Development of the GABAergic phenotype in murine spinal cord-dorsal root ganglion cultures

Murine spinal cord and dorsal root ganglion GABAergic neurons, derived from 12-day-old fetuses, were examined autoradiographically, biochemically and immunocytochemically in vitro to determine the timecourse of appearance and maturation of this phenotype and the extent and mode of its innervation of target neurons. Specific 3H-GABA uptake into spinal cord neurons was the first property to develop and was present at the earliest time studied, one day in vitro. Immunocytochemical localization of glutamic acid decarboxylase (GAD) revealed positively stained neurons beginning at four days. At five days in vitro, electron microscopic immunocytochemistry revealed GAD-immunoreactive (GAD-IMR) boutons investing neuronal perikarya as well as neuronal processes. By one week in vitro, GAD-IMR neurons constituted 27% of the total population and GAD-IMR boutons could be seen contacting every cell with a neuronal morphology. The mode of investment of target neurons by GAD-IMR boutons was not circumscribed to either soma or dendrites but usually involved the entire neuronal perimeter and did not change with time in culture. Three morphologically distinct types of GAD-IMR neurons were evident: a small, bipolar type; a medium-sized multipolar neuron which was the most common and a large, multipolar type, resembling a motoneuron. A small population (8%) of dorsal root ganglion neurons was found to contain GAD both biochemically and immunocytochemically but was never invested by GAD-IMR boutons. GAD activity in vitro paralleled in vivo levels with maximal activity being reached at four weeks in vitro and 10 days postnatally in the intact mouse spinal cord. Murine spinal cord GABAergic neurons are a morphologically diverse and abundant neuronal population with extensive, precocious innervation of all other neuronal phenotypes in vitro suggesting that GABA has a widespread influence over other developing neuronal systems in the murine spinal cord.