tetrodotoxin and Hirschsprung-Disease

The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Movement; Chick Embryo; Cobalt; Disease Models, Animal; Hirschsprung Disease; Intestines; Muscle, Smooth; Myenteric Plexus; Neural Crest; Organ Culture Techniques; Peristalsis; Tetrodotoxin; Time-Lapse Imaging

It is not clear what contribution the internal anal sphincter makes to the impaired motility observed in patients with Hirschsprung's disease (HD). Neuropeptides have recently been shown to be neurotransmitters in the nonadrenergic, noncholinergic inhibitory and excitatory nerves in the human gut. To clarify the physiologic significance of vasoactive intestinal polypeptide and substance P in the internal anal sphincter of HD (aganglionosis), we investigated the enteric nerve responses on lesional and normal internal anal sphincter muscle strips above the dentate line.. The lesional and normal internal anal sphincter muscle strips above the dentate line were derived from patient with HD (9 cases) and patients who underwent rectal amputation for low rectal cancers (8 cases). A mechanographic technique was used to evaluate in vitro muscle responses to these peptides of adrenergic and cholinergic nerves before and after treatment with various autonomic nerve blockers.. Nonadrenergic, noncholinergic inhibitory nerves were found to act on the normal internal anal sphincter but had no effect on the enteric nerves in aganglionosis. Peptidergic (vasoactive intestinal polypeptide and substance P) nerves were found to act on normal colon, but no effect was observed in the aganglionic internal anal sphincter.. These findings suggest that peptidergic nerves play an important role in the impaired motility observed in the internal anal sphincter with HD.

Topics: Aged; Anal Canal; Atropine; Electric Stimulation; Female; Ganglia, Autonomic; Gastrointestinal Motility; Hirschsprung Disease; Humans; In Vitro Techniques; Infant; Male; Middle Aged; Muscle Contraction; Muscle, Smooth; Phenoxybenzamine; Propranolol; Rectal Neoplasms; Substance P; Tetrodotoxin; Vasoactive Intestinal Peptide

Responses of isolated muscle strips from the rat and the dog internal anal sphincter (IAS) to drugs and electrical field stimulation (EFS) were investigated in vitro for the purpose of clarifying a manner of neural control of IAS. Also, responses of muscle strips from IAS of the patients with Hirschsprung's disease were compared with those of muscle strips from human control IAS. Muscle strips from the dog and human IAS as normal control showed contractions to norepinephrine (NE), which were abolished in the presence of phentolamine and relaxations to isoproterenol. EFS (less than 1 msec) induced relaxations of the muscle strips. These responses to EFS were not affected by either one of phentolamine, propranolol and atropine but were inhibited by tetrodotoxin. Muscle strips from IAS in Hirschsprung's disease contracted to both NE and EFS, the responses of which were abolished in the presence of phentolamine. But no relaxation to EFS of muscle strips from IAS in Hirschsprung's disease was observed. These findings revealed that normal IAS is pharmacologically innervated by alpha-adrenergic excitatory nerve, beta-adrenergic inhibitory nerve and non-adrenergic, non-cholinergic inhibitory nerve and suggested that IAS in Hirschsprung's disease is also affected by alpha-adrenergic excitatory nerve but inhibitory neural control is absent.

Topics: Anal Canal; Animals; Child, Preschool; Dogs; Electric Stimulation; Female; Hirschsprung Disease; Humans; In Vitro Techniques; Infant; Isoproterenol; Male; Muscle Contraction; Muscle Relaxation; Norepinephrine; Phentolamine; Rats; Rats, Inbred Strains; Sympathetic Nervous System; Tetrodotoxin

This study examined the function in vitro of aganglionic colon musculature in mice with hereditary aganglionosis--a strain of animals used as a model of Hirschsprung's disease. Double sucrose gap recordings from the muscle strips of both normal and aganglionic colon showed bursts of spike potentials with muscle contraction. Intracellular recordings of the membrane potentials of the circular muscle cells of normal, aganglionic and oligo-ganglionic colon had no statistical difference. Microelectrode recordings from the circular muscle cells of normal siblings, in the presence of nifedipine, irregular ongoing fluctuations in membrane potential, which were abolished by tetrodotoxin and reduced by d-tubocurarine or apamin. The fluctuations were less effected by atropine. These observations suggest that there is ongoing inhibitory neural activity to the circular smooth muscle of normal colon. These ongoing fluctuations were not recorded from the cells of aganglionic and oligo-ganglionic colon of affected animals. Although transmural stimulation of the intrinsic nerves produced cholinergic excitatory and inhibitory junction potentials in normal colon, no junction potentials were evoked by transmural stimulation in aganglionic colon. It was concluded that the ongoing tonic inhibitory activity may contribute to the compliance of the normal mouse colon and lack of the compliance may affect functional intestinal obstruction of the aganglionic colon in Hirschsprung's disease.

