G.M. Lees

Morphological studies of electrophysiologically-identified myenteric plexus neurons of the guinea-pig ileum.

The morphology of neurons in the myenteric plexus of the guinea-pig ileum has been studied by means of the intracellular application of Procion Yellow. Sixty-six electrophysiologically-unidentified cells showed a great variety of soma shapes and number of processes, the vast majority of the longer of which were circumferentially-orientated. The electrophysiological properties of an additional 47 neurons were ascertained; 29 were neurons with a slow after-hyperpolarization (AH-neurons), 14 showed fast excitatory synaptic potentials (S-neurons) and 4 were of neither category. Twenty-two of the AH-neurons had a smooth soma outline and, on average, each had 5 processes, of which the great majority of long processes were circumferentially-orientated and intraganglionic. The projection ratios of oral:circumferential:aboral processes were 6:61:9. Branching was a prominent feature of the processes. In contrast, a large soma with many broad, short processes was a feature of 8 of 14 S-neurons studied. The average number of processes was 8.6 per cell and relatively more of them were aborally-directed, giving projection ratios of 2:21:7. There was, however, such a variation and overlap in the morphology of AH- and S-neurons that it was not possible to achieve a simple, reliable classification. It is concluded that many neuronal processes may be intraganglionic and that longitudinal ones are mainly aboral. From the varied morphological characteristics of AH-neurons, it is unlikely that these neurons subserve a single function in the plexus. For the same reasons S-neurons may fulfil different physiological roles.

Effects of cannabinoid receptor ligands on electrophysiological properties of myenteric neurones of the guinea-pig ileum

1 The effect of cannabinoid receptor agonists was studied in guinea‐pig myenteric neurones in vitro by use of conventional intracellular recording techniques. 2 Exposure of myenteric neurones of the S‐cell type to the cannabinoid receptor agonists WIN 55,212‐2 (100 nM) and CP 55,940 (100 nM) reversibly and significantly depressed the amplitude of fast excitatory synaptic potentials (fast e.p.s.ps) by 46% and 37%, respectively. 3 The depressant effect of WIN 55,212‐2 and CP 55,940 on fast e.p.s.p. amplitude (expressed as the area above the amplitude‐time curve (mVs)) was significantly greater than that of the vehicle, Tween 80, which had no detectable effect. 4 The inhibitory effect of WIN 55,212‐2 appeared to be concentration‐dependent over the range 1–100 nM. WIN 55,212‐3, its (−)‐enantiomer (100 nM), was inactive. 5 The cannabinoid CB1 receptor antagonist, SR141716A (1 μM), reversed the inhibitory effects of WIN 55,212‐2 on fast e.p.s.ps in 38% of neurones tested (3/8) and acetylcholine (ACh)‐induced depolarizations in 42% of neurones tested (5/12). 6 When tested on its own, SR141716A (1 μM) caused a 40–50% reduction in the amplitude of fast e.p.s.ps (n=9). 7 WIN 55,212‐2 reversibly depressed the amplitude of the slow e.p.s.p. and, in 2 out of 7 neurones, this effect was reversed by SR141716A (1 μM). 8 It is concluded that cannabinoid‐induced inhibition of fast cholinergic synaptic transmission occurred by reversible activation of both presynaptic and postsynaptic CB1 receptors and that slow excitatory synaptic transmission can also be reversibly depressed by cannabinoids. Furthermore, it would seem that subpopulations of myenteric S‐neurones and their synapsing cholinergic and non‐cholinergic, non‐adrenergic terminals are not endowed with cannabinoid receptors.

Myenteric neurons of the rat descending colon: electrophysiological and correlated morphological properties.

