Zebra fish depressive disorder induction and traditional Chinese medicine high-throughput screening platform

Abstract

The invention relates to the field of biomedical technology and automation equipment, and discloses a zebra fish depressive disorder induction and traditional Chinese medicine high-throughput screening platform which comprises a high-throughput behavior observation module, an image data acquisition module and a data processing and analysis module. The method comprises the following steps: performing grouped administration treatment on zebrafish in a high-throughput behavior observation module; real-time image data of the zebra fish is collected in a non-interference mode through an image data collection module; the data processing and analysis module automatically calculates the behavioral parameters such as the total movement distance and the wall taxis index, so that the efficacy of the traditional Chinese medicine is objectively evaluated. The module can also carry out registration and analysis on zebra fish brain images so as to evaluate neuron activity of a specific brain region. Through automatic and integrated design, high throughput of drug screening and objective quantification of evaluation results are realized, and a correlation analysis path of behavioral phenotypes and neural mechanisms is established.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology and automated equipment, specifically to a zebrafish depression induction and high-throughput screening platform for traditional Chinese medicine. Background Technology

[0002] Depressive disorders, as a common neuropsychiatric illness, have complex pathogenesis and impose a heavy burden on patients and society. Therefore, the development of novel and effective antidepressants is of significant practical importance. In the preclinical stage of drug development, large-scale efficacy screening and evaluation using animal models is a crucial step in discovering lead compounds. Zebrafish, as an emerging model vertebrate, is playing an increasingly important role in the screening research of neuropsychiatric drugs due to its high genetic homology with humans, strong reproductive capacity, rapid development, individual transparency, and suitability for live observation.

[0003] Currently, the conventional technical approach for screening antidepressants using zebrafish typically involves establishing a depressive-like behavioral model in zebrafish using chemical inducers (such as reserpine), followed by observing and recording behavioral changes in zebrafish before and after drug administration in specific experimental paradigms (such as new environment exploration experiments) to preliminarily evaluate drug efficacy.

[0004] However, existing technical solutions still face some technical bottlenecks in practical applications. On the one hand, traditional behavioral observation and recording methods have limitations in processing capacity. Their experimental setups often cannot support the simultaneous execution of large-scale samples, resulting in low overall screening throughput. This is inefficient when dealing with large compound libraries, especially complex traditional Chinese medicine libraries. On the other hand, the evaluation process of zebrafish behavior may introduce subjectivity and uncertainty due to the lack of unified and automated quantitative standards. Even with video recording, subsequent data analysis may be affected by human intervention, impacting the objectivity and reproducibility of the results. More importantly, conventional behavioral screening mainly focuses on the phenotypic level of "whether it is effective," and cannot directly reveal which brain regions or neural circuits the drug acts on. To delve deeper into the neurobiological mechanisms of action, it is usually necessary to rely on histological or molecular biological techniques that are independent of behavioral experiments. This makes the process of analyzing the correlation between behavioral phenotypes and neural mechanisms fragmented and inefficient, limiting the rapid and in-depth understanding of drug mechanisms of action. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-throughput screening platform for zebrafish depression induction and traditional Chinese medicine, which solves the problems of low screening throughput, insufficient objectivity of behavioral evaluation, and difficulty in establishing the correlation between phenotype and neural mechanisms in existing drug screening methods.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a high-throughput screening platform for zebrafish depression induction and traditional Chinese medicine, comprising: The first aspect of this invention provides a zebrafish depression induction and high-throughput screening system for traditional Chinese medicine.

[0007] The system includes: a high-throughput behavior observation module, an image data acquisition module, and a data processing and analysis module.

[0008] The high-throughput behavioral observation module is internally configured to accommodate a perforated plate and provide a controllable culture environment for zebrafish within the plate. In one specific embodiment, the high-throughput behavioral observation module includes an environmental control unit for setting and controlling the light, temperature, and sound of the culture environment.

