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 antibody 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. These findings suggested that gradually declines by 26 hpf (1). We aimed to visualize the activity GCaMP-HS may be used to detect Ca2+ transient where Ca2+ concentrations are low. of the motor circuit during the spontaneous contraction. To isolate Second, we tested the cellular performance of GCaMP-HS. a driver line that expresses Gal4FF in the primary motor neurons, Tol2 The expression plasmids pN1-GCaMP-HS and pN1-GCaMP2 we performed genetic screens using the -based gene-trap were used to transfect HEK293 cells. The cells transfected with constructs T2KSAGFF and T2TSAIGFF. We observed offspring ∼ fi fi pN1-GCaMP-HS showed ∼1.6-fold higher fluorescence at the from 500 sh injected with these constructs and identi ed the resting levels (Fig. 1 E and F), and 1.4-fold intensity changes when activated with 100 μM carbachol (Fig. 1G). We analyzed the cellular protein levels by Western blotting and found that the levels of the GCaMP-HS protein were constantly 1.7-fold higher A C than those of the GCaMP2 protein (Fig. 1H), suggesting that the higher folding activity increased the cellular expression level, and hence increased the basal fluorescence of GCaMP-HS.

Construction of the UAS:GCaMP-HS Transgenic Zebraﬁsh. We aimed

to apply the new GCaMP-HS to in vivo imaging of neuronal ac-

tivities in zebrafish. To take advantage of the Gal4FF-UAS system B we described previously (11), we constructed the T2RUASG- * CaMP-HS plasmid that carried the GCaMP-HS gene downstream of the upstream activation sequence (UAS). The T2RUASG- CaMP-HS plasmid was injected with the transposase mRNA to fertilized eggs, and the injected fish were crossed with the hspGGFF27A fish that expressed the Gal4FF-GFP fusion protein Fig. 2. GFP expression in the SAIGFF213A;UAS:GFP double transgenic em- fi bryos. (A) A side view at 24 hpf. GFP is expressed in the central nervous in the central nervous system or with the SAGFF73A sh that μ expressed Gal4FF ubiquitously (11, 13). All of four injected fish system. (Scale bar, 100 m.) (B) A side view of the spinal cord at 24 hpf observed with a confocal microscope. Arrowheads indicate CaP motor neurons. used for the mating produced transgenic F1 embryos that carried In some cases, two CaP neurons are found in one spinal segment (asterisks). the T2RUASGCaMP-HS transgene in their offspring. We ana- (Scale bar, 100 μm.) (C) The soma (arrowhead) and axon of the GFP- lyzed the F1 fish by Southern blot hybridization and determined the expressing single CaP neuron at 24 hpf. The CaP neurons project their axons number of insertions carried by these fish. Then we crossed the F1 to the ventral muscle (arrow). Dotted line shows the boundary between fish further with wild-type fish, and finally identified transgenic fish dorsal and ventral muscles. (Scale bar, 50 μm.)

