Responses of myenteric AH and S neurons to local application of chemicals to the mucosa of the guinea pig small intestine were obtained using conventional intracellular recording techniques. Preparations were dissected to reveal the myenteric plexus over one-half of the circumference of the gut with intact mucosa on the the half. Neurons were impaled within the exposed one-half, whereas potential stimulants, in buffered saline, were transiently applied to the mucosa, 1-1.5 mm circumferential from the impalement. The stimulants elicited action potentials (AP) in AH neurons that did not arise from synaptic activity. AH neurons also responded with slow excitatory postsynaptic potentials (EPSP). S neurons were activated synaptically, via fast and slow EPSP, but not nonsynaptically. Mucosal application of solutions of a low pH (3-5) or a high pH (9-11) were both effective stimulants. Solutions of a neutral pH, which was also a control for mild mechanical stimulation, were usually ineffective. Both a short-chain fatty acid, acetate (pH 7.2), and 5-hydroxytryptamine elicited responses in each neuronal type. We conclude that myenteric AH neurons of the guinea pig distal ileum are primary afferent neurons that respond to a variety of mucosally applied chemical stimuli with burst of AP. In addition, the physiologically evoked transmission of slow EPSP to AH neurons suggests that primary afferent neurons interconnect in a self-reinforcing network. S neurons are second or higher order neurons in the reflex pathways.

The aim of the present paper was to determine the electrotonic properties of myenteric neurones, using patchclamp recording from non-dissociated myenteric neurones, with emphasis on the identification and quantitation of the ionic currents that modulate the resting membrane potential of AH neurones. Recordings were made with a technique we have recently developed for patch-clamp recording from intact ganglia (Kunze et al. 2000).The electrophysiological properties of myenteric neurones in intact ganglia from the small intestine of the guinea-pig have been investigated previously by intracellular recordings in myenteric plexus/longitudinal muscle preparations. The first intracellular recording studies were performed using duodenal (Hirst et al. 1974) and ileal tissue (Nishi & North, 1973). In both of these parts of the intestine, the studies separated the myenteric neurones into two groups, S and AH neurones, terms that were introduced in 1974 for duodenal neurones (Hirst et al. 1974). S neurones were so named because they received prominent fast synaptic inputs, while AH neurones did not. AH neurones were so named because the action potential is followed by a long-lasting afterhyperpolarization (AHP). It was later demonstrated that both cell types receive slow EPSPs (Wood & Meyer, 1978; Johnson et al. 1980 Johnson et al. , 1981 Bornstein et al. 1984). Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact gangliaFrançois Rugiero, Maurice Gola, Wolf A. A. Kunze *, Jean-Claude Reynaud, John B. Furness * and Nadine Clerc Laboratoire 'Intégration des Informations Sensorielles' (ITIS), CNRS, Bâtiment LNB, No. 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France and *Department of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC 3010, Australia Whole-cell patch-clamp recordings taken from guinea-pig duodenal myenteric neurones within intact ganglia were used to determine the properties of S and AH neurones. Major currents that determine the states of AH neurones were identified and quantified. S neurones had resting potentials of _47 ± 6 mV and input resistances (R in ) of 713 ± 49 MV at voltages ranging from _90 to _40 mV. At more negative levels, activation of a time-independent, caesium-sensitive, inwardrectifier current (I Kir ) decreased R in to 103 ± 10 MV. AH neurones had resting potentials of _57 ± 4 mV and R in was 502 ± 27 MV. R in fell to 194 ± 16 MV upon hyperpolarization. This decrease was attributable mainly to the activation of a cationic h current, I h , and to I Kir . Resting potential and R in exhibited a low sensitivity to changes in [K + ] o in both AH and S neurones. This indicates that both cells have a low background K + permeability. The cationic current, I h , contributed about 20 % to the resting conductance of AH neurones. It had a half-activation voltage of _72 ± 2 mV, and a voltage sensitivity of 8.2 ± 0.7 mV per e-fold change. I h has relatively fast, voltage-dependent kinetics, with on and off time constants in the range ...

1. The process by which stretch of the external muscle of the intestine leads to excitation of myenteric neurons was investigated by intracellular recording from neurons in isolated longitudinal muscle-myenteric plexus preparations from the guinea-pig. 2. Intestinal muscle that was stretched by 40% beyond its resting size in either the longitudinal or circular direction contracted irregularly. Both multipolar, Dogiel type II, neurons and uniaxonal neurons generated action potentials in stretched tissue. Action potentials persisted when the membrane potential was hyperpolarized by passing current through the recording electrode for 10 of 14 Dogiel type II neurons and 1 of 18 uniaxonal neurons, indicating that the action potentials originated in the processes of these neurons. For the remaining four Dogiel type II and 17 uniaxonal neurons, the action potentials were abolished, suggesting that they were the result of synaptic activation of the cell bodies. 3. Neurons did not fire action potentials when the muscle was paralysed by nicardipine (3 ìÒ), even when the preparations were simultaneously stretched by 50% beyond resting length in longitudinal and circular directions. Spontaneous action potentials were not recorded in unstretched (slack) tissue, but when the L-type calcium channel agonist (−)-Bay K 8644 (1 ìÒ) was added, the muscle contracted and action potentials were observed in Dogiel type II neurons and uniaxonal neurons. 4. The proteolytic enzyme dispase (1 mg ml¢) added to preparations that were stretched 40% beyond slack width caused the myenteric plexus to lift away from the muscle, but did not prevent muscle contraction. In the presence of dispase, the neurons ceased firing action potentials spontaneously, although action potentials could still be evoked by intracellular current pulses. After the action of dispase, (−)_Bay K 8644 (1 ìÒ) contracted the muscle but did not cause neurons to fire action potentials. 5. Gadolinium ions (1 ìÒ), which block some stretch activated ion channels, stopped muscle contraction and prevented action potential firing in tissue stretched by 40%. However, when (−)-Bay K 8644 (1 ìÒ) was added in the presence of gadolinium, the muscle again contracted and action potentials were recorded from myenteric neurons. 6. Stretching the tissue 40% beyond its slack width caused action potential firing in preparations that had been extrinsically denervated and in which time had been allowed for the cut axons to degenerate. 7. The present results lead to the following hypotheses. The neural response to stretching depends on the opening of stretch activated channels in the muscle, muscle contraction in response to this opening, and mechanical communication from the contracting muscle to myenteric neurons. Distortion of sensitive sites in the processes of the neurons opens channels to initiate action potentials that are propagated to the soma, where they are recorded. Neurons are also excited indirectly by slow synaptic transmission from neurons that respond directly to distortion.

No abstract

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 microM). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 microM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50-100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by niflumic acid (50-100 microM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary conductance (10-17 pS) and open with a high probability (