Slow Excitatory Postsynaptic Potentials Research Articles

Estradiol elicits distinct firing patterns in arcuate nucleus kisspeptin neurons of females through altering ion channel conductances.

Hypothalamic kisspeptin (Kiss1) neurons are vital for pubertal development and reproduction. Arcuate nucleus Kiss1 (Kiss1ARH) neurons are responsible for the pulsatile release of Gonadotropin-releasing Hormone (GnRH). In females, the behavior of Kiss1ARH neurons, expressing Kiss1, Neurokinin B (NKB), and Dynorphin (Dyn), varies throughout the ovarian cycle. Studies indicate that 17β-estradiol (E2) reduces peptide expression but increases Vglut2 mRNA and glutamate neurotransmission in these neurons, suggesting a shift from peptidergic to glutamatergic signaling. To investigate this shift, we combined transcriptomics, electrophysiology, and mathematical modeling. Our results demonstrate that E2 treatment upregulates the mRNA expression of voltage-activated calcium channels, elevating the whole-cell calcium current and that contribute to high-frequency burst firing. Additionally, E2 treatment decreased the mRNA levels of Canonical Transient Receptor Potential (TPRC) 5 and G protein-coupled K+ (GIRK) channels. When TRPC5 channels in Kiss1ARH neurons were deleted using CRISPR, the slow excitatory postsynaptic potential (sEPSP) was eliminated. Our data enabled us to formulate a biophysically realistic mathematical model of the Kiss1ARH neuron, suggesting that E2 modifies ionic conductances in Kiss1ARH neurons, enabling the transition from high frequency synchronous firing through NKB-driven activation of TRPC5 channels to a short bursting mode facilitating glutamate release. In a low E2 milieu, synchronous firing of Kiss1ARH neurons drives pulsatile release of GnRH, while the transition to burst firing with high, preovulatory levels of E2 would facilitate the GnRH surge through its glutamatergic synaptic connection to preoptic Kiss1 neurons.

Vagally-mediated heart block after myocardial infarction associated with plasticity of epicardial neurons controlling the atrioventricular node.

Imbalances in the opposing actions of sympathetic and parasympathetic nerves controlling the heart enhance risk for arrhythmia and sudden cardiac death after myocardial infarction (MI). Plasticity in peripheral neuron function may underlie the observed changes in cardiomotor nerve activity. We studied vagal control of the heart in pigs after chronic infarction of the left ventricle. Stimulation of the cervical vagus nerve produced greater bradycardic responses 8-weeks after MI. Recordings of epicardial electrocardiograms demonstrate increased severity and duration of atrioventricular (AV) block in MI-pigs during 20 Hz vagal stimulation. Intracellular voltage recordings from isolated neurons of the inferior vena cava-inferior left atrium (IVC-ILA) ganglionated plexus, a cluster of epicardial neurons receiving innervation from the vagus known to regulate the AV node, were used to assess plasticity of membrane and synaptic physiology of intrinsic cardiac neurons (ICNs) after MI. Changes to both passive and active membrane properties were observed, including more negative resting membrane potentials and greater input resistances in MI-pig ICNs, concomitant with a depression of neuronal excitability. Immunoreactivity to pituitary adenylate cyclase-activating polypeptide (PACAP), a cardiotropic peptide known to modulate cardiac neuron excitability, was localized to perineuronal varicosities surrounding pig IVC-ILA neurons. Exogenous application of PACAP increased excitability of control but not MI-ICNs. Stimulation (20 Hz) of interganglionic nerves in the ex vivo whole-mount preparations elicited slow excitatory postsynaptic potentials (sEPSPs) which persisted in hexamethonium (500 μM), but were blocked by atropine (1 μM), indicating muscarinic receptor-mediated inhibition of M-current. Extracellular application of 1 mM BaCl2 to inhibit M-current increased neuronal excitability. The muscarine-sensitive sEPSPs were observed more frequently and were of larger amplitude in IVC-ILA neurons from MI animals. In conclusion, we suggest the increased probability of muscarinic sEPSPs play a role in the potentiation of the vagus nerve mediated-slowing of AV nodal conduction following chronic MI. We identify both a novel role of a muscarinic sensitive current in the regulation of synaptic strength at ICNs projecting to the AV node, and demonstrate changes to both intrinsic plasticity and synaptic plasticity of IVC-ILA neurons which may contribute to greater risk for heart block and sudden cardiac death after MI.