5426 | www.pnas.org/cgi/doi/10.1073/pnas.1000887108 Muto et al. Downloaded by guest on September 30, 2021 SAIGFF213A fish. In the SAIGFF213A;UAS:GFP double- A B transgenic embryos, GFP was expressed rather ubiquitously in the central nervous system at 24 hpf (Fig. 2A). In the spinal cord, several different types of neurons, including Rohon-Beard neurons, subpopulations of interneurons, and a set of neurons that were located in the ventral spinal cord, one per each spinal segment, that extended their axons to the ventral muscles were identifiable (Fig. 2 B and C). The zebrafish embryos have three types of the primary motor neurons in the spinal cord, middle primary neurons (MiP), rostral primary neurons (RoP), and caudal primary neurons (CaP). Among them, only the CaP motor neurons extend their axons to the ventral muscles (14). From the location and the morphology, we C concluded that the SAIGFF213A;UAS:GFP expressed GFP in the CaP motor neurons, and MiP and RoP, which should be detected adjacent to CaP, did not express GFP at the detectable levels. In 3 4 some cases, two CaP neurons were observed in one segment, as 2 reported previously (15) (Fig. 2B). The GFP expressing CaP motor 1 neurons were detectable as early as 17 hpf (the 16-somites stage) and observable at 2 dpf, and then the expression was gradually de- creased and hardly detected at 7 dpf, although GFP expression in D the other neurons were still observable (Fig. S1). We analyzed the 1 Tol2 insertion in the SAIGFF213A line and found that the insertion was mapped close to the prdm14 gene on the chromosome 24. 2 Spontaneous Contractions and Detection of the Activity of the CaP Primary Motor Neurons. We observed the spontaneous contractions in our laboratory conditions (∼28 °C). The spontaneous con- 3 tractions started around 19 hpf, showed the maximum frequency (0.6 Hz) at 21 hpf, and gradually ceased by 27 hpf (Fig. 3A and Movie S2). The 2-h delay of the start time in comparison with the 4 previous work (1) may be a result of slightly lower temperatures in our laboratory condition or to a difference in the fish line we used. To analyze how motor circuits are activated during this behavior, E we crossed the UAS:GCaMPHS4A fish with the SAIGFF213A 1 fish and created double-transgenic embryos. Then we carried out imaging of the Ca2+ transient in the soma of the CaP neurons 2 between 21 and 27 hpf. We found that the maximal changes of the fluorescence increased from 53% (21 hpf) to 400% (27 hpf) as the * fl F * * motor neurons matured; the basal uorescence was nearly con- 1 * * stant (Fig. 3B). 2 20% First, we analyzed the activity of the CaP neurons at early de- 2 sec velopmental stages. At 18.5 hpf, an embryo contracted its trunk muscles at a frequency less than 0. 1 Hz (Fig. 3A). We performed imaging of the SAIGFF213A;UAS:GCaMPHS4A double trans- Fig. 3. Imaging of the activity of CaP motor neurons in the SAIGFF213A; genic embryo embedded in agarose at 18.5 hpf (Fig. 3C and Movie UAS:GCaMPHS double-transgenic embryo during spontaneous contractions. S3). We used axons of the CaP neurons as ROIs (regions of in- (A) Time course of the frequency of spontaneous contractions between 17 fl and 27 hpf. (B) The increase of the maximal fluorescence changes (Upper) terest) because the uorescence changes were obvious in the fl axons but were hardly detected in the soma at this stage. Periodic and the uorescence at the resting state (Lower) of GCaMP-HS in the soma fl of the CaP motor neuron between 21 and 27 hpf. Means and SD are shown uorescence changes at a frequency of 0.03 Hz were detected in (n = 5). (C) A side view of the double-transgenic embryo at 18.5 hpf. Anterior about half of single CaP neurons observed (ROI-2 and ROI-4 in to the left. GCaMP fluorescence was detected in the CaP neurons and some Fig. 3 C and D). The fluorescence changes in the other half of CaP other neurons. The axons of the CaP neurons were used as ROI (1–4). (Scale neurons were rarely detected (ROI-1 and ROI-3 in Fig. 3 C and bar, 50 μm.) (D)Thefluorescence changes in the ROI-1, -2, -3, and -4 are D). We found that the periodic fluorescence changes in different shown as graphs. Downward peaks at the left ends of ROI-1, -2, and -3 are ipsilateral CaP neurons were synchronized at the temporal reso- artifacts caused by the movement of the embryo. (E) A dorsal view of the lution of 10 fps (frames per second) (ROI-2 and ROI-4 in Fig. SAIGFF213A;UAS:GCaMPHS4A embryo at 24 hpf. Anterior to the left. The D CaP neurons that are circled and numbered were used as ROIs. (Scale bar, 3 ). Moreover, nonperiodic activation of a CaP neuron also oc- μ fl curred simultaneously with activation of the other synchronized 200 m.) (F)The uorescence changes in the ROI-1 (dotted line) and ROI-2 D (solid line). Two consecutive peaks detected in ROI-2 are marked with CaP neurons (ROI-1 in Fig. 3 ), suggesting these neurons are asterisks. physically connected or receiving common inputs at this stage. It should be noted that actual muscle contractions were not associated with 46% (18 of 39) of the cases where the fluorescence ipsilateral CaP neurons were detected in the embryos embedded in increases were detected in 10 different CaP neurons. These ± n findings indicate that the motor network to generate periodic and agarose with a frequency of 0.41 0.24 Hz ( = 20 embryos) (ROI- E F synchronized activation starts to form before the muscle con- 1 and -2 in Fig. 3 and ,andMovie S4). It should be noted that tractions are fully coupled with the activity of the motor network. the muscle contractions occurred concomitantly with the fluores- Second, we carried out imaging at 10 fps using the same cence increases of GCaMP-HS at this stage. Occasionally, two

