A device for studying the behavior of zebrafish in relation to nicotine intake and a method of use

Abstract

The application discloses a device for studying the intake behavior of zebrafish to nicotine compounds, characterized in that a non-test area (A) and a plurality of test areas (B) are divided in a test area, the area of the non-test area (A) is larger than that of the test area (B), water can flow freely between the non-test area (A) and the test area (B), and zebrafish can swim freely between the non-test area (A) and the test area (B); nicotine compounds are contained in part of the test areas (B); an LED lamp plate (31) and a video acquisition device (32) are arranged above or below the test area. The application also discloses a use method of the device for studying the intake behavior of zebrafish to nicotine compounds. The application provides a simple and effective device for the "active drug intake" of zebrafish, and provides a basis for perfecting the experimental research on the intake preference of zebrafish.

Description

Technical Field

[0001] This invention belongs to the field of tobacco technology, specifically relating to a research device and method for studying the intake behavior of zebrafish on nicotine compounds. Background Technology

[0002] Zebra danios are bony fish belonging to the genus *Brutalina* in the family Cyprinidae of the subclass Pleuronectiformes. Sexing is relatively easy; males have yellowish blue stripes interspersed with lemon-colored stripes, while females have brighter, more bluish blue stripes interspersed with silver-gray stripes. Females are fuller and more robust than males, and their fins are shorter. Zebra danios are cold- and low-oxygen fish, exhibiting strong tolerance to both cold and heat. They are easy to keep, not demanding in terms of water quality, requiring neutral water at a temperature of 22-26℃. They can be kept with other small fish. Zebra danios are prolific breeders, with a breeding temperature of 24℃. The breeding cycle lasts approximately 7 days, and they can breed 6-8 times a year. Each female lays about 300 eggs per clutch, with larger females sometimes laying over a thousand. Sexing can be determined after about 2 months, and they reach sexual maturity at 5 months. Compared with mammalian model organisms such as rats and mice, zebrafish have mating behavior controlled by photoperiod, produce more eggs, fertilize eggs in vitro, have a faster reproductive cycle, are easier to raise, and their early embryos are transparent and easy to observe in vivo. Moreover, compared with lower animals such as Xenopus and fruit flies, zebrafish have a relatively clear genetic map with a similarity of more than 86% to humans, while rats have a similarity of 90%. They also have the same opioid receptors and neurotransmitters such as dopamine as humans.

[0003] Compared to rodents such as mice and rats commonly used in drug dependence research, zebrafish have the following advantages: 1. The good balance between the simplicity and complexity of organs and systems in adult zebrafish allows for a more accurate reflection of the effects of drugs on animal behavior. For behavioral changes and physiological manifestations that are difficult to observe in mice and rats, such as stress behavior, diving behavior, aggression, and phototaxis, zebrafish show more obvious and easily observable results; 2. Zebrafish are small and lightweight, requiring less medication, significantly reducing the amount of addictive substances used in experiments and conserving drug resources; 3. Zebrafish have a high reproductive rate and a short reproductive cycle, significantly shortening the experimental period; 4. Zebrafish embryos are transparent, facilitating live observation. Due to their advantages such as short passage time, ease of rearing, low cost, and suitability for large-scale screening, zebrafish are increasingly recognized by researchers for their potential applications in toxicology and behavioral science, and are gradually being used in drug dependence research to reduce the use of mammals such as mice and rats and accelerate the experimental process.

[0004] Common methods of drug administration in zebrafish include: (1) immersion administration, where small molecules dissolved in the aquatic environment can be directly absorbed through the zebrafish's skin and gills; (2) intraperitoneal administration, where zebrafish can swallow 72 hours after fertilization, and can also be administered orally after this stage; and (3) microinjection administration, where drugs that are poorly soluble in water and macromolecular drugs can be administered via microinjection into the yolk sac, sinus venosus, or circulatory system. Adult zebrafish can be administered via oral cannulation. The above three methods of drug administration have significant inconveniences in actual experiments: immersion administration and intraperitoneal administration are passive drug administration, where the test drug needs to be directly dissolved in the aquatic environment, and only a very small portion is directly absorbed, resulting in waste and water pollution. Only comparisons before and after administration can be made, making it difficult to observe the dynamic drug administration process. Microinjection administration can improve drug utilization and avoid waste and water pollution; however, microinjection is more difficult to operate and requires specialized training and equipment. All three methods of drug administration share a common drawback: they cannot be used for active drug administration experiments in zebrafish, which is a major limitation for behavioral research. Some researchers have tried embedding test drugs in feed pellets, which can achieve a preliminary "active drug administration" effect. However, drugs embedded in feed are easily soaked and diffused into the entire water body, making it difficult to continue the experiment.