Computational simulations and Ca2+ imaging reveal that slow synaptic depolarizations (slow EPSPs) inhibit fast EPSP evoked action potentials for most of their time course in enteric neurons.

Transmission between neurons in the extensive enteric neural networks of the gut involves synaptic potentials with vastly different time courses and underlying conductances. Most enteric neurons exhibit fast excitatory post-synaptic potentials (EPSPs) lasting 20-50 ms, but many also exhibit slow EPSPs that last up to 100 s. When large enough, slow EPSPs excite action potentials at the start of the slow depolarization, but how they affect action potentials evoked by fast EPSPs is unknown. Furthermore, two other sources of synaptic depolarization probably occur in enteric circuits, activated via GABAA or GABAC receptors; how these interact with other synaptic depolarizations is also unclear. We built a compartmental model of enteric neurons incorporating realistic voltage-dependent ion channels, then simulated fast EPSPs, slow EPSPs and GABAA or GABAC ligand-gated Cl- channels to explore these interactions. Model predictions were tested by imaging Ca2+ transients in myenteric neurons ex vivo as an indicator of their activity during synaptic interactions. The model could mimic firing of myenteric neurons in mouse colon evoked by depolarizing current during intracellular recording and the fast and slow EPSPs in these neurons. Subthreshold fast EPSPs evoked spikes during the rising phase of a slow EPSP, but suprathreshold fast EPSPs could not evoke spikes later in a slow EPSP. This predicted inhibition was confirmed by Ca2+ imaging in which stimuli that evoke slow EPSPs suppressed activity evoked by fast EPSPs in many myenteric neurons. The model also predicted that synchronous activation of GABAA receptors and fast EPSPs potentiated firing evoked by the latter, while synchronous activation of GABAC receptors with fast EPSPs, potentiated firing and then suppressed it. The results reveal that so-called slow EPSPs have a biphasic effect being likely to suppress fast EPSP evoked firing over very long periods, perhaps accounting for prolonged quiescent periods seen in enteric motor patterns.

Augmentation of M‐Current and Increased Synaptic Efficacy at Intrinsic Cardiac Neurons Contributes to an Enhanced Cardiac Responsiveness to Vagal Nerve Stimulation in the Infarcted Porcine Heart

Imbalances to autonomic regulation of cardiac function contribute to risk for arrhythmia after myocardial infarction (MI). We tested the hearts responsiveness to vagal nerve stimulation (VNS), 8 wks after MI, to determine if parasympathetic control of the heart is altered in MI pigs. A significantly greater bradycardic response was elicited in MI animals during 20Hz stimulation. Intracellular voltage recordings from whole‐mount preparations of intrinsic cardiac neurons (ICNs) were subsequently used to assess whether alterations in neuronal membrane properties or synaptic transmission underlie the observed increase in responsiveness to VNS in MI animals. General membrane properties (resting membrane potential, input resistance, action potential amplitude/duration, afterhyperpolarization amplitude/duration) of ICNs from control and MI pigs were not different, but ICN excitability was significantly depressed in MI pigs. Testing synaptic efficacy revealed a greater proportion of strong (high AP probability) vs weak synapses (low AP probability) in MI animals. Tetanic stimulation (20Hz) of interganglionic nerve bundles elicited slow excitatory postsynaptic potentials (sEPSPs) in a significantly greater subset of MI ICNs (10/22) vs controls (4/26). The sEPSP persisted in hexamethonium (500uM) but was blocked by atropine (1uM), indicating mediation by cholinergic inhibition of M‐current. Application of 1mM Ba2+ increased excitability of ICNs from both control and MI animals further supporting a role for M‐current in the regulation of porcine ICN excitability. In hexamethonium, the amplitude of the sEPSP was significantly greater in MI ICNs. In conclusion, we identify a important role of M‐current in the regulation of the excitability of porcine ICNs, and demonstrate enhanced membrane depolarization of ICNs from MI animals after presynaptic stimulation due, in part, to greater inhibition of IM during tetanic nerve stimulation which may underlie the enhanced sensitivity to VNS in MI pigs.Support or Funding InformationThis work was supported in part by NIH grant OT2OD023848.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscarinic receptors in adrenal chromaffin cells: physiological role and regulation of ion channels