SAIGFF213A;UAS:GCaMPHS4A double-transgenic embryo at consecutive contractions occurred at the same side. In such cases, NEUROSCIENCE 24 hpf. The periodic synchronized ﬂuorescence changes in the we observed two consecutive peaks in the ipsilateral CaP neurons

Muto et al. PNAS | March 29, 2011 | vol. 108 | no. 13 | 5427 Downloaded by guest on September 30, 2021 (asterisks in Fig. 3F). The synchronized activation of contralateral detected the fluorescence change with a frequency of 0.39 ± 0.10 Hz motor neurons occurred alternately with approximately regular (n =6)(0.41± 0.24 Hz in uncut embryos), suggesting that the intervals (Fig. 3F). surgery had little effect on the frequency. We applied the cross-correlogram, which is commonly used to Spatiotemporal Activation of the Motor Network: Synchronization, analyze correlation of timing of spike trains (16), to the analysis Alternation, and Propagation. As it was difficult to calculate the of the calcium signals (Fig. 4 C and D). The cross-correlogram of brightness of ROIs precisely in embryos that moved in accordance two ipsilateral neurons created a peak at zero, indicating that the with muscle contractions, we paralyzed the embryo with a neuro- activation of ipsilateral neurons signals were synchronized (Fig. muscular junction blocker, D-tubocurarine, and performed imaging 4C). The synchronized activation of the right and left side oc- at 10 fps. Because it is not cell-permeable, the embryos were soaked curred alternately in both tubocurarine-untreated and treated in 100 μM D-tubocurarine and their tail tips were cut. The periodic embryos. The cross-correlogram of pairs of contralateral neurons and synchronized fluorescence changes with a frequency of 0.67 ± showed the peak was shifted by a 0.46 to 0.53 cycle (n = 6 pairs 0.11 Hz (n = 5 embryos) were detected in the tubocurarine-treated from 6 embryos), indicating relatively precise alternate activation embryos, which was comparable but slightly faster than the fre- of the contralateral neurons (Fig. 4D). quencies detected in the untreated embryos (ROI-1, -2, -3, and -4 in Although the imaging at 10 fps demonstrated synchronized ac- Fig. 4 A and B and Movie S5). We also imaged embryos whose tails tivation of ipsilateral CaP neurons, one could imagine activation of were cut but not treated with D-tubocurarine as a control, and motor neurons in a rostral-to-caudal sequence, which was detected during a swimming behavior in later developmental stages (17). To test if propagation of the CaP activation occurs during the spontaneous contractions and if GCaMP-HS can detect such rapid A 1 2 propagation, we performed imaging at 20 fps. Although time resolution of 20 fps was still too low to directly observe the propaga- 3 4 tion during a single activation event, the averaged timing of the peaks of each CaP neuron during a total of 7-min imaging revealed delayed activation of CaP neurons located caudally (Fig. 4E). With linear regression [the coefficient of determination (R2) was in the B 1 range of 0.8–0.85], the velocity of the propagation of CaP activation 2 was calculated as 5.6 μm/ms ± 0.8 (mean ± SE, n = 4 embryos). Finally, to get insight into correlation between Ca2+ imaging 3 using GCaMP-HS and spikes of the CaP neurons, we performed 2+ 4 Ca imaging at 8 fps and electrophysiological recording simultaneously using ∼24-hpf embryos. On average, 53.5 ± 9.0 peaks per burst (n = 20 bursts from two embryos) were detected (Fig. 4F). 20% R2 > 2 sec With linear regression, we calculated a linear relationship ( 0.99) between the fluorescence change and the number of spikes C E that had occurred to give rise to the change in the rising phase (Fig. S2). The increment of the fluorescence changes per spike was calculated as 0.29% ± 0.019 (mean ± SE, n = 21). Because, in our imaging, a range of the noise or a fluctuation of the base line were typically within 1%, it is reasonable to assume that neuronal activation composed of at least four spikes could be detected by using the current GCaMP technology. D Discussion In this study, we analyzed spontaneous contractions, the first locomotive behavior in zebrafish, by combination of an improved GCaMP technology and the Gal4FF-UAS system. We successfully imaged Ca2+ signals in the motor circuit during the natural

