Single neuron studies of opiate action in the guinea pig myenteric plexus.

The action of narcotics and other drugs on electrical activity of neurons in the guinea pig myenteric plexus was examined by extracellular recording with a suction electrode. Morphine, in a stereospecific and naloxone-sensitive action, inhibits spontaneous electrical activity of many neurons, and antagonizes an increased firing rate caused by serotonin or nicotine. The inhibition by morphine of spontaneous electrical activity occurs under conditions of synaptic transmission blockade, which renders unlikely several possible synaptic mechanisms in the primary effect of opiates. Morphine was found not to alter conduction velocity of myenteric neurons. It is concluded that morphine probably acts to reduce the excitability of a class of myenteric plexus neurons, perhaps by hyperpolarizing or stabilizing the membrane potential.     

Action of morphine on guinea-pig myenteric plexus and mouse vas deferens studied by intracellular recording.

Morphine reduces the output of transmitter from the myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum and from the mouse vas deferens. Intracellular recordings were made from ganglion cells of the myenteric plexus and smooth muscle cells of the vas deferens. Synaptic transmission within the myenteric plexus was blocked by hexamethonium. Morphine did not change the properties of the ganglion cells, nor did it affect synaptic potentials. 5-Hydroxytryptamine inhibited acetylcholine release at intraganglionic synapses by an action which was unaffected by morphine. In the vas deferens, excitatory junction potentials were elicited by stimulation of postganglionic adrenergic nerve fibres. The junction potentials were depressed by morphine and levorphanol but not by dextrorphan. This depression was reversed by naloxone. The results indicate that morphine acts directly to reduce transmitter release at the neuro-effector junctions in the myenteric plexus-longitudinal muscle preparation and in the vas deferens in these species.     

Comparison of the effect of morphine on locus coeruleus noradrenergic and ventral tegmental area dopaminergic neurons in vitro

Extracellular single-cell recordings were performed on rat brain slices to compare the effects of morphine on noradrenergic neurons of the locus coeruleus (LC) and on dopaminergic neurons of the ventral tegmental area (VTA). Morphine inhibited the firing of LC neurons at very low concentrations. The mean IC50 was 13.4 +/- 1nM (mean +/- SEM) (n = 7). Moreover, the inhibitory effect of morphine was identical in slices obtained from rats anesthetized with chloral hydrate or from non-anesthetized rats. On the contrary, morphine did not have any influence on the firing of most VTA neurons (N = 20) up to 100 microM, and did not modify the sensitivity of their autoreceptors (N = 8). It is concluded that morphine potently inhibits the firing of LC neurons in vitro both in slices of anesthetized and not anesthetized animals and has no direct excitatory effect on VTA dopaminergic neurons of the rat.     

Morphine Decreases Enteric Neuron Excitability via Inhibition of Sodium Channels

Gastrointestinal peristalsis is significantly dependent on the enteric nervous system. Constipation due to reduced peristalsis is a major side-effect of morphine, which limits the chronic usefulness of this excellent pain reliever in man. The ionic basis for the inhibition of enteric neuron excitability by morphine is not well characterized as previous studies have mainly utilized microelectrode recordings from whole mount myenteric plexus preparations in guinea pigs. Here we have developed a Swiss-Webster mouse myenteric neuron culture and examined their electrophysiological properties by patch-clamp techniques and determined the mechanism for morphine-induced decrease in neuronal excitability. Isolated neurons in culture were confirmed by immunostaining with pan-neuronal marker, β-III tubulin and two populations were identified by calbindin and calretinin staining. Distinct neuronal populations were further identified based on the presence and absence of an afterhyperpolarization (AHP). Cells with AHP expressed greater density of sodium currents. Morphine (3 µM) significantly reduced the amplitude of the action potential, increased the threshold for spike generation but did not alter the resting membrane potential. The decrease in excitability resulted from inhibition of sodium currents. In the presence of morphine, the steady-state voltage dependence of Na channels was shifted to the left with almost 50% of channels unavailable for activation from hyperpolarized potentials. During prolonged exposure to morphine (two hours), action potentials recovered, indicative of the development of tolerance in single enteric neurons. These results demonstrate the feasibility of isolating mouse myenteric neurons and establish sodium channel inhibition as a mechanism for morphine-induced decrease in neuronal excitability.     