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K+ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K+ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.

Pursuit of Neurotransmitter Functions: Being Attracted with Fascination of the Synapse.

In the beginning of the 1970s, only two chemical substances, acetylcholine and γ-aminobutyric acid (GABA), had been definitely established as neurotransmitters. Under such circumstances, I started my scientific career in Professor Masanori Otsuka's lab searching for the transmitter of primary sensory neurons. Until 1976, lines of evidence had accumulated indicating that the undecapeptide substance P could be released as a transmitter from primary afferent fibers into spinal synapses, although the substance P-mediated synaptic response had yet to be identified. Peripheral synapses could serve as a good model and thus, it was demonstrated in the prevertebral sympathetic ganglia by1985 that substance P released from axon collaterals of primary sensory neurons acts as the transmitter mediating non-cholinergic slow excitatory postsynaptic potential (EPSP). At that time, we also found that autonomic synapses were useful to uncover the transmitter role of the opioid peptide enkephalins, whose functions had been unknown since their discovery in 1975. Accordingly, enkephalins were found to serve a transmitter role in mediating presynaptic inhibition of cholinergic fast and non-cholinergic slow transmission in the prevertebral sympathetic ganglia. In 1990s, we attempted to devise a combined technique of brain slices and patch-clamp recordings. We applied it to study the regulatory mechanisms that operate around cerebellar GABAergic inhibitory synapses, because most of the studies then had centered on excitatory synapses and because inhibitory synapses are crucially involved in brain functions and disorders. Consequently, we discovered novel forms of heterosynaptic interactions, dual actions of a single transmitter, and receptor crosstalk, the details of which are described in this review.

A model of the enteric neural circuitry underlying the generation of rhythmic motor patterns in the colon: the role of serotonin.

We discuss the role of multiple cell types involved in rhythmic motor patterns in the large intestine that include tonic inhibition of the muscle layers interrupted by rhythmic colonic migrating motor complexes (CMMCs) and secretomotor activity. We propose a model that assumes these motor patterns are dependent on myenteric descending 5-hydroxytryptamine (5-HT, serotonin) interneurons. Asynchronous firing in 5-HT neurons excite inhibitory motor neurons (IMNs) to generate tonic inhibition occurring between CMMCs. IMNs release mainly nitric oxide (NO) to inhibit the muscle, intrinsic primary afferent neurons (IPANs), glial cells, and pacemaker myenteric pacemaker interstitial cells of Cajal (ICC-MY). Mucosal release of 5-HT from enterochromaffin (EC) cells excites the mucosal endings of IPANs that synapse with 5-HT descending interneurons and perhaps ascending interneurons, thereby coupling EC cell 5-HT to myenteric 5-HT neurons, synchronizing their activity. Synchronized 5-HT neurons generate a slow excitatory postsynaptic potential in IPANs via 5-HT7 receptors and excite glial cells and ascending excitatory nerve pathways that are normally inhibited by NO. Excited glial cells release prostaglandins to inhibit IMNs (disinhibition) to allow full excitation of ICC-MY and muscle by excitatory motor neurons (EMNs). EMNs release ACh and tachykinins to excite pacemaker ICC-MY and muscle, leading to the simultaneous contraction of both the longitudinal and circular muscle layers. Myenteric 5-HT neurons also project to the submucous plexus to couple motility with secretion, especially during a CMMC. Glial cells are necessary for switching between different colonic motor behaviors. This model emphasizes the importance of myenteric 5-HT neurons and the likely consequence of their coupling and uncoupling to mucosal 5-HT by IPANs during colonic motor behaviors.

