Labeling and Identification of Living Donor Cells in Brain Slices of Recipient Hemiparkinsonian Model Rats for Physiological Recordings: Methods for Physiological Assessments of Neural Transplantation

Abstract

Physiological properties of grafted neurons, such as membrane and intracellular properties, have not been reported. To fill this lack in knowledge, physiological recordings from the identified grafted cells are required. Fluorescent latex microspheres (FLM) are nontoxic and stable, and thus seem suitable for long-term labeling of donor cells. Therefore, we tested the feasibility of labeling with FLM to identify living donor cells in the recipients' brain slices. We also tested if physiological recordings from the identified cells are possible or not. Cell suspensions were prepared from the substantia nigra (SN) of Embryonic Days 15 or 16 rats with enzymatic and mechanical trituration. Cell suspensions were then incubated with 0.5% FLM for 30 min to 2 h. The longer cells were incubated, the more FLM were taken up. The FLM-labeled SN cell suspensions were injected in the striatum of the hemiparkinsonian model rats. Eight to 13 weeks later, 150-μm thick coronal brain slices including the graft track were prepared from the recipients. Slices were keptin vitrofor several hours. Grafted cells could be clearly identified in the slice preparations by the uptake of FLM under a fluorescence microscope. Voltage-dependent currents and intracellular Ca2+transients were successfully recorded from the identified grafted neurons. It is suggested that labeling and identification of living donor cells with FLM is feasible and thus can provide a powerful tool to study the mechanisms underlying graft-induced amelioration of neurological deficits in parkinsonism by enabling physiological assessments of grafted cells.