Delay (sec) Delay vertebrate behavior and demonstrate how the motor neurons are coordinately activated. Distance (um) Improvement of GCaMP. GCaMP-HS was generated by introducing

F ﬂuorescence Normalized substitutions described in the GFP superfolder mutant (12) into the previous version of GCaMP, GCaMP2. As expected, the substitutions conferred the higher refolding activity after heat-

Voltage (x0.5mV) denaturation to GCaMP-HS. Consistent with this, the GCaMP-HS protein became more stable and showed stronger fluorescence than GCaMP2 in transfected cells, suggesting that the substitutions Time (sec) indeed affected in vivo activities. Moreover, GCaMP-HS showed 2+ 2+ Fig. 4. Spatiotemporal patterns of activation of the CaP motor neurons. the increased sensitivity to Ca [i.e., the affinity for Ca (Kd = (A) A dorsal view of the SAIGFF213A;UAS:GCaMPHS4A embryo at 24 hpf 102 nM) was higher than that of GCaMP2 (Kd = 146 nM) (9)], and treated with 100 μM D-tubocurarine. (Scale bar, 200 μm.) (B) The fluorescence Hill coefficient became 5.0 although it is 3.8 in GCaMP2. How is changes in ROIs in A.(C) Cross-correlogram of the ipsilateral CaP neurons. (D) the sensitivity to Ca2+ affected by the “superfolder” substitutions Cross-correlogram of the contralateral CaP neurons. (E) The averaged timing in the GFP domain? It has been shown by X-ray crystallography of the peaks of the ipsilateral CaP neurons located in a rostral (Left)-to-caudal A (Right) sequence. Data from four embryos are shown. (F) A whole cell-patch that Arg-377 (position 74 of the calmodulin domain in Fig. 1 ), recording and simultaneous calcium imaging of the CaP neurons during which is located close to the second EF hand of the calmodulin, is spontaneous contractions. Action potentials (gray) and calcium signals (black) interacting with the GFP chromophore (18). Therefore, we infer are shown. that the structural changes in the GFP chromophore affected the

