Bidirectional alterations in cerebellar synaptic transmission of tottering and rolling Ca2+ channel mutant mice

Abstract

Hereditary ataxic mice, tottering (tg) and rolling Nagoya (tg(rol)), carry mutations in the P/Q-type Ca(2+) channel alpha(1A) subunit gene. The positions of the mutations and the neurological phenotypes are known, but the mechanisms of how the mutations cause the symptoms and how the different mutations lead to various onset and severity have remained unsolved. Here we compared fundamental properties of excitatory synaptic transmission in the cerebellum and roles of Ca(2+) channel subtypes therein among wild-type control, tg, and tg(rol) mice. The amplitude of EPSC of the parallel fiber-Purkinje cell (PF-PC) synapses was considerably reduced in ataxic tg(rol). Although the amplitude of the parallel fiber-mediated EPSC was only mildly decreased in young non-ataxic tg mice, it was drastically diminished in adult ataxic tg mice of postnatal day 28-35, showing a good correlation between the impairment of the PF-PC synaptic transmission and manifestation of ataxia. In contrast, the EPSC amplitude of the climbing fiber-Purkinje cell (CF-PC) synapses was preserved in tg, and it was even increased in tg(rol), which was associated with altered properties of the postsynaptic glutamate receptors. The climbing fiber-mediated EPSC was more dependent on other Ca(2+) channel subtypes in mutant mice, suggesting that such compensatory mechanisms contribute to maintaining the CF-PC synaptic transmission virtually intact. The results indicate that different mutations of the P/Q-type Ca(2+) channel not only cause the primary effect of different severity but also lead to diverse additional secondary effects, resulting in disruption of well balanced neural networks.

Fig. 1.

PF-PC synaptic transmission in mutant mice.A, PF-EPSC peak amplitudes were plotted against the intensity of stimulation for wt and mutant mice at P14–20. Mean peak amplitudes ± SEM from 10 wt, 7 tg, and 7tg^rol Purkinje cells are shown.Insets show traces of PF-EPSCs from wt (left), tg (middle), andtg^rol (right) Purkinje cells. Each trace is an average of 10 current recordings.B, The PF-EPSC peak amplitude–stimulus intensity relationship, as in A, from 8 wt and 10tg Purkinje cells (P28–35).Insets show traces of PF-EPSC from wt (left) and tg (right) Purkinje cells evoked by 10 V stimulation. Each trace is an average of 10 current recordings.

Fig. 2.

Paired-pulse facilitation in PF-EPSC.A, The PF-EPSC peak amplitude–stimulus intensity relations for paired-pulse stimulation (interval 50 msec) of wt and mutants. Mean peak amplitudes evoked by the first stimulation (○) and the second stimulation (●) were obtained from 10 measurements. Calibration is common for all traces. Insets show typical current traces evoked by 10 V stimulation (average of 10 recordings). B, Mean paired-pulse ratio (second EPSC/first EPSC) from wt, tg, andtg^rol at P14–20 (10 Purkinje cells each). C, Mean paired-pulse ratio from wt andtg at P28–35 (10 Purkinje cells each). Error bars represent SEM in A–C.

Fig. 3.

Toxin sensitivity of PF-EPSC. A,B, Time courses of the peak PF-EPSC amplitude in response to application of 0.2 μm ω-Aga-IVA (A) and 3 μm ω-CgTx and subsequent 0.2 μm ω-Aga-IVA (B).Insets show current traces at the time indicated by thenumbers. Each trace is an average of 10 recordings.C, D, The ω-Aga-IVA-sensitive (C) and the ω-CgTx-sensitive (D) components (mean ± SEM) of PF-EPSC of wt, tg, and tg^rol from three to six measurements.

Fig. 4.

CF-PC synaptic transmission in mutant mice.A, The distribution of the CF-EPSC peak amplitude from 40 wt, 30 tg, and 31tg^rol Purkinje cells at P14–20. All Purkinje cells were mono-innervated. Insets show typical traces of CF-EPSC from wt, tg, andtg^rol Purkinje cells at P14–20. Three to five traces were averaged. B, Mean peak amplitudes of CF-EPSC of wt, tg, andtg^rol at P14–20. Values are presented as mean ± SEM. *p < 0.05. C, CF-EPSCs to pairs of stimuli separated by 50 msec in mono-innervated Purkinje cells of wt, tg, andtg^rol at P14–20. Three to five traces were averaged. D, Paired-pulse ratios (second EPSC/first EPSC; mean ± SEM) from 17 wt, 10 tg, and 10tg^rol Purkinje cells. Note that the value of tg is larger than wt ortg^rol (*p < 0.05).

Fig. 5.

Toxin sensitivity of CF-EPSC. A,B, Time course of the peak CF-EPSC amplitude in response to application of 0.2 μm ω-Aga-IVA and 3 μm ω-CgTx (A) and to application of those blockers in the reverse order (B).Insets show current traces at the time indicated by thenumbers. Each trace is an average of five recordings.C, D, The ω-Aga-IVA-sensitive (C) and the ω-CgTx-sensitive (D) components (mean ± SEM) of CF-EPSC of wt, tg, and tg^rol from three to six measurements. E, The remaining components after application of ω-Aga-IVA and ω-CgTx from six to eight measurements. *p < 0.05.

Fig. 6.

Effect of lowering the external Ca²⁺ concentration on EPSC amplitude. PF-EPSC amplitude (○) and CF-EPSC amplitude (●) were plotted as a function of the external Ca²⁺ concentration. The current amplitude was normalized to that at 2 mm external Ca²⁺. Values are presented as mean ± SEM from five measurements each. PF-EPSC amplitude was fit by a power relation,y = 0.30 ∗ x^m, where m = 1.75. CF-EPSC was fit by the Hill's equation, y = 1.0068 ∗x^m/(x^m+ d′^m), where m = 3.78 and d′ = 0.59. The relative half-saturation concentration d = 0.59 mm/2 mm = 0.29.

Fig. 7.

Effects of a partial AMPA receptor antagonist γ-DGG on CF-EPSCs. The graph shows concentration–inhibition curves for wt (white circles), tg(gray diamonds), andtg^rol (black triangles). The ordinate in the graph indicates percentage of the control CF-EPSC amplitude after application of γ-DGG. Values are presented as mean ± SEM from three to five measurements. The curves were fit by the Hill's equation. The half inhibitory concentrations were 3.94 ± 0.36 mm in wt, 4.98 ± 2.26 mm in tg, and 4.80 ± 0.43 mm intg^rol.

Fig. 8.

Altered postsynaptic glutamate sensitivity.A, Traces of miniature CF-EPSCs in response to CF stimulation, recorded from wt, tg, andtg^rol Purkinje cells in the presence of extracellular Sr²⁺. Peak CF-EPSC was cropped to illustrate asynchronous quantal events in the tail. B, The decay time constants (mean ± SEM) of wt, tg, and tg^rol; 190–270 events were averaged. C, The AMPA concentration–response curves of wt (white circles), tg(gray diamonds), andtg^rol (black triangles). Data from each cell were normalized to theI_max value obtained from the Hill equation. Values are presented as mean ± SEM from 8–12 measurements.