Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish

Abstract

Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.

Fig. 1.

Construction and analysis of GCaMP-HS. (A) The structure of GCaMP-HS. The positions of the amino acid substitutions are indicated. (B) Excitation (blue) and emission (green) spectra of GCaMP-HS in Ca²⁺-chelated (20 mM BAPTA; dotted lines) and saturated (300 nM Ca²⁺; solid lines) solutions. Excitation and emission are maximum at 488 nm and 509 nm, respectively. (C) Restoration of the fluorescence of GCaMP-HS (solid line) and GCaMP2 (dotted line) after heat-denaturation. (D) Titration assay with defined Ca²⁺concentrations. Comparison of GCaMP-HS (solid line) and GCaMP2 (dotted line). Means and SD are shown (n = 3). (E) GFP fluorescence in HEK cells transfected with pN1-GCaMP2 (Left) and pN1-GCaMP-HS (Right). (Scale bar, 30 μm.) (F) The basal fluorescence intensity in HEK cells transfected with the GCaMP2 and GCaMP-HS plasmids. (G) The fluorescence changes upon addition of 100 μM carbachol to the GCaMP2- and GCaMP-HS–transfected cells. Means and SD from three independent transfection experiments. (H) Western blot analysis using an anticalmodulin antibody in three independent transfection experiments. Protein levels are increased in the GCaMP-HS–transfected cells.

Fig. 2.

GFP expression in the SAIGFF213A;UAS:GFP double transgenic embryos. (A) A side view at 24 hpf. GFP is expressed in the central nervous system. (Scale bar, 100 μm.) (B) A side view of the spinal cord at 24 hpf observed with a confocal microscope. Arrowheads indicate CaP motor neurons. In some cases, two CaP neurons are found in one spinal segment (asterisks). (Scale bar, 100 μm.) (C) The soma (arrowhead) and axon of the GFP-expressing single CaP neuron at 24 hpf. The CaP neurons project their axons to the ventral muscle (arrow). Dotted line shows the boundary between dorsal and ventral muscles. (Scale bar, 50 μm.)

Fig. 3.

Imaging of the activity of CaP motor neurons in the SAIGFF213A;UAS:GCaMPHS double-transgenic embryo during spontaneous contractions. (A) Time course of the frequency of spontaneous contractions between 17 and 27 hpf. (B) The increase of the maximal fluorescence changes (Upper) and the fluorescence at the resting state (Lower) of GCaMP-HS in the soma of the CaP motor neuron between 21 and 27 hpf. Means and SD are shown (n = 5). (C) A side view of the double-transgenic embryo at 18.5 hpf. Anterior to the left. GCaMP fluorescence was detected in the CaP neurons and some other neurons. The axons of the CaP neurons were used as ROI (–4). (Scale bar, 50 μm.) (D) The fluorescence changes in the ROI-1, -2, -3, and -4 are shown as graphs. Downward peaks at the left ends of ROI-1, -2, and -3 are artifacts caused by the movement of the embryo. (E) A dorsal view of the SAIGFF213A;UAS:GCaMPHS4A embryo at 24 hpf. Anterior to the left. The CaP neurons that are circled and numbered were used as ROIs. (Scale bar, 200 μm.) (F) The fluorescence changes in the ROI-1 (dotted line) and ROI-2 (solid line). Two consecutive peaks detected in ROI-2 are marked with asterisks.

Fig. 4.

Spatiotemporal patterns of activation of the CaP motor neurons. (A) A dorsal view of the SAIGFF213A;UAS:GCaMPHS4A embryo at 24 hpf treated with 100 μM d-tubocurarine. (Scale bar, 200 μm.) (B) The fluorescence changes in ROIs in A. (C) Cross-correlogram of the ipsilateral CaP neurons. (D) Cross-correlogram of the contralateral CaP neurons. (E) The averaged timing of the peaks of the ipsilateral CaP neurons located in a rostral (Left)-to-caudal (Right) sequence. Data from four embryos are shown. (F) A whole cell-patch recording and simultaneous calcium imaging of the CaP neurons during spontaneous contractions. Action potentials (gray) and calcium signals (black) are shown.