Innervation of enteric mast cells by primary spinal afferents in guinea pig and human small intestine.

Mast cells express the substance P (SP) neurokinin 1 receptor and the calcitonin gene-related peptide (CGRP) receptor in guinea pig and human small intestine. Enzyme-linked immunoassay showed that activation of intramural afferents by antidromic electrical stimulation or by capsaicin released SP and CGRP from human and guinea pig intestinal segments. Electrical stimulation of the afferents evoked slow excitatory postsynaptic potentials (EPSPs) in the enteric nervous system. The slow EPSPs were mediated by tachykinin neurokinin 1 and CGRP receptors. Capsaicin evoked slow EPSP-like responses that were suppressed by antagonists for protease-activated receptor 2. Afferent stimulation evoked slow EPSP-like excitation that was suppressed by mast cell-stabilizing drugs. Histamine and mast cell protease II were released by 1) exposure to SP or CGRP, 2) capsaicin, 3) compound 48/80, 4) elevation of mast cell Ca²⁺ by ionophore A23187, and 5) antidromic electrical stimulation of afferents. The mast cell stabilizers cromolyn and doxantrazole suppressed release of protease II and histamine when evoked by SP, CGRP, capsaicin, A23187, electrical stimulation of afferents, or compound 48/80. Neural blockade by tetrodotoxin prevented mast cell protease II release in response to antidromic electrical stimulation of mesenteric afferents. The results support a hypothesis that afferent innervation of enteric mast cells releases histamine and mast cell protease II, both of which are known to act in a diffuse paracrine manner to influence the behavior of enteric nervous system neurons and to elevate the sensitivity of spinal afferent terminals.

Angiotensin behaves like a paracrine mediator in guinea pig enteric nervous system (874.1)

Enteric mast cell hyperplasia occurs in inflammatory bowel disease and the irritable bowel syndrome. Mast cell proteases are catalysts for conversion of angiotensin I to angiotensin II (AT‐II). We applied AT‐II to ENS neurons, while recording neuronal electrical and synaptic behavior. The AT‐II acted to elevate or suppress neuronal excitability. Suppression of was reversed by noradrenergic alpha2 receptor blocking drugs. AT‐II enhanced electrically‐evoked noradrenergic inhibitory postsynaptic potentials and suppressed fast and slow excitatory postsynaptic potentials. Selective AT‐II1 receptor blocking drugs, but not selective AT2 receptor antagonists, suppressed all actions of AT‐II. AT2 receptor protein and the AT2 mRNA transcript were expressed in the ENS. The AT1 receptor was not expressed. Immunoreactivity for the AT1 receptor was expressed by ENS neurons with AH‐type 2 and S‐type electrophysiological behavior. Our results predict that mast cell‐induced AT‐II formation will have paracrine function in the ENS that includes enhanced release of norepinephrine from sympathetic postganglionic axons. Elevation of norepinephrine represses excitatory neurotransmission in the neural networks of the brain‐in‐the‐gut and suppresses neurogenic secretion thru inhibition of excitability of secretomotor neurons in the submucosal division of the ENS.Grant Funding Source: Supported by National Institutes of Health, R01DK037238