5428 | www.pnas.org/cgi/doi/10.1073/pnas.1000887108 Muto et al. Downloaded by guest on September 30, 2021 Ca2+ interacting part of the calmodulin domain, causing the GCaMP Technology and the Gal4FF-UAS System. In vivo calcium 2+ fi change in the Ca sensitivity. This Kd value is closer to the resting imaging of neuronal activities has been carried out in zebra sh level of estimated intracellular Ca2+ concentrations (∼100 nM) larvae. Bulk loading of the fluorescent calcium-indicator dyes has (19, 20), suggesting that GCaMP-HS is better suited to detect Ca2+ been used to record the activity of a neuronal ensemble in the transients in vivo. spinal cord (4). However, with this method, neuronal subtypes Recently, Tian et al. (10) reported construction of GCaMP3 by could not be specified. To overcome this problem, Cameleon, introducing amino acid substitutions into GCaMP2. None of these a FRET-based GECI, was used in zebrafish (25). However, until substitutions is overlapping with the substitutions introduced in now it has not been widely used probably because it is not easy to GCaMP-HS. Therefore, it will be interesting to combine these analyze the calcium transient with a poor signal-to-noise ratio. substitutions and test whether they show additive effects in de- GCaMP1.6 and GCaMP2 were successfully used to detect cal- fi tecting cellular Ca2+ transients. cium signals in the neuropil of the zebra sh tectum, which con- sisted of axonal arbors of retinal ganglion cells and dendritic Alternating and Synchronized Periodic Activation of the Motor arbors of the tectal cells (26, 27). In contrast to these studies, our Network. The activity of the primary neurons during the sponta- present study clearly demonstrated that GCaMP-HS can detect fi neous contraction has been analyzed by electrophysiology. Periodic calcium signals in both the cell bodies and neurites. This nding depolarization of single primary motor neurons and interneurons, is important because axonal imaging does not immediately allow synchronized depolarization of a pair of ipisilateral neurons of one us to identify neurons that are being activated, especially when side, and alternating activation of a pair of contralateral neurons at multiple neurons are imaged. 19 to 24 hpf have been observed (2, 3). However, it has been difficult It has been shown that GCaMP has faster kinetics in calcium to analyze multiple neuronal activities by the electrophysiological ion binding than other GECIs (28). In our recording of action potentials and simultaneous calcium imaging (at 8 fps), we could approach. In this article, we demonstrated the periodic and syn- detect the fluorescence change even on the same frame as the first chronized activation of the right and left sets of the ipsilateral motor spike was generated (Fig. S2). Furthermore, the half-life of the neurons and their alternating activation. decay of the maximal fluorescence change was 0.92 ± 0.13 second Our present system enabled analysis of the onset of the sponta- (mean and SD, n = 20 events) (Fig. S2), which was about 0.8 s with neous contraction in early stages. We found periodic and synchro- a calcium-indicator dye Oregon Green BAPTA (26). From these nized activation of ipsilateral CaP neurons in the very beginning of findings, we think that time resolution of GCaMP-HS in vivo is the spontaneous contractions. Although the soma of CaP neurons fl similar to that of calcium-indicator dyes. can be uorescently labeled by GCaMP-HS even before 18 hpf in We are currently constructing transgenic fish that express Gal4FF fl the double-transgenic embryo, the uorescence changes in the soma in various types of specific neural circuits (11, 29). By combining these were hardly detected at this stage. In contrast, axonal Ca2+ tran- 2+ with the present GCaMP technology, we expect that neuronal ac- sients were observable at 18.5 hpf. Larger Ca transients in the tivities of various neural circuits will be imaged and studies of func- axons than in the soma were detected in developing Purkinje neu- tional neural circuits in a living vertebrate will be facilitated. rons in the rat by using a calcium-indicator dye (21), and it was shown that Ca2+ channels are functioning in the axons of developing Materials and Methods chick motor neurons (22). Although it remains to be proven, these Construction and Analysis of GCaMP-HS. We used pN1-GCaMP2 (9) as a template findings suggest that voltage-gated Ca2+ channels may be more for mutagenesis. S30R (TCC to CGC), Y39N (TAC to AAC), N105T (AAC to AAC), abundant in the axonal membranes in developing neurons. We and A206V (AAA to GTA) were introduced by using QuikChange Multi Site- found that the Ca2+ transients in the CaP neurons were not always directed Mutagenesis Kit (Agilent Technologies), giving rise to pN1-GCaMP-HS. associated with muscle contractions at this stage, demonstrating that The SalI-MluI fragment from pN1-GCaMP-HS was cloned into pRSETb, giving rise to pRSETb-GCaMPHS that was used to produce the His-tagged GCaMP-HS periodic and synchronized activation of the CaP neurons precedes protein in Escherichia coli. For calcium titration assay, the His-tagged GCaMP-HS its functional coupling to muscle contractions. The clustering of protein was purified by metal affinity chromatography, and fluorescence (F) of 2+ acetylcholine receptors and formation of neuromuscular junctions GCaMP-HS was measured in different Ca concentrations. Fmin and Fmax were proceed as the formation of the myotomes proceeds in a rostral-to- measured in Ca2+-chelated buffer (20 mM BAPTA) and Ca2+-saturated buffer caudal direction between 16 and 20 hpf (23) (Fig. S3). Therefore, in (300 nM Ca2+), respectively. All of the measurements were carried out in trip- − − 2+ these stages, the connection between the motor circuits and muscle licate. (F Fmin)/(Fmax Fmin) was plotted against Ca concentrations (X). Kd (dissociation constant) and n (Hill coefficient) were calculated by applying the cells may not be complete or the activity of the motor circuit may not n n n real data to the Hill equation, Y = X /(Kd + X ). To test the refolding activity, the be strong enough to reliably contract muscles. It has not been known purified proteins were denatured at 95 °C for 5 min, cooled down to 25 °C, and how the rhythmicity emerges in the early motor circuit. Although it measured for restoration of the fluorescence every 17 s in KM buffer (100 mM still remains to be elucidated, our present study demonstrated that KCl, 20 mM Mops pH 7.5) in the presence of 1 mM EGTA. The fluorescence was the periodic and synchronized activity is generated by a network normalized by the initial fluorescence before heat-denaturation. The pN1- involving a small number of CaP neurons before synchronization of GCaMP2 and pN1-GCaMP-HS plasmids were transfected to HEK cells, as de- all of the CaP neurons. scribed previously (7). In three independent transfection experiments, the fluorescence intensity was calculated by measuring those of 150 cells per dish. For Western blot analysis, anticalmodulin antibody (Upstate #05–173) was used Propagation of Activation of CaP Motor Neurons. We found prop- to detect both GCaMP2 and GCaMP-HS. agation of activation of the CaP neurons along a rostrocaudal axis in the spinal cord during spontaneous contractions. The calcu- Construction of the UAS:GCaMPHS4A and SAIGFF213A Transgenic Zebrafish. lated velocity was 5.6 μm/ms. A similar activity, the rostrocaudal The GCaMP-HS gene was cloned into the EcoRI site of pT2MUASMCS (11), delay, was detected during a swimming behavior in later stages giving rise to pT2RUASGCaMP-HS. The DNA construct was injected with (17). However, its velocity was much faster (about 20-fold faster) transposase mRNA into zebrafish embryos at one-cell stage (30). The injec- than those in the spontaneous contractions. It will be interesting ted fish were crossed with the hspGGFF27A fish or with the SAGFF73A;UAS: fi to disclose what makes this difference. Interestingly, a similar slow RFP sh (11). Among the offspring, UASGCaMP-HS;hspGGFF27A double- transgenic embryos were identifiable because they showed much brighter rostrocaudal wave has been reported in the neonatal mouse spinal fl μ green uorescence than the Gal4FF-GFP transgene alone, and UASGCaMP- cord (24). The calculated velocity was 13 to 18 m/ms, which is HS;SAGFF73A double-transgenic embryos were distinguishable because of faster but closer to that we observed for the CaP motor neurons. the different color of the fluorescence.

