Distributed Representation of Chemical Features and Tunotopic Organization of Glomeruli in the Mouse Olfactory Bulb

Distributed representation of chemical features and tunotopic organization of glomeruli in the mouse olfactory bulb Limei Maa,1, Qiang Qiua, Stephen Gradwohla,2, Aaron Scotta,3, Elden Q. Yua, Richard Alexandera, Winfried Wiegraebea, and C. Ron Yua,b,1 aStowers Institute for Medical Research, Kansas City, MO 64110; and bDepartment of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS 66160 Edited by John R. Carlson, Yale University, New Haven, CT, and accepted by the Editorial Board February 14, 2012 (received for review October 24, 2011) In the mammalian brain, similar features of the sensory stimuli are features, but recent experiments have provided little support of this often represented in proximity in the sensory areas. However, how notion (7, 23, 24). These studies have suggested that chemotopy at chemical features are represented in the olfactory bulb has been the fine scale does not exist and that the olfactory system violates controversial. Questions have been raised as to whether specific the organization principle found in other sensory systems (7, 25, chemical features of the odor molecules are represented by spatially 26). Moreover, the topographic organization in the bulb is dis- clustered olfactory glomeruli. Using a sensitive probe, we have persed when the projection reaches the piriform cortex (PirC). analyzed the glomerular response to large numbers of odorants at Whereas the mitral cell in the bulb receives input from a single single glomerulus resolution. Contrary to the general view, we find glomerulus, its projection into the PirC extends into a wide area, and a confined cortical region receives input from mitral cells across that the representation of chemical features is spatially distributed in – the olfactory bulb with no discernible chemotopy. Moreover, odor- the entire bulb (27 29). Accordingly, odors evoke a sparsely distributed ensemble of neurons in the PirC (30, 31). This distributed evoked pattern of activity does not correlate directly with odor odor representation removes an a priori reason for a spatial orga- structure in general. Despite the lack of spatial clustering or prefer- nization of the glomeruli. Serious question as to whether any or- ence with respect to chemical features, some structurally related ganizational principle exists for the glomeruli has been raised. NEUROSCIENCE odors can be similarly represented by ensembles of spatially distrib- In this study, we reevaluate the spatial representation of chemical uted glomeruli, providing an explanation of their perceptual similar- features with systematic investigations of odor responses in the dorsal ity. Whereas there is no chemotopic organization, and the glomeruli olfactory bulb in mice expressing the Ca2+ sensor G-CaMP2 (32). are tuned to odors from multiple classes, we find that the glomeruli These mice provide unprecedented sensitivity and single glomerular arehierarchicallyarrangedintoclustersaccordingtotheirodor- resolution that allow us to examine large numbers of odor stimuli and tuning similarity. This tunotopic arrangement provides a framework compare odor response patterns within individual animals. This to understand the spatial organization of the glomeruli that conforms approach overcomes several previous shortfalls by eliminating the to the organizational principle found in other sensory systems. requirement of collecting and assembling data from multiple animals, as in studies using [3H]2-deoxyglucose (2-DG) uptake or im- GCaMP2 | calcium imaging | topographic map mediate early gene expression (8–11).Italsoallowsustouse relatively low odor concentrations and to examine sparsely activated regions, which are likely neglected in previous studies. Our results or most external senses, sensory areas in the brain are spa- fi Ftially organized according to certain features of the stimuli. nd little support of chemotopy. Instead, we provide an alternative Visual and somatosensory information maps topographically in framework to explain the spatial organization of the glomeruli. the thalamus and cortex (1, 2), and frequency tuning maps of Results sound are found in various stages from the cochlear to the au- ditory cortex (3). In chemical senses, the submodalities of taste Odor Response in G-CaMP2 Animals. We imaged compound heter- OMP-tTA/tetO-G-CaMP2 are represented in different parts of the gustatory cortex (4). The ozygotic mice (32), which expressed G- CaMP2 in the OSNs without affecting their projection patterns topographic representation of sensory features allows the pro- Δ – cessing of information through parallel channels while preserving (Fig. S1). G-CaMP2 signals ( F/F up to 25 40%; Fig. 1) were much larger than intrinsic signals (ΔF/F ≈1%) and signals from syn- the next-neighbor relationship to enable computations such as – contrast enhancement and fill-in (5). aptopHluorin or Oregon Green (1 5%) (23, 24, 33). Using an automated olfactometer, we