Membrane potential imaging method for zebrafish individuals

Abstract

To provide a method capable of non-invasively visualizing and recording membrane potentials of neuronal populations and individual neurons in zebrafish in real time and over a long period of time.SOLUTION: A method involves observing embryos obtained by crossing zebrafish individuals which express ArcLight at high brightness selected from an ArcLight gene-introduced zebrafish population. These embryos are reared at 23°C under either a 14-hour light / 10-hour dark cycle or continuous darkness for 24 hours, and 30 minutes prior to sample preparation on the day of recording, the rearing temperature is changed to 28.5°C. The embryos are treated with a muscle relaxant to prevent movement during observation, mounted in low-melting-point agarose, and membrane potentials are observed and recorded using a fluorescence microscope.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to a method for non-invasively visualizing and recording the membrane potential of zebrafish neuronal populations and neurons in real time over long periods of time. [Background technology]

[0002] Nervous tissues such as the brain and spinal cord consist of numerous neurons with various functions. These neurons are generated during the process of biological development and communicate with each other to form functional neural networks. To gain a deeper understanding of brain formation and function, it is essential to understand how the brain is formed and organized. However, this is difficult due to the enormous number of neurons. Optical methods are a powerful way to study the processes of neural tissue formation and organization because they allow non-invasive observation, i.e., without destroying the target neural tissue. Electrophysiological methods, which are often used to study nervous tissue, cannot approach neurons without destroying the target neural tissue.

[0003] Membrane potential imaging, a technique for measuring the membrane potential of cells using light, enables rapid, direct, and real-time detection of the membrane potential of neuronal groups, making it an effective method for analyzing neural circuits (Non-Patent Document 1). Because membrane potential imaging is fast and direct, it enables the detection of action potentials, hyperpolarization, and weak responses. These detections are difficult to achieve with calcium imaging, which has been used in neuronal activity research. This is because the indicator and calcium fluctuations in calcium imaging are slow. Voltage-sensitive dye (VSD) imaging began with recording activity using voltage-sensitive dyes (VSDs) (Non-Patent Document 2). Subsequently, various types of VSDs were developed, including those with improved brightness and signal-to-noise ratios and those with various colors, which helped advance research on the development of various brain regions and neural networks.

[0004] In recent years, the development of genetically encoded membrane potential indicators (GEVIs) has progressed rapidly, enabling cell-type-specific membrane potential imaging, which was previously difficult with VSDs. GEVIs respond more rapidly and emit brighter fluorescence, significantly improving the previous limitations of reactivity and signal-to-noise ratio (Non-Patent Document 3). There are several types of GEVIs with different structures. One type is a mosaic protein that utilizes the voltage-sensitive domain of a voltage-sensitive phosphatase. Another type of GEVI utilizes the voltage-sensitive domain of microbial rhodopsin. These have been used to study the function of various brain regions; however, they have not yet been used to study neural development.

[0005] Until now, electrophysiological methods have been used to observe membrane dynamics in the spinal cord. However, because this method is invasive, stable recording of neuronal observations was only possible for a short period of time, about 30 minutes (Non-Patent Document 4). After this time, it is thought to be difficult to record normal conditions due to damage to the cell membrane and changes in intracellular fluid. Furthermore, observations were limited to a single cell (Non-Patent Document 4). Calcium imaging has also been used to analyze developmental processes. However, calcium imaging has not been able to record membrane potential dynamics (Non-Patent Document 5).

[0006] Zebrafish, a small tropical fish measuring approximately 5 cm in length, is an ideal model organism for embryonic development and neuroscience research. The relatively small and transparent brain of zebrafish, combined with the availability of various transgenic strains, make zebrafish an ideal system for analyzing the functional organization of the brain in various behavioral modalities through whole-brain recording. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Cohen, L.B. and B.M. Salzberg, Optical measurement of membrane potential. Rev Physiol Biochem Pharmacol, 1978. 83: p. 35-88。

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

[0008] The problem to be solved by the present invention is to provide a method for non-invasively visualizing and recording the membrane potential of zebrafish neuronal populations and neurons in real time over a long period of time. [Means for solving the problem]

[0009] As a result of extensive research, the inventors of the present application have concluded that in order to elucidate the mechanisms of neuronal development and the construction of neural tissue networks, it is necessary to visualize and record the membrane potential of neurons and neuronal populations non-invasively in real time over long periods of time, and have established a method that makes this possible.

