Catecholamine neurons of the central nervous system in organotypic culture

A standardized dissection has been designed to produce explants from the locus coeruleus of the newborn mouse brain. This area, termed the peduncular region, may be explanted with or without cerebellar neurons. Organotypic cultures with peduncular cells contain a cluster of 20-50 (or more) closely-packed large neurons, characterized by intracellular refractile granules in the living state. When treated with the glyoxylic acid method, somata of the granule-containing neurons exhibited catecholamine fluorescence, and a profuse plexus of varicose fluorescent fibers permeated the explant and its outgrowth. Intracellular staining of these cells with horseradish peroxidase demonstrated bipolar and multipolar neurons (25-30 micrometers), with stout dendrites and varicose axons. Granule-containing neurons stained for acetylcholinesterase. Electron microscopically, large dense granules were seen, which appear to correspond to the granules seen in living neurons. Cultures with these cells also have a high concentration of catecholamines in the medium. It is concluded that the granule-bearing neurons of the living culture are in fact neurons of the locus coeruleus.