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Figure 1. HVA Ca2+ channels in ORNs
(A-C) Patch-clamp configuration in a slice of the olfactory mucosa. A, C, wide-field image, fluorescence image of individual ORN; B, overlay of A and C. (D) Upper, depolarization pulse; middle, Ca2+ current at 0 mV. Ten current segments in 20-ms intervals are marked as 1-10. Lower, current mean of each segment with standard error. Segment 11 shows the current averaged over the period 20 - 200 ms of the depolarization. (E) Variance plotted against the current mean over the period 20 - 200 ms of the depolarization pulse (segment 11) (n=32). Linear regression fit with slope k. (F) Upper, voltage clamp test-pulse stepping from +50 mV to -70 mV. Middle, Ca2+ tail current and, lower, the variance of the current. (G) Variance versus current mean (dots) and quadratic fit (solid). 15 trials, excluding data from the first 1-3 ms of capacitative transient. (H, I) Occurrence, f, of the number of channels, m, and the single channel current, ICa. (n=39).
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Figure 2. Coefficient of variation of fluorescence in Ca2+ channel clusters
(A) Average fluorescence image taken during depolarization subtracted by the images taken just prior to the depolarization. Scale, 2 μm. (B) Correlation map of A, showing pixel-wise temporal cross-correlation with the test pulses in (D). (C) Image mask (two ROIs) obtained from B and used for evaluation. (D) Raw fluorescent signals of ROI 1 (blue), ROI 2 (green), and background (red) taken
outside the neuron. Symbols at the traces, circles indicate data taken before and triangles data taken during the depolarizing voltage test-pulses (top, black trace). (E) Variance of fluorescence plotted against mean fluorescence for the data before (circles) and during (triangle) depolarization (same symbols as in D, connected by gray lines) from ROI 1 (blue), 2 (green) and the background (red). Rhomboids, variance/mean relationship obtained by imaging the patch pipette while varying the illumination intensity. Horizontal dashed line, variance due to dark noise; solid line, variance expected from photon shot noise. fu, fluorescence unit.
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Figure 3. Co-localization of BK channels and Ca2+ channel clusters
(A) Image of patch-clamped cell in tissue slice showing Ca2+ channel clusters (arrows) during depolarization, control images taken prior to depolarization subtracted. (B) Same tissue slice stained with Alexa 488 - labeled IbTX prior to patch clamping. (C) Merge of A and C. Images are maximum projections of z-stacks. Arrows indicate co-localization of BK channels and Ca2+ channel clusters in the patch-clamped cell. Green spots outside this cell presumably indicate BK channels on neighboring neurons. Scale, 5 μm.
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Figure 4. Blocking BK channels slows the falling phase of spontaneous action potentials
ORNs recorded in whole-cell current-clamp mode. (A) Example voltage traces with action potentials before (control, pre) and 3 min after IbTX incubation. Lower, superimposed action potentials with decay times of falling phase fitted as single-exponentials. Fitted decay time constants, when compared to control (lower, right, light blue) are longer after IbTX application. (B) Phase plots, du/dt (u), of action potentials before (blue) and after ChTX (red) application. Arrow, repolarization phases (ie, negative du/dt) are clearly separated. (C) Comparison of decay times before (Pre) and 3 min after IbTX (* t8=2.3, P=0.046) or ChTX (** t10=2.18, P=0.014) incubation. Nondirectional Student's t-test.
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Figure 5. Blocking BK channels reduces the firing rate on odorant responses
ORNs of mucosa slices were recorded in cell-attached configuration of the patch clamp technique. (A) From top to bottom, current trace of spontaneous firing (control), response to an application (bar over trace) of an amino acid mixture (AA), response after incubation in IbTX, response to amino acid mixture together with IbTX (IbTX + AA), response after 5 min of washing with bath solution, and response to the same amino acid mixture. (B) Summary statistics for average firing rates of spontaneous activity (before, white) and odorant induced responses (AA, gray), with or without (control) incubation of IbTX (* t18=2.34, P=0.031, nondirectional Student's t-test). (C-D) Interspike interval (ISI) distributions from 15 ORNs, calculated either from spontaneous activity (C) or odorant responses (D) with or without IbTX treatment. Data for AA induced response was taken within the first 30s after stimulation.