[0010] Zebrafish were used as a model organism. A population of zebrafish transfected with the ArcLight gene at the 15- to 18-somite stage was screened to select individuals expressing ArcLight. High-brightness individuals were then selected from the population, and the resulting embryos were used for observation.

[0011] To observe embryos at early stages, they are reared at 23°C under a 14-hour light / 10-hour dark cycle or a 24-hour dark cycle, and then switched to 28.5°C 30 minutes before sample preparation on the day of recording.

[0012] To prepare the samples, zebrafish embryos 16 to 24 hours after fertilization, in which ArcLight expression was observed, were first treated with a muscle relaxant. The number of hours after fertilization to use depends on the developmental stage of the embryo to be observed. Prior to treatment, a wound is made at the tip of the tail to improve the penetration of the muscle relaxant. This increases the muscle relaxant's effectiveness and enables prolonged suppression of movement. After treatment, the embryos are mounted in 1.7% low-melting-point agarose containing a muscle relaxant. After mounting, the agarose from the tail is removed with a scalpel, and a small amount of muscle relaxant solution is added from above to suppress movement during imaging. Examples of muscle relaxants that can be used include tubocurarine and bungarotoxin.

[0013] The embryos mounted in the low-melting-point agarose are observed under a fluorescence microscope, and the observed images are recorded.

[0014] The first invention is a method for non-invasively visualizing and recording the neuronal populations and membrane potentials of zebrafish neurons in real time over an extended period of time, characterized in that zebrafish embryos into which the ArcLight gene has been introduced are used as the observation subject, the embryos are treated with a muscle relaxant, the treated embryos are mounted in low-melting-point agarose containing the muscle relaxant, the embryos mounted in the low-melting-point agarose are observed under a fluorescence microscope, and the observed images are recorded.

[0015] The second invention relates to a method for non-invasively visualizing and recording the neuronal population and membrane potential of zebrafish according to the first invention in real time over an extended period of time. This method comprises screening a population of zebrafish at the 15- to 18-somite stage into which the ArcLight gene has been introduced to select a population expressing ArcLight, selecting high-brightness individuals from the population, and mating these selected individuals to obtain embryos for observation. The embryos are reared at 23°C under a light-dark cycle of 14 hours light and 10 hours dark or a 24-hour dark cycle, and are then changed to a rearing temperature of 28.5°C 30 minutes before sample preparation on the day of recording. A wound of approximately 50 micrometers in diameter is made at the tip of the embryo's tail, and the embryo is treated with 0.5 mM tubocurarine for 20 minutes. The treated embryo is mounted in 1.7% low-melting-point agarose containing 0.20 mM tubocurarine. The agarose from the tail of the mounted embryo is removed, and the embryo is then placed in a 0.2 mm thick tubocurarine-containing 1.7% low-melting-point agarose solution. The agarose from the tail of the mounted embryo is then removed and placed in a 0.2 mm thick tubocurarine-containing 1.7% low-melting-point agarose solution. After adding approximately 2 mL of 10 mM tubocurarine solution, the embryo mounted in the low-melting-point agarose is observed under a fluorescence microscope, and the observed images are recorded. [Effects of the Invention]

[0016] The present invention makes it possible to visualize and record the membrane potential of zebrafish neuronal populations and neurons non-invasively in real time over long periods of time. [Brief explanation of the drawings]

