Broadcasting of cortical activity to the olfactory bulb

Abstract

Odor representations are initially formed in the olfactory bulb, which contains a topographic glomerular map of odor molecular features. The bulb transmits sensory information directly to piriform cortex, where it is encoded by distributed ensembles of pyramidal cells without spatial order. Intriguingly, piriform cortex pyramidal cells project back to the bulb, but the information contained in this feedback projection is unknown. Here, we use imaging in awake mice to directly monitor activity in the presynaptic boutons of cortical feedback fibers. We show that the cortex provides the bulb with a rich array of information for any individual odor and that cortical feedback is dependent on brain state. In contrast to the stereotyped, spatial arrangement of olfactory bulb glomeruli, cortical inputs tuned to different odors commingle and indiscriminately target individual glomerular channels. Thus, the cortex modulates early odor representations by broadcasting sensory information diffusely onto spatially ordered bulbar circuits.

Figure 1. Cortical feedback inputs have diverse response properties

A₁) Left, viral vectors expressing GCaMP6s and tdTomato are injected in piriform cortex (PCx) to label olfactory bulb projections. Right, schematic of 2-photon imaging via a cranial window over the ipsilateral olfactory bulb (OB) in awake, head-fixed mice. A₂) Left, tdTomato expression in PCx axons from a coronal OB slice. Axonal projections are most prevalent in the granule cell and glomerular layer. Right, In vivo 2-photon image of tdTomato (red) and GCaMP6s (green) expressing boutons from the OB glomerular layer of an awake mouse. B) Odor-evoked GCaMP6s activity in individual boutons reveals a wide range of response properties. B₁) Responses from a glomerular layer imaging field show that boutons tuned to different odors are intermingled. Left, Image of GCaMP6s expression (white) shows ROIs (red outlines) drawn around individual boutons. Right, Responses of four boutons (rows) to four odors (columns). Grey lines are individual trails and black lines show the average response to each odor. Filled circles above each trace indicate a significant response (excitation: red, inhibition: blue) and the colored circles to the left of traces indicate ROIs marked in the GCaMP6s image. B₂) Responses in the granule cell layer from the same animal. C) Dynamics of odor-evoked feedback activity. Top: heat maps of the activity of all responsive bouton-odor pairs showing excitation (3855 bouton-odor pairs) or suppression (1907 bouton-odor pairs), sorted by their onset times (50% of peak). Bottom: histograms of the onset times of all responsive bouton-odor pairs show that excitatory responses are temporally more diverse in the awake state, while inhibitory responses are more time locked to odor onset. Vertical lines indicate the odor period. D₁) Odor tuning curve for boutons responding with excitation (red) or suppression (blue). Inset, proportion of all boutons with no response (NR), excitation only (E), suppression only (S), or both excitation and suppression (E&S). D₂) All odors have an equal probability of eliciting excitatory responses (n = 13 imaging fields). Dashed line indicates expected value (14.3%) if each odor randomly activates boutons with equal probability.

Figure 2. Cortical feedback activity is enhanced during wakefulness

A) Anesthesia reduces spontaneous activity. Heat map of dF/F values for 180 boutons from one imaging field in the awake state (left) and during ketamine anesthesia (right). B) Odor-evoked cortical feedback activity is reduced in the anesthetized state. Representative average responses of four boutons (rows) to four odors (black bars) from a single imaging field in the awake (black) and anesthetized state (red). BA; butyric acid, 2-4DM; 2-4 dimethylthiazole, Ani; anisole, ET; ethyl tiglate. C) Odor-evoked bouton excitation is stronger (C₁) and more broadly tuned (C₂) in the awake state. D) Odor-evoked excitation is more prolonged in the awake vs. anesthetized state. D₁) Heat maps of the activity of bouton-odor pairs showing excitation from the same animals in the awake (left) and anesthetized (right) state, aligned to their onset times and ordered by duration. Numbers of responsive bouton-odor pairs in each condition are in parentheses. D₂) Average time course of excitatory responses peak normalized and aligned by their rise times for the awake (black) and anesthetized (red) state. Shading, SEM. D₃) and D₄) Results for bouton-odor pairs showing odor-evoked suppression.

Figure 3. Cortical feedback inputs representing different odors are diffusely distributed at the spatial scale of individual glomeruli

A) Boutons responding to particular odors are not spatially segregated. A₁) Results from one granule cell layer imaging field showing all bouton ROIs (left) and map of the preferred odor for each responsive bouton (right). Color scale indicates each of the seven tested odors (Cin; cineole, Cit; citral, Hept; heptanal, BA; butyric acid, 4H; heptan-4-on, IA; isoamyl acetate, ET; ethyl tiglate. ROIs are shown enlarged for clarity. A₂) Cumulative frequency distribution of the pairwise distance between all boutons in A₁ with the same (Matched, n=296) or different (Mismatched, n=1702) odor preference. The two distributions are not significantly different (p=0.29, KS test). B) Summary data (grey, n=22 fields) reveals no significant difference in the mean distance between matched and mismatched responsive boutons (red, average±SEM). C) Rank-ordering orders by their probability of eliciting excitatory responses in individual imaging fields indicates that the fraction of responses elicited by any odor is random. Red circles, responses to rank ordered odors for both granule cell layer and glomerular imaging fields (mean ± SEM, n = 22 fields). The observed values fall within the curves expected by chance for the largest (n=406) and smallest (n=23) number of responsive boutons per imaging field (dotted lines). Inset, response probability distribution for the most represented odor (Odor #1) derived from random subsampling using the sample sizes from 22 imaging fields. Experimentally measured probability (red line) falls within central 90% of the distribution (dotted lines).

Figure 4. Glomerular layer targeting of feedback inputs is unrelated to glomerular odor specificity

A) Schematic. B₁) Intrinsic signal optical imaging from one mouse showing glomerular responses to three different odors (Ethyl tiglate (ET), Anisole (Ani), 2-Hexanone (Hex)) and the activity map superimposed on the olfactory bulb surface vasculature (bottom right). Colored circles highlight activated glomeruli; boxes represent fields selected for bouton imaging. Field 1 is centered over a glomerulus responding to ethyl tiglate and Field 2 indicates a region without a response to the odors. B₂) Targeted 2-photon imaging beneath an identified glomerulus. Top, blow up of region around Field 1 from B₁ showing overlay of intrinsic optical signal (red, ethyl tiglate) and surface vasculature. Bottom, 2-photon image stack of Texas red dextran-filled vessels aligned with the vasculature in the image above. B₃) Map of glomerular layer boutons responding with excitation within fields indicated in B₁. Colors indicate boutons responding selectively to individual odors (red, green, blue), boutons with overlapping responses to two odors (yellow, magenta, cyan), and boutons responsive to all three (white). ROIs are shown enlarged for clarity. B₄) Numbers of boutons responding with excitation to each odor for the fields in B₃. C) Summary of results (mean ± SEM) from all experiments using three odors indicates that individual odors were equally likely to activate boutons regardless of the responses of the overlying glomerulus. We calculated a variation index for each odor ((Observed fraction of responsive boutons / 0.33) – 1) for fields centered on an odor-responsive glomerulus (white circles) and fields within regions that did not show a glomerular response to any of the three odors (black circles). Variation index=0 if each odor (1 out of 3) has an equal probability of eliciting bouton responses in an imaging field. Odors were grouped between those that did (Glom+) or did not (Glom-) activate the odor-responsive glomerulus. In all cases, odors consistently had a variation index near zero.