Mathematical modelling of enteric neural motor patterns

1. The enteric nervous system modulates intestinal behaviours, such as motor patterns and secretion. Although much is known about different types of neurons and simple reflexes in the intestine, it remains unclear how complex behaviours are generated. 2. Mathematical modelling is an important tool for assisting the understanding of how the neurons and reflexes can be pieced together to generate intestinal behaviours. 3. Models have identified a functional role for slow excitatory post-synaptic potentials (EPSPs) by distinguishing between fast and slow EPSPs in the ascending excitation reflex. These models also discovered coordinated firing of similarly located neurons as emergent properties of feed-forward networks of interneurons in the intestine. A model of the recurrent network of intrinsic sensory neurons identified important control mechanisms to prevent uncontrolled firing due to positive feedback and that the interaction between these control mechanisms and slow EPSPs is necessary for the networks to encode ongoing sensory stimuli. This model also showed that such networks may mediate migrating motor complexes. 4. A network model of vasoactive intestinal peptide neurons in the submucosal plexus found this relatively sparse recurrent network could produce uncontrolled firing under conditions that appear to be related to cholera toxin-induced hypersecretion. 5. Abstract modelling of the intestinal fed-state motor patterns has identified how stationary contractions can arise from a polarized network. 6. These models have also helped predict and/or explained pharmacological evidence for two rhythm generators and the requirement of feedback from contractions in the circular muscle.

Lack of Kinase Regulation of Canonical Transient Receptor Potential 3 (TRPC3) Channel-dependent Currents in Cerebellar Purkinje Cells

Canonical transient receptor potential (TRPC) channels are widely expressed in the brain and play several roles in development and normal neuronal function. In the cerebellum, Purkinje cell TRPC3 channels underlie the slow excitatory postsynaptic potential observed after parallel fiber stimulation. In these cells TRPC3 channel opening requires stimulation of metabotropic glutamate receptor 1, activation of which can also lead to the induction of long term depression (LTD), which underlies cerebellar motor learning. LTD induction requires protein kinase C (PKC) and protein kinase G (PKG) activation, and although PKC phosphorylation targets are well established, virtually nothing is known about PKG targets in LTD. Because TRPC3 channels are inhibited after phosphorylation by PKC and PKG in expression systems, we examined whether native TRPC3 channels in Purkinje cells are a target for PKG or PKC, thereby contributing to cerebellar LTD. We find that in Purkinje cells, activation of TRPC3-dependent currents is not inhibited by conventional PKC or PKG to any significant extent and that inhibition of these kinases does not significantly impact on TRPC3-mediated currents either. Based on these and previous findings, we propose that TRPC3-dependent currents may differ significantly in their regulation from those overexpressed in expression systems.

Cholinergic modulation on spike timing-dependent plasticity in hippocampal CA1 network

Cholinergic inputs from the medial septum are projected to pyramidal neurons in the hippocampal CA1 region and release acetylcholine (ACh) from their terminals. The cholinergic inputs are considered to be integrated with sensory inputs and to play a crucial role in learning and memory. Meanwhile, it has been reported that the relative timing between pre- and post-synaptic spiking determines the direction and extent of synaptic changes in a critical temporal window, a process known as spike timing-dependent plasticity (STDP). Positive timing where excitatory postsynaptic potential (EPSP) precedes the postsynaptic action potential induces long-term potentiation (LTP) while negative timing where EPSP follows the action potential induces long-term depression (LTD). To investigate the influence of muscarinic activation by cholinergic inputs on synaptic plasticity, STDP-inducing stimuli were applied during the muscarinic induction of a slow EPSP followed by repetitive stimulation in the stratum oriens. As a result, LTP was facilitated and LTD was abolished by the muscarinic activation. Furthermore, interestingly, LTP was also facilitated and LTD was switched to LTP with an increase in ACh concentration following application of the cholinesterase inhibitor eserine. These results indicate that the orientation of plasticity was shifted for potentiation by muscarinic activation. On the other hand, the application of excess ACh concentration completely suppressed STDP, LTP and LTD. In addition, STDP was suppressed in the presence of atropine, a muscarinic ACh receptor antagonist. Taken together, the findings suggest that synaptic plasticity modulation depends on the amount of cholinergic inputs. The modulation of synaptic plasticity by muscarinic activation might be an important stage in the integration of top-down and bottom-up information in hippocampal CA1 neurons.

