GCaMP5G is a highly improved genetically encoded calcium indicator (Akerboom et al. 2012, submitted). It exhibits a significantly enhanced level of fluorescence change upon binding calcium as compared to previous versions (Nakai 2001, Tian et al. 2009). Using the Gal4/UAS system (Brand and Perrimon 1993), we expressed GCaMP5G in all central neurons where a 7.4 kb cholinergic promoter is active (Salvaterra and Kitamoto 2001). To prepare the CNS capsule of a third instar larva for live-imaging we used a semi-intact preparation called “CNS-hoisting” (Iyengar 2012). The movie above shows rhythmic GCaMP fluorescence oscillations in the thoracic compartment of the ventral cord. Despite a noticeable level of movement one can identify several interesting patterns of localized calcium transients within the neuropile and cell bodies. One key feature is the neuronal activity in what appears to be a fiber tract near the midline of the ventral cord. This may represent an ascending interneuronal fiber whose cell body is located at the posterior tip of the ventral cord (Iyengar et al. 2011). This video was captured using a 20X water-immersion objective at 4 fps (played back at 8 fps) using a wide-field calcium imaging microscope with an Andor Technology EMCCD camera (see Chouhan et al. 2012 for details). The ventral cord is oriented with anterior to the top. The authors would like to thank Adam Rossano and Greg Macleod (UTHSCSA) for the technical help. References: Akerboom J, Chen TW, Wardill TJ, Tian L, Marvin JS, Mutlu S, Calderon NC, Esposti F, Borghuis BG, Sun XR, Gordus A, Orger MB, Portugues R, Engert F, Macklin JJ, Filosa A, Aggarwal A, Kerr R, Takagi R, Kracun S, Shigetomi E, Khakh BS, Baier H, Lagnado L, Wang SSH, Bargmann CI, Kimmel BE, Jayaraman V, Svoboda K, Kim DS, Schreiter ER, Looger LL. 2012. Submitted. Optimization of a GCaMP calcium indicator for neural activity imaging. Akerboom J, Rivera JD, Guilbe MM, Malavé EC, Hernandez HH, Tian L, Hires SA, Marvin JS, Looger LL, Schreiter ER. 2009. J Biol Chem. 284:6455-64. Crystal structures of the GCaMP calcium sensor reveal the mechanism of fluorescence signal change and aid rational design. PMID:19098007 Brand AH, Perrimon N. 1993. Development. 118:401-15. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. PMID:8223268 Chouhan AK, Ivannikov MV, Lu Z, Sugimori M, Llinas RR, Macleod GT. 2012. J Neurosci. 32:1233-43. Cytosolic calcium coordi...