These ﬁndings may suggest the existence of a common mechanism For gene trapping, the Tol2 constructs T2KSAGFF (11) and T2TSAIGFF were NEUROSCIENCE for motor circuits in the vertebrate developing spinal cord. used. T2TSAIGFF was constructed by cloning a splice acceptor, the Gtx IRES

Muto et al. PNAS | March 29, 2011 | vol. 108 | no. 13 | 5429 Downloaded by guest on September 30, 2021 sequence (31) and the Ga4FF gene into a minimal Tol2 vector (32). The gene- throughout the body. The GFP-expressing CaP neurons were observed with trap screens were performed essentially as described previously (33). The a confocal laser microscope Zeiss LSM510Meta. Southern blot hybridization, inverse PCR and adaptor-ligation PCR were carried out as described previously (34). Electrophysiological Recording. The SAIG213A;UAS:GCaMPHS embryos at 24 hpf were immobilized by treatment of Tricaine and D-tubocrarine and whole- Calcium Imaging and Microscopy. A Zeiss Axiovert upright microscope with cell recording of the activity of CaP neurons was performed essentially as a10×/NA0.3 lens and a water-immersion 40×/NA0.8 lens, equipped with a described previously (25). Simultaneously, the image of the CaP neurons were high-sensitivity cooled CCD camera (Hamamatsu Photonics, ORCA-R2), was taken under 100-ms exposure at 8 fps by a cooled CCD camera (ORCA-R2) with used for calcium imaging. Each frame was taken under 100-ms exposure at a software Metamorph (Molecular Devices). The TTL pulses from the camera 10 fps or 50-ms exposure at 20 fps using the manufacturer’s image-acquisition for each frame were recorded in the electrophysiology measurements. software AquaCosmos. Images were analyzed off-line with custom-made softwares developed on LabVIEW (National Instruments) and Matlab ACKNOWLEDGMENTS. We thank M. Suster for helpful discussion; N. Mouri, M. Mizushina, M. Suzuki, Y. Kanebako, and A. Ito for ﬁsh room works; and (Mathworks). Zebraﬁsh embryos were embedded in 5 mL of 2% low-melting M. Itoh-Hiratani and H. Takakubo for technical assistance. This work was agarose in a lid of a 6-cm dish covered with 5 mL E3 water with or without supported in part by the Mitsubishi Foundation (A.M.) and the National 100 μM D-tubocurarine chloride hydrate (Sigma; T2379). Because tubocrarine BioResource Project, and grants from the Ministry of Education, Culture, is not cell-permeable, their tail tips were cut for tubocurarine to permeate Sports, Science, and Technology of Japan.

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