The Journal of Neuroscience, February 15, 2001, 21(4):1351–1360 Tuning and Topography in an Odor Map on the Rat Olfactory Bulb Markus Meister1,2 and Tobias Bonhoeffer2 1Harvard University, Cambridge, Massachusetts 02138, and 2Max Planck Institute of Neurobiology, D-82152 Munich- Martinsried, Germany The sense of smell originates in a diverse array of receptor centration dependence and yields the effective affinity with neurons, comprising up to 1000 different types. To understand which a glomerulus responds to an odorant. When tested with how these parallel channels encode chemical stimuli, we re- aliphatic molecules of increasing carbon chain length, many corded the responses of glomeruli in the olfactory bulbs of the glomeruli were sharply tuned for one or two adjacent chain anesthetized rat, by optical imaging of intrinsic signals. Odor lengths. Glomeruli with similar tuning properties were located stimulation produced two kinds of optical responses at the near each other, producing a systematic map of molecular surface of the bulb: a broad diffuse component superposed by chain length on the surface of the olfactory bulb. Given local discrete small spots. Histology showed that the spots corre- inhibitory circuits within the olfactory bulb, this can account for spond to individual glomeruli, and that ϳ400 of them can be the observed functional inhibition between related odors. We monitored in this way. Based on its wavelength-dependence, explore several parallels to the function and architecture of the this optical signal appears to derive from changes in light visual system that help interpret the neural representation of scattering during neural activity. Pure odorants generally acti- odors. vated several glomeruli in a bilaterally symmetric pattern, whose extent varied greatly with concentration. A simple for- Key words: olfaction; receptor; glomerulus; map; tuning; malism for ligand binding accounts quantitatively for this con- imaging Our olfactory system senses chemicals in the ambient air through pounds, even structurally very dissimilar molecules (Gesteland et specialized receptor cells that are located in the neural epithelium al., 1963; Duchamp et al., 1974; Sicard and Holley, 1984; lining the upper reaches of the nasal cavity. Each of these olfac- Duchamp-Viret et al., 1999). For technical reasons, these exper- tory neurons is thought to express a single type of receptor iments used a limited set of stimuli, sometimes at rather high odor protein that spans the plasma membrane and whose binding concentrations, to ensure that responses could be detected at all. properties determine the interaction with extracellular ligands Thus, it remains unclear for any olfactory receptor what the full (Buck and Axel, 1991; Buck, 1996; Mombaerts, 1999). In this range of active ligands is, whether there is one with specially high respect, olfactory receptors are similar to photoreceptors: each affinity, and which ligand is dominant under natural conditions of photoreceptor cell makes a single type of opsin protein, whose stimulation. Still, it is safe to assume that individual odorants— absorption properties determine the interaction of the cell with and certainly the mixtures of compounds that characterize real photons of different wavelength. However, photoreceptors come olfactory objects—generate a pattern of activity across several in only a handful of varieties, whereas mammals seem to have up receptors. To understand this code, it would obviously be useful to 1000 types of olfactory neurons (Mombaerts, 1999). Therefore, to monitor in parallel the responses of many receptor types. fully 1% of our genome may be dedicated to generating the Among the tens of millions of neurons in the olfactory epithe- diversity of olfactory receptors. In this great variety of primary lium, the different receptor types are widely interspersed: each receptors, the olfactory system is unique compared with all other type is restricted to one of four coarse zones of expression, but senses. How is sensory information processed in a system that there is no apparent order within each zone (Buck, 1996). A from the very outset uses such a large number of parallel receptor cell projects its axon to the olfactory bulb, where it channels? terminates in a small ball of neuropil, a glomerulus. The rat The number of distinct organic structures that elicit a smell in olfactory bulb has ϳ2400 of these glomeruli, close-packed in a humans is clearly much larger than the number of olfactory layer just beneath the surface (Meisami and Sendera, 1993). receptor genes (Mori et al., 1998). Therefore, many of the ϳ1000 receptors must be promiscuous and bind to several different Olfactory neurons of a given receptor type send their axons to ligands. In fact, physiological experiments have shown that a just two glomeruli in the bulb (Ressler et al., 1994; Vassar et al., given olfactory neuron can often be stimulated by many com- 1994; Mombaerts et al., 1996a). Given that there are approxi- mately twice as many glomeruli as