Topics: Animals; Atropine; Colon; Disease Models, Animal; Female; Ganglia; Hirschsprung Disease; In Vitro Techniques; Male; Membrane Potentials; Mice; Muscle Denervation; Muscle, Smooth; Tetrodotoxin; Tubocurarine

A true role of extrinsic nerve fibres proliferating abundantly in the aganglionic colon of Hirschsprung's disease has not yet been established. Electrophysiological and pharmacological studies were carried out on rectal muscle strips of piebald lethal mice and their siblings. Muscle strips of both aganglionic and normal colon obtained from fifteen patients with Hirschsprung's disease were also investigated for the purpose of determining whether these extrinsic nerve fibres innervate the smooth muscle cells in the aganglionic colon or not. Junction potentials and contractile tensions evoked by electric stimulations were recorded employing the double-sucrose gap method, and examined the effects of nerve blocking agents on junction potentials. In the normal colon, excitatory junction potentials and inhibitory junction potentials were recorded following stimulation in both man and mouse. Excitatory junction potentials were accompanied by muscle contraction and abolished by atropine or tetrodotoxin. Inhibitory junction potentials were resistant to phentolamine and propranolol, and abolished by tetrodotoxin. It was concluded that the normal colon of both mouse and man is innervated by cholinergic excitatory and non-adrenergic inhibitory nerves. In the aganglionic colon, on the other hand, neither junction potentials nor contractions were evoked by stimulations in both mouse and man. These evidences led to the conclusion that in the aganglionic colon in Hirschsprung's disease of man and murine model, there may be a deficiency in neuronal regulation.

Topics: Adolescent; Adult; Animals; Atropine; Child; Child, Preschool; Colon; Culture Techniques; Electric Stimulation; Female; Hirschsprung Disease; Humans; Infant; Male; Membrane Potentials; Mice; Mice, Neurologic Mutants; Muscle Contraction; Myenteric Plexus; Neuromuscular Junction; Phentolamine; Propranolol; Synaptic Transmission; Tetrodotoxin

In order to assess abnormalities in the adrenergic mechanism in the intestine of Hirschsprung's disease, catecholamine concentrations, alpha-adrenoceptors, and the effect of electrical field stimulation were examined in aganglionic segments of colon or rectum. The aganglionic segment had a higher concentration of norepinephrine, assayed with high performance liquid chromatography with an electrochemical detector, whereas concentrations of epinephrine or dopamine were similar in normal and pathological segments. In four patients with extensive aganglionosis, the norepinephrine concentration in aganglionic colon segments decreased progressively in descending, transverse, and ascending colon. The tissue content of alpha-adrenoceptors and their affinity assayed from the specific binding of [3H]dihydro-alpha-ergocryptine appeared similar in normal and aganglionic segments of the rectosigmoidal colon. Electrical field stimulation of normal rectosigmoidal colon segments caused relaxation at low frequencies and contraction at a very high frequency. Relaxation was not abolished by blocking concentrations of propranolol or phentolamine. In aganglionic segments, the predominant response to electrical field stimulation was contraction, which was inhibited by either atropine or tetrodotoxin. These results indicate that an alpha-adrenergic system and cholinergic innervation apparently exist in aganglionic colon segments and that dysfunction of the colon appears to result from lack of a nonadrenergic inhibitory system.

Topics: Atropine; Catecholamines; Chromatography, High Pressure Liquid; Colon; Dopamine; Electric Stimulation; Epinephrine; Ganglia, Parasympathetic; Hirschsprung Disease; Humans; Norepinephrine; Phentolamine; Propranolol; Receptors, Adrenergic, alpha; Tetrodotoxin

This study examined the projections in vitro of excitatory and inhibitory colonic motor nerves in the unaffected siblings of mice with hereditary aganglionic colon--a strain of animals used as a model of Hirschsprung's disease. Microelectrode recordings from the circular muscle layer showed ongoing fluctuations in membrane potential (presumably junction potentials) that were sensitive to tetrodotoxin. Transmural electrical stimulation of the intrinsic nerves produced cholinergic excitatory and inhibitory junction potentials. The latter responses were more prolonged when recorded aboral to the stimulating electrodes. Apamin reduced, but did not block, these inhibitory potentials. Cholinergic excitatory junction potentials were abolished by d-tubocurarine when recording 2 mm from the stimulating electrodes; inhibitory junction potentials were recorded up to 10 mm in the presence of this drug. Thus, the intrinsic excitatory cholinergic motor neurons have relatively short projections to the circular smooth muscle compared with the projections of the inhibitory motor neurons.

