Summary of Work: The embryonic and early postnatal mammalian central nervous system (CNS) dynamically changes in cell number and anatomical structure as cells proliferate, die, migrate and differentiate into neurons and glia. This project line complements research conducted in project Z01 NS 02019-27 (Physiological properties developing on CNS cells), most of which involves direct recording of cell physiology in real time (electrophysiology), or near real-time (membrane potential and calcium imaging). Our long-term goal involves explanation of specific steps inCNS morphogenesis. This includes: 1) characterization of the physiological correlates of cell lineage progression emerging in vivo using surface labeling, physiological indicator dyes and flow cytometry and 2) the roles of transmitters and their corresponding receptors in mitogenic, motogenic and morphogenic activities. Previously, we used immunocytochemistry of surface epitopes (gangliosides) in conjunction with flow cytometry to characterize the cellular distribution of physiological properties emerging in vivo among precursors and neuronal and glial progenitors at the end of neurogenesis in the embryonic rat cortex. These results demonstrated contrasting patterns of transmitter receptor and ion channel expression emerging in vivo among proliferating precursors and cells progressing along neuronal and glial lineages. During FY99, we developed a novel FACS strategy to isolate for the first time in a prospective manner proliferating precursor (stem) cells at the beginning of neurogenesis in the cortex. We found that cells, which did not express surface epitopes characteristic of either neurons or glia or apoptosis (naturally occurring cell death), accounted for about 20% of the total. After sorting, the unlabeled cells were cultured in a serum-free, defined medium where they rapidly proliferated and then began to differentiate into neurons, astrocytes or oligodendrocytes. The cells could be frozen, thawed and cultured again with the same outcome as occurred with the primary sort. These self-renewing and multipotential characteristics identify the unlabeled population as neural stem cells. We used Ca2+ imaging to survey the cellular distribution of physiological properties expressed by stem cells and their progeny in vitro. Stem cells initially did not respond to transmitters from all the known classes. Over 24 hours, cytosolic Ca2+ (Ca2+c) responses to acetylcholine (ACh) and adenosine triphosphate (ATP) appeared, which were blocked by atropine and suramin, respectively. As cells progressed along neuronal and glial lineages they expressed contrasting and changing patterns of physiological properties similar to those identified in vivo at the end of neurogenesis. Thus, in vitro stem cells can differentiate in a defined medium into the three major cell types, recapitulating the developmental appearance of their physiological properties emerging in vivo. The early appearance and widespread cellular distribution of atropine-sensitive ACh receptors, identifying them as muscarinic in type, led us to investigate their role in neurogenesis. We treated explants of the early embryonic cortex with atropine, then used immunocytochemistry and flow cytometry to quantify the development of precursors and progenitors. Blockade of muscarinic receptors expressed by cells in cortical explants decreased the relative number of proliferating (BrdU+) precursors and neuronal and glial progenitors. These effects were closely mimicked by exposing the explants to BAPTA-AM, which blocks muscarinic receptor-mediated Ca2+c responses. Thus, endogenous cholinergic signaling at muscarinic receptors regulates proliferation during early neurogenesis. We used immunocytochemistry and flow cytometry to identify subpopulations of cells with choline acetyltransferase (ChAT), the enzyme that synthesizes ACh, in dissociates of the early embryonic cortex. ChAT+ cells were primarily restricted to cells progressing along a neuronal lineage. Together, these results demonstrate a major mitogenic role for ACh during the earliest phases of neurogenesis in the cortex. Previously, we used flow cytometry and immunocytochemistry to quantify the developmental appearance and cellular distribution of GABAergic system components among neurons in the embryonic rat cortex. During neurogenesis, neuronal progenitors migrate from the ventricular zone to the cortical plate. We found that GABA was a primary chemoattractant in the rat, while glutamate was a primary motogen in the mouse. In FY99, we found that GABAergic neurons dissociated from the cortical plate region of the rat directed the migration of neuronal progenitors dissociated from the ventricular zone via GABA(B) receptor-coupled pathways involving pertussis toxin-sensitive G proteins, Ca2+ fluctuations, and mitogen-activated phosphorylating enzyme activity. Since cortical plate neurons also secrete other amino acids like taurine and beta-alamine, we tested their chemoattractant activities and found both to be effective. Although both amino acids have demonstrated efficacies at GABA(A) receptor/chloride channels we found that their chemoattractant effects occurred via GABA(B) receptors. These results thus implicate an array of amino acids in cortical cell migration. We studied GABA?s morphogenic roles in neurite outgrowth of cortical plate neurons, which are transiently GABAergic. In vitro, neurite outgrowth was eliminated by suppressing GABA synthesis or blocking GABA(A) receptor/chloride channels. It could largely be restored in neurons no longer synthesizing GABA by addition of agonists at GABA(A) receptors, by astrocyte-conditioned medium, which contains GABA, taurine and beta-alanine and by modest elevation in K+, which, like agonists at GABA(A) receptor/chloride channels, depolarizes neurons, thereby activating Ca2+ entry via L-type channels. Hence, the transient and widespread appearance of GABA in neurons throughout the embryonic CNS coincides with, and likely promotes neurite outgrowth.