CN116171885A - Closed loop ultrasonic stimulation system and method for free activity mouse behavior state dependence - Google Patents
Closed loop ultrasonic stimulation system and method for free activity mouse behavior state dependence Download PDFInfo
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Abstract
The invention provides a closed loop ultrasonic stimulation system and a method for free activity mouse behavior state dependence, which belong to the field of biomedicine, and comprise an ultrasonic stimulation control platform, wherein a miniature head-mounted ultrasonic stimulation device is used for applying ultrasonic stimulation to a mouse; the animal behavior acquisition device is used for acquiring behavior information of the mice, including video information of the plantar track of the mice; the visual analysis platform is used for analyzing the behavior information of the mice and obtaining behavior characteristic data of the mice, and the behavior characteristic data comprise information such as the movement speed, the stride, the pressure, the running time, the residence time and the like of the mice. The visual analysis platform provided by the invention can analyze the behavior information of the mice sent by the animal behavior acquisition device in real time and obtain the behavior characteristic data of the mice. The ultrasonic stimulation control platform applies different ultrasonic stimulations to the mice according to the behavior characteristic data of the mice presented by the visual analysis platform, and behavior information of the stimulated mice is fed back to the visual analysis platform.
Description
Technical Field
The invention relates to the biomedical field, in particular to a closed loop ultrasonic stimulation system and method for free movement mouse behavior state dependence.
Background
Currently, most ultrasound transducers used in ultrasound neuromodulation studies are cumbersome and require fixation to a stent for use, while experimental animals are anesthetized and fixed to a brain stereotactic apparatus. The cumbersome ultrasound probe and anesthetized and tethered limbs mounted atop anesthetized animals limit the development of certain experimental studies involving sensory, cognitive and behavioral assessment. The experimental animal is frightened by the constraint of limbs, and the nerve activity is inhibited by deep anesthesia, so that the experimental result of ultrasonic regulation is affected.
At present, the mode aiming at observing the ultrasonic stimulation effect mainly comprises electrophysiological signal feedback and behavioral feedback. The electrophysiological signals mainly comprise brain electrical signals, electromyographic signals and the like, and have the defect of abstract feedback and non-intuitiveness, thereby influencing the judgment of the ultrasonic stimulation effect.
In the patent application document of the invention with the publication number of CN105684934A, an animal behavior analysis method and an animal behavior analysis system are disclosed, wherein in the example, shape detection is respectively carried out on experimental animals at different moments, and first shape information and second shape information of the animals are obtained. And then comparing the first shape information with the second shape information to obtain the change information of the animal shape. And then the comparison of the database can be carried out to obtain the behavior change of the animal. The method is too rough for detecting the behavior change of the animal, can only effectively identify the large-amplitude motion of the animal, including standing, squatting and the like, and cannot effectively detect the fine motion of the experimental animal. In this example, the behavior change of the animal cannot be detected to form a closed loop, which is not beneficial to the experimenter to adjust at any time.
In the patent application document of the invention with the publication number of CN109977768A, a closed-loop feedback type animal behavior analysis system, a method and a device are disclosed. In the system, a camera is placed above a mine, and behavior characteristic data of experimental mice are analyzed by capturing motion trajectories of the experimental mice in the mine. And comparing the behavior characteristic data of the experimental mouse at the previous moment with the behavior characteristic data of the experimental mouse at the next moment to obtain the parameter control command of the stimulation platform. The method can still only roughly obtain the exercise behaviors of the experimental mice, including exercise tracks, running time, running speed, the times of meeting other mice, residence time and the like. Finer data such as running posture of an experimental mouse, body twisting angle during running, lifting height of limbs during running, and pressure of the limbs on a runway cannot be obtained. And the ultrasonic stimulation of the experimental mice in this example failed to form an effective device or method that did not affect the free-play status of the free-play mice during the application of the ultrasonic stimulation. Therefore, the device or method provided by the embodiment is unfavorable for accurately reflecting the movement state of the experimental mouse, and can influence the experimenter to observe the movement change of the mouse and control the command of the stimulation parameters in time.
Disclosure of Invention
The present application aims to solve the above problems, and provides a closed loop ultrasonic stimulation system and method for the behavior state dependence of a free-moving mouse, which aims to provide a closed loop feedback for ultrasonic stimulation by means of behavior information such as plantar motion track of the free-moving mouse, and can solve the problem of fine movements (including lifting amplitude of limbs, body torsion angle, pressure of limbs to a runway, etc.) of experimental mice in the motion process of the above-mentioned invention patent, and autonomously design a miniature head-mounted ultrasonic stimulator for the ultrasonic stimulation of the free-moving mouse, which can solve the problem that the application of ultrasonic stimulation to the experimental mouse in the above-mentioned invention patent must fix the mouse and cannot make the mouse in the free-moving state (as shown in fig. 3).
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a free-living mouse behavior state dependent closed loop ultrasound stimulation system comprising: the ultrasonic stimulation control platform is used for applying ultrasonic stimulation to the mice;
the animal behavior acquisition device is used for acquiring behavior information of the mice in real time and acquiring motion track videos of soles of the mice;
the visual analysis platform is connected with the animal behavior acquisition device and used for analyzing the behavior information of the mice and calculating behavior characteristic data of the mice, including the information of the movement speed, the stride, the pressure, the running time and the residence time of the mice.
The technical scheme of the invention is further improved as follows: the animal behavior acquisition device comprises a motor driving system, an animal runway system and an optical imaging system; the ultrasonic stimulation control platform is used for receiving ultrasonic stimulation parameter control commands and generating ultrasonic stimulation in different forms according to the commands, wherein the ultrasonic stimulation comprises intensity, interval time, duration and brain action area; and the ultrasonic stimulation control platform generates ultrasonic stimulation to different forms of mice according to the received ultrasonic stimulation parameter control command.
