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, 13 (11), 1404-12

Non-redundant Odor Coding by Sister Mitral Cells Revealed by Light Addressable Glomeruli in the Mouse


Non-redundant Odor Coding by Sister Mitral Cells Revealed by Light Addressable Glomeruli in the Mouse

Ashesh K Dhawale et al. Nat Neurosci.


Sensory inputs frequently converge on the brain in a spatially organized manner, often with overlapping inputs to multiple target neurons. Whether the responses of target neurons with common inputs become decorrelated depends on the contribution of local circuit interactions. We addressed this issue in the olfactory system using newly generated transgenic mice that express channelrhodopsin-2 in all of the olfactory sensory neurons. By selectively stimulating individual glomeruli with light, we identified mitral/tufted cells that receive common input (sister cells). Sister cells had highly correlated responses to odors, as measured by average spike rates, but their spike timing in relation to respiration was differentially altered. In contrast, non-sister cells correlated poorly on both of these measures. We suggest that sister mitral/tufted cells carry two different channels of information: average activity representing shared glomerular input and phase-specific information that refines odor representations and is substantially independent for sister cells.


Figure 1
Figure 1. OMP-ChR2 (ORC) transgenic expression pattern; Laser scanning photo-stimulation (LSPS) identifies parent glomeruli for mitral cells in ORC mice olfactory bulb slices
a. Confocal micrograph of olfactory bulb sagittal section from ORC mice showing EYFP fluorescence; GL – glomerular layer, MCL – mitral cell body layer, AOB – accessory olfactory bulb, A-anterior, P-posterior, D-dorsal, V-ventral; scale bar is 100 μm. (Inset) higher magnification view: arrow indicates olfactory sensory neuron axons; scale bar is 50 μm. b. Bath application of glutamate receptor antagonists CNQX and APV; the three traces for each of the conditions correspond to three adjacent photo-stimulation foci 50 μm apart; red trace – before drug application; black trace – during drug application; arrow indicates the time of photo-stimulation. c. Phase (Top left) and red fluorescence image (Bottom left) of one mitral cell filled with Alexa 546; box indicates the field of photo-stimulation; (Top right) Primary dendrite and tuft projecting to a glomerulus. (Bottom right) Matching two-dimensional light activation map (16 X16 2DLAM); inter-foci distance = 15 μm; photostimulation duration = 1 ms. Note the fiduciary circle on the two panels. Color represents peak amplitude of the light induced currents; ONL – olfactory nerve layer; scale bar is 100 μm. d. Currents recorded in the mitral cell by LSPS in c are shown at locations corresponding to each point in the 16 × 16 grid; traces were averaged across 4 repeats; 1.6 mW laser power was used. Scale bars are 50 pA and 100 ms respectively.
Figure 2
Figure 2. DMD patterned illumination in ORC mice maps the parent glomeruli of M/T cells in vivo
a. (Left) Schematics of DLP projector based photostimulation setup (Top panel right) Dorsal surface of the bulb with a tetrode positioned in the mitral cell layer. One square light spot can be seen projected onto the bulb surface; scale bar, 500 microns. (Inset) Cartoon schematic of glomeruli showing a sub-glomerular size light spot and dual-tetrodes positioned in the mitral cell layer. i. DLP projector ii. focusing lens, iii. blue excitation filter, iv. dichroic mirror, v. emission filter, vi. CCD camera, vii. dual-tetrode, viii. olfactory bulb. b. (Top) Raw voltage traces corresponding to the four channels of a tetrode during photostimulation. (Center) Raster plot of spikes from an isolated single unit; (Bottom) Peri-stimulus time histogram (PSTH) with 25 ms time bins. c. (Left) Example spike waveform of a single unit across the eight channels of a dual-tetrode. Dark traces - individual spikes; white line - average waveform. (Center) 2DLAM showing the change in firing rate of the M/T unit during photostimulation over 10 repeats; scale bar, 100 μm; light spot size, 50 μm. (Right) 2DLAM re-sampled by interpolation. d. 2DLAMs obtained at different stimulation intensities (spot size, 50 μm). All maps were normalized to the highest bin in the 20.8 mW/mm2 2DLAM. e. Distribution of 2DLAM hotspot widths (FWHM) for all units (N = 40) obtained in a minimal photo-stimulation regime (black bars). Distribution of synaptopHluorin labeled glomerular widths (FWHM) from OMP-spH mice (Red line, N=572).
