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Keywords:

  • dissociated cultures;
  • calbindin D-28K;
  • glia;
  • Purkinje cell;
  • voltage-dependent conductance

Abstract

The development of the major morphological and electrophysiological properties of presumptive Purkinje cells (PCs) was studied in primary cultures of rat cerebellum dissociated on the 14th embryonic day, when PCs are minimally differentiated and migrate in vivo PCs were identified with a specific antibody to calbindin D-28K (CaBP), which allowed visualization of the different morphological types of PCs between 3 and 29 days in vitro (DIV). CaBP-immunopositive cells were first detected at 3 DIV. Thereafter, the shape of these cells resembled some of those described in viva After 20 DIV, 95% of the CaBP-immunopositive cells had characteristic PC dendritic trees, although they were very atrophic. Glial cells immunopositive for the glial fibrillary acidic protein (GFAP) were first seen at 3 DIV. Thereafter GFAP-immunopositive cells resembled Bergmann cells or velate astrocytes. Neurons regarded as PCs were studied electrophysiologically using the patch-clamp whole-cell configuration. Voltage-dependent, tetrodotoxin-sensitive fast inward currents were virtually absent at 2–4 DIV, but increased between 7 and 14 DIV to reach two-thirds of the amplitude obtained after 15 DIV. These currents were large enough to give rise to overshooting spikes as early as 7 DIV in the current-clamp mode. This time schedule is in keeping with that of PCs developed in situ. The tetraethylammonium-sensitive, slowly inactivating outward currents had reached two-thirds of the amplitude obtained after 15 DIV by 3–4 DIV. Their amplitude remained stable between 4 and 7 DIV, and increased to their maximal value during 7–14 DIV, with a marked shortening of action potentials. 4-Aminopyridine-sensitive, fast-inactivating outward currents might also be associated with development, since they were present in 66% of the cells between 7 and 14 DIV but in only 39% from 15 to 29 DIV; however, their amplitude did not vary with time. Presumptive PCs bore l-glutamate-activated receptors, which preceded the emergence of kynurenate-sensitive, spontaneous synaptic currents at 7 DIV. These currents were sometimes intermingled with inhibitory currents, although presumptive PCs were sensitive to γ-aminobutyrate at 7 DIV. The present model represents some unequivocal features of PC development, although the PCs used had undergone minimal differentiation in vivo and were cultured in a very disturbed cellular environment.