INTEGRATION OF CO 2 AND ODORANT SIGNALS IN THE MOUSE OLFACTORY BULB L. GAO, a,b J. HU, b C. ZHONG b AND M. LUO b,c * a Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101 PR China b National Institute of Biological Sciences, Beijing, 102206 PR China c Department of Biological Sciences and Biotechnology, Tsinghua Uni- versity, 100084 PR China Abstract—Carbon dioxide (CO 2 ) is an important environmen- tal cue for many animal species. In both vertebrates and invertebrates, CO 2 is detected by a specialized subset of olfactory sensory neurons (OSNs) and mediates several ste- reotypical behaviors. It remains unknown how CO 2 cues are integrated with other olfactory signals in the mammalian ol- factory bulb, the first stage of central olfactory processing. By recording from the mouse olfactory bulb in vivo, we found that CO 2 -activating neurons also respond selectively to odor- ants, many of which are putative mouse pheromones and natural odorants. In addition, many odorant-responsive bul- bar neurons are inhibited by CO 2 . For a substantial number of CO 2 -activating neurons, binary mixtures of CO 2 and a spe- cific odorant produce responses that are distinct from those evoked by either CO 2 or the odorant alone. In addition, for a substantial number of CO 2 -inhibiting neurons, CO 2 addition can completely block the action potential firing of the cells to the odorants. These results indicate strong interaction be- tween CO 2 signals and odorant signals in the olfactory bulb, suggesting important roles for the integration of these two signals in CO 2 -mediated behavioral responses. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: mitral/tufted cells, pheromone, mixture, lateral inhibition, olfactory coding. CO 2 is one of the major byproducts of cellular metabolism. Although the basal CO 2 level in the air is relatively low (0.038%), local atmospheric CO 2 level can fluctuate dra- matically with the metabolic activity of organisms. Ambient CO 2 level can signal the presence of food, predators, or environmental stress and mediate stereotypical behaviors for animals across phyla (Stange and Stowe, 1999; Suh et al., 2004; Thom et al., 2004; Jones et al., 2007; Hallem and Sternberg, 2008; Luo et al., 2009). Recent studies have shown that, for both vertebrates and invertebrates, CO 2 is detected by a specialized subset of olfactory sensory neu- rons (OSNs) (Suh et al., 2004; Thom et al., 2004; Luo et al., 2009). Although CO 2 is odorless to humans, it is sen- sitively detected by a specialized olfactory subsystem in mice (Hu et al., 2007; Sun et al., 2009). The CO 2 -respon- sive OSNs in mammals uniquely express several signaling molecules, such as guanylyl cyclase-D (GC-D) and phos- phodiesterase 2A (PDE2A) that suggest their use of guanosine 3=,5=-cyclic monophosphate (cGMP) as the second messenger (Fulle et al., 1995; Juilfs et al., 1997; Sun et al., 2009). In addition, they project to a set of the so-called necklace glomeruli at the caudal end of the main olfactory bulb (MOB) (Juilfs et al., 1997; Hu et al., 2007; Walz et al., 2007). Because GC-D + OSNs are not known to express signaling components in the canonical trans- duction pathway within the typical OSNs, such as G olf and ACIII (Juilfs et al., 1997; Meyer et al., 2000), it is likely that GC-D + neurons represent a specialized channel dedi- cated to detecting CO 2 but not typical olfactory signals. A key question that remains unresolved is whether CO 2 -activating bulbar neurons can also respond to other odorants. CO 2 activates bulbar neurons that extend their dendrites into the necklace glomeruli (Hu et al., 2007). However, necklace glomeruli are intermingled with other glomeruli in the main olfactory bulb, suggesting potential interaction between CO 2 signals and olfactory signals in the MOB. A recent tracing study suggests that at least some necklace glomeruli receive inputs from GC-D - OSNs (Cockerham et al., 2009). Thus, the CO 2 -responsive bulbar neurons may also be excited by odorants that acti- vate these GC-D - OSNs. Although mitral/tufted (M/T) cells—the projection neurons of the bulb— extend their primary dendrite into only one glomerulus, they also inter- act extensively with their basal dendrites (Shepherd et al., 2004). Inhibitory lateral connections are believed to shape odorant responses (Mori et al., 1999; Tan et al., 2010). However, it remains unclear whether CO 2 -activating cells can be inhibited by odorants and conversely whether typ- ical odorant-responsive M/T cells can be inhibited by CO 2 . An interesting feature of CO 2 signaling lies in the fact that CO 2 levels are correlated with the metabolic activity of organisms including plants and animals. It is conceivable that CO 2 cues are often simultaneously released with other odorants in natural environment. It remains untested how CO 2 -responsive cells in the bulb respond to the mixtures of CO 2 and odorants. In this study, we carried out in vivo physiological re- cordings from the mouse olfactory bulb to address these questions. We find that a majority of CO 2 -responsive neu- rons are selectively activated by other odorants. Many of these odorants are putative mouse pheromones and nat- *Correspondence to: M. Luo, NIBS, #7 Science Park Road, Zhong- guancun Life Science Park, Beijing, 102206, PR China. Tel: +86-10- 80726688-8320; fax: +86-10-80723342. E-mail address: luominmin@nibs.ac.cn (M. Luo). Abbreviations: CAII, carbonic anhydrase II; cGMP, guanosine 3=,5=- cyclic monophosphate; CNG channel, cyclic nucleotide-gated chan- nel; GC-D, guanylyl cyclase-D; MOB, main olfactory bulb; M/T cell, mitral/tufted cell; OSN, olfactory sensory neuron; PDE2A, phosphodi- esterase 2A; PSTHs, peri-stimulus time histograms; 2,5-DMP, 2,5- dimethyl pyrazine. Neuroscience 170 (2010) 881– 892 0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.08.006 881