Topics: Animals; Apamin; Atropine; Colon; Disease Models, Animal; Electric Stimulation; Evoked Potentials; Hirschsprung Disease; Membrane Potentials; Mice; Mice, Mutant Strains; Microelectrodes; Motor Neurons; Muscle, Smooth; Neuromuscular Junction; Nifedipine; Tetrodotoxin; Tubocurarine

These studies were directed toward better characterization of the abnormalities of motor function in the large intestine of mutant mice with congenital aganglionosis and megacolon. Analysis of pressure-volume relations in the megacolon and aganglionic terminal segment showed increased intestinal wall compliance in the dilated colon and reduced wall compliance in the aganglionic region as compared to normal littermates. Migrating contractile complexes occurred spontaneously in ganglionated regions of the large intestine of both normal and mutant mice, but never propagated into the aganglionic segment of the abnormal bowel. Tetrodotoxin eliminated the migrating complexes and increased random spontaneous contractions in all areas except the aganglionic region. Circular muscle tension was reduced by electrical field stimulation, and poststimulus rebound contractions occurred in all ganglionated regions of the intestine of both normal and mutant mice. No responses to electrical stimulation occurred in the aganglionic segments of the preparations from mutant mice. The poststimulus responses "fatigued" at a faster rate in the megacolonic region of the abnormal bowel than in the equivalent region of the normal bowel, when evoked repetitively over prolonged time periods. There were no differences between the intestines of normal and mutant mice in latency, amplitude, duration, or area under the contractile curves of the poststimulus responses. Intracellular electrical recording from circular muscle fibers revealed slow depolarizing potentials with action potentials at the crests in all regions of the large bowel from both normal and abnormal mice. It also showed excitatory and inhibitory junction potentials in response to electrical stimulation. Inhibitory junction potentials summated during repetitive stimulation and postinhibitory rebound excitation occurred after offset of the stimulation. Stimulus-evoked junction potentials were recorded in all regions of the large intestine except in the aganglionic segment of the mutant mice. We concluded that most of the electrical and mechanical behavior of the aganglionic terminal segment reflected the absence of inhibitory innervation of the musculature in this region.

Topics: Animals; Disease Models, Animal; Electric Stimulation; Epinephrine; Hirschsprung Disease; Intestine, Large; Lidocaine; Membrane Potentials; Mice; Mice, Mutant Strains; Microelectrodes; Muscle Contraction; Tetrodotoxin; Time Factors; Transducers, Pressure

Pharmacological agents that might influence the poststimulus rebound contractile response (PSRR) through an action on the intrinsic inhibitory innervation of the circular muscle coat were studied in the large intestine of the piebald mouse model for Hirschsprung's disease. Tetrodotoxin (0.3-3 microM) suppressed, in a dose-dependent manner, the amplitude, duration, and area under the contractile traces of the PSRR and increased the latency for the response in ganglionated segments of the large intestine of piebald and normal mice. Sensitivity to tetrodotoxin was greater in the megacolon of piebald mice than in the colon of normal mice. Norepinephrine (6-360 microM) suppressed all parameters of the PSRR in ganglionated regions of the large intestine of abnormal mice. Sensitivity to norepinephrine was greater in the megacolon than in the colon of normal mice. Norepinephrine abolished spontaneous contractile behavior in ganglionated regions of the large intestine of normal and diseased mice and in the aganglionic segment of the piebald mice. Suppression of the PSRR by phentolamine and propranolol were interpreted as nonspecific local anesthetic effects. Atropine (20-200 microM) increased the area under the contractile traces in the midcolon of the intestine of both normal and piebald mice and had no effects on the terminal segment of the large intestine of normal mice. Spontaneous contractile activity and baseline tension were increased by atropine (350 microM) in all preparations. The nicotinic agonist, DMPP (3-157 microM), suppressed the PSRR in the ganglionated regions of the bowel from both normal and abnormal mice. DMPP relaxed resting tension in ganglionated regions, but did not affect the aganglionic terminal segment of the piebald mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Dimethylphenylpiperazinium Iodide; Guanethidine; Hirschsprung Disease; Indomethacin; Intestine, Large; Mice; Mice, Mutant Strains; Muscle Contraction; Norepinephrine; Phentolamine; Propranolol; Tetrodotoxin

Total Intestinal Aganglionosis is a rare, uniformly fatal condition with absence of ganglia from the duodenum to the rectum. A neonate with this extreme form of Hirschsprung's disease is presented with a review of 12 previously reported cases. Smooth muscle strips from this infant's gastrointestinal tract demonstrated viable cholinergic receptors, absence of intrinsic neuronal innervation, and colonic contractile activity to the purported peptidergic neurotransmitter cholecystokinin. Four aspects of total intestinal aganglionosis are noteworthy: (1) one-third of patients presented between four and eight days of age after passing meconium on the first day of life; (2) at laparotomy, no intestinal distention, obstruction, or transition zone was evident; (3) hypertrophic nerve fibers seen in classic Hirschsprung's disease were absent in one-quarter of patients; and (4) a high incidence of affected siblings occurred in the previously reported cases. This form of Hirschsprung's disease may represent a distinct entity with autosomal recessive inheritance and significant risk for recurrence.

Topics: Acetylcholine; Colon; Electric Stimulation; Hirschsprung Disease; Humans; In Vitro Techniques; Infant, Newborn; Intestines; Jejunum; Male; Muscle Contraction; Sincalide; Tetrodotoxin