Research ArticleNEUROSCIENCE

A BK channel–mediated feedback pathway links single-synapse activity with action potential sharpening in repetitive firing

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Science Advances  04 Jul 2018:
Vol. 4, no. 7, eaat1357
DOI: 10.1126/sciadv.aat1357
  • Fig. 1 Mimicking spike-evoked Ca2+ rises in presynaptic structures with targeted uncaging.

    (A) Confocal projection of target neuron expressing (left) tagRFP and (right) cartoon schematic. Inset shows green sypI-SypHer2 expression indicating target node (“n”) and bouton (“b”). Scale bar, 10 μm. (B) Cartoon reconstruction of neuron used for imaging with Ca2+-sensitive indicator OGB1. Bottom panel shows site of line scan (ls) (yellow) transecting the bouton and node. Scale bar, 10 μm. (C) Ca2+ transients in bouton and node evoked by a single AP. Pseudocolor plot (middle) and traces (bottom) of relative change in fluorescence (ΔF/F) for line scan in (b). (D) Ca2+ uncaging with DMNP-EDTA (top) evokes Ca2+ rise with equivalent amplitude to first AP in spike train (middle). Bottom: Detail of Ca2+ rises evoked by uncaging (blue) and APs (red) from above.

  • Fig. 2 Single spike–evoked Ca2+ rises in first node of Ranvier and proximal bouton drive AP narrowing.

    (A) Schematic for testing impact of Ca2+ uncaging on spike width showing uncaging targets (squares). (B and C) AP waveforms in neurons after uncaging at the node (B, green) or proximal bouton (C, blue) versus “external-to-neuron” control (red). Insets are compressed time scales of evoked spikes with uncaging and current injection indicated. (D) Mean ± SEM histogram for each condition. *P < 0.05 (n = 5, repeated-measures ANOVA, pairwise Tukey’s tests). (E) Limited Ca2+ spread after targeted uncaging in the node. Image shows OGB2 fluorescence used for the readout of Ca2+ dynamics. (F) Left: Averaged traces (100-Hz acquisition) from ROIs (~15-μm length) along the axon during Ca2+ uncaging at the axonal node [white square in (E)]. Right: Somal AP–induced Ca2+ signals at same ROIs shown as a reference. (G) Plot summarizing Ca2+ signal propagation from the uncaging site in the node toward the AIS (n = 4 cells) at different ROIs. Each point shows the amplitude of uncaging-induced Ca2+ signal. A single exponential fit of the data (blue line) indicates that diffusion of uncaged Ca2+ from the node to the AIS was negligible.

  • Fig. 3 Spike modulation is mediated by BK channels.

    (A) Example confocal projection showing local application of the BK channel blocker IbTx (red) to the first node and bouton region and (B) effect on AP width. Control is current-evoked spike alone. Scale bar, 10 μm. (C) Image shows alternative IbTx application site near dendrite and remote from bouton. Scale bar, 10 μm. (D) Summary histogram shows normalized AP half width versus pretreatment control spikes. Axon, *P < 0.01 (paired t test, n = 6); dendrite, *P < 0.05 (paired t test, n = 6); axon versus dendrite, *P < 0.05 (unpaired t test, n = 6). (E and F) Traces and summary histograms reveal no Ca2+ uncaging-mediated spike narrowing in the presence of IbTx. *P < 0.05 (n = 6, repeated-measures ANOVA, pairwise Tukey’s tests).

  • Fig. 4 Synaptic activation–driven spike modulation influences only the repolarization phase and does not depend on synaptic transmission.

    (A) Comparison of AP velocities of depolarization and repolarization measured at half peak for control current-evoked spike versus condition where current-evoked spike is preceded by Ca2+ uncaging. *P < 0.01 (n = 7, paired t test). (B) Summary histogram of AP half widths for control current-evoked spike, current-evoked spike with cocktail of transmission blockers [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoate (AP5), α-methyl-4-carboxyphenylglycine (MCPG), and α-cyclopropyl-4-phosphonophenylglycine (CPPG)], and current-evoked spike with cocktail of transmission blockers preceded by Ca2+ uncaging. *P < 0.05 (n = 7, repeated-measures ANOVA, pairwise Tukey’s tests).

  • Fig. 5 BK channel activity constrains spike broadening in stimulus trains and supports high-frequency spiking.

    (A) Example traces showing that spike broadening varies with spike interval after evoked doublet (inset on right for intervals 300 and 75 ms) and (B) summary plot (n = 15 neurons, repeated-measures ANOVA, P < 0.0001). (C) Scatter plot showing that individual neurons have different intrinsic BK channel function based on an index quantifying the degree of spike broadening in the presence of IbTx versus baseline. (D) Scatter plot showing robust inverse relationship (one-phase exponential decay) when AP half width is plotted against BK channel function for a given interval (275 ms; goodness of fit, R2 = 0.71; Spearman’s correlation, −0.80; P < 0.0003; n = 15). (E) Surface plot showing relationship between BK channel function and spike broadening for all intervals tested (75 to 300 ms). (F) Histogram showing interval between first and second spikes in a depolarizing current-evoked burst in control and IbTx-treated neurons. *P < 0.01 (paired t test; n = 12). (G) Experimental example showing that IbTx applied locally to proximal bouton region (blue traces) extends interspike interval versus pretreatment controls (red and gray traces).

  • Fig. 6 Spike-evoked activation of BK channels limits ongoing synaptic release.

    (A) Experimental approach based on dual patch-clamp recording from monosynaptically connected L5 pyramidal neurons. Scale bar, 25 μm. (B) Typical traces showing EPSP responses to evoked spike pairs with 10-ms interspike interval for pretreatment control and post-IbTx. (C) Sample traces illustrate spike broadening effect of IbTx treatment on AP width for the second evoked spike. Inset: Example showing increased EPSP amplitude persists in the third spike–evoked response. Scale bars, 1 mV (top) and 50 mV (bottom). (D and E) Summary histogram of second EPSP amplitude (D) and coefficient of variation (CV) analysis for same data (E) plotted as the CV2Control/CV2IbTx ratio against the mean amplitude ratio for IbTx versus control. Points lying above a 1:1 ratio signify a presynaptic change. Unfilled circles are individual cell pairs. Filled circle is mean ± SEM. (F) Histogram of EPSP#2-to-EPSP#1 amplitude ratio for pretreatment control and IbTx (*P < 0.05, paired t test, n = 4).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/7/eaat1357/DC1

    Fig. S1. Single spike–evoked Ca2+ rises in the second node of Ranvier drive AP narrowing.

    Fig. S2. Photoactivation of the bouton/node with targeted ChR2 uncaging leads to significant spike narrowing.

    Fig. S3. Determining parameters of IbTx application for local BK channel inhibition.

    Fig. S4. Mimicking spike-evoked Ca2+ rises in distal AIS with targeted Ca2+ uncaging.

    Fig. S5. Ca2+ uncaging at the bouton leads to significant spike narrowing in 4AP perfused neurons.

  • Supplementary Materials

  • This PDF file includes:
    • Fig. S1. Single spike–evoked Ca2+ rises in the second node of Ranvier drive AP narrowing.
    • Fig. S2. Photoactivation of the bouton/node with targeted ChR2 uncaging leads to significant spike narrowing.
    • Fig. S3. Determining parameters of IbTx application for local BK channel inhibition.
    • Fig. S4. Mimicking spike-evoked Ca2+ rises in distal AIS with targeted Ca2+ uncaging.
    • Fig. S5. Ca2+ uncaging at the bouton leads to significant spike narrowing in 4AP perfused neurons.

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