Temperature control behavioral detection device for small-model organisms

Abstract

The utility model provides a temperature control behavioral detection device for small-model organisms. The device mainly comprises three systems, namely a behavioral monitoring system, a temperature control system and an optical genetic control system. Wherein the behavioral monitoring system consists of a porous culture plate and an infrared camera device, and is used for recording behaviors of small-model organisms; the temperature control system adopts a 24-hole, 48-hole or 96-hole thermosensitive metal plate structure, the temperature is accurately controlled through a programmable multi-section temperature controller, and a water cooling system is arranged to realize rapid heat dissipation; the optical genetic control system uses an LED lamp with a specific wavelength to realize accurate regulation and control of neurons. According to the device, through the structural design that the thermosensitive metal plate is tightly attached to the culture plate, accurate regulation and control of the culture environment temperature are achieved, and the technical problems that a traditional incubator is slow in temperature response and large in fluctuation are solved. The device is compact in structure, accurate in temperature control and convenient to operate, and is particularly suitable for behavioral research of small model organisms such as drosophila melanogaster.

Description

Technical Field

[0001] The utility model belongs to the technical field of neurobiology, and particularly relates to a temperature-controlled ethology detection device for small model organisms. Background Art

[0002] The temperature control method of a traditional constant temperature incubator usually blows the heat energy and cold energy generated by a heating and cooling module into the chamber through a fan, so as to change the temperature of the chamber. This kind of temperature rise and fall by blowing air usually has poor control over temperature stability and often causes large temperature fluctuations. In addition, another disadvantage of the air-blowing temperature control is that it is difficult to control the temperature to the set effect in a short time, and the temperature control method is single and constant, and cannot perform temperature control linearly and in multiple segments. Therefore, it cannot meet the experimental requirements of complex multi-segment temperature changes. The temperature change in nature is usually slow and linear, so the fixed temperature control of the traditional incubator has great limitations for most biological experiments.

[0003] The TRP channel protein (Transient receptor potential channels) is a cation-selective channel protein composed of six transmembrane domains, especially selectively permeable to Ca2+ and Na+ ions. At the same time, the TRP channel protein is highly conserved among various species. In genetic manipulation, dTrpA1 is often used to excite the activity of nerve cells, and the activation of dTrpA1 needs to be achieved by heating. At present, in the field of Drosophila ethology, the activation of dTrpA1 is generally achieved by heating the incubator. Due to the large space of the incubator, the air in the whole incubator needs to be heated each time when heating to reach the preset heating temperature. Therefore, using the incubator to heat has limitations such as high cost, slow heating time, large temperature fluctuations and difficulty in freely and accurately controlling the temperature. The limitations of heating the incubator make it difficult to achieve the expected effect in the application of dTrpA1 in small model animal ethology experiments, and the repeatability of the experiment is poor.

[0004] The literature search of the prior art found that the patents closely related to the present utility model are "Drosophila Ethology Research System and Research Method, (Application No.: CN101869093B)", and one is "Systems and methods for monitoring and controlling Drosophila activity, (Application No.: US11058093B2). The former focuses on the development of Drosophila ethology paradigms, especially the social behavior of Drosophila, while the latter developed Drosophila ethology monitoring system mainly involves the rhythm field. Although the latter in DAM ( DrosophilaBased on the Activity Monitor system, the Flybox, a high-throughput screening system for the behavioral detection of small model organisms, was further developed. However, from the publicly available specifications and claims, since the Flybox is a sealed box, when controlling the temperature, the entire Flybox is usually placed in an incubator. It is difficult to heat the small model organisms in the Flybox, which to a certain extent affects the repeatability and accuracy of the experimental results. At the same time, these two utility model patents only focus on the research of Drosophila behavior, and their universality and extensiveness still need to be further developed. In addition, due to the limitations of the technology and experimental conditions at that time, the inventors did not pay attention to the externally applied temperature genetics conditions, so temperature genetics could not be well applied to the small model organism behavioral detection system. This system can not only achieve complex multi-segment linear temperature control, but also can be adjusted to 24-well, 48-well or 96-well plates according to the size of small model organisms, and can perform temperature-controlled behavioral manipulation and monitoring on larger insects such as mosquitoes, ants, house flies and zebrafish larvae. Summary of the Invention

[0005] In view of the limitations of temperature genetics technology in the current small model organism behavior science, the present utility model develops a small model organism behavior temperature control system that integrates optogenetic control system, temperature genetics control system and temperature control system, which is simple to operate, stable in the long term and has wide practicability.

