ABSTRACT
The development of cholinergic synapses in the rat olfactory bulb was investigated by measuring changes in the activity of choline acetyltransferase (ChAT; EC 2.3.1.6.), a presynaptic cholinergic marker, and in the concentration of muscarinic receptors, components of cholinoceptive membranes. Three biochemical properties of the muscarinic system also were examined for possible differentiation: ligand binding, molecular weight, and isoelectric point. Receptors from embryonic (day 18), neonatal (postnatal day 3), and adult rat olfactory bulbs exhibited identical complex binding (nH = 0.45) of the agonist carbachol. For each age, the relative proportions of high-affinity (Ki approximately equal to 1.0 microM) and low-affinity (Ki approximately equal to 100 microM) binding states were 60% and 40%, respectively. The antagonist pirenzepine also bound to high-affinity (Ki approximately equal to 0.15 microM, RH approximately equal to 70%) and low-affinity (Ki approximately equal to 2.0 microM, RL approximately equal to 30%) sites in neonatal and adult rats. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis of [3H]propylbenzilylcholine mustard-labeled receptors from neonatal and adult rats showed a single electrophoretic form with an apparent molecular weight of 65,000. In contrast, analytical isoelectric focusing indicated high pI (4.50) and low pI (4.00) receptor forms were present. Neonatal rats contained approximately equal proportions of the two receptor forms, whereas adult rats contained mainly the low pI form, indicating that molecular alteration of the receptor population had occurred during development. Comparison of postnatal changes in acetylcholine receptors and ChAT activity showed a striking correlation between the development of cholinergic terminals and muscarinic receptors. Throughout the first postnatal week, ChAT activity remained at 5% of adult levels; activity began to rise on postnatal day 6 and gradually reached adult levels (56 +/- 4 mumol of [3H]acetylcholine/h/g) during the fourth week. Similarly, muscarinic receptor concentration was low (30-50 fmol/mg) throughout the first week, began to rise at postnatal day 7; and reached 90% of adult levels (317 +/- 17 fmol/mg) by the fourth week. In contrast, there was little increase in the concentration of nicotinic acetylcholine receptors (30 fmol/mg) during this period. The parallel postnatal development of ChAT activity and muscarinic receptors suggests the existence of factors that couple the differentiation of presynaptic cholinergic terminals and postsynaptic cholinoceptive elements.
