Fast EPSPs Research Articles

Electrophysiological and morphological features of myenteric neurons of human colon revealed by intracellular recording and dye fills.

Ex vivo intracellular recordings and dye fills, combined with immunohistochemistry, are a powerful way to analyze the enteric nervous system of laboratory animals. Myenteric neurons were recorded in isolated specimens of human colon. A key determinant of successful recording was near-complete removal of circular muscle from the surface of ganglia. Treatment with a collagenase/neutral protease mix before dissection significantly improved recording success and reduced damage to the plexus. Carboxyfluorescein in microelectrodes allowed recorded neurons to be routinely labeled, analyzed, and subjected to multi-layer immunohistochemistry. Carboxyfluorescein revealed morphological details that were not detected by immunohistochemical methods. Of 54 dye-filled myenteric neurons (n= 22), 45 were uni-axonal and eight were multi-axonal. There was a significant bias toward recordings from large neural somata. The close association between morphology and electrophysiology (long after-hyperpolarizations and fast EPSPs) seen in mice and guinea pigs did not hold for human myenteric neuron recordings. No slow EPSPs were recorded; however, disruption to the myenteric plexus during dissection may have led the proportion of cells receiving synaptic potentials to be underestimated. Neurons immunoreactive for nitric oxide synthase were more excitable than non-immunoreactive neurons. Distinctive grooves were observed on the serosal and/or mucosal faces of myenteric neurons in 3D reconstructions. These had varicose axons running through them and may represent a preferential site of synaptic inputs. Human enteric neurons share many features with laboratory animals, but the combinations of features in individual cells appear more variable.

HCN Channel Blockers Act Pre‐ and Post‐Synaptically to Inhibit Synaptic Transmission and Gain in the Rat Superior Cervical Sympathetic Ganglion

Hyperpolarization‐activated cation channels encoded by HCN subunits are permeable to Na+ and K+ ions and carry inward h‐currents. The h‐current (Ih) was originally discovered in the heart, where it is the principal pacemaker current. Ivabradine, a selective blocker of Ih, was approved in the US in 2015 as an adjunct to β‐adrenergic blockade for clinical management of heart failure. Recently we observed that sympathetic neurons in the rat superior cervical ganglion (SCG) express prominent Ih responses that can be blocked by ZD7288, another selective antagonist. The goal of the present study was to test the hypothesis that Ih modulates synaptic transmission in sympathetic ganglia. We did this using dissociated SCG neurons and using acutely isolated intact ganglia, treated with enzymes to permit whole‐cell recording, together with simultaneous presynaptic stimulation and extracellular recording. When synaptic transmission was elicited by submaximal 1 Hz stimulation in the isolated intact SCG, the extracellular postsynaptic compound action potential was inhibited by about 20% by 10–40 μM Ivabradine and by 10–20 μM ZD7288. In dissociated SCG neurons, we measured Ih under voltage clamp, fit the data to a Hodgkin‐Huxley model and then used the model to implement virtual Ih under dynamic clamp. When neurons were probed with virtual nicotinic fast EPSPs created with dynamic clamp, blocking Ih with 10–15 μM ZD7288 increased the threshold‐synaptic conductance (thresh‐gsyn) from 7.0 ± 0.9 nS to 12.1 ± 2.0 nS (p<0.0001) and decreased synaptic gain from 2.2 to 1.7 in response to noisy patterns of virtual synaptic activity that were designed to mimic physiological activity in vivo. This indicates that the native postsynaptic Ih normally enhances synaptic amplification to increase postsynaptic spike output by the SCG. When the native Ih was reconstituted with virtual Ih, synaptic amplification was restored. Shifting the voltage‐dependence of activation to mimic binding of cyclic AMP to HCN channels further modulated thresh‐gsyn and synaptic gain. To assess presynaptic actions of Ih, we then measured nicotinic cholinergic synaptic currents evoked by minimal presynaptic stimulation of the intact SCG. Ivabradine and ZD7288 both reduced peak EPSC amplitude and total charge by 40 to 50% while also increasing the failure rate. This indicates that native Ih normally enhances the probability of acetylcholine release and the amount of release by preganglionic terminals. Taken together the results suggest that clinical use of Ivabradine may have the heretofore unknown effect of reducing peripheral sympathetic activity through its pre‐ and postsynaptic effects on ganglionic transmission. This may have beneficial therapeutic consequences by countering the sympathetic hyperactivity that can drive human hypertension and that exacerbates the progression of heart failure.Support or Funding InformationSupported by the Great Rivers Affiliate of the American Heart Association through Grant‐in‐Aid 13GRNT1685007

