Figure 7

Absence of inhibitory basket cell terminals and Purkinje cell axon pathology in Neurod2^−/− mutants. (A,B), Absence of basket cell terminals at the soma (pericellular basket) and the AIS (pinceau formation) of Purkinje cells (dashed areas in A, B) in Neurod2^−/− mutants. Fluorescent immunostaining for (A) GAD67/HCN1 and (B) PSD95/PV; ANK3/PV (P25). Note that HCN1 and PSD95 mark basket cell terminals, whereas GAD67 and PV label both basket cell terminals and Purkinje cells. Immunostaining for the AIS marker ANK3 indicates unaltered AIS assembly in Purkinje cells of Neurod2^−/− mutants (arrowhead) compared to controls (arrow). Scale bar, 20 µm (A), 10 µm (B). (C) Targeted basket cell axon growth and formation of inhibitory presynaptic terminals is permanently abolished in Neurod2^−/− mutants. Confocal analysis of fluorescent immunostaining for PSD95 and NFH at P75. Basket cell axons are reduced in number and show undirected trajectories (asterisks mark Purkinje cell somata). Scale bar, 20 µm. (D) Schematic of electrophysiological recordings of mIPSCs in Purkinje cells and MLIs. (E) Left: Representative traces of Purkinje cell mIPSC recordings reveal reduced mIPSC frequency in Neurod2^−/− mutants (red, bottom) compared to control (black, top). Middle: Cumulative probability histogram indicates longer mIPSC inter-event intervals for Neurod2^−/− mutants. Right: Cumulative probability histogram of control (n = 16 cells) and mutant Purkinje cell mIPSCs (n = 15 cells) indicates similar mIPSC amplitude distributions in Neurod2^−/− mutants and controls. Inset, average mIPSCs of control (black) and Neurod2^−/− mutant (red). (F) Left: Representative traces of MLI mIPSC recordings show reduced mIPSC frequency in Neurod2^−/− mutants (red, bottom) compared to control (black, top). Middle: Cumulative probability histogram indicates longer mIPSC inter-event intervals for Neurod2^−/− mutants compared to control. Right: Cumulative probability histogram of control (n = 8 cells) and mutant MLI mIPSCs (n = 9 cells) indicates similar mIPSC amplitude distributions in controls and Neurod2^−/− mutants. Inset, average MLI mIPSCs of control (black) and Neurod2^−/− mutant (red). (G) Axonal swellings proximal to the AIS in Purkinje cells of Neurod2^−/− mutants. Fluorescent immunostaining for PV at P25. Scale bar, 50 µm.