The stop signal revised: immature cerebellar granule neurons in the external germinal layer arrest pontine mossy fiber growth.

During the formation of neuronal circuits, afferent axons often enter target regions before their target cells are mature and then make temporary contacts with nonspecific targets before forming synapses on specific target cells. The regulation of these different steps of afferent-target interactions is poorly understood. The cerebellum is a good model for addressing these aspects, because cerebellar development is well defined and identified neurons in the circuitry can be purified and combined in vitro. Previous reports from our laboratory showed that cultured granule neurons specifically arrest the extension of their pontine mossy fiber afferents, leading us to propose that granule cells arrested growth of their afferents as a prelude to synaptogenesis. However, we knew little about the differentiation state of the cultured granule cells that mediate afferent arrest. In this study, we better define the purified granule cell fraction by marker expression and morphology, and demonstrate that only freshly plated granule cells in the precursor and premigratory state arrest mossy fiber outgrowth. Mature granule cells, in contrast, support extension, defasciculation, and synapse formation, as in vivo. In addition, axonal tracing in vivo during the first postnatal week indicates that immature mossy fibers extend into the Purkinje cell layer but never into the external germinal layer (EGL), where precursors of granule cell targets reside. We found that the stop-growing signals are dependent on heparin-binding factors, and we propose that such signals in the EGL restrict the extension of mossy fiber afferents and prevent invasion of proliferative regions.