Geoffrey Pearson

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

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).

A reduction in interstitial cells of Cajal in horses with equine dysautonomia (grass sickness).

Equine dysautonomia (grass sickness) is a common, frequently fatal disease of horses characterised by dysfunction of the gastrointestinal tract. Interstitial cells of Cajal are the c-Kit-immunoreactive cells responsible for the generation of pacemaker activity in gastrointestinal smooth muscle. Impairment of this pacemaker action has been implicated in several motility disorders in humans and laboratory mammals. The aim of this study was to test the hypothesis that changes in interstitial cells of Cajal may be involved in the pathophysiology of the intestinal dysfunction observed in equine grass sickness. Interstitial cells of Cajal were identified using immunohistochemical labelling with an anti-c-Kit antibody and their density was assessed using a semi-quantitative grading system. Segments of ileum were examined from 24 horses free from gastrointestinal disease and compared to tissues from 28 horses with grass sickness. Segments of the pelvic flexure region of the large colon were examined from 13 horses free from gastrointestinal disease and compared to tissues from 10 horses with grass sickness. In horses with grass sickness, interstitial cells of Cajal were significantly decreased in both the myenteric plexus and circular muscle regions of both ileum and pelvic flexure compared to normal animals. Therefore, it is possible that the decline in interstitial cells of Cajal may be an important factor in the development of intestinal dysmotility observed in grass sickness.

Mouse pancreatic acinar cells: effects of electrical field stimulation on membrane potential and resistance.

1. Intracellular micro‐elctrode recordings of acinar cell membrane potential and resistance were made from the mouse pancreas superfused in vitro. The acinar cells under investigation were stimulated by electrical field stimulation using two platinum wire electrodes and by micro‐ionophoretic acetylcholine (ACh) application from an extracellular AChCl‐filled micro‐electrode. 2. Field stimulation evoked membrane depolarization and reduction in input resistance. Maximal effects were observed at 20‐40 Hz frequency, 1‐2 msec pulse width and 8‐20 V amplitude. The mean latency for the field stimulation‐evoked depolarization was 900 msec. Field stimulation responses were seen at low frequency levels of stimulation, the majority of cells responding at 5 Hz and some at 2 Hz. The physiological significance of the low frequency stimulation is discussed. 3. The field stimulation effects resembled those induced by ACh ionophoresis and were abolished by atropine. The equilibrium potentials for both field stimulation and ACh ionophoresis were identical at about ‐15 mV. The field stimulation response was selectively abolished by tetrodotoxin and by superfusion with Na‐free or Ca‐free media, while the ACh ionophoretic response persisted. Field stimulation therefore initiated nerve action potentials and consequent ACh release. 4. Spontaneous miniature depolarizations observed in some preparations were not abolished by tetrodotoxin and woult therefore seem to be a result of quantal release of ACh from nerve terminals. 5. There is no indication from the present studies of the existence of neurotransmitters other than ACh. No inhibitory effects have been observed. 6. All preparations studied to date have responded to field stimulation and it is concluded that all acinar cells are potentially under cholinergic neural influence.

Presence of in vitro electrical activity in the ileum of horses with enteric nervous system pathology: equine dysautonomia (grass sickness)

Equine dysautonomia (grass sickness) is a frequently fatal disease of horses characterised by intestinal stasis. Interstitial cells of Cajal (ICC) are the pacemakers and mediators of neurotransmission in the gastrointestinal tract. Impaired ICC-mediated control of motility has been implicated in intestinal disorders in laboratory mammals, humans and in equine grass sickness. The aim of this study was to compare the in vitro electrical properties of ileum from grass sickness cases with horses free from gastrointestinal disease. Intracellular microelectrode recordings were made from smooth muscle cells in cross-sectional preparations of equine ileum, superfused in vitro. Samples were taken from six horses with grass sickness and from eight horses free from gastrointestinal disease, all euthanised on humane grounds. Ileal tissues were processed for haematoxylin and eosin histology, and c-Kit immunohistochemistry. Membrane potential oscillations were recorded in the ileal preparations from four of the six horses with grass sickness and from all of the normal horses. A waxing and waning pattern of the membrane potential oscillation activity was noted in some cells. In comparison to the normal horses, the membrane potential oscillations in grass sickness horses had a significantly reduced frequency (P = 0.0001) and increased duration (P = 0.0002). Immunohistochemistry revealed the presence but reduction of ICC in grass sickness. Histological assessment of the same tissues used for analysis of the ICC showed the depletion and pathology of the enteric neurons in grass sickness. Therefore, the majority of ileal preparations from grass sickness-affected horses exhibited prominent membrane potential oscillation activity suggesting that, although the neural elements are damaged severely, the ICC-mediated pacemaker function remains intact.

