Fig 1.
Dual imaging of the dOB and lOB in GCaMP6f reporter mice.
(A) Schematic representation of the dual microscope imaging setup. Illustrating the plane of the dorsal and lateral images. Inset: schematic of the odor delivery assembly relative to the nostrils, illustrating the odor and vacuum flow channels. (B) Schematic of the mouse genotype. (C) Top: resting light image of the dOB. Bottom: resting light image of the lateral region of the OB. (D) Top: odor-induced activation map of the dOB. Arrows highlight 4 individual glomeruli. Bottom: odor-induced activation map of the lOB. Arrows point to the same 4 glomeruli as top. (E) Left: glomerular response traces from the 4 glomeruli imaged with the dorsal camera in response to 1% heptanone. Middle: responses from the 4 glomeruli imaged with the lateral camera in response to 1% heptanone. Right: glomerulus 4 imaged from the dorsal and lateral camera, overlaid. (F) Top: an example of the ROI selection on the dorsal bulb for 1 animal. Bottom: an example of the ROI selection for the lateral bulb for 1 animal. (G) Top: % ΔF/F responses for the dorsal ROIs in panel F (top) selection of first odor response is indicated (1 animal). Middle: % ΔF/F responses for the lateral ROIs in panel F (Bottom) (1 animal). Bottom: a sniffing trace from the same animal. Inhalation is the upward inflection (see Methods for details). Underlying data for this figure can be found in S1 Data. dOB, dorsal olfactory bulb; lOB, lateral olfactory bulb; OB, olfactory bulb; ROI, region of interest.
Fig 2.
Simultaneous odor mapping of the dOB and lOB.
(A) Odor-induced activation maps for AA, carvone, heptanol, heptanone, hexanal, and MV. Top row: the dorsal region of the OB. Bottom row: the lateral region of the OB (1 animal, average of 3 trials). (B) Spatial odor map correlations between response amplitudes and the location along each spatial dimension of all identified glomeruli. Left: dorsal region of the OB. Right: the lateral region of the OB. Error bars are SEM (AA, heptanol, and hexanal 5 animals; all others 6 animals). (C) Across-glomerular response pattern similarities, assessed by Pearson correlations across glomerular response amplitudes. Left: for the dOB. Right: for the lOB (AA, heptanol, and hexanal 5 animals; all others 6 animals). (D) Average z-scores of glomerular response amplitudes (relative to pre-odor breathing response amplitudes). z-Scores are organized relative to MV. Left represents the dorsal region of the OB. Right: the lateral region of the OB (1 animal, 3 trials, same trials as shown in 2A). Underlying data for this figure can be found in S1 Data. AA, amyl acetate; dOB, dorsal olfactory bulb; lOB, lateral olfactory bulb; MV, methyl valerate; OB, olfactory bulb.
Fig 3.
Temporal dynamics of the dOB and lOB.
(A) Color-scaled T90 of responses by ROIs for AA and MV. Top: the dorsal region of the OB. Bottom: the lateral region of the OB (1 animal). (B) Comparison of spatiotemporal odor dynamics, i.e., correlation between T90s of glomeruli and their location along each dimension across the dOB of AA (n = 5 mice) and MV (n = 6 mice) from pre-odor (breathing response to clean air) to odor onset (1st odor response peak). Arrow represents the direction from pre-odor to odor on. Top: the dorsal region of the OB, data are represented in both the A-P directions and the M-L as shown in panel A. Bottom: the lateral region of the OB, data are represented in both the A-P directions and the D-V as shown in panel A. Error bars are SEM. (C) Spatiotemporal odor map dynamics for all odors. Top: the dorsal region of the OB. Bottom: the lateral region of the OB (AA, heptanol, and hexanal 5 animals; all others 6 animals). (D) T90 correlations with odor amplitude (ΔF/F). Left: the dorsal region of the OB. Right: the lateral region of the OB. Error bars are SEM (AA, heptanol, and hexanal 5 animals; all others 6 animals). (E) Left: correlation of glomerular response amplitudes to MV sorted from anterior to posterior in the dOB (r2 = 0.24, slope = −0.08 ± 0.02, 1 animal, 57 glomeruli). Right: correlation of glomerular T90 responses to MV sorted from anterior to posterior in the dOB (r2 = 0.18, slope = −0.0002 ± 0.0009, 1 animal, 57 glomeruli). (F) Left: amplitude of glomeruli represented on a color scale. Red indicates high responses and blue low. Right: T90 of glomeruli represented on a color scale. Red indicates the slowest responses and blue the fastest (1 animal [same as panel E], 57 glomeruli). Underlying data for this figure can be found in S1 Data. A-P, anterio-posterior; AA, amyl acetate; D-V, dorso-ventral; dOB, dorsal olfactory bulb; lOB, lateral olfactory bulb; M-L, medio-lateral; MV, methyl valerate; OB, olfactory bulb; ROI, region of interest.
Fig 4.
Glomerular odor concentration dependence.