Purinergic signalling in the enteric nervous system (An overview of current perspectives).

Purinergic Signalling in the Enteric Nervous System involves the regulated release of ATP (or a structurally-related nucleotide) which activates an extensive suite of membrane-inserted receptors (P2X and P2Y subtypes) on a variety of cell types in the gastrointestinal tract. P2X receptors are gated ion-channels permeable to sodium, potassium and calcium. They depolarise cells, act as a pathway for calcium influx to activate calcium-dependent processes and initiate gene transcription, interact at a molecular level as a form of self-regulation with lipids within the cell wall (e.g. PIP2) and cross-react with other membrane-inserted receptors to regulate their activity (e.g. nAChRs). P2Y receptors are metabotropic receptors that couple to G-proteins. They may release calcium ions from intracellular stores to activate calcium-dependent processes, but also may activate calcium-independent signalling pathways and influence gene transcription. Originally ATP was a candidate only for NANC neurotransmission, for inhibitory motoneurons supplying the muscularis externa of the gastrointestinal tract and bringing about the fast IJP. Purinergic signalling later included neuron-neuron signalling in the ENS, via the production of either fast or slow EPSPs. Later still, purinergic signalling included the neuro-epithelial synapse-for efferent signalling to epithelia cells participating in secretion and absorption, and afferent signalling for chemoreception and mechanoreception at the surface of the mucosa. Many aspects of purinergic signalling have since been addressed in a series of highly-focussed and authoritative reviews. In this overview however, the current focus is on key aspects of purinergic signalling where there remains uncertainty and ambiguity, with the view to stimulating further research in these areas.

Impact of basal forebrain cholinergic inputs on basolateral amygdala neurons.

In addition to innervating the cerebral cortex, basal forebrain cholinergic (BFc) neurons send a dense projection to the basolateral nucleus of the amygdala (BLA). In this study, we investigated the effect of near physiological acetylcholine release on BLA neurons using optogenetic tools and in vitro patch-clamp recordings. Adult transgenic mice expressing cre-recombinase under the choline acetyltransferase promoter were used to selectively transduce BFc neurons with channelrhodopsin-2 and a reporter through the injection of an adeno-associated virus. Light-induced stimulation of BFc axons produced different effects depending on the BLA cell type. In late-firing interneurons, BFc inputs elicited fast nicotinic EPSPs. In contrast, no response could be detected in fast-spiking interneurons. In principal BLA neurons, two different effects were elicited depending on their activity level. When principal BLA neurons were quiescent or made to fire at low rates by depolarizing current injection, light-induced activation of BFc axons elicited muscarinic IPSPs. In contrast, with stronger depolarizing currents, eliciting firing above ∼ 6-8 Hz, these muscarinic IPSPs lost their efficacy because stimulation of BFc inputs prolonged current-evoked afterdepolarizations. All the effects observed in principal neurons were dependent on muscarinic receptors type 1, engaging different intracellular mechanisms in a state-dependent manner. Overall, our results suggest that acetylcholine enhances the signal-to-noise ratio in principal BLA neurons. Moreover, the cholinergic engagement of afterdepolarizations may contribute to the formation of stimulus associations during fear-conditioning tasks where the timing of conditioned and unconditioned stimuli is not optimal for the induction of synaptic plasticity.

