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Genetic Visualization with an Improved Gcamp Calcium Indicator Reveals Spatiotemporal Activation of the Spinal Motor Neurons in Zebrafish

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Genetic Visualization with an Improved Gcamp Calcium Indicator Reveals Spatiotemporal Activation of the Spinal Motor Neurons in Zebrafish Genetic visualization with an improved GCaMP calcium indicator reveals spatiotemporal activation of the spinal motor neurons in zebrafish Akira Mutoa, Masamichi Ohkurab, Tomoya Kotania,1, Shin-ichi Higashijimac, Junichi Nakaib,2, and Koichi Kawakamia,d,2 aDivision of Molecular and Developmental Biology, National Institute of Genetics, and dDepartment of Genetics, Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka 411-8540, Japan; bBrain Science Institute, Saitama University, Sakura-ku, Saitama 338-8570, Japan; and cNational Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, Okazaki, Aichi 444-8787, Japan Edited by Thomas M. Jessell, Columbia University College of Physicians and Surgeons, New York, NY, and approved February 8, 2011 (received for review January 23, 2010) Animal behaviors are generated by well-coordinated activation of (6, 7). Previously we created a fusion of the calmodulin-binding neural circuits. In zebrafish, embryos start to show spontaneous domain from the myosin light-chain kinase (which is called an M13 muscle contractions at 17 to 19 h postfertilization. To visualize how peptide), permutated EGFP, and the calmodulin, and named it motor circuits in the spinal cord are activated during this behavior, GCaMP (7). GCaMP increases its intensity of green fluorescence we developed GCaMP-HS (GCaMP-hyper sensitive), an improved upon binding to calcium ions. Since the first version was created, version of the genetically encoded calcium indicator GCaMP, and efforts have been made to generate brighter and more sensitive created transgenic zebrafish carrying the GCaMP-HS gene down- versions of GCaMP by introducing amino acid substitutions (8–10). stream of the Gal4-recognition sequence, UAS (upstream activation In this study, we aim to visualize a motor network during the sequence). Then we performed a gene-trap screen and identified the first behavior in zebrafish. For this purpose, we used the GCaMP SAIGFF213A transgenic fish that expressed Gal4FF, a modified ver- technology and the Gal4FF-UAS (upstream activator sequences) sion of Gal4, in a subset of spinal neurons including the caudal pri- system, which also was described recently in zebrafish (11). First, mary (CaP) motor neurons. We conducted calcium imaging using the we constructed an improved version of GCaMP, GCaMP-HS SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the (GCaMP-hyper sensitive), which is brighter at the resting level spontaneous contractions. We demonstrated periodic and synchro- and more sensitive to the change of cellular Ca2+ concentrations nized activation of a set of ipsilateral motor neurons located on the than the previous version. Second, to express GCaMP-HS under right and left trunk in accordance with actual muscle movements. the control of the Gal4FF transcription activator, we constructed The synchronized activation of contralateral motor neurons oc- an effector fish line that carried the GCaMP-HS gene down- curred alternately with a regular interval. Furthermore, a detailed stream of the Gal4 recognition sequence UAS. Third, we per- analysis revealed rostral-to-caudal propagation of activation of the formed gene-trap screens and isolated a driver line that expressed ipsilateral motor neuron, which is similar to but much slower than Gal4FF in a specific subset of motor neurons. Here we demon- the rostrocaudal delay observed during swimming in later stages. strate successful imaging of the activity of the motor neurons Our study thus demonstrated coordinated activities of the motor during the spontaneous contractions in the vertebrate embryos. neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a pow- Results fi erful tool to study functional neural circuits in zebra sh. Development of GCaMP-HS. Previously, it was shown that amino acid substitutions in GFP could enhance the folding activity and thereby neuronal activity | calcium transient | transgenesis | Tol2 | gene trapping increase its fluorescence. This more recent version of GFP was called “superfolder GFP” (12). To develop a better GECI, we tested if the ertebrate behaviors are generated by coordinated activation substitutions described in the superfolder GFP can improve GCaMP Vof neural networks. The zebrafish provides an excellent sys- in terms of the brightness and the signal amplitude. We introduced tem to study the neural activities of vertebrates because of its four