[0006] A temperature-controlled behavior detection device for small model organisms, the device includes an optogenetic control system, a small model organism behavior monitoring system and a temperature control system, characterized in that: the temperature control system heats or cools the small model organisms in the small model organism behavior monitoring system through a metal hole plate.

[0007] The small model organism behavior detection system includes a small model organism behavior recording system and a small model organism culture system. The small model organism behavior detection system provides supplementary light to the small model organisms through an 850nm bottom infrared light source and records the movement of the small model organisms in real time through a camera, and the infrared cut-off filter inside the camera needs to be removed. The small model organism culture system includes a white polystyrene 24-well, 48-well or 96-well plate, food and a multi-well plate sealing film. The composition of the food is: 50 g / L of sucrose, 10 g / L of agar powder, and 3 g / L of methylparaben; several small holes need to be poked in the multi-well plate sealing film with tweezers to ensure the air required for the survival of small model organisms.

[0008] The temperature control system includes a multi-segment temperature controller, a heating and cooling module, a temperature sensor, a heat dissipation module, a thermosensitive metal multi-well plate (24-well, 48-well or 96-well plate) and a water cooling system. The size of the thermosensitive metal multi-well plate is shown in Figure 1 、 2And 3, the thermosensitive metal porous plate controls the heating and cooling module through a multi-stage thermostat for linear or constant cooling or heating; the heat generated by the operation of the heating and cooling module is dissipated by a water block; the heat dissipation module diffuses the temperature generated by the heating and cooling module; the water block is connected to the water cooling system through a silicone tube for heat dissipation, and the water cooling system includes a water pump and a water chiller.

[0009] The multi-stage thermostat receives the real-time temperature information of the metal porous plate through a temperature sensor, and controls heating and cooling and their amplitudes by a bidirectional reversing electrode and outputting voltages and currents with different ratios. The temperature sensor should be closely attached to the groove (30*4.8) in the metal porous plate.

[0010] The heating and cooling module should be fixed at both ends of the metal porous plate to provide thermal or cold power for the metal porous plate.

[0011] After the heating and cooling modules are connected in parallel, they should be connected to the current output port of the multi-stage thermostat.

[0012] The water block should be closely attached to the heating and cooling module and is connected to the water pump and the water chiller through a silicone tube to dissipate the heat generated by the operation of the cooling fin.

[0013] The food in the white polystyrene 24-, 48-, and 96-well plates in the culture system should cover the entire small hole to ensure that the activity space of the small model organisms is only within the range of the small holes in the metal hole plate.

[0014] The optogenetic control system includes red or green LED lights and manipulates the optogenetic elements in the neurons of small model organisms through the red or green LED lights.

[0015] Specifically, closely attach the white polystyrene 24-well, 48-well or 96-well plate filled with food to the metal porous plate, and there should be no gap between the two plates to prevent small model organisms from flying out; place the small model organisms to be tested in the small holes of the metal porous plate, and attach the sealed film with small holes to the metal porous plate to maintain the air required for the survival of the small model organisms; closely attach the heating and cooling modules to both ends of the metal porous plate, connect them in parallel and then connect them to the current output port of the multi-stage thermostat to provide thermal or cold power for the metal porous plate; closely fix the water block to the heating and cooling module and connect it to the water pump and the water chiller through a silicone tube to dissipate the heat generated by the operation of the cooling fin through water circulation; fix the temperature sensor in the groove at any one end of the metal porous plate and connect it to the multi-stage thermostat to collect the temperature information of the metal porous plate in real time. The multi-stage thermostat receives the surface temperature of the metal porous plate collected by the temperature sensor in real time and converts and outputs the control voltage and current ratio in real time through the PID (Process Identification) algorithm to quickly reach the preset temperature and stably maintain it. Fix two red or green LED lights diagonally above the metal porous plate to ensure that the light of the LED lights can evenly cover the entire metal 2 porous plate.

