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. 2017 Apr 4;8:14912. doi: 10.1038/ncomms14912

Figure 1. An in vivo conditional genetic strategy for silencing neurotransmission at excitatory olivocerebellar axon terminals in mice.

Figure 1

(a) Ptf1aCre can be used to target the inferior olivary nucleus as demonstrated by tdTomato expression. Scale bar, 200 μm. (b) Using the same Ptf1aCre driver, exon 2 of Vglut2 can be selectively removed and Vglut2 deleted from the inferior olive. (c) An antisense in situ probe for Vglut2 mRNA reveals its removal from the inferior olive by postnatal day 0. Scale bar, 200 μm. (d) Ptf1aCre-driven tdTomato expression compared with the distribution of NeuN-positive cells in the inferior olive, as schematized in the right panel. Scale bar, 100 μm. Schematic reproduced in part, with permission, from Reeber et al.78. (e) Quantification of the recombination efficiency of Ptf1aCre by dividing the number of tdTomato-positive cells by the number of NeuN-positive cells in the inferior olive in Ptf1aCre;Rosalox-stop-lox-tdTomato mice; n=3 mice. (f) Quantification of VGLUT2 protein expression in the molecular layer of lobules in all four transverse zones of the cerebellum show a significant reduction in Ptf1aCre;Vglut2fx/fx mice (P=9.294 × 10−16; Student's unpaired t-test); n=4 mice of each genotype. ***P<0.001. (g) Examples of expression of VGLUT2 protein in the molecular layer of lobule VI in a Vglut2fx/fx and a Ptf1aCre;Vglut2fx/fx mouse. Scale bar, 50 μm. (h) CART expression in climbing fibres in the molecular layer of lobule IX in a Vglut2fx/fx and a Ptf1aCre;Vglut2fx/fx mouse. Note that although CART expression in lobules IX and X is predominant and heaviest in climbing fibres, in other lobules it does label a subset of mossy fibres and scattered beaded fibres19. Arrowheads point to climbing fibres. Scale bar, 20 μm. (i) Electron microscopy showing the ultrastructure of climbing fibre synapses onto large, Purkinje cell dendritic branches in a Vglut2fx/fx and a Ptf1aCre;Vglut2fx/fx mouse. Scale bar, 200 nm; n=4 mice of each genotype. (j) Schematic depicting the outcome of genetically silencing climbing fibre terminals. CF, climbing fibre; gl, granular layer; pcl, Purkinje cell layer; ml, molecular layer; PCd, Purkinje cell dendrite; CART, cocaine- and amphetamine-related transcript.