Electrical Stimulation Of Intrinsic Nerves Research Articles

Translational neuropharmacology: the use of human isolated gastrointestinal tissues

Translational sciences increasingly emphasize the measurement of functions in native human tissues. However, such studies must confront variations in patient age, gender, genetic background and disease. Here, these are discussed with reference to neuromuscular and neurosecretory functions of the human gastrointestinal (GI) tract. Tissues are obtained after informed consent, in collaboration with surgeons (surgical techniques help minimize variables) and pathologists. Given the difficulties of directly recording from human myenteric neurones (embedded between muscle layers), enteric motor nerve functions are studied by measuring muscle contractions/relaxations evoked by electrical stimulation of intrinsic nerves; responses are regionally dependent, often involving cholinergic and nitrergic phenotypes. Enteric sensory functions can be studied by evoking the peristaltic reflex, involving enteric sensory and motor nerves, but this has rarely been achieved. As submucosal neurones are more accessible (after removing the mucosa), direct neuronal recordings are possible. Neurosecretory functions are studied by measuring changes in short-circuit current across the mucosa. For all experiments, basic questions must be addressed. Because tissues are from patients, what are the controls and the influence of disease? How long does it take before function fully recovers? What is the impact of age- and gender-related differences? What is the optimal sample size? Addressing these and other questions minimizes variability and raises the scientific credibility of human tissue research. Such studies also reduce animal use. Further, the many differences between animal and human GI functions also means that human tissue research must question the ethical validity of using strains of animals with unproved translational significance.

High spatial resolution studies of muscarinic neuroeffector junctions in mouse isolated vas deferens

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca2+ dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and α,β methylene ATP (α,β-MeATP) (1 μM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 μM) elicited Ca2+ transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 μM). Ca2+ transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 μM). Stimulation produced a marked increase in the frequency and number of Ca2+ transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The α2-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca2+ transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional α2-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca2+ events, with no effect on Ca2+ transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca2+ transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M3 muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca2+ dynamics in this tissue.

Hypertrophic smooth muscle in the partially obstructed opossum esophagus: Excitability and electrophysiological properties

Partial obstruction of the opossum esophagus leads to thickening of the circular muscle, hypertrophy of smooth muscle cells, and diminution of the extracellular space. The pharmacological and electrophysiological properties of this hypertrophied muscle were studied. Carbachol produced phasic and tonic contractions of the circular muscle. The EC50 for tonic contractions was greater for hypertrophied than for normal muscle (21.1 ± 3.9 μmol/L vs. 4.8 ± 2.2 μmol/L; P < 0.05). The resting membrane potential difference of hypertrophied muscle (−50.8 ± 0.2 mV) was similar to that of normal muscle (−50.0 ± 0.2 mV). Electrical stimulation of intrinsic nerves in the normal muscle produced a hyperpolarization followed by a depolarization of smooth muscle membrane potential. Hypertrophied muscle responded either with an attenuated hyperpolarization or no hyperpolarization, both of which were followed by a depolarization. The space constant in the long axes of the hypertrophied circular muscle cells was greater than normal (4.4 ± 0.2 mm vs. 3.4 ± 0.1 mm; P < 0.001). The threshold potential for initiation of action potentials was more negative for hypertrophied (−43.2 ± 0.4 mV) than for normal circular muscle (−41.6 ± 0.2 mV; P < 0.005). These data indicate that alterations in neuromuscular function accompany the hypertrophy of esophageal smooth muscle.

Inhibitory innervation of colonic smooth muscle cells and interstitial cells of Cajal

The effect of neural inhibition on the electrical activities of circular and longitudinal colonic smooth muscle was investigated. In addition, a comparative study was carried out between circular muscle preparations with and without the "submucosal" and "myenteric plexus" network of interstitial cells of Cajal (ICC) to study innervation of the "submucosal" ICC and to investigate whether or not the ICC network is an essential intermediary system for inhibitory innervation of smooth muscle cells. Electrical stimulation of intrinsic nerves in the presence of atropine caused inhibitory junction potentials (ijps) throughout the circular and longitudinal muscle layers. The ijp amplitude depended on the membrane potential and not on the location of the muscle cells with respect to the ICC network. Neurally mediated inhibition of the colon resulted in a reduction in amplitude and duration of slow wave type action potentials in circular and abolishment of spike-like action potentials in longitudinal smooth muscle, both resulting in a reduction of contractile activity. With respect to mediation by ICC, the study shows (i) "submucosal" ICC receive direct inhibitory innervation and (ii) circular smooth muscle cells can be directly innervated by inhibitory nerves without ICC as necessary intermediaries. The reversal potential of the ijp in colonic smooth muscle was observed to be approximately -76 mV, close to the estimated potassium equilibrium potential, suggesting that the nerve-mediated hyperpolarization is caused by increased potassium conductance.