Conventional intracellular electrophysiological recordings were made from 502 myenteric neurons of the rat descending colon. Myenteric neurons could be classified into three groups on the basis of distinct electrophysiological properties. The first group of neurons (51% of all neurons) fired tetrodotoxin-sensitive action potentials in response to direct somal depolarization and the majority (98%) of this group generated fast cholinergic excitatory synaptic potentials in response to focal stimulation and were therefore designated S/Type 1 neurons. The second group (40%) of neurons fired tetrodotoxin-insensitive action potentials which were followed by long-lasting membrane afterhyperpolarizations, hence were termed AH neurons. These neurons did not receive fast cholinergic synaptic inputs but ionophoretic application of acetylcholine induced rapid nicotinic cholinoceptor-mediated depolarizations. The final group of neurons (9%), named Type 3 neurons, received fast cholinergic synaptic inputs but could never be made to fire action potentials. Rundown in amplitude of successive fast excitatory synaptic potentials evoked by a short train of presynaptic nerve stimuli was observed in only a small proportion of neurons (8/37; 22%) with the majority of neurons (29/37; 78%) showing no such decrease in amplitude, even at frequencies of stimulation as high as 10 Hz. Superfusion of 5-hydroxytryptamine could induce both an inhibition and a facilitation of cholinergic fast synaptic transmission. Evidence was adduced that these presynaptic inhibitory and facilitatory actions appeared to be mediated via 5-hydroxytryptamine 1A and 5-hydroxytryptamine 4 receptors, respectively. Muscarinic slow excitatory synaptic potentials were not detected (9/9 neurons tested) and non-cholinergic slow excitatory synaptic potentials following repetitive focal presynaptic nerve stimulation were observed in only 39/502 (8%) of all neurons. In those neurons in which a demonstrable change in membrane input resistance was detectable, slow excitatory potentials were accompanied by an increased input resistance. In addition, in a small subset (4%) of S/Type 1 neurons, slow membrane hyperpolarizations accompanied by an increased membrane input resistance were observed following tetanic presynaptic nerve stimulation. Superfusion of 5-hydroxytryptamine induced both membrane depolarizations and hyperpolarizations. Membrane depolarizations were observed in 40% of all neuronal types (34% of S/Type 1 neurons, 58% of AH neurons and 11% of Type 3 neurons) and were accompanied by an increased membrane input resistance and occasionally, in S/Type 1 and AH neurons, by anodal break excitation or spontaneous action potential firing. Membrane hyperpolarizations were observed in S/Type 1 neurons (5%) only and were accompanied, unexpectedly, by an increased membrane input resistance. In those neurons that responded both to application of 5-hydroxytryptamine and tetanic presynaptic nerve stimulation, 5-hydroxytryptamine always mimicked the slow synaptic response indicating that 5-hydroxytryptamine may function as a slow synaptic mediator in some myenteric neurons. Myenteric neurons identified by intracellular injection of the neuronal marker Neurobiotin TM were found to conform to the morphological classification schemes proposed for myenteric neurons of the guinea-pig and porcine intestine, that is, Dogiel Types I and II and Stach Type IV neurons were present. Simultaneous electrophysiological recording and intracellular staining techniques revealed that a correlation existed between the electrophysiological and morphological properties of myenteric neurons of the rat colon, with electrophysiological classified S/Type 1 neurons having Dogiel Type I morphologies (95/108 neurons; 88%) and electrophysiological classified AH neurons having Dogiel Type II morphologies (87/94 neurons; 93%)...

Depressant effects of hypoxia and hypoglycaemia on neuro-effector transmission of guinea-pig intestine studied in vitro with a pharmacological model.

Since intermittent ischaemia may play an important role in the ætiology of Inflammatory Bowel Disease, particularly Crohns Disease, a pharmacological model of neuronal ischaemia was applied to guinea‐pig isolated intestinal preparations to mimic the acute effects of reduced blood flow on intestinal motility. Neuro‐effector transmission and smooth muscle performance were examined in myenteric plexus‐longitudinal muscle preparations of guinea‐pig ileum exposed to sodium cyanide (NaCN), in order to inhibit oxidative phosphorylation, or to iodoacetic acid (IAA), to block glycolysis. Comparisons were made with the effects due to simple deprivation of oxygen or glucose. Depressions of cholinergic neuro‐effector transmission induced by hypoxia or NaCN (effective concentration range 0.1–3 mm), given as separate treatments, singly or repetitively over 60–90 min, were apparent within 30 s and were reversible. The maximum inhibition was 90% and the IC50 for NaCN was 0.3 mm. A conspicuous component of these inhibitions was prejunctional. Non‐cholinergic neuro‐effector contractions were inhibited by up to 90% by anoxia or NaCN but recovery was incomplete and slower than with cholinergic contractions. Glucose‐free solutions also caused a reversible failure of cholinergic neuro‐effector transmission but of slower onset. In contrast, IAA (0.06–1 mm) abolished contractions irreversibly, apparently by a direct depressant effect on smooth muscle contraction. Unlike NaCN, IAA caused an initial potentiation of electrically‐induced contractions, partly by a prejunctional potentiation of cholinergic neuro‐effector transmission. It is concluded that a disruption of intestinal activity in pathological conditions associated with intestinal ischaemia may result from disturbances in the function of enteric neurones.

