Alterations in intracellular calcium ion concentrations in cerebellar granule cells of the CACNA1A mutant mouse, leaner, during postnatal development

Abstract

Maintaining calcium ion (Ca²+) homeostasis is crucial for normal neuronal function. Altered Ca²+ homeostasis interferes with Ca²+ signaling processes and affects neuronal survival. In this study, we used homozygous leaner and tottering mutant mice, which carry autosomal recessive mutations in the gene coding for the α(1A) pore forming subunit of Ca(V)2.1 (P/Q-type) voltage-gated calcium channels (VGCC). Leaner mice show severe ataxia and epilepsy, while tottering mice are less severely affected. Leaner cerebellar granule cells (CGC) show extensive apoptotic cell death that peaks at postnatal (P) day 20 and continues into adulthood. Intracellular Ca²+ ([Ca²+](i)) concentrations in leaner and tottering mouse Purkinje cells have been described, but [Ca²+](i) concentrations have not been reported for granule cells, the largest neuronal population of the cerebellum. Using the ratiometric dye, Fura-2 AM, we investigated the role of Ca²+ homeostasis in CGC death during postnatal development by demonstrating basal [Ca²+](i), depolarization induced Ca²+ transients, and Ca²+ transients after completely blocking Ca(V)2.1 VGCC. From P20 onward, basal [Ca²+](i) levels in leaner CGC were significantly lower compared to age-matched wild-type CGC. We also compared basal [Ca²+](i) levels in leaner and wild-type CGC to basal [Ca²+](i) in tottering CGC. Potassium chloride induced depolarization revealed no significant difference in Ca²+ transients between leaner and wild-type CGC, indicating that even though leaner CGC have dysfunctional P/Q-type VGCC, Ca²+ transients after depolarization are the same. This suggests that other VGCC are compensating for the dysfunctional P/Q channels. This finding was further confirmed by completely blocking Ca(V)2.1 VGCC using ω-Agatoxin IV-A.

Fig. 1

Mean basal [Ca²⁺]_i in cerebellar granule cells (CGC) during postnatal development. Graph shows mean basal [Ca²⁺]_i data from isolated CGC of wild-type (Wild), leaner (Leaner), and tottering (Tottering) mice (n = 6). The basal [Ca²⁺]_i level in wild-type CGC was significantly higher than observed in leaner and tottering CGC at P20 and P30 (P < 0.001). There also were significant differences between wild-type and leaner basal [Ca²⁺]_i at P40 (P < 0.001). However, no significant difference in basal [Ca²⁺]_i between wild-type and leaner CGC at P10 was observed. “*” indicates significant differences between wild-type and other genotypes and “#” indicates significant differences within the wild-type mouse groups. Data were analyzed using GLM-univariate analysis of variance at α = 0.05

Fig. 2

Mean calcium transients in cerebellar granule cells (CGC) of wild-type (Wild) and leaner (Leaner) mice during postnatal development. Graph shows mean Ca²⁺ transient data in leaner and wild-type CGC after they were depolarized using 40 mM KCl. A minimum of 3–4 cells from each mouse were analyzed for each age group and genotype. There were no significant differences between wild-type and leaner CGC for all age groups observed. However, Ca²⁺ transients in P10 wild-type CGC were different from the entire wild-type group (“*”, P < 0.05) and Ca²⁺ transients in P10 leaner CGC were different from P30 and P40 (“**”, P < 0.05). Data were analyzed using GLM-univariate analysis of variance at α = 0.05

Fig. 3

Mean Ca²⁺ transients data from leaner and wild-type cerebellar granule cells (CGC) after they were incubated in the presence or absence of ω-Agatoxin IV-A and depolarized with KCl. Reduced Ca²⁺ transients were observed only in wild-type (Wild) but not in leaner (Leaner) CGC after incubation with the Ca_V2.1 channel specific blocker ω-Agatoxin IV-A (P < 0.05; n = 30). Data were analyzed using GLM-univariate analysis of variance at α = 0.05