Brain-wide Ca2+ imaging in transgenic zebrafish during wakefulness, anesthesia, and recovery states

The experiment utilized a double transgenic zebrafish line, Tg(elavl3:GCaMP6f)jf1Tg;Tg(pou4f3:GAP-GFP)s356Tg, to achieve pan-neuronal calcium indicator expression and sparse labeling of neuromast cells as registration landmarks. The zebrafish was placed in a custom quartz behavioral arena without physical restraint. The recording protocol proceeded sequentially: a 3-minute awake state recording , perfusion of 60 µmol/L propofol to induce anesthesia with a 3-minute recording immediately preceding the loss of righting reflex (LORR) , and a final 3-minute recording following washout with system water and natural recovery. All experiments were conducted on juvenile zebrafish at 30 days post-fertilization (dpf). Zebrafish were maintained under standard conditions (14 h light/10 h dark cycle, 26–28 °C) prior to the experiments. A custom built wide-field fluorescence imaging system was used for simultaneous behavioral tracking and calcium imaging. The system consisted of a sCMOS camera (Kinetix, Teledyne) mounted on an MVX10 macroscope (Olympus), capturing images at 250 Hz. Illumination was provided by two 465 nm line-shaped lasers positioned bilaterally to ensure uniform excitation of GCaMP6f fluorescence. A custom C#-based synchronization program, interfaced with data acquisition boards (PXIe-5711 and PXI-62020, JYTEK), coordinated the timing of camera triggering and perfusion control.Raw image stacks were first processed to correct for motion artifacts. Using the stable GFP signal from neuromast cells as fiducial landmarks, two or three rounds of rigid image registration were performed with ANTs software. After registration, the image stacks were upsampled by a factor of two using bilinear interpolation to enhance spatial resolution. Neuronal activity signals (ΔF/F0) and spatial coordinates of individual neurons were extracted using the CaImAn algorithm package (CNMF method). Behavioral data were derived from the same raw image stacks. The centroid of the zebrafish brain (identified as the maximum connected region of GCaMP6f fluorescence) was tracked across frames to generate swimming trajectories. The coordinate update interval for each frame in the data file was 0.004 seconds.All data are stored in standard formats to ensure accessibility and reproducibility. The dataset contains three recording sequences. Under each of the three state (awake, anesthesia, recovery), a sequence consists of a 3-minute recording with a sampling rate of 250 Hz, resulting in 45,000 image frames in each sequence. The images are cropped and saved as TIFF image stacks, while the extracted calcium signal and coordinate of neurons are saved in plain text csv format. The provided data include raw segmented TIFF images with brain region masks, which have not been aligned across states; extracted and denoised calcium transients (ΔF/F0); neuronal spatial coordinates calculated after aligning all images to a common coordinate system; and behavioral tracking coordinates of the zebrafish.We recommend using the ΔF/F0 files (dff.csv) for analysis, as the neuron IDs in these csv files correspond directly to those in the neuron coordinate files (co.csv), allowing data users to combine the spatial coordinates with signal fluctuations to map the functional network structures of neurons into anatomical space for high-resolution spatiotemporal dynamic analysis. Furthermore, the high dimensionality and variability inherent to large-scale neural networks may make cluster boundaries and node alignment sensitive to the choice of hyperparameters. We recommend that users experiment with more robust soft clustering or advanced network alignment algorithms.The dataset is organized as follows:f1-<state>.tar.part: Split TIFF file archives wide-field fluorescent microscopy image data, with brain region mask for zebrafish.f1-<state>-dff.csv: calcium signal (ΔF/F0) of index neurons, corresponding to neuronal activities.f1-<state>-co.csv: Coordinates in brain for the indexed neuron.f1-motion-<state>-co.txt: Zebrafish behavioral tracking data.