Functional coupling between mGluR1 and Cav3.1 T-type calcium channels contributes to parallel fiber-induced fast calcium signaling within Purkinje cell dendritic spines

Abstract

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Ca(v)3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Ca(v)3.1 gene knock-out animals show that Ca(v)3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Ca(v)3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.

Figure 1.

mGluR1 differentially modulates T-type Ca²⁺ channels. A–C, Left, Representative voltage-clamped current traces during depolarizing pulses from −110 to −30 mV (or −40 mV for C) in HEK 293 cells coexpressing recombinant mGluR1a and the indicated T-type isoform. Activation of mGluR1a with 100 μm glutamate (gray line) caused a potentiation of Ca_v3.1 (A) and Ca_v3.2 (B) currents and an inhibition of Ca_v3.3 (C) currents. Right, Normalized peak current levels during perfusion of control recording solution (2 mm Ca²⁺), followed by 100 μm glutamate for Ca_v3.1 (A), Ca_v3.2 (B), and Ca_v3.3 (C) currents. The potentiation shown for Ca_v3.1 and Ca_v3.2 channels was observed in ∼30% of cells tested, whereas the mGluR1a-induced inhibition of Ca_v3.3 currents was observed in ∼70% of cells. mGluR1a activation caused an average potentiation (at equilibrium for each cell; see Materials and Methods) of Ca_v3.1 currents by 29.6 ± 5.8% (n = 8), potentiation of Ca_v3.2 currents by 41.8 ± 8.8% (n = 6), and inhibition of Ca_v3.3 currents by 28.7 ± 1.9% (n = 33). For Ca_v3.1 and Ca_v3.2 currents, glutamate had no effect on all excluded cells, whereas for Ca_v3.3, 100 μm glutamate caused either no effect or inhibition followed by a stimulation/recovery in the cells not shown (data not shown; for additional details, see Materials and Methods).

Figure 2.

T-type Ca²⁺ currents are potentiated by mGluR1 activation in young cerebellar Purkinje neurons. A, Representative voltage-clamped current traces for rat PC T-type currents during depolarizing pulses from −90 to −43 mV before (1), during (2), and after (3) activation of endogenous mGluR1s with 20 μm DHPG. B, Normalized peak current time course for the same cell in A demonstrating the reversibility of DHPG-induced potentiation. C, DHPG stimulates T-type currents specifically through mGluR1s. Blocking mGluR1 with perfusion of 100 μm LY367385 (n = 8) for 20 min before and during DHPG application abolished potentiation. D, Average normalized peak T-type current time course during control perfusion followed by either bath perfusion of 20 μm DHPG (black plot; T-type currents increased by 51 ± 7%, n = 19 after 100–120 s of DHPG application) or a picospritzer puff (50 ms, 10 psi) of DHPG (100 μm) that shows a rapid potentiation of T-type current amplitude by 45 ± 12% (n = 6; blue plot). E, Representative current traces during depolarizations from −80 to −40 mV in WT (black) and Ca_v3.1^−/− (red) mice. F, Left, Quantification of peak PC T-type currents in Ca_v3.1^−/− mice (n = 4) compared with WT mice (n = 4) during depolarizing steps to −40 mV. Right, DHPG (20 μm) causes robust and equal T-type potentiation in WT mice (n = 3) and Ca_v2.3^−/− mice (n = 4). G, Normalized I–V curve showing that DHPG (red plot) increases maximal current, resulting in a significant potentiation of T-type currents at potentials between −45 and −20 mV (n = 7). *p < 0.05 compared with control current values. Inset, Representative voltage-clamped current traces from rat PCs during depolarizations to potentials ranging from −60 to −30 mV before (left) and after (right) mGluR1 was activated with 30 μm DHPG. H, Normalized conductance curves for activation and steady-state inactivation fitted with Boltzmann equations (see Materials and Methods). DHPG application caused a small but significant (p < 0.02) shift of ∼2 mV in the V _50act and had no significant (p > 0.05) effect on the steady-state inactivation (V _50inact) of T-type currents within PCs (supplemental Table 1, available at www.jneurosci.org as supplemental material).

Figure 3.