[0009] The image data acquisition module, linked to the high-throughput behavior observation module, is used to acquire real-time image data of each zebrafish within the perforated plate. In one specific embodiment, the image data acquisition module includes an infrared light source and a high-resolution camera. The infrared light source provides illumination for the high-resolution camera in dark conditions to record the behavior of the zebrafish in the absence of visible light.

[0010] The data processing and analysis module is electrically connected to the image data acquisition module and is configured to receive the real-time image data and calculate zebrafish behavioral parameters from it for evaluating the efficacy of traditional Chinese medicine.

[0011] Specifically, the data processing and analysis module is configured to calculate the behavioral parameters, including at least the total distance traveled and the average speed. The total distance traveled (… The calculation of ) follows the formula below: ; in, This represents the total distance traveled. This refers to the total number of video frames acquired by the image data acquisition module. This is the frame number of the video. For zebrafish in the first The center point coordinates at frame time; For zebrafish in the first The coordinates of the center point in the frame.

[0012] In a further embodiment, the data processing and analysis module is further configured to virtually divide the observation area of ​​a single well in the porous plate into a central region and an edge region, and calculate the wall-attracting index and the percentage of residence time in the central region as the behavioral parameters. Wherein, the wall-attracting index (… The calculation of ) follows the formula below: ; in, It is the wall-climbing index; The total time the zebrafish stayed in the edge region; The total observation time used to acquire the real-time image data.

[0013] In an optional technical solution, the data processing and analysis module is further configured to receive a three-dimensional image of the zebrafish brain and perform image registration between the three-dimensional image and a preset digital brain atlas to identify specific brain regions. After completing the image registration, the data processing and analysis module is further configured to quantify the fluorescence signal intensity within the specific brain region to assess the neuronal activation level.

[0014] The second aspect of this invention provides a method for inducing depression in zebrafish and high-throughput screening of traditional Chinese medicine.

[0015] This method uses the aforementioned system and includes the following steps: S1: In the high-throughput behavioral observation module, zebrafish are grouped into at least a blank control group, a model group treated with reserpine, and a traditional Chinese medicine group treated with traditional Chinese medicine extracts. S2: The image data acquisition module acquires real-time image data of each group of zebrafish in S1 within a preset time period. S3: The real-time image data is processed through the data processing and analysis module to calculate various behavioral parameters used to characterize zebrafish activity, and the efficacy of the traditional Chinese medicine is evaluated based on the differences in the behavioral parameters among the groups.

[0016] This invention provides a high-throughput screening platform for inducing depression in zebrafish and using traditional Chinese medicine. It has the following beneficial effects: 1. This invention utilizes a high-throughput behavioral observation module capable of accommodating multi-well plates, combined with a data processing and analysis module capable of parallel processing of multi-channel image data, to achieve simultaneous observation and automated analysis of multiple zebrafish samples. This design transforms traditional, individual animal behavioral experiments into a high-throughput screening process, significantly improving the overall efficiency and processing capacity of drug screening.

[0017] 2. This invention uses an image data acquisition module to record the movement trajectory of zebrafish with high fidelity, and a data processing and analysis module to quantitatively calculate behavioral parameters based on preset mathematical formulas. This approach transforms the evaluation of animal behavior from traditional subjective observation to objective, quantifiable data analysis, eliminating observer bias and ensuring the accuracy and repeatability of experimental results.

