The enteric neural network in the proximal murine colon shows a regularly occurring hypoganglionic region, which is here characterized by using anatomical and electrophysiological techniques. Staining with NADPH diaphorase, methylene blue, and cuprolinic blue in standard whole mounts and three-dimensional gut preparations of the murine proximal colon consistently revealed two hypoganglionic areas surrounded by a dense clustering of enteric neurons. This irregularity in the ganglionic plexus was found to be present in mice of three different genetic backgrounds, as well as in rats. The lack of myenteric ganglia in these regions was associated with an absence of the longitudinal muscle layer, as shown in cross sections. Histochemical identification of interstitial cells of Cajal in Kit W-lacZ/ϩ transgenic mice showed Kit-positive cells oriented parallel to both muscle layers of the colon. Kit-positive cells oriented parallel to the longitudinal muscle layers were absent in the hypoganglionic area described. Electrical field stimulation elicited TTX-sensitive inhibitory junction potentials (IJPs), which showed region-specific characteristics. The initial partly apamin-sensitive hyperpolarization was present in all parts of the murine colon, whereas a second sustained N G -nitro-L-argininesensitive hyperpolarization was absent in the cecum and decreased from the proximal to the distal colon. Dissecting the hypoganglionic area from the surrounding tissue abolished the otherwise normal inhibitory neurotransmission to the circular muscle (1.6 Ϯ 1.4 and 2.6 Ϯ 1.7 mV for the fast and slow component of IJP amplitude in the hypoganglionic area vs. 16.5 Ϯ 1.9 and 23.7 Ϯ 2.7 mV for the fast and slow component of IJP amplitude in the neuron-rich area, respectively, P Ͻ 0.01, n ϭ 6), whereas dissection of an area of identical size with an intact myenteric network showed normal inhibitory neurotransmission, indicating that the hypoganglionic area receives essential functional neural input from the neuron-rich surrounding tissue. In summary, in the murine and rat proximal colon, a constant and distinct hypoganglionic region is described with important concomitant changes in local electrophysiology. mouse; enteric nervous system; smooth muscle; inhibitory junction potentials SINCE THE ORIGINAL DESCRIPTION of the enteric nervous system in the small bowel by Auerbach (1), it has been believed that the enteric ganglia form a regular network over the entire length of the gut wall. Distribution of subgroups of enteric neurons is known to vary along the longitudinal axis of the gut as demonstrated, for example, for nitric oxide (NO) synthase (NOS)-containing neurons (25, 26), but it is assumed that there is a homogeneous distribution of the neural network within a short gut segment. The shape of the small and large intestine in mammals resembles an elongated hollow organ. In contrast to simple tubes, however, the gut has to generate the force needed to ensure intraluminal transport (6,14). Accordingly, a sophisticated neural and muscul...

The aim of the present study was to investigate the effects of melatonin on non-adrenergic, non-cholinergic (NANC) relaxant neurotransmission in the gastrointestinal tract, which is mainly mediated by nitrergic and peptidergic mechanisms. Melatonin (10(-7)-10(-3) M) had no effect on the basal tonus of the rat gastric fundus smooth muscle. Relaxant responses following electrical stimulation(40 V; 0.5 ms pulse duration; 10 s stimulation duration) under NANC conditions on a 5-hydroxytryptamine (5-HT, 10(-7) M) contraction plateau were elicited at frequencies in the range of 0.5-16 Hz. Melatonin significantly reduced these inhibitory NANC responses (16 Hz without melatonin: -103 +/- 6.3%; melatonin 10(-5) M: -80.4 +/- 7.5%; melatonin 10(-4) M: -39.1 +/- 17.1%). Intracellular recording was carried out in a mouse colonic preparation. Electrical neural stimulation of the mouse colonic neurons caused biphasic intracellular hyperpolarization in smooth-muscle cells. The initial fast component is apamin-sensitive, and the following slow component is dependent on nitrergic mechanisms, as it is abolished in the presence of NG-nitro-L-arginine (L-NNA). Melatonin significantly reduced the nitric oxide-dependent slow component of neurally transmitted hyperpolarization, whereas the initial fast component was left unchanged. In a synaptosomal preparation of the enteric nervous system of rat intestine, enzymatic nitric oxide synthase (NOS) activity was significantly reduced by melatonin at concentrations ranging from 10(-7) to 10(-4) M (basal preparation including cofactors: 61.2 +/- 9.4 fmol/mg; melatonin 10(-4) M: 39.2 +/- 6.9 fmol/mg). Reverse transcriptase-polymerase chain reaction (RT-PCR) studies were conducted to investigate the melatonin receptors (mt(1), MT(2) and MT(3)) present in the esophagus, stomach and ileum of the rat. The presence of mt1 mRNA expression alone, but not of mRNA expression for MT(2) or MT(3), was demonstrated in the tissues. In conclusion, this study demonstrates that melatonin reduces the functional inhibitory NANC response. It shows that this effect may be the result of a reduction of the nitrergic component of the smooth-muscle inhibitory junction potential (IJP) and related to direct inhibition of NOS activity in enteric synaptosomes. The presence of mt1 receptor transcripts adds supportive evidence for a possible physiological role of melatonin within the enteric nervous system.