The technical scheme of the invention is further improved as follows: the motor driving system comprises a driving motor for driving the animal runway system to move, a motor fixing plate, a motor controller and a belt conveying device; the animal runway system comprises rubber rollers, a transparent runway, baffle plates and runway cleaning brushes, wherein the rubber rollers comprise a driving wheel rubber roller and a driven wheel rubber roller, the transparent runway is sleeved on the two rubber rollers separated by a certain distance, the belt conveying device drives the driving wheel rubber roller to operate so as to drive the transparent runway to operate, supporting plates are arranged on the front side and the rear side of the two rubber rollers, and the baffle plates which can be inserted and pulled are arranged in the middle of the supporting plates on the transparent runway to form a mouse movable chamber.
The technical scheme of the invention is further improved as follows: the belt conveying device is characterized in that the belt synchronous wheels are in transmission with a belt, the belt synchronous wheels are respectively fixed on an output shaft of a driving motor and a driving wheel rubber roller in a key groove and jackscrew mode, the two belt synchronous wheels are connected in a belt connection mode, and the tooth ratio of the two synchronous wheels is 1: and 3, fixing the driving motor on a motor fixing plate, and controlling the power by adjusting the motor controller so as to control the transparent runway.
The technical scheme of the invention is further improved as follows: the front support plate and the rear support plate are provided with roller transfer plates at corresponding positions, the driven wheel rubber rollers are fixed on sliding blocks of the roller transfer plates, and the positions of the sliding blocks are adjusted through adjusting screws, so that the positions of the driven wheel rubber rollers are adjustable; a semi-cylindrical boss is arranged behind the baffle plate of the mouse movable chamber and used for driving a mouse to run according to experimental expectations, and a runway cleaning brush is arranged above the transparent runway and can be contacted with the transparent runway.
The technical scheme of the invention is further improved as follows: the optical imaging system comprises a reflector platform, a light source and a camera box body, wherein the reflector platform is arranged in the middle of an upper runway and a lower runway of the transparent runway, a reflector is fixed on the reflector platform, the reflector reflects the plantar track of a mouse on the transparent runway into the camera box body, the camera box body is arranged in front of a mouse movable chamber, the camera box body is made of opaque materials, an observation window is arranged on the upper half part of the camera box body and is used for receiving behavior information of the mouse, a groove guide rail is arranged in the middle of the camera box body and used for installing the camera and adjusting focal length, and the plantar track of the mouse reflected by the reflector is captured.
The technical scheme of the invention is further improved as follows: the ultrasonic stimulation control platform comprises a function signal generator, a radio frequency power amplifier and a miniature head-mounted ultrasonic stimulator, wherein the miniature head-mounted ultrasonic stimulator is fixed on the head of a mouse in a sleeved mode, the function signal generator generates an excitation signal of ultrasonic waves, the excitation signal is amplified through the radio frequency power amplifier, and finally the miniature head-mounted ultrasonic stimulator is driven to generate the required ultrasonic waves; and adjusting the ultrasonic characteristics generated by the miniature head-mounted ultrasonic stimulator by adjusting the signal generator and the radio frequency power amplifier at any time according to the mouse behavior characteristic data generated by the visual analysis platform.
The technical scheme of the invention is further improved as follows: the ultrasound characteristics include ultrasound sound pressure amplitude, stimulus duration, stimulus repetition period, and stimulus "off" time.
The technical scheme of the invention is further improved as follows: the miniature head-mounted ultrasonic stimulator comprises a power line, a metal shell, a gasket, piezoelectric ceramics, a focusing acoustic lens and a skull base, wherein the gasket is fixed between the metal shell and the piezoelectric ceramics, the skull base is used for fixing the ultrasonic stimulator and the head of a mouse, a periodic excitation signal generated by a function signal generator is amplified by a radio frequency power amplifier to act on the piezoelectric ceramics, periodic vibration with fixed frequency, namely ultrasonic wave, is generated, and then is focused in the skull of the mouse through the acoustic lens to be further transmitted to a specific brain region of the mouse.
A method of closed loop ultrasound stimulation of behavior state dependence of a free-living mouse, using the closed loop ultrasound stimulation system of behavior state dependence of a free-living mouse of any one of the preceding claims, comprising the steps of:
s1, an animal behavior acquisition device acquires behavior information of a mouse in ultrasonic stimulation in real time, mainly a plantar track video of the mouse, which is called first behavior information, wherein the first behavior information can be transmitted to the visual analysis platform as real-time data to calculate behavior characteristic data of the mouse, and can be temporarily stored as an offline video for later analysis;
s2, when the first behavior information is transmitted to the visual analysis platform as real-time data, the visual analysis platform analyzes the first behavior information by using a preset visual analysis algorithm and obtains first behavior characteristic data of the mice;
s3, judging whether the first behavior feature data meets the requirements according to the expected requirements of the experiment; if the ultrasonic stimulation control platform meets the requirements, the control parameters of the ultrasonic stimulation control platform are kept unchanged; if the first behavior characteristic data does not meet the requirements, comparing the first behavior characteristic data with the experimental pre-requirements, generating an ultrasonic stimulation parameter control command, and transmitting the ultrasonic stimulation parameter control command to the ultrasonic stimulation control platform;
s4, the ultrasonic stimulation control platform generates ultrasonic stimulation to different forms of mice according to the received ultrasonic stimulation parameter control command;
s5, capturing behavior information of the mice under the ultrasonic stimulation in different forms by using an animal behavior acquisition device, wherein the behavior information is called second behavior information; the visual analysis platform analyzes the second behavior information and generates corresponding second behavior characteristic data, the visual analysis platform compares the first behavior characteristic data with the second behavior characteristic data, generates an ultrasonic stimulation control parameter command and sends the ultrasonic stimulation control parameter command to the ultrasonic stimulation control platform to generate ultrasonic stimulation for the mice, and meanwhile, the second behavior characteristic data is stored as the first behavior characteristic data so as to achieve closed-loop ultrasonic stimulation depending on behavior states of the free-moving mice.
