Establishment of a long-term primary culture of striatal neurons

A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical, pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.