DIFFERENTIATION OF HIPPOCAMPAL PROGENITOR CELLS IN-VITRO - TEMPORAL EXPRESSION OF INTERCELLULAR COUPLING AND VOLTAGE-GATED AND LIGAND-GATEDRESPONSES

Mechanisms regulating the expression of intercellular coupling, develo pment of membrane excitability, and cellular responsiveness to neurotr ansmitters during neuronal ontogeny are largely unknown. To define the temporal relationship among these properties during neurogenesis, mur ine embryonic hippocampal progenitor cells immortalized with a tempera ture-sensitive allele of the SV40 large T-antigen were examined during successive stages of neuronal differentiation in. vitro using patch c lamp, dye coupling, and Ca2+ imaging techniques. Electrotonic and dye coupling between untreated neuroblasts were frequent in cells maintain ed at the temperature (39 degrees C) nonpermissive for T-antigen expre ssion. However, as neuroblasts differentiated into neurons under the i nfluence of interleukin-7 added alone or concurrently with transformin g growth factor-alpha after basic fibroblast growth factor, both junct ional conductance and the extent of dye coupling progressively decreas ed. Voltage-dependent inward currents were present within 2 to 6 days after differentiating treatments began. During intermediate developmen tal stages (3 to 5 days in culture), cells became responsive to GABA ( greater than or equal to 100 mu M) but not to glutamate, glycine, or t o acetylcholine (less than or equal to 1 mM), as indicated by [Ca2+](i ) measurements and patch clamp recordings. In contrast, voltage- and l igand-gated responses but not electrotonic coupling were frequently ob served in mature neuronal primary cultures. Together, these results in dicate that certain cytokines may orchestrate the progressive expressi on of functional neuronal phenotypes in vitro, in which the gradual di sappearance of intercellular coupling parallels the onset of voltage-d ependent responses and both of which precede the expression of neurotr ansmitter chemosensitivity. (C) 1995 Academic Press, Inc.