Fig 1.
Interglomerular pathways of the main olfactory bulb circuit.
Schematic of the main olfactory bulb circuit with the (top) interglomerular-interneuron and (bottom) mitral-granule-mitral pathways highlighted. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; PG, periglomerular cell; SA, short axon cell; ET, external tufted cell; MC, mitral cell; GC, granule cell.
Fig 2.
GABAAR antagonist enhances lateral flavoprotein signal spread in the GL and EPL.
(A and B) Representative stimulus-dependent flavoprotein response before treatment (A) and during bath application of 10 μM GBZ (B). (top) Representative overlaid, pseudo-colored ΔF/F images at 2.5s after stimulus initiation (stimulation initiated at 0s). Representative point ROIs (regions of interest; white squares) from stimulated glomerulus, central EPL and neighboring rostral/caudal areas. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; R, rostral; C, caudal. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Scale bars = 400 μm. (A and B, right) Heat map of the vertical ROI signal response over time. The late flavoprotein signal seen at the top of the image comes from the targeted glomerulus as visualized through the lateral aspect of the MOB slice. (A and B, bottom) Heat maps of the horizontal ROI signal response from the GL and EPL over time. (C and D) Mean signal peak of the (C) GL (F(2,16) = 49.2, p = 0.002, n = 9) and (D) EPL (F(2,16) = 5.4, p<0.001, n = 9) at 360 μm rostral (black), center (white), and 360 μm caudal (gray) areas as indicated in (A). (E and F) Following GBZ treatment, the mean FWHM lateral spread in the (E) GL (F(2,8) = 14.0, p = 0.018, n = 9) and (F) EPL (F(2,8) = 21.2, p = 0.009, n = 9) increased when compared to pretreated slices. Differences in the duration of the flavoprotein signal in the rostral and caudal dimensions were not quantified.
Fig 3.
Lateral signal spread is dependent on interglomerular pathway.
(A) Schematic of the MOB circuitry with GL surgical microcut, and preservation of the mitral-granule-mitral pathway (red highlights). ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; PG, periglomerular cell; SA, short axon cell; ET, external tufted cell; MC, mitral cell; GC, granule cell. (B) Fluorescence image of MOB slice. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Representative point ROIs from neighboring rostral/caudal areas (squares). Scale bar = 400 μm. (C). Overlaid pseudo-colored ΔF/F stimulus-dependent responses before (top) and during (bottom) GBZ treatment at 2.5s after stimulus initiation. (D and E) Mean signal peak of the GL (D) (F(2,6) = 10.02, p = 0.012, Bonferroni t-test, n = 4) and EPL (E) (F(2,6) = 9.72, p = 0.013, Bonferroni t-test, n = 6) at 360 μm rostral (black) and 360 μm caudal (gray) of the stimulated glomerulus. (F and G) Normalized horizontal signal traces in the GL (F) and EPL (G), before (black) and during (red) GBZ treatment. (H and I) Mean FWHM signal spread in the GL (H) (F(2,5) = 7.2, p<0.001, n = 6) and EPL (I) (F(2,5) = 0.7, p = 0.02, n = 6).
Fig 4.
Lateral signal spread is dependent on the mitral-granule-mitral pathway.
(A) Schematic of the MOB circuitry with EPL surgical microcut, and preservation of the interglomerular-interneuron pathway (red highlights). ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; PG, periglomerular cell; SA, short axon cell; ET, external tufted cell; MC, mitral cell; GC, granule cell. (B) Fluorescence image of MOB slice indicating MOB layers, microcut location. Asterisk, tip of the stimulating electrode. Targeted glomerulus indicated by dashed circle. Representative point ROIs from neighboring rostral/caudal areas (squares). Scale bar = 400μm. (C) Overlaid pseudo-colored ΔF/F stimulus-dependent responses before (top) and during GBZ treatment (bottom) at 2.5s after stimulus initiation. (D and E) Mean signal peak of the (D) GL (F(2,12) = 8.32, p = 0.005, Bonferroni t-test, n = 7) and (E) EPL (F(2,12) = 14.85, p<0.001, Bonferroni t-test, n = 7) at 360 μm rostral (black) and 360 μm caudal (gray) of the activated glomerulus. (F and G) Normalized horizontal signal traces in the GL (F) and EPL (G), before (black) and during (red) GBZ treatment. (H and I) Mean FWHM signal spread in the (H) GL (F(2,8) = 12.6, p<0.001, n = 7) and (I) EPL (F(2,8) = 6.6, p<0.001, n = 7).
Fig 5.
Mapping of intrinsic flavoprotein and NAD(P)H fluorescence signals following necklace glomerulus stimulation.
(A) (left) Necklace glomeruli (NGs) innervated by GC-D+ OSNs (arrows) from Gucy2d-GFP mice. Anterior olfactory nucleus, AON; lateral olfactory tract, LOT; anterior piriform cortex, aPCX. Scale bar = 1 mm. (right) Schematic of angled coronal slice, where the slices were made 60° from the ventral surface of the main olfactory bulb (MOB). (B) Fluorescence image of coronal MOB slice, with the stimulus electrode on a single NG (asterisk). Scale bar = 400 μm. (C) Representative stimulus-dependent flavoprotein (top) and NAD(P)H (bottom) response at 1.5s after stimulus initiation. (D and E) Mean signal peak of the (D) GL (F(2,60) = 5.92, p = 0.004) and (E) EPL (F(2,66) = 5.92, p = 0.002) from stimulated NG, and CGs from coronal slices (C-CG) and horizontal slices (H-CG).
Fig 6.
Gabazine treatment enhances lateral signal spread following necklace glomerulus stimulation.
(A) Fluorescence image of coronal MOB slice with stimulus electrode on a single NG (asterisk). Scale bar = 400μm. (B and C) Gabazine (GBZ)-treated slices exhibit increased stimulus-dependent flavoprotein (B) and NAD(P)H (C) signal response and spread. (D and E) Mean intrinsic signal peak of the (D) GL (F(1,2) = 18.8, p<0.004) and (E) EPL (F(1,2) = 21.4, p<0.001) from the stimulated necklace (NG) and canonical glomeruli (CG).
Fig 7.
Necklace glomeruli are not preferentially connected to other necklace glomeruli.
(A) Schematic indicating stimulation of a central necklace glomerulus (NG) and ROIs (squares) taken from the neighboring canonical glomerulus (nCG, blue) and neighboring necklace glomerulus (nNG, green), and their respective EPL areas. ONL, olfactory nerve layer; GL, glomerular layer; EPL, external plexiform layer; MCL, mitral cell layer; IPL, internal plexiform layer; GrL, granule cell layer; PG, periglomerular cell; SA, short axon cell; ET, external tufted cell; MC, mitral cell; GC, granule cell. (B) Normalized signal peak from nNG (white) and nCG (gray), following individual necklace glomeruli stimulation. (C) Normalized mean signal peak from the EPL below the nNG and nCG.