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1 Previous studies suggested that nitric oxide (NO) may cause hyperpolarization and relaxation of canine colonic smooth muscle by both cGMP-dependent and cGMP-independent mechanisms. This hypothesis was tested using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a novel inhibitor of NO-stimulated guanylate cyclase. 2 In the presence of histamine (30 mM), atropine and indomethacin (both at 1 mM), electrical ®eld stimulation of intrinsic neurons (EFS; 5 Hz) produced inhibition of phasic contractile activity that is due to NO synthesis. ODQ caused a concentration-dependent block of this response (10 nM to 10 mM). 3 Inhibitory junction potentials (IJPs) due to NO synthesis were recorded from muscle cells located near the myenteric border of the circular muscle layer, using intracellular microelectrodes. IJPs were abolished by ODQ (1 ± 10 mM). 4 EFS (10 ± 20 Hz) produced frequency-dependent inhibition of electrical slow waves recorded from cells located near the submucosal surface of the circular muscle layer. This inhibition is due to NO synthesis, and it was abolished by ODQ (1 ± 10 mM). 5 Hyperpolarization and relaxation produced by an NO donor, sodium nitroprusside, were abolished by ODQ pretreatment (1 ± 10 mM). In contrast, inhibitory responses to 8-Br-cGMP (1 mM) were unaected by ODQ. 6 ODQ alone (1 ± 10 mM) had no signi®cant eect on spontaneous electrical or phasic contractile activity. In tissues pre-treated with L-NAME (300 mM), ODQ decreased the amplitude of spontaneous or histamine-stimulated phasic contractile activity. 7 These results suggest that electrical and mechanical eects of endogenously released and exogenously applied NO in canine colon are largely due to cGMP synthesis by ODQ-sensitive soluble guanylate cyclase. No evidence to support a direct (cGMP-independent) mechanism of NO action was found. ODQ also appears to cause a non-speci®c inhibition of muscle contractile activity; however, this eect does not contribute to block of NO-dependent eects.

1. In order to investigate purinergic effects on rat ileal smooth muscle, we used alpha,beta-methylene ATP (alpha,beta-MeATP), ATP, ADP and UTP. alpha,beta-Methylene ATP and ATP were the only agonists that caused a concentration-dependent inhibition of carbachol-precontracted smooth muscle. The inhibitory effect of alpha,beta-MeATP was completely blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (3 x 10(-5) mol/L), a selective antagonist of the P2X > > P2Y receptor. 2. Using reverse transcription-polymerase chain reaction we demonstrated the presence of both, P2X and P2Y receptor mRNA within the rat ileal longitudinal muscle/myenteric plexus layer preparation. 3. The alpha,beta-MeATP-induced inhibition was blocked in a concentration-dependent manner in the presence of the K+ channel blocker apamin, but was unaffected by other K+ channel blockers, such as charybdotoxin (10(-7) mol/L), 4-aminopyridine (10(-4)mol/L), glibenclamide (10(-5) mol/L) and tetraethylammonium (10(-3) mol/L). 4. The alpha,beta-MeATP-induced inhibition was unaffected by pretreatment with atropine (10(-6) mol/L), phentolamine (10(-6) mol/L), propranolol (10(-6) mol/L), nitrendipine (10(-7) mol/L), pertussis toxin (10(-6) mol/L) NG-nitro-L-arginine (3 x 10(-4) mol/L) and tetrodotoxin (10(-6) mol/L), excluding an involvement of adrenergic, cholinergic, neural, nitrinergic or G-protein involvement in purinergic-mediated inhibition. 5. In order to investigate whether the internal Ca2+ stores participated in the inhibitory effect observed, we depleted internal Ca2+ stores with cyclopiazonic acid, a specific Ca2+-ATPase inhibitor. The inhibitory effect of alpha,beta-MeATP was completely abolished after depletion of the intracellular Ca2+ stores. 6. This is in contrast with the effects seen for neurotensin, where neurotensin-induced inhibition was unchanged after depletion of intracellular Ca2+ stores, suggesting at least two different pathways of apamin-sensitive non-adrenergic, non-cholinergic inhibition in rat ileal smooth muscle. 7. According to our results, the inhibitory effect of alpha,beta-MeATP in rat ileum longitudinal smooth muscle is mediated via a P2 purinoceptor, most likely a P2X receptor, involves G-protein-independent activation of an apamin-sensitive K+ channel and requires filled intracellular Ca+ stores.