Calbindin in cerebellar Purkinje cells is a critical determinant of the precision of motor coordination

Abstract

Long-term depression (LTD) of Purkinje cell-parallel fiber synaptic transmission is a critical determinant of normal cerebellar function. Impairment of LTD through, for example, disruption of the metabotropic glutamate receptor-IP3-calcium signaling cascade in mutant mice results in severe deficits of both synaptic transmission and cerebellar motor control. Here, we demonstrate that selective genetic deletion of the calcium-binding protein calbindin D-28k (calbindin) from cerebellar Purkinje cells results in distinctly different cellular and behavioral alterations. These mutants display marked permanent deficits of motor coordination and sensory processing. This occurs in the absence of alterations in a form of LTD implicated in the control of behavior. Analysis of synaptically evoked calcium transients in spines and dendrites of Purkinje cells demonstrated an alteration of time course and amplitude of fast calcium transients after parallel or climbing fiber stimulation. By contrast, the delayed metabotropic glutamate receptor-mediated calcium transients were normal. Our results reveal a unique role of Purkinje cell calbindin in a specific form of motor control and suggest that rapid calcium buffering may directly control behaviorally relevant neuronal signal integration.

Fig. 1.

Generation and characterization of the conditional mutant. A, Targeting strategy. B, Western blot of equal amounts of protein from the indicated tissues fromCalb^tm2/Calb^tm2and wild-type mice detected by a monoclonal calbindin antibody.C, RT-PCR of calbindin transcripts in cerebellum, cortex, and hippocampus. Glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. D, Western blot analysis of calbindin expression (as in B) of recombined and control mice. Cb28kD, Positions of calbindin protein or amplification product. E–P, Localization of calbindin in recombined (F, H, J,L, N, P) and not-recombined (E, G, I,K, M, O) mice by immunohistochemistry using a monoclonal calbindin antibody.E, F, Cerebellar cortex;G, H, cerebellar cortex double-stained for calbindin (green) and parvalbumin (red); I,J, deep cerebellar nuclei; K,L, cerebral cortex; M, N, striatum; O, P, inferior olive. Scale bars: E–H, M–P, 20 μm;I, J, 40 μm; K,L, 80 μm.

Fig. 2.

Limb coordination. A, Five day runway task on the 2-cm-wide bar (not recombined, n= 19; recombined, n = 21; *p ≤ 3.1 × 10⁻⁶). B, Balance rod test (not recombined, n = 9; recombined,n = 7; *p ≤ 3.2 × 10⁻²). C, Ten-day runway task on the 1-cm-wide bar (not recombined, n = 9; recombined,n = 10; *p ≤ 9.2 × 10⁻⁵).

Fig. 3.

Compensatory eye movements. The gain values of recombined mice (n = 11) were significantly smaller than those of the control animals (n = 10) during both OKR (A) and VOR-L (C) at all tested frequencies. In contrast, the phase values (B, D) did not differ significantly at any of the frequencies.

Fig. 4.

LTD in not-recombined mice (A) and recombined mice (B). Top, PF-EPSCs (average of 12 traces for A, a single experiment for B) recorded 5 min before the start of LTD induction (control) and 50 min after the end of LTD induction (50 min). The superposition shows the reduction in amplitude after pairing. On average, the pairing protocol induced a reduction of ∼40% in the amplitude of PF-EPSCs in both groups (middle, summary of four experiments; error bars represent SEM). The series resistance (Rs; bottom) remained well within the range of 10% change compared with the baseline value in all experiments.

Fig. 5.

Synaptic Ca²⁺ signaling.A, Top, Line scan recording of early synaptic calcium transients evoked by single parallel fiber stimulation (ESCT) in a spine and the adjacent dendrite of a not-recombined mouse. Left, Image of the spiny dendrite and the line and regions of interest chosen for the experiment. Scale bar, 1 μm; d, dendrite; s, spine. Below, Current (EPSC) evoked by parallel fiber stimulation (stimulation intensity, 30 V). Right, ESCT in the spine and the dendrite. Broken red lines represent monoexponential decay functions fitted to the data points. Time constants (τ) were 348 msec (spine) and 363 msec (dendrite). Bottom, ESCT in a spine and the adjacent dendrite of a recombined mouse. Left (compare with top), Stimulation intensity was 10 V. Right, Despite the smaller EPSC amplitude, ESCT amplitudes were much larger than in the not-recombined animal. Time constants were significantly faster. B, Top, Line scan recording of early synaptic calcium transients evoked by climbing fiber stimulation in a spine and the adjacent dendrite of a not recombined mouse. Left (compare with A, below), Complex spike evoked by climbing fiber stimulation. Right, Calcium transients in the spine and the dendrite and time constants calculated from monoexponential decay functions (compare with A). Bottom, Calcium transients in a spine and the adjacent dendrite of a recombined mouse. Right, Despite the similar electrical response, amplitudes of calcium transients were much larger than in not recombined animal. Time constants were significantly faster. Calcium transients represent the averages of three consecutive trials; arrowhead indicates the time of synaptic stimulation. C, Histograms of ESCT amplitudes and decay time constants in spines (s) and dendrites (d) of not-recombined and recombined animals (parallel fiber stimulation: black bars, n = 21 dendrites, 30 spines; gray bars,n = 17 dendrites, 44 spines; climbing fiber stimulation: black bars, n = 11 dendrites, 26 spines; gray bars, n = 18 dendrites, 35 spines;). Error bars represent SEM. D, mGluR-mediated synaptic calcium signaling. Right, Local dendritic Ca²⁺signals mediated by repetitive parallel fiber stimulation (marked by arrowheads, 5 pulses, 50 Hz) in the presence of 40 μmCNQX. Images demonstrate activated dendritic regions. Scale bars, 20 μm. Traces show relative changes in fluorescence in regions of interest within these active regions. Broken red lines represent monoexponential decay functions fitted to the data points. Time constants were 806 msec (not recombined) and 811 msec (recombined). Below, Histogram of dendritic decay time constants of the delayed synaptic calcium transients evoked by repetitive parallel fiber stimulation (DSCT) [n = 5 for not recombined;n = 7 for recombined; averages are not significantly different (t test, p< 0.01)]. Error bars represent SEM.