As previously described for IR25a ( Benton et al., 2009), IR8a is detected in both the cell body and sensory dendritic endings,

but not in axon termini, consistent RO4929097 concentration with a role in peripheral odor detection (Figures 1C and 3B; data not shown). To confirm the specificity of these antibodies and initiate functional analysis of these receptors, we generated null mutations in IR8a by the same gene-targeting strategy used for IR25a ( Benton et al., 2009) ( Figure 2A). Homozygous IR8a, IR25a, and IR8a/IR25a double mutant animals are viable and fertile, and all corresponding immunoreactivity in the antenna is abolished ( Figure 2A). The broad antennal expression and cilia localization of IR8a and IR25a indicated that these receptors might have a widespread role in odor Epacadostat clinical trial detection in IR neurons. We tested this hypothesis by performing extracellular electrophysiological recordings of odor-evoked neuronal responses in individual coeloconic sensilla in IR8a and IR25a mutants. Four classes of coeloconic sensilla have been defined, named ac1 through ac4 ( Benton et al., 2009 and Yao et al., 2005). These house neurons that express different combinations of IRs and display distinct odor sensitivities ( Figure 2B) ( Benton et al., 2009 and Yao et al., 2005), although matching of the specific ligands to receptors has yet to be determined in most cases. We tested a panel of seven odors, representing the

best sensilla-specific agonists identified in previous or on-going ligand screens ( Yao et al., 2005; R. Rytz and R. Benton, unpublished data), which we assume are recognized by different IRs. The ac4 sensilla contain three neurons and are stimulated by phenylacetaldehyde and phenylethyl amine (Figure 2B). We observed that IR8a mutant ac4 sensilla lack all responsiveness to phenylacetaldehyde, while phenylethyl amine responses are unchanged ( Figures

2B and 2C). Spontaneous activity in IR8a mutant ac4 sensilla (25 ± 4 spikes/s [mean ± SEM]; n = 13) is also markedly reduced compared with the wild-type (82 ± 1 spikes/s; n = 11). These electrophysiological phenotypes resemble those of mutants in IR84a (Y. Grosjean Idoxuridine and R.B., unpublished data), suggesting a role for IR8a in acting with this receptor in mediating both basal and evoked responses in IR84a neurons. By contrast, mutation of IR25a had no effect on phenylacetaldehyde responses but abolished phenylethyl amine-evoked activity ( Figures 2B and 2C), suggesting an essential function in a different IR neuron. Spontaneous activity in IR25a mutant ac4 sensilla was also reduced (43 ± 6 spikes/s; n = 18). The ac3 sensilla house two neurons, one of which expresses three IRs (IR75a, IR75b, and IR75c) and responds to propionic acid (Benton et al., 2009 and Yao et al., 2005). Responses to this ligand are abolished in IR8a, but not IR25a, mutants ( Figure 2B).