Blocks of embryonic rat cerebral cortex and mesencephalon were cryopreserved and stored for up to 1 year in liquid nitrogen at -196 degrees C with 7.5% dimethylsulfoxide (DMSO) as cryoprotectant. After thawing, these tissues were only mechanically dissociated and the cells were cultured for 2-7 weeks before immunocytochemical staining with anti-GABA (gamma-aminobutyric acid) antibodies. The freeze-stored GABA-immunoreactive (IR) mesencephalic neurons were compared, with computerized morphometry, to fresh mesencephalic cells and to their fresh and frozen cerebral cortical counterparts. A part of the cortical cells was treated with thienyl-phencyclidine (TCP) in order to assess the potential morphological effects of this neuroprotective agent upon these cortical neurons. Two types of GABA-IR neurons (small and large neuritic field cells) could be evidenced in both structures without any difference between fresh and frozen materials, but with significant quantitative morphological differences linked to their anatomical source. GABAergic phenotype is expressed similarly in fresh and frozen cultured neurons with intrinsically programmed morphological features and only minor influences of epigenetic factors. Small and large neuritic field GABA-IR neurons represent, respectively, local and long-range circuits of inhibition, strongly reminiscent of those described in vivo and which remain unchanged in culture even after freeze-storage.