ANTIBIOTIC‐ASSOCIATED COLITIS: AN in vitro INVESTIGATION OF THE EFFECTS OF ANTIBIOTICS ON INTESTINAL MOTILITY

1 Nine antibiotic compounds in common use were studied to determine their ability to affect intestinal motility in vitro, in the guinea‐pig ileum and rabbit colon. 2 Ampicillin, doxycycline, mecillinam and metronidazole were without effect over a concentration range which included typical serum levels found when these drugs are used therapeutically. 3 Clindamycin, gentamicin, kanamycin, pivmecillinam and trimethoprim were all found to inhibit evoked and reflex responses of the guinea‐pig ileum but only clindamycin and trimethoprim also affected evoked responses of the rabbit colon. 4 Kanamycin and gentamicin appeared to have a predominantly prejunctional action, pivmecillinam and trimethoprim a predominantly post‐junctional action. Clindamycin had a prejunctional action at low concentrations and long exposure times, and a post‐junctional action at high concentrations and short exposure times. 5 The concentration of each antibiotic required to inhibit the peristaltic reflex of the guinea‐pig ileum was less than that required to inhibit its responses to electrical stimulation or exogenous acetylcholine or histamine but greater than the serum levels associated with their respective use in therapeutic doses. 6 A sequence of events whereby antibiotic‐induced alterations in gastro‐intestinal motility could lead to the development of pseudomembranous colitis is proposed.

Slow intracellular potentials in AH-neurons of the myenteric plexus evoked by repetitive activation of synaptic inputs

In after-hyperpolarization-neurons of guinea-pig myenteric plexus, several types of slow responses to repetitive activation of synaptic inputs were recorded. There were at least two types of slow excitatory depolarizing potential, both associated with an increased input resistance, but in one there was an abolition or marked attenuation of the amplitude of the slow after-hyperpolarization of the neuron, whereas in the other type the slow after-hyperpolarization was not significantly affected. Slow depolarizing and hyperpolarizing synaptic potentials associated with a decrease input resistance were also recorded in some neurons following transmural stimulation. The neural processes responsible for these slow potentials in after-hyperpolarization-neurons showed no preferential projection within the myenteric plexus.

Reappraisal of the innervation of rat intestine by vasoactive intestinal polypeptide and neuropeptide Y-immunoreactive neurons

The occurrence and distribution of neurons and nerve fibres showing vasoactive intestinal polypeptide-like and neuropeptide Y-like immunoreactivity were re-examined in the enteric nervous system of the small and large intestine of the adult rat using dual-labelling indirect immunofluorescence histochemistry to detect the co-existence of these neuropeptides. In the myenteric plexus of both small and large intestine, a population of neuropeptide Y-immunoreactive neurons that did not contain vasoactive intestinal polypeptide was noted; it accounted for 29-53% of neuropeptide Y neurons. Such neurons were also found in the submucosa but there they constituted at most 2% of neuropeptide Y-immunoreactive neurons. In both myenteric and submucous plexuses, regional variations were observed in the number of immunoreactive neurons and in the proportion of dual-labelled neurons. In the myenteric plexus, for example, the density of neurons with immunoreactivity to these two neuropeptides was constant throughout the small intestine, whereas it progressively increased distally within the colon. In addition, a distinct but small subset of immunoreactive myenteric neurons was found to have a novel soma morphology, unclassifiable according to the criteria used for porcine or guinea-pig enteric neurons. Such neurons had one or more conspicuous processes, which were much longer than the short, lamellar somal processes of typical Dogiel Type 1 neurons; moreover, these protruded from an essentially smooth soma and terminated at distances of up to two cell diameters from their point of origin. Thus, our results suggest that the organization of the enteric nervous system of the rat differs from that of other species and indicate that investigation of the co-localizations of neuropeptides and biologically active mediators in the intestinal tract would be incomplete without reference to regional differences in the incidence and distribution of such neurochemicals.