By adopting the technical scheme, the invention has the following technical effects:
according to the mouse behavior characteristic data presented by the visual analysis platform, the ultrasonic stimulation control platform of the application achieves closed-loop ultrasonic stimulation depending on the behavior state of the mouse by adjusting the ultrasonic stimulation parameters of the mouse in the next period in real time according to the expected expectations of the system.
Drawings
FIG. 1 is a schematic diagram of the composition of a method and apparatus for closed loop ultrasonic stimulation of free-living mouse behavior dependence in accordance with the present invention;
FIG. 2 is a flow chart of a closed loop ultrasonic stimulation method of the present invention that is dependent on the behavior of a free-living mouse;
FIG. 3 is a schematic diagram of a device of a free-moving mouse behavior dependent closed-loop ultrasound stimulation method of the present invention;
FIG. 4 is a schematic diagram of an animal behavior acquisition device for acquiring behavior information of an experimental mouse according to the present invention;
FIG. 5 is an internal cross-sectional view of an animal behavior acquisition device;
FIG. 6 is a diagram of a pluggable cage for mouse exercise;
FIG. 7 is a schematic diagram of an optical imaging for reflecting mouse plantar information;
FIG. 8 is a flowchart of an image algorithm for identifying the sole of a mouse;
FIG. 9 is a schematic diagram of the ultrasound stimulation control platform composition;
wherein, 1, a supporting plate, 2, a driving wheel rubber roller, 3, a driven wheel rubber roller, 4, a transparent runway, 5, a baffle plate, 6, a driving motor, 7, a motor fixing plate, 8, a reflecting mirror, 9, a reflecting mirror platform, 10 and a belt, 11, a camera platform, 12, a camera, 13, a roller mobilizing plate, 14, adjusting screws, 15, a miniature head-mounted ultrasonic stimulator, 16, a strut, 17, a camera box, 18, a background light plate, 19 and a mouse movable chamber.
Detailed Description
The technical scheme of the invention is described in detail below with reference to the specific embodiments.
A free-moving mouse behavior state dependent closed loop ultrasound stimulation system, characterized by: comprising the following steps: the ultrasonic stimulation control platform is used for applying ultrasonic stimulation to the mice;
the animal behavior acquisition device is used for acquiring behavior information of the mice in real time and acquiring motion track videos of soles of the mice;
the visual analysis platform is connected with the animal behavior acquisition device and used for analyzing the behavior information of the mice and calculating behavior characteristic data of the mice, including the information of the movement speed, the stride, the pressure, the running time and the residence time of the mice.
Animal behavior acquisition device
The animal behavior acquisition device mainly comprises a motor driving system, an animal runway system, an optical imaging system and the like, as shown in fig. 4. The animal behavior acquisition device mainly depends on two support plates 1 for connecting all systems, the two support plates 1 are separated by a plurality of struts 4, and the support plates 1 are provided with screw holes, rubber roll openings, observation windows and other parts for connecting all other systems.
The motor driving system is composed of a motor fixing plate 7, a driving motor 6, a motor controller, a belt conveying device and the like, as shown in fig. 5. The motor fixing plate 7 is fixed at the bottom end of the front supporting plate 1 according to the corresponding position, two notches with the same diameter as the motor base screw are formed in the motor fixing plate 7, the driving motor 6 is fixed on the motor fixing plate 7, the motor can be fixed, and the flexibility of adjusting the position of the motor during installation is ensured. The belt conveying device adopts a belt synchronous wheel to drive with a belt, the belt synchronous wheel is fixed on the motor output shaft and the driving wheel rubber roll 2 in a key slot and jackscrew mode respectively, and the two belt synchronous wheels are connected in a belt connection mode for power transmission. The ratio of the teeth of the two said synchronizing wheels is 1:3, the motor rotation speed can be carried out
Is introduced into the animal runway system. After the driving motor 6 is fixed on the motor fixing plate 7, the power of the driving motor is reduced through the belt conveying device and then is transmitted into the runway system, and the motor controller can be adjusted to realize the speed reductionThe purpose of controlling the power is achieved, and the speed of the runway is further controlled.
The runway system consists of rubber rollers, a transparent runway 4, a baffle plate 5, runway cleaning and the like. And the belt synchronous wheel is arranged at one end of the driving wheel rubber roll 2, so that the power of the motor driving system is transmitted to the animal runway system.
The device for adjusting the positions of the rubber rollers is arranged on the front supporting plate 1 and the rear supporting plate 1, as shown in fig. 4, the driven rubber roller 3 is fixed with the sliding block through a bearing, the position of the sliding block can be adjusted through an adjusting screw 14, the purpose of adjusting the tightness of the transparent runway 4 is achieved, and a roller adjusting plate 13 is arranged outside the sliding block and fixed with the front supporting plate 1 and the rear supporting plate 1, so that the function of fixing the sliding block can be achieved.