MGluR1/TRPC3-mediated Synaptic Transmission and Calcium Signaling in Mammalian Central Neurons

Metabotropic glutamate receptors type 1 (mGluR1s) are required for a normal function of the mammalian brain. They are particularly important for synaptic signaling and plasticity in the cerebellum. Unlike ionotropic glutamate receptors that mediate rapid synaptic transmission, mGluR1s produce in cerebellar Purkinje cells a complex postsynaptic response consisting of two distinct signal components, namely a local dendritic calcium signal and a slow excitatory postsynaptic potential. The basic mechanisms underlying these synaptic responses were clarified in recent years. First, the work of several groups established that the dendritic calcium signal results from IP(3) receptor-mediated calcium release from internal stores. Second, it was recently found that mGluR1-mediated slow excitatory postsynaptic potentials are mediated by the transient receptor potential channel TRPC3. This surprising finding established TRPC3 as a novel postsynaptic channel for glutamatergic synaptic transmission.

R-type Ca2+ channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine

R-type Ca(2+) channels are expressed by myenteric neurons in the guinea pig ileum but the specific function of these channels is unknown. In the present study, we used intracellular electrophysiological techniques to determine the function of R-type Ca(2+) channels in myenteric neurons in the acutely isolated longitudinal musclemyenteric plexus. We used immunohistochemical methods to localize the Ca(V)2.3 subunit of the R-type Ca(2+) channel in myenteric neurons. We also studied the effects of the non-selective Ca(2+) channel antagonist, CdCl₂ (100 μmol L⁻¹), the R-type Ca(2+) channel blockers NiCl₂ (50 μmol L⁻¹) and SNX-482 (0.1 μmol L⁻¹), and the N-type Ca(2+) channel blocker x-conotoxin GVIA (CTX 0.1 μmol L⁻¹) on action potentials and fast and slow excitatory postsynaptic potentials (fEPSPs and sEPSPs) in S and AH neurons in vitro. Ca(V)2.3 co-localized with calretinin and calbindin in myenteric neurons. NiCl₂ and SNX-482 reduced the duration and amplitude of action potentials in AH but not S neurons. NiCl₂ inhibited the afterhyperpolarization in AH neurons. x-conotoxin GVIA, but not NiCl₂, blocked sEPSPs in AH neurons. NiCl₂ and SNX-482 inhibited cholinergic, but not cholinergic/purinergic, fEPSPs in S neurons. These data show that R-type Ca(2+) channels contribute to action potentials, but not slow synaptic transmission, in AH neurons. R-type Ca(2+) channels contribute to release of acetylcholine as the mediator of fEPSPs in some S neurons. These data indicate that R-type Ca(2+) channels may be a target for drugs that selectively modulate activity of AH neurons or could alter fast synaptic excitation in specific pathways in the myenteric plexus.

Slow synaptic transmission in myenteric AH neurons from the inflamed guinea pig ileum