[0017] [Figure 1] This is a lateral image of a 1 dpf embryo expressing ArcLight. [Figure 2] This is a dorsal view of the neural tube of an embryo at 1 dpf. [Figure 3] Fig. 1d shows an image of the ventral region of the spinal cord at 20 hpf and a schematic diagram of the imaging conditions. [Figure 4] Fluctuations in membrane potential on the right and left sides of an embryo and images of the ventral region of the embryo's spinal cord at a certain point in time. [Figure 5] Dorsal view of the spinal cord expressing ArcLight at 20 hpf. [Figure 6] FIG. 6 shows membrane potential activity of neurons 1-6 in FIG. 5 at 20 hpf, 21 hpf, 22 hpf and 23 hpf. [Figure 7] FIG. 1 shows the results of cross-correlation analysis of the activity of ipsilateral and contralateral pairs of spinal cord neurons at 20 hpf and 23 hpf. DETAILED DESCRIPTION OF THE INVENTION

[0018] Next, an embodiment of the present invention will be described, but it goes without saying that various changes and modifications can be made without departing from the technical scope of the present invention. [Example]

[0019] Zebrafish embryos were raised in an incubator at 23°C or 28°C. Developmental stages were expressed in terms of hours post-fertilization (hpf) or days post-fertilization (dpf). Some of the transgenic lines (Tg) used were transparent and had a genetic background of nacre, a mutant that lacks pigment cells derived from neural crest cells.

[0020] A double transgenic line, Tg(elavl3:GAL4-VP16;UAS:ArcLight), was obtained by crossbreeding the recombinant zebrafish lines Tg(UAS:ArcLight) and Tg(elavl3:GAL4-VP16) (Non-Patent Documents 6, 7). From the resulting double transgenic line, Tg(elavl3:GAL4-VP16;UAS:ArcLight), we screened embryos at the 15- to 18-somite stage to select individuals expressing ArcLight. High-brightness individuals were then selected from this lineage, and embryos obtained by crossbreeding these selected individuals were used for observation. Note that "Tg" refers to a transgenic line incorporating the gene sequence shown in parentheses following "Tg." UAS is a yeast transcriptional regulatory sequence that activates downstream gene expression upon binding of the GAL4 transcription factor. elavl3 is a neuron-specific transcriptional regulatory region. GAL4 is a yeast-derived transcriptional gene that binds to UAS and activates transcription. GAL4-VP16 is a protein that combines the DNA binding domain of GAL4 with the transcription activation domain derived from VP16 (Herpes simplex virus protein 16). UAS is a yeast transcriptional regulatory sequence that activates the expression of downstream genes when the GAL4 transcription factor binds to it. ArcLight is a DNA sequence containing a membrane potential-sensitive domain.

[0021] To observe the embryos at an early stage, they were reared at 23°C with a 14-hour light / 10-hour dark cycle or a 24-hour dark cycle, and then changed to 28.5°C approximately 30 minutes before sample preparation on the day of recording.

[0022] For sample preparation, 16- to 24-hpf zebrafish embryos in which ArcLight expression was observed were treated with 0.5 mM tubocurarine (Sigma, T2379) for 20 minutes. Prior to treatment, a 50-micrometer incision was made at the tip of the tail with a surgical scalpel (Feather scalpel No. 11, FEATHER) to enhance the penetration of the tubocurarine solution. This enhanced the effect of tubocurarine and resulted in prolonged suppression of movement. After treatment, the embryos were mounted in 1.7% low-melting-point agarose (Sigma) containing 0.20 mM tubocurarine. After mounting, the agarose from the tail was removed with a scalpel, and approximately 2 mL of 0.2 mM tubocurarine solution was added from above to suppress movement during imaging.

[0023] High-speed imaging was performed using a widefield microscope (FN-1, NIKON, 40x water immersion lens: NIR Apo 40x / 0.8 NA) equipped with a cMOS camera (ORCA-Flash 4.0; Hamamatsu Photonics). A 0.7x relay lens (NIKON) was used as needed. For both microscopes, high-speed imaging was performed using the NIS-Elements program (NIKON). Images were acquired at high speeds of 50–200 Hz with the widefield microscope. To minimize photobleaching and phototoxicity of the ArcLight signal due to high-intensity excitation light, each high-speed imaging session lasted 1 min, with a 30-min wait between sessions. A heated stage (Thermo Plate TPi-110RX, TOKAI HIT) was used during the observations, and the water temperature was constantly adjusted to 28°C.

