The restoration of functional circuitry in damaged adult mammalian cerebral cortex remains the goal of the proposed research. During the previous grant period we showed that solid grafts consisting of thousands of neurons can be induced to form functional circuits with the host brain, so that graft cells can be driven by activity in the adult host sensory pathways. However, despite the fact that neurons in the solid grafts develop extensive internal circuitry, they form only weak connections with the host brain. That is, graft cells do not show normal levels of spontaneous activity and host-graft synapses do not show normal synaptic drive. The hypothesis that arises from these previous results is that the maturation of embryonic neurons grafted into adult cerebral cortex must be assisted in specific ways during sequential phases of differentiation: namely 1) during initial cell survival and process outgrowth, 2) during synaptogenesis and onset of neural activity and 3) during the activity-dependent (normally postnatal) phase of excitatory synapse adjustment. The proposed experiments will institute different procedures to facilitate each of these phases, with the ultimate goal of optimizing NMDA-receptor mediated plasticity in the host-graft synapses.
In Specific Aim 1 the effect of growth factors will be tested on the initial survival of the dissociated embryonic cells and on the elongation of their neurites.
In Specific Aim 2 the effect of modulatory neurotransmitters (NE + Ach,) on the graft cells will be tested after process formation has occurred to determine the modulators effect on initial synaptic activity.
In Specific Aim 3 graft cells will be stimulated in ways that synchronize afferent sensory fiber activity and depolarization of the grafted neurons. Stimulation of sensory inputs will allow us to assay the effect of activity on the development of synaptic properties in the grafted neurons and on the induction of NMDA receptor function. The main series of experiments will be carried out in vivo, with quantitative studies of growth factors, modulators and stimulation dose-response effects being quantified in cultures of dissociated neurons. In parallel with the cellular analysis, a final aim (4) will be to assay the ability of the graft to restore specific behavioral deficits caused by the cortical lesion. The results of these studies should lead to a clearer understanding of the optimal strategy for enhancing the development of plasticity mechanisms and for restoring function in damaged cortical circuits.
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