Figure 3
Figure 3. Functional hotspots correspond to anatomically identified glomeruli
a. (Left) Functional hotspot from representative two dimensional light activated map (2DLAM); (Center) Z-stack image projection of anatomical glomeruli from the same field of view as the 2DLAM obtained via multiphoton microscopy. (Right) Overlay of the 2DLAM and the z-projection. Yellow dotted lines indicate the boundaries of the 2DLAM. b. Hotspot FWHMs plotted against corresponding anatomical glomerular widths. c. Normalized spatial jitter between the centroids of functional hotspots and the corresponding anatomical glomeruli plotted against anatomical glomerular widths. The spatial jitter was normalized by the mean width of the anatomical glomerulus and the hotspot (a value of 1 corresponds to jitter of 1 glomerular width). d. Example z-stack image projections of OMP-ChR2-YFP glomeruli obtained via multiphoton microscopy. Each image shown is a 20 μm thick projection, taken 20 μm apart in the z-axis from the subsequent one. Drawing illustrates contours of the glomeruli in the field of view. Arrows indicate overstacked glomeruli.
Figure 4
Figure 4. Light mapping sorts M/T cells into sister and non-sister pairs
a. Example spike waveforms on individual tetrode channels for two isolated non-sister M/T cell units. b. 2DLAMs for the units shown in A at different light intensities used for stimulation. The color scale bar range to the right of the highest intensity maps indicates the range of firing rate changes with respect to baseline. All light maps for a particular unit are scaled to this range. Difference map refers to the difference between the normalized 2DLAMs of the two units, plotted for each of light intensities used; spatial scale bar is 100 μm. c. Example waveforms for two isolated sister M/T cell units. d. 2DLAMs for the units shown in c at different light intensities used for photo-stimulation. Color scale bar range, as described in b; spatial scale bar is 100 μm. e. Cartoon schematic of parent glomerular connectivity for sister and non-sister M/T cells. f. Separation of M/T cells into sisters and non-sisters based on Euclidean distance between the centers of light hotspots on 2DLAMs obtained in a minimal photo-stimulation regime. The distance between the centers of hotspots is expressed in units of the mean full width at half maximum (FWHM) of Gaussian fits to the two hotspots for each pair. Dotted line marks the separation between sister and non-sisters M/T cells and is placed at 1 FWHM.
Figure 5
Figure 5. Examples of similarities and differences in odor responses of sister M/T cells
a. Example odor response of an M/T unit. (Top) Five odor stimulation trials are shown for this unit; vertical lines mark the time of a spike occurrence. Shadowed area indicates odor presentation window (5 s). (Bottom) Respiration trace. One respiratory cycle (labeled 0 to 2π) was typically ~500 ms long. Inset: expanded traces showing three respiratory cycles during air and odor presentation periods for one trial. b. (Top) Phase-time plot of the odor response of the same M/T unit in a, shown over five repeats of allyl tiglate. Note the change in the preferred phase during odor stimulation (shadowed area). On the right of the phase-time plot are shown the phase tuning curves calculated during air (dotted line) and odor (continuous line) presentation where each respiratory cycle was divided into 5 time bins. (Bottom) PSTH of the same unit showing a drop in firing rate triggered by odor onset; bin width, 500 ms; NSC- normalized spike count. c. Example odor responses to p-anis aldehyde, heptanal and 2-heptanone for two sister M/T cells (Unit 1 and Unit 2) shown as phase plots, PSTHs and phase tuning curves as in b. (Left) note the strong increase in firing, spread across all respiration cycle phases for both units; (Center) note the excitatory versus inhibitory response triggered by odor onset in the two units; (Right) note the change in preferred phase of Unit 2 triggered by the odor onset.
