Consistent with a previous report BMS-354825 in vivo ( Okada et al., 2009), we found that the vast majority of Mz699+ ventral PNs were

GABAergic based on GABA staining ( Figure 1F; 87.6% ± 2.5% GABA positive, from 8 antennal lobes with an average of 55 cells per lobe). As described later, the ventral PNs provide inhibition that requires GABA synthesis. Thus, we refer to them hereafter as inhibitory PNs (iPNs) to distinguish them from the excitatory PNs (ePNs) from the anterodorsal and lateral lineages. Additionally, we examined Mz699+ vlpr neurons that also project to the lateral horn as putative higher-order neurons in the olfactory pathway. Neuroblast and single-cell clone analyses of vlpr neurons showed that a subset projected to the

lateral horn as well as the vlpr neuropil (Figures 1D and 1E). Both processes were enriched Galunisertib for Syt-HA (Figures S1D and S1E), similar to several other lateral horn neurons, including those that connect the lateral horn to the vlpr (Jefferis et al., 2007). Furthermore, in the single-cell clone of vlpr neurons, Syt-HA+ puncta are distributed through the processes in the vlpr neuropil including most of the terminals. In the lateral horn, however, the neural processes end with fine branches without Syt-HA puncta (Figure S1E). This result suggests that Mz699+ vlpr neurons mostly send information from the lateral horn to the vlpr neuropil. Thus, these lateral horn-projecting Mz699+ vlpr neurons represent a subset of putative third-order neurons in the lateral horn. None of the Mz699+ vlpr neurons were GABA positive (Figure 1G). Below, we used their odor-evoked response as a means to investigate the role of iPN function in olfactory signal processing. To investigate the function of iPNs, we first

examined their odor responses utilizing two-photon Sclareol Ca2+ imaging in alert flies labeled by Mz699-GAL4 driving UAS-GCaMP3 ( Tian et al., 2009). When we applied 500 ms pulses of 0.1% isoamyl acetate (IA), a major component of the banana odor, to the antennae, we observed a robust increase of Ca2+ signals in the antennal lobe ( Figure 1H; Figure S2A), the lateral horn ( Figure 1I), and the mACT tract before it enters the lateral horn neuropil ( Figure 1I, arrow). Application of 1% apple cider vinegar gave similar results (data not shown; see below). We further tested Ca2+ response of iPNs to IA applied at different concentrations in the antennal lobe ( Figures S2C and S2E). At low IA concentration, the response was sparse and weak. As the odor concentration increased, more glomeruli were recruited with elevated Ca2+ signals, similar to the concentration-dependent odor responses of ORN and ePN ( Hallem and Carlson, 2006 and Wang et al., 2003).