of the six superfolder mutations, S30R, Y39N, N105T in the transparency and external development. It has been reported that N-terminus half of GFP, and A206V in the C-terminus half of GFP, zebrafish embryos show a first locomotive activity as early as 17 h which were shown to have more effects in folding (12), into the postfertilization (hpf), which is called spontaneous contraction previous version of GCaMP, GCaMP2 (9), and named the new or coiling; namely, they spontaneously contract the left and right version of GCaMP GCaMP-HS (Fig. 1A). trunk muscles alternatively without any external stimulus. This First, we purified the GCaMP-HS protein produced in bac- behavior precedes touch-evoked escape response and swimming, teria and characterized it in vitro. Spectrum analysis of GCaMP- which emerge in later developmental stages (1). The neuronal HS showed maximal excitation at 488 nm and maximal emission activities during the spontaneous contractions were analyzed by at 509 nm in the presence of 300 nM Ca2+, which were similar to electrophysiological approaches, and periodic and synchronized depolarization of the spinal neurons has been observed (2, 3). However, it is still challenging to analyze the activity of a neural Author contributions: A.M., M.O., J.N., and K.K. designed research; A.M., M.O., T.K., network by electrophysiology because it requires recording from S.-i.H., J.N., and K.K. performed research; A.M., M.O., T.K., J.N., and K.K. contributed new reagents/analytic tools; A.M., M.O., S.-i.H., J.N., and K.K. analyzed data; and A.M., multiple neurons by using multiple electrodes. To understand M.O., J.N., and K.K. wrote the paper. how functional neural circuits are formed and operated, a new The authors declare no conflict of interest. technology to monitor the activity of multiple neurons is desired. This article is a PNAS Direct Submission. An alternative approach to record activities from multiple 1 neurons is to detect Ca2+ influx associated with generation of ac- Present address: Laboratory of Reproductive and Developmental Biology, Faculty of fl Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan. tion potentials. For this purpose, uorescent calcium-indicator 2 To whom correspondence may be addressed. E-mail: [email protected] or jnakai@ dyes, which should be introduced into cells by microinjection or by mail.saitama-u.ac.jp. bulk-loading using their acetoxymethyl ester forms (4, 5), or ge- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NEUROSCIENCE netically encoded calcium indicators (GECIs) have been used 1073/pnas.1000887108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1000887108 PNAS | March 29, 2011 | vol. 108 | no. 13 | 5425–5430 Downloaded by guest on September 30, 2021 A E GCaMP2 GCaMPHS BCD FG H GCaMP2 GCaMPHS 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)GFPfluorescence 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 anti- body in three independent transfection experiments. Protein levels are increased in the GCaMP-HS–transfected cells. those of GCaMP2 (Fig. 1B). To test the effect of the superfolder that carried a single transposon insertion and showed the brightest mutations on the folding activity, we analyzed restoration of the fluorescence when crossed with the Gal4FF-expressing fish lines. fluorescence after heat-denaturation of the GCaMP proteins. We named the fish line and the insertion UAS:GCaMPHS4A. The The GCaMP-HS showed higher efficiencies of restoration (Fig. SAGFF73A;UAS:GCaMPHS4A double-transgenic embryo was 1C), indicating that the substitutions introduced in GCaMP-HS embedded in agarose at 5 d postfertilization (dpf) and observed fl increased the refolding activity. Then we measured their uo- under a fluorescence microscope. We could detect the fluores- rescence intensities at various Ca2+ concentrations. From this 2+ cence changes in the trunk muscles in accordance with twitch- Ca titration assay, we calculated that Kd and Hill coefficient of D K ing (Movie S1). Therefore, the UAS:GCaMPHS4A transgenic GCaMP-HS were 102 nM and 5.0, respectively (Fig. 1 ). d and fish was used for further studies. Hill coefficient of GCaMP2 were 146 nM and 3.8, respectively, fi 2+ indicating that GCaMP-HS has a higher af nity to Ca ions and Identification of a Driver That Expressed Gal4FF in a Subset of the a higher cooperativity in binding to Ca2+. The actual values of − fl fl Primary Motor Neurons in the Spinal Cord. Spontaneous contraction (Fmax Fmin)/Fmin (maximal uorescence minus minimal uo- is the first behavior of a zebrafish embryo. It can be observed as rescence per minimal fluorescence) were comparable: 4.1 in early as 17 hpf, reaches the maximum frequency at 19 hpf, and GCaMP-HS and 3.9 in GCaMP2.
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