Smooth muscle from aganglionic bowel in Hirschsprung's disease impairs neuronal development in vitro

Hirschsprung's disease results from the congenital absence of enteric neurons in human distal colon. The reason for aganglionosis is unknown but may reflect an unfavourable microenvironment for neuronal development. We asked if smooth muscle cells from the aganglionic region could affect neuronal development in vitro. Neurons from neonatal mouse superior cervical ganglia were added to cultures of smooth muscle obtained from normal or aganglionic regions of five patients with Hirschsprung's disease. Although neurons initially showed more rapid attachment to aganglionic smooth muscle, this was equal by 60 min and thereafter. Progressive increase in the diameter of the nerve cell body was characteristic of normal maturation in vitro. This was consistently inhibited by 15–22% in neurons grown on aganglionic muscle compared with normal controls over the 6-day test period (P<0.05). This phenomenon was preserved when the smooth muscle cells were lysed by brief exposure to distilled water before initiation of coculture (16–18% inhibition; P<0.05). These data imply that smooth muscle of the aganglionic colon is less favourable for neuronal development than the normally innervated region and suggest a membrane-linked factor. Clearly, this persists in postnatal life and in vitro and may reflect an abnormality of cellular interaction causing Hirschsprung's disease.     

Interstitial cells of Cajal and electrical activity in ganglionic and aganglionic colons of mice

An antibody directed against Kit protein was used to investigate the distribution of interstitial cells of Cajal (ICC) within the murine colon. The ICC density was greatest in the proximal colon and decreased along its length. The distribution of the different classes of ICC in the aganglionic colons of lethal spotted (ls/ls) mice was found to be similar in age-matched wild-type controls. There were marked differences in the electrical activities of the colons from ls/ls mutants compared with wild-type controls. In ls/ls aganglionic colons, the circular muscle was electrically quiescent compared with the spontaneous spiking electrical activity of wild-type tissues. In ls/ls aganglionic colons, postjunctional neural responses were greatly affected. Inhibitory junction potentials were absent or excitatory junction potentials inhibited by atropine were observed. In conclusion, the distribution of ICC in the ganglionic and aganglionic regions of the colons from ls/ls mutants appeared similar to that of wild-type controls. The electrical activity and neural responses of the circular layer are significantly different in aganglionic segments of ls/ls mutants.     

Segmental distribution of colonic neuropeptides in Hirschsprung's disease.

Despite continued research, the pathophysiologic mechanism responsible for functional obstruction in the aganglionic segment of bowel in Hirschsprung's disease remains controversial. Narrowing of the affected segment is thought by many investigators to be the result of loss of intrinsic inhibitory innervation. For this hypothesis to be consistent, inhibitory neuropeptides should be present in the dilating, transitional segment of bowel. In order to quantitate reported changes in peptidergic nerve staining in Hirschsprung's disease, we measured concentrations of five neuropeptides (vasoactive intestinal peptide, peptide histidine-methionine, met5-enkephalin, substance P and bombesin-like immunoreactivity) by radioimmunoassay in the affected segments of bowel from six patients with Hirschsprung's disease. Tissue extracts were prepared using gut obtained at surgery from the: (1) constricted, aganglionic segment, (2) dilating, aganglionic transitional segment and (3) dilated, proximal ganglionic segment. Concentrations of vasoactive intestinal peptide, peptide histidine-methionine, substance P and met5-enkephalin were significantly reduced in both the muscularis externa and the mucosal-submucosal layers from the constricted aganglionic segment. By contrast, concentrations of the candidate inhibitory neuropeptides, vasoactive intestinal peptide and peptide histidine-methionine, were minimally reduced in the dilating, aganglionic transitional segment. These results are consistent with the hypothesis that constriction of the aganglionic segment is due to loss of intrinsic inhibitory innervation. Concentrations of bombesin-like immunoreactivity were similar in the three segments of human gut, suggesting the presence of this immunoreactive neuropeptide in extrinsic nerve fibers.     

Fine structure of neurons synthesizing vasoactive intestinal peptide in the human colon from patients with Hirschsprung's disease

Summary The fine structure of neuronal perikarya and processes containing VIP-like immunoreactive material in the colon of patients with Hirschsprung's disease was investigated by immunoelectron microscopy. No VIP-like immunoreactive terminals were found in Auerbach's plexus of the ganglionic segment. However, VIP-like immunoreactive preterminal axons were frequently found to make synaptic contact with both immunoreactive and non-immunoreactive elements within Meissner's plexus. Therefore, the function of the VIP neurons in Auerbach's plexus seems to differ from that in Meissner's plexus. In the oligoganglionic segment, there were a few VIP-like immunoreactive processes, but no VIP-like immunoreactive synaptic formations. VIP-like immunoreactive processes were rarely encountered in the aganglionic segment. In both the oligo-and aganglionic segments, bowel relaxation is considered to be disturbed due to the lack of synaptic contacts of VIP-like immunoreactive neurons with other neuronal components.This work was supported in part by a grant (no. 57370002) from the Ministry of Education, Science and Culture, Japan     