One of the two rubber rollers is a driving wheel rubber roller 2, drives a driven wheel rubber roller 3 to rotate, supports a transparent runway 4, and transmits the power of the driving wheel rubber roller 2 to the driven wheel rubber roller 3 by means of friction between the transparent runway 4 and the rubber rollers, so that the runway runs smoothly. The baffle plates with the protrusions are arranged on the front side and the rear side of each rubber roller, so that the runway can be prevented from deviating from the rubber rollers during operation, and smooth operation of an animal runway system is ensured. The pluggable baffle plate 5 is added above the runway and is used for defining the running area of the mice, the structure can not only prevent the mice from escaping, but also adapt to the experimental mice with different sizes by plugging the baffle plate, and a semi-cylindrical boss is arranged at the rear of the baffle plate 5 and is used for driving the mice to run according to experimental expectations as shown in fig. 6.
When the mouse runs, the mouse can generate stains including excrement and mud points on the transparent runway 4, so that a hairbrush is arranged above the transparent runway 4 and is fixed through screw holes provided by the supporting plate 1 for cleaning the transparent runway in real time.
The optical imaging system consists of a reflector platform 9, a light source, a camera box 17 and the like.
A reflecting mirror platform 9 is installed in the middle of the upper runway and the lower runway of the transparent runway 4, the reflecting mirror platform 9 is fixed through a threaded hole provided by the supporting plate 1, and a reflecting mirror 8 is fixed on the reflecting mirror platform 9 and used for reflecting the plantar track of a mouse on the runway into a camera box 17. The camera box 17 is made of opaque materials, interference of other light is prevented, an observation window is formed in the upper half portion of the box, the observation window is used for receiving mouse behavior information, a camera platform 11 is arranged in the camera box 17, a groove guide rail is arranged on the camera platform 11, the camera 12 can be installed, the focal length can be adjusted, and the mouse plantar track reflected by the reflecting mirror is captured, as shown in fig. 7. Screw holes for fixing the camera body 17 and the support plate 1 are reserved on the lower side of the camera body, so that the camera 12 needs to be refocused due to the change of relative positions of the runway system and the optical imaging system. A background light plate 18 is arranged above the baffle plate 5 and can emit green background light, white light supplementing lamps are respectively arranged on the left side and the right side of the baffle plate 5, and the light intensity of each light source is adjusted in real time through a light controller, so that convenience is brought to subsequent video analysis.
Visual analysis platform
The visual analysis platform is a computer desktop application program, a personal computer is connected with an industrial camera through a USB data line for data transmission, and the visual analysis platform is used for processing animal behavior information provided by the animal behavior acquisition device, processing and analyzing the animal behavior information by using computer vision knowledge, and finally obtaining mouse behavior characteristic data including running speed, stride, pressure, running time, intermittent time and other information. The specific steps of extracting and identifying the mouse sole (comprising a right forefoot, a left forefoot, a right hind foot and a left hind foot) are as follows (as shown in figure 8):
the video is acquired, the high definition color camera is adopted in the embodiment to acquire the motion video of the mouse, and the camera can be started or stopped by using an API provided by opencv through a visual analysis platform. And the camera is started to record the running track of the mouse, because the video output by the camera is an analog signal, in order to ensure that the image can be processed by using the knowledge of digital image processing, namely opencv processing function, the acquired video needs to be transcoded, namely, the analog signal is converted into a digital signal, and the conversion process is completed by an analog-digital conversion chip in the camera. And because the camera outputs the RGB model adopted by the image, and the image is stored and processed in opencv by using the BGR model, the image is used for processing opencv (the effective number of the image adopted in this example is eight bits) after being converted by codes:
firstly, deeply copying an original image in a temporary variable, then, converting the image from an RGB model to a BGR model by utilizing an API function cvtColor () function in opencv, wherein the API function in opencv is self-contained, and finally, ensuring that the image can be processed and operated by using the API algorithm function in opencv.
Firstly, obtaining plantar motion video of a mouse, recording a runway video of the mouse which is not put in advance, transcoding the video according to a video transcoding mode, and then carrying out a process of transcoding according to a formula (B in the formula n (x, y) represents the background image, N represents the number of frames in a sequence of images, image i (x, y) represents the (x, y) pixel point in the ith frame sequence image
All background images can be obtained by changing the values of x and y, and the background images are saved. And simultaneously acquiring real-time video data with the movement track of the mouse sole, namely the first behavior data.
Since only the sole portion of the mouse is analyzed when it is in motion, a target tracking algorithm is first performed on the moving mouse in video. Because the camera acquires color images (R red, G green and B blue, and three channels), the data size is large, and the performance cost of a computer is large in the image processing and storage process, the image is subjected to gray processing, the gray image refers to that each pixel of the image only contains one color information, namely, the image is stored and processed through only one channel, and the colors can have different shades of gray, which is called gray level. The gray scale is represented by an integer between 0 and 255. The present example implements the graying process of a color image using an average method (wherein V Ash of ash Representing a pixel at a point in a grayed imageGray value of (2)
The image is preprocessed, and because the camera is affected by light and environmental problems such as runway stains when acquiring the image, the image needs to be preprocessed first.
