Morphological and physiological differentiation of Purkinje neurons in cultures of rat cerebellum

Abstract

During ontogeny, vertebrate CNS neurons differentiate from relatively simple stem cells to complex units that express unique morphological and electrophysiological characteristics. We have examined several aspects of this developmental process in an identified CNS neuronal type, the Purkinje neuron (PN) of the cerebellum. Our approach has included the use of a tissue culture preparation and immunohistochemical and electrophysiological techniques. Using immunohistochemical techniques, we have identified immature PNs in culture and examined their morphological and synaptic development. These studies have shown that PNs undergo extensive morphological and synaptic development in culture, the morphological characteristics of the immature PNs in culture and the developmental sequence and time course are reflective of that described for PNs in vivo, synapse formation is initiated at an early stage of PN development in culture and proceeds concurrently with the morphological development, and the main period of synapse formation is associated with the main period of dendritic development, reflecting the preferential location of synaptic sites at the dendritic region of mature PN. Using electrophysiological techniques, we have examined the physiological development of PNs in culture and have correlated the stages in physiological, morphological, and synaptic development. Results from these studies show the following. Mature PNs in culture exhibit complex electrophysiological properties, including the ability to generate 2 types of spike events, simple and complex spikes, and endogenously generated activity. Expression of electrophysiological properties begins at an early stage in PN development, when the PNs consist of little more than a soma with a few fine perisomatic processes. The earliest physiological characteristics to be expressed by the PN include sensitivity to transmitters, the ability to respond to synaptic input, and the ability to generate simple spikes. Synaptic input produces spontaneous activity in young PNs, but the patterns of activity change during development as mechanisms underlying endogenously generated activity and complex spike generation are expressed, and synapse formation proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)

Glial cell line-derived neurotrophic factor promotes the survival and morphologic differentiation of Purkinje cells.

“…2B). Between 4 and 10 days in vitro, a regression of the primitive processes occurs and short perisomatic processes appear (stage C; In control cultures, the time course of changes in Purkinje cell morphology was consistent with previous reports (16,17). At day 4, the distribution of cells in categories A-D was 51%, 44%, 5%, and 0%, whereas at day 6 it was 22%, 51%, 25%, and 3%.…”

“…Examination of CaBP+ cells revealed four general morphologies, previously described as characteristic of Purkinje cells between days 4 and 14 in culture (16,17). These morphologies resemble those displayed in vivo between embryonic day 17 and postnatal day 10.…”

“…Immunopositive cells were assigned to one of five categories, based on neuritic morphology and cell shape. These categories have been shown to correspond to sequential stages in the normal development of Purkinje cells both in vitro (16,17) and in vivo (refs. 18-24; reviewed in ref.…”

In vivo analysis of Purkinje cell firing properties during postnatal mouse development

“…Several previous studies have followed Purkinje cell firing in developing rodents. In vitro analysis in mice and rats demonstrated using slices, cell culture, and tissue explants that Purkinje cells can establish simple spike and complex spike firing activity in the absence of the highly structured architecture of the intact cerebellum (Bishop 2002;Gruol and Franklin 1987;Guan et al 2006;Hockberger et al 1989;Mariani and Changeux 1981). Furthermore, in acute cerebellar slices a trimodal pattern of firing becomes prominent in the second week of life (Womack and Khodakhah 2002).…”

Resurgent Sodium Current and Action Potential Formation in Dissociated Cerebellar Purkinje Neurons

“…Spontaneous firing is maintained in cerebellar brain slice preparations (Hounsgaard, 1979;Llinás and Sugimori, 1980a) and in cultured Purkinje neurons (Gruol and Franklin, 1987), even when synaptic activity is blocked. Although complex spikes apparently occur only after excitation of climbing fibers when Purkinje neurons are studied in situ (Eccles et al, 1966;Stuart and Häusser, 1994), multipeaked action potentials are also seen in cultured Purkinje cells in the absence of climbing fiber or other synaptic input (Gruol and Franklin, 1987;Gruol et al, 1992). Llinás and Sugimori (1980a,b) attributed the distinctive firing properties of Purkinje neurons to a variety of intrinsic membrane conductances in the neurons, including voltage-activated calcium conductances and a persistent sodium conductance.…”