[0018] 3. This invention establishes a direct link between macroscopic behavioral phenotypes and microscopic neural circuit activity changes by integrating the registration function of three-dimensional brain images and digital brain atlases, as well as the ability to quantitatively analyze the activity of neurons in specific brain regions, into the data processing and analysis module. This design not only evaluates the effectiveness of drugs but also provides multi-dimensional analytical tools for exploring and elucidating the neurobiological mechanisms of drug action, deepening our understanding of drug action mechanisms. Attached Figure Description

[0019] Figure 1 This is a system architecture diagram of the present invention; Figure 2 This is a flowchart of the method of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example: Please see the appendix Figure 1 This invention provides a high-throughput screening platform for zebrafish depression induction and traditional Chinese medicine, comprising: The high-throughput behavior observation module is internally configured to accommodate a perforated plate and to provide a controlled culture environment for zebrafish within the perforated plate. This embodiment describes in detail the high-throughput behavioral observation module in the zebrafish depression induction and traditional Chinese medicine high-throughput screening system provided by the present invention. This module is the basic platform for realizing drug treatment and subsequent behavioral observation of zebrafish. Its structure and function are designed to provide standardized, low-interference experimental subjects and environment for the subsequent image data acquisition module and data processing and analysis module.

[0022] The core structure of the high-throughput behavioral observation module is an observation chamber capable of accommodating and precisely positioning a standard multiwell plate. The multiwell plate is preferably a 24-well, 48-well, or 96-well plate. Using such standardized multiwell plates enables simultaneous processing and observation of multiple zebrafish samples, which is the technological foundation for high-throughput screening. Each well serves as an independent observation chamber for a single zebrafish, thus avoiding interference from interactions between fish groups on individual behavioral data.

[0023] To ensure the consistency and reproducibility of experimental conditions, the high-throughput behavioral observation module integrates an environmental control unit. This environmental control unit is crucial in ensuring that the only variable between all experimental groups and the control group is the administered substance.

[0024] Specifically, the environmental control unit includes a temperature control subunit. Since zebrafish are poikilothermic animals, their behavior and metabolic activities are highly sensitive to environmental temperature. To eliminate the interference of temperature fluctuations on subsequent behavioral parameter calculations, the temperature control subunit uses built-in heating and / or cooling elements, combined with a high-precision temperature sensor, for closed-loop feedback control. This design allows the temperature of the culture water within the perforated plate to be precisely maintained at a preset constant value, thus providing a consistent physiological environment for all tested zebrafish.

[0025] In addition, the environmental control unit includes a lighting control subunit. This subunit simulates the light-dark cycle required for zebrafish survival and is implemented using a programmable LED array. The LED array allows for precise programming control of the lighting's on / off timing, duration, and intensity. This is not only significant for studying the effects of drugs on zebrafish's diurnal rhythms but also a necessary technical means to apply precise environmental stimuli in the execution of specific behavioral paradigms.

[0026] To further reduce random interference from the external environment and ensure that the collected real-time image data accurately reflects the behavioral changes in zebrafish caused by drug intervention, the outer shell of the observation chamber is made of a material with sound insulation and light-blocking properties. In some preferred embodiments, the observation chamber can be placed entirely on an active or passive vibration-damping platform to maximize the isolation of possible sound and physical vibrations in the experimental environment, ensuring that the zebrafish are in a quiet and stable environment.

[0027] To ensure seamless integration with the subsequent image data acquisition module, the observation chamber was designed with an adaptive physical structure. Specifically, the bottom of the chamber supporting the perforated plate is made of a highly transparent optical material. This design ensures that the high-resolution camera in the image data acquisition module located beneath the observation chamber can capture the complete movement trajectory of each zebrafish within the perforated plate without obstruction or distortion.

[0028] Meanwhile, to improve image data quality, the inner wall surface of the observation box is treated with a low-reflectivity material or coating, typically a diffuse white or gray. This aims to eliminate or reduce the interference of stray light reflection on the image recognition algorithm and avoid deviations in zebrafish center point localization caused by artifacts formed by reflections. High-quality, high signal-to-noise ratio raw image data is essential for subsequent data processing and analysis modules to accurately calculate the total movement distance (…). ), wall-tendency index ( Prerequisites for key behavioral parameters such as total distance traveled ( ). For example, in calculating the total distance traveled ( ) During this process, the data processing and analysis module needs to accurately identify the coordinates of the zebrafish's center point in each video frame. Clear, interference-free images are the foundation for achieving this precise positioning.

