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The functional significance of the slow excitatory synaptic potentials (EPSPs) in myenteric neurones is unknown. We investigated this using intracellular recording from myenteric neurones in guinea-pig ileum, in vitro. In all, 121 neurones responded with fast EPSPs to distension of the intestine oral to the recording site. In 28 of these neurones, distension also evoked depolarizations similar to the slow EPSPs evoked by electrical stimulation in the same neurones. Intracellular injection of biocytin and immunohistochemistry revealed that neurones responding to distension with slow EPSPs were descending interneurones, which were immunoreactive for nitric oxide synthase (NOS). Other neurones, including inhibitory motor neurones and interneurones lacking NOS, did not respond to distension with slow EPSPs, but many had slow EPSPs evoked electrically. Slow EPSPs evoked electrically or by distension in NOS-immunoreactive descending interneurones were resistant to blockade of NK 1 or NK 3 tachykinin receptors (SR 140333, 100 n; SR 142801, 100 n, respectively) and group I metabotropic glutamate receptors (PHCCC, 10-30 m), when the antagonists were applied in the recording chamber of a two-chambered organ bath. However, slow EPSPs evoked electrically in inhibitory motor neurones were substantially depressed by SR 140333 (100 n). Blockade of synaptic transmission in the stimulation chamber of the organ bath abolished slow EPSPs evoked by distension, indicating that they arose from activity in interneurones, and not from anally directed, intrinsic sensory neurones. Thus, distension evokes slow EPSPs in a subset of myenteric neurones, which may be important for intestinal motility. lasting at least 10 s and mediated by a decrease in potassium conductance, slow EPSPs (Wood & Mayer, 1978; Johnson et al. 1980; Bornstein et al. 1984). However, there have been no reports of distension evoking slow EPSPs in the myenteric neurones that make up the descending reflex pathways (Hirst et al. 1975; Smith et al. 1992).The aim of the present study was to systematically explore the possibility that distension evokes slow EPSPs in neurones of the descending reflex pathways in the guineapig ileum. Neurones in the descending pathway were identified because they responded to distension with fast EPSPs and the stimulus was then repeated under recording conditions that optimized the possibility of detecting slow EPSPs. Impaled neurones were injected with an intracellular marker, biocytin, so that their morphologies, projections and immunoreactivity for nitric oxide synthase (NOS) could be used to determine to which functional class they belonged (Stebbing & Bornstein, 1996). The pharmacology of slow EPSPs evoked by distension was explored with specific antagonists acting at NK 1 and NK 3 tachykinin receptors, because there is evidence that these two classes of receptor are involved in the descending inhibitory pathway (Johnson et al. 1996(Johnson et al. , 1998. The effect of a group 1 metabotropic glutamate receptor (mGluR1) antagonist,...

BackgroundThe nature of synaptic transmission at functionally distinct synapses in intestinal reflex pathways has not been fully identified. In this study, we investigated whether transmission between interneurons in the descending inhibitory pathway is mediated by a purine acting at P2Y receptors to produce slow excitatory synaptic potentials (EPSPs).Methodology/Principal findingsMyenteric neurons from guinea-pig ileum in vitro were impaled with intracellular microelectrodes. Responses to distension 15 mm oral to the recording site, in a separately perfused stimulation chamber and to electrical stimulation of local nerve trunks were recorded. A subset of neurons, previously identified as nitric oxide synthase immunoreactive descending interneurons, responded to both stimuli with slow EPSPs that were reversibly abolished by a high concentration of PPADS (30 μM, P2 receptor antagonist). When added to the central chamber of a three chambered organ bath, PPADS concentration-dependently depressed transmission through that chamber of descending inhibitory reflexes, measured as inhibitory junction potentials in the circular muscle of the anal chamber. Reflexes evoked by distension in the central chamber were unaffected. A similar depression of transmission was seen when the specific P2Y1 receptor antagonist MRS 2179 (10 μM) was in the central chamber. Blocking either nicotinic receptors (hexamethonium 200 μM) or 5-HT3 receptors (granisetron 1 μM) together with P2 receptors had no greater effect than blocking P2 receptors alone.Conclusions/SignificanceSlow EPSPs mediated by P2Y1 receptors, play a primary role in transmission between descending interneurons of the inhibitory reflexes in the guinea-pig ileum. This is the first demonstration for a primary role of excitatory metabotropic receptors in physiological transmission at a functionally identified synapse.

The plant lectin Bandeiraea simplicifolia I-isolectin B4 (BSI-B4) identifies a galactose-containing, membrane-associated glycoconjugate expressed by a discrete subpopulation of unmyelinated primary sensory neurones in the rat. We have previously suggested that BSI-B4 selectively binds to primary sensory neurones that innervate the skin. However, in that study, the tracer diamidino yellow was applied to the cut ends of peripheral nerves to identify neurones innervating particular target tissues. In this study, we have avoided axotomy by retrogradely labelling primary sensory neurones from peripheral tissues using the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbacyanine perchlorate (DiI). DiI was injected into the plantar skin, gastrocnemius muscle, and pyloric region of the stomach in rats. Corresponding ganglia were sectioned, incubated in BSI-B4 conjugated to fluorescein isothiocyanate, and examined with a fluorescence microscope. DiI-labelled cells were identified by red fluorescence within the cytoplasm, whereas cells binding BSI-B4 displayed green fluorescence associated with the plasma membrane and Golgi apparatus. Quantitative analysis revealed that 36.2% of cutaneous neurones, 7.6% of muscle neurones, and 6.8% of visceral neurones expressed the BSI-B4-binding site, indicating that a small but significant proportion of small-diameter primary sensory neurones innervating muscle and viscera also express BSI-B4-binding sites.

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