[0005] Therefore, finding a simpler and more effective method for "active drug administration" in zebrafish is key to improving experimental research on zebrafish intake preferences. This invention is proposed for this purpose. Summary of the Invention

[0006] This invention provides a research apparatus for studying the intake behavior of zebrafish to nicotinic compounds and a method for using the apparatus. The apparatus of this invention solves the current difficulty in "active drug administration management" of zebrafish in research, providing strong hardware and software support for related research fields.

[0007] The technical solution adopted in this invention is as follows:

[0008] The first aspect of this invention discloses a research device for zebrafish ingestion of nicotinic compounds. The experimental area is divided into a non-test area A and several test areas B. The area of ​​non-test area A is larger than the area of ​​test area B. Water can flow freely between non-test area A and test area B, and the zebrafish can swim freely between non-test area A and test area B. Some test areas B contain nicotinic compounds. An LED light panel 31 and a video acquisition device 32 are set above or below the experimental area.

[0009] Preferably, the test area is the area of ​​water in the first water tank 1, and the test area B is the area of ​​water in the second water tank 2; the second water tank 2 is in the first water tank 1; the height of the side wall of the first water tank 1 is higher than the height of the side wall of the second water tank 2; the height of the water surface is not less than 10 mm higher than the height of the side wall of the second water tank 2.

[0010] Preferably, the height of the bottom wall 21 of the second water tank is greater than the height of the bottom wall 11 of the first water tank.

[0011] Preferably, the height of the side wall of the second water tank 2 is not less than 7 mm; the height of the bottom wall 21 of the second water tank is 5 mm higher than the height of the bottom wall 11 of the first water tank.

[0012] Preferably, a nicotine compound release needle 41 is provided on the bottom wall of test area B, and the nicotine compound release needle 41 is connected to a nicotine compound container 43 through a power transmission device 42.

[0013] Preferably, the power delivery device 42 is one of a peristaltic pump, a gear pump, a plunger pump, or a syringe pump, and the power delivery device 42 is electrically connected to the automatic control device 5; the video acquisition device 32 is electrically connected to the automatic control device 5.

[0014] Preferably, the first water tank 1 is a cuboid container, with an inlet 111 and an outlet 112 provided at both ends of the diagonal of the bottom wall of the first water tank.

[0015] The second aspect of this invention discloses a method for using the apparatus for studying the intake behavior of zebrafish to nicotinic compounds, comprising the following steps:

[0016] Water is supplied to the first water tank through the inlet 111, so that the water level is slightly lower than the height of the side wall of the first water tank 1; under the control of the automatic control device 5, nicotine compounds are released into the test area B in a pulse mode with different durations through the power delivery device 42 and the nicotine compound release needle 41.

[0017] Several adult zebrafish were selected and placed in the water of the first tank 1. The LED light panel 31 and the video acquisition device 32 were activated to track the movement trajectory of the zebrafish in the test area and the time spent in the test area B. Then, the zebrafish’s intake behavior of nicotinic compounds was analyzed and studied.

[0018] The beneficial effects of this invention are:

[0019] The apparatus for studying zebrafish nicotinic compound intake behavior of this invention provides a simple and effective device for "active drug intake" in zebrafish, and provides a basis for improving experimental research on zebrafish intake preferences. In the non-test area outside the test area of ​​this apparatus, due to the unidirectional inflow and outflow of water in the experimental tank, the drug in the water in this non-test area is sufficiently diluted and carried away, resulting in an extremely low drug concentration, which can be considered close to a "blank" state. The test area of ​​this apparatus is set in a shallow water zone, and the intensity of the zebrafish's water depth aversion response is used to determine the intake preference intensity of the tested nicotinic compound. Attached Figure Description

[0020] Figure 1 This is a front view of the apparatus used in Example 1 to study the zebrafish's intake behavior of nicotine compounds.

