristic of the M-potassium current. Twenty per cent of responding cells displayed no M-current but only a voltage-independent current consistent with a leak current. In the latter type of cells, the muscarine-evoked inward currents reversed near E K and became outward at more hyperpolarized potentials. Analysis of steady state I-V relationships before and after bath application of muscarine showed that the two muscarine-sensitive potassium currents were distributed differently among three types of cells: (i) with M-current (18%); (ii) with leak current (18%); and (iii) with M-current and with leak current (64%). Cesium and barium were used to differentiate the M-current and the muscarine-sensitive leak current. Barium (2 mM) reduced the M-current and the leak potassium current, whereas cesium (2 mM) reduced the M-current but did not affect leak current. Thus, barium reduced the amplitude of muscarinic responses by 79% but cesium reduced them by only 14%. We conclude that muscarinic responses in guinea-pig celiac neurons are produced by suppression of two K + currents: the M-current and a muscarine-sensitive leak current. These two currents are differentially susceptible to the potassium channel blockers barium and cesium.

Electrophysiological analysis of the convergence of peripheral inputs onto neurons of the coeliac ganglion in the guinea pig

The convergence of intestinofugal axons from different intestinal regions onto individual neurons in the coeliac ganglion of the guinea pig was investigated using intracellular recording methods in vitro. Peripheral nerve trunks from the distal ileum, the most proximal colon and the colon near the colonic flexure were electrically stimulated along with preganglionic fibres running in the splanchnic nerve. Fast cholinergic excitatory synaptic potentials (EPSPs) were seen in ganglion cells in response to stimulation of each nerve trunk. Roughly half of 78 neurons impaled received inputs from stimulation of peripheral nerves, and almost all of these received input from the proximal colon. Most cells responded to stimulation of more than one peripheral nerve indicating that coeliac neurons receive converging inputs from intestinofugal neurons located in more than one intestinal region. In a second series of experiments, segments of intestine were left attached to the ganglion and distended with saline to stimulate peripheral mechanosensory input to the coeliac ganglion. In each experiment, two segments were stimulated. A subgroup of ganglion cells exhibited spontaneous fast EPSPs and the frequency of these potentials was increased by distension of one or other of the attached intestinal segments. However, few neurons responded to distension of both of the attached intestinal segments suggesting that some of the intestinofugal inputs to the coeliac ganglion identified by electrical stimulation may be sensitive to sensory modalities other than distension. Hexamethonium (0.5 mM) applied to the intestine, and not to the coeliac ganglion, reduced the frequency of the spontaneous synaptic potentials seen in coeliac ganglion cells, but did not abolish the response to distension of the colon ( n = 8). When the Ca 2+ concentration of the solution bathing the proximal colon was reduced to block all synaptic transmission in the enteric plexuses the background synaptic input was further depressed, but again the response to distension was little changed ( n = 4). This suggests that at least some of the neurons projecting from the colon to the coeliac ganglion are first order mechanosensory neurons.

Neurotransmitter properties of guinea-pig sympathetic neurons grown in dissociated cell culture—II. Fetal and embryonic neurons: Regulation of neuropeptide Y expression

We report here the neurotransmitter characteristics of neurons cultured from the same ganglia of fetal and embryonic guinea-pigs. Both the celiac ganglion and the superior mesenteric ganglion were examined. In a previous paper we described the neurotransmitter properties of adult guinea-pig prevertebral sympathetic neurons grown in dissociated cell culture, including the expression by these cells of immunoreactivity for tyrosine hydroxylase, neuropeptide Y and somatostatin. Tyrosine hydroxylase immunoreactivity was ubiquitously expressed in all fetal embryonic cultures, as was the case for adult neurons. Fetal-derived celiac and superior mesenteric gangli neurons displayed neuropeptide Y and somatostatin immunoreactivity in the same percentage of neurons as in adult cultures but at markedly lower levels. Embryonic neurons also expressed somatostatin immunoreactivity in roughly the same proportion of neurons as in adult and fetal cultures; however, the expression of neuropeptide Y immunoreactivity in both celiac and superior mesenteric gangli cultures was significantly different. Specifically, neuropeptide Y immunoreactivity in embryonic celiac cultures was greatly reduced in both the number of positive-labeled neurons and the amount of immunoreactive product, while neuropeptide Y immunoreactivity in embryonic superior mesenteric gangli cultures was markedly increased compared to their adult and fetal counterparts. The expression of neuropeptide Y immunoreactivity in celiac neurons was found to be specifically elevated by culturing the neurons in medium conditioned by disassociated vascular cells, this treatment having no effect on tyrosine hydroxylase or somatostatin immunoreactivity. Heart cell-conditioned medium did not effect neuropeptide Y or somatostatin immunoreactivity, although it did result in a significant reduction of tyrosine hydroxylase immunoreactivity and an increase in 5-hydroxytryptamine immunoreactivity. We conclude that the expression of neuropeptide Y immunoreactivity develops independently in cultures of adult and near-term fetuses but that embryonic neurons require interactions with target cells to express this phenotype. Neuropeptide Y immunoreactivity can be induced in embryonic sympathetic neurons by a target-derived factor(s).

