Narrowly Confined and Glomerulus-Specific Onset Latencies of Odor-Evoked Calcium Transients in the Juxtaglomerular Cells of the Mouse Main Olfactory Bulb

Activity (ΔF/F₀) maps color-coded by the phase of response in wide-field and two-photon imaging. A1, Surface image for wide-field (single-photon) imaging. L: lateral, A: anterior, M: medial, P: posterior. Scale bar, 500 µm. A2–A4, Maps of odor-evoked response to 4CHO, 5CHO, and 6CHO, respectively. Each map is a synthesis of red, green, and blue maps that represent the early (0.5–2.5 s), intermediate (2.5–4.5 s), and late (4.5–6.5 s) periods after stimulus onset, respectively. The duration of stimulus was 2 s. This visualization allows us to map the heterogeneity of response time courses. For example, glomeruli that declined quickly after the stimulation appear in red (e.g., lateral glomeruli in A2–A4), whereas those with persistent response appear in yellow or white as they maintained the high-level calcium over the first two or all three periods (e.g., subsets of medial glomeruli in A2–A4). Glomeruli that appear in bluish colors are slow-rising ones. B1, A two-photon image of the selected area, indicated by the black box in A1 and by white corners in A2–A4. Scale bar, 50 µm. B2–B4, Maps of odor-evoked response, with the colors representing the same periods as in A2–A4.

Odor-evoked calcium transients and grouping of JG cells by response profile. A, An example dataset from a single trial of random-access scanning of 15 ROIs (cells). Left traces are responses to a 2 s odor stimulus, and right traces are responses to a single-respiration-cycle stimulus. The bottom black traces show the respiration signal. Gray horizontal bars above the respiration signals indicate the timing of valve opening for odor presentation. Note that actual odor presentation lags ∼0.1 s behind the valve opening. The ordering of the ROIs is intentional, based on the result of grouping shown in C. B, Two-photon image of the recording site (B1) and the ROI indexes (B2). To obtain the two-photon image of GCaMP6f (green), all XYT imaging data for all odors were averaged and then all poststimulus frames were averaged, solely for making the GCaMP fluorescence more visible. The tdTomato image (magenta) was obtained with a similar procedure, but an average of prestimulus frames was used. Dots and contours represent the ROIs and glomeruli, respectively. Non-white colors of dots and contours in B2 indicate the groups presented in C. Scale bar, 50 μm. C, Odor-evoked responses of the same ROIs to five odors (single-cycle stimulus). The left block shows the response time courses. The middle block shows the areas under the time courses as bar charts. The right block shows the difference in areas between every possible pair of odors. Colored vertical lines at the right indicate the groups of cells putatively associated with the same glomerulus (see text for details). Scale bars: horizontal, 3 s; vertical, 100% ΔF/F₀.

Characterization of calcium signals in the prestimulus period. A, An example data of the prestimulus period and the initial part of odor-evoked response in a trial. Green traces are the signal from the green channel (GCaMP6f), and magenta traces are the signal from the red channel (tdTomato). Apparent odor-evoked changes in some of the magenta traces (e.g., ROI 10) suggest a minor contribution of the GCaMP signal to the red channel. The relative contribution from the GCaMP signal may vary depending on the relative expression levels of GCaMP and tdTomato at an individual ROI. Note clear respiration-coupled modulations of the odor-evoked response in the subset of traces. B, Two-photon image of the recording site. The image was obtained with the same procedure for the image in Figure 3B1. ROIs were indicated by white dots and the index of each dot is shown on the right of image. Scale bar, 50 μm. C, Stacked histogram of the ratio between peak response amplitude and range (the distance between the 1st and 99th percentiles) in the prestimulus period. Note the logarithmic scale on the x-axis. The response is much larger than the fluctuations in the prestimulus period in the vast majority of cases. D, Histograms showing the distribution of pairwise correlation coefficients for the time courses of activity during the prestimulus period. Top and bottom histograms show data from cell pairs putatively associated with the same glomerulus and pairs from different glomeruli, respectively.