We investigated the effect of inflammation on slow synaptic transmission in myenteric neurons in the guinea pig ileum. Inflammation was induced by the intraluminal injection of trinitrobenzene sulfonate, and tissues were taken for in vitro investigation 6-7 days later. Brief tetanic stimulation of synaptic inputs (20 Hz, 1 s) induced slow excitatory postsynaptic potentials (EPSPs) in 49% and maintained postsynaptic excitation that lasted from 27 min to 3 h in 13% of neurons from the inflamed ileum. These neurons were classified electrophysiologically as AH neurons; 10 were morphological type II neurons, and one was type I. Such long-term hyperexcitability after a brief stimulus is not encountered in enteric neurons of normal intestine. Electrophysiological properties of neurons with maintained postsynaptic excitation were similar to those of neurons with slow EPSPs. Another form of prolonged excitation, sustained slow postsynaptic excitation (SSPE), induced by 1-Hz, 4-min stimulation, in type II neurons from the inflamed ileum reached its peak earlier but had lower amplitude than that in control. Unlike slow EPSPs and similar to SSPEs, maintained excitation was not inhibited by neurokinin-1 or neurokinin-3 receptor antagonists. Maintained postsynaptic excitation was not influenced by PKC inhibitors, but the PKA inhibitor, H-89, caused further increase in neuronal excitability. In conclusion, maintained excitation, observed only in neurons from the inflamed ileum, may contribute to the dysmotility, pain, and discomfort associated with intestinal inflammation.

Electrical stimulation of the mucosa evokes slow EPSPs mediated by NK1 tachykinin receptors and by P2Y1 purinoceptors in different myenteric neurons

Slow excitatory postsynaptic potentials (EPSPs) in enteric neurons arise from diverse sources, but which neurotransmitters mediate specific types of slow EPSPs is unclear. We investigated transmitters and receptors mediating slow EPSPs in myenteric neurons evoked by electrical stimulation of the mucosa in guinea pig small intestine. Segments of ileum or jejunum were dissected to allow access to the myenteric plexus adjacent to intact mucosa, in vitro. AH and S neurons were impaled with conventional intracellular electrodes. Trains of stimuli delivered to the mucosa evoked slow EPSPs in AH neurons that were blocked or depressed by the neurokinin-1 (NK1) tachykinin antagonist SR140333 (100 nM) in 10 of 11 neurons; the NK3 tachykinin receptor antagonist SR142801 (100 nM) had no effect on slow EPSPs in seven of nine AH neurons. Single pulses to the mucosa evoked fast EPSPs and slow depolarizations in S neurons. The depolarizations were divided into intermediate (durations 300-900 ms) or slow (durations 1.3-9 s) EPSPs. The slow EPSPs were blocked by pyridoxal phosphate-6-axophenyl-2-4-disulfonic acid (30 microM, N = 3) or the specific P2Y(1) antagonist MRS 2179 (10 microM, N = 6) and were predominantly in anally projecting S neurons that were immunoreactive for nitric oxide synthase (NOS). In contrast, intermediate EPSPs were predominantly evoked in NOS-negative neurons; these were abolished by MRS 2179 (N = 8). Thus activation of pathways running from the mucosa excites three different types of slow EPSP in myenteric neurons, which are mediated by either a tachykinin (NK1, AH neurons) or a purine nucleotide (P2Y(1), S neurons).

NaV‐igating excitement in the enteric nervous system

Na_V‐igating excitement in the enteric nervous system

MGluR1 Receptors Contribute to Non-Purinergic Slow Excitatory Transmission to Submucosal VIP Neurons of Guinea-Pig Ileum

Vasoactive intestinal peptide (VIP) immunoreactive secretomotor neurons in the submucous plexus are involved in mediating bacterial toxin-induced hypersecretion leading to diarrhoea. VIP neurons become hyperexcitable after the mucosa is exposed to cholera toxin, which suggests that the manipulation of the excitability of these neurons may be therapeutic. This study used standard intracellular recording methods to systematically characterize slow excitatory postsynaptic potentials (EPSPs) evoked in submucosal VIP neurons by different stimulus regimes (1, 3 and 15 pulse 30 Hz stimulation), together with their associated input resistances and pharmacology. All slow EPSPs were associated with a significant increase in input resistance compared to baseline values. Slow EPSPs evoked by a single stimulus were confirmed to be purinergic, however, slow EPSPs evoked by 15 pulse trains were non-purinergic and those evoked by 3 pulse trains were mixed. NK1 or NK3 receptor antagonists did not affect slow EPSPs. The group I mGluR receptor antagonist, PHCCC reduced the amplitude of purinergic and non-purinergic slow EPSPs. Blocking mGluR1 receptors depressed the overall response to 3 and 15 pulse trains, but this effect was inconsistent, while blockade of mGluR5 receptors had no effect on the non-purinergic slow EPSPs. Thus, although other receptors are almost certainly involved, our data indicate that there are at least two pharmacologically distinct types of slow EPSPs in the VIP secretomotor neurons: one mediated by P2Y receptors and the other in part by mGluR1 receptors.