[0024] The images were analyzed using NIS-Elements (NIKON) to define the region of interest (ROI) and calculate the rate of change in fluorescence intensity (ΔF / F0). If the zebrafish moved during imaging, Turboreg in the Fiji program was used to correct each frame image by translating the average image of all frames, as necessary.

[0025] Membrane potential imaging was performed, and the extracted ΔF / F0 data was subjected to signal processing and baseline correction using Origin Pro (registered trademark, Originlab Corporation) for data significantly affected by background noise and bleaching. For signal processing, a 5-50 Hz low-pass filter was applied using the Origin Pro FFT filter function. Baseline correction was performed using the peak analyzer function (baseline subtraction), which automatically set anchor points on the baseline and subtracted the baseline calculated based on the anchor points from the original data. [Example]

[0026] We generated a transgenic zebrafish line, Tg(elavl3:GAL4-VP16;UAS:ArcLight;UAS:lyn-RFP). Here, lyn-RFP is a sequence consisting of a cell membrane localization signal (Lyn) and RFP (red fluorescent protein). Embryos from this line were raised as described in Paragraph 18, samples were prepared as described in Paragraph 21, and observed as described in Paragraph 22. In this line, gal4 is expressed in neurons, and the cell membrane is labeled with Lyn-RFP. At 1 dpf, ArcLight was observed widely throughout the neural tube and colocalized with Lyn-RFP (Figures 1 and 2). Figure 1 shows a lateral image of a 1 dpf embryo expressing ArcLight. The top row of Figure 1 is a fluorescent image, and the bottom row is a superimposed image of the fluorescent image and bright-field image. ArcLight is shown to be widely distributed throughout the neural tube. Figure 2 shows a dorsal image of the neural tube of this embryo at 1 dpf. The lower panel shows high-magnification images, which show that ArcLight co-localizes with Lyn-RFP within the neural tube (arrowheads). These observations indicate that ArcLight is properly distributed to the cell membrane within the neural tube. [Example]

[0027] Embryos of the recombinant zebrafish line Tg(elavl3:GAL4-VP16;UAS:ArcLight) described in Paragraph 0019 were reared using the method described in Paragraph 0018, samples were prepared using the method described in Paragraph 0021, and observed using the method described in Paragraph 0022. Spinal cord neurons were shown to spontaneously fire early in development (neuronal firing refers to the phenomenon in which an action potential is generated in a postsynaptic cell when a depolarizing potential change occurs in the postsynaptic membrane as a result of synaptic transmission and exceeds a threshold). This coordinated activity of neurons began at 17 hpf. At 20 hpf, oscillatory fluctuations in ArcLight fluorescence intensity were detected in the ventral region from the third to eighth somites at a frequency of 0.14 ± 0.10 Hz (Figures 3 and 4). The phases of the fluorescence fluctuations were different and alternated between the right and left sides of the spinal cord. Figure 3 shows an image of the ventral region of the spinal cord of an embryo at 20 hpf. The left side is the head side, and somites 3 to 8 are shown. A schematic diagram of the imaging system is shown on the right side of the figure. The upper right side of Figure 4 shows the fluctuations in AtcLight fluorescence (-ΔF / F0) on the right and left sides of the embryo. The upper left side of Figure 4 shows an image of the embryo at the time indicated by the black arrow on the right side of Figure 4. The white arrow indicates an activated neuron. At this point, neurons on the left side of the embryo are activated, while neurons on the right side are inactive. The region of interest is located in somites 5 to 7, and is the area enclosed by a square in Figure 3; the upper square represents the right side of the embryo, and the lower square represents the left side of the embryo. A 1-micromolar tetrodotoxin solution was then administered to the embryo by perfusion. The lower part of Figure 4 shows an image of the embryo after tetrodotoxin treatment (left) and the fluctuations in AtcLight fluorescence (right). Tetrodotoxin treatment abolished neuronal activity. These results demonstrate that ArcLight can detect spontaneous firing of neurons in the spinal cord of developing zebrafish. [Example]

