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, 113 (4), 1062-7

Calcium Sensor Regulation of the CaV2.1 Ca2+ Channel Contributes to Short-Term Synaptic Plasticity in Hippocampal Neurons

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Calcium Sensor Regulation of the CaV2.1 Ca2+ Channel Contributes to Short-Term Synaptic Plasticity in Hippocampal Neurons

Evanthia Nanou et al. Proc Natl Acad Sci U S A.

Abstract

Short-term synaptic plasticity is induced by calcium (Ca(2+)) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated CaV2.1 Ca(2+) channels by Ca(2+) sensor proteins induces facilitation of Ca(2+) currents and synaptic facilitation in cultured neurons expressing exogenous CaV2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca(2+) sensor binding site. This mutation does not alter voltage dependence or kinetics of CaV2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼ 50%. In the presence of EGTA-AM to prevent global increases in free Ca(2+), the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca(2+) is dependent upon regulation of CaV2.1 channels by Ca(2+) sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10-20 Hz. Evidently, regulation of CaV2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.

Keywords: calcium channel; calcium sensor proteins; calmodulin; hippocampus; synaptic facilitation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
IM-AA mutation in CaV2.1 channels does not alter the kinetics of P/Q Ca2+ currents in cultured hippocampal neurons. (A) Mean-normalized CaV2.1-mediated ICa from WT (black, n = 23) and IM-AA (red, n = 27) evoked by 1-s depolarizing test pulse from −80 to +10 mV. The traces shown are averages ± SEM. Enlarged time scale of the peak CaV2.1-mediated ICa is shown in a dashed-line box. (B) Average peak CaV2.1-mediated ICa current density from WT (black, n = 23) and IM-AA (red, n = 27). (C) Voltage dependence of activation of ICa from WT (black, n = 23) and IM-AA (red, n = 27) tail currents measured from a holding potential of −80 mV to the indicated potential. n indicates the number of cells recorded in each genotype.
Fig. 2.
Fig. 2.
IM-AA mutation in CaV2.1 channels abolishes paired-pulse facilitation but does not affect spontaneous neurotransmitter release in cultured hippocampal synapses. (A) Example mEPSCs from WT and IM-AA in the presence of 1 μM ω-conotoxin GVIA. (B) Average mEPSC amplitude from WT (n = 36) and IM-AA (n = 26). (C) Average mEPSC frequency from WT (black, n = 36) and IM-AA (red, n = 26). (D) Example paired action currents followed by EPSCs from WT (black) and IM-AA (red) hippocampal neurons evoked by 1-ms depolarizing stimuli at a 50-ms ISI in the presence of 1 μM ω-conotoxin GVIA. (E) Average amplitude of first EPSC from WT (black, n = 21) and IM-AA (red, n = 20). (F) Average PPR plotted against ISI from WT (black, n = 21) and IM-AA (red, n = 20). n indicates the number of cells recorded in each genotype. *P < 0.05; **P < 0.01.
Fig. 3.
Fig. 3.
IM-AA mutation in CaV2.1 channels does not alter spontaneous release in SC-CA1 synapses. (A) Example mEPSCs from WT (black) and IM-AA (red) in the presence of 1 μM ω-conotoxin GVIA and 1 μM tetrodotoxin. (B) Average mEPSC amplitude from WT (black, n = 24) and IM-AA (red, n = 34) (Left), and average mEPSC frequency from WT (black, n = 24) and IM-AA (red, n = 34) (Right). n indicates the number of 4-min recordings in each genotype.
Fig. 4.
Fig. 4.
IM-AA mutation reduces paired-pulse facilitation in SC-CA1 synapses. (A) Example evoked EPSCs from WT (black) and IM-AA (red) pyramidal neurons in response to incremental stimulation of SC fibers. (B) Average peak amplitude of evoked EPSCs plotted as a function of stimulus intensity from WT (black, n = 8) and IM-AA (red, n = 7). (C) Percentage of evoked synaptic response blocked by application of 1 μM ω-conotoxin GVIA from WT (black, n = 10) and IM-AA (red, n = 9). (D) Example paired EPSCs from WT (black) and IM-AA (red) pyramidal neurons at a 50-ms ISI from WT and IM-AA. Stimulus artifacts were blanked for clarity. (E) PPR plotted as a function of ISI from WT (black, n = 12) and IM-AA (red, n = 11). All recordings were made in the presence of 1 μM ω-conotoxin GVIA, 50 μM APV, 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride. (F) Mean amplitudes of EPSCs in response to the first stimulus from WT (black, n = 12) and IM-AA (red, n = 11). n indicates the number of cells recorded in each genotype. *P < 0.05; **P < 0.01.
Fig. 5.
Fig. 5.
IM-AA mutation abolishes paired-pulse facilitation in SC-CA1 synapses buffered with the slow Ca2+ chelator EGTA. (A) Example paired EPSCs from WT (black) and IM-AA (red) pyramidal neurons at a 50-ms ISI in the presence (EGTA-AM) and absence (Control) of the calcium chelator EGTA-AM (100 μM). Stimulus artifacts were erased for clarity. (B) Percentage blocked of evoked synaptic responses by application of 100 μM EGTA-AM from WT (n = 8) and IM-AA (n = 8). (C) PPR from WT (gray, n = 8) and IM-AA (pink, n = 8) in the presence of 100 μM EGTA-AM. All recordings were made in the presence of 1 μM ω-conotoxin GVIA, 50 μM APV, 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride. n indicates the number of cells recorded in each genotype. ***P < 0.001.
Fig. 6.
Fig. 6.
IM-AA mutation shifts the timing of facilitation and depression during high-frequency trains in SC-CA1 synapses. (A and B) Examples of WT (A, black) and IM-AA (B, red) EPSCs recorded during a 20-Hz stimulus train. The shaded gray (WT) and red (IM-AA) areas represent asynchronous release (31). Stimulus artifacts were blanked for clarity. (C–F) Average normalized peak amplitude of evoked EPSCs during trains from WT (black) and IM-AA (red). (C) 5 Hz: WT, n = 13; IM-AA, n = 9. (D) 10 Hz: WT, n = 13; IM-AA, n = 10. (E) 20 Hz: WT, n = 14; IM-AA, n = 16. (F) 50 Hz: WT, n = 9; IM-AA, n = 9. All recordings were made in the presence of 1 μM ω-conotoxin GVIA, 50 μM APV, 50 μM picrotoxin, and 10 μM CGP55845 hydrochloride. n indicates the number of cells recorded in each genotype. *P < 0.05; **P < 0.01; ***P < 0.001.

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