Onset latencies of odor-evoked calcium transients are heterogeneous across JG cells. A, Graphic representation of the definition of onset latency. Aa, Baseline, defined as the mean of the prestimulus period signal. Ab, Threshold, defined as 2.5 times the SD of the prestimulus period signal. Ac, Time point at which the signal exceeds the threshold. Ad, Time window of the first 100 ms above the threshold. Ae, Regression line of the signal in the 100 ms time window. Af, The point where the regression line crosses the baseline. This time point is considered the onset of the calcium transient. Ag, Onset of the first inhalation with the odor stimulus. Ah, Onset latency, defined as the distance between the onset of inhalation and the onset of the calcium transient. Ai, One cycle of respiration. B, Distribution of onset latencies. Each row corresponds to a single cell–odor pair and the distribution of onset latencies across repetitions is presented as a box-and-whisker plot (inset). Dots in the box-and-whisker plots represent the median, and expression of the GAD2 marker tdTomato is indicated by the dot color (red, tdTomato⁺; black, tdTomato⁻). Cell–odor pairs are arranged according to median onset latency and every other pair is presented for clarity (83 cell–odor pairs from 8 recording sites, out of all 165 pairs, are presented). Each box-and-whisker plot represents data from 5–17 trials. C, Overlapping histograms showing the distribution of medians shown in B for tdTomato⁺ (red) and tdTomato⁻ (gray) cell populations. Triangles in the top indicates median for each population (tdTomato⁺: 84 ms, 105 cell–odor pairs; tdTomato⁻: 108 ms, 60 cell–odor pairs; p = 0.012, Mann–Whitney U test).

JG-cell onset latency strongly depends on the putative glomerular association. A subset of the box-and-whisker plot from Figure 5B is presented, rearranged according to glomerulus–odor pairs. To clarify, cell–odor pairs in this figure include those not actually presented in Figure 5B, where only half of the cell–odor pairs are shown for clarity. As in Figure 5B, each row represents a single-odor pair, in which distribution of onset latency across repetitions was presented as box-and-whisker plots. Different glomeruli are presented in different colors. In some of the glomeruli, data from more than one odorant were available. Dots in the box-and-whisker plots represent the median, and expression of the GAD2 marker tdTomato is indicated by the dot color (red, tdTomato⁺; black, tdTomato⁻). No statistical difference was observed between the onset latencies of tdTomato⁺ and tdTomato⁻ cells, considering the glomerulus-odor pairs (see text). Mouse and glomerulus identity are at the left. Odorant is indicated at the right.

Detailed analysis of onset latency across cells putatively associated with the same glomerulus. The onset latencies of cell–odor pairs from a glomerulus in Mouse 4 (Fig. 6) are presented with an alternative visualization. A1, A2, Reconstruction of plots in Figure 6, except that each trial was explicitly plotted as circles. Each row represents an individual cell, as in Figure 6. The median and IQR for each cell are presented as the accompanying vertical and horizontal lines, respectively. Colors represent individual cells and are preserved across all graphs in A and B. The cells are sorted by their median onset latency. Left and right graphs show the responses to two different odors. Bottom, The black box-and-whisker plots show the distribution of median onset latency across cells. A3, A4, The same data as in A1 and A2, but rearranged so that each row represents an individual trial. Note that the variances within individual rows are markedly smaller than those in A1 and A2, suggesting that the primary cause of intra-cell deviation in A1 and A2 are trial-by-trial variability and not random errors. Trials are sorted according to their median onset latency, not by the order of acquisition. Bottom, The gray box-and-whisker plots show the distribution of median onset latency across trials. B1–A2, Onset latencies are plotted as in A1 and A2, except for that the contribution of trial-by-trial variance was removed. Specifically, the median onset latency across the in-group cells in the corresponding trial is subtracted from each data point. C, IQRs of median onset latency across cells (black circles, corresponding to the black box-and-whisker plots in A1, A2) and across trials (gray circles, corresponding to the gray box-and-whisker plots in A3, A4) in each glomerulus-odor pairs are compared for all glomerulus-odor pairs shown in Figure 6. IQRs across cells are smaller in nearly all cases. Red triangle indicates the IQR of onset latency across all glomerulus–odor pairs involved in this analysis (50 ms). (The onset latency of a glomerulus-odor pair is defined as the median of medians across involved cells.) This value is much larger than IQRs across cells in each glomerulus–odor pairs (black circles), but is close to the IQRs across all cell–odor pairs pooled (blue triangle, 55 ms, 88 cell–odor pairs), suggesting that the spread of IQRs across all cell–odor pairs are primarily accounted for by the spread across glomerulus-odor pairs.