examined glomerular responses to How odor stimulus is mapped topographically in the olfactory ≈ ≈ system has been controversial. In the olfactory bulb, axons of ol- 200 odors, among which 60 were selected for further study because they activated the dorsal glomeruli (Dataset S1). Different factory sensory neurons (OSNs) expressing the same odorant re- – ≈ ceptor (OR) gene converge onto two stereotypically positioned odors evoked distinct patterns of activity in 60 100 out of 200 glomeruli in the imaged area (Fig. 1 C–E). Responses were glomeruli (6). The topographic map of the olfactory glomeruli shows > remarkable stereotypy among animals of the same species and recorded across 1,000-fold change in odor concentrations (Fig. 1, sometimes across species (6, 7). Numerous studies have examined Fig. S2,andDataset S2), and the patterns to the same odor how chemical features are represented in the olfactory bulb (8–18). The prevailing hypothesis posits that different chemical features are represented by compartmentalized regions in the olfactory bulb to Author contributions: L.M. and C.R.Y. designed research; L.M. and Q.Q. performed research; “ ” L.M., Q.Q., S.G., A.S., E.Q.Y., and C.R.Y. contributed new reagents/analytic tools; L.M., Q.Q., form a chemotopic map (19, 20). The chemotopic hypothesis E.Q.Y., R.A., W.W., and C.R.Y. analyzed data; and L.M. and C.R.Y. wrote the paper. suggests that the olfactory glomeruli can be grouped according to the fl chemical features of the odorants that activate them and are spatially The authors declare no con ict of interest. located in clustered regions (8–16, 21, 22). This article is a PNAS Direct Submission. J.R.C. is a guest editor invited by the Editorial Board. This hypothesis, however, has been challenged. Quantitatively, 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. its framework does not specify how many features are represented 2Present address: Washington University School of Medicine, St. Louis, MO 63110. in a segregated manner, nor does it specify the size and permissible 3Present address: University of Missouri School of Medicine, Columbia, MO 65212. overlap among the clusters. Qualitatively, chemotopy requires This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nearby glomeruli be tuned to odors that share common molecular 1073/pnas.1117491109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1117491109 PNAS Early Edition | 1of6 Downloaded by guest on October 7, 2021 A C Methyl valerate A Cyclohexylamine Hexylamine Isoamylamine Triethylamine 0.25% 0.75% 2.5% B D Pentanone 25% 20% Isoamyl Acetate Ethyl tiglate Vinyl butyrate Methyl propionate 15% B 10% 5% 0.25% 0.75% 2.5% 0% ΔF/F E 10 20 30 C Acid Aldehyde Amine Ester 40 50 60 Glomerulus 25% 70 20% 15% 80 10% 5% 90 0% ΔF/F Ether Hydrocarbon Ketone Thiazole de er on ole st ther rb tone z Acid E E a e Amine c K hia ldehy ro T Max A d y H Fig. 1. Glomerular response to odor stimulation in G-CaMP2 animal. (A and B) Confocal image stack projections showing G-CaMP2 expression in the 0 main olfactory epithelium (A) and the main olfactory bulb (B)inanOMP- IRES-tTA/ tetO-G-CaMP2 animal. (Scale bars, 30 μminA, 300 μminB.) (C and R D) Glomerular responses patterns to methyl valerate (C) and 2-pentanone (D) at 0.25%, 0.75%, and 2.5% saturated vapor (S.V.), respectively. Bright spots are activated glomeruli. (E) Heatmap of the glomerular response D d1 E patterns to a panel of 59 odors (0.25% S.V.) from eight different chemical d2 1 classes. Each pixel represents the peak response of a single glomerulus to d4 d3 a single odor stimulus. Rows represent the response of single glomeruli, and d5 d6 columns represent the pattern of activation by individual odors. Odor classes 0.1 are indicated in the heatmap. Names and structures of the odors are in- d_WITHIN= 1/n Σ di e dicated in Dataset S1. alu v 0.01 d1’ p d2’ d3’ threshold =0 d4’ 1E-3 threshold =0.3 stimulation within individual animals were highly reproducible threshold =0.5 (Fig. S3). d5’ d6’ 0 50 100 150 200 Representation of Chemical Features. We examined the represen- d_BETWEEN= 1/n’ Σ di’ D(μm) = d_BETWEEN - d_WITHIN tation of molecular features using odorants from eight chemical classes. Because the position of glomerulus expressing the same Fig. 2. Representation of odor classes in the bulb. (A–C) Each dot represents OR varied from animal to animal (34), all comparisons were the location of a glomerulus, with the brightness indicating the response am- conducted within individual mice. Although odors belonging to plitude. (A) Patterns evoked by four amine odors (0.25% S.V.). (B)Patterns the same chemical class could activate a similar set of glomeruli, evoked by four ester odors (0.025% S.V.). (C) Each panel represents the patterns of activation by odorants belonging to a single class of chemicals. The intensity these glomeruli were also activated by odors from a distinct class E of each glomerulus shows its strongest responses to any odor belonging to (rows in Fig.

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