Firstly, filtering is adopted to reduce noise in an image, trivial details in the image are removed, a subsequent algorithm is simplified, and the effects of smoothing curves and softening line-to-line friction are achieved. The example adopts a weighted average filtering algorithm, which belongs to a linear algorithm, and can ensure the local characteristics of signals while ensuring the filtering advantages. The method is characterized in that the weight is reasonably weighted according to the distance from the center point of the pixel, the weight is higher as the distance is closer, and otherwise, the weight is lower as the distance from the center point of the pixel is farther. Sliding formwork with size of 3*3
The image is subjected to sliding processing within the template range, and the gray scale of a pixel point of the template is I (x, y), so that the gray scale value after filtering is +.>
And extracting a background picture and a foreground object in the first behavior data, namely the sole motion track of the mouse by adopting an image difference method. The specific measures are that the first behavior data and the background picture are differenced according to the following formula to obtain a frame difference image D (x, y) of two images, wherein F (x, y) is a mouse motion video captured by a camera, namely the first behavior data of the mouse, and Fb (x, y) is the background picture
D(x,y)＝|F(x,y)-Fb(x,y)|
Extracting the body coverage area of the mouse, namely a foreground object, according to a connected domain formula (wherein R (x, y) is the result of the connected domain, and T is a preset threshold value)
The example calculates the threshold T by adopting an optimal threshold method, so as to distinguish foreground objects from background pictures and divide images. Let the gray level normal probability density function of the foreground pixel be p (z), the mean be μ, and the variance be δ 2 . The gray level normal probability density function of the background pixel is q (z), the mean value is v, and the variance is tau 2 . And the foreground object occupies the total image surface position to be lambda, and the background image occupies the total image area to be (1-lambda). Therefore, the pixel gray scale formula of the total image can be obtained as follows: λ is p (z) + (1- λ) q (z).
Assuming that a threshold T, i.e. pixels with gray levels smaller than T, are foreground regions, the rest of the pixel sets are background regions, and the probability of misjudging the pixels as foreground is set to be Q 1 (T) setting the probability of misjudging the pixel as background as Q 2 (T) the formula lambda Q can be obtained 2 (T)+(1-λ)Q 1 (T)＝λ[1-p(T)]+(1-λ)*Q 1 (T),
The differentiation of the above equation shows that (1- λ) q (T) =λ×p (T), and q (T) and p (T) are both subject to normal distribution, so that the threshold T can be obtained according to the above equation, and finally the binarization operation of the image is completed.
After the grey-scale and binarization of the image, the body contour of the mouse needs to be detected. The essence is to distinguish and realize the segmentation of the image based on the abrupt change of the gray value, wherein the section of the gray edge can be divided into three types of mathematical models: step edge model, ramp edge model, roof edge model. In order to detect the gray level change of an image, the edge of the image is detected and positioned by taking the first derivative or the second derivative, and the expression is that
Gradient->
The image edge intensity and direction representing the position of any point (x, y). Vector->
Has important geometric meaningI.e. the direction of maximum rate of change of the image at the (x, y) position. Vector->
The size of (2) is represented by M (x, y): />
M (x, y) is the value of the gradient vector direction change rate, the gradient vector direction is as follows:
in image processing, computing the gradient partial derivatives for each pixel in an image requires filtering the entire image using a filter template, which essentially is a convolution operation of values in an array of digital images. The image filter template is called an edge detection operator, and the recognition and extraction of the mouse body coverage area are finally completed by adopting a Canny operator in the embodiment.
And extracting a body coverage area of the mouse according to a binarization algorithm, calculating a head centroid coordinate, a tail centroid coordinate and a body centroid coordinate of the mouse according to a contour area of the mouse, connecting the head centroid coordinate and the tail centroid coordinate, and making a perpendicular line perpendicular to the straight line through the body centroid coordinate to perform cross segmentation on the area.
The centroid method is adopted to identify the centroid of the image, the S set is set as an edge set, and I (I, j) is the pixel point of the image after being segmented, so that the centroid of the image can be obtained according to the following formula.
1. Drawing closed curves sequentially on the mouse sole part of the identified mouse body coverage area by using a mouse, and calculating the average value of R, G, B components of all pixel points in the area according to a formula (wherein N pixel points in the S set closed curves are arranged and R is the total number of the pixel points in the S set closed curves i Within S setThe red component of the ith pixel, R Left forefoot Is the average value of the red component of the pixels in the region, B Left forefoot 、G Left forefoot Two-component same principle
And sequentially selecting the left forefoot, the right forefoot, the left rearfoot and the right rearfoot of the mouse in a frame way to obtain the characteristics of each foot of the mouse. And obtaining the foot characteristics of the mice according to the formula
And according to the cross dividing line, judging that each sole of the mouse is divided into a left front foot, a left rear foot, a right front foot and a right rear foot according to the positions.
2. The characteristics of each sole of the mouse are sequentially extracted manually, and the three primary colors of RGB of each sole mainly comprise right front foot, left front foot, right rear foot and left rear foot, are respectively ten times, are averaged, and are stored as the characteristics of the corresponding sole. And then judging the areas, which are areas with the body area conforming to the characteristics, of the mouse, namely the areas with the sole in cross segmentation, as corresponding right forefoot, left forefoot, right hindfoot and left hindfoot according to the characteristics of each sole. So that each sole of the mouse is identified as a left forefoot, a right forefoot, a left hind foot, and a right hind foot.
According to the steps, each sole of the mouse can be identified from the exercise video of the mouse, and as the RGB value of each sole of the mouse is greatly different in the landing and suspending states in the exercise process of the mouse, whether the sole of the mouse is in a suspending or landing state can be judged according to the threshold value, and further the exercise amplitude, running speed, running time, body twisting angle and the like of the legs of the mouse can be judged.
Ultrasonic stimulation control platform
The ultrasonic stimulation control platform mainly comprises a function signal generator, a radio frequency power amplifier and a miniature head-mounted ultrasonic stimulator 15. Fig. 9 shows a schematic diagram of the implementation of neuromodulation of free-living mice using an ultrasound stimulation device with a miniature head-mounted ultrasound stimulator as the core.
