Second, Shh stimulates OPC proliferation. Our results confirm the mitogenic effect of Shh on OPCs. These results also suggest that changes in Shh expression may control the timing of OPC differentiation. Thus, Purkinje cells, through Shh secretion, participate in the control of OPC proliferation in the cerebellum. The addition of vitronectin to immature Albaspidin-AA slices enhanced oligodendrocyte differentiation and vitronectin-blocking antibodies inhibited the effects of mature-slice induced differentiation on immature slices. Furthermore, when the slices cultures contained more Purkinje cells, levels of vitronectin were elevated. Only a slight difference between control and axotomized slices was detected likely because we are closed to the threshold of detection. Thus, our results clearly demonstrate that vitronectin is required for mature slices to promote oligodendrocyte differentiation in immature slices. Previous studies reported no effect of vitronectin on OPC differentiation. Moreover, it has been shown that the CG4 cell lines grown on vitronectin proliferate more that those grown on polylysine. However, combinations of vitronectin and mitogenic factors stimulate the differentiation of embryonic stem cells into oligodendrocytes, whereas vitronectin alone has no effect. Interestingly, Vitronectin inhibits the effects of Shh in other systems, through biochemical interactions in the spinal cord or the signaling pathway induced by its binding to integrin in cerebellar granule cells. Nevertheless, Shh and vitronectin may also control OPC differentiation through different mechanisms. Organotypic culture is an integrated system, in which cell interactions mimic those occuring in vivo, and is easier to manipulate than in vivo models. Furthermore only one type of neuron is myelinated in this system: the Purkinje cell. Using this system, we showed that the maturation of the Purkinje cell is involved in Tulathromycin B controlling the timing of oligodendrocyte differentiation. Indeed, our results suggest that Purkinje cells release different factors during their maturation, which have opposing effects on oligodendrocyte differentiation. This temporal regulation probably synchronizes the differentiation of oligodendrocytes and Purkinje cells. However, as discussed above, oligodendrocyte differentiation occurs even when the number of Purkinje cells is reduced. This suggests the presence of other differentiating factors in cultured slices or medium. Many of the factors known to affect oligodendrocyte development, such as TGF?, IGF-1, and progesterone, are present in the cerebellum. Oligodendrocyte differentiation 
rates were lower in serum-free medium than in the presence of serum. Thyroid hormones are crucial for both oligodendrocyte and Purkinje cell differentiation. Among the factors involved in controlling OPC proliferation and differentiation, our study identifies two molecules with developmentally regulated expression patterns which are involved in the switch between OPC proliferation and oligodendrocyte differentiation. Vitronectin has been detected in active multiple sclerosis plaques and a decrease in Shh levels has been observed in the white matter of patients with multiple sclerosis. These actors are thus present, but it is unclear as to whether they are correctly synchronized. Determining how the timing of the various steps leading to myelination or remyelination is controlled is therefore still a major challenge.