On the excitatory effects of ATP and its role as a neurotransmitter in coeliac neurons of the guinea‐pig.

1. The effects of ATP on neurons from guinea-pig coeliac ganglia were studied to evaluate the possibility that this nucleotide acts as an excitatory neurotransmitter substance. 2. In experiments with intracellular microelectrodes, ATP (> or = 10 nM) depolarized coeliac neurons from the resting potential and produced an increase in the membrane conductance. These excitatory effects of ATP were observed in isolated coeliac ganglia, in acutely dissociated neurons or in cultured neurons. ATP also produced membrane conductance increases in neurons clamped at the resting potential using a single electrode voltage clamp. 3. When studied in the whole-cell configuration of the patch clamp (intracellular Cs+ to block K+ currents; -50 mV holding potential), ATP evoked inward currents in a manner more potent and efficacious than acetylcholine (ACh). 4. Whole-cell currents induced by ATP were inwardly rectifying and reversed at -13 mV in normal Na+ solutions. Changes in extracellular Na+ concentration altered the reversal potential in a manner predicted by the Goldman-Hodgkin-Katz bi-ionic equation with a ratio of Na+ to Cs+ permeability (PNa/PCs) = 0.6. 5. Single channel currents were evoked by ATP in excised (outside-out) patches. Current-voltage relationships for single channel currents exhibited inward rectification. The mean single channel conductance was 22 pS at -50 mV. 6. Antagonists of ATP-gated channels (suramin, Reactive Blue 2) reduced the effects of ATP but not ACh. 7. Antagonists at nicotinic receptors/ion channels (hexamethonium or tubocurarine) reduced the effects of ACh but not ATP. 8. Excitatory synaptic currents were observed in cultures of coeliac neurons. Synaptic currents possessed similar current-voltage relationships to currents produced by ATP, were increased in frequency by K+ depolarization in a Ca(2+)-dependent manner, and were selectively antagonized by ATP antagonists. 9. Local K+ depolarization of the ends of neurites evoked single channel currents characteristic of ATP in outside-out patches when patches were positioned near the region of apparent synaptic contact but not when patches were positioned at remote regions. 10. The results suggest that ATP receptors are linked to ion channels and mediate excitatory synaptic transmission between coeliac neurons.

Potassium currents and their modulation by muscarine and substance P in neuronal cultures from adult guinea pig celiac ganglia.

