Measurement of choline acetyltransferase with [14C]acetate by a cycling procedure.

A multiple enzyme and multisubstrate cycling system is described for the radiometric determination of cholineacetyltransferase (ChAT) activity in crude tissue homogenates. The methods employs [14C]acetate coupled with the enzymes acetate kinase (AK) and phosphotransacetylase (PTA) for the generation of [14C]acetyl CoA. By recycling it was possible to avoid product inhibition of ChAT by CoA, ATP was maintained constant by rephosphorylation of ADP. Kinetics of the individual enzyme reactions were studied and the parameters obtained were used to select appropriate conditions to maintain linearity of varying amounts ChAT activity over a sixty minute time course. The sensitivity of the method is limited only by the specific activity of commercially available isotope labeled acetate.

Synaptogenesis in chick paravertebral sympathetic ganglia: a morphometric analysis.

Synaptogenesis was studied in lumbar sympathetic ganglia of chicken by light and electron microscopic morphometric methods. At 10 days in ovo, fewer than 1% of the adult number of synapses are present. The total numbers of synapses and of synaptic vesicles per ganglion increase progressively with age; however, the majority of both are formed after 30 days after hatching. The average number of synaptic vesicles per synapse increases several fold after hatching. The numbers of synapses and of synaptic vesicles per ganglion increase roughly in concert with biochemical markers of presynaptic development (activity of choline acetyltransferase and levels of acetylcholine) as well as postsynaptic development (tyrosine hydroxylase; based on biochemical data reported elsewhere). The amount of acetylcholine and activity of choline acetyltransferase per synaptic vesicle at 10 days in ovo are 8 and 27 times the corresponding adult values. By 1 day after hatching, these ratios have fallen to near adult levels. These data are consistent with the early presence of cholinergic neuroblasts, as suggested by others, and suggest further that such cholinergic neuroblasts are eliminated, or their cholinergic properties suppressed, before hatching.