Markers for Gene Expression in Cultured Cells from the Nervous System

Abstract

Methodsare presented for preparation of extracts from cultured cells from the nervous system and for study of choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase, and catechol 0-methyltransferase activities.These enzyme activities are markers that can be used for studying gene expression in neurons.The methods are sufficiently sensitive so that all assays can be performed with protein harvested from one Petri dish.Activities of the marker enzymes were assessed in surface cultures of newborn mouse brain cells, and in glial and nonbrain cell lines.Low activities of choline acetyltransferase, acetylcholinesterase, and glutamate decarboxylase were detected in all the cells tested.All of these activities, and particularly glutamate decarboxylase, were higher in cultured brain cells from newborn animals than in non-neuronal cell lines.Glutamate decarboxylase activity in glial cells and in brain cells was inhibited more than 95 % by 1 mM amino-oxyacetic acid.Techniques have been developed in this laboratory and others for culture of differentiated neurons (l-lo).Activities of enzymes important in neuronal cell metabolism are useful parameters for following cell maturation and exploring steps in differentiation in such cultures.The purpose of this communication is to describe a set of methods used to explore the expression of genes that determine the metabolism of molecules involved in intercellular communication in the nervous system.Simple, convenient methods are presented for preparing cell-free extracts from surface cultures