Muscarinic acetylcholine receptors are expressed and enriched in growth cone membranes isolated from fetal and neonatal rat forebrain: Pharmacological demonstration and characterization

Abstract

Nerve growth cones, the motile tips of growing neuntes, are closely related to the exact pathway finding, and their roles for synaptogenesis have been proposed to be modified by some neurotransmitters. In the present study, to clarify the expression and the ontogeny of muscarinic acetylcholine receptors in growth cones, growth cone membranes from fetal and neonatal rat forebrain were isolated, and muscarinic receptors in growth cone membrane were pharmacologically characterized, by using the [ 3H]quinuclidinyl benzilate as a labeled ligand. The specific binding sites for [ 3H]quinuclidinyl benzilate had already been detected in growth cone membrane on embryonic day (E)17 ( B max = 557fmol/mg protein: K D = 19.7pM) and gradually increased in amount without significant changes in the K D values from E17 to postnatal day (P)5. [ 3H]Quinuclidinyl benzilate binding sites in growth cone membrane were several times higher than that in the P 2-fraction-derived membranes, and in perinuclear membranes. Competitive inhibition studies showed that the proportion of high-affinity sites for pirenzepine (M1-subtype) to total [ 3H]quinuclidinyl benzilate binding sites in growth cone membrane was significantly lower than that in adult synaptic plasma membranes. In contrast, the proportion of high-affinity sites for AF-DX 116 (M2-subtype) was significantly higher than that in adult synaptic plasma membranes (E17 growth cone membrane: M1, 29.5%; M2, 56.9%; adult synaptic plasma membrane: M1, 63.6%, M2, 5.9%). Electron micrographic examination revealed that there were no significant morphological differences among growth cone particle fractions at the developmental stages which we examined, and that mature synaptic elements did not contaminate the growth cone particle fractions. Biochemical examination by electrophoresis and the phosphorylation study of the growth cone particle fractions showed that the protein composition and the phosphoprotein pattern did not change markedly during these stages. Our results suggest that muscarinic receptors were expressed and more concentrated in growth cone membrane than in other membrane portions from perinatal rat forebrain, and that they may play some role in the axonal guidance in growth cone via receptor subtype-specific signal transduction mechanisms.