[0021] Figure 2 This is a top view of the apparatus used in Example 1 to study the zebrafish's intake behavior of nicotine compounds.

[0022] Figure 3 This is a front view of the apparatus used to study the zebrafish's intake behavior of nicotinic compounds in Example 2; test area B has a shallow water zone.

[0023] The attached diagram is labeled as follows: 1. First water tank; 11. Bottom wall of the first water tank; 111. Inlet; 112. Outlet; 2. Second water tank; 21. Bottom wall of the second water tank; 31. LED light panel; 32. Video acquisition device; 41. Nicotine-based compound release needle; 42. Power transmission device; 43. Nicotine-based compound container; 5. Automatic control device; A. Non-testing area; B. Testing area. Detailed Implementation

[0024] The present invention will be further described in detail below through specific embodiments, but it should not be construed as limiting the scope of the invention to the following examples. Various substitutions and modifications made based on ordinary technical knowledge and conventional methods in the art without departing from the spirit of the invention should be included within the scope of the invention.

[0025] Example 1: Nicotine Preference Test in Zebrafish

[0026] The apparatus used in this embodiment to study the zebrafish's intake behavior of nicotine compounds is as follows: Figure 1 and Figure 2 As shown in the diagram, the device consists of a square polymethyl methacrylate (plexiglass) water tank (60 cm long x 40 cm wide x 15 cm high) as the first water tank 1. Two circular test areas (B) with a diameter of 10 cm and a baffle wall height of 7 cm are located at either end of one diagonal of the first water tank, forming the second water tank 2. The center distance between the two test areas is 56 cm. The remaining areas within the first water tank are non-test areas (A). An inlet 111 and an outlet 112 are located at either end of the other diagonal. An LED light panel 31 and a video acquisition device 32 are mounted on top of the entire device; the video acquisition device 32 is connected to an automatic control device 5 (not shown in the diagram). Two nicotine compound release needles 41 are located at the center of the test areas, with the needle tips 1 cm from the bottom of the water tank.

[0027] The nicotine compound release needle 41 is connected to two sets of power delivery devices 42 micro-injection pumps (not shown in the figure), and to the nicotine compound container 43, and to the automatic control device 5 (not shown in the figure); the water tank circulation system is started, water is introduced into the water tank (first water tank) through the inlet 111 to make the water depth in the water tank (first water tank) 14 cm, and water is discharged from the outlet 112 to circulate the water in the first water tank 1; the LED light source is turned on. The nicotine compound release needle in the first test area provides 30 mg / L nicotine solution at a flow rate of 1 ml / min, and the injection needle in the second test area provides blank solution at a flow rate of 1 ml / min.

[0028] Three 3-month-old adult zebrafish were placed in the first tank 1. The video acquisition device 32 was activated to track the zebrafish's movement trajectory in the experimental area and the time spent in test area B. The zebrafish's behavior regarding the intake of nicotinic compounds was then analyzed. Each test lasted for one hour, after which the three zebrafish were returned to the normal tank. This cycle was repeated for five days.

[0029] Experimental results: On the first day, the three zebrafish spent almost the same amount of time in the two test areas; however, by the fifth day, the three zebrafish basically only gathered in the test area that provided nicotine solution, and almost completely ignored the control test area that did not provide nicotine.

[0030] Example 2: Nicotine Preference Test in Zebrafish with Superimposed Shallow-Water Anxiety

[0031] Zebrafish typically avoid shallow waters, presumably to avoid becoming targets for birds and other predators. Therefore, if the drug causes zebrafish to spend more time in shallow water after administration, it suggests that the drug effectively reduces their shallow-water danger anxiety through a reward effect.

[0032] The water tank in Example 1 was modified by raising the interior of the bottom wall 21 of the second water tank, which has two circular test areas with a diameter of 10 cm and a baffle wall height of 7 cm, by 5 cm. The position of the nicotine compound release needle 41 was changed accordingly so that it was 1 cm away from the bottom of the raised test area.

