Development of neural receptors for serotonin in the murine bowel

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Abstract
During the ontogeny of the enteric nervous system, the varicosities of mature neurons contact dividing neural precursors, which persist in the developing murine gut for several weeks postnatally. This phenomenon has led to the hypothesis that the release of transmitter from mature neurons may influence the subsequent development of uncommitted neuroblasts. In order to test this hypothesis, it is necessary to know the timing of the expression of postsynaptic receptors for enteric neurotransmitters. Since serotoninergic neurons are among the earliest of enteric neurons to develop (appearing on day E12 of development in the mouse), the ontogeny of enteric neural serotonin receptors (5‐HTR) was studied. These receptors have previously been characterized in adult guinea pigs and rabbits. In the current experiments, 5‐HTR were identified in the adult murine bowel; their properties were compared with the 5‐HTR of guinea pig and rabbits; their ontogeny was followed throughout the length of the developing mouse gut; and the properties of 5‐HTR in the developing murine bowel were compared with those of the mature gut. The 5‐HTR were assayed by measuring the binding of 3H‐serotonin (3H‐5‐HT) to isolated enteric membranes by rapid filtration, and to frozen sections of bowel by radioautography. A single saturable, high affinity 3H‐5‐HT binding site was found in membranes from the adult mouse gut (KD = 3.9 ± 0.5 nM; Bmax = 1.6 ± 0.3 pmoles/mg protein). Binding of 3H‐5‐HT at this site was not antagonized by compounds known to be antagonists at receptors for other neurotransmitters or at the 5‐HT1 or 5‐HT2 class of CNS 5‐HTR. Hydroxylation of the indole ring of analogues of serotonin was required for affinity at the enteric 3H‐5‐HT binding site. Binding of 3H‐5‐HT was inhibited by N‐acetyl‐5‐hydroxytryptophyl‐5‐hyroxytryptophan dipeptide, a compound that has been demonstrated to antagonize those responses of myenteric neurons to serotonin that resemble slow excitatory postsynaptic potentials, but not by ICS 205–930 (Sandoz), a serotonin antagonist that does not block these responses. All of these properties of adult murine 3H‐5‐HT binding sites are virtually identical of those of guinea pigs and rabbits, which have previously been shown to be 5‐HTR; therefore, murine enteric 3H‐5‐HT binding sites are probably 5‐HTR as well. The 5‐HTR were found radioautographically in both enteric plexuses and in the mucosa of all regions of the adult murine bowel with the exception of the fundus (rumen) of the stomach. During ontogeny the binding of 3H‐5‐HT, evaluated radioautographically, appeared for the first time on day E14, when it was found in the stomach and small intestine from the pylorus to the level of the mid‐jejunum. The 3H‐5‐HT binding sites then spread proximodistally, reaching the ileocolic sphincter on day E15, the proximal colon on days E16–18, and the distal colon on day P2. The acquisition of 3H‐5‐HT binding sites by the rectum occurred entirely postnatally and was not complete until day P22. The properties of the 3H‐5‐HT binding sites of developing animals were not distinguishable from those of adults, and thus were considered to be 5‐HTR. Additional 'H‐5‐HT binding sites were found in the perianal skin of adult mice. The characteristics of 3H‐5‐HT binding sites to these cutaneous sites were similar to those of the gut, which suggests that there are 5‐HTR of the enteric type in the skin. These observations indicate that enteric neural 5‐HTR make their appearance late indevelopment than do serotoninergic neurons; however, 5‐HTR are present during the period when proliferating neural precursors are present in the enteric nervous system. It seems unlikely, therefore, that 5‐HTR on precursors are important in determining the serotoninergic phenotype. Yet, the observations are consistent with the hypothesis that serotoninergic neurons influence the development of other types of neuron that sequentially follow the appearance of serotoninergic neurons in development.