Research ArticleNeuroscience

Hippocampal mGluR1-dependent long-term potentiation requires NAADP-mediated acidic store Ca2+ signaling

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Science Signaling  27 Nov 2018:
Vol. 11, Issue 558, eaat9093
DOI: 10.1126/scisignal.aat9093
  • Fig. 1 NAADP causes membrane depolarization in pyramidal neurons of the hippocampus in a manner dependent on acidic store signaling, intracellular Ca2+, and RyR.

    (A) Diagram showing the experimental configuration to record membrane potential of CA1 or CA3 pyramidal neurons in hippocampal slices while NAADP-AM was applied locally. (B) Example voltage traces recorded while applying NAADP-AM, NAADP, or vehicle. Arrowheads indicate the start of delivery, and gray bar indicates the total time of application. (C) Transient membrane depolarization (ΔVM) upon application of NAADP-AM (300 μM; n = 12 cells), NAADP (300 μM; n = 5), or vehicle (n = 6). Data are means ± SEM. (D) Mean ΔVM upon application of NAADP-AM (300 μM; n = 11 cells) alone or (left to right) in combination with a desensitizing concentration of NAADP (1 mM) inside the internal solution of the patch pipette (n = 6) after preincubation with the NAADP antagonist Ned-19 (100 μM, 40 min; n = 6) and preincubation with the vacuolar H+-ATPase inhibitor bafilomycin (4 μM, 40 min; n = 5), and with BAPTA (15 mM) inside the internal solution of the patch pipette (n = 5) after preincubation with ryanodine (30 μM, 40 min; n = 4). Significance was assessed with Kruskal-Wallis and post hoc Dunn’s tests. Data are means ± SEM. n = single cells. ***P < 0.005 by Kruskal-Wallis and post hoc Dunn’s tests.

  • Fig. 2 NAADP is unique among second messengers in its ability to depolarize hippocampal pyramidal neurons.

    (A) Example voltage traces for dialysis of CA1 pyramidal neurons patched with internal solutions containing various concentrations of the Ca2+-mobilizing second messengers NAADP, IP3, and cADPR. Changes to membrane potential were recorded over time as the second messengers dialyzed into the patched cell. (B) Transient membrane depolarization (ΔVM) of the cells described in (A) in response to increasing concentrations of the Ca2+-mobilizing second messengers. Data are means ± SEM. n = single cells, indicated above each column; n > 4 for all concentrations of second messengers. ***P < 0.005 and *P <0.05 by Kruskal-Wallis and post hoc Dunn’s tests.

  • Fig. 3 Activation of mGluR1 in CA1 pyramidal neurons causes a membrane depolarization that depends on NAADP signaling and acidic store Ca2+ signaling.

    (A) Diagram showing the experimental configuration to record membrane potential of CA1 pyramidal neurons in hippocampal slices while mGluRs were pharmacologically isolated (50 μM AP5, 10 μM NBQX, 100 μM picrotoxin, and 2 μM CGP 55845) and electrical stimulation was applied to Schaffer collaterals (four pulses, 20 Hz) (n = 11 cells). (B) Typical voltage recordings from single cells upon electrical stimulation with pharmacological isolation of mGluRs or plus antagonism of group II and III mGluRs (100 nM LY341485; n = 6), pan-mGluRs (100 μM LY341485; n = 6), mGluR5 (10 μM MPEP; n = 5), or mGluR1 (300 nM JNJ16259685; n = 5) (top to bottom). The red lines indicate where membrane potentials were compared before and after stimulation. (C and D) Transient membrane depolarization (ΔVM) of CA1 pyramidal neurons after electrical stimulation alone (control; n = 7 cells) or with the presence of (C) the mGluR antagonism described in (B) or (D) pan-mGluR antagonist [100 μM LY341485; n = 6; cells and dataset are independent from those in (C)] with a desensitizing concentration of NAADP inside the internal solution of the patch pipette (1 mM; n = 6) and NAAD inside the internal solution of the patch pipette (1 mM; n = 6), preincubation with the NAADP antagonist Ned-19 (100 μM, 40 min; n = 6), or acute administration of the lysosomal disrupting agent GPN (200 μM; n = 5). Data are means ± SEM. n = single cells. **P < 0.01 and *P < 0.05 by Kruskal-Wallis and post hoc Dunn’s tests.