[0033] The system is connected to two sets of power delivery devices 42 micro-injection pumps (not shown in the figure), a nicotine compound container 43, and an automatic control device 5 (not shown in the figure); the water tank circulation system is activated, making the water depth in the first water tank 1 14 cm; the LED light source is turned on. The nicotine compound release needle in the first test area provides a 30 mg / L nicotine solution at a flow rate of 1 ml / min, while the injection needle in the second test area provides a blank solution at a flow rate of 1 ml / min. At this time, the water depth in non-test area A is 14 cm, and the water depth in test area B is 9 cm.

[0034] Three 3-month-old adult zebrafish were placed in the first tank 1. The video acquisition device 32 was activated to track the zebrafish's movement trajectory in the experimental area and the time spent in test area B. The zebrafish's behavior regarding the intake of nicotinic compounds was then analyzed. Each test lasted for one hour, after which the three zebrafish were returned to the normal tank. This cycle was repeated for five days.

[0035] Experimental results: On the first day, the swimming time of the three zebrafish in the two test areas B was almost the same, but the cumulative time was less than 1 minute, mainly concentrated in the deep water area of ​​non-test area A; by the fifth day, the three zebrafish basically only gathered in the test area provided with nicotine solution, almost completely ignoring the control test area without nicotine and the deep water area.

[0036] This invention employs a "localized" drug delivery design, where the drug concentration outside the test area is considered "zero" due to rapid dilution by the water flow, creating a local dynamic drug concentration-gradient region. This effectively improves active drug administration experiments in zebrafish, and the test results can more accurately correlate with zebrafish's preference behavior for specific compounds. By activating the video acquisition device 32, real-time monitoring of zebrafish entering the test area is achieved, and this real-time signal triggers the infusion pump to deliver fluid in various modes, thus meeting the needs of more complex experimental studies on zebrafish preference behavior.

[0037] The embodiments described are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for using a research apparatus for studying the nicotinic acid intake behavior of zebrafish with superimposed shallow-water anxiety, characterized in that, The research apparatus includes a non-test area (A) and several test areas (B) in the experimental area, wherein the area of ​​the non-test area (A) is larger than the area of ​​the test area (B), water can flow freely between the non-test area (A) and the test area (B), and zebrafish can swim freely between the non-test area (A) and the test area (B); some test areas (B) contain nicotinic compounds; and LED light panels (31) and video acquisition devices (32) are installed above or below the experimental area. The test area is the area of ​​water in the first water tank (1), and the test area (B) is the area of ​​water in the second water tank (2); the second water tank (2) is in the first water tank (1); the first water tank (1) is 15 cm high, and the second water tank (2) is a circular test area (B) with a baffle wall height of 7 cm. Nicotine compound release needle (41) is set on the bottom wall of the test area (B), and the nicotine compound release needle (41) is connected to the nicotine compound container (43) through a power transmission device (42). The power delivery device (42) is one of a peristaltic pump, a gear pump, a plunger pump or an injection pump, and the power delivery device (42) is electrically connected to the automatic control device (5); the video acquisition device (32) is electrically connected to the automatic control device (5). The first water tank (1) is a rectangular container, with an inlet (111) and an outlet (112) set at both ends of the diagonal of the bottom wall of the first water tank. The method of using the research device includes the following steps: The interior of the bottom wall (21) of the second water tank in the circular test area is raised by 5 cm, and the needle position of the nicotine compound release needle (41) is 1 cm away from the bottom of the raised test area; water is supplied to the first water tank through the inlet (111) so that the water level is slightly lower than the height of the side wall of the first water tank (1); water is discharged from the outlet (112) to circulate the water in the first water tank (1); Under the control of the automatic control device (5), nicotine compounds are released into the test area (B) in pulse mode with different durations through the power delivery device (42) and the nicotine compound release needle (41); Several adult zebrafish were selected and placed in the water of the first tank (1). The LED light panel (31) and video acquisition device (32) were activated to track the movement trajectory of the zebrafish in the test area and the time spent in the test area (B). Then the zebrafish’s intake behavior of nicotine compounds was analyzed.

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