of Work:The physiological correlates of the major stages in the morphogenesis of the mammalian central nervous system, including proliferation, apoptosis, migration and differentiation remain to be elucidated. Relatively few extramural research programs are aimed in this direction, although the field is growing. We have used electrophysiological recordings and digital videomicroscopic and imaging techniques, which complement those employed in Z01 NS 02230-22 (Biological properties developing in central nervous system cells), to study the development, differentiation and cellular distribution of physiological properties expressed by precursor, progenitor and differentiating embryonic rat central nervous system cells in culture. The cellular distribution and developmental appearance of physiologically relevant properties among cortical stem cells and their progeny was investigated using simultaneous imaging of cytosolic Ca2+ (Ca2+c) levels in up to 100 cells. Stem cells, the majority of which were proliferating when initially sorted and cultured, exhibited Ca2+-dependent Ca2+c levels but did not respond to all the known major classes of transmitter. However, all responded to unknown components in low and high molecular weight fractions of fetal calf serum, a well as to cerebrospinal fluid, which in vivo contacts the proliferating neuroepithelium composed of stem cells and their progeny. Over 24 hours, Ca2+c responses to acetylcholine (ACh) and adenosine triphosphate (ATP) appeared, which were blocked by atropine and suramin, respectively. Progressively more cells responded to both ACh and ATP while still immature (nestin+) and proliferating (BrdU+) before the emergence of epitopes characteristic of neurons or glia. As neuron-specific epitopes (tetanus toxin, cholera toxin, beta-tubulin) appeared in stem cell progeny a stereotypical pattern of excitability emerged, including GABA(A) receptor/Cl- channels and CNQX-sensitive glutamate receptor/cation channels, voltage-dependent, tetrodotoxin-sensitive Na+ channels and Na+-Ca2+ exchange activity and voltage-dependent L-type Ca2+ channels. Stem cell progeny expressing epitopes characteristic of astrocytes (GFAP) or oligodendrocytes (O4) did not express these other properties, but exhibited functional ACh, ATP and catecholamine responses. These contrasting patterns of excitability were similar to those identified in vivo during neurogenesis using indicator dyes and immunocytochemistry combined with flow cytometry or imaging. Thus, quite different physiological properties emerge among neuronal and glial phenotypes derived from stem cells sorted at the beginning of neurogenesis and cultured in a serum-free, defined medium and these largely recapitulate those appearing in vivo. Many of the neurons descended from sorted stem cells are also GABA-immunoreactive, again recapitulating observations made in vivo during neurogenesis. Dual-imaging (membrane potential, Ca2+c) of GABA-immunoreactive neurons isolated from the cortical plate/subplate region at the end of neurogenesis revealed a baseline membrane potential which depended on continual synthesis and secretion of GABA followed by activation of GABA(A) receptor/Cl- channels. The concurrent activity of Na+ K+ 2Cl- co-transporters maintained elevated Cl- levels that served to provide a depolarizing component to the baseline and this in turn stimulated Ca2+ entry via L-type Ca2+ channels. The autocrine circuit involving GABAergic stimulation of Ca2+ entry was critical for neurite outgrowth and decreased progressively as neurites grew into functional circuits and networks. Morphological differentiation of neurites by cortical plate/subplate neurons consistently occurred in a serum-free, defined medium (Neurobasal with, but not without B27 supplement). However, astrocyte-conditioned Neurobasal completely supported neuritogenesis and network formation in the absence of B27 supplement, while the more minimal astrocyte-conditioned saline effectively sustained the earliest phases of process formation. Electrophysiological recording and Ca2+ imaging of cortical plate/subplate neurons demonstrated that astrocyte-conditioned saline tonically activated GABA(A) receptor/Cl- channels and elevated Ca2+c levels via Ca2+ entry through L-type Ca2+ channels. This may help to explain why astrocytes can support neurite outgrowth. They add to the depolarizing GABAergic signals emanating from the cortical plate/subplate neurons, thus sustaining the Ca2+ entry necessary for their morphological differentiation. Astrocytes can synthesize and secrete a variety of substances active at GABA(A) receptor/Cl- channels including GABA, taurine, beta-alanine and steroid metabolites. One or more of these substances could mediate the differentiating effects of astrocytes on the neurons. The effects of the amino acids were immediate, while those of 3 alpha-substituted metabolites also contained a delayed component similar to that recorded with astrocyte-conditioned saline. The mechanism of tonic and transient forms of GABAergic signaling at GABA(A) receptor/Cl- channels was studied using embryonic neurons cultured from the hippocampus and cortex. Both tonic and transient forms of signaling could be rapidly and reversibly altered or eliminated by gentle streams of saline, indicating that they were both derived from surface-accessible sources. A ?false? GABAmimetic neurotransmitter (isoguvacine) applied to the surface of differentiating neurons was immediately able to substitute for endogenous GABA in mediating both tonic and transient forms of GABAergic signaling. Hence, GABAergic signaling at GABA(A) receptor/Cl- channels differentiating on embryonic central neurons arises from surface-accessible sources. The discovery that GABAergic transients also arise from the surface challenges the long-standing notion that such transients are derived from all-or-none release of transmitter following vesicular exocytosis. Rather, exocytosis may supply transmitter like GABA, which is complexed to a matrix lining the vesicle. Discharge of transmitter packets could result from Ca2+-dependent enzyme activity or electrostatic effects.
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