The objective of this proposal is to analyze the anatomical and physiological integration of neurons from dissociated fetal hippocampus which have been grafted into normal and lesioned adult hippocampus. The synaptic integration of the transplants into the hippocampal circuitry of the recipient will be assessed in the following specific aims.
The first aim i s to directly test the hypothesis that transplants of dissociated fetal hippocampus become integrated into the normal host hippocampus and develop synaptic connections with host neurons. This will be accomplished by pre-labeling fetal neurons with Rhadomaine microspheres to identify the grafted neurons in an in vitro slice preparation so that extracellular and intracellular physiological recording techniques can be utilized to assess synaptic integration of identified grafted cells. The density and quality of synaptic interactions will be used to evaluate the physiological integration of the grafted neurons which will be filled with the intracellular stain, Lucifer yellow, to precisely assess the degree of their anatomical integration into the host parenchyma. Ingrowth of graft processes into appropriate and/or inappropriate cytoarchitectonic areas will be used to measure the anatomical integration of the grafted neurons. In a second series of experiments the principal investigator will establish the specificity and strength of physiological connectivity between graft and host by selectively removing only the grafted neurons from hippocampal slices during physiological recording sessions. To accomplish this the grafted neurons will be pre-labeled with a laser sensitive photo-activated dye, chlorin e6 attached to latex microspheres. After physiological recording are made in hippocampal slices the tissue will be exposed to laser illumination from an infrared laser which will produce selective loss of only the labeled grafted neurons. It is stated that this approach will directly remove only grafted neurons and their synaptic connections and lead to a specific test of the physiological mechanism of graft interaction with the host hippocampus. In the final specific aim the investigators will test the hypothesis that synaptic integration and survival of fetal hippocampal grafts varies as a function of both the severity and time course of lesions to the host brain. This will be accomplished by using unilateral kainic acid lesions involving the CA3 region of the hippocampus as a more severe target lesion to remove the Schaffer collateral input onto the dendrites of CA1 pyramidal neurons and to compare the effects of this lesion on graft survival and integration with a less extensive and more selective novel laser induced lesion of CA3 neurons possessing commissural projections. This second lesion will be produced by infrared laser illumination of one hippocampus in which the commissural projecting CA3 neurons have been retrogradely labeled with photo-activated dye containing microspheres injected into the contralateral hippocampus. Replacement of synaptic circuitry in the CA1 area will be analyzed after grafting of pre-labeled fetal hippocampal cells into the denervated CA1 region at various time points after either lesion. Analysis of the time course of enhancement of graft integration will critically test the hypotheses that both neurotrophic factors induce by the lesions and vacated synaptic sites resulting from the denervation influence graft survival and integration.
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