[0029] ; In summary, the high-throughput behavioral observation module in this embodiment, through its structural design and integrated environmental control unit, provides zebrafish with a standardized, controllable, and high-throughput behavioral research platform, laying a solid technical foundation for the subsequent data acquisition and analysis process of the system.

[0030] The image data acquisition module, in conjunction with the high-throughput behavior observation module, is used to acquire real-time image data of each zebrafish within the multi-well plate. This embodiment describes in detail the image data acquisition module of the zebrafish depression induction and traditional Chinese medicine high-throughput screening system provided by the present invention. This module plays a crucial role in the entire system by converting the biological behavior of zebrafish into raw digital signals that can be analyzed, serving as a bridge connecting the physical experimental environment and subsequent data quantification analysis. Its design aims to capture all dynamic information of zebrafish within the high-throughput behavioral observation module without interference and with high fidelity.

[0031] The image data acquisition module and the high-throughput behavior observation module are physically linked. In a preferred embodiment, the image data acquisition module is positioned entirely below the high-throughput behavior observation module, directly opposite the optically transparent bottom of the perforated plate that supports the observation module. This layout ensures that the acquisition field of view completely covers all preset observation areas within the perforated plate, thereby enabling synchronous and parallel data acquisition of all zebrafish samples within the plate.

[0032] Specifically, the image data acquisition module includes a high-resolution camera. The purpose of using a high-resolution camera is to ensure that each frame of real-time image data has sufficient pixel detail to support accurate image recognition and feature extraction in subsequent data processing and analysis modules. A high-resolution image is fundamental for accurately segmenting the zebrafish's outline, distinguishing its body from the background, and ultimately calculating its geometric center point coordinates. The accuracy of these center point coordinates directly determines the reliability of all subsequent kinematic parameter calculations. For example, in calculating the zebrafish's total movement distance (… In this case, it is precisely by relying on the continuous image frame sequence captured by the camera that a high-precision center point coordinate sequence can be extracted. This ensures the accuracy of the calculation results.

[0033] ; To achieve uninterrupted recording of zebrafish's natural behavior under different lighting conditions, especially in dark environments, the image data acquisition module further includes an infrared light source. The wavelength of light emitted by this infrared source is outside the visible spectrum range of zebrafish. Therefore, even in completely dark environments for zebrafish, this infrared source can provide sufficient and uniform illumination for the high-resolution camera without stimulating the zebrafish visually, thus avoiding the light source itself acting as a distracting variable to affect their behavior. This technical feature is crucial for studying the effects of drugs on zebrafish's diurnal rhythms and anxiety-like behaviors (such as wall-to-wall behavior), which are closely related to lighting conditions.

[0034] During system operation, the infrared light source and the high-resolution camera work together. The camera continuously captures the observation area illuminated by the infrared light source at a preset, constant frame rate, generating a video stream. This video stream is the real-time image data and is transmitted to the data processing and analysis module in real time.

[0035] The quality of real-time image data directly affects the accuracy of subsequent behavioral parameter analysis. For example, in analyzing the wall-tendency index (WTI), ... When zebrafish are in the central or edge region of a virtual area, the data processing and analysis module needs to determine whether they are located at any given time. A high-contrast, low-noise image provided by this module makes this location determination algorithm more robust, thus accurately accumulating the total time the zebrafish spends in the edge region. This ensures the validity of the final calculation results.

[0036] ; In summary, the image data acquisition module in this embodiment, through the combination of high-resolution imaging and non-invasive infrared illumination technology, can record the natural behavior of zebrafish in a high-throughput environment around the clock with high fidelity. This provides high-quality, unbiased raw data input for the entire screening system, which is the fundamental guarantee for achieving accurate quantification of subsequent behavioral studies.

