These findings indicate that the response differences between these deeper neurons are location dependent. To better understand the anatomical and

functional organization of a single glomerular module, we studied the anteromedial area of the dorsal OB because odorants that yield strong activation of this area have been identified (see Figure S1A available online; Uchida et al., 2000; Wachowiak and Cohen, 2001). Heterozygous knockin mice that expressed the synapto-pHluorin (spH) protein (a genetically encoded pH-sensitive fluorescent protein that reports synaptic vesicle fusion) under control of the olfactory marker protein promoter (OMP-spH mice) were used to visualize glomeruli (Bozza et al., 2004). A glass pipette filled with a calcium indicator Saracatinib concentration dye (dextran-conjugated Oregon Green BAPTA-1) was guided by two-photon imaging and used to penetrate a target glomerulus. The neurons that were associated with the single glomerulus were labeled by our previously established electroporation method (Figures 1A, 1B, and S1B; Nagayama et al., 2007). Using this method, we were able to clearly visualize multiple neurons that were all associated with a single target glomerulus in the glomerular layer (GL), external plexiform layer (EPL), and even

in the mitral cell layer (MCL) (Figures 1C–1F and Movie S1; 11.4 ± 1.8 cells per glomerulus, mean ± standard error of the mean [SEM]). As can be observed in Movie S1, the labeled dendrites were heterogeneous within the glomerulus U0126 nmr and there were small parts of the glomerulus that did not appear to be labeled. These data support the idea of anatomical compartmentalization within glomerular formations (Kasowski et al., 1999). Representative examples of glomerular structure and component neurons are shown in Figures 1D–1E. The labeled cells were grouped based on layers of soma locations, cell shapes, cell sizes,

and whether lateral dendrites were present (L-Dends; Figures 1F and S1C–S1E; Table S1). Although only a small population of neurons within a glomerular module was labeled in each trial, these data enabled us to visualize the anatomical connectivity within a single glomerulus module and to compare odorant response properties and between multiple neuronal subtypes associated with the same glomerulus. To investigate the anatomical architecture of a glomerular module, we first analyzed distribution patterns of cells associated with a single glomerulus (263 cells in 23 glomeruli). The labeled cells in each layer were plotted on an x-y horizontal plane that was centered on the glomerulus that had been injected with dye (Figures 2A–2C). Labeled neurons were observed in every direction from the glomerulus, and were particularly prominent in the GL and EPL. However, closer observation showed that the distribution was not isotropic, as more neurons were observed in the caudomedial area (Figure 2D).