1. Double-Nanodomain Coupling of Calcium Channels, Ryanodine Receptors, and BK Channels Controls the Generation of Burst Firing 
Tomohiko Irie, Laurence O. Trussell 
Neuron 
Volume 96, Issue 4, Pages e4 (November 2017)
DOI: /j.neuron
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2. Figure 1 Blockade of CICR Induces Spontaneous Spike Bursts
(A) Loose cell-attached recordings of a spontaneously firing cell in the presence of synaptic blockers. (Ai, left) Control, all action potentials are simple spikes. (Ai, right) The boxed region (Ai, left) with expanded time base is shown. (Aii, left) Bursting (∗) is observed in the presence of 20 μM ryanodine. (Aii, right) The boxed region (Aii, left) shows one burst of 5 spikelets.
(B) Instantaneous firing frequency over time. The data were obtained from the same cell as in (A). Ryanodine was bath-applied during the time marked by the gray box.
(C) Summarized data of instantaneous frequencies in ryanodine or CPA (10 μM). Simple, data from spontaneous simple spike-firing cells; burst, data from spontaneous burst-firing cells; simple + burst, pooled data from both firing types of cells. Here and elsewhere, error bars indicate SEM, and statistical significance was tested using paired t test unless otherwise stated (significance at p < 0.05). ∗p < 0.05 and ∗∗∗p <
(D) Normalized histograms of instantaneous spike frequencies from cells showing only simple spikes in the absence of ryanodine. Black arrowhead, the main peak of the event in control at 20 Hz (23.2%); gray and white arrowheads, peaks at 40 Hz (10.9%) and 398 Hz (10.7%) in ryanodine, respectively. Here and elsewhere, numbers in parentheses indicate the number of cells.
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3. Figure 2 Ryanodine Induces an Increase in IPSC Bursts
(A) Spontaneous glycinergic IPSCs recorded in the presence of NBQX, MK-801, and 10 μM SR The cell was voltage-clamped at −65 mV and filled with a CsCl-based internal solution so that IPSCs were inward. Spike bursts led to IPSCs with multiple inflections on their rising phase. In control traces, one high-frequency IPSC burst can be seen (∗ in left traces in Ai), whereas 10 IPSC bursts are seen in ryanodine (Aii, ∗). Right panels in (Ai) and (Aii) show expanded sIPSCs from corresponding boxed regions of left traces.
(B) Summary of instantaneous frequencies of spontaneous spike-driven IPSCs. Miniature IPSCs were excluded (see the STAR Methods).
(C) Normalized histograms of instantaneous frequencies of action potential-evoked spontaneous IPSCs. White arrowhead indicates a peak at 251 Hz in ryanodine (4.4% of total events), similar to the spikelet frequency within spike bursts (Figures 1 and S1).
Error bars indicate ± SEM.
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4. Figure 3
The Effects of Ryanodine and IbTX on Action Potential Properties
(A and B) Evoked action potentials recorded in perforated patch mode. (A) Resting potential was slightly hyperpolarized by injecting negative current to suppress spontaneous firing. Traces recorded in control (black) and ryanodine (gray) are superimposed. Injected currents in (Ai) and (Aii) were 500 and 900 pA, respectively. Duration is 5 ms. Asterisk indicates fAHP between first and second spikelets. The boxed region in (Ai) is expanded in the inset. The inset in (Aii) includes a longer segment of the recording to illustrate the slow afterpotential. (Aiii) Summary of the change of burst-firing probability by ryanodine. Three to four successive trials were used to obtain averaged probability in each experiment. (B) The effect of ryanodine was occluded by IbTX. Bath application of IbTX (100 nM) alone broadened first action potentials (Bi and Bii, gray traces) and made the fAHP less negative (Bii, asterisk). Subsequent application of ryanodine in the presence of IbTX did not affect the waveform (Bi’ and Bii’, black traces). Inset in (Bii) is the same sweep but displayed with a longer time base, with spikes truncated. (Biii) Summary of the change of burst firing probabilities by IbTX is shown. Statistical significance was tested between control and IbTX. Error bars indicate ± SEM.