Jejunal circular muscle motility is decreased in nematode-infected rat

Jejunal circular muscle motility was studied in vitro in rats 8–10 days after inoculation with the inflammation-inducing nematode Nippostrongylus brasiliensis. The passive properties of the muscle, i.e., the development of passive tension and the optimal amount of stretch for active contractions, were unchanged by infection. Infection decreased the development of active resting tension, spontaneous contractions, muscle contraction to muscarinic receptor activation, and direct electrical stimulation. Relaxation to β-adrenergic stimulation was also decreased in tissues from infected animals. Response to cholinergic stimulation, spontaneous contractions, and active resting tension were completely dependent on extracellular calcium. The dominant response to electrical stimulation of intrinsic nerves was relaxation in control tissue and contraction in tissue from infected rats. In the presence of atropine, all tissues from control rats but only 33% of the tissues from infected rats relaxed, suggesting a marked difference in functional inhibitory innervation. The inflammation may have either decreased the circular muscle responsiveness to the inhibitory transmitter or decreased the release of this transmitter. Thus, a nematode infection produces decreased responsiveness of the intestinal circular muscle to both contracting and relaxing stimuli and causes a reduction in functional inhibitory innervation in this layer. These changes suggest mechanisms for the reduction of intestinal transit observed after some nematode infections.

Pacemaker activity in the proximal lower oesophageal sphincter of the dog.

1. The electrical and mechanical activities of different regions of the canine lower oesophageal sphincter were measured using the single sucrose gap technique. 2. Spontaneous electrical activity was found in the region 0-6 mm oral to the squamocolumnar border. 3. The electrical activity consisted of bursts of spikes superimposed on slow waves. The slow-wave frequency ranged from 0.6 to 5 min-1 in different muscle strips. 4. The slow wave-spike complex and associated contraction were insensitive to tetrodotoxin and atropine. 5. In the pacemaker region, electrical stimulation of intrinsic nerves evoked excitatory junction potentials (atropine sensitive), inhibitory junction potentials (non-adrenergic) and post-stimulus excitation. 6. Increase in the frequency of the slow waves was obtained by muscarinic receptor stimulation (carbachol 10(-7) M) and 10 mM-KCl. 7. The distal lower oesophageal sphincter exhibited a high basal tension but did not show spontaneous electrical activity and stimulation of intrinsic nerves revealed only non-cholinergic, non-adrenergic inhibition. 8. The electrical slow-wave activity observed in the proximal sphincter may constitute the control mechanism for the phasic nature of the contractile activity seen during both the postprandial period and phase III of the interdigestive migrating myoelectric complex. 9. The neural cholinergic activity present in the proximal lower oesophageal sphincter suggests the possibility of neural modulation of the myogenic control activity.

Ion transport in human cecum, transverse colon, and sigmoid colon in vitro: Baseline and response to electrical stimulation of intrinsic nerves

In a flux chamber study of ion transport in human colon, we compared baseline rates with those measured during electrical stimulation of intrinsic nerves. In baseline studies, sodium was absorbed throughout, but maximally in transverse colon. In cecum, sodium absorption accounted for the short circuit current and chloride was not absorbed. Chloride was absorbed in transverse and sigmoid colon, however. Residual current was minimal in cecum and transverse colon, but increased in sigmoid colon. Neural stimulation caused chloride secretion in cecum, reduced chloride absorption in sigmoid colon, but caused no change in transverse colon; sodium absorption decreased in cecum. A neurotransmitter of unknown identity affects baseline short circuit current in sigmoid colon. Half of the increase in short circuit caused by neural stimulation in sigmoid colon is mediated by muscarinic receptors. The identity of the other transmitter(s) is not known. It is not substance P or histamine. The three divisions of the colon differ in relative rates of baseline ion transport and in their transport responses to intrinsic nerve stimulation.

