Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses

Abstract

We studied how Ca2+ influx through different subtypes of Ca2+ channels couples to release at a calyx-type terminal in the rat medial nucleus of the trapezoid body by simultaneously measuring the presynaptic Ca2+ influx evoked by a single action potential and the EPSC. Application of subtype-specific toxins showed that Ca2+ channels of the P/Q-, N-, and R-type controlled glutamate release at a single terminal. The Ca2+ influx through the P/Q-type channels triggered release more effectively than Ca2+ influx through N- or R-type channels. We investigated mechanisms that contributed to these differences in effectiveness. Electrophysiological experiments suggested that individual release sites were controlled by all three subtypes of Ca2+ channels. Immunocytochemical staining indicated, however, that a substantial fraction of N- and R-type channels was located distant from release sites. Although these distant channels contributed to the Ca2+ influx into the terminal, they may not contribute to release. Taken together, the results suggest that the Ca2+ influx into the calyx via N- and R-type channels triggers release less effectively than that via P/Q-type because a substantial fraction of the N- and R-type channels in the calyx is localized distant from release sites.

Fig. 1.

Effect of Aga on presynaptic Δ[Ca²⁺] and EPSC. A, Aga (100 nm) reduced concurrently both the Δ[Ca²⁺] and the EPSC evoked by single presynaptic action potentials. The values of Δ[Ca²⁺] and of the EPSCs are given relative to their respective average values before application of the toxin.B, Sample traces of the presynaptic Ca²⁺ influx (left), the presynaptic action potential (top right), and the EPSC (bottom right) before (a) and after (b, as indicated in A) Aga application. The stimulus was given at time 0. Each trace was taken from a single sweep (same for all the following figures).C, D, EPSCs plotted against the Δ[Ca²⁺] from panel A on linear (C) and double logarithmic (D) scales. The slope of the linear regression line in D was 3.5. E, Summary of the dose-dependent effect of Aga on the Δ[Ca²⁺] and the EPSC (each value was obtained from three to nine cells).

Fig. 2.

Effect of Ctx on presynaptic Δ[Ca²⁺] and EPSC. A, Ctx (1 μm) reduced both the Δ[Ca²⁺] and the EPSC evoked by single presynaptic action potentials with a similar time course. Amplitudes of both the Δ[Ca²⁺] and the EPSC were normalized to the values during the control period.B, Sample traces of the presynaptic Ca²⁺ influx (left), the presynaptic action potential (top right), and the EPSC (bottom right) before (a) and after (b, as indicated in A) Ctx application. The stimulus was given at time 0. C, EPSCs plotted against the Δ[Ca²⁺] fromA on double logarithmic scales. The slope of the linear regression line was 1.3. D, Summary of the effect of Ctx on the Δ[Ca²⁺] and the EPSC. Each value was obtained from 3–11 cells. The effects of Ctx at 1 and 3 μm were not significantly different (p > 0.5; t test).

Fig. 3.

Presynaptic Ca²⁺ currents elicited by an action potential waveform command (I_Ca(AP)) before and after Ctx application. A, Application of Ctx (1 μm) reduced the I_Ca(AP). B, Sample traces of the I_Ca(AP) before (Ctrl) and after Ctx application, the latter of which was also scaled (Ctx scaled) for comparison with the Ctrl trace. C, TheI–V relation before (Ctrl) and after Ctx application. The difference between these two I–V curves yielded the Ctx-sensitive I–V curve (Diff), which activated at potentials more positive than −30 mV. In contrast, the threshold for activation of Ctx-insensitive currents (Ctx) was at approximately −40 mV.D, Sample Ca²⁺ currents elicited by a 10 msec step pulse to −30 and 0 mV before (left) and after (right) Ctx application. Note that currents elicited at −30 mV did not change significantly after Ctx application.

Fig. 4.