Light Microscopy of the Enteric Nervous System of Horses with or without Equine Dysautonomia (Grass Sickness): its Correlation with the Motor Effects of Physostigmine

Light microscopy was undertaken on sections from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold in 5 control horses, 5 horses with acute grass sickness (AGS), and 5 horses with chronic grass sickness (CGS). With the exception of the ileal submucous plexus of the CGS group, the AGS group had the lowest number of neurons as measured using a subjective scoring scheme. The proportion of abnormal neurons in the AGS group was similar in both plexuses and both regions, whereas the values for the CGS group were much higher in the duodenal region than in the ileal region. The motility of tissue adjacent to that used for histology was measured isometrically in vitro. The increase in the rate of contractions following exposure to physostigmine was greatest for the AGS group, both from the duodenal and from the ileal region. The latency was longest for the AGS group, suggesting that the material from this group had the least number of active cholinergic neurons. The studies with physostigmine thus indicated that the most severe functional damage occurred in cases of AGS. These findings confirm that extensive damage occurs in the enteric neurons in equine grass sickness. There was good correlation between the functional cholinergic responses and the extent of neuronal degeneration.

In vitro microelectrode study of the electrical properties of smooth muscle in equine ileum

Intracellular microelectrode recordings were made from smooth muscle cells in cross-sectional preparations of equine ileum, superfused in vitro. Membrane potential oscillations and spike potentials were recorded in all preparations, but recordings were made more readily from cells in the longitudinal muscle layer than from cells in the circular layer. The mean (se) resting membrane potential (RMP) of smooth muscle cells in the longitudinal muscle layer was -51.9 (1.2) mV, and the membrane potential oscillations in this layer had a mean amplitude of 4.8 (0.4) mV, a frequency of 9.0 (0.1) cycles per minute and a duration of 5.8 (0.2) seconds. The membrane potential oscillations were preserved in the presence of tetrodotoxin. A waxing and waning pattern of membrane potential oscillation activity was observed. Nifedipine abolished the spiking contractile activity of the smooth muscle, did not abolish the membrane potential oscillations but did alter their temporal characteristics.

Electrophysiological effects of field stimulation on mouse pancreatic acinar cells.

Ellory, 1974). Transport systems in human red cells studied by this technique include the sodium pump and its partial reactions assayed as Na, K-activated ATPase, K-dependent p-nitrophenylphosphatase (PNPPase) or ouabain-binding, and the nucleoside transport system, assayed by the specific component of nitrobenzylthioinosine binding (Jarvis & Young, 1978). Irradiation of the sodium pump assayed as Na, K-activated ATPase yields a component of molecular weight 330 + 30 x 103 (12) daltons, with both ouabain-binding and K-dependent PNPPase activity inactive with a target size of 180 + 28 x 103 (12) daltons. For the nucleoside transport system, results plotted as log activity vs. dose consistently yield biphasic curves, where 30-45 % of the binding inactivates with an apparent high molecular weight > 106 daltons, the remaining 55-70% ofthe activity giving a target size of 128 + 25 x 103 (5) daltons.

ELECTROPHYSIOLOGICAL CLASSIFICATION OF SUBMUCOSAL PLEXUS NEURONES IN THE JEJUNUM OF THE NEWBORN PIG

Intracellular recordings were made from the internal and external submucosal ganglia of the porcine small intestine and neuronal properties were classified using two existing schemes for guinea-pig enteric neurones. In the first analysis, 77% of cells were designated as Type 4 since they were a heterogeneous population of neurones with the overlapping properties of S/Type 1 and AH/Type 2. The simplicity and usefulness of the second classification scheme was due to its emphasis on a single electrophysiological event, namely, the long-lasting after-hyperpolarization (AH) following the action potential. Eighty-eight percent of the cells studied were thus categorized as either AH (with an AH) or S (without an AH). All S neurones displayed fast synaptic potentials in response to stimulation of interganglionic fibre strands. AH neurones were subdivided into two groups dependent on whether they received fast synaptic inputs. Only by employing the second scheme of classification were differences in the neuronal characteristics and synaptic profiles between the two submucosal plexuses detected. It is concluded that the S and AH system of classification is the most appropriate method for the analysis of intracellular recordings from submucosal neurones in the porcine small intestine.

Synaptic Communication Between External and Internal Submucosal Plexus Neurones in the Jejunum of the Newborn Pig

Intracellular recordings were made from neurones in the internal submucosal plexus (ISP) of porcine small intestine and synaptic inputs were investigated by focal stimulation of nerve fiber tracts. Nicotinic fast excitatory potentials (e.p.s.p.s) were recorded in all neurones, but slow e.p.s.p.s and slow inhibitory potentials (i.p.s.p.s) were rarely seen. Membrane potential changes similar to those occurring during the slow e.p.s.p. and slow i.p.s.p. could be evoked by exogenous application of neurotransmitters, even in neurones failing to display a nerve-mediated response. We suggest that the predominant source of the slow synaptic inputs to the ISP may be the neurones of the external submucosal plexus (ESP). The failure to record slow e.p.s.p.s and i.p.s.p.s could be a consequence of the anatomical arrangement of the submucosal plexuses whereby interconnecting strands between the ISP and ESP are inaccessible to the focal stimulation.