[0028] Fluctuations in spontaneous firing in the spinal cord were observed. Embryos of the double transgenic line Tg(elavl3:GAL4-VP16;UAS:ArcLight) described in paragraph 0019 were raised in the manner described in paragraph 0018, samples were prepared in the manner described in paragraph 0021, and observed in the manner described in paragraph 0022. Membrane potential imaging was performed from 20 hpf to 23 hpf. Figure 5 shows dorsal images of an ArcLight-expressing spinal cord at 20 hpf. Active neuronal population activity was observed. Numbers 1–6 in Figure 5 refer to the cells whose membrane potential activity was measured, as shown in Figure 6. Figure 6 shows the membrane potential activity of neurons 1–6 in Figure 5 at 20 hpf, 21 hpf, 22 hpf, and 23 hpf. The active neuronal population activity observed at 20 hpf attenuated by 23 hpf. The depolarization pattern also changed. Firing duration gradually lengthened, and the alternating firing pattern between the left and right sides was lost (Figure 6). Figure 7 shows the results of cross-correlation analysis of the activity of ipsilateral and contralateral pairs of spinal neurons at 20 hpf and 23 hpf. At 20 hpf, ipsilateral and contralateral pairs of neurons exhibited high synchrony and periodicity. However, by 23 hpf, these synchrony and periodicity were attenuated, except for the synchrony of the ipsilateral pair (Figure 7). These results indicate that rhythmic activity and left-right synchronization decrease with the developmental stage, but synchrony between ipsilateral neurons is maintained. In this study, we were able to visualize and record the membrane potential of a zebrafish neuronal population for a long period of time (3 hours). [Industrial Applicability]

[0029] The method of the present invention has the potential to be used in the development of therapeutic drugs for nervous system diseases and for early detection and rapid response to water pollution in lakes, rivers, etc.

Claims

1. A method for non-invasively visualizing and recording the membrane potential of a zebrafish neuronal population and neuronal cells over a long period of time in real time, comprising: The zebrafish embryos transfected with the ArcLight gene were used as the observation subjects. treating the embryos with a muscle relaxant; The treated embryos are mounted in low-melting-point agarose containing a muscle relaxant, The embryos mounted in the low-melting-point agarose are observed under a fluorescence microscope, and the observed images are recorded. A method for non-invasively visualizing and recording the membrane potential of zebrafish neuronal populations and neurons in real time over an extended period of time.

2. 2. A method for non-invasively visualizing and recording the membrane potential of a zebrafish neuronal population and neurons over a long period of time in real time according to claim 1, comprising: A population of zebrafish introduced with the ArcLight gene at the 15- to 18-somite stage is screened to select individuals expressing ArcLight, and high-brightness individuals are selected from the population. Embryos obtained by mating the selected individuals are then used as the subject of observation. The embryos were reared at 23°C under a 14-hour light / 10-hour dark cycle or a 24-hour dark cycle, and then reared at 28.5°C 30 minutes before sample preparation on the day of recording. After making a wound of approximately 50 micrometers at the tip of the tail of the embryo, the embryo was treated with 0.5 mM tubocurarine for 20 minutes. After the treatment, the embryos were mounted in 1.7% low-melting-point agarose containing 0.20 mM tubocurarine. The agarose was removed from the tail of the mounted embryo, and approximately 2 mL of 0.2 mM tubocurarine solution was added from above. The embryos mounted in the low-melting-point agarose are observed under a fluorescence microscope, and the observed images are recorded. A method for non-invasively visualizing and recording the membrane potential of zebrafish neuronal populations and neurons in real time over an extended period of time.

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