receptor types, it is plausible Received Sept. 15, 2000; revised Dec. 4, 2000; accepted Dec. 5, 2000. that each glomerulus receives axons from just one receptor type, M.M. was supported by grants from the Office of Naval Research (N00014-98-1- although direct evidence on this point is still lacking (Mombaerts, 0829) and the National Science Foundation (IBN 9453317), and T.B. was supported by the Max Planck Gesellschaft. We thank Imke Goedecke, Iris Kehrer, and Frank 1999). If so, then the olfactory bulb effectively acts as a switch- Sengpiel for experimental advice and assistance and Catherine Dulac and members board, collecting the signals of a given receptor type from a broad of our laboratories for comments on this manuscript. zone of the epithelium into a small brain region ϳ150 ␮min Correspondence should be addressed to Markus Meister, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138. E-mail: [email protected]. diameter. The structure of this switchboard is stereotyped and Copyright © 2001 Society for Neuroscience 0270-6474/01/211351-10$15.00/0 precise: glomeruli for the various receptor types are generally 1352 J. Neurosci., February 15, 2001, 21(4):1351–1360 Meister and Bonhoeffer • An Odor Map on the Rat Olfactory Bulb located at mirror-symmetric positions in the two bulbs, and their tutes of Health guidelines and were approved by Harvard University’s Ani- arrangement is reproducible across individuals (Ressler et al., mal Care and Use Committee, as well as the Regierung von Oberbayern. Stimulation. Odors were delivered from a stimulator with eight tanks 1994; Vassar et al., 1994; Mombaerts et al., 1996b). Thus, the containing different odorants (AutoMate Scientific, San Francisco, CA). projection to the olfactory bulb maps every possible odor into a Humidified air passed through a tank containing filter paper soaked with two-dimensional image of neural activity in the layer of glomeruli. odorant diluted in mineral oil. This air was diluted fivefold with clean air These neural images elicited by odors on the olfactory bulb and directed at the nose with a final flow rate of 2500 ml/min. A four-way have been a subject of great interest (Shepherd, 1994; Xu et al., valve close to the nose switched between this stream of odorized air and a stream of clean air. The animal breathed through the nose at a stable 2000). Early experiments showed that odorants with very different rate of ϳ2 inspirations/sec. chemical properties also produce different activity patterns (Stew- The odor concentration was controlled by varying the v/v dilution of art et al., 1979; Jourdan et al., 1980; Cinelli et al., 1995). More odorant in mineral oil, D, over the range from 0.0001 to 0.1. The final importantly, molecules with similar chemistry tend to produce vapor concentration was measured with a flame ionization detector (MicroFID; Pine Environmental, Cranbury, NJ), and found to be ap- related patterns. Certain regions of the bulb appear to be dedi- proximately proportional to the dilution ratio D. To obtain the odor cated to odorants of a particular chemical class (Imamura et al., concentration as a fraction of saturated vapor, for the aldehyde with L 1992; Katoh et al., 1993; Johnson et al., 1999; Johnson and Leon, carbons at dilution D, multiply D by 0.063 (L ϭ 4), 0.12 (L ϭ 5), 0.24 (L ϭ 2000). Odorant molecules with several different functional groups 6), 0.47 (L ϭ 7), 0.93 (L ϭ 8), or 1.8 (L ϭ 9). may activate several regions in a combinatorial manner (Friedrich The data acquisition computer controlled the eight valves to select the active odor tank and the four-way valve that determined onset and offset and Korsching, 1997; Johnson et al., 1998). Collectively, these of odor stimulation. Generally, a 12 sec odor exposure was followed by 12 studies suggest that there is a coarse topography of chemical sec of clean air, followed by the next odor. The sequence of presentations properties on the surface of the bulb, although the rules of that was pseudorandom, ensuring that each odor was presented exactly once map remain to be discovered. Many recent contributions in this in a cycle. One of the tanks of the stimulator was left empty, providing a control condition of stimulation without odorant. area relied on the method of 2-deoxyglucose labeling, which Imaging. Images of the brain surface were recorded through the agar marks cells in the brain that have increased metabolic activity window as described elsewhere (Bonhoeffer and Grinvald, 1996). In during exposure to an odor. A great boon of this technique is that brief, the brain was illuminated with 707 nm light using a stable incan- it allows observation of all glomeruli simultaneously, and thus descent source and an interference filter (20 nm pass band). A cooled gives access to the entire odor-evoked neural image. A drawback CCD camera (ORA 2001; Optical Imaging, Germantown, NY) equipped with two front-to-front photo objectives recorded images (resolution is that one can inspect the image for only one odor at one 192 ϫ 144) in 0.5 sec frames.