UTILIZATION OF 3-DIMENSIONAL CULTURE FOR EARLY MORPHOMETRIC AND ELECTROPHYSIOLOGICAL ANALYSES OF SOLITARY CEREBELLAR NEURONS

When cells from the cerebellum of late-gestation rats were grown at low density (50-100 cells/mm2) in a three-dimensional culture system, they rapidly expressed morphological and electrophysiological properties of neurons. Growth and differentiation of the population of solitary neurons as a whole was statistically assessed at 6, 12, 18, 24, 48 and 72 h using data obtained from image analysis. Mean length of axons and dendrites increased 17- and 220-fold, respectively, from 6 to 72 h. Average number of branch points rose 35-fold. Other indices of complexity increased 2- to 3-fold. Whole-cell voltage clamp revealed that as early as 24-30 h the cells displayed a tetrodotoxin-sensitive Na2+ current (I(Na)), a nifedipine-sensitive high-threshold Ca2+ current, a Ni2+-sensitive low-threshold Ca2+ Current, and two voltage-dependent K+ outward currents consisting of a 4-aminopyridine-sensitive fast transient outward current and a CsCl-sensitive slow delayed component. These observations correlate closely with voltage-activated currents previously recorded in neonatal or young rat cerebellum, and demonstrate that the culture model is useful for analyzing the early rapid growth, differentiation and intrinsic ionic currents of these neurons as single cells.