Transgenic mouse expressing synaptopHluorin (spH) in mitral and tufted cells. (A, B) Fluorescence of whole-mount brain of Tbx-spH transgenic mouse viewed from ventral (A) and lateral side (B). (C1-C3) spH signal increase in the piriform cortex is dependent on the pulse numbers of electrical stimulation onto olfactory bulb (OB) glomeruli. ΔF/F fluorescence change images (C1), time course (C2) and pulse-response relationship (C3). (D1, D2) spH signal increase is dependent on the amplitude of electrical stimulation onto OB glomeruli. Time course (D1) and amplitude-response relationship (D2). (E1-F3) spH signal increase upon the electrical stimulation of trimethylthiazoline (TMT; E1-E3) and heptanoic acid (HA)-responsive (F1-F3) glomeruli. Odourant-responsive glomeruli were identified by the intrinsic signal imaging of the dorsal surface of OB and stimulated with a microelectrode (E1, F1). The electrical stimulation of these two glomeruli leads to the spH signal increase in distinct subregions of the piriform cortex (E2, E3, F2, F3). (G1) Similarity and dissimilarity between fluorescent responses evoked by the OB electrical microstimulation. The imaged region was divided into four subregions (APCd, APCv, PPCd, PPCv: dorsal and ventral parts of the anterior and posterior piriform cortex) [40, 43]. Significantly responsive areas (P < 0.01, yellow-to-red areas in E2-3 and F2-3) evoked by electrical stimulation of TMT- and HA-responsive glomeruli were plotted (n = 3). (G2) Cluster analysis of spH responses. The linkage distance was 4.95 ± 1.70 between any two TMT stimulation images and 22.4 ± 4.53 between any two HA stimulation images. In contrast, the distance between any TMT stimulation image and any HA stimulation image was significantly larger (58.0 ± 1.95; P< 0.00001). (H1, H2) spH signal increase in the piriform cortex upon odourant stimulation. TMT (H1) or HA (H2) applied into mouse nostril leads to the spH signal increase in overlapping but distinct regions of the piriform cortex.