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5. Figure 4 CICR Triggers BK Channel-Mediated Transient Outward Currents
(A) IbTX-sensitive transient outward currents. (Ai) In control, transient currents followed by sustained currents were evoked by depolarizing voltage steps (−30 to −10 mV from −70 mV holding potential, 10-mV increment). All transient currents were inhibited by 100 nM IbTX (traces in IbTX and subtraction). IbTX-sensitive currents were obtained by subtracting traces in IbTX from control traces. (Aii) Summary of the peak current densities (left panel) and the rise time and decay time constant of IbTX-sensitive currents (right panel) is shown. In (A) and (B), capacitive artifacts were blanked for clarity. Here and in the following figures, dashed lines in current traces indicate zero current levels.
(B) RyRs are involved in the transient outward currents. Same voltage protocol is shown as in (A). (Bi) Note that some transient outward currents remain in ryanodine (Bi, ryanodine). (Bii) Summary of peak current densities (left panel) and the rise time and decay time constant of ryanodine-sensitive currents (right panel) is shown.
(Ci) Most of the transient current is suppressed by ω-Agatoxin-IVA (Aga-IVA, a P/Q-type blocker, 200 nM; trace in Aga-IVA and the Aga-IVA-sensitive current). Subsequent application of nonspecific Cav channel blockers (200 μM CdCl2 and 500 μM NiCl2) blocked transient currents almost completely (traces in lower panel in Ci). Data in (C) were recorded in the presence of TTX, synaptic blockers, and 1 mM 4-AP. (Cii) Summary of peak current densities (left panel) and the rise time and decay time constant of Aga-VIA-sensitive currents (right panel) is shown. (Ciii) Summary of effects of subtype-specific Cav blockers on transient currents is shown. Aga-VIA inhibited transient currents more potently than nimodipine or TTA-P2. Effects are expressed as 100 × (selective blocker-sensitive current)/(nonspecific Cav blockers-sensitive current). ∗∗∗p < 0.001, unpaired t test.
Error bars indicate ± SEM.
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6. Figure 5
Action Potential-Induced CICR at Somatic Plasma Membrane, but Not AIS or Dendrites
(A) Two-photon Ca2+ imaging at the somatic plasma membrane. (Ai) Maximum intensity projection of Alexa-594-filled cartwheel cell is shown. The red boxed region is enlarged (Ai, inset). Regions of interest for segmented line scans are indicated by red lines. C, cytosolic side; M, membrane side. (Aii, top and middle panels) Spike trains evoked by current injection (top) elicited an increase of Fluo-5F fluorescence with no change in Alexa-594. (Aii, bottom) Ca2+ transients induced by spike trains (6 simple spikes at 50 Hz) are shown. The transients are expressed as ΔG/R (change in Fluo-5F intensity divided by Alexa-594 intensity). Black, control; blue, in ryanodine. (Aiii) Averaged Ca2+ transients from 10 regions of interest of 5 cells are shown. Single spike or trains of simple spikes (6 spikes at 50 Hz) were evoked by current injection. (Aiv) Summary of the changes in Ca2+ transients is shown. ∗∗∗p < 0.001, unpaired t test.
(B) Ca2+ imaging at AIS. (Bi) Single-image plane of cell body and AIS is shown. (Bii) Ca2+ transients at AIS in (Bi) were induced by spike trains (6 spikes at 50 Hz). (Biii) Summary of the changes of Ca2+ transients is shown.
(C) Ca2+ imaging at dendrites. (Ci) Maximum intensity projection of a cartwheel cell is shown. Spines in the region of interest are numbered. (Cii, top) Ca2+ transients at the dendritic shaft in (Ci) are shown. (Cii, bottom) Summary of the changes of Ca2+ transients at shafts is shown. (Ciii, top) Ca2+ transients at the spine 2 (Ci) are shown. (Ciii, bottom) Summary of Ca2+ transients at spines is shown.
Error bars indicate ± SEM.
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7. Figure 6 SMOCs Are Induced by CICR Triggered by P/Q-type Ca2+ Channels
(A–C) Representative current traces containing SMOCs evoked by 10-mV depolarization from −70 mV in the presence of TTX and synaptic blockers. SMOCs were blocked completely by IbTX (A), ryanodine (B), and Aga-IVA (C, P/Q-type Ca2+ blocker, 200 nM).
(D) Summary of change of SMOC frequency (Di) and amplitude (Dii). Frequencies and amplitudes were normalized by using control data obtained before drug application. ∗∗p < 0.01 and ∗∗∗p < 0.001, one-sample t test.