(A) Color-scaled response amplitudes for chosen ROIs for 0.1% heptanone. Top: the dorsal region of the OB. Bottom: the lateral region of the OB. (B) Color-scaled responses amplitude for chosen ROIs for 1% heptanone. Top: the dorsal region of the OB. Bottom: the lateral region of the OB. (C) Percent of glomeruli that are significantly activated by 0.1% (AA, heptanol, hexanal, and MV 5 animals; all others 6 animals) compared to the higher concentration of 1% (AA, heptanol, and hexanal 5 animals; all others 6 animals). The number of responding glomeruli to the higher odor concentration has been normalized to 100%. Top: the dorsal region of the OB. Bottom: the lateral region of the OB. Statistics represent two-way ANOVA (odor × concentration) per OB region with Bonferroni’s multiple comparisons test for concentration effect. Error bars are SEM. *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001. (D) Average ΔF/F responses for odors comparing 0.1% and 1% concentration. Top: the dorsal region of the OB. Bottom: the lateral region of the OB. Statistics as in panel C. Additional statistics represent two-way ANOVA (odor × OB region) with Bonferroni’s multiple comparisons test for OB region effect at 1% s.v. ♦P < 0.05, ♦♦P < 0.01, ♦♦♦P <0.001, ♦♦♦♦P < 0.0001. (E) Odor-induced activation maps for 0.1% heptanone presentation in the dorsal region of the OB, arrows indicate 4 glomeruli. Left: during odor presentation. Right: after the odor is removed by a vacuum. Arrows indicate the same glomeruli as left. (F) Glomerular response traces for the 4 glomeruli in panel E. Vertical blue bars indicate response and reference frames to compute response maps. (G) Odor-induced activation maps for 0.1% heptanone presentation in the lateral region of the OB, arrows indicate 7 glomeruli. Left: during odor presentation. Arrows show 4 of the glomeruli. Right: after the end of odor presentation. Arrows represent 3 additional glomeruli. (H) Glomerular response traces for the 7 glomeruli in panel F. Glomeruli 5–8 (reds) respond during odor presentation, and glomeruli 9–11 respond when the odor is removed by vacuum (blues). I–L same as E–H but for 1% heptanone. Underlying data for this figure can be found in S1 Data. AA, amyl acetate; MV, metyl valerate; OB, olfactory bulb; ROI, region of interest.
Fig 5.
The lOB displays mechanosensitive activation beyond breathing.
(A) Raw traces (% ΔF/F) of two glomerular breathing responses during presentation of clean air flow rates (0.5, 0.25, 0.1, 0.05, and 0.005 L/min) and vacuum rates (2.5, 1.25, and 0 L/min) and room air with no external flows (1 animal, 2 glomeruli). These are presented as examples of the trial stimulus; green represents a glomerulus in the dorsal region of the lOB, blue in the ventral (maps shown in panel B). Red bar illustrates the period in which only the air is presented (“Vacuum off”), and grey is when the vacuum is also turned on (“Vacuum on”); height indicates flow rates relative to other trials. Vertical blue bars indicate response and reference frames to compute response maps. (B1, vacuum off response; B2, vacuum on response; maps shown in panel B). (B) Activation map in response to inspiration during only clean air flow rate of 0.5 L/min (Left top) and additionally the vacuum 2.5 L/min (Left bottom). Arrows indicate the ROI displayed in panel A. B1 and B2 indicate the respective time points in panel A (0.5 L/min airflow: vacuum 2.5 L/min). Right: activation map of all glomeruli chosen to clean air flow rate of 0.5 L/min (Top) and vacuum 2.5 L/min (Bottom). (C) The z-scores of the lOB glomerular responses during only clean air flow (red) and also vacuum (grey), organized from dorsal to ventral, for different clean air flow rates (0.5, 0.25, 0.1, 0.05, and 0.005 L/min) and vacuum rates (2.5, 1.25, and 0 L/min) and room air (3 animals, 47 glomeruli). Linear correlation fits are indicated. (D) Top left: histogram of the ratio of vacuum off responses relative to room air breathing responses in the lateral bulb. Top right: histogram of the ratio of vacuum on breathing responses relative to room air breathing responses in the lateral bulb (3 animals, 47 glomeruli, total 329 glomerular responses across all flow rates). Number of responses (n) was divided into number of glomerular responses <1.6 and >1.6 times above the room air breathing response. Bottom: same as top but in the dorsal bulb. Underlying data for this figure can be found in S1 Data. lOB, lateral olfactory bulb; OB, olfactory bulb; ROI, region of interest.
Fig 6.
The lOB is sensitive to differential pressure.
(A) Mean z-scored OB responses to different flow rates in the ventral lOB. (B) Mean z-scored OB responses to different flow rates in the dOB. (C) Mean z-scored OB responses to different pressure changes in the lOB. (D) Mean z-scored OB responses to different pressure changes in the lOB. Left: the ventral lOB. Right: the dorsal lOB. (E) Mean z-scored OB responses to different pressure changes in the dOB. (F) Mean z-scored OB responses to different pressure changes in the dOB. Left: the anterior dOB. Right: the posterior dOB: 3 animals; dOB: 99 glomeruli; and lOB: 47 glomeruli. Graphs are reorganized from S4C and S4D Fig, all offset by respective z-scores for no–flow-change condition (0–0, room; see Methods). Only the lOB—and the vlOB in particular—shows a strong and positive response to an intermediary drop in pressure at the odor delivery tube. The dOB only shows mild response suppression, particularly by intermediate changes in pressure. Pressure unit is relative only. Underlying data for this figure can be found in S1 Data. dOB, dorsal olfactory bulb; lOB, lateral olfactory bulb; OB, olfactory bulb; vlOB,