THE ONTOGENY OF M1-M5 MUSCARINIC RECEPTOR SUBTYPES IN RAT FOREBRAIN

The ontogeny of muscarinic receptor subtypes in rat forebrain has been determined utilizing a panel of antisera recognizing unique epitopes of the m1-m5 receptor proteins. Total receptor density in forebrain, as measured by the non-selective antagonist, [H-3]quinuclidinyl benzilate (QNB), was found to increase from 507 fmol/mg in neonates (3.5 days) to 1727 fmol/mg in mature animals (45 days). Adult levels were reached two weeks post-partum. A recently developed precipitation protocol was then used to further characterize receptor ontogeny. Cumulatively, 90% of [H-3]QNB labelled muscarinic receptors from adult forebrain were immunoprecipitated with subtype selective antibodies (m1-m5). In 3- to 4-day-old neonates, ml receptors are expressed at 31% of adult levels, m2 receptors at 32% of adult levels. m3 receptors at 36% of adult levels. and m4 receptors at 20% of adult levels. In mature animals, ml receptors were equal to 35%, m2 equal to 18%, m3 equal to 11%, and m4 equal to 26%, of total receptors, respectively. Levels of m5 receptors are consistently very low at all ages tested (less-than-or-equal-to 1% of total receptor density). Although there were subtle differences in the time courses of development between muscarinic receptor subtypes, in general, they developed with similar ontogenic profiles. Subtypes coupled preferentially to inhibition of adenylate cyclase (m2 and m4) comprised 35% of total receptors expressed in neonates. The combined m2/m4 receptor density increased at a uniform rate during development from 173 to 757 fmol/mg. Receptors preferentially coupled to phosphoinositide turnover (ml, m3, and m5) were found in newborns at approximately 270 fmol/mg. Maximum expression of phosphoinositide-linked receptors was observed in 26-day-old rats and declined to 90% of maximum levels by day 45. In agreement with reported levels of M1 class receptors in forebrain, m1 and m4 subtypes (which exhibit high affinity for pirenzepine), comprise approximately 54% of total receptors in newborns and 60% of total forebrain receptors in adults.