In a medium that can propagate mechanical vibration, a mechanical wave generated by mechanical vibration with a vibration frequency greater than 20kHz is called an ultrasonic wave. The source for generating mechanical vibration is called a wave source, and the interaction force of the wave source and particles in the medium enables the particles to periodically vibrate back and forth by taking the self balance position as the center, so that the energy transmission is realized. The ultrasonic wave has thermal effect, cavitation effect, radiation force and the like, and can be well applied to the aspect of nerve regulation, wherein the ultrasonic wave entering the cranium and brain in the application of ultrasonic nerve regulation is mainly longitudinal wave. According to the spatial position of the stimulation target point and the acoustic parameters of the biological tissue where the stimulation target point is positioned, a proper acoustic wave propagation path is designed, so that the ultrasonic energy of specific parameters can be effectively transmitted to the target point area, and the key of effectively developing the ultrasonic nerve regulation and control application is realized. The one-dimensional wave equation describing planar ultrasound is the following equation (where p is sound pressure, t is time, x is the position of the target in the one-dimensional plane, and C is the speed of sound).
Solving the above equation to obtain the sound pressure with the one-dimensional space coordinate x at the time t as the following equation (wherein p a For sound pressure amplitude at the source, ω is angular frequency and k is wavenumber
p(x,t)＝p a e j(wt-kx)
w＝2πf,
The miniature head-mounted ultrasonic stimulator 15 for applying ultrasonic stimulation to the free-moving mice is designed, and is fixed to the heads of the experimental mice through a head cover mode, so that the free-moving state of the experimental mice can not be influenced while ultrasonic stimulation is applied to the experimental mice, the situation that the traditional ultrasonic stimulation method needs to fix the experimental mice in a heavy ultrasonic transducer in an anesthesia mode is avoided, and the effective real-time observation and feedback of the behaviors of the experimental mice subjected to ultrasonic stimulation are ensured to the greatest extent. The miniature head-mounted ultrasonic stimulator 15 mainly comprises a power line, a metal shell, a gasket, lead zirconate titanate piezoelectric ceramics, a focusing acoustic lens, a skull base and the like. The flexible cable is used as a power supply line, the gasket is fixed between the metal shell and the piezoelectric ceramic, the stimulator can work in the environment with air serving as a back lining, and the skull base is used for fixing the ultrasonic stimulator and the head of the mouse. The piezoelectric ceramic converts the periodic voltage generated by the function signal generator through the power amplifier into periodic vibration with fixed frequency, namely ultrasonic wave, and then acts on the inside of the skull of the mouse through the focusing of the acoustic lens so as to reach the specific brain region of the mouse.
The miniature head-mounted ultrasonic stimulator is arranged on the skull of the freely movable mouse, the function signal generator generates an excitation signal of ultrasonic waves, the excitation signal is amplified by the radio frequency power amplifier, and finally the miniature head-mounted ultrasonic stimulator is driven to generate the required ultrasonic waves. And the ultrasonic characteristics generated by the miniature head-mounted ultrasonic stimulator are regulated at any time by regulating the signal generator and the radio frequency power amplifier according to the mouse behavior characteristic data generated by the visual analysis platform, wherein the ultrasonic characteristics comprise ultrasonic sound pressure amplitude, stimulation duration time, stimulation repetition period, stimulation closing time and the like.
A closed loop ultrasound stimulation method for free-living mice behavior state dependence, comprising the steps of:
s1, an animal behavior acquisition device is used for acquiring behavior information of a mouse in real time, mainly comprising a plantar track video of the mouse when running, and testing the behavior characteristic data change of the mouse by adjusting the speed of a running machine. Firstly, the animal behavior acquisition device can acquire behavior information of a mouse in ultrasonic stimulation in real time, mainly a plantar track video of the mouse, and the behavior information is called first behavior information. The first behavior information can be used as real-time data to be transmitted to the visual analysis platform for calculating the behavior characteristic data of the mice, and can also be temporarily stored as offline video for later analysis.
S2, the visual analysis platform connected with the animal behavior acquisition device is used for analyzing behavior information (mouse plantar track video) of the mouse and obtaining behavior characteristic data of the mouse: running speed, stride, pressure, running time, residence time, etc. When the first behavior information is transmitted to the visual analysis platform as real-time data, the visual analysis platform analyzes the first behavior information by using a preset visual analysis algorithm and obtains first behavior characteristic data of the mice.
S3, judging whether the first behavior feature data meets the requirements or not according to the expected requirements of the experiment. Meets the requirements, and keeps the control parameters of the ultrasonic stimulation control platform unchanged; and if the first behavior characteristic data does not meet the requirements, comparing the first behavior characteristic data with the experimental pre-requirements, generating an ultrasonic stimulation parameter control command, and transmitting the ultrasonic stimulation parameter control command to the ultrasonic stimulation control platform.
And S4, the ultrasonic stimulation control platform is used for receiving ultrasonic stimulation parameter control commands and generating ultrasonic stimulation in different forms according to the commands, wherein the ultrasonic stimulation comprises intensity, interval time, duration, brain action area and the like. And the ultrasonic stimulation control platform generates ultrasonic stimulation to different forms of mice according to the received ultrasonic stimulation parameter control command.
