Fig. 3.

Mtss1 prevents SFK-dependent firing defects and ataxia. (A) Confocal projection of an individual Purkinje cell filled with biocytin and fluorescent dye to visualize morphology. (B–E) Measurement of dye-filled Purkinje neurons shows MIM^EX15 mutants have reduced arbor volume (n = 3 each genotype) (B) and reduced dendritic spine density (C) but no change in dendritic spine length (D) and no change in dendritic spine width (E). MIM^EX15/+, n = 3 neurons, 1,720 spines; MIM^EX15, n = 3 neurons, 1,454 spines. *P < 0.05, Student’s t test. Error bars indicate SEM. (F) Western blot for active SFK-Y416 phosphorylation with actin as a loading control. Cerebellar lysate was collected from MIM^EX15 mice and age-matched controls at the indicated times between postnatal day 15 (P15) and postnatal day 30 (P30). (G) Slow EPSP in wild-type (Left) and MIM^EX15 (Center) cells elicited by the stimulation of parallel fibers with 10-pulse trains at 100 Hz in the presence AMPA, NMDA, and GABA receptor antagonists (control conditions). Corresponding intracellular Ca²⁺ signals (ΔF/F) for responses for wild-type and MIM^EX15 mGluR EPSPs are illustrated. EPSPs and corresponding Ca²⁺ signals are blocked by mGluR1 antagonist CPCCOEt. (Right) Summary data of intracellular Ca²⁺ signals (ΔF/F) for responses for WT and MTSS1^EX15 in control conditions and in the presence of CPCCOEt are shown. (H) Percent histograms of Purkinje neuron mean firing frequencies (Left), examples of extracellular recordings of 1-s duration of a spontaneously spiking Purkinje neuron in the respective condition (Center), and histograms of interspike intervals (ISIs) calculated for the 2-min recording periods of the same neuron (Right) are shown for the wild-type, MIM^EX15, wild-type+dasatinib, and MIM^EX15+dasatinib conditions. (I) Summary of data presented in H. *P = 6.1E-14; **P = 1E-13; one-way ANOVA, Tukey post hoc test. (J) Direct cerebellar administration of dasatinib maintains rotarod performance, slowing the progressive ataxia in MIM^EX15 mice. q = 0.006, two-stage step-up Benjamini, Krieger, Yekutieli method. Error bars indicate SEM.