(E) Caffeine-induced (10 mM) changes of SMOC frequencies. In (E), SMOCs were recorded in the presence of TTX, synaptic blockers, and 1 mM 4-AP. (Eii) Summary of caffeine-induced changes of frequencies is shown. ∗p < 0.05 and ∗∗∗p < 0.001, one-sample t test.
Error bars indicate ± SEM.
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8. Figure 7 Activation of BK Channels Mediated by Nanodomain Coupling
(A) Experimental configuration. Recordings were made in the presence of TTX, synaptic blockers, 100 nM apamin, and 1 mM 4-AP.
(B) No effects of intracellularly applied 10 mM EGTA on SMOCs. Representative traces before rupturing the membrane patch with the second pipette (top) and 10 min after rupturing (bottom) are shown. In (B) and (C), capacitive artifacts were blanked for clarity.
(C) The effects of 10 mM BAPTA on SMOCs. Traces before break-in (top) and 2 min (middle) and 5 min (bottom) after break-in are shown.
(D) Time course of SMOC frequencies recorded from cells dialyzed with EGTA or BAPTA. The dialysis was done at 0 min (break-in). The frequencies were normalized to the average frequency between −5 and 0 min. ∗∗p < 0.01 and ∗∗∗p < 0.001, one-sample t test.
(E) No effect of EGTA on SMOC amplitude. Average amplitude of SMOCs between 8 and 10 min after break-in was normalized to the average between −5 and 0 min.
(F) No effects of EGTA on BK transient currents. The currents were evoked by depolarization from −70 mV to −60 mV, and leak and capacitive currents were subtracted by using the p/4 protocol with opposite polarity. Control, trace before break-in (black); EGTA, trace recorded 10 min after break-in (gray).
(G) Time-dependent effects of BAPTA on the transient currents. Traces before break-in (control) and 1 min (BAPTA 1 min) and 3 min (BAPTA 3 min) after break-in are shown.
(H) Time course of peak amplitudes of the transient currents. Amplitudes were normalized to the average amplitude between −5 and 0 min. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, one-sample t test.
(I) Subsurface ER cisterns expressing RyRs are located just beneath somatic plasma membrane. The distances between RyRs and P/Q-type Ca2+ channels or BK channels are less than 100 nm. Ca2+ influx likely reaches both RyR and BK channels, but Ca2+ is boosted significantly by RyR channel activation.
Error bars indicate ± SEM.
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9. Figure 8 Colocalization of BK Channels and RyRs at Somatic Membranes
Glycine transporter 2 (GlyT2)-Cre;Ai32 mouse brainstem sections containing DCN were triple-immunofluorescence labeled for EYFP (green), BK channels (yellow), and RyRs (magenta) and imaged with a Zeiss Airyscan super-resolution confocal microscope.
(A) Single optical section images containing three cartwheel cells (asterisks). BK and RyR signals are merged in (Aii). The regions marked “B” and “C” are expanded in (B) and (C).
(B and C) Expanded images of cell body (B) and dendrite (C). Arrows in (B) indicate obvious colocalization of BK and RyR signals. Signal intensities between (a) and (b) in (Biii) along the cell membrane and between (a’) and (b’) in (Ciii) were plotted in (Biv) and (Civ), respectively. The cell membrane marked by EYFP, which was expressed as a fusion protein with a membrane protein ChR2. (Bi and Ci) Labeling for BK is shown. (Bii and Cii) Labeling for RyRs is shown. (Biii and Ciii) Merged image of BK and RyR labeling is shown. (Biv and Civ) Plots of signal intensities along somatic membrane (Biv) and dendritic membrane (Civ) are shown. The overlapping of the two signals is highlighted by an asterisk.
(D and E) Colocalization of BK and RyR signals with membrane EYFP. Images in (D) and (E) were expanded from the marked regions in Figure S4B. Regions of interests (transparent white lines) were drawn perpendicular to cell membranes labeled by EYFP. (Di and Ei) Labeling for EYFP is shown. (Dii and Eii) Merged image of BK and RyR labeling is shown. (Diii and Eiii) Labeling for BK is shown. (Div and Eiv) Labeling for RyRs is shown. (Dv and Ev) Plots of signal intensities along the regions of interests are shown.
(F) Average signal intensities versus location. (Fii) The intensity around the peaks was fitted by sixth-order polynomial regression curves.
Error bars indicate ± SEM.
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