5-hydroxytryptamine (5-HT) is a neurotransmitter in the enteric nervous system (ENS). Two types of enteric neural 5-HT receptor have been identified, 5-HT1P and 5-HT3. The 5-HT1P receptor is labeled by 3H-5-HT; antagonists at 5-HT1P receptors include N- acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP) and BRL 24924. During developments 5-HT1P receptors arise after the appearance of serotonergic neurons. 5-HT3 receptors are not labeled by 3H-5-HT; antagonists at 5-HT3 receptors include ICS 205- 930, BRL 43694 (granisetron) and GR 65630. Neither the distribution in the wall of the bowel, nor the ontogeny of 5-HT3 receptors have previously been studied. It is now proposed to study enteric 5-HT3 receptors in the murine gut by radioligand binding techniques, using both rapid filtration of enteric membranes and radioautography with 3H-granisetron and 3H-GR 65630. Specific binding will be defined as that displaced by ICS 205-930. The location of 5-HT3 receptors will be determined in the adult and fetal bowel and the timing of their appearance in ontogeny will be related to that of 5-HT1P receptors. Selective destruction of serotonergic neurites with 5,7-dihydroxytryptamine (5,7-DHT) will be used to explore their influence on the development, maturation, and maintenance of 5-HT1P receptors. The ability of 5-HT itself to affect 5-HT1P receptor development will be separately explored. The role of 5-HT will be studied by examining 5-HT1P receptor development and maturation in the presence of specific antagonists and after depletion of 5-HT by inhibition of tryptophan hydroxylase. Experiments will be done with explants of fetal gut grown in organotypic tissue culture and in vivo with developing hindgut, in which 5-HT1P receptors develop during the first 3 weeks of life. Chemical sympathectomy with 6-hydroxydopamine (6-OHDA) induces a rapid upregulation of 5-HT1P receptors. The effect of 6-OHDA on 5-HT3 receptors will now be ascertained. In addition, studies will be done to determine whether the effect of 6-OHDA on enteric neural 5-HT receptors is due to loss of norepinephrine (NE), and is specifically related to destruction of the noradrenergic innervation of serotonergic neurons. These experiments will utilize tyrosine hydroxylase inhibition to deplete BE and the quantitative cytochemical demonstration of cytochrome oxidase activity to evaluate effect of sympathectomy on the tonic activity of serotonergic neurons. Finally, since both serotonergic neurons and 5-HT1P receptors are already present while the gut contains the proliferating precursors of neurons that contain vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) immunoreactivity the possibility exists that serotonergic neurons may influence the phenotypic expression of these late-developing neurons. This possibility will be tested by determining the effects of 5,7-DHT or chronic inhibition of 5-HT1P and/or 5-HT3 receptors on the birthdays and ultimate numbers of VIP- and CGRP-immunoreactive neurons. The effect of these treatments on the earlier-developing neuropeptide Y-immunoreactive neurons will also be examined as a control. Finally, if effects ar found, studies will be done to determine whether affected neurons actually receive serotonergic synapses. These experiments will enhance understanding of the pathogenesis of congenital defects of enteric neuromuscular development and will be important in evaluating the safety of psychoactive drugs that affect serotonergic neurons of 5-HT receptors in pregnancy.