The actions of opiates in the rat substantia nigra: An electrophysiological analysis

Single unit recording and micropressure ejection techniques were used to investigate the actions of opiates on dopaminergic and non-dopaminergic neurons in the rat substantia nigra. Systemic administration of morphine, 1 to 4 mg/kg, led to a naloxone-reversible increase in firing rate of all zona compacta dopaminergic (ZC) neurons examined (n=10). In a specifically defined subpopulation of non-dopaminergic nigral zona reticulata (ZR) neurons, systemically administered morphine led to a naloxone reversible decrease in activity (n=9). D-Ala²-d-leu⁵ (DADL)-enkephalin, when applied directly onto ZC neurons by micropressure ejection techniques, had no effect on their firing rate. In contrast, micropressure ejection of DADL enkephalin onto ZR neurons produced a decrease in firing rate which was blocked by systemically administered naloxone. Morphine sulfate applied by pressure ejection onto both ZC and ZR neurons produced mixed results which were not always blocked by naloxone. These results suggest that one of the mechanisms by which opiates increase dopaminergic neurotransmission is through disinhibition of dopaminergic neurons in the substantia nigra.     

Morphine: Ability to block neuronal activity evoked by a nociceptive stimulus

The effect of morphine on the neuronal activity evoked by a nociceptive stimulus, a foot pinch, was studied in the dorsal raphe nucleus (DR) and in the mesencephalic reticular formation (MRF) of the rat. In the MRF and adjacent areas, neuronal firing was accelerated by the nociceptive stimulus. Morphine blocked this acceleration when administered either microintophoretically or i.v. Three lines of evidence indicate that this is a specific narcotic effect. First, naloxone, a specific narcotic antagonist, antagonized the effect of morphine. Secondly, two morphine agonists, oxymorphone and methadone, blocked the evoked neuronal acceleration like morphine when administered either microiontophoretically or i.v.; naloxone also blocked the effects of the two agonists. Finally, two non-opioid CNS depressants did not block the acceleration in neuronal firing even though microintophoretic ejection currents 2–5 times greater than those for morphine were used. In contrast, neuronal firing in the DR was rarely altered by the nociceptive stimulus or by morphine, administered either microiontophoretically or i.v. Furthermore, morphine did not affect the inhibition produced by 5-HT on neurons in the DR.It is concluded from this study that the MRF is a possible site of action for the antinociceptive effects of morphine. It is also concluded that morphine does not affect the spontaneous neuronal firing rate in the DR and that the DR is not a site of action of the antinociceptive effects of morphine when a foot pinch is used as the nociceptive stimulus.     

The action of substance P on neurons of the myenteric plexus of the guinea-pig small intestine.

Extracellular and intracellular recordings were made in vitro from single neurons of the myenteric plexus of the guinea-pig small intestine. Synthetic substance P was applied to the neurons by means of the perfusing solution or by electrophoresis from micropipettes. Extracellular recording showed that substance P (100 pm-30 nm), applied by perfusion, increased the firing rate of myenteric neurons. Intracellular recording indicated that perfusion with substance P caused a dose-dependent membrane depolarization which was unaffected by hexamethonium, hyoscine, naloxone or baclofen. The depolarization was also evoked by electrophoretic application of substance P. It was associated with an increase in membrane resistance, augmented by membrane depolarization and reduced by membrane hyperpolarization. The relation between the substance P reversal potential and the logarithm of the extracellular potassium concentration was linear with a slope of 54 mV/log10[K+], which indicates that substance P inactivates the resting potassium conductance of the myenteric neurons. This effect on ion conductance is the same as that of an unknown substance that mediates slow synaptic excitations with the myenteric plexus.     

DETECTION OF A SOLUBLE SEROTONIN-BINDING PROTEIN IN THE MAMMALIAN MYENTERIC PLEXUS AND OTHER PERIPHERAL SITES OF SEROTONIN STORAGE

Groups of neurons intrinsic to the mammalian myenteric plexus have been shown to have both tryptophan hydroxylase and a specific uptake mechanism for serotonin. They are probably serotonergic. A soluble protein with a high binding affinity for serotonin, similar to a protein previously found in rat brain by TAMIR & HUANG (1974), has now been found in the myenteric plexus of both rabbit and guinea pig. Partial purification of the protein from the rabbit's myenteric plexus by ammonium sulfate fractionation increased the ratio of specific to nonspecific serotonin binding almost 3-fold. Two dissociation constants for serotonin binding were obtained by equilibrium dialysis: 6.7 × 10^?10 M and 4.8 × 10^?7 M. The protein was similar to the soluble serotonin-binding protein of CNS: the indole derivatives 5, 6- and 5, 7-dihydroxytryptamine, and 6-hydroxytryptamine inhibited serotonin binding by 50% at 10^?7 M; norepinephrine was a poor inhibitor of serotonin binding; most of the serotonin-protein complex had a very high molecular weight and did not penetrate a 6.5% acrylamide gel. The appearance of the serotonin binding protein during development of the intestine in fetal rabbit correlates closely with the development of a serotonin uptake mechanism by nerves of this tissue and precedes the ingrowth of the adrenergic innervation. In-vitro administration of 6-hydroxydopamine to adult animals has no effect on the binding capacity for serotonin. Binding activity in denervated preparations is only 1/5 that of innervated tissue. It is concluded that the serotonin-binding protein, which has been found associated with serotonergic pathways in the CNS, is found associated with serotonergic neurons in the periphery as well. Since a similar serotonin-binding protein is also found in sheep thyroid, which stores but does not take up serotonin, the protein may be a component of the serotonin storage mechanism.     