[0037] The data processing and analysis module is electrically connected to the image data acquisition module. It is configured to receive real-time image data and calculate zebrafish behavioral parameters from it for evaluating the efficacy of traditional Chinese medicine.

[0038] This embodiment elaborates on the data processing and analysis module of the zebrafish depression induction and traditional Chinese medicine high-throughput screening system provided by the present invention. This module, as the computational core of the system, is responsible for transforming the massive, unstructured real-time image data provided by the image data acquisition module into a series of structured quantitative indicators that objectively reflect the physiological and behavioral state of zebrafish.

[0039] The data processing and analysis module, in terms of hardware, can be composed of a general-purpose computer or an embedded processor. It receives real-time image data in the form of video streams from the image data acquisition module via electrical connection. After receiving the data, the module's built-in algorithm program will automatically initiate a series of processing and analysis procedures.

[0040] First, the module processes the input video stream frame by frame. For each frame, the image recognition algorithm within the module automatically identifies and locates the zebra goby within each hole of the perforated plate. This process typically includes image preprocessing, target segmentation, and morphological calculations, ultimately accurately extracting the center point coordinates of each zebra goby in the current frame's image coordinate system. ,in Represents the frame number of the video.

[0041] Based on this, the data processing and analysis module uses the center point coordinate information between consecutive frames to calculate the basic kinematic parameters of the zebrafish. These parameters are fundamental to evaluating the impact of drugs on the overall activity level of zebrafish. One core parameter is the total movement distance (…). This parameter reflects the total activity of zebrafish throughout the observation period. The calculation follows this formula: ; in, This represents the total distance traveled. This represents the total number of frames in the video. This is the frame number of the video. For zebrafish in the first The center point coordinates at frame time; For zebrafish in the first The coordinates of the center point in the frame.

[0042] By accumulating the displacement between each frame, this module can obtain a quantitative value that objectively reflects the intensity of zebrafish activity.

[0043] To further assess the exploratory behavior and anxiety levels of zebrafish, the data processing and analysis module was also configured to perform spatial location-related behavioral analyses. This module first, at the software level, virtually divides the circular or square observation area of ​​a single hole in the multi-well plate into a central region and a peripheral region. This division serves as the basis for analyzing approach-avoidance behavior. Subsequently, the module tracks the zebrafish's center point coordinates and their residence within different virtual regions.

[0044] Based on this spatial partitioning, the module calculates the wall-tethering index (…). This index is a classic indicator for measuring anxiety-like behavior in rodents and fish. The calculation follows this formula: ; in, It is the wall-climbing index; This represents the total duration during which the zebrafish's center point is located in the edge region, calculated by accumulating video frames. The total observation time used to acquire real-time image data. The calculation of this index provides crucial quantitative data for assessing whether traditional Chinese medicine has anti-anxiety effects.

[0045] In an optional, more advanced technical solution, the functionality of the data processing and analysis module is further expanded to establish the link between behavioral phenotypes and neural mechanisms. In this solution, the module is configured to receive and process three-dimensional fluorescence images of zebrafish brains acquired after behavioral experiments. The module first executes an image registration algorithm to precisely spatially align the three-dimensional brain images of the experimental samples with a standard, pre-defined digital brain atlas.

[0046] After image registration is completed, specific brain regions associated with emotion and stress response, such as the habenula and hypothalamus, can be automatically identified in the brain images of the experimental samples. Subsequently, the module quantifies the fluorescence signal intensity in these specific brain regions to assess the activation level of their neurons. To eliminate background noise and ensure comparability of data between different samples, the module preferably calculates a standardized mean fluorescence intensity (...). ): ; in, Standardized average fluorescence intensity; It is the average fluorescence intensity measured within a specific brain region (ROI); It is the average background fluorescence intensity measured in signal-free regions of brain tissue; It refers to the area or volume of that specific brain region.