  • Fig. 4 In CA1 pyramidal neurons, mGluR1-dependent membrane depolarization and Ca2+ release require acidic store signaling and Ca2+ release from the ER via RyRs but not IP3Rs.

    (A) Diagram showing the experimental configuration. The membrane potential of CA1 pyramidal neurons in hippocampal slices was recorded while mGluR1 was pharmacologically isolated and extracellular glutamate was applied. (B) Typical voltage recordings recorded upon bath application of glutamate (300 μM, 120 s) or the vehicle. (C) Columns show mean ΔVM of CA1 pyramidal neurons before and after extracellular glutamate application under control conditions (n = 8) and in the presence of the lysosomal disrupting agent GPN (200 μM; n = 6), RyR antagonist ryanodine (40 μM, 15 min; n = 6), IP3R antagonist xestospongin C (2 μM, 15 min; n = 6), “fast” Ca2+ chelator BAPTA (20 μM, 15 min; n = 6), or the “slow” Ca2+ chelator EGTA (20 μM, 15 min; n = 6). (D) Time-series images of CA1 neurons filled with Ca2+ indicator OGB-1 (1 mM) were recorded while mGluR1 was pharmacologically isolated (50 μM AP5, 10 μM NBQX, 100 μM picrotoxin, and 2 μM CGP 55845) and electrical stimulation was applied (four pulses, 20 Hz). Images (from top to bottom) of z stack of the dendritic branch being imaged (green), Ca2+ signal at baseline before stimulation, Ca2+ signal 300 ms after stimulation, and subtraction of Ca2+ at 300 ms from baseline (purple). Scale bar, 0.5 μm. (E) ΔF/F over the imaging time course where mGluR1 was pharmacologically isolated (n = 20 cells) in combination with acute application of LY341495 (100 μM, 10 min; n = 5), preincubation with Ned-19 (100 μM, 1 hour; n = 6), or acute application of ryanodine (20 μM, 10 min; n = 5), xestospongin C (2 μM, 15 min; n = 5), or 2-aminoethoxydiphenyl borate (2-APB) (50 μM, 15 min; n = 5). (F) Columns show mean ΔF/F before and after electrical stimulation for each pharmacological manipulation undertaken. Significance was assessed with Kruskal-Wallis and post hoc Dunn’s tests. Error bars denote SEM. n = single cell. Significant differences indicated by asterisks where ***P < 0.005 and *P < 0.05.

  • Fig. 5 In CA1 pyramidal neurons, mGluR1-dependent depolarization occurs through the inactivation of SK channels by possibly PP2A.

    (A and B) Representative voltage recordings (A) and mean ΔVM (B) upon electrical stimulation (four pulses, 20 Hz) of CA1 neurons while mGluR1 was pharmacologically isolated, then subsequent addition of apamin (200 nM, 15 min) and, last, GPN (200 μM, 10 min; n = 6 cells). (C and D) Representative voltage recordings (C) and mean Δ VM (D) upon bath application of glutamate (red arrowhead; 300 μM, 120 s) of CA1 neurons while mGluR1 was pharmacologically isolated, then subsequent addition of apamin (200 nM, 15 min) and, last, GPN (200 μM, 10 min; n = 6 cells). (E and F) Representative voltage recordings (E) and mean ΔVM (F) upon electrical stimulation (four pulses, 20 Hz) of CA1 neurons while mGluR1 was pharmacologically isolated in the absence or presence of okadaic acid (100 nM, 15 min, n = 6 cells). Data are means ± SEM, each from n = 6 single cells. *P < 0.05 (relative to mGluR1 isolation-alone condition) by Freidman’s test with post hoc Dunn’s tests (B and D) or a Wilcoxson pair-matched signed-rank test (F). n.s., no significant difference.

  • Fig. 6 In CA1 pyramidal neurons, mGluR1-dependent synaptic plasticity requires inhibition of SK channels via NAADP signaling.

