Topographical and electrophysiological characteristics of highly excitable S neurones in the myenteric plexus of the guinea-pig ileum.

Most intracellular electrical recordings from myenteric neurones have been made from the centre of large ganglia. In this study, we examined the electrophysiological properties of neurones at the corners of large ganglia close to internodal strands and in microganglia. Of 150 neurones in these locations: 111 were tonic S neurones; 9 were phasic S neurones and 30 were AH neurones. Tonic S neurones were characterized by: (i) low resting membrane potentials (−50 ± 1 mV, mean ± s.e.m.); (ii) high input impedance (522 ± 23 MΩ); (iii) low threshold for action potential (AP) generation (0.012 ± 0.004 nA); (iv) firing of APs throughout a depolarizing pulse (duration ≤ 1 s) and one to four APs following a hyperpolarizing pulse and (v) spontaneous fast excitatory postsynaptic potentials (FEPSPs). A substantial proportion of tonic S neurones (43 %) also fired APs spontaneously (7.6 ± 0.6 Hz; range, 0.3–19 Hz). All APs were blocked by tetrodotoxin (1 μm). Tonic S neurones were subclassified, according to their post-stimulus responses, as SAH or SAD neurones. Following a burst of APs, SAH neurones exhibited a prominent after-hyperpolarization (duration, 711 ± 10 ms) and SAD neurones an after-depolarization (duration, 170 ± 10 ms). The after-hyperpolarization was reduced in four of ten neurones by apamin (0.3 μm). FEPSPs were evoked in 20 of 38 S neurones by electrical stimulation applied both oral and anal to the recording site. Repetitive stimuli evoked slow excitatory postsynaptic potentials (SEPSPs) in some tonic S neurones. Three functional classes of S neurones were identified after injection of neurobiotin through the recording microelectrode: (i) longitudinal muscle motor neurones, (ii) short circular muscle motor neurones, and (iii) ascending interneurones. In conclusion, there appears to be topographical organization of highly excitable, tonic S neurones within the myenteric plexus, since, in contrast to other S neurones, they can be readily impaled in myenteric ganglia close to internodal strands and in microganglia. The enteric nervous system (ENS) consists of a large number of functionally diverse neurones arranged in circuits that regulate peristalsis in the gastrointestinal tract (see Hirst & McKirdy, 1974; Hirst et al. 1975; Smith & Furness, 1988; Bornstein et al. 1991; Smith et al. 1992; Smith & Robertson, 1998). These circuits are organized so that during peristalsis they produce synchronous contraction and relaxation of the longitudinal and circular muscle layers (Bayliss & Starling, 1899; Smith & Robertson, 1998; Smith & McCarron, 1998; Yamomoto et al. 1998). At other times the muscle layers may move independently (Yokoyama & Ozaki, 1990), since they are innervated by different populations of motor neurones (Bornstein et al. 1991; Smith et al. 1992; Costa et al. 1996). Many, if not all, of the neurones regulating propulsion in the small intestine are in the myenteric plexus (Hirst et al. 1974, 1975; Smith & Furness, 1988; Smith et al. 1990, 1992; Bornstein et al. 1991; Costa et al. 1996). Myenteric neurones are generally classified electrophysiologically into AH/type II and S/type I neurones (Nishi & North, 1973; Hirst et al. 1974). Action potentials in AH neurones are calcium dependent. It is the influx of calcium with the action potential which leads to the opening of calcium-dependent potassium channels that are responsible for the long after-hyperpolarization (AH) which characterizes these neurones (Nishi & North, 1973; Hirst et al. 1974). The calcium-activated potassium current renders the soma refractory to further stimuli (Nishi & North, 1973; Hirst et al. 1974). In contrast, action potentials in S neurones are dependent on Na+ influx since they are completely blocked by tetrodotoxin (Nishi & North, 1973; Hirst et al. 1974; Shuttleworth & Smith, 1999). Fast excitatory postsynaptic potentials (FEPSPs) are readily evoked in S neurones (S for synaptic input), but rarely in AH neurones (Nishi & North, 1973; Hirst et al. 1974, 1975). Prominent slow excitatory postsynaptic potentials (SEPSPs), however, can be readily generated in AH neurones by