Morphology and electrophysiology of guinea-pig paratracheal neurones.

Although guinea‐pig tracheal preparations are used as models of asthma, the morphological and electrophysiological characteristics of its associated ganglion neurones (paratracheal neurones) have not been characterized.

Regional differences in the sympathetic innervation of the guinea pig large intestine by neuropeptide Y- and tyrosine hydroxylase-immunoreactive nerves of divergent extrinsic origin

Region‐specific patterns of nerves with immunoreactivity to neuropeptide Y (NPY) have been described previously in the submucous plexus of guinea pig large intestine. Because these may have functional significance, the possibility of similar, characteristic variations of NPY‐like immunoreactivity (NPY‐ir) in the myenteric plexus was explored. Regional differences were found in the occurrence and pattern of distribution of NPY‐ir in the myenteric plexus of the guinea pig large intestine. NPY‐ir was present rarely within neuron somata in any region of the large intestine, and NPY‐ir nerve fibers were present only within the distal large intestine, increasing progressively in density from the distal spiral to the rectum. Lesion of the colonic nerves, but not the hypogastric, intermesenteric, or lumbar splanchnic nerves, resulted in a loss of NPY‐ir in the distal spiral and transverse colon but not in the descending colon or rectum. Ring myotomies in the descending colon resulted in a loss of NPY‐ir proximal to the lesion. Dual‐labeling immunohistochemical studies revealed that the NPY‐ir nerve fibers rarely contained immunoreactivity for tyrosine hydroxylase (TH). Extrinsic nerve lesions resulted in an unequivocal reduction in NPY‐ir in intraganglionic fibers of the submucosal plexuses of the transverse colon and a partial loss in the distal spiral and descending colon: the rectum was unaffected; in only a minority of guinea pigs, however, was any decrease in the NPY‐ir innervation of submucosal blood vessels detected. The principal projections of NPY‐ir nerves were from and through the inferior mesenteric ganglion; however, NPY‐ir was not colocalized with TH‐ir. It is proposed that nonnoradrenergic, NPY‐containing neurons located in the inferior mesenteric ganglion project through the colonic nerves and that these proximally directed fibers innervate the transverse colon and the distal spiral. Nonnoradrenergic, NPY‐ir neurons lying in the pelvic ganglia or sacral sympathetic chain may make an important contribution to the innervation of the myenteric plexus of the rectum and the descending colon. J. Comp. Neurol. 410:515–530, 1999.

Synaptic potentials recorded by the sucrose‐gap method from the rabbit superior cervical ganglion

1 Compound ganglionic potentials evoked by stimulation of the preganglionic nerves to the superior cervical ganglion of the rabbit were recorded by the sucrose‐gap method. 2 When the distal part of the ganglion was bathed in flowing isotonic sucrose solution or sodium‐deficient solutions, ganglionic action potentials were no longer evoked, only large synaptic potentials. 3 The compound synaptic potential, which remained unaltered for more than 1 h, originated in a population of cells at the interface between the Krebs and sucrose solutions. Hexamethonium reduced the size but did not alter the time course of the synaptic potential. 4 It is suggested that a higher concentration of sodium ions is required for the generation of ganglionic action potentials than for either conduction in the postganglionic axons or production of synaptic potentials. 5 When lithium replaced sodium in the solution bathing the distal part of the ganglion, the synaptic potential was greatly reduced in amplitude. Impulse propagation in the postganglionic axons was only slightly impaired when lithium replaced sodium in the solution bathing the axons. 6 A quantitative assessment of the potency of the ganglion‐blocking drugs nicotine, pentolinium, hexamethonium and pempidine was made by measuring the depression of the synaptic potentials produced by bathing the distal part of the ganglion in flowing isotonic sucrose solution. The concentrations which produced a 50% depression were 8.1 μM nicotine, 26.5 μM pentolinium, 111 μM hexamethonium and 22.2 μM pempidine.