Development of P2X receptor clusters on smooth muscle cells in relation tonerve varicosities in the rat urinary bladder

Postnatal development of the distribution of different isoforms of purinerg ic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary blad der has been determined with immunofluorescence and confocal microscopy. An tibodies against the extracellular domains of the P2X(1) to P2X(6) receptor s were used to detect the receptors in the bladder. Several other antibodie s were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X(1) to P2X(6) receptor subtypes (about 0.4 mu m diameter) were obser ved in the muscle which were independent of each other, and sometimes juxta posed to the rare isolated varicosity strings. At D4 large numbers of strin gs of varicosities could be discerned throughout the detrusor. Most of thes e clouds of small P2X(1) to P2X(6) receptor clusters in their immediate vic inity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to t yrosine hydroxylase. Up to D14 there was a gradual coalescence of many of t he isolated P2X(1)-6 small receptor clusters so that they became colocalise d, often at varicosities. Most of the varicosities in isolated strings poss essed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P 2X small receptor clusters or possessing such clusters in colocalisation. H owever, large numbers of small P2X receptor clusters, many of which consist ed of a mixture of isoforms, could be found spatially unrelated to nerve va ricosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or th ese were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different comb inations of colocalised P2X receptor isoforms could still be discerned in s mall clusters unrelated to varicosities. These observations are discussed i n relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.