S5, the animal behavior acquisition device captures behavior information of the mice under the ultrasonic stimulation in different forms, and the behavior information is called second behavior information. And the visual analysis platform analyzes the second behavior information and generates corresponding second behavior characteristic data. The visual analysis platform compares the first behavior characteristic data with the second behavior characteristic data, generates an ultrasonic stimulation control parameter command and sends the ultrasonic stimulation control parameter command to the ultrasonic stimulation control platform to generate ultrasonic stimulation for the mice, and simultaneously stores the second behavior characteristic data as the first behavior characteristic data so as to achieve closed-loop ultrasonic stimulation depending on behavior states of the free-moving mice.
The invention provides a closed loop ultrasonic stimulation method and device for free activity mice behavior state dependence, comprising an animal behavior acquisition device, a visual analysis platform and an ultrasonic stimulation control platform. And the visual analysis platform analyzes the first behavior information of the animal provided by the animal behavior acquisition device and obtains corresponding first behavior characteristic data. Analyzing whether the first characteristic data meets expected experimental expectations or not, applying ultrasonic stimulation to the mice by the ultrasonic stimulation control platform according to an ultrasonic stimulation control parameter command, collecting behavioral response of the mice by the animal behavior collecting device and the visual analysis platform, calculating behavioral characteristic data of the mice as second behavioral characteristic data, comparing the behavioral characteristic data with the first behavioral characteristic data, and changing stimulation parameters of the ultrasonic stimulation device according to the behavioral characteristic data, so that closed-loop ultrasonic stimulation depending on behavior states of the freely movable mice is achieved.
Claims (10)
1. A free-moving mouse behavior state dependent closed loop ultrasound stimulation system, characterized by: comprising the following steps: the ultrasonic stimulation control platform is used for applying ultrasonic stimulation to the mice;
the animal behavior acquisition device is used for acquiring behavior information of the mice in real time and acquiring motion track videos of soles of the mice;
the visual analysis platform is connected with the animal behavior acquisition device and used for analyzing the behavior information of the mice and calculating behavior characteristic data of the mice, including the information of the movement speed, the stride, the pressure, the running time and the residence time of the mice.
2. A free-living mouse behavior state dependent closed loop ultrasound stimulation system according to claim 1, wherein: the animal behavior acquisition device comprises a motor driving system, an animal runway system and an optical imaging system; the ultrasonic stimulation control platform is used for receiving ultrasonic stimulation parameter control commands and generating ultrasonic stimulation in different forms according to the commands, wherein the ultrasonic stimulation comprises intensity, interval time, duration and brain action area; and the ultrasonic stimulation control platform generates ultrasonic stimulation to different forms of mice according to the received ultrasonic stimulation parameter control command.
3. A free-living mouse behavior state dependent closed loop ultrasound stimulation system according to claim 2, wherein: the motor driving system comprises a driving motor (6) for driving the animal runway system to move, a motor fixing plate (7), a motor controller and a belt conveying device; the animal runway system comprises rubber rollers, a transparent runway (4), baffle plates (5) and runway cleaning brushes, wherein the rubber rollers comprise a driving wheel rubber roller (2) and driven wheel rubber rollers (3), the transparent runway (4) is sleeved on the two rubber rollers separated by a certain distance, the belt conveying device drives the driving wheel rubber roller (2) to operate so as to drive the transparent runway (4) to operate, the supporting plates (1) are arranged on the front side and the rear side of the two rubber rollers, and the baffle plates (5) which can be inserted and pulled are arranged in the middle of the supporting plates (1) on the transparent runway (4) to form a mouse movable chamber (19).
4. A free-living mouse behavior state dependent closed loop ultrasound stimulation system according to claim 3, wherein: the belt conveying device is characterized in that the belt synchronous wheels are in transmission with a belt (10), the belt synchronous wheels are respectively fixed on an output shaft of a driving motor (6) and a driving wheel rubber roller (2) in a key groove and jackscrew mode, the two belt synchronous wheels are connected in a belt (10) connection mode, and the tooth ratio of the two synchronous wheels is 1: and 3, the driving motor (6) is fixed on the motor fixing plate (7), and the power is controlled by adjusting the motor controller so as to control the transparent runway (4).
5. A free-living mouse behavior state dependent closed loop ultrasound stimulation system according to claim 3, wherein: the front support plate (1) and the rear support plate (1) are correspondingly provided with roller transferring plates (13), the driven roller rubber rollers (3) are movably fixed on the roller transferring plates (13), and the positions of the driven roller rubber rollers (3) on the roller transferring plates (13) are adjustable; the back of the baffle plate (5) of the mouse movable chamber (19) is provided with a semi-cylindrical boss for driving the mouse to run according to experimental expectation, and the runway cleaning hairbrush is arranged above the transparent runway and can be contacted with the transparent runway (4).
6. A free-living mouse behavior state dependent closed loop ultrasound stimulation system according to claim 2, wherein: the optical imaging system comprises a reflector platform (9), a light source and a camera box body (17), wherein the reflector platform (9) is arranged in the middle of an upper runway and a lower runway of the transparent runway (4), a reflector (8) is fixed on the reflector platform (9), the reflecting mirror (8) reflects the plantar track of a mouse on the transparent runway (4) into the camera box body (17), the camera box body (17) is arranged in front of a mouse movable chamber (19), the camera box body (17) is made of an opaque material, an observation window is arranged at the upper half part of the camera box body, and is used for receiving behavior information of the mouse, a groove guide rail is arranged in the middle of the camera box body (17) and used for installing a camera (12) and adjusting focal length, and the plantar track of the mouse reflected by the reflecting mirror is captured.
