Short-term potentiation of mEPSCs requires N-, P/Q- and L-type Ca2+ channels and mitochondria in the supraoptic nucleus

Abstract

The glutamatergic synapses of the supraoptic nucleus display a unique activity-dependent plasticity characterized by a barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) persisting for 5-20 min, causing postsynaptic excitation. We investigated how this short-term synaptic potentiation (STP) induced by a brief high-frequency stimulation (HFS) of afferents was initiated and maintained without lingering presynaptic firing, using in vitro patch-clamp recording on rat brain slices. We found that following the immediate rise in mEPSC frequency, STP decayed with two-exponential functions indicative of two discrete phases. STP depends entirely on extracellular Ca(2+) which enters the presynaptic terminals through voltage-gated Ca(2+) channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na(+)-Ca(2+) exchanger. Initiation of STP is largely mediated by any of the N-, P/Q- or L-type channels, and only a simultaneous application of specific blockers for all these channels attenuates STP. Furthermore, the second phase of STP is curtailed by the inhibition of mitochondrial Ca(2+) uptake or mitochondrial Na(+)-Ca(2+) exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca(2+). In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca(2+) channels while the second phase depends on mitochondrial Ca(2+) sequestration and release. Since glutamate influences unique firing patterns that optimize hormone release by supraoptic magnocellular neurons, a prolonged barrage of spontaneous excitatory transmission may aid in the induction of respective firing activities.

Figure 1. Presynaptic high-frequency stimulation (HFS) results in a short-term potentiation of mEPSCs in magnocellular neurons

A, voltage-clamp traces from a representative cell showing STP induced by 50 Hz × 1 s HFS (arrow). Lower panels show expanded traces at various time points as indicated. Both the frequency and amplitude of mEPSCs show significant increase. B, time-effect plot of mEPSC frequency following HFS, fitted with two-exponential decay curves. HFS was applied at time 0. C and D, half-life of 1st and 2nd phase exponential decays. E, a typical cell showing STP with smooth transition from the 1st to 2nd phase. F and G, examples of STP that display a clear second peak (arrowhead).

Figure 2. STP is dependent on presynaptic Ca²⁺ entry

Aa, time-effect plot of mEPSC frequency in response to HFS, applied in the absence (0 mm) or presence of 2 mm Ca²⁺ in the bath. Arrows indicate HFS. Note that this cell shows robust STP in the presence of extracellular Ca²⁺. Ab, evoked EPSCs recorded in the absence or presence of Ca²⁺. Ac, sample traces prior to (control) and immediately after HFS recorded with no extracellular Ca²⁺. B, group data showing that HFS induces no change in mEPSC frequency in the absence of Ca²⁺. C, cumulative plots of mEPSC amplitude from representative cells; before (continuous line: control) and after HFS (dashed line: post-HFS). In 2 mm Ca²⁺ (left panel), mEPSC amplitude distribution shifts to the right, indicating appearance of larger events. Without extracellular Ca²⁺ (0 mm Ca²⁺; right panel), there is no change in the amplitude. D, EGTA application into the postsynaptic cell via recording pipette does not alter STP. E, holding the postsynaptic cell at 0 mV during HFS has no effect on STP.

Figure 3. STP is dependent on high-voltage-gated Ca²⁺ channels

A, representative time-effect plot showing the effect of Cd²⁺ on STP. In the presence of Cd²⁺, STP is largely abolished. Arrows indicate HFS. Right panel, evoked EPSC is completely blocked by Cd²⁺. B, group data showing the significant attenuation of STP by 200 μm Cd²⁺. C, low concentration of Cd²⁺ (50 μm) also significantly reduces STP. There is no significant difference between the effect of 50 and 200 μm Cd²⁺. D, Ni²⁺ has no effect on STP. E, sample traces showing mEPSCs prior to HFS in the presence of Cd²⁺ (top), immediately (middle) and 40 s following HFS (bottom). Note the delayed increase in mEPSCs observed in the bottom trace. Result of ANOVA, where significant, is indicated in the graphs. ***P < 0.001, Bonferroni post test.

Figure 4. Low level of Ca²⁺ influx occurs during HFS

A, synaptic responses during HFS, recorded in control condition (normal aCSF), Ca²⁺ free (0 mm Ca²⁺), in Cd²⁺ or KB-R7943 + Cd²⁺, as indicated. Blocking VGCCs and NCX does not completely eliminate synaptic transmission. Stimulus artifacts are blanked for the purpose of clear presentation. B, frequency of mEPSCs after HFS in the presence of Cd²⁺ or KB-R7943 + Cd²⁺. HFS was applied at time 0. Note the Y-axis is linear, not in logarithm. C, representative traces showing mEPSCs in KB-R7943 + Cd²⁺, immediately or 1 min after HFS, as indicated. mEPSC increase was minimal immediately following the stimulation, but potentiation appeared with a delay.

Figure 5. Specific Ca²⁺ channel blockers fail to block STP

ω-Conotoxin GVIA (ω-CTx) (A), ω-agatoxin TK (ω-Aga) (B) or nicardipine (C) applied alone has no effect on STP. Pairs of specific Ca²⁺ channel blockers also do not inhibit STP. Two blockers are simultaneously applied: D, ω-CTx and ω-Aga; E, ω-Aga and nicardipine; F, ω-CTx and nicardipine. Blockers are administered prior to (until their effect on evoked EPSC, if any, reached a plateau) and during HFS (time 0).

Figure 6. Ca²⁺ channels redundantly mediate the induction of STP

A, HFS induces a robust STP in a representative cell, but its effect is attenuated in the presence of specific blockers for N-, P/Q- and L-type channels. Arrows indicate HFS. Inset, evoked EPSC is completely abolished by the cocktail of 3 blockers. B, time-effect plot of control STP and in the presence of ω-CTx, ω-Aga and nicardipine (3 blockers). Result of ANOVA is indicated in the graph. ***P < 0.001, Bonferroni post test. C and D, half-life of exponential decays (1st and 2nd phase) in control condition and in the presence of 3 Ca²⁺ channel blockers.

Figure 7. Endoplasmic reticulum Ca²⁺ store is not involved in STP

A, a representative cell showing that application of thapsigargin for 30 min has no effect on the magnitude of STP. Arrows indicate HFS. B, group data indicating no change in STP in the presence of thapsigargin.

Figure 8. Mitochondrial Ca²⁺ sequestration underlies the second phase of STP

A, a representative cell that showed a robust STP. Co-application of CCCP and oligomycin caused a faster return of mEPSC frequency to baseline following HFS (arrows). Note that the initial increase is intact. Following prolonged washout of the drugs, the 2nd phase shows a sign of recovery (last arrow). B, half-life of 1st and 2nd phase exponential decays. *P < 0.05. C, group result of the time course of STP in control and in the presence of CCCP and oligomycin. Compared to control, the initial potentiation is similar but the overall duration is curtailed. D, oligomycin alone has no effect on STP. E, CGP37157 also shortens the duration of STP. F, SPBN does not alter STP. Result of ANOVA, where significant, is indicated in the graphs.