Experimental device for detecting learning memory of insects

Abstract

The utility model relates to an experimental device for detecting learning memory of insects. The experimental device comprises a smell conveying device and a conditional training device. The smell conveying device comprises an air pump which is connected through a hose and located outside the box body, and an air filter, an air humidifier / flowmeter, a smell source chamber and a smell release pipe which are sequentially connected through the air pump through hoses, the conditioned training device is composed of an isolation chamber and an insect body fixator, the isolation chamber is of a hexahedron structure, the right face of the isolation chamber is provided with an opening, and the opening face is provided with an air outlet; the odor releasing pipe penetrates through the left side face of the isolation chamber and forms an included angle of 15-45 degrees with the bottom face of the isolation chamber, the left side, the front side, the rear side and the upper face of the isolation chamber are composed of light transparent thin plates, the bottom face is wrapped with a film, a micro-hole is formed in the center of the film on the bottom face, the insect body fixing device is located below the micro-hole of the isolation chamber, and an exhaust fan is arranged on the right side wall of the box body. The device is reasonable in structure, and can evaluate whether insects can establish the combined learning and memory ability of conditional odor stimulation and volatile compound or non-volatile food intake.

Description

Technical Field

[0001] The utility model belongs to the technical field of insect behavior science and relates to an experimental device for detecting insect learning and memory, in particular to a detection device for insect learning and memory driven by volatile chemical substances. Background Art

[0002] Olfactory conditioning of the proboscis extension reflex (Olfactory conditioning of PER) is a form of associative learning and memory. The establishment of this conditioned reflex requires the integration of two different types of external sensory information in the brain, namely the olfactory information of the odor, the taste brought by the ingestion of chemicals, and other brain action information. Since Kimihisa Takeda (1961) successfully developed the device and method for insect olfactory conditioned proboscis extension reflex, only sucrose food can be used as an unconditioned stimulus (US) paired with a conditioned stimulus (CS, odor) for conditioning training to evaluate the insect olfactory learning and memory ability driven by food goals. This classic method has been widely used in many insect groups such as fruit flies and bees. For example, Yu Jinxin (2019) used the classical olfactory conditioning training method to conduct odor and food joint learning training on male adults of the orange fruit fly indoors, that is, reward training combined with 10% sucrose solution (non-volatile food, US) and sweet orange essential oil (volatile odor, CS), using the proboscis reflex behavior as the criterion for learning or not, and measuring the selection behavior and tendency behavior of the orange fruit fly after learning. In other words, existing domestic and foreign technologies can only evaluate the association between non-volatile food (US) and volatile odor stimulation (CS), thereby evaluating the olfactory learning and memory ability of insects driven by non-volatile substances. However, to date, people have overlooked a basic fact, that is, in nature, there are still a large number of volatile chemicals (either food or drugs), which not only release odors for insects to perceive, but can also be actively orally ingested by insects to improve their own survival and reproduction capabilities. For example, volatile aliphatic compounds, phenylpropanoid derivatives, monoterpenes, sesquiterpenes and their oxygen-containing derivatives, and volatile fatty acids in the roots, stems, leaves, flowers, fruits, and peels of plants. However, it is still unclear whether insects can establish an association between volatile US and volatile CS in the insect brain. These ingested chemicals are volatile, so they can also be used as odor substances. Because of this, using existing device technology, the insect antennae olfactory system receives the odor of conditioned odor stimulation (CS, such as ethyl acetate, etc.) and unconditioned appetite stimulants almost simultaneously. The insect cannot determine which odor is the conditioned stimulus, so the insect cannot establish the classic CS-US joint learning and memory. In other words, the volatility of unconditioned appetite stimulants hinders the establishment of conditioned odor stimulation CS in the insect brain in our experiment. So far, people have not been able to evaluate the insect olfactory learning and memory ability driven by volatile appetite stimuli US, and people can only stay at the traditional classic CS-US (sucrose) olfactory learning and memory level research.

[0003] Therefore, it is necessary to develop a new insect conditioned proboscis reflex device, which can theoretically test the association between any odor (CS) and any volatile appetite stimulant (US, such as methyl eugenol), thereby more deeply evaluating the insects' learning and memory ability and survival ability, and serving the protection of insect biodiversity and human well-being. Utility Model Content

[0004] In view of the above technical problems, the utility model provides a device for detecting insect learning memory (insect conditioned proboscis reflex), and its technical solution is as follows:

[0005] The invention discloses an experimental device for detecting insect learning and memory, wherein the device comprises a box body made of an opaque material, a drawer door is arranged on the front of the box body, and an odor delivery device and a conditioning training device are arranged in the box body; the odor delivery device is an air pump located outside the box body connected by a hose, and the air pump is sequentially connected to an air filter, an integrated air humidifier / flow meter, an odor source chamber, and an odor release tube through the hose; the conditioned training device consists of an isolation chamber and an insect body fixer; the isolation chamber is a hexahedral structure, with an opening on the right side, and the opening side is an air outlet; the odor release tube runs through the left side of the isolation chamber and forms an angle of 15-45 degrees with the bottom surface of the isolation chamber, and the isolation chamber must be kept horizontal to facilitate the fine operation of the insect's tiny antennae; the left, front, back and top of the isolation chamber are composed of lightweight transparent thin plates, the bottom surface is wrapped with a film, and a micro hole is arranged in the middle of the bottom film; the insect body fixer is located below the micro hole of the isolation chamber, and an exhaust fan is arranged on the right side wall of the box body.

[0006] Furthermore, the insect body fixer is a small fixed tube with a backrest and a bracket with holes for stabilizing the small fixed tube.

[0007] Furthermore, the odor release tube is a hard tube with a diameter of ≤10 mm.

[0008] Furthermore, a white light and an infrared light are arranged on the top of the box.

[0009] Furthermore, the size of the box is 60*40*40 cm, and the size of the isolation chamber is 40*30*30 mm.

[0010] Furthermore, the box top is also provided with an adjustable fixing frame with a clamp for fixing the odor release tube.

[0011] Preferably, a universal exhaust hood is also provided outside the box to avoid odor accumulation in the room.

[0012] When in use, put the insect into the insect body holder, carefully guide the insect's tiny antennae into the micro-holes at the bottom of the isolation chamber, keep the antennae vertical and extended, and seal the gap with high vacuum silicone grease; after 1 hour of adaptation, gently touch the front feet of the fruit fly in the device with cotton wool soaked in pure water to observe whether it has a proboscis reflex, remove the insect individuals that still extend their proboscis after contacting pure water, and use the remaining individuals for subsequent experiments. All conditioned training processes are carried out under infrared light; absorb the conditioned stimulus odor source substance and place it on filter paper (4×1cm), and put it into the odor source chamber; turn on the air pump, and the odor in the filter paper in the odor source chamber is blown to the antennae of the fruit fly by the airflow (1L / min). At the same time, open the box Multiple exhaust fans are set on the side wall of the body to immediately blow away various odors and keep the odors away from the insect body to ensure the success of the experiment. After each training, the filter paper is replaced; 3 seconds after the conditioned stimulus odor source substance is released, the fruit fly appetite stimulating substance solution is given as a reward at the same time, and the beak of the fruit fly is gently touched with soft absorbent cotton soaked in the fruit fly appetite stimulating substance solution for 3 seconds; the odor release is maintained for 6 seconds; in the training of the CS-US paired group, the fruit flies are subjected to a total of 5 conditioned trainings, each training interval is 10 minutes, and the beak extension reflex to the odor source substance is recorded in each experiment; the appetite stimulating substance solution includes volatile drugs, etc., and non-volatile food.

[0013] After the training, the fruit flies were taken out of the isolation room and placed in a room under an infrared lamp, maintained at a temperature of 25-26°C and a relative humidity of 50-60%, and fed a small amount of sucrose solution every 1 hour to prevent the fruit flies from starving and dying. Then, memory retrieval experiments were conducted 10 minutes, 24 hours, and 48 hours later. The fruit flies that showed a proboscis reflex within 3 seconds of the odor provision were considered to have remembered the relationship between the conditioned stimulus (CS: odor source substance) and the unconditioned appetitive stimulus, that is, the CS became a signal predicting the reward and was recorded as "learned", while the other fruit flies were recorded as "not learned".

[0014] When working in an indoor laboratory, you also need to open the universal exhaust hood in the room to prevent the smell from spreading indoors and causing the experiment to fail. When working in an outdoor laboratory, you only need to turn on the exhaust fan on the box.

[0015] The film has good air-impermeability and hydrophobicity, and is used to install and fix the insect antennae upward. The size of the isolation chamber can be determined according to the size of the insect antennae and the convenience of operation. It is composed of a lightweight transparent plate and a wrapped film. A circular hole is opened at one end of the rectangular structure, which is just put on the circular pipe wall of the odor releaser and fixed. The other end is not equipped with a transparent plate, which serves as an air outlet to keep the air path unobstructed. The bottom of the rectangular structure is not installed with a lightweight transparent plate, but is wrapped with a special film to form the bottom surface.

[0016] The micro-holes support the antennae to be lifted up and fixed, and the isolation chamber isolates the space where the tiny antennae (smell) of the insect's head are located from the space of other head organs (such as the tiny beak), which can not only ensure that the antennae can only receive the stimulation of conditioned odors, but also block the chemical odors that serve as unconditioned appetite stimulants from diffusing to the antennae. The gap between the micro-holes and the antennae is sealed with high vacuum silicone grease. During operation, special attention should be paid to avoid damaging the insect antennae, otherwise it will affect subsequent experiments; because the antennae and beak are both on the head and the distance between the two is very close, the space for hand operation is very narrow.

[0017] Compared with the prior art, the utility model has the following technical effects:

[0018] The utility model has a system for simultaneously testing the insect olfactory conditioned proboscis reflex driven by volatile or non-volatile appetitive substances, and further uses this system to establish a training paradigm for conditional PER driven by volatile unconditioned appetitive stimuli. This provides a new tool for scientists and technicians around the world to evaluate the joint learning and memory status of insects' CS (conditioned odor)-US (volatile unconditioned stimulus), thereby leading and opening up new research directions around the world. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a structural schematic diagram of an insect learning and memory detection device; wherein, 1-box, 11-air pump, 12-air filter, 13-integrated air humidifier / flow meter, 14-odor source chamber, 15-odor release tube, 16-isolation chamber, 17-insect body fixer, 18-micropores, 19-exhaust fan, 20-adjustable fixing frame with clamp, 171-fixed small tube, 172-perforated bracket, 21-white light, 22-infrared light. DETAILED DESCRIPTION

[0020] Embodiment 1 A device for detecting insect learning and memory

[0021] An experimental device for detecting insect learning and memory, the device comprises a box body 1 made of an opaque material, a drawer door is arranged on the front of the box body 1, and an odor delivery device and a conditioning training device are arranged in the box body; one end of the odor delivery device is an air pump 11 located outside the box body connected by a hose, and the other end