Hydrogen Sulfide Selectively Potentiates Central Preganglionic Fast Nicotinic Synaptic Input in Mouse Superior Mesenteric Ganglion

Hydrogen sulfide (H2S) plays important roles in the enteric system in the wall of the gastrointestinal tract. There have been no studies on whether H2S is endogenously generated in peripheral sympathetic ganglia and, if so, its effect on synaptic transmission. In this study, we examined the effect of H2S on cholinergic excitatory fast synaptic transmission in the mouse superior mesenteric ganglion (SMG). Our study revealed that NaHS and endogenously generated H2S selectively potentiated cholinergic fast EPSPs (F-EPSPs) evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. The H2S-producing enzyme cystathionine-γ-lyase (CSE) was expressed in both neurons and glial cells. The CSE blocker PAG (dl-propargylglycine) significantly reduced the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Inhibiting the breakdown of endogenously generated H2S with stigmatellin potentiated the amplitude of F-EPSPs evoked by splanchnic nerve stimulation but not F-EPSPs evoked by colonic nerve stimulation. Splanchnic F-EPSPs but not colonic F-EPSPs were reduced in CSE knock-out (KO) mice. Functional studies showed that NaHS enhanced the inhibitory effect of splanchnic nerve stimulation on colonic motility. Colonic motility in CSE-KO mice was significantly higher than colonic motility in wild-type mice. We conclude that endogenously generated H2S acted selectively on presynaptic terminals of splanchnic nerves to modulate fast cholinergic synaptic input and that this effect of H2S modulates CNS control of gastrointestinal motility. Our results show for the first time that the facilitatory effect of endogenous H2S in the mouse SMG is pathway specific.

183 Glial Transmission in Human Enteric Nervous System

the circumferentially projecting axons of DII neurons achieved full length by P10-11, but the orally and anally projecting axons of uniaxonal neurons grew significantly between P1011 and adult. Neither increase closely matched the increase in size of the small intestine. Uniaxonal neurons underwent major changes in dendritic morphology. While most adult uniaxonal neurons (>70%) had only lamellar dendrites, P0 uniaxonal neurons rarely had lamellar dendrites but have filamentous dendrites instead. Electrophysiological properties of postnatal DII neurons differed markedly from their adult counterparts. At P0, DII neurons rarely had AHPs; and at P10-11, AHPs were only evoked by trains of action potentials. Postnatal DII neurons had prominent Ca2+-mediated afterdepolarizing potentials (ADPs, up to 70 mV in amplitude from a starting membrane potential of -100 mV) and rarely exhibited slow EPSPs. Adult AH/DII neurons displayed AHPs and slow EPSPs, and only a small ADP. Like adult S neurons, at P10-11, S neurons exhibited fast EPSPs which were mainly mediated by nicotinic receptors. Conclusions: This is the first study to examine the postnatal development of electrophysiological andmorphological properties in the intact ENS. At P0, functional synapses are present and neurons can be differentiated electrophysiologically and morphologically into two classes resembling adult classifications, but major modifications occur later in development. When compared their adult counterparts, P10-11 DII neurons are mature morphologically, but not electrophysiologically, while the morphology of S/uniaxonal neurons remains immature.

182 Neural Circuit Behavior in Enteric Neural Plexus Developed From Neural Crest-Derived Stem Cells (ENPCs)

the circumferentially projecting axons of DII neurons achieved full length by P10-11, but the orally and anally projecting axons of uniaxonal neurons grew significantly between P1011 and adult. Neither increase closely matched the increase in size of the small intestine. Uniaxonal neurons underwent major changes in dendritic morphology. While most adult uniaxonal neurons (>70%) had only lamellar dendrites, P0 uniaxonal neurons rarely had lamellar dendrites but have filamentous dendrites instead. Electrophysiological properties of postnatal DII neurons differed markedly from their adult counterparts. At P0, DII neurons rarely had AHPs; and at P10-11, AHPs were only evoked by trains of action potentials. Postnatal DII neurons had prominent Ca2+-mediated afterdepolarizing potentials (ADPs, up to 70 mV in amplitude from a starting membrane potential of -100 mV) and rarely exhibited slow EPSPs. Adult AH/DII neurons displayed AHPs and slow EPSPs, and only a small ADP. Like adult S neurons, at P10-11, S neurons exhibited fast EPSPs which were mainly mediated by nicotinic receptors. Conclusions: This is the first study to examine the postnatal development of electrophysiological andmorphological properties in the intact ENS. At P0, functional synapses are present and neurons can be differentiated electrophysiologically and morphologically into two classes resembling adult classifications, but major modifications occur later in development. When compared their adult counterparts, P10-11 DII neurons are mature morphologically, but not electrophysiologically, while the morphology of S/uniaxonal neurons remains immature.