[0016] The beneficial effects of the present utility model are as follows: to provide a small model organism behavioral temperature control system that integrates a optogenetic control system, a temperature genetic control system, and a behavioral monitoring system, which is simple to operate, convenient to use, has a stable system, and is widely applicable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 are the three-view dimension diagrams of a metal 96-well plate (unit: mm)

[0018] Figure 2 are the three-view dimension diagrams of a metal 48-well plate (unit: mm)

[0019] Figure 3 are the three-view dimension diagrams of a metal 24-well plate (unit: mm)

[0020] Figure 4 is the schematic diagram of the small model organism behavioral temperature control device

[0021] Figure 5 is the real-time acquisition diagram of linear multi-segment temperature change

[0022] Figure 6 is the comparison diagram of real-time temperature monitoring between the small model organism Drosophila behavioral temperature control device and the incubator (using a high-precision thermometer to monitor the real-time temperature of the Drosophila behavioral temperature control device and the incubator in real time. The results show that the Drosophila behavioral temperature control system has a good temperature control effect)

[0023] Figure 7 is the activity statistical chart of promoting active neurons by heating (using the Drosophila behavioral temperature control device and the incubator to heat for a long time to activate the promoting active nerve to monitor the activity of Drosophila. The results show that the Drosophila behavioral temperature control device has a better heating effect than the incubator when heating) DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The present utility model will be further described below in conjunction with specific embodiments, but the protection scope of the present utility model is not limited thereto:

[0025] Embodiment 1: A small model organism behavioral temperature control device

[0026] A temperature control behavioral detection device for small model organisms, the device includes an optogenetic control system, a small model organism behavioral monitoring system, and a temperature control system. The temperature control system heats or cools small model organisms in the small model organism behavioral monitoring system through a metal 96-well plate.

[0027] The temperature control system includes a thermosensitive metal 96-well plate, a heating and cooling module (12V 5A), a multi-stage temperature controller (TCM-1031) (purchased from Chengdu Yexian Technology Co., Ltd.), and a water cooling system. The dimensions of the thermosensitive metal 96-well plate are shown in Figure 1 , the thermosensitive metal 96-well plate controls the heating and cooling of the heating and cooling module through a multi-stage temperature controller. The heat dissipation module diffuses the temperature generated by the heating and cooling module, and the refrigeration chip dissipates heat through the water cooling system. The water cooling system includes a water pump, a water block, and a water chiller.

[0028] The multi-stage temperature controller receives the real-time temperature information of the metal 96-well plate through a temperature sensor, and controls the heating and cooling and their amplitudes by reversing the two-way electrode and outputting voltages and currents with different ratios. The temperature sensor is fixed in the groove at any end of the metal 96-well plate and is connected to the multi-stage temperature controller, collecting the temperature information of the metal 96-well plate in real time, and converting and outputting the control voltage and current ratio in real time through the PID (Process Identification) algorithm, so as to quickly reach the preset temperature and maintain it stably.

[0029] The heating and cooling module is closely attached to both ends corresponding to the metal 96-well plate, providing a heat source or a cold source for the metal 96-well plate.

[0030] The heat dissipation module is closely attached above the heating and cooling module, diffusing the temperature generated by the heating and cooling module to ensure that the air around the temperature control system is consistent with the temperature of the metal multi-well plate.

[0031] The water block should be closely attached to the heating and cooling module and is connected to the water pump and the water chiller through a silicone tube to dissipate the heat generated by the refrigeration chip during operation.

[0032] The food in the white polystyrene 96-well plate in the culture system needs to cover the entire small hole to ensure that the activity space of the small model organisms is only within the range of the small holes in the metal 96-well plate.

[0033] The optogenetic control system includes red or green LED lights and manipulates the optogenetic elements in the neurons of small model organisms through the red or green LED lights.