repetitive nerve stimulation (see Wood, 1994b). AH neurones are usually large multipolar neurones (Dogiel type II morphology) which send processes down to the mucosa and appear to be primary afferent neurones (Furness et al. 1990). They respond to a variety of mucosal stimuli such as serotonin and HCl (Smith, 1994; Kunze et al. 1995). AH neurones communicate with S neurones and other AH neurones mainly via slow excitatory postsynaptic potentials (SEPSPs) (Kunze et al. 1993), whereas, S neurones communicate with other S neurones mainly via FEPSPs (Hirst et al. 1975; Bornstein et al. 1991; Smith et al. 1992). As a group, S neurones are functionally more diverse than AH neurones. They are uniaxonal neurones, which exhibit a variety of dendritic morphologies and soma sizes, and comprise the motor neurones and interneurones within the reflex pathways that supply both the longitudinal and circular muscle layers (Bornstein et al. 1991; Smith et al. 1992; Costa et al. 1996). S neurones, which respond to mucosal stimulation or distension with bursts of FEPSPs, usually lie mainly within either ascending or descending nervous pathways, although some S neurones (e.g. longitudinal muscle motor neurones) are common to both pathways (Smith et al. 1992). In recognition of this wide functional diversity amongst myenteric neurones, some investigators have tried to subdivide them further into several electrophysiological classes (see Wood, 1994a). Under typical recording conditions, most (85–92 %) myenteric S neurones exhibit a phasic or rapidly adapting firing pattern, whereas some others have a slowly adapting firing pattern (reviewed in Bornstein et al. 1994; Kunze et al. 1994; Kunze et al. 1997). Although, Brookes et al. (1997) recently reported that ascending interneurones, identified by 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) retrograde labelling techniques, were tonic S neurones. Early studies using extracellular electrodes have reported that many myenteric neurones fired action potentials spontaneously and that this activity increased with stretch (reviewed in Wood, 1994b). Since spontaneous action potentials were rarely, if ever, observed in myenteric neurones with intracellular microelectrodes it has been suggested that this activity resulted from the mechanical disturbance applied by pressure or suction electrodes to mechanically sensitive somas (North & Williams, 1977). Later Wood & Mayer (1978), using intracellular recording techniques, found a number of myenteric S type neurones that fired action potentials spontaneously. They attributed these differences to the fact that they did not use drugs to paralyse the muscle or because they sampled neurones in most regions of the ganglia. More recently it has been shown that some phasic neurones increase their firing rates in response to stretch applied to the tissue in the absence of drugs to paralyse the muscle (Kunze et al. 1998). The idea of a topographical organization of myenteric neurones that is related to function, as in the spinal cord or spinal ganglia of invertebrates (see Levitan & Kaczmarek, 1991), is very attractive but in the past has not gained much support. Immunohistochemical and DiI retrograde labelling studies in recent years, however, suggest the possibility that some degree of neuronal organization occurs within myenteric ganglia. Motor neurones that innervate the longitudinal muscle are generally found at the edges of myenteric ganglia near the junction with internodal strands or in microganglia (Bornstein et al. 1991; Brookes et al. 1992). Also, calretinin (a calcium-binding protein) immunoreactive neurones, which include both cholinergic longitudinal muscle motor neurones and ascending interneurones, are often located near the edges of ganglia and are often seen in clusters of two to four cells near the internodal strands (Brookes et al. 1991a). In this study, we examined the electrophysiological and morphological characteristics of S neurones located in the corners of large ganglia close to where internodal strands emerge from the ganglion, and in microganglia formed at the intersection of several internodal strands. We report that highly excitable tonic S neurones can be readily accessed in these locations.