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Figure 6. IbTX affects glomerular odor response activity patterns
(A) Images of ORNs (left), single glomerulus (GL, middle), and mitral cells (MC, right), after maximum time projection. Scale, 10 μm, 2 μm, and 20 μm, respectively. Three exemplary ROIs are indicated in red, green and blue. Stimulus, mixture of amino acids (100 μM, left and right) and L-arginine (100 μM, middle) (B) Odor-induced fluorescent traces corresponding to ROIs shown in A. Each response onset time is indicated by a dashed line. All responses before/after IbTX application are depicted in dark/light color (Control/IbTX). (C) Pooling of latency vectors. Response onset times tij of ROI ensembles (vertical axis) recorded for m repetitions (horizontal axis) under control conditions (amino acids, left box) and n repetitions under IbTX condition (amino acids with IbTX application, right box) are pooled into latency vectors, mean-subtracted, and compared using normalized cross-correlation (CC). Resulting CCs of latency vector pairs from control conditions are pooled into the âControl CCâ value in D, while CCs from mixed control and IbTx conditions are pooled into the âIbTx CCâ value. (D) Pooled results (from 55, 116 and 79 ROIs, respectively) for response amplitudes (left panel) and latency correlation coefficients (CC,
right panel). IbTX amplitudes are reduced for ORNs (t393=7.06, P=7.8*10^(-12)) and glomerular compartments (t975=2.95, P=0.0032), while IbTX CC is reduced for MCs (t234=3.23, P=0.0014). * P<0.01, two-sample nondirectional Student's t-test, data represented as mean ± SEM.
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Figure S1. HVA current in ORNs
(A, B) Whole-cell Ca2+ currents (A) and IV curves (B) in ORNs. Macroscopic Ca2+ current
(bottom) induced by depolarizing pulses from -60 to 40 mV for 50 ms (top). IV curves recorded
with internal solution containing Fluo-5F (n=6). (C) Two additional examples of variance versus
current mean (dots) and quadratic fit (solid) as in Fig. 1G.
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Figure S2. Ca2+ channel clusters in single neurons
Neurons were depolarized for 10 ms to -10 mV. Six optical sections at Dz = 1 μm were recorded
and labeled 1 through 6. (A, B) Images taken during (A) and after (B) depolarization, with
background images taken immediately before depolarization subtracted. (C) Mask from difference
of A and B (A-B > 0), showing the sites of Ca2+ entry. Scale, 5 μm.
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Figure S3. BK channels and their currents in ORNs
(A-C) Staining of BK channels in live slices. (A) Fluorescence images of a slice stained Alexa-488-
labeled IbTX. (B) Same slice, after 15 min of dye washout. (C) control image from a slice stained
with Alexa-488 alone. Scale, 5 μm. (D-E) Whole-cell currents were induced by depolarizing pulses
in 10 mV steps from -60 to +50 mV for 100 ms. (D) Total current recorded in standard bath
solution, after incubation with IbTX (500 nM, 3 min), and after washout (Wash). The BK channel
current, IBK, was obtained by subtracting the current after IbTX incubation from the total current.
(E) Average BK channel current as a function of clamped voltage (IV, n=4).
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Figure S4. Fluorescence of Alexa-labeled Iberiotoxin
(A) Amino acid sequence diagram of Iberiotoxin and five possible residues (red circles) for
labeling. (B) Image of Alexa 488 spin-coated onto a cover slip. Scale, 0.5 μm. Two ROIs (1, 2)
indicated as red circles. (C) Fluorescent signals, F, of ROI 1 and 2 in B. (D) Spots of Alexa-labeled
IbTX (Alexa-IbTX). (E) Time course of the fluorescence of the encircled spot. Dashed lines,
starting from 0, in multiples of the unitary fluorescent signal detected. (F) Frequency distribution, f,
of the background-corrected fluorescence, F, of 270 Alexa dye spots. (G) Frequency distribution, f,
of the background-corrected fluorescence, F, of 360 Alexa-labeled IbTX spots.
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Figure S1. HVA current in ORNs
(A, B) Whole-cell Ca2+ currents (A) and IV curves (B) in ORNs. Macroscopic Ca2+ current
(bottom) induced by depolarizing pulses from -60 to 40 mV for 50 ms (top). IV curves recorded
with internal solution containing Fluo-5F (n=6). (C) Two additional examples of variance versus
current mean (dots) and quadratic fit (solid) as in Fig. 1G.
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