Synaptically evoked Ca2+release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones

The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

Habenula “Cholinergic” Neurons Corelease Glutamate and Acetylcholine and Activate Postsynaptic Neurons via Distinct Transmission Modes

Acetylcholine is an important neurotransmitter, and the habenulo-interpeduncular projection is a major cholinergic pathway in the brain. To study the physiological properties of cholinergic transmission in the interpeduncular nucleus (IPN), we used a transgenic mouse line in which the light-gated cation channel ChannelRhodopsin-2 is selectively expressed in cholinergic neurons. Cholinergic axonal terminals were activated by light pulses, and postsynaptic responses were recorded from IPN neurons. Surprisingly, brief photostimulation produces fast excitatory postsynaptic currents that are mediated by ionotropic glutamate receptors, suggesting wired transmission of glutamate. By contrast, tetanic photostimulation generates slow inward currents that are largely mediated by nicotinic acetylcholine receptors, suggesting volume transmission of acetylcholine. Finally, vesicular transporters for glutamate and acetylcholine are coexpressed on the same axonal terminals in the IPN. These results strongly suggest that adult brain "cholinergic" neurons can corelease glutamate and acetylcholine, but these two neurotransmitters activate postsynaptic neurons via different transmission modes.

Effects of Ethanol on Persistent Activity and Up‐States in Excitatory and Inhibitory Neurons in Prefrontal Cortex

Elucidating mechanisms that underlie the neural actions of ethanol is critical for understanding how this drug affects behavior. Increasing evidence suggests that, in addition to mid-brain dopaminergic regions, higher cortical structures play an important role in the pathophysiology associated with alcohol abuse. Previous studies from this laboratory used a novel slice co-culture system to demonstrate that ethanol reduces network-dependent patterns of activity in excitatory pyramidal neurons of the prefrontal cortex (PFC). In the present study, we examine the effect of ethanol on the activity of fast-spiking (FS) interneurons, a sub-population of neurons critically involved in shaping cortical activity. Slices containing the dorsolateral PFC were prepared from neonatal C57 mice and maintained in culture. After 2 to 3 weeks in vitro, whole-cell patch-clamp electrophysiology was used to monitor spontaneous episodes of persistent activity in prelimbic PFC neurons. In some experiments, the use-dependent NMDA receptor blocker, MK801, was included in the pipette recording solution to assess the contribution of NMDA receptors to up-states. MK801 reduced up-state amplitude and revealed underlying fast EPSPs in excitatory pyramidal neurons while having little effect on these parameters in FS interneurons. Despite this difference, ethanol (44 mM), significantly reduced up-state duration and up-state area in both pyramidal and FS interneurons. These results suggest that ethanol reduces the activity of FS interneurons due to disruption of network-dependent activity. This would be expected to further impair the ability of PFC networks to carry out their normal function and may contribute to the adverse effects of ethanol on PFC-dependent behaviors.

Multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the myenteric plexus of the guinea pig ileum

An important feature of the enteric nervous system (ENS) is its capability to respond to mechanical stimulation which, as currently suggested for the guinea-pig ileum, is encoded by specialized intrinsic primary afferent neurons (IPANs). We used von Frey hairs or intraganglionic volume injections to mimic ganglion deformation as observed in freely contracting preparations. Using fast voltage-sensitive dye imaging we identified rapidly adapting mechanosensitive enteric neurons (RAMEN, 25% of all neurons) in the myenteric plexus of the guinea pig ileum. RAMEN responded with phasic spike discharge to dynamic changes during ganglion deformation. This response was reproducible and increased with increasing forces. Deformation-evoked spike discharge was not changed by synaptic blockade with hexamethonium, omega-conotoxin or low Ca(2+)/high Mg(2+), defunctionalization of extrinsic afferents with capsaicin or muscle paralysis with nifedipine, suggesting direct activation of RAMEN. All RAMEN received hexamethonium-sensitive fast EPSPs, which were blocked by omega-conotoxin and low Ca(2+)/high Mg(2+). Seventy-two per cent of RAMEN were cholinergic, 22% nitrergic, and 44% were calbindin and NeuN negative, markers used to identify IPANs. Mechanosensitivity was observed in 31% and 47% of retrogradely traced interneurons and motor neurons, respectively. RAMEN belong to a new population of mechanosensitive neurons which differ from IPANs. We provided evidence for multifunctionality of RAMEN which may fulfil sensory, integrative and motor functions. In light of previously identified mechanosensitive neuron populations, mechanosensitivity appears to be a property of many more enteric neurons than generally assumed. The findings call for a revision of current concepts on sensory transmission within the ENS.

Mitochondrial Reactive Oxygen Species Inactivate Neuronal Nicotinic Acetylcholine Receptors and Induce Long-Term Depression of Fast Nicotinic Synaptic Transmission

Neuronal nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels implicated in a variety of cognitive, motor, and sensory behaviours, are targeted to compartments rich in mitochondria, particularly postsynaptic domains and presynaptic terminals, exposing these receptors to reactive oxygen species (ROS) generated by oxidative phosphorylation. In addition, these receptors can become exposed to ROS during the progression of certain neurodegenerative diseases. Because ROS are known to modify several membrane proteins, including some types of ion channels, it raises the question of whether elevations in cytosolic ROS alter the function of nAChRs. To address this, we elevated ROS in cultured sympathetic neurons, directly by perfusing neurons intracellularly with ROS, indirectly by blocking the mitochondrial electron transport chain, or noninvasively by transient NGF removal; we then simultaneously measured changes in cytosolic ROS levels and whole-cell ACh-evoked currents. In addition, we elevated cytosolic ROS in postganglionic neurons in intact ganglia and measured changes in nerve-evoked EPSPs. Our experiments indicate that mild elevations in cytosolic ROS, including that produced by transient interruption of NGF signaling, induce a use-dependent, long-lasting rundown of ACh-evoked currents on cultured sympathetic neurons and a long-lasting depression of fast nerve-evoked EPSPs. We show that these effects of cytosolic ROS are specific to nAChRs on neurons and do not cause rundown of ACh-evoked currents on muscle. Our results demonstrate that elevations in cytosolic ROS inactivate neuronal nAChRs in a use-dependent manner and suggest that mild oxidative stress impairs mechanisms mediated by cholinergic nicotinic signaling at neuronal-neuronal synapses.

Suprachiasmatic Modulation of Noradrenaline Release in the Ventrolateral Preoptic Nucleus

As the major brain circadian pacemaker, the suprachiasmatic nucleus (SCN) is known to influence the timing of sleep and waking. We thus investigated here the effect of SCN stimulation on neurons of the ventrolateral preoptic nucleus (VLPO) thought to be involved in promoting sleep. Using an acute in vitro preparation of the rat anterior hypothalamus/preoptic area, we found that whereas single-pulse stimulations of the SCN evoked standard fast ionotropic IPSPs and EPSPs, train stimulations unexpectedly evoked a long-lasting inhibition (LLI). Such LLIs could also be evoked in VLPO neurons by pressure application of NMDA within the SCN, indicating the specific activation of SCN neurons. This LLI was shown to result from the presynaptic facilitation of noradrenaline release, because it was suppressed in presence of yohimbine, a selective antagonist of alpha2-adrenoreceptors. The LLI depended on the opening of a potassium conductance, because it was annulled at E(K) and could be reversed below E(K). These results show that the SCN can provide an LLI of the sleep-promoting VLPO neurons that could play a role in the circadian organization of the sleep-waking cycle.

Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system

Nicotinic transmission in the enteric nervous system (ENS) is extensive, but the role of individual nicotinic acetylcholine receptor (nAChR) subtypes in the functional connectivity of its plexuses has been elusive. Using monoclonal antibodies (mAbs) against neuronal alpha3-, alpha4-, alpha3/alpha5-, beta2-, beta4- and alpha7-subunits, combined with radioimmunoassays and immunocytochemistry, we demonstrate that guinea-pig enteric ganglia contain all of these nAChR-subunits with the exception of alpha4, and so, differ from mammalian brain. This information alone, however, is insufficient to establish the functional role of the identified nAChR-subtypes within the enteric networks and, ultimately, their specific contributions to gastrointestinal physiology. We have used voltage-sensitive dyes and a high-speed CCD camera, in conjunction with specific antagonists to various nAChRs, to elucidate some of the distinct contributions of the individual subtypes to the behaviour of enteric networks. In the guinea-pig, the submucous plexus has the extraordinary advantage that it is virtually two-dimensional, permitting optical recording, with single cell resolution, of the electrical activity of all of its neurones. In this plexus, the block of alpha3beta2-, alpha3beta4- and/or alpha7-nAChRs always results in a decrease in the magnitude of the synaptic response. However, the magnitude of the fast excitatory post-synaptic potentials (epsps) evoked by electrical stimulation of a neighbouring ganglion varies from cell to cell, reflecting the differential expression of subunits already observed using mAbs, as well as the strengths of the activated synaptic inputs. At the same time, we observe that submucous neurones have a substantial mecamylamine (Mec)-insensitive (non-nicotinic) component to their fast epsps, which may point to the presence of purinergic or serotonergic fast epsps in this system. In the myenteric plexus, on the other hand, the antagonist-induced changes in the evoked synaptic response vary depending upon the location of the stimulating electrode with respect to the ganglion under study. The range of activity patterns that follows sequential pharmacological elimination of individual subtypes suggests that nAChRs may be capable of regulating the activity of both excitatory and inhibitory pathways, in a manner similar to that described in the central nervous system.

Synaptic transmission mediated by ionotropic glutamate, glycine and GABA receptors in the rat’s ventral nucleus of the lateral lemniscus

The synaptic pharmacology of the ventral nucleus of the lateral lemniscus (VNLL) was investigated in brain slices obtained from rats of 14–37 days old using intracellular recording techniques. Excitatory and inhibitory synaptic potentials (EPSPs and IPSPs) were elicited by electrical stimulation of the lemniscal pathway and recorded from neurons with five types of intrinsic firing patterns (onset, pause, adapting, regular and bursting types). Synaptic receptors that mediated the EPSPs and IPSPs were identified using AMPA, NMDA, GABA A and glycine receptor antagonists. The early/short EPSPs were mediated by AMPA receptors. The late/long EPSPs, encountered only in neurons of younger animals, were mediated by NMDA receptors. The IPSPs in most neurons were mediated by glycine receptors. In some neurons the IPSPs were mediated by GABA A receptors or both glycine and GABA A receptors. The temporal dynamics of fast AMPA EPSPs and glycinergic IPSPs were very similar. AMPA EPSPs and glycinergic (and/or GABAergic) IPSPs could be encountered in a single neuron. The results suggest that the VNLL not only relays incoming signals rapidly from the lower brainstem to the inferior colliculus, but also integrates excitatory and inhibitory inputs to modify and process auditory information.

PACAP modulation of the colon-inferior mesenteric ganglion reflex in the guinea pig.

