The fluorescent images were split by dual-view with a CFP/YFP filter set and projected onto the CCD camera operated by Volocity (Improvision, Lexington, MA)

or MicroManager (http://micro-manager.org). The 4×-binned images were obtained at 50–100 ms GSK126 mw exposures, 10–20 fps for 5 min. The YFP and CFP fluorescent intensities were measured by in-house-developed ImageJ plugins (http://rsb.info.nih.gov/ij). Calcium imaging experiments were performed either by manually recentering moving animals on the stage or through an in-house-developed acquisition software that controls the camera and motorized stage through Micromanager and ImageJ. Each frame of the acquired images was subjected to real-time processing to detect targeted cells, track objects, record stage positions, and recenter the tracked object. During postimaging processing,

two regions of interest Transmembrane Transporters activator were set to detect the anterior-posterior axis. VA8 and DB6 were used as anterior and posterior cells, respectively, in motoneuron imaging. AVA/AVE or AVB and cluster of cells were used in interneuron imaging. The cell position at each time point was determined based on the coordinates of the stage position and cell position in the field of view, and the velocity was calculated by changes in the cell position between each frame. The forward and backward directions were determined by comparing changes in the anterior-posterior axis. Interneuron imaging was performed under two different conditions. (1) Imaging when animals were allowed relatively free movement (Figures 1D, 1E, and 6). This condition allows correlation between motion and changes in calcium

signals. Animals were placed on freshly made 2% Amisulpride wet agarose pads, mounted with a few microliters of M9 buffer, and imaged with a 16× objective through the automated tracking system. We imaged multiple interneurons as a single ROI in the head region of hpIs190 (AVA and AVE) or a single interneuron as an ROI in hpIs179 (AVB). (2) Imaging when animals were allowed restricted movement. Single-neuron imaging of AVA or AVE with hpIs157 and hpIs190 ( Figure 1C; Figures S1B, S1C, S3, and S7), and AVB and AVE simultaneous imaging in a strain carrying both hpIs157 and hpIs179 ( Figure 1F), was carried out under this condition. In both hpIs157 and hpIs190, the closely spaced cell bodies prevented precise tracking at individual neuron resolution when animals were allowed free movement. Animals were mounted on dried 5% agarose pads with a few microliters of M9 buffer, covered by a coverslip, and imaged with a 63× objective.