1. Intracellular microelectrode and whole-cell patch-clamp recordings were obtained from adult guinea pig celiac ganglion neurons grown in tissue culture for 7-14 days. Over 90% of neurons showed phasic-type action-potential discharge with the use of either type of recording electrode; they stained immunohistochemically for catecholamines, tyrosine hydroxylase, and neuropeptide Y. Input resistance (140 M omega) and action-potential amplitude (103 mV) were significantly greater with whole-cell than with microelectrode recordings, but other passive electrical properties were similar. 2. Five potassium currents were characterized: an apamin-sensitive after hyperpolarizing current (IAHP), an apamin and tetraethylammonium-insensitive slow IAHP, an M-like current, a transient outward IA current, and a delayed rectifier IK current. A hyperpolarization-activated cationic Ih current was also present. The first three currents were not observed with whole-cell recordings. 3. Cadmium (200 microM), cobalt (1 mM), lanthanum (30 microM), or a low calcium/high magnesium solution blocked both IAHPS and the M-like current; barium (1 mM) also blocked these currents. 4. Kinetics of the M-like current were best described by a double exponential fit to deactivating tail currents with time constants of 50 and 390 ms at -50 mV. The apamin-sensitive and slow IAHP decayed exponentially with time constants of 145 ms and 3.5 s, respectively. There was no correlation between occurrence of M-like current (95% of neurons) and slow IAHP (40% of neurons), nor any correlation between magnitude of M-like current and IAHP in those cells exhibiting both currents. 5. Muscarine and substance P (SP) caused depolarizations or inward currents (under voltage clamp) at the resting potential (-55 mV) associated with a decreased membrane conductance. The slow IAHP and the M-like current, but not the apamin-sensitive IAHP nor the IA, were blocked by muscarine and SP (IC50 3 microM and 100 nM, respectively). Muscarine and SP also decreased a "leak" potassium current. 6. We conclude that celiac neurons express two calcium-dependent IAHP currents and a calcium-dependent M-current; these are seen by fine-tipped intracellular microelectrodes but not by whole-cell patch electrodes. These currents are not required for spike frequency accommodation. Muscarine and SP reduce these currents, as well as voltage-independent leakage potassium current.

Calcium induced calcium release is involved in the afterhyperpolarization in one class of guinea pig sympathetic neurone

The mechanisms underlying two potassium conductances which are activated by Ca 2+ influx during the action potential in ympathetic prevertebral neurones of guinea pigs have been investigated pharmacologically. One Ca-activated K + conductance, which is present in all mammalian sympathetic postganglionic neurones, is maximal after the action potential and decays exponentially with a time constant of about 130 ms; this conductance was inhibited by apamin (50–100 nM) consistent with the involvement of SK channels. A second Ca-activated K + conductance with much slower kinetics is present in a large subpopulation of coeliac neurones. This conductance was resistant to apamin but markedly inhibited by application of ryanodine (5–20 μM). suggesting that Ca 2+ influx during the action potential triggers release of Ca 2+ from intracellular stores which in turn activates a different class of K + channel. Noradrenaline (100 μM) depressed the second K + conductance selectively.

ATP mediates excitatory synaptic transmission in mammalian neurones.

Adenosine 5'-triphosphate (ATP, 0.1-100 microM), produced inward currents in patch-clamped coeliac neurones from guinea-pig when studied in either the whole cell configuration or in excised (outside-out) patches. The P2-purinoceptor antagonists suramin (80-230 microM) or reactive blue 2 (2-20 microM) depressed the ATP-induced currents but not those produced by acetylcholine. Excitatory post-synaptic currents (e.p.s.cs) were observed in cultured neurones. E.p.s.cs had similar current-voltage relationships to currents evoked by ATP in excised patches and were reduced by suramin or reactive blue 2 to a similar extent as ATP currents. The results suggest that ATP is the excitatory neurotransmitter in cultures of these neurones.

Effects of modifications of gastric motility on the membrane potential of the coeliac ganglionic neurons in the rabbit

AbstractThe involvement of gastric motility in the slow variations of membrane potential of sympathetic ganglionic neurons was investigated in rabbit.The present study was performed in vitro on the coeliac plexus connected to the stomach. The electrical activity of ganglionic neurons was investigated using intracellular recording techniques. Spontaneous increases in basal intragastric pressure triggered slow hyperpolarizations of the ganglionic neurons (n = 7), the duration of which was similar to that of the increase in the basal pressure. Conversely, cooling of the nerve trunks connecting the coeliac plexus to the stomach induced a slow depolarization of the ganglionic neurons (n = 3). Carbachol‐induced increases in basal intra‐gastric pressure modified the membrane potential of ganglionic neurons in several ways: hyperpolarization (n = 6), depolarization (n = 4), hyperpolarization followed by depolarization (n = 2), and these modifications lasted longer than the modifications of gastric motility. After cooling of the nerve trunks connecting the coeliac plexus to the stomach, the carbachol‐induced variations of gastric motility were no longer able to modify the membrane potential of the ganglionic neurons.These results demonstrate that modifications of the gastric motility can trigger slow modifications of the resting membrane potential of coeliac neurons and therefore could modify their excitability. This suggests that gastric motility can modify at the postsynaptic level the transmission of central or peripheral inputs to ganglionic neurons.