Anatomical and functional reconstruction of the nigrostriatal system in vitro: Selective innervation of the striatum by dopaminergic neurons

To study development of the nigrostriatal pathway in an in vitro model syst em, organotypic slices obtained from rat pups (P4) and containing the stria tum and the cortex were grown together with apposed embryonic (E13.5) mesen cephalic blocks according to the static slice culture method of Stoppini et al, (1991; J. Neurosci, Methods 37:173-182), Under these conditions, mesen cephalic dopaminergic (DA) fibers rapidly grow through the slice, preferent ially its striatal portion. This innervation provides a true synaptic inner vation to the striatum, as shown by the presence of DA terminals on striata l neurons. DA fibers are able to exert a functional influence, as seen by t heir ability to modulate c-Fos expression in striatal neurons in the same w ay as in vivo. Thus, blockade, under basal conditions, of the effect of spo ntaneously released dopamine by the D2 receptor antagonist haloperidol lead s to the activation of c-Fos expression in the striatum, Furthermore, stimu lation of DA release by amphetamine induces striatal c-Fos expression in a D1 receptor-dependent manner. Next, the mechanisms of the selective striata l innervation were examined. Indeed, DA fibers innervated specifically the striatum, avoiding the cortical portion of the slice. This selectivity seem s to be specific for DA neurons; no selectivity could be observed when nora drenergic neurons were substituted for DA neurons. Short-term cocultures in a collagen gel of mesencephalic blocks with striatal blocks failed to reve al any oriented outgrowth of DA fibers from the mesencephalon, suggesting t hat the selective innervation observed in the organotypic slices results fr om some contact-dependent, presumably adhesive interactions rather than fro m the presence of some diffusible substance orienting the growth of DA fibe rs towards the striatum. On the other hand, DA neurons seeded onto striatal slices did not attach selectively onto the striatal portion of the slice, indicating that the putative specific adhesive interactions governing the s elective striatal innervation are not the same as those determining the adh esion of the DA neurons. These results show that cocultures of cortex-stria tum and mesencephalic slices result in a system that displays a number of t he morphological and functional traits of the normal nigrostriatal system a nd that can be relied on as a good in vitro model of in vivo development. J , Neurosci. Res. 58:553-566, 1999. (C) 1999 Wiley-Liss, Inc.