Flow cytometry, discontinuous-gradient cell isolation, aminoacid analysis, dissociated cell culture, immunoblots, cell migration, immunochemistry, in situ hybridization and PCR methods are applied to embryonic/early postnatal rat CNS tissues to study the development, differentiation and cellular distribution of transmitter, transmitter-related enzymes and their corresponding receptors. During the past several years, we have focussed intensely on GABA, which is abundantly but transiently expressed during CNS development before it becomes restricted to fast inhibitory synapses in the adult. In FY 92 we investigated the following: 1) quantitative yield of embryonic (E) CNS cells by enzymatic digestion and mechanical dissociation resulting in complete enumeration of all cells during the embryonic period; 2) the absolute number of cells recovered from cortex, hypothalamus, midbrain, brainstem and spinal cord regions increases 50-fold over E11-13; 3) cord and brainstem cell abundance plateaus over E13-16, while other regions show parallel, 5-fold increases; 4) cord cells decline by approximately 90% during E15-22 along a rostrocaudal gradient while subcortical cells wane, and cortical cells increase 10-fold in number; 5) quantitative flow cytometric analysis of embryonic CNS cells exposed in vivo to bromodeoxyuridine (BrdU) reveals anatomic gradients of cellular proliferation and differentiation and their change during CNS development; 6) the percentage of BrdU + cells reaches a nadir first at El 5 in brainstem, at E 1 6 in cervical spinal cord, then mid-brain/thoracic cord, followed by the rest of the CNS; 7) discontinuous Percoll gradients permit isolation and enumeration of all cells and their subcellular elements according to their buoyant density; 8) quantitative flow cytometric analysis of Percoll-isolated cells exposed to BrdU on Ell in vivo using a pulse-chase-pulse paradigm reveals characteristic patterns in cellular proliferation and differentiation correlated with cellular buoyancy; 9) sections of the embryonic CNS reveal the anatomic distribution of cells labelled in the pulse-chase-pulse protocol; 10) at E12-14, cells labelled on Ell have migrated from proliferative zones, while cells labelled 1 hr before sacrifice remain confined to these zones; 11) cellular buoyancy corresponds to proliferation/differentiation status and to anatomic location in vivo; 12) flow cytometric analysis of gradient-separated cells stained with molecular probes reveals characteristic patterns of physiological properties and receptor/ion-channel expressions differentiating at cellular and subcellular levels; 13)differentiating E13-15 spinal neurons spontaneously migrate through pores as well as in response to gradients of GABA and NGF; 14) transcripts encoding specific GABA receptor subunits appear at E12/13 in the cervical spinal cord and become colocalized during embryogenesis in anatomically distinct patterns throughout the CNS.
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