We investigated the effect of pituitary adenylate cyclase activating peptide (PACAP) on the colon-inferior mesenteric ganglion (IMG) reflex loop in vitro. PACAP27 and PACAP38 applied to the IMG caused a prolonged depolarization and intense generation of fast EPSPs and action potentials in IMG neurones. Activation of PACAP-preferring receptors (PAC1-Rs) with the selective agonist maxadilan or vasoactive intestinal peptide (VIP)/PACAP (VPAC) receptors with VIP produced similar effects whereas prior incubation of the IMG with selective PAC1-R antagonists PACAP6-38 and M65 inhibited the effects of PACAP. Colonic distension evoked a slow EPSP in IMG neurones that was reduced in amplitude by prolonged superfusion of the IMG with either PACAP27, maxidilan, PACAP6-38, M65 or VIP. Activation of IMG neurones by PACAP27 or maxadilan resulted in an inhibition of ongoing spontaneous colonic contractions. PACAP-LI was detected in nerve trunks attached to the IMG and in varicosities surrounding IMG neurones. Cell bodies with PACAP-LI were present in lumbar 2-3 dorsal root ganglia and in colonic myenteric ganglia. Colonic distension evoked release of PACAP peptides in the IMG as measured by radioimmunoassay. Volume reconstructed images showed that a majority of PACAP-LI, VIP-LI and VAChT-LI nerve endings making putative synaptic contact onto IMG neurones and a majority of putative receptor sites containing PAC1-R-LI and nAChR-LI on the neurones were distributed along secondary and tertiary dendrites. These results suggest involvement of a PACAP-ergic pathway, operated through PAC1-Rs, in controlling the colon-IMG reflex.

Presynaptic modulation of cholinergic and non-cholinergic fast synaptic transmission in the myenteric plexus of guinea pig ileum.

Abstract These studies investigated receptors modulating release of mediators of fast excitatory postsynaptic potentials (fEPSPs) in guinea pig ileum myenteric plexus using electrophysiological methods. Fast EPSPs inhibited by >95% by hexamethonium (100 micromol L(-1)) were cholinergic; mixed fEPSPs were inhibited <95% by hexamethonium. Non-cholinergic fEPSPs were studied in the presence of hexamethonium. The alpha2-adrenergic receptor agonist UK 14304 inhibited cholinergic (maximum inhibition = 76%, EC(50) = 18 nmol L(-1)), mixed (81%, 21 nmol L(-1)) and non-cholinergic (76%, 44 nmol L(-1)) fEPSPs equally. The 5-HT(1) receptor agonist 5-carboxamidotryptamine inhibited cholinergic, mixed and non-cholinergic fEPSPs equally. Renzapride, increased non-cholinergic (33%) less than mixed (97%, 13 micromol L(-1)) fEPSPs. Renzapride inhibited the purely cholinergic fEPSPs (-29%) but potentiated the cholinergic component of mixed fEPSPs (39%). Prucalopride potentiated all fEPSPs equally (30-33%). 5-HT (0.1 micromol L(-1)) induced potentiation of cholinergic (75%), mixed (97%) and non-cholinergic (84%) fEPSPs was not statistically different. The potentiating effects of renzapride and 5-HT on fEPSPs were inhibited by the 5-HT(4) receptor antagonist, SB 204070 (10 nmol L(-1)). Renzapride (0.3 micromol L(-1)) blocked 5-HT-induced increases in cholinergic fEPSPs. alpha2-Adrenergic and 5-HT(1) receptors mediate inhibition of transmitter release from cholinergic and mixed terminals. 5-HT and prucalopride, acting at 5-HT(4) receptors, facilitate all fEPSPs; renzapride facilitates the cholinergic and non-cholinergic components of mixed fEPSPs but not purely cholinergic fEPSPs. Cholinergic synapses may express few 5-HT(4) receptors or a renzapride-insensitive 5-HT(4) receptor isoform.

Cyclooxygenase-2 contributes to dysmotility and enhanced excitability of myenteric AH neurones in the inflamed guinea pig distal colon.

