Figure 3.

Detailed analysis of calbindin transgenic mice. (A) Confocal image of a stained section of cerebellum. The Purkinje cells express high levels of GFP–TTC (arrow) and are double-labeled with an antibody to β-gal (red) that, as a stationary marker, identifies the cell where GFP–TTC protein is produced. Transfer of GFP–TTC can be visualized to cerebellar granule cells, the small ovoid neurons with dark nuclei lying below the Purkinje cells in their characteristic internal granular layer of the cerebellum, see F. Individual GFP positive granule cells are marked by arrowheads. (Scale bar, 20 μm.) (B) Transfer of GFP from a double-labeled Purkinje cell to an adjacent, strongly labeled basket cell, arrowhead. The putative site of synaptic contact between the two cells is marked by an arrow. Analysis was as in A. (Scale bar, 20 μm.) (C) Confocal analysis of a cerebellar Golgi neuron labeled with GFP. It is identified by its location and specific morphology. Note the absence of β-gal in the nucleus, arrow. Analysis as in A. (Scale bar, 20 μm.) (D) In situ hybridization (16) with a riboprobe complementary to GFP. Only the Purkinje cells are labeled. The Purkinje cell layer is outlined by arrowheads. Note the absence of signal elsewhere. (Scale bar, 200 μm.) (E) A section of brainstem analyzed as in Fig. 2C. Strong signal is detectable in the inferior olive, outlined by arrowheads, a major source of afferents to cerebellar Purkinje cells, see Fig. 3F. (Scale bar, 100 μm.) Asterisk marks basilar artery. (F) A scheme summarizing the retrograde transsynaptic transfer in the cerebellar system (modified from ref. 23), with an indication of the different cerebellar cell layers on the right, i.e., Molecular layer, Purkinje cell layer, and granular layer.