Theophylline-induced changes in the presynaptic effects of adenosine, baclofen, clofelin and morphine in the myenteric plexus of the guinea pig

The experiments on the isolated guinea-pig ileum have shown that the contractions caused by low frequency transmural stimulation are decreased by adenosine, baclofen, clofelin and morphine. Theophylline is a competing antagonist of adenosine and a noncompeting antagonist of baclofen, clofelin and morphine. The effects of the latter are not altered by 1,3-dipropyl-8-phenylxanthine suppressing adenosine effects. It is concluded that presynaptic effects of baclofen, clofelin and morphine depend on cAMP level in cholinergic neurons of myenteric plexus.     

Projections of neurochemically specified neurons in the porcine colon

The intramural projections of nerve cells containing serotonin (5-HT), calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP) and nitric oxide synthase or reduced nicotinamide adenine dinucleotide phosphate diaphorase (NOS/NADPHd) were studied in the ascending colon of 5- to 6-week-old pigs by means of immunocytochemistry and histochemistry in combination with myectomy experiments. In control tissue of untreated animals, positive nerve cells and fibres were common in the myenteric and outer submucous plexus and, except for 5-HT-positive perikarya, immunoreactive cell bodies and fibres were also observed in the inner submucous plexus. VIP- and NOS/NADPHd-positive nerve fibres occurred in the ciruclar muscle layer while VIP was also abundant in nerve fibres of the mucosal layer. 5-HT- and CGRP-positive nerve fibres were virtually absent from the aganglionic nerve networks. In the submucosal layer, numerous paravascular CGRP-immunoreactive (IR) nerve fibres were encountered. Myectomy studies revealed that 5-HT-, CGRP-, VIP- and NOS/NADPHd-positive myenteric neurons all displayed anal projections within the myenteric plexus. In addition, some of the serotonergic myenteric neurons projected anally to the outer submucous plexus, whereas a great number of the VIP-ergic and nitrergic myenteric neurons send their axons towards the circular muscle layer. The possible function of these nerve cells in descending nerve pathways in the porcine colon is discussed in relation to the distribution pattern of their perikarya and processes and some of their morphological characteristics.     

Properties of synaptic inputs from myenteric neurons innervating submucosal S neurons in guinea pig ileum

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating "myenteric chamber" from the recording side "submucosal chamber," all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.     

Dopamine Affects the Change of Pain-Related Electrical Activity Induced by Morphine Dependence

Morphine is among the most effective analgesics. However, many evidences suggest that, besides the well-know analgesic activity, repeated opioids treatment can induce some side effects such as dependence, hyperalgesia and tolerance. The mechanism of noxious information transmission in the central nervous system after dependence is not clear. An important neurotransmitter, dopamine (DA) participates not only in the process of opioid dependence but also in pain modulation in the central nervous system. In the present study we observed changes of electrical activities of pain-excitation neurons (PENs) and pain-inhibition neurons (PINs) in the caudate nucleus (Cd) following the development of morphine dependence. We also observed the role of DA on these changes. Our results revealed that both the latency of PEN discharges and the inhibitory duration of PIN discharges decreased, and the net increased values of PEN and PIN discharges increased in the Cd of morphine dependent rats. Those demonstrated that electrical activities of both PENs and PINs increased in morphine dependent rats. DA inhibited the electrical activities of PENs and enhanced those of PINs in morphine dependent rats.     

Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections

Summary The distribution of nerve cells with immunoreactivity for the calcium-binding protein, calbindin, has been studied in the small intestine of the guinea-pig, and the projections of these neurons have been analysed by tracing their processes and by examining the consequences of nerve lesions. The immunoreactive neurons were numerous in the myenteric ganglia; there were 3500±100 reactive nerve cells per cm² of undistended intestine, which is 30% of all nerve cells. In contrast, reactive nerve cells were extremely rare in submucous ganglia. The myenteric nerve cells were oval in outline and gave rise to several long processes; this morphology corresponds to Dogiel's type-II classification. Processes from the cell bodies were traced through the circular muscle in perforating nerve fibre bundles. Other processes ran circumferentially in the myenteric plexus. Removal of the myenteric plexus, allowing time for subsequent fibre degeneration, showed that reactive nerve fibres in the submucous ganglia and mucosa came from the myenteric cell bodies. Operations to sever longitudinal or circumferential pathways in the myenteric plexus indicated that most reactive nerve terminals in myenteric ganglia arise from myenteric cell bodies whose processes run circumferentially for 1.5 mm, on average. It is deduced that the calbindin-reactive neurons are multipolar sensory neurons, with the sensitive processes in the mucosa and with other processes innervating neurons of the myenteric plexus.     

Sensory peptide neurotransmitters mediating mucosal and distension evoked neural vasodilator reflexes in guinea pig ileum

The aim was to determine the role CGRP and/or tachykinins released from sensory neural mechanisms in enteric neural vasodilator pathways. These pathways project through the myenteric plexus to submucosal vasodilator neurons. Submucosal arterioles were exposed in the distal portion of an in vitro combined submucosal-myenteric guinea pig ileal preparation, and dilation was monitored with videomicroscopy. Vasodilator neural reflexes were activated by gently stroking the mucosa with a fine brush or by distending a balloon placed beneath the flat-sheet preparation in the proximal portion. Dilations evoked by mucosal stroking were inhibited 64% by the CGRP 8-37 and 37% by NK3 (SR 142801) antagonists. When the two antagonists were combined with hexamethonium, only a small vasodilation persisted. Balloon distension-evoked vasodilations were inhibited by NK3 antagonists (66%) but were not altered by CGRP 8-37. In preparations in which myenteric descending interneurons were directly activated by electrical stimulation, combined application of CGRP 8-37 and the NK antagonists had no effect. Stimulation of capsaicin sensitive nerves in the myenteric plexus did not activate these vasodilator reflexes. These findings suggest that mucosal-activated reflexes result from the release of CGRP and tachykinins from enteric sensory neurons. Distension-evoked responses were significantly blocked by NK3 antagonists, suggesting that stretch activation of myenteric sensory neurons release tachykinins that activate NK3 receptors on myenteric vasodilator pathways.     

Myenteric denervation differentially reduces enteroendocrine serotonin cell population in rats during postnatal development

Summary The enteric nervous and enteroendocrine systems regulate different processes in the small intestine. Ablation of myenteric plexus with benzalkonium chloride (BAC) stimulates epithelial cell proliferation, whereas endocrine serotonin cells may inhibit the process. To evaluate the connection between the systems and the influence of myenteric plexus on serotoninergic cells in rats during postnatal development, the ileal plexus was partially removed with BAC. Rats were treated at 13 or 21 days and sacrificed after 15 days. The cell bodies of myenteric neurons were stained by β NADH-diaphorase to detect the extension of denervation. The number of enteroendocrine cells in the ileum was estimated in crypts and villi in paraffin sections immunostained for serotonin. The number of neurons was reduced by 27.6 and 45% in rats treated on the 13th and 21st days, respectively. We tried to establish a correlation of denervation and the serotonin population according to the age of treatment. We observed a reduction of immunolabelled cells in the crypts of rats treated at 13 days, whereas this effect was seen in the villi of rats denervated at 21 days. These results suggest that the enteric nervous system might control the enteroendocrine cell population and this complex mechanism could be correlated to changes in cell proliferation.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.     

Effects of morphine on electrical activity of the emotion-producing zones in the hypothalamus of adult and aged rats

In the experiments performed on adult and aged rats, the effect of morphine on the electrical activity, recorded from the emotion-producing zones of the hypothalamus, the ventromedial nucleus (VMN), and the lateral hypothalamic area (LHA), was studied. In thein vitro experiments, an age-dependent reduction of background impulse activity (BIA) was found in the VMN single neurons, but not in the LHA neurons. Morphine reduced BIA in most of the VMN neurons, but enhanced it in the LHA neurons of adult rats, and enhanced BIA in the neurons of both structures of the aged rats. The inhibitory effect of morphine on the VMN and LHA neurons and its excitatory effect on the LHA neurons decreased with age. In thein vivo experiments, an age-dependent reduction of the background field electrical activity (background electrogram, BEG) was found in the neurons of both emotion-producing zones. Morphine reduced the BEG magnitude in the VMN and LHA more effectively in the aged rats than in the adult rats. The results allow us to suggest that both the opiate regulation of hypothalamic functions and formation of an opiate dependence in the adult rats essentially differ from those in the aged rats.Neirofiziologiya/Neurophysiology, Vol. 27, No. 2, pp. 126–133, March–April, 1995.