Fine structure of neurons synthesizing vasoactive intestinal peptide in the human colon from patients with Hirschsprung's disease

The fine structure of neuronal perikarya and processes containing VIP-like immunoreactive material in the colon of patients with Hirschsprung's disease was investigated by immunoelectron microscopy. No VIP-like immunoreactive terminals were found in Auerbach's plexus of the ganglionic segment. However, VIP-like immunoreactive preterminal axons were frequently found to make synaptic contact with both immunoreactive and non-immunoreactive elements within Meissner's plexus. Therefore, the function of the VIP neurons in Auerbach's plexus seems to differ from that in Meissner's plexus. In the oligoganglionic segment, there were a few VIP-like immunoreactive processes, but no VIP-like immunoreactive synaptic formations. VIP-like immunoreactive processes were rarely encountered in the aganglionic segment. In both the oligo- and aganglionic segments, bowel relaxation is considered to be disturbed due to the lack of synaptic contacts of VIP-like immunoreactive neurons with other neuronal components.     

Hirschsprung's disease and congenital deafness

Summary A family is described showing deafness in three consecutive generations. Hirschsprung's disease was present in at least two of the affected patients and a history of bowel dysfunction was present in the third. The association of the two disorders in this family may be due to a single autosomal dominant gene and in this regard differs from previously reported isolated patients with Hirschsprung's disease and deafness.     

Molecular heterogeneity of RET loss of function in Hirschsprung's disease.

The RET proto-oncogene encodes a receptor with tyrosine kinase activity (RET) that is involved in several neoplastic and non-neoplastic diseases. Oncogenic activation of RET, achieved by different mechanisms, is detected in a sizeable fraction of human thyroid tumors, as well as in multiple endocrine neoplasia types 2A and 2B (MEN2A and MEN2B) and familial medullary thyroid carcinoma tumoral syndromes. Germline mutations of RET have also been associated with a non-neoplastic disease, the congenital colonic aganglionosis, i.e. Hirschsprung's disease (HSCR). To analyse the impact of HSCR mutations on RET function, we have introduced into wild-type RET and activated RET(MEN2A) and RET(MEN2B) alleles three missense mutations associated with HSCR. Here we show that the three mutations caused a loss of function of RET when assayed in two model cell systems, NIH 3T3 and PC12 cells. The effect of different HSCR mutations was due to different molecular mechanisms. The HSCR972 (Arg972-->Gly) mutation, mapping in the intracytoplasmic region of RET, impaired its tyrosine kinase activity, while two extracellular mutations, HSCR32 (Ser32-->Leu) and HSCR393 (Phe393-->Leu), inhibited the biological activity of RET by impairing the correct maturation of the RET protein and its transport to the cell surface.     

Conditional ablation of GFRalpha1 in postmigratory enteric neurons triggers unconventional neuronal death in the colon and causes a Hirschsprung's disease phenotype

The regulation of neuronal survival and death by neurotrophic factors plays a central role in the sculpting of the nervous system, but the identity of survival signals for developing enteric neurons remains obscure. We demonstrate here that conditional ablation of GFRalpha1, the high affinity receptor for GDNF, in mice during late gestation induces rapid and widespread neuronal death in the colon, leading to colon aganglionosis reminiscent of Hirschsprung's disease. Enteric neuron death induced by GFRalpha1 inactivation is not associated with the activation of common cell death executors, caspase-3 or -7, and lacks the morphological hallmarks of apoptosis, such as chromatin compaction and mitochondrial pathology. Consistent with these in vivo observations, neither caspase inhibition nor Bax deficiency blocks death of colon-derived enteric neurons induced by GDNF deprivation. This study reveals an essential role for GFRalpha1 in the survival of enteric neurons and suggests that caspase-independent death can be triggered by abolition of neurotrophic signals.     

Pharmacological and electrophysiological characteristics of neurones in the paramedian reticular nucleus.

1. Neurones in the paramedian reticular nucleus of decerebrate, unanaesthetised cats have been identified by microelectrode recording combined with antidromic activation of their axons in the ipsilateral inferior cerebellar peduncle. Most paramedian reticular neurones were not influenced by somatic stimulation. 2. When applied by iontophoresis from multibarrelled micropipettes acetylcholine and 5-hydroxytryptamine excited all but a few paramedian reticular neurones while l-noradrenaline inhibited almost all the neurones investigated. The excitatory response to acetylcholine could be antagonised by gallamine. 3. The paramedian reticular nucleus appears to be a relatively homogeneous group of neurones, pharmacologically as well as anatomically.