Here we overcome this challenge by using chronic two-photon calcium imaging with the genetically encoded calcium indicator GCaMP3. By selectively imaging the activity of ensembles of mitral cells and inhibitory granule cells, we show that the transition from the awake to anesthetized brain state modulates olfactory bulb circuits and odor coding. Furthermore, we monitored the dynamics Proteasome inhibitor of odor responses of the same populations of mitral cells over months in awake mice to test how odor experience affects olfactory bulb odor representations. These approaches revealed a surprisingly dynamic nature of odor representations, which is sensitive to brain state and experience. We expressed

GCaMP3 (Tian et al., 2009) specifically in olfactory bulb principal cells (mitral and tufted cells) by injecting a Cre recombinase-dependent viral vector (Atasoy et al., 2008) into the olfactory bulbs of protocadherin-21 (PCdh21)-Cre mice, which express Cre exclusively in olfactory bulb principal cells (Nagai et al., 2005). Several weeks after injection, virtually all glomeruli had GCaMP3-expressing dendrites, and immunostaining with a mitral/tufted cell-specific antibody (Tbx21) (Yoshihara et al., 2005) showed that the majority of Tbx21-positive cells (67%, n = 3 mice, 644 cells) express GCaMP3

(Figure 1B). Consistent with the selective expression in mitral/tufted cells, all GCaMP3-expressing cells (n = 2 mice, 323 cells) lacked immunoreactivity for GAD67, a marker for GABAergic interneurons (data not shown). We next performed simultaneous patch-clamp recording and two-photon Metalloexopeptidase imaging in olfactory bulb slices to test the ability of GCaMP3 to report click here action potential firing in mitral cells (GCaMP3-expressing cells in the mitral cell layer). We measured GCaMP3 fluorescence changes in mitral cells in response to spikes elicited at 50–100 Hz via brief depolarizing current steps (1 nA, 3 ms). In agreement with previous findings in cortical pyramidal cells (Tian et al., 2009), we found that increases in GCaMP3 fluorescence

had a relatively linear relationship with the number of action potentials evoked in mitral cells (Figures 1C and 1D). To optically monitor mitral cell activity in vivo, we implanted a cranial window (Holtmaat et al., 2009) over the dorsal olfactory bulb of GCaMP3-expressing mice. Using two-photon imaging selectively in the mitral cell layer allowed us to repeatedly image the same sets of up to 100 mitral cells over months in awake, head-fixed mice (Dombeck et al., 2007; Komiyama et al., 2010) (Figures 1E and 1F). Consistent with previous electrophysiological studies in anesthetized animals (Bathellier et al., 2008; Davison and Katz, 2007; Dhawale et al., 2010; Fantana et al., 2008; Meredith, 1986; Mori et al., 1992; Tan et al., 2010), passive application of structurally diverse odors elicited activity revealed by increases in GCaMP3 fluorescence in overlapping but distinct ensembles of mitral cells (Figure 1G).