7. A free-living mouse behavior state dependent closed loop ultrasound stimulation system according to claim 2, wherein: the ultrasonic stimulation control platform comprises a function signal generator, a radio frequency power amplifier and a miniature head-mounted ultrasonic stimulator (15), wherein the miniature head-mounted ultrasonic stimulator (15) is fixed on the head of a mouse in a sleeved mode, the function signal generator generates an excitation signal of ultrasonic waves, the excitation signal is amplified through the radio frequency power amplifier, and finally the miniature head-mounted ultrasonic stimulator is driven to generate the required ultrasonic waves; and the ultrasonic characteristics generated by the miniature head-mounted ultrasonic stimulator (15) are adjusted at any time by adjusting the signal generator and the radio frequency power amplifier according to the mouse behavior characteristic data generated by the visual analysis platform.
8. A free-living mouse behavior state dependent closed loop ultrasound stimulation system as claimed in claim 7 wherein: the ultrasonic characteristics include ultrasonic sound pressure amplitude, stimulus duration, stimulus repetition period, and stimulus "off" time; the miniature head-mounted ultrasonic stimulator (15) comprises a power line, a metal shell, a gasket, piezoelectric ceramics, a focusing acoustic lens and a skull base, wherein the gasket is fixed between the metal shell and the piezoelectric ceramics, the skull base is used for fixing the ultrasonic stimulator and the head of a mouse, a periodic excitation signal generated by a function signal generator is amplified by a radio frequency power amplifier to act on the piezoelectric ceramics, periodic vibration with fixed frequency, namely ultrasonic waves, are generated, and then are focused by the acoustic lens to act on the skull of the mouse so as to reach a specific brain region of the mouse.
9. A method for closed-loop ultrasonic stimulation of behavior state dependence of a free-living mouse, characterized by being applied to the closed-loop ultrasonic stimulation system of behavior state dependence of the free-living mouse according to any one of the above claims, comprising the following steps:
s1, an animal behavior acquisition device acquires behavior information of a mouse in ultrasonic stimulation in real time, wherein the behavior information is mainly a plantar track video of the mouse, namely first behavior information, and the first behavior information can be transmitted to a visual analysis platform as real-time data to calculate behavior characteristic data of the mouse or can be temporarily stored as an offline video for later analysis;
s2, when the first behavior information is transmitted to the visual analysis platform as real-time data, the visual analysis platform analyzes the first behavior information by using a preset visual analysis algorithm and obtains first behavior characteristic data of the mice;
s3, judging whether the first behavior feature data meets the requirements according to the expected requirements of the experiment; if the ultrasonic stimulation control platform meets the requirements, the control parameters of the ultrasonic stimulation control platform are kept unchanged; if the first behavior characteristic data does not meet the requirements, comparing the first behavior characteristic data with the experimental pre-requirements, generating an ultrasonic stimulation parameter control command, and transmitting the ultrasonic stimulation parameter control command to the ultrasonic stimulation control platform;
s4, the ultrasonic stimulation control platform generates ultrasonic stimulation to different forms of mice according to the received ultrasonic stimulation parameter control command;
s5, capturing behavior information of the mice under the ultrasonic stimulation in different forms by using an animal behavior acquisition device, wherein the behavior information is called second behavior information; the visual analysis platform analyzes the second behavior information and generates corresponding second behavior characteristic data, the visual analysis platform compares the first behavior characteristic data with the second behavior characteristic data, generates an ultrasonic stimulation control parameter command and sends the ultrasonic stimulation control parameter command to the ultrasonic stimulation control platform to generate ultrasonic stimulation for the mice, and meanwhile, the second behavior characteristic data is stored as the first behavior characteristic data so as to achieve closed-loop ultrasonic stimulation depending on behavior states of the free-moving mice.
10. A method of closed loop ultrasound stimulation dependent on the behavior state of a free-living mouse as claimed in claim 9 wherein: the specific method for analyzing the first behavior information by the visual analysis platform in the step S2 by utilizing a preset visual analysis algorithm and obtaining the first behavior characteristic data of the mice is as follows:
sequentially drawing closed curves on the mouse sole part of the identified mouse body coverage area by using a mouse, and calculating the average value of R, G, B components of all pixel points in the area according to a formula, wherein N pixel points are arranged in the S set closed curves, and R is the total number of the pixel points in the S set closed curves i For the red component of the ith pixel in the S set, R Left forefoot Is the average value of the red component of the pixels in the region, B Left forefoot 、G Left forefoot The two components are the same as each other,
sequentially selecting the left forefoot, the right forefoot, the left hind foot and the right hind foot of the mouse in a frame mode to obtain the characteristics of each foot of the mouse, and obtaining the foot characteristics of the mouse according to a formula:
and according to the cross dividing line, judging that each sole of the mouse is divided into a left front foot, a left rear foot, a right front foot and a right rear foot according to the positions;
sequentially manually extracting the characteristics of each sole of a mouse, namely, three primary colors of RGB of each sole, including a right forefoot, a left forefoot, a right hindfoot and a left hindfoot, respectively, taking an average value of the three primary colors, storing the three primary colors as the characteristics of the corresponding soles, and then judging the areas, which are areas with the characteristics, of the body area of the mouse, namely, the areas with the sole in cross segmentation, as the corresponding right forefoot, left forefoot, right hindfoot and left hindfoot according to the characteristics of each sole, so that each sole of the mouse is identified as the left forefoot, the right forefoot, the left hindfoot and the right hindfoot respectively;
according to the steps, each sole of the mouse can be identified from the exercise video of the mouse, and as the RGB value of each sole of the mouse is greatly different in the landing and suspending states in the exercise process of the mouse, whether the sole of the mouse is in a suspending or landing state can be judged according to the threshold value, and then the exercise amplitude, running speed, running time and body twisting angle of the legs of the mouse can be judged.
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