Electrophysiology of the mammalian cerebellar cortex in organ culture

Abstract

A direct comparison was made between the electrical properties of rat Purkinje cells in cerebellar organotype cultures and those in acute slices from age-matched animals. Cultures were prepared from 9–11-day-old animals. Intracellular recordings were made 5–12 days later, at which time the folia architecture of the cerebellum was still well preserved. The resting membrane potentials and input resistances of Purkinje cells in cultured and acute slice preparations from young animals were comparable to those of mature Purkinje cells in slices. Neurons from animals younger than 14 days differed from mature Purkinje cells in that they fired at low frequencies in response to outward current pulses. The latter property was found in all cultured neurons studied, independent of their time in culture. These action potentials were generated by Na + and Ca 2+ conductances as shown by the application of selective channel blockers. Cultured or acute slice preparations from animals younger than 11 days shared other immature electroresponsive features. In both groups, Na +-dependent plateau depolarizations were observed in less than 10% of Purkinje cells unless K-conductances were blocked, and considerable membrane depolarization was often required to elicit Ca 2+-dependent action potentials. These findings are compatible with the relative prominence of voltage-dependent outward currents in immature Purkinje cells, a property which may be enhanced in culture. The injection of hyperpolarizing current pulses revealed a marked time-dependent anomalous rectification in all Purkinje cells. At the breaks of such pulses, several events were observed. In all cells, a rebound conductance was identified which could generate post-anodal spike bursts. In cultured neurons, however, hyperpolarizing pulses were also followed by a slow return to resting potential. This membrane potential profile was similar to that produced by the activation of an A conductance. Experiments on acute slices from animals of different ages (P9–P17) showed that this A-like conductance was expressed only during a brief period in Purkinje cell development. A higher level of spontaneous synaptic activity was observed in cultured than in acute slice preparations. Both unitary excitatory postsynaptic potentials and inhibitory postsynaptic potentials could be elicited in the former by parallel fiber stimulation, and could be fully reversed by outward or inward transmembrane current injections, respectively. The inhibitory postsynaptic potentials were blocked by the GABA antagonist picrotoxin, but their reversal properties differed from those of inhibitory postsynaptic potentials recorded in vivo. Inhibitory postsynaptic potentials were also followed by oscillations in the membrane potential compatible with the presence of rebound inward currents. Parallel fiber stimulation generated prolonged plateau-like depolarizations when cultures were exposed to picrotoxin. These plateaus were shown to be caused by a dendritic, voltage-dependent Ca 2+-conductance similar to that which underlies the climbing fiber response in vivo. We conclude that the firing properties of immature Purkinje cells differ from those in adult animals. Further, the electrical properties of these neurons did not develop in slice culture despite evidence that cell growth and synaptogenesis continue in culture.