Carbon dioxide (CO2) is an important environmental cue for many organisms but is odorless to humans. It remains unclear whether the mammalian olfactory system can detect CO2 at concentrations around the average atmospheric level (0.038%). We demonstrated the expression of carbonic anhydrase type II (CAII), an enzyme that catabolizes CO2, in a subset of mouse olfactory neurons that express guanylyl cyclase D (GC-D+ neurons) and project axons to necklace glomeruli in the olfactory bulb. Exposure to CO2 activated these GC-D+ neurons, and exposure of a mouse to CO2 activated bulbar neurons associated with necklace glomeruli. Behavioral tests revealed CO2 detection thresholds of approximately 0.066%, and this sensitive CO2 detection required CAII activity. We conclude that mice detect CO2 at near-atmospheric concentrations through the olfactory subsystem of GC-D+ neurons.

The mammalian olfactory system consists of two anatomically segregated structures, the main olfactory system and the vomeronasal system, which each detect distinct types of chemical stimuli in the environment. During development, sensory neurons establish precise axonal connections with their respective targets within the olfactory bulb. The specificity of the odorant or vomeronasal receptor expressed by the sensory neuron is crucial in this process, yet it is less clear which of the more conventional axon guidance molecules are involved. Here, we show that neuropilin-2, a coreceptor for some of the class 3 semaphorins, is expressed in subpopulations of olfactory and vomeronasal sensory neurons. We generated a knock-out mutation in the neuropilin-2 gene by gene targeting in embryonic stem cells. Neuropilin-2 mutant mice exhibit profound and distinct effects on target innervation within the olfactory bulb. In the main olfactory system, axons of olfactory sensory neurons penetrate into the deeper layers of the main olfactory bulb. In the vomeronasal system, axonal fasciculation within the vomeronasal nerve is affected; some axons are misrouted and innervate glomeruli in an ectopic domain of the accessory olfactory bulb.

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.

Our understanding of mammalian olfactory coding has been impeded by the paucity of information about the odorant receptors (ORs) that respond to a given odorant ligand in awake, freely behaving animals. Identifying the ORs that respond in vivo to a given odorant ligand from among the ϳ1100 ORs in mice is intrinsically challenging but critical for our understanding of olfactory coding at the periphery. Here, we report an in vivo assay that is based on a novel gene-targeted mouse strain, S100a5-tauGFP, in which a fluorescent reporter selectively marks olfactory sensory neurons that have been activated recently in vivo. Because each olfactory sensory neuron expresses a single OR gene, multiple ORs responding to a given odorant ligand can be identified simultaneously by capturing the population of activated olfactory sensory neurons and using expression profiling methods to screen the repertoire of mouse OR genes. We used this in vivo assay to re-identify known eugenol-and muscone-responsive mouse ORs. We identified additional ORs responsive to eugenol or muscone. Heterologous expression assays confirmed nine eugenol-responsive ORs (Olfr73, Olfr178, Olfr432, Olfr610, Olfr958, Olfr960, Olfr961, Olfr913, and Olfr1234) and four muscone-responsive ORs (Olfr74, Olfr235, Olfr816, and Olfr1440). We found that the human ortholog of Olfr235 and Olfr1440 responds to macrocyclic ketone and lactone musk odorants but not to polycyclic musk odorants or a macrocyclic diester musk odorant. This novel assay, called the Kentucky in vivo odorant ligand-receptor assay, should facilitate the in vivo identification of mouse ORs for a given odorant ligand of interest.

+ neurons. Some glomeruli homogeneously innervated by axons of GC-D + neurons form ectopically within the glomerular layer, across wide areas of the main olfactory bulb. Similarly, axonal wiring of some vomeronasal sensory neurons is perturbed by a deficiency of Nrp2 or Sema3f, but not Sema3b or Sema3c. Our findings provide genetic evidence for a Nrp2-Sema3f interaction as a determinant of the wiring of axons of GC-D + neurons into the unusual configuration of necklace glomeruli.