Phenotypic characterization of septal neurons in culture: immunohistochemistry of GABA, calbindin D-28k and choline acetyltransferase, and histochemistry of acetylcholinesterase

Abstract

Phenotypes of septal neurons, dissociated from 19-day-old fetal rat brains and then cultured in a medium containing nerve growth factor for 4 weeks, were examined using gamma-aminobutyric acid (GABA), calbindin D-28k, parvalbumin and choline acetyltransferase immunohistochemistry, and acetylcholinesterase histochemistry. There were primarily four groups of neurons identified in this septal culture: the first group (12.7% of 212 neurons examined) displayed a cholinergic, but not GABAergic, phenotype and had an average diameter of 13.6±2.7 μm (mean±S.D.); the second group (31.6%) displayed both cholinergic and GABAergic phenotypes and had a diameter of 12.2±2.8 μm; the third group (31.0%) displayed only a GABAergic phenotype and had a diameter of 10.4±2.3 μm; and the fourth group (24.7%) displayed neither a GABAergic nor cholinergic phenotype and had a diameter of 10.4±2.1 μm. Neurons in the first two groups described were significantly larger than those in the second two groups; neurons in the third and fourth groups were the same size. Calbindin D-28k was expressed in some neurons of each group (31.3%, 18.8%, 9.6% and 15.7%, respectively). These results demonstrate that septal neurons have the ability to express a variety of phenotypes when grown in vitro. This culture will be a useful tool for studying mechanisms of phenotype expression in septal neurons.