[0034] The small model organism behavior detection system includes a small model organism behavior recording system and a small model organism culture system. The small model organism behavior detection system provides supplementary light to the small model organisms through an 850nm bottom infrared light source and records the movement of the small model organisms in real time through a camera. The infrared cut-off filter inside the camera needs to be removed. The small model organism culture system includes food, a white polystyrene 96-well plate, and a 96-well plate sealing film. The composition of the food is: 50g / L sucrose, 10g / L agar powder, 3g / L methyl paraben; several small holes need to be poked in the 96-well plate sealing film with tweezers to ensure the air required for the survival of small model organisms.

[0035] Example 2: Evaluation of the Heating and Cooling Performance of the Small Model Organism Ethology Device

[0036] The utility model evaluates the heating and cooling effects of the small model organism ethology temperature control device by comparing the heating and cooling of a metal 96-well plate in an ordinary incubator.

[0037] 1 Heating and Cooling of the Incubator

[0038] Place the small model organism ethology temperature control device in an incubator (purchased from Ningbo Saifu Experimental Instrument Co., Ltd.), turn on the water cooling system in the temperature control equipment, but do not turn on the multi-stage thermostat. Fix the metal probe of the temperature recorder in the groove of the metal 96-well plate, and set to record once every 1 second; set the incubator temperature to 24 °C and keep it for 1 hour. After 1 hour, immediately raise the temperature to 30 °C or lower the temperature to 18 °C and continue for 1 hour.

[0039] 2 Heating and Cooling of the Small Model Organism Ethology Temperature Control Equipment

[0040] Place the small model organism ethology temperature control device in an incubator, turn on the water cooling system in the temperature control equipment, but do not turn on the incubator temperature. Fix the metal probe of the temperature recorder in the groove of the metal 96-well plate, and set to record once every 1 second; set the multi-stage thermostat temperature to 24 °C and keep it for 1 hour. After 1 hour, immediately raise the temperature to 30 °C or lower the temperature to 18 °C and continue for 1 hour.

[0041] From Figure 5 the real-time temperature acquisition, we can see that the temperature control of the utility model has a significant stable temperature control effect both in the short term and in the long term, and can achieve multi-stage temperature change control both in the short term and in the long term.

[0042] From Figure 6 it can be seen that the small model organism ethology temperature control device of the utility model not only has a significantly shorter heating and cooling time than the incubator, but also has a significantly lower temperature fluctuation of the small model organism ethology temperature control device under long-term continuous constant temperature conditions than the incubator temperature fluctuation. This shows that the utility model provides a heating and cooling method that is more efficient and rapid than the prior art, greatly avoiding the experimental error caused by the difference between the target heating and cooling time and the actual heating and cooling time, and facilitating users to obtain more real and reliable experimental data.

[0043] Example 3: Application and Effect Evaluation of the Small Model Organism Ethology Device in Small Model Organism Ethology

[0044] 1 Hybridization and Collection of Experimental Drosophila

[0045] Drosophila carrying UAS-dTrpA1 with an activation element and specifically expressed in dopaminergic (TH-D4 -gal4, a nerve cell that promotes Drosophila locomotion) Drosophila hybrids of projection neurons, and UAS-dTrpA1 and TH-D were respectively used. 4 -Gal4 was crossed with wild-type Drosophila (wcs), and the male offspring were collected. All hybrid lines and offspring were cultured at a constant temperature of 22 °C, 50% humidity, and a 12-hour light: 12-hour dark cycle.

[0046] 2 Incubator heating to activate locomotion-promoting neurons

[0047] The male offspring of the experimental group and the control group collected were placed in the small holes of a metal 96-well plate above a white polystyrene 96-well plate covered with food, and sealed with a 96-well plate sealing film with small holes. The small model organism culture system was placed directly above an 850 nm bottom infrared light source and directly below a camera. The water cooling system was turned on but the multi-stage temperature controller was not turned on. The incubator temperature was set at 22 °C and continuously incubated at a constant temperature for three days. On the fourth day, the incubator temperature was raised to 30 °C to activate specifically labeled neurons. Throughout the experiment, 50% humidity and a 12-hour light: 12-hour dark cycle were given. The camera was set to record every 10 s for four days, and the movement or sleep of small model organisms was processed and analyzed by pysolo software.