Effect of Ctx on Δ[Ca²⁺] and EPSC in the presence of Aga. A, An experiment showing that Ctx (1 μm) reduced both the Δ[Ca²⁺] and the EPSC in the presence of Aga (100 nm). In the bottom panel, the EPSC is plotted on a different scale. If the maximum amplitude of the postsynaptic current was less than twice the SD of the baseline noise, the response was classified as a failure. B, In the presence of Aga (100 nm), the block (%) of the Δ[Ca²⁺] and of the EPSC by Ctx (1 μm) were significantly different (p < 0.01; paired t test). The percentages were normalized to the values immediately before Ctx application.

Fig. 5.

Effect of Ctx on EPSC after EGTA was dialyzed into the terminal. A, While the EPSC was recorded, a cell-attached recording was made on the presynaptic terminal with a pipette containing 10 mm EGTA. A suction pulse (arrow) was applied to establish the whole-cell configuration, allowing EGTA to diffuse from the pipette into the terminal. The EPSC decreased rapidly and reached a new baseline value. Ctx (1 μm) was then applied to the bath, which further decreased the EPSC. B, Comparison of the block of the EPSC by Ctx (1 μm) in the control (Ctrl; n = 10) and after EGTA (n = 5) was dialyzed into the presynaptic terminal. In both cases, the block was normalized to the value immediately before Ctx application.

Fig. 6.

Distribution of the α₁ subunit of classes A, B, and E Ca²⁺ channels in synapses in the MNTB. A, B, Tissue section double-labeled using antibodies to the α₁ subunit of class A Ca²⁺ channels (A,arrows) and anti-synaptotagmin (B), illustrating their distribution in the synapse surrounding neurons located in the MNTB. C, Merged image of A and B illustrating regions of colocalization (yellow;arrowheads) between the α₁ subunit of class A Ca²⁺ channels (green) and synaptotagmin (red), demonstrating the presence of these channels at the release face of the synapse.D, E, Tissue section double-labeled using antibodies to the α₁ subunit of class B Ca²⁺ channels (D,arrows) and anti-synaptotagmin (E), illustrating their distribution in the synapse surrounding MNTB neurons. F, Merged image of the staining observed in D and E, illustrating few regions of colocalization (yellow, arrowheads) between class B Ca²⁺ channels and synaptotagmin in the synapse and the presence of class B Ca²⁺ channels in other regions of the synapse. G, H, Tissue section double-labeled with antibodies to the α₁ subunit of class E Ca²⁺ channels (G,arrows) and anti-synaptotagmin antibodies (H), illustrating the presence of synaptotagmin at the release face of the synapse and the distribution of class E channels at other sites in the synapse. I, Merged image of the staining shown in G andH, illustrating very few sites of colocalization (yellow) of these two proteins.Arrows illustrate sites of class E Ca²⁺ channels outside the release face of the synapse. Scale bars: A–F, 10 μm;G–I, 5 μm.

Fig. 7.

Double labeling of class A and class B Ca²⁺ channels in the MNTB.A, B, Double labeling of a tissue section from the MNTB using antibodies to the class B (A, arrows) and class A (B, arrows) α₁ subunits of Ca²⁺ channels. C, Merged image of staining shown in A and B to illustrate regions of only class B staining (green), or regions of only class A staining (red) and regions of colocalization (yellow,arrowheads) of these two channels in the synapses and neurons located in the MNTB. Scale bar:A–C, 10 μm.

Fig. 8.

A schematic view of the distribution of Ca²⁺ channels in the calyx of Held (not drawn to scale). Within a release site, three subtypes of channels, P/Q-, N-, and R-type channels are equally close to the docked vesicle. The local Ca²⁺ domains created by the opening of channels overlap within the release site. Some Ca²⁺ channels, labeled as “distant Ca²⁺ channel”, are located so far from this or other release sites that their Ca²⁺ domains do not contribute to release. A larger fraction of N- and R-type than P/Q-type channels are distant Ca²⁺ channels.