T-type Ca²⁺ currents are potentiated by mGluR1 through a signaling pathway that involves G-proteins, intracellular Ca²⁺, and tyrosine phosphatases. A, Top left, Control normalized time course showing the effects of DHPG application on voltage-clamped rat PC T-type currents in the absence of any other signaling antagonists (during depolarizations from −90 mV to between −50 and −40 mV). Top right, Potentiation of T-type currents via mGluR1 requires G-protein activation. Substitution of 2 mm GDP-β-S (n = 5) for GTP in the intracellular pipette solution for 10 min in the whole-cell conformation eliminated DHPG-induced potentiation. Bottom left, Potentiation of T-type currents by DHPG is enhanced by blocking tyrosine kinase activity. Blocking Src-family tyrosine kinases with inclusion of 10 μm PP1 in the pipette (n = 5) augmented the DHPG-induced increase. Bottom right, Potentiation of T-type currents by DHPG requires tyrosine phosphatase activity. Blocking tyrosine phosphatases via perfusion of 100 μm bpV(phen) (n = 6) for 10 min before and during DHPG application attenuated the potentiation effect. B, Histogram showing potentiation values compared with the control (DHPG) potentiation value for the above results as well as for other antagonists. Blocking group I mGluRs with 500 μm MCPG (n = 6), buffering intracellular Ca²⁺ through inclusion of 20 mm BAPTA (n = 6) in the pipette, and blocking tyrosine phosphatases with 1 mm Na₃VO₄ (n = 5) all significantly (gray bars; p < 0.02) reduced the DHPG-induced increase. Blocking Src-family tyrosine kinases with inclusion of 10 μm PP2 in the pipette (n = 6) significantly (p < 0.02) augmented the DHPG-induced increase. Blocking phospholipase C with 1 μm U73122 (n = 6) or 10 μm edelfosine (n = 7), serine/threonine kinases (such as protein kinase C) with 1–2.5 μm staurosporine (n = 8), IP₃Rs with 1 μm xestospongin C (n = 6), and sEPSC currents with 250 μm IEM 1460 (n = 6) or 100 μm NA-spermine (n = 5) all caused no significant (p > 0.05) change in the level of DHPG-mediated increase in T-type currents. All potentiation values were calculated 100–120 s after initiation of DHPG application, except for NA-spermine and edelfosine, in which the effect was calculated 60–80 s into potentiation (equivalent time from start of potentiation to other groups), because the effect was delayed. *p < 0.02.

Figure 4.

Immunofluorescence showing predominant distribution of Ca_v3.1 in the dendritic spines of cerebellar Purkinje cells. A, B, Specificity of Ca_v3.1 immunohistochemistry in the mouse brain. Note intense immunofluorescence labeling in the cerebellum (Cb) and thalamus (Th) of WT (A) but not Ca_v3.1^−/− mice (B). CPu, Caudate–putamen; Cx, cortex; Hi, hippocampus; Mb, midbrain; MO, medulla oblongata; Po, pons. Scale bar, 1 mm. C, Single immunofluorescence for Ca_v3.1 in the cerebellar cortex. GL, Granular layer; ML, molecular layer. Scale bar, 20 μm. D–H, Double immunofluorescence for Ca_v3.1 (red) and other molecules (green) in the cerebellar molecular layer. Asterisks in H₁ indicate shaft dendrites of PCs. Note punctate Ca_v3.1 labeling in the neuropil, which overlaps extensively with calbindin (calb; D) and mGluR1 (H) in putative dendritic spines but not with VGluT1 (E), VGluT2 (F), or VGAT (G) in excitatory and inhibitory presynaptic terminals. Scale bars, 10 μm.

Figure 5.

Immunoelectron microscopy showing predominant localization of Ca_v3.1 on the extrasynaptic surface of dendritic spines in cerebellar Purkinje cells. A–C, Immunoperoxidase. Note intense labeling of Ca_v3.1 (arrowheads) in dendritic spines and shafts of PCs. D–F, Preembedding silver-enhanced immunogold showing predominant surface labeling in dendritic spines, in contrast to intracellular labeling in dendritic shafts. G–I, Postembedding immunogold. Arrowheads and arrows indicate gold particles associated with the cell membrane or distributed intracellularly, respectively. PCD, Purkinje cell dendrite. Scale bars: A, C–E, 500 nm; B, F, I, 1 μm; G, 200 nm.

Figure 6.