The effect of pertussis toxin on alpha-2-adrenoceptor-mediated pigment migration in fish melanophores

The aggregation of melanin-granules within fish pigment cells (melanophores) can be elicited either by electrical stimulation of intrinsic nerves or by the addition of adrenergic agonists. The pigment aggregation seems to be mediated by alpha-2-adrenoceptors. In this investigation we have used various agonists and antagonists (noradrenaline, (+)- and (−)-adrenaline, isoprenaline, yohimbine and prazosin) to further characterize the pigment-aggregating receptor of Labrus ossifagus. All the results obtained support the notion of alpha-2-adrenoceptor-mediated pigment aggregation. The pertussis toxin, islet-activating protein (IAP), is known to inhibit the alpha-2-adrenoceptor-mediated signal transduction in mammals. We have used IAP to investigate whether fish melanophore alpha-2-adrenoceptors are also inhibited by this toxin. We found that IAP inactivated the alpha-2-adrenoceptor-mediated pigment aggregation in a dose-dependent manner. The inhibitory IAP- effect had a remarkably short onset-time in the melanophores (maximal effect was obtained within 10 min of incubation). Interestingly, binding of an agonist (noradrenaline) to the receptors prevented IAP from exerting its inhibitory action, whereas binding of an antagonist (yohimbine) gave no protection against the IAP-inactivation. In conclusion, the pigment-aggregating receptors of melanophores of L. ossifagus are very similar to the mammalian alpha-2-adrenoceptors. It is possible to inactivate the melanophore receptor system with IAP and this inactivation has a remarkably short onset-time. Stimulation of the alpha-2-adrenoceptors prevents IAP from inactivating the receptor system.

Depression of mechanical and electrical activity in muscle strips of opossum stomach and esophagus by acidosis

We studied the effect of acidosis on nerve-muscle preparations from the opossum stomach and esophagus. All muscle strips were mounted in superfusion chambers and spontaneous contractile activity was recorded from longitudinal and transverse gastric muscle strips. Mechanical activity in esophageal and oblique gastric muscle strips was elicited by electrical stimulation of intrinsic nerves. The contractile activity of all types of strips changed when the pH of the superfusate was lowered for 30 min from 7.4 to 7.0. The baseline tension rose in strips from the lower esophageal sphincter and in oblique gastric muscle strips, and a baseline tension occurred de novo in longitudinal esophageal strips. Muscle relaxation was also impaired in these three types of strips. The amplitude of contractions declined in transverse strips from the esophageal body and in transverse and longitudinal strips from the stomach. The frequency of spontaneous contractions and of the pacesetter potential as recorded by extracellular electrodes was also reduced by acidosis in muscle strips from the stomach. Similar but less marked changes occurred when the pH was lowered from 7.4 to 7.2 for 30 min. Most changes related to a pH of 7.0 became irreversible after superfusion periods of more than 2 h. These findings indicate that acidosis alters a variety of neuromuscular functions and that its effects are determined by the electrical and contractile characteristics and the intrinsic innervation of individual segments and layers of the gut muscle coat.

Transmembrane voltage of opossum esophageal smooth muscle and its response to electrical stimulation of intrinsic nerves

This study was designed to examine the transmembrane potential of esophageal circular smooth muscle both before and in response to electrical stimulation of the intrinsic neural plexus, and to determine if regional differences are present in these potentials. Transverse strips of the opossum esophagus were prepared from sites located 1,3,5, and 7 cm above the gastroesophageal junction and subjected to 1-s trains of square-wave pulses. The effects of varying pulse frequency, voltage, and duration were assessed by conventional microelectrode techniques. The latency of the electrical off response ranged from a mean value of 1.08 s at 5 Hz to 1.0 s at 16 Hz when measured in cells from strips 5 cm above the gastroesophageal junction. Associated with this increase in latency is both an increase in the mean amplitude of hyperpolarization and a fall in the rate of the ensuing depolarization. In contrast, an increase in pulse voltage or duration had no effect on electrical off response latency. Resting potential analyzed on a regional basis ranged from a mean of −52.8 mV to −43.5 mV in strips 7 cm and 1 cm above the gastroesophageal junction respectively. Electrical off response latencies also demonstrated regional differences ranging from 1.06 to 1.50 s in strips taken 7 cm and 3 cm above the gastroesophageal junction respectively. Associated with the prolonged latencies noted in the more aboral strips was a decrease in the average rates of both membrane hyperpolarization and ensuing depolarization. It can be concluded from these studies that (a) within a given region an increase in stimulus frequency increases the mean amplitude of hyperpolarization, decreases the rate of the ensuing depolarization, and prolongs the electrical off response latency; (b) mean resting potential of esophageal circular smooth muscle cell becomes gradually less negative in an oral to aboral direction; and (c) cells of the more aboral smooth muscle segments studied demonstrated a decrease in the average rates of both membrane hyperpolarization and ensuing depolarization, and a prolonged electrical off response latency.