Organotypic slice cultures of the rat striatum—I. A histochemical and immunocytochemical study of acetylcholinesterase, choline acetyltransferase, glutamate decar☐ylase and GABA

Abstract

Slices of striatal tissue from newborn to eight-day-old rats were cultured for six to 47 days. Cholinergic neurons and fibres were then visualized by histochemical staining for acetylcholinesterase or immunocytochemical staining for choline acetyltransferase. GABA-containing neurons and fibres were visualized by immunocytochemical staining for glutamate decar☐ylase or GABA. Corresponding to the normal postnatal development in vivo, acetylcholinesterase staining of the striatal tissue progressed from a “patchy” distribution in the six to 14 days old cultures to an almost even distribution of high acetylcholinesterase activity after 18–27 days. Extrinsic afferents were accordingly not necessary for the maintenance of a patch-matrix-like, acetylcholinesterase distribution during the first one to two weeks in culture, just as a subsequent, normal developmental change of the acetylcholinesterase staining pattern into a more homogeneous distribution also occurred without such afferents. Cholinergic, choline acetyltransferase-immunoreactive neurons were evenly distributed within the cultured striatal tissue, like in vivo, but the density of the neurons appeared to be higher in the cultures. The neurons had a morphology corresponding to the “classical”, large-sized, aspiny, cholinergic interneurons in the adult rat striatum. Glutamate decar☐ylase-immunoreactive and GABA-immunoreactive neurons were either lightly or darkly stained and of medium size, but some large, lightly stained glutamate decar☐ylase-immunoreactive and GABA-immunoreactive neurons were also found. The difference in staining density among the medium-sized cells was observed with both antisera and hence provide evidence for the existence of two populations of medium-sized GABAergic neurons, which in vivo are intensely stained interneurons and more weakly stained, spiny projection neurons. Fibres stained better for glutamate decar☐ylase than for GABA and outgrowth of glutamate decar☐ylase-immunoreactive nerve fibres from the striatal slice cultures onto the coverslip was often observed. The presence at all culture periods of “protospines” on cell bodies and proximal dendrites of some glutamate decar☐ylase-immunoreactive, and in particular some GABA-immunoreactive neurons, suggested that at least some developmental characteristics might be maintained for extended periods in culture. In several cultures, groups of small GABA-immunoreactive cells were observed. Similar groups were also found by staining for glutamate decar☐ylase, but a smaller proportion of the cells were then positively stained. In view of their immature appearance with few or no processes, the known presence of GABA in neuroblast-like cells, and the recent demonstration of neuronal and glial progenitor cells in the adult mouse striatum, the small cells might belong to a population of undifferentiated cells surviving in the slice cultures. In conclusion, the study demonstrates that striatal cholinergic interneurons and GABA-containing interneurons and projection neurons can survive in slice cultures for at least seven weeks without the reinstated or continous presence of normal extrinsic afferents. During this period the neurons attain a basically normal morphological and neurochemical differentation. Based on the results we will proceed to use slice cultures for experimental neurotoxic and neurotrophic studies, related to striatal neurodegenerative diseases, like Huntington's chorea, Parkinson's disease and cerebral ischemia.