DHPG mediates an increase in T-type Ca²⁺ transients in Purkinje cells. A, Two-photon image of a patch-clamped rat cerebellar PC. The dendritic regions outlined by circles are sites of imaging. Scale bar, 10 μm. B, DHPG causes an increase in voltage-clamped low-threshold Ca²⁺ transients in spines and proximal dendrites when IP₃Rs are blocked. The Ca²⁺ transients (ΔF/R; see Materials and Methods) at individual POIs during depolarizing steps from −80 to −45 mV at the soma are shown before (black) and after (red) application of 20 μm DHPG. Numbers refer to labels from A. Traces were smoothed. The dotted vertical line indicates onset of the depolarizing pulse. Recordings were performed in the presence of 4 mg/ml heparin to block IP₃Rs. C, The increase in low-threshold Ca²⁺ transients by mGluR1 activation coincides with the potentiation of T-type somatic currents. Three successive depolarizing pulses at the onset of the DHPG effect, starting 2 min after the beginning of the application, with 1 min between pulses. Average Ca²⁺ transients in all the POIs in the PC are shown in red. The red dotted line represents the standardized baseline Ca²⁺ level before the pulse; the red dashed line identifies the peak of fluorescence before the DHPG effect. In black, current recorded at the soma. The black dashed line identifies the peak current before the DHPG effect. D, Top, The average Ca²⁺ transient in all imaged spines at all time points (10 s between sweeps) during the control period is shown in black (10 min total duration), and the average Ca²⁺ transient after onset of the DHPG effect is shown in red (5 min total duration). Middle, Mean current in control period (black) and during DHPG application (red). Bottom, Holding potentials. All data shown above in this figure are from the same cell. E, Left, DHPG causes a potentiation of voltage-clamped T-type Ca²⁺ transients in the spines and proximal dendrites of PCs when IP₃Rs are blocked with heparin inclusion (4 mg/ml) in the patch pipette. Proximal dendrites, n = 33, 5 cells; distal dendrites, n = 5, 5 cells; spines, n = 84, 5 cells; soma, n = 9, 5 cells. Right, DHPG causes a potentiation of T-type Ca²⁺ transients in the spines, distal dendrites, and proximal dendrites of PCs when IP₃Rs are not blocked. Proximal dendrites, n = 20, 5 cells; distal dendrites, n = 40, 5 cells; spines, n = 55, 5 cells; soma, n = 6, 5 cells. **p < 0.01, ****p < 0.001 (Wilcoxon's rank test). The values for %ΔF/R Normalized are derived from the variation of the peak Ca²⁺ transient (ΔF/R) normalized to the density of current at the soma (under control conditions; see Materials and Methods) in different compartments of the cell during the control period (black bars) and DHPG application (white bars).

Figure 7.

Parallel fiber stimulation activates mGluR1-potentiated T-type Ca²⁺ transients. A, Left, Schematic drawing of experimental arrangement in mature mice (P15–P25). Inset, Representative EPSP recorded at the soma after PF stimulation. Right, Fluorescence transients (ΔF/R) in spiny branchlets evoked by PF stimulation positioned on a two-photon section. In red, average fluorescence of three to seven POIs in the region of interest outlined with white dashed lines. Note the local and graded Ca²⁺ transients. The two black dashed lines indicate the duration of the PF stimulations (13 stimulations/100 Hz). B, Fluorescence transients (ΔF/R) in spiny branchlets evoked by 1 pulse (left) and three pulses (right) of PF stimulation at V _h = −70 mV (black) or with no injected current (blue). Right, There was no significant difference in the mean ΔF/R between control conditions (black) and depolarized conditions (blue, no current injected) (control, ΔF/R = 3.7 ± 0.4%; depolarized, ΔF/R = 4.1 ± 0.4%, n = 5). C, Mean ΔF/R in control conditions (black) or during JNJ16259685 application for one, three, four, or six pulses in the PF train (n = 3). D, Effect of JNJ16259685 application on the change of Ca²⁺ fluorescence in WT (n = 6) and Ca_v3.1^−/− mice (n = 6). *p < 0.05, Wilcoxon's matched-pairs signed-ranks test. **p < 0.01, Wilcoxon's test. E, Left, The PF-induced Ca²⁺ transient is attenuated after perfusion of JNJ16259685 (1.5 μm) (red dashed line). Each trace is an average of five consecutive trials. Fluorescence traces are an averaging of all responsive spiny branchlets. Right, Time course of the size of the first EPSP in the train (black) and the change of Ca²⁺ fluorescence (red) after perfusion of JNJ16259685 (1.5 μm) in the same set of cells tested with 11 pulses at 100 Hz (n = 6).

Figure 8.

Spine-specific mGluR1 potentiation of T-type Ca²⁺ channels. A, Left, Mean ΔF/R for all POIs in a cell in the control condition (black) or during JNJ application (red) correlated to mean EPSPs recorded at the soma. Middle, ΔF/R for individual spines in the control condition (black) or in JNJ (red) for the same cell. Orange labels refer to the POIs highlighted in the inset. Scale bar, 5 μm. Right, Plot of the ΔF/R for each POI in JNJ versus the control condition. Each ΔF/R is a mean of five consecutive trials at 0.05 Hz. The black dashed line represents the bisector line. B, Plot of the ΔF/R for each POI in JNJ versus the control condition for of all the cells tested in WT (left; n = 224 POIs, 5 cells) and Ca_v3.1 KO (right; n = 274 POIs, 5 cells) mice. Each ΔF/R is a mean of five consecutive trials at 0.05 Hz. A green point illustrates a significant (p < 0.05) difference between control and JNJ application ΔF/R values. Black dashed line, Bisector line. Gray dashed line and rectangle highlight POIs for which control ΔF/R signal is above 9%. C, Example of ΔF/R for labeled spines in the control condition (black) or in JNJ (red), demonstrating the activation and potentiation of a Ca²⁺ transient that is specific to an individual spine (label 2).