[0048] 2 Heating of the temperature control device for small model organism behavior to activate locomotion-promoting neurons

[0049] The male offspring of the experimental group and the control group collected were placed in the small holes of a metal 96-well plate above white polystyrene covered with food, and sealed with a 96-well plate sealing film with small holes. The small model organism culture system was placed directly above an 850 nm bottom infrared light source and directly below a camera. The water cooling system was turned on but the incubator temperature was not turned on. The multi-stage temperature controller was set at 22 °C and continuously incubated at a constant temperature for three days. On the fourth day, the metal 96-well plate was heated to 30 °C by setting the multi-stage temperature controller to activate specifically labeled neurons. Throughout the experiment, 50% humidity and a 12-hour light: 12-hour dark cycle were given. The camera was set to record every 10 s for four days, and the movement or sleep of Drosophila was processed and analyzed by pysolo software.

[0050] From Figure 7 It can be seen that by heating to 30 °C for long-term activation of dopamineergic neurons that promote locomotion, with the same heating setting, the temperature control device can promote Drosophila locomotion significantly more than the incubator heating, indicating that during long-term heating, the heating effect of the Drosophila behavior temperature control device of the present utility model is better than that of the incubator, and it has better timeliness, stability, persistence, and universality.

Claims

1. A temperature control behavioral detection device for small model organisms, the device comprising a small model organism behavioral monitoring system, characterized in that: The small-model organism behavior detection system includes a small-model organism behavior recording system, a small-model organism culture system and an optogenetic control system; the small-model organism behavior recording system provides supplementary light through an 850nm bottom infrared light source and records the movement of the small-model organism in real time through a camera, and the camera needs to remove the infrared cutoff filter inside the camera; the small-model organism culture system includes food and a white polystyrene 24-well, 48-well or 96-well plate and a sealing film, and the sealing film needs to be pierced with a number of small holes with tweezers to ensure the air required for the survival of the small-model organism; the optogenetic control system includes a red or green LED light; The temperature control system includes a multi-stage thermostat, a hot and cold module, a temperature sensor, a heat dissipation module, a thermosensitive metal porous plate and a water cooling system. The thermosensitive metal porous plate is a 24-hole, 48-hole or 96-hole plate; the thermosensitive metal porous plate controls the hot and cold module through the multi-stage thermostat to perform linear or constant cooling or heating; the heat generated by the hot and cold module is dissipated through a water cooling head; the heat dissipation module diffuses the temperature generated by the hot and cold module; the water cooling head is connected to the water cooling system through a silicone tube to dissipate heat, and the water cooling system includes a water pump and a water chiller.

2. A temperature control behavior detection device for small model organisms as claimed in claim 1, characterized in that: The multi-stage temperature controller receives the real-time temperature information of the metal porous plate through the temperature sensor, and controls the heating and cooling and their amplitudes through bidirectional reversal electrodes and output voltages and currents of different proportions. The temperature sensor should be tightly attached to the 30*4.8 groove in the metal porous plate.

3. A temperature control behavior detection device for small model organisms as claimed in claim 1, characterized in that: The hot and cold modules should be fixed at both ends of the metal porous plate to provide thermal power or cold power for the metal porous plate.

4. A temperature control behavior detection device for small model organisms as claimed in claim 1, characterized in that: The hot and cold modules should be connected in parallel and then connected to the current output port of the multi-stage temperature controller.

5. A temperature control behavior detection device for small model organisms as claimed in claim 1, characterized in that: The water cooling head should be close to the hot and cold modules and connected to the water pump and the water cooling machine through a silicone tube.

6. A temperature control behavior detection device for small model organisms as claimed in claim 1, characterized in that: The heat dissipation module is closely attached to the top of the cold and hot modules to diffuse the temperature generated by the cold and hot modules.

7. A temperature control behavior detection device for small model organisms as claimed in claim 1, characterized in that: The food in the white polystyrene 24-well, 48-well or 96-well plate in the culture system should almost cover the entire small holes to ensure that the activity space of the small model organisms is limited to the small holes in the metal porous plate.

8. The temperature control behavior detection device for small model organisms according to claim 1, characterized in that: The optogenetic control system manipulates optogenetic elements in neurons of small model organisms using red or green LED light.

Images