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. 2022 Mar 23;11:e76993. doi: 10.7554/eLife.76993

Figure 3. Low ΔCaAP dendrites contain voltage-gated calcium channels.

(A) Calcium-dependent fluorescence transients evoked by 1 bAP or 5 bAPs at 150 Hz in high (black) and low (blue) ΔCaAP dendrites. (B) Comparison of peak calcium influx evoked by 1 bAP or 5 bAPs in each dendrite. Gray and blue patches indicate selection of high (gray) and low (blue) ΔCaAP spines used in panel A. The line y = 6 x is plotted for reference. N = 109/33/9, R2 = 0.609, F = 166, p = 1.5 × 10–23.(C) Calcium-dependent fluorescence transients evoked by 1 bAP before and after application of 2 mM 4-AP in high (black) and low (blue) ΔCaAP branches. Inset: average AP waveform before and after application of 4-AP. (D) Comparison of peak calcium influx evoked by 1 bAP before and after application of 2 mM 4-AP. Gray and blue patches indicate selection of high (gray) and low (blue) ΔCaAP spines used in panel C. There was no significant correlation: N = 22/7/3, R2 = 0.135, F = 3.11, p = 0.093. (E) Maximum z-projection of Alexa 594 fluorescence from a L2/3 pyramidal cell with differential-interference contrast image overlaid showing glutamate puffing pipette. The full apical dendritic morphology was not imaged. (F) Calcium-dependent fluorescence transients evoked by 1 bAP, 5 bAPs at 150 Hz, and a 1 mM glutamate puff in the example branch from panel E. (G) Average calcium-dependent fluorescence transients evoked by 1 bAP, 5 bAPs at 150 Hz, and a 1 mM glutamate puff across all branches recorded. (H) Comparison of peak calcium influx evoked by bAP, 5 bAPs at 150 Hz, and a 1 mM glutamate puff in all branches. The response to 5 bAPs was not recorded in all dendrites (N = 20/6/3).

Figure 3.

Figure 3—figure supplement 1. Glutamate puff-evoked calcium signals are mediated by voltage-gated calcium channels.

Figure 3—figure supplement 1.

(A) Dendritic calcium influx evoked by picospritzer puff of either 1 mM glutamate (N = 22) or ACSF (N = 4). (B) Comparison of peak calcium influx evoked by 1 mM glutamate and ACSF. No calcium influx was observed in response to ACSF, indicating that the calcium signal was not due to mechanical displacement evoked by the picospritzer. (C) Example traces of calcium influx evoked by 1 mM glutamate puff for one dendrite across all trials, shifted along the x-axis to visualize each trial clearly. (D) Dendritic calcium influx evoked by 1 mM glutamate for each trial, normalized to first trial amplitude. If glutamate was out-competing CPP (a competitive antagonist) at NMDAR’s binding sites, then the presence of MK-801, a use-dependent blocker, should reduce NMDAR conductance in each trial. No systematic reduction in the calcium signal was observed, indicating that NMDARs were fully blocked. Colored points indicate example used in panel C.