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. 2020 Jul 16;14:220. doi: 10.3389/fncel.2020.00220

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Copyright © 2020 Ackels, Jordan, Schaefer and Fukunaga.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Divergent sniff-locking of tufted cells (TCs) and mitral cells (MCs) is observable by imaging and electrophysiology. (A–D) Electrophysiology. (A) Experimental scheme; whole-cell patch-clamp recording was performed in mice anesthetized with ketamine/xylazine. Examples of reconstructed TC (top) and MC (bottom) morphology (from Fukunaga et al., 2012). (B) Top left: average membrane potential triggered by inhalation onset, for example, TC. The dotted line represents the start of inhalation. Action potentials (APs) had been clipped. Bottom left: raster plot of AP occurrences for a 700 ms window from the inhalation onset for the same example TC as the top panel. Right: the same but for an example MC. (C) Peristimulus time histogram of APs for all morphologically identified TCs (left) and MCs (right). The histogram height is normalized by the number of inhalation onsets, and bin size = 20 ms. (D) Top: warped subthreshold Vm for example TC (blue) and example MC (red) in polar coordinates. Mean ± SEM shown. Arrows indicate the resultant vectors for the example TC (blue) and MC (red). Middle and bottom: distribution of resultant vector directions for all morphologically identified TCs (blue) and MCs (red) for subthreshold Vm (middle) and AP histogram (bottom). (E–I) Imaging from M/TCs. (E) Experimental scheme; two-photon imaging of GCaMP6f in TCs and MCs from anesthetized mice. Middle and bottom: example field of view showing tufted cells (middle) and mitral cells (bottom). Scale bar = 100 μm and 50 μm for top and bottom. (F) Left: inhalation-triggered averages from all TCs that couple significantly to sniff for anesthetized mice; the fluorescence fluctuation is normalized in amplitude and shown with grayscale, and ROI index sorted by the time of peak. Right: same but for MCs. (G) Left: distribution of peak times for inhalation-triggered average for TCs. N = 863 ROIs, 15 mice. Right: same, but for MCs. N = 315 ROIs. (H) Average fluorescence transients when “warped” and shown concerning the phase of the sniff cycle in a polar plot. Top: normalized waveform for an example TC (blue) and an example MC (red), with corresponding resultant vector. Bottom: averages of normalized waveforms from all significantly coupled TCs (blue) and MCs (red). (I) Distribution of resultant vector directions plotted as a polar histogram. Tick marks correspond to proportions of ROIs.