    (A) A causal STDP protocol was used to induce mGluR1-dependent LTP, in which one causal presynaptic stimulation is paired with two backpropagating action potentials (bAPs) (100 Hz) at a 10-ms interval. The induction protocol is delivered, where t = 0, indicated by the black triangles. Example excitatory postsynaptic potential (EPSP) traces before (black) and after (red) STDP induction are shown at the top right of each graph. Scale bar, 5 mV by 50 ms. This STDP protocol produces LTP lasting at least 30 min (n = 7 cells). (B) LTP in the STDP protocol described in (A) with mGluR1-specific antagonism with JNJ16259685 (300 nM; n = 5 cells). (C) LTP as described in (A) upon prevention of NAADP/acidic store Ca2+ signaling with a desensitizing concentration of NAADP (5 mM; n = 5 cells). (D) Magnitude of LTP upon induction of STDP in the presence of SK channel antagonist apamin (200 nM; n = 7 cells). (E) LTP as described in (A) in the presence of apamin and JNJ16259685 (300 nM; n = 6 cells). (F) Mean change in synaptic strength at 25 to 30 min, expressed as a percentage of the baseline. Data are means ± SEM. n = single cell. *P < 0.05 by Kruskal-Wallis and post hoc Dunn’s tests.

  • Fig. 7 In CA1 pyramidal neurons, TPCs are required for mGluR1-mediated membrane depolarization and mGluR1-dependent LTP.

    (A) Representative average (five traces) voltage recordings from CA1 neurons in hippocampal slice preparations from WT, Tpcn1−/−, and Tpcn2−/− mice (n = 6 mice each). Recordings were obtained upon pharmacological isolation of mGluR1 (50 μM AP5, 10 μM NBQX, 100 nM LY341495, 10 μM MPEP, 100 μM picrotoxin, and 2 μM CGP 55845) and electrical stimulation (four pulses, 20 Hz) of afferent fibers in stratum radiatum. Solid red lines indicate where membrane potentials were compared before and after stimulation. (B) Columns show mean ΔVM of CA1 pyramidal neurons before and after electrical stimulation described in (A). (C) A causal STDP protocol was used to induce mGluR1-dependent LTP after a baseline of EPSPs were recorded for 5 min (indicated by marker at 0 min) in WT (n = 4), Tpcn1−/− (n = 5), and Tpcn2−/− (n = 7) animals. One casual presynaptic stimulation is paired with two bAPs (100 Hz) at a 10-ms interval. (D) Mean change in synaptic strength at 25 to 30 min shown/described in (C), expressed as a percentage of the baseline (red dashed line). Data are means ± SEM. n = single cell. **P < 0.01 and *P < 0.05 by Kruskal-Wallis and post hoc Dunn’s tests.

  • Fig. 8 Proposed model for mGluR1-dependent plasticity.

    (A) Model of SK channel activation, wherein (i) synaptic glutamate activates GluA (AMPA) receptors to produce (ii) membrane depolarization and (iii) Ca2+ entry via VDCCs. This causes (iv) activation of SK channels and local hyperpolarization, resulting in inhibition of GluNs (NMDAs) by reinstating Mg2+ block, thereby reducing Ca2+ entry through the GluNs and reducing the probability of LTP induction. Where synaptic activity is sufficiently strong, the mGluR1 receptors are recruited. (B) The proposed model for SK channel inhibition mediated by mGluR1 signaling; GluA/VDCC regulation of SK channels is also present but not shown. (i) Glutamate activates mGluR1 receptors and causes (ii) NAADP synthesis, which results in (iii) acidic store Ca2+ release, which is amplified through activation of RyRs in the ER. This somehow inactivates SK channels (iv), which in turn prevents local hyperpolarization and (v) allows greater Ca2+ entry through the GluN receptors, which facilitates the induction of LTP.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/558/eaat9093/DC1

    Fig. S1. Xestospongin C inhibits somatic IP3-mediated Ca2+ release in CA1 pyramidal neurons in the hippocampus.

    Fig. S2. Apamin does not affect resting membrane potential of CA1 pyramidal neurons.

    Fig. S3. mGluR1-mediated depolarization unlikely to occur via TRP channel activation.

    Fig. S4. NAADP-mediated membrane depolarization in CA1 pyramidal neurons requires Tpc1 and Tpc2.

  • This PDF file includes:

    • Fig. S1. Xestospongin C inhibits somatic IP3-mediated Ca2+ release in CA1 pyramidal neurons in the hippocampus.
    • Fig. S2. Apamin does not affect resting membrane potential of CA1 pyramidal neurons.
    • Fig. S3. mGluR1-mediated depolarization unlikely to occur via TRP channel activation.
    • Fig. S4. NAADP-mediated membrane depolarization in CA1 pyramidal neurons requires Tpc1 and Tpc2.

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