[0047] Finally, the data processing and analysis module integrates all calculated behavioral parameters and optional neuronal activity data, and outputs them in a structured data format. These outputs provide direct and objective data support for subsequent statistical analysis and drug efficacy evaluation.

[0048] Please see the appendix Figure 2 A method for inducing depression in zebrafish and high-throughput screening of traditional Chinese medicine, comprising: S1: In the high-throughput behavioral observation module, zebrafish are grouped into at least a blank control group, a model group treated with reserpine, and a traditional Chinese medicine group treated with traditional Chinese medicine extracts. S2: The image data acquisition module acquires real-time image data of each group of zebrafish in S1 within a preset time period. S3: Through the data processing and analysis module, real-time image data is processed to calculate various behavioral parameters used to characterize zebrafish activity, and the efficacy of traditional Chinese medicine is evaluated based on the differences in behavioral parameters among groups.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-throughput screening platform for inducing depression in zebrafish and using traditional Chinese medicine, characterized in that, include: A high-throughput behavior observation module is configured to accommodate a perforated plate and to provide a controlled culture environment for zebrafish within the perforated plate. The image data acquisition module, linked with the high-throughput behavior observation module, is used to acquire real-time image data of each zebrafish in the multi-well plate. The data processing and analysis module is electrically connected to the image data acquisition module and is configured to receive the real-time image data and calculate zebrafish behavioral parameters from it for evaluating the efficacy of traditional Chinese medicine.

2. The platform according to claim 1, characterized in that, The high-throughput behavior observation module includes an environmental control unit for providing controllable lighting, temperature, and sound.

3. The platform according to claim 1, characterized in that, The image data acquisition module includes an infrared light source and a high-resolution camera, wherein the infrared light source is used to provide illumination for the high-resolution camera in dark conditions.

4. The platform according to claim 1, characterized in that, The data processing and analysis module is configured to calculate at least the behavioral parameters, including total distance traveled and average speed.

5. The platform according to claim 4, characterized in that, When calculating the total distance traveled, the data processing and analysis module follows this formula: ； in, This represents the total distance traveled. This refers to the total number of video frames acquired by the image data acquisition module. This is the frame number of the video. For zebrafish in the first The center point coordinates at frame time; For zebrafish in the first The coordinates of the center point in the frame.

6. The platform according to claim 1, characterized in that, The data processing and analysis module is further configured to virtually divide the observation area of ​​a single hole in the porous plate into a central region and an edge region, and calculate the wall-tendency index and the percentage of residence time in the central region as the behavioral parameters.

7. The platform according to claim 6, characterized in that, When calculating the wall-tendency index, the data processing and analysis module follows the formula below: ； in, It is the wall-climbing index; The total time the zebrafish stayed in the edge region; The total observation time used to acquire the real-time image data.

8. The platform according to claim 1, characterized in that, The data processing and analysis module is also configured to receive three-dimensional images of zebrafish brains and perform image registration between the three-dimensional images of zebrafish brains and preset digital brain atlases to identify specific brain regions.

9. The platform according to claim 8, characterized in that, After completing the image registration, the data processing and analysis module is further configured to quantify the fluorescence signal intensity in the specific brain region to assess the neuronal activation level.

10. A method for inducing depression in zebrafish and high-throughput screening of traditional Chinese medicine, comprising a zebrafish depression induction and traditional Chinese medicine high-throughput screening platform according to any one of claims 1-9, characterized in that, Includes the following steps: S1: In the high-throughput behavioral observation module, zebrafish are grouped into at least a blank control group, a model group treated with reserpine, and a traditional Chinese medicine group treated with traditional Chinese medicine extracts. S2: The image data acquisition module acquires real-time image data of each group of zebrafish in S1 within a preset time period. S3: The real-time image data is processed through the data processing and analysis module to calculate various behavioral parameters used to characterize zebrafish activity, and the efficacy of the traditional Chinese medicine is evaluated based on the differences in the behavioral parameters among the groups.

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