Flow cytometry, buoyant density-gradient cell fractionation, dissociated cell culture, cell migration, immunocytochemistry and in situ hybridization 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 focused primarily on GABA, which is transiently expressed in a widespread manner during CNS development before it becomes relatively restricted to fast- transmitting synapses in the adult where it often functions in an inhibitory manner. In FY95 we investigated the following: 1) transcripts encoding three GABA-synthesizing GAD enzymes and those encoding most GABA/A receptor subunit proteins were detected by in situ techniques in progressively more regions of the developing CNS; 2) specific subunits are expressed at all levels of the embryonic neuraxis beginning during the period of intense neuroblast proliferation; 3) distinct patterns of GAD and GABA transcript coexpressions are apparent: one almost exclusively in cells of the neuroepithelial proliferative zone, one in many, if not most differentiating cells during embryogenesis and one differentiating during the postnatal period; 4) some transcripts are only transiently detected for variable periods while others persist, becoming restricted to subpopulations; 5) transcripts encoding GAD and GABA receptors are more abundant and widely distributed during embryogenesis than in the adult, implying """"""""morphogenic"""""""" roles; 6) GAD and GABA receptor subunit family proteins were detected in cells and processes, indicating that the transcripts are functional; 7) continuous-gradient centrifugation of embryonic CNS cells reveals that subpopulations accumulate in visible bands of specific buoyant density beginning during the logarithmic growth period; 8) FACS analyses of fractionated cells show that highly proliferative cells populate the dense region while primarily post-mitotic elements in G0 or G1 compose the visible bands; 9) FACS studies show that differentiation marker+, GAD+, GABA+, GABA/A receptor subunit+ cells exhibit characteristic patterns of expression according to their buoyant density; 10) GABA+ immunoreactivity can be eliminated by ionophore treatment, indicating dynamic lability in the immunodetectable signals; 11) FACS recordings of membrane potential and Cac2+ levels reveal characteristic patterns of functional GABA, glutamate and ACh receptor-coupled responses exhibited by different subpopulations cortical cells show that gradient-directed migration occurs at femtomolar GABA, while gradient-independent motility requires micromolar GABA; 13) structure-activity studies show that A, B and C are implicated; 14) GABA loaded with BAPTA-AM, indicating Cac2+-dependent signal transduction; 15) GABA+ spinal and hippocampal neurons differentiate in culture independently of the initial presence of cortical astrocytes.
| Li, Y X; Schaffner, A E; Barker, J L (1999) Astrocytes regulate the developmental appearance of GABAergic and glutamatergic postsynaptic currents in cultured embryonic rat spinal neurons. Eur J Neurosci 11:2537-51 |
| Zhang, L; Ma, W; Barker, J L et al. (1999) Sex differences in expression of serotonin receptors (subtypes 1A and 2A) in rat brain: a possible role of testosterone. Neuroscience 94:251-9 |
| Chandross, K J; Cohen, R I; Paras Jr, P et al. (1999) Identification and characterization of early glial progenitors using a transgenic selection strategy. J Neurosci 19:759-74 |
| Behar, T N; Scott, C A; Greene, C L et al. (1999) Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration. J Neurosci 19:4449-61 |
| Silverman, W F; Alfahel-Kakunda, A; Dori, A et al. (1999) Separation of dorsal and ventral dopaminergic neurons from embryonic rat mesencephalon by buoyant density fractionation: disassembling pattern in the ventral midbrain. J Neurosci Methods 89:1-8 |