Figure 6
Figure 6. Sister M/T cells have correlated changes in odor induced firing rates
a. and b. Examples of firing rate odor response spectra (F-ORS) obtained using a set of 42 odors for three pairs of sister (a) and three pairs of non-sister M/T cells (b). Arrows in a denote differential responses across pairs of sister units. c. A scatter plot of the similarity (correlation coefficient) of odor-induced firing rate change against the Euclidean distance between the centers of the hotspots in the 2DLAMs for each pair of M/T units considered. Gray indicates non-sister M/T cells; black indicates sister M/T cells. The marginal distributions are shown as histograms on the top and right axes. (Top) Separation of units into sister and non-sister M/T cells, as shown in Fig. 3f. (Right) Histograms of sister (N = 20) and non-sister pairs (N = 15) F-ORS correlations. d. Average F-ORS correlations for sister and non-sister M/T cells; `self' refers to the same unit (N = 40) probed across different blocks of odor repeats by splitting the total number of trials in two; # marks p < 0.05 for F-ORS correlations, across groups with respect to sister M/T pairs (e.g. sister versus non-sister M/T units). Error bars represent standard error of the mean (s.e.m.).
Figure 7
Figure 7. Odors disrupt phase correlations of sister and non-sister M/T cells
a. Example phase tuning curves for two M/T units (Unit 1 and Unit 2) during Air and Odor. b. Phase response spectra for one representative sister M/T unit pair. Arrows indicate example mismatches between the spectra. c. Average phase response spectra correlations between sister and non-sister M/T pairs. d. Example phase similarity spectra for two sister M/T cells during Air (blue) and Odor (red) for 42 stimuli. e. Histograms of phase similarity during Air (blue) and Odor (red) for all sister (N = 20) (Top) and non-sister (N = 15) (Bottom) M/T unit pairs for 42 stimuli. f. Average phase similarity for sister and non-sister M/T pairs. g. Example phase tuning curves for two M/T units (Unit 1 and Unit 2, different from panel a) during Air and Light activation of single parent glomeruli. Note that light induces similar changes in phase for both units. h. (Left) Average phase response between Air and Light for individual sister M/T units; (Right) Average phase similarity between sister M/T pairs during Air (dotted line) and Light (continuous line). `self' refers to similarity between phase tuning curves generated from the same unit, by splitting the number of trials into two; * p < 0.05 for comparisons within the same group (sister or non-sister M/T units) across conditions (Odor vs. Air); # p < 0.05 for same condition (Air or Odor), across groups (sister versus non-sister M/T units) with respect to sister M/T pairs.
Figure 8
Figure 8. Odors trigger firing rate and phase changes in an independent manner
a. Average number of odors that induced differential responses in units of the same pair, considered in terms of firing rate changes or phase similarity; `overlap' refers to number of odors that induced significant differential changes in both firing rate and phase between units; `expected overlap' refers to the number of odors that would induce significant differential changes in both firing rate and phase, if the two were independent. Data is shown for sister (Left) and non-sister (Right) M/T pairs; * marks p < 0.05. b. (Left) Cartoon representation of the diversity of sister M/T cell surround fields. (Right) Example spike trains of two model sister M/T cells (red and blue) during Air and Odor periods with respect to the respiratory cycle (top trace); Stimulus 1 elicits different phase shifts between the two neurons, but their firing rates are unchanged. Stimulus 2 elicits similar firing rate changes in both neurons, and their firing times remain correlated. Stimulus 3 elicits the same firing rate change in both neurons, but different phase shifts.

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