Trung V. Nguyen

Phenotypic changes of morphologically identified guinea-pig myenteric neurons following intestinal inflammation

We investigated the responses of morphologically identified myenteric neurons of the guinea‐pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin‐resistant component, and a prolonged after‐hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca2+‐dependent K+ channel blocker TRAM‐34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin‐resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (Ih) antagonist Cs+. It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

Myenteric neurons of the mouse small intestine undergo significant electrophysiological and morphological changes during postnatal development

•  Different functional types of neurons within the gut wall form circuits that regulate intestinal motility. •  To examine the postnatal development of electrical properties of different classes of enteric neurons, we performed intracellular recordings from neurons in the mouse duodenum at three ages: postnatal day (P)0, P10–11 and adult. •  Like adults, two main morphological classes of neurons are present at P0 and P10–11. P0 and P10–11 neurons with Dogiel type II (DII) morphology had multiple long processes that achieved their adult projection length by P10–11. However, they differed electrophysiologically from adult DII neurons in that they displayed prominent afterdepolarizing potentials. •  Most electrical properties of neurons with a single long process were mature by P10–11. However, these neurons showed significant postnatal changes in morphology and projection length. •  Major morphological and electrophysiological changes in enteric neurons occur postnatally, which could underlie changes in gut motility during development.

Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system

Hyperpolarisation-activated non-specific cation currents (Ih currents) are important for the regulation of cell excitability. These currents are carried by channels of the hyperpolarisation-activated nucleotide-gated (HCN) family, of which there are four known subtypes. In the enteric nervous system (ENS), the Ih current is prominent in AH neurons. We investigated the expression and localization of HCN isoforms in the ENS of mice, rats and guinea-pigs. HCN1, HCN2 and HCN4 were expressed in enteric neurons. Immunoreactivity for HCN1 was observed on neuronal cell membranes of Dogiel type II neurons in rat and mouse. HCN2 channel immunoreactivity occurred in the majority of enteric neurons in the guinea-pig, rat and mouse. Immunoreactivity for HCN4 protein was revealed on the cell membranes of many neurons, including Dogiel type II neurons, in the guinea-pig. HCN4 was expressed by glial cells in guinea-pig. There was no evidence of HCN3 channel protein in any species with either immunohistochemistry or Western analysis. RT-PCR (polymerase chain reaction) using mouse HCN primers revealed mRNA for all four channels in the longitudinal muscle plus myenteric plexus of mouse distal colon. Sequencing confirmed the identity of the mRNA. Quantitative PCR demonstrated that HCN2 was the most highly expressed HCN channel subtype in the myenteric plexus of mouse distal colon. HCN1 and HCN4 were expressed at lower levels. HCN3 subtype mRNA was 0.2% of HCN2. We used intracellular recording to identify neurons having Ih currents and intracellular dye filling to locate the neurons for the immunohistochemical determination of channel expression. AH neurons with Ih currents were HCN2 and HCN4 channel positive. There was no correlation between the magnitude of the Ih and intensity of channel immunoreactivity. Our results indicate that HCN1, 2 and 4 genes and protein are expressed in the ENS. AH/Dogiel type II neurons, which have a prominent Ih, express HCN2 and 4 in guinea-pig and HCN1 and 2 in mouse and rat.

The role of neural activity in the migration and differentiation of enteric neuron precursors.

Background  As they migrate through the developing gut, a sub‐population of enteric neural crest‐derived cells (ENCCs) begins to differentiate into neurons. The early appearance of neurons raises the possibility that electrical activity and neurotransmitter release could influence the migration or differentiation of ENNCs.

Sites of action of ghrelin receptor ligands in cardiovascular control.

Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.

Investigation of PKC isoform‐specific translocation and targeting of the current of the late afterhyperpolarizing potential of myenteric AH neurons

AH neurons in the enteric nervous system play an essential role in initiating intestinal reflexes and factors that control AH neuron excitability therefore influence the state of the digestive system. Prominent afterhyperpolarizations that follow action potentials in these neurons strongly affect their excitability. In the present work, we have investigated the regulation of the afterhyperpolarizing current (IAHP) by protein kinase C (PKC). Electrophysiological responses and protein translocation were investigated in AH neurons of freshly dissected preparations of myenteric ganglia from the guinea‐pig ileum. The activator of conventional and novel PKCs, phorbol dibutyrate, but not the activator of novel PKCs, ingenol, blocked the IAHP. Phorbol dibutyrate had no effect on the hyperpolarization‐activated current (Ih) or on the A current (IA). Stimulation of synaptic inputs to the neurons also reduced the IAHP, and had no effect on Ih or IA. Phorbol dibutyrate also reduced a background outward current that was present after the IAHP current had been blocked by clotrimazole. Both phorbol dibutyrate and ingenol caused translocation of the novel PKC, PKCε, in these neurons. Only phorbol dibutyrate caused translocation of PKCγ, a conventional PKC. The studies thus indicate that the activation of PKC by phorbol esters and by nerve stimulation affects AH neurons in a similar way, and that PKC activation targets both the IAHP and another background K+ current. The IAHP is targeted by a conventional PKC, suggested to be PKCγ, as this is the only conventional PKC that is prominent in AH neurons.

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.

Hypotensive effects of ghrelin receptor agonists mediated through a novel receptor

Some agonists of ghrelin receptors cause rapid decreases in BP. The mechanisms by which they cause hypotension and the pharmacology of the receptors are unknown.

Identification of subunits of voltage-gated calcium channels and actions of pregabalin on intrinsic primary afferent neurons in the guinea-pig ileum

Background  The intrinsic primary afferent neurons (IPANs) in the intestine are the first neurons of intrinsic reflexes. Action potential currents of IPANs flow partly through calcium channels, which could feasibly be targeted by pregabalin. The aim was to determine whether pregabalin‐sensitive α2δ1 subunits associate with calcium channels of IPANs and whether α2δ1 subunit ligands influence IPAN neuronal properties.

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 after hyperpolarizing 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 muM), 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.