Morphometric characteristics of cryopreserved mesencephalic dopamine neurons in culture

Blocks of embryonic rat mesencephalon were freeze-stored for 1-2 years in liquid nitrogen at -196 degrees C with 7.5% dimethyl sulfoxide (DMSO) as cryoprotectant. After thawing, pooled mesencephalic tissues were mechanically dissociated. The cells, plated at two different densities (4.10[5] and 2.10[5]/cm2) were cultured in a serum-supplemented medium for at least 2 weeks before immunocytochemical staining with highly specific antidopamine (DA) antibodies. The cryopreserved DA-immunoreactive (IR) neurons were compared, by means of computerized morphometry, to the fresh ones plated at the same densities. A separate analysis of the dendritic and axonal morphometric parameters revealed that the cryopreserved DA-IR cells, whatever the experimental conditions, had significantly larger dendritic fields and, less significantly, larger axonal fields than their fresh counterparts. A principal component analysis, mainly based on the dendritic morphometric parameters, allowed to individualize only two populations (cryopreserved and fresh) among the four groups studied. These findings underline the role of dendrites as potential sites of release and/or re-uptake of dopamine and their possible implications in functionally effective cryopreserved nigral grafts.