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. 1997 Feb 18;94(4):1488-93.
doi: 10.1073/pnas.94.4.1488.

Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene

M S Airaksinen  1 , J Eilers, O Garaschuk, H Thoenen, A Konnerth, M Meyer
Affiliations

Ataxia and altered dendritic calcium signaling in mice carrying a targeted null mutation of the calbindin D28k gene

M S Airaksinen et al. Proc Natl Acad Sci U S A. .

Abstract

Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiological role of a major endogenous calcium-binding protein, calbindin D28k (calbindin) in vivo, we have generated calbindin null mutant mice by gene targeting. Surprisingly, calbindin deficiency does not affect general parameters of development and behavior or the structure of the nervous system at the light microscopic level. Null mutants are, however, severely impaired in tests of motor coordination, suggesting functional deficits in cerebellar pathways. Purkinje neurons, the only efferent of the cerebellar cortex, and inferior olive neurons, the source of the climbing fiber afferent, have previously been shown to express calbindin. Correlated with this unusual type of ataxia, confocal calcium imaging of Purkinje cells in cerebellar slices revealed marked changes of synaptically evoked postsynaptic calcium transients. Their fast, but not their slow, decay component had larger amplitudes in null mutant than in wild-type mice. We conclude that endogenous calbindin is of crucial importance for integrated nervous system function.

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Figures

Figure 1
Figure 1
Targeted disruption of the calbindin gene. (A) The targeting vector replaces part of the promoter and first exon with a neo cassette. H, HindIII. (B) Southern blot analysis of progeny from a heterozygote intercross. The 7.8- and 5.5-kb fragments indicate the wild-type and mutated alleles, respectively. (C) Calbindin protein is absent in homozygous and reduced in heterozygous mutant mice. (D) Western blot analysis of brain without cerebellum, cerebellum, and kidney of adult wild-type and mutant mice using a polyclonal calbindin antiserum. (E) Western blot analysis (Upper) and calcium-overlay assay (Lower) of adult cerebellum, brain without cerebellum, and kidney. Note the disappearance of the 28-kDa calbindin band (arrow) also in the Coomassie stained gel (Upper). Protein standards: Cb, calbindin; Pv, parvalbumin. +/+, Wild type; +/−, heterozygous; −/−, homozygous mutant.
Figure 4
Figure 4
Synaptically evoked calcium transients in cerebellar Purkinje neurons from mice lacking calbindin. (A) Confocal fluorescence image of a Purkinje neuron in a cerebellar slice obtained from a 24-day-old null mutant mouse (−/−). The neuron was loaded through the recording patch pipette with the calcium indicator dye, Calcium Green-1. The region delimited by the dashed line indicates a typical dendritic area from which the fluorescence data (C and D) were obtained. (B) Excitatory postsynaptic potential (complex spike) evoked by extracellular stimulation of the afferent climbing fiber. Recording from a null mutant mouse, membrane potential (Vm) was −70 mV. (C and D) Dendritic calcium transients evoked by climbing fiber stimulation recorded in null mutant mice (C, −/−, average trace from n = 7 cells in 4 mice) and wild-type mice (D, +/+, average trace from n = 6 cells in 4 animals). The curves above each fluorescence trace represent superpositions of the corresponding double-exponential time-course fit (continuous line) with the fast (tf, dotted line) and the slow (ts, dashed line) components depicted separately. (E) Af amplitude components (corresponding to tf) of the synaptically induced calcium signals in wild-type, heterozygous (+/−) and null mutant mice, respectively. (F) Free intracellular calcium concentration ([Ca2+]i) under resting conditions measured in somata of fura-2 loaded cells (50 μM) of wild-type and null mutant mice. In E and F, values are the means ± SEM. The asterisk indicates significant difference to wild type (Student’s t test, P < 0.05). Arrowheads mark the time points of single-shock synaptic stimulation.
Figure 2
Figure 2
Histological analysis. Midsagittal section of wild-type (A) and calbindin null mutant (B) mice stained with cresyl violet. Purkinje cells of wild-type (C) and null mutant (D) mice stained with antiserum to calbindin. (E) Golgi staining of Purkinje cell dendrites shows normal spine morphology and density. Purkinje cells of wild-type (F) and mutant (G) mice visualized with antiserum to parvalbumin.
Figure 3
Figure 3
Behavioral analysis. Performance of adult (7-month-old; A and B) and young adult (6-week-old; C) mice walking over a 1-m-long, 2-cm-wide runway. Four sessions on consecutive days are documented in A and B; the day 1 and 2 sessions after which no further changes were observed are shown in C. (A) Average number of slips counted on one side. (B) Percentage of drops during passage. In case of no drops, the value 0 is given at the position of the bar in A. Except for 129Sv mice (mean ± range), values are the means ± SEM. (D) Performance of 2-month-old males in the thin horizontal stationary rod test. The time spent on the rod up to a limit of 60 sec was measured. Values are the means ± SEM. ∗, P < 0.05; ∗∗, P < 0.005 compared with wild-type mice.
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