Electrophysiology and morphology of vasoactive‐intestinal‐peptide‐immunoreactive neurones of the guinea‐pig ileum.

Abstract

Simultaneous intracellular staining and electrophysiological recording techniques have been applied to neurones of guinea-pig myenteric plexus-longitudinal muscle preparations. With micro-electrodes filled with a solution of the fluorescent dye Lucifer Yellow, neurones were first characterized morphologically and electrophysiologically, and subsequently subjected to an indirect immunohistochemical method for the detection of vasoactive intestinal peptide (VIP)-like immunoreactivity. Cross-correlations of morphology, electrophysiology and VIP immunoreactivity were successfully achieved in a total of 164 neurones. Sixty-three had the slow after-hyperpolarization characteristic of AH neurones; 101 cells displayed fast excitatory post-synaptic potentials (e.p.s.p.s) in response to transmural or focal stimulation and were therefore, by definition, S neurones. Unequivocal VIP immunoreactivity was observed in 25 (25%) S neurones, which, with only one exception, had Dogiel Type I morphology (i.e. many short soma processes and a single long process). In contrast, AH neurones had Dogiel Type II morphology (i.e. smooth soma with several long processes) and none showed VIP immunoreactivity. In addition to cholinergic fast e.p.s.p.s., non-cholinergic slow synaptic inputs were evoked in seventeen of the twenty-two VIP-immunoreactive S neurones tested. Both the fast and slow e.p.s.p.s could be elicited by stimulation of the preparation, oral or aboral to the site of recording. These observations demonstrate that, in the guinea-pig ileum, myenteric plexus neurones showing VIP immunoreactivity are of a single electrophysiological type (S neurones) and belong to essentially one morphological class (Dogiel Type I).

Morphology and electrophysiology of guinea-pig paratracheal neurones

“…This is a non-toxic, fluorescent dye which can stain living neural elements without affecting synaptic transmission or the ability of neurons to generate action potentials (Magrassi et al, 1987;Hanani, 1992; Lees, unpublished observations on guinea-pig enteric neurones). The yellowish fluorescent staining of the principal nerve bundles and of the plexus on the trachealis muscle with this agent, was revealed with the same filter set as required for Lucifer Yellow (Lees and Gray, 1982;Katayama et al, 1986;Lees, 1993). If, however, there were doubt as to the presence of neurone somata among or adjacent to adipocytes, it was found useful to use a rhodamine filter set (Zeiss No.…”

“…At the end of an experiment, the preparation was pinned to a sheet of either thin plastic or of Sylgard and immersed in Zamboni fixative solution at 4°C for 15-20 h. The tissue was then cleared in either dimethylsulphoxide (Sigma) or ethanol (Bornstein et al, 1984(Bornstein et al, , 1991Katayama et al, 1986;Lees, 1993) and washed in phosphate buffered saline (PBS; pH 7.1) and mounted on a glass slide in buffered glycerol (pH 8.6). In the case of biocytin-filled cells, the preparations were subsequently subjected to an indirect immunohistochemical technique to reveal the neurone morphology by exposing them to a solution of streptavidin coupled to the fluorescent probe, Texas Red (Sigma) for 20 min; they were washed in PBS and mounted, as above.…”

“…Once located, neurones were impaled with glass microelectrodes (60-200MV) filled with 2.0% (w/v) solution of Lucifer Yellow CH (LY; Aldrich) for morphological studies only or with either a 0.5% solution of Lucifer Yellow dissolved in 0.5 M KCl (Bornstein et al, 1984;Katayama et al, 1986) or 2M KCl for intracellular electrophysiological recording. In a few experiments, neurones were filled with 4% biocytin (Sigma) and 0.5% LY (w/v) dissolved in 0.5 M KCl buffered to pH 7.4 with 50 mM Tris (Sigma) (Bornstein et al, 1991).…”

Long aboral projections of Dogiel type II, AH neurons within the myenteric plexus of the guinea pig small intestine

“…A variety of characteristics can be used to identify AH cells; chief among them is the long afterhyperpolarization that follows their action potentials (see Wood, 1994, for reviews). Many AH cells have been demonstrated to have Dogiel type II morphological features (Bornstein et al, 1984;Erde et al, 1985;Katayama et al, 1986;Iyer et al, 1988;Hendriks et al, 1990;Bornstein et al, 1991) with large round or oval cell bodies and several long, tapering processes that run circumferentially out of the ganglion of origin and which give rise to varicose endings in surrounding myenteric ganglia (Bornstein et al,199 I ). A subtype of Dogiel type II neuron, with multiple, short dendritic processes, has also been described (Stach, 1989) and shown to have a different pattern of local projections to other Dogiel type II neurons .…”

Electrophysiological properties of neurones in the internal and external submucous plexuses of newborn pig small intestine.

“…About half of the AH neurones in the pig submucous plexus received fast synaptic input. In the guinea-pig small intestine, such inputs have been noted for AH neurones in the myenteric plexus (Iyer et al 1988;Katayama, Pearson & Lees, 1986; but have been reported as very rare (Hirst & McKirdy, 1975) or absent (Surprenant, 1984 (Tamura, 1992). Low incidence of slow EPSPs and IPSPs The rare occurrence of both slow EPSPs and IPSPs in the pig was surprising in view of the prominence of these synaptic potentials in neurones of the guinea-pig submucous plexus (Hirst & McKirdy, 1975;Surprenant, 1984b).…”