Synaptic transmission from the submucosal plexus to the myenteric plexus in Guinea‐pig ileum

Stimulation of the myenteric plexus results in activation of submucosal neurons and dilation of arterioles, one way that motility and secretion can be coupled together. The present study aimed to examine the converse, whether myenteric neurons receive synaptic input from the submucosal plexus (SMP). Intracellular recordings were made from guinea-pig ileal myenteric neurons while the SMP was electrically stimulated. Of the 29 neurons studied (13 S and 16 AH neurons), stimulation of the SMP evoked a synaptic potential in only seven cells, or 24% of neurons. When the SMP was situated oral to the myenteric plexus, 4 of 13 (31%) myenteric neurons had synaptic input. When it was situated circumferential, 2 of 8 (25%) had input, and when the SMP was situated anal 1 of 8 (13%) had input. Overall, 5 of the 13 (38%) S neurons responded with fast excitatory post-synaptic potentials (EPSPs), one of which also showed a slow EPSP, while 2 of the 16 (13%) AH neurons responded with a slow EPSP. This study indicates that the synaptic input from the SMP to myenteric neurons is relatively sparse. Whether this input is less important than the myenteric to submucosal input or simply represents a more selective form of control is unknown.

Evidence that TASK1 channels contribute to the background current in AH/type II neurons of the guinea-pig intestine

Neurons that have AH (designation of neurons with a prominent and prolonged afterhyperpolarizing potential that follows the action potential) electrophysiological characteristics and type II morphology (AH/type II neurons) are the first neurons in reflex circuits in the small intestine. Thus, the state of excitation of these neurons strongly influences the properties of enteric reflexes. The resting outward current in the type II neurons is reduced, causing depolarization and increased excitability, when protein kinase C (PKC) or synaptic inputs are activated, suggesting that regulation of background channels is an important determinant of the state of excitability of these neurons. However, the channels that carry the background current are not yet identified. We used intracellular microelectrodes to record from myenteric AH/type II neurons of the guinea-pig ileum, immunohistochemistry to localize channels and reverse transcriptase–polymerase chain reaction (RT-PCR) to characterize channel transcripts. The blockers of TASK1 channels, bupivacaine (1 mM) and methanandamide (10 μM), depolarized AH/type II neurons by 11.6 mV and 7.9 mV, respectively, and increased resting input resistance by about 30%. The reversal potential determined for the effect of bupivacaine was −92 mV, indicating that bupivacaine acts at K + channels, without significant action on other channel types that are open at rest. The membrane potential of type II neurons was depolarized by acidification to pH 6.4, but this depolarization was associated with decreased input resistance and was not reduced by bupivacaine. Thus an unidentified current that is activated by reduced pH masks effects on TASK channels. Slow excitatory post-synaptic potentials in the neurons were reduced in amplitude by methanandamide, suggesting that they are generated in part by closure of TASK1 channels. TASK1 immunoreactivity occurred in all type II neurons (determined by double labeling for IB4 and NeuN), but no type II neurons were immunoreactive for TASK2 or TASK3. These latter channels were localized to non-type II neurons. Transcripts for TASK1, TASK2, TASK3 and other two-pore-domain potassium channels were found in ganglion extracts. It is concluded that TASK1 channels contribute to the resting outward current in AH/type II neurons, and that neurotransmitters that evoke slow depolarizations in these neurons do so through the closure of resting K + channels that include TASK1 channels.