We have previously demonstrated that trinitrobenzene sulphonic acid (TNBS)-induced colitis in guinea pig is associated with hyperexcitability of myenteric AH neurones, enhanced synaptic activity in the myenteric plexus, increased serotonin (5-HT) availability in the mucosa, and decreased propulsive motor activity. The current study tested the hypothesis that the activation of cyclooxygenase (COX) contributes to these alterations in bowel functions. DFU inhibition of COX-2, but not SC-560 inhibition of COX-1, restored to normal levels the electrical properties of myenteric AH neurones, the proportion of S neurones exhibiting slow EPSPs, and the rate of propulsive motor activity. Neither inhibitor was effective in altering the level of inflammation, the increased availability of mucosal 5-HT, or the enhanced fast EPSPs in myenteric AH and S neurones. COX-2 expression is enhanced in the myenteric plexus and cells within the smooth muscle layers during colitis, possibly reflecting the site at which COX-2 inhibition acts to allow recovery of motor function. In support of this concept, COX-1, but not COX-2, inhibition was effective in restoring normal mucosal prostaglandin levels. These results indicate that the various changes that occur in the motor neural pathways of the distal colon in TNBS-induced colitis do not involve a single neuroimmune mechanism. COX-2 activation is a critical step in the enhanced excitability of AH neurones as well as diminished propulsive motility in TNBS colitis, whereas other yet to be resolved pathways, that do not involve COX-1 or COX-2 activation, lead to altered 5-HT content in the mucosa and an augmentation of fast EPSPs.

Actions of orexin-A in the myenteric plexus of the guinea-pig small intestine.

Orexins and orexin-receptors are localized by displaying their immunoreactivity in the enteric nervous system. Intracellular recordings were made from isolated myenteric neurons to investigate actions of orexin-A in the myenteric plexus of the guinea-pig ileum. Superfusion of orexin-A caused membrane depolarizations in a subset of S and AH neurons. Orexin-A responses were preserved in Ca2+ free/high Mg2+ solution and associated with an increase in input membrane resistance; their reversal potential was about -90 mV. Orexin-A augmented nicotinic fast EPSPs, whereas it did not affect the postsynaptic sensitivity to acetylcholine; this indicates that orexin-A increased the presynaptic release of acetylcholine. In conclusion, orexin-A contributes in the regulation of gut motility via its pre- and postsynaptic actions in the myenteric plexus.

Mast cell tryptase and proteinase-activated receptor 2 induce hyperexcitability of guinea-pig submucosal neurons.

Mast cells that are in close proximity to autonomic and enteric nerves release several mediators that cause neuronal hyperexcitability. This study examined whether mast cell tryptase evokes acute and long-term hyperexcitability in submucosal neurons from the guinea-pig ileum by activating proteinase-activated receptor 2 (PAR2) on these neurons. We detected the expression of PAR2 in the submucosal plexus using RT-PCR. Most submucosal neurons displayed PAR2 immunoreactivity, including those colocalizing VIP. Brief (minutes) application of selective PAR2 agonists, including trypsin, the activating peptide SL-NH2 and mast cell tryptase, evoked depolarizations of the submucosal neurons, as measured with intracellular recording techniques. The membrane potential returned to resting values following washout of agonists, but most neurons were hyperexcitable for the duration of recordings (> 30 min-hours) and exhibited an increased input resistance and amplitude of fast EPSPs. Trypsin, in the presence of soybean trypsin inhibitor, and the reverse sequence of the activating peptide (LR-NH2) had no effect on neuronal membrane potential or long-term excitability. Degranulation of mast cells in the presence of antagonists of established excitatory mast cell mediators (histamine, 5-HT, prostaglandins) also caused depolarization, and following washout of antigen, long-term excitation was observed. Mast cell degranulation resulted in the release of proteases, which desensitized neurons to other agonists of PAR2. Our results suggest that proteases from degranulated mast cells cleave PAR2 on submucosal neurons to cause acute and long-term hyperexcitability. This signalling pathway between immune cells and neurons is a previously unrecognized mechanism that could contribute to chronic alterations in visceral function.