Functional ionotropic glutamate receptors emerge during terminal cell division and early neuronal differentiation of rat neuroepithelial cells

Ionotropic glutamate receptors mediate fast forms of excitatory synaptic tr ansmission in mature neurons and may play critical roles in neuronal develo pment. However, the developmental stage at which neuronal cells begin to ex press functional receptors and their roles in lineage progression remain un clear. In the present study, neural precursor cells were isolated from the cortical neuroepithelium of embryonic day 13 rats, and rapidly expanded in serum-free medium in response to basic fibroblast growth factor, RT-PCR rev ealed the presence of mRNAs encoding AMPA(A), AMPA(C), KA(1), KA(2), NMDA(1 ), and NMDA(2D) subunits after 3 days in culture, The functional expression of AMPA/kainate and NMDA receptors was investigated using Ca2+ imaging and whole-cell patch-clamp recording techniques in cells pulse-labeled with br omodeoxyuridine (BrdU) for 1-4 hr. The recorded cells were then double-immu nostained for BrdU incorporation and neuron-specific p-tubulin (TuJ1), The results show that AMPA/kainate and NMDA induced increases in cytosolic Ca2 and inward currents only in differentiating neurons. In contrast, prolifer ating BrdU(+)TuJ1(-) cells failed to respond to any ionotropic glutamate re ceptor agonists, Interestingly, Ca2+ imaging revealed that a subpopulation of BrdU(+)TuJ1(+) cells also responded to AMPA, indicating the emergence of functional ionotropic AMPA/kainate receptors during terminal cell division and the earliest commitment to neuronal cell lineage. These in vitro resul ts were supported by flow cytometric sorting of AMPA-responsive cells pulse -labeled with BrdU for I hr in vivo, which revealed that functional AMPA re ceptors appear in BrdU(+)TuJ1(+) cells under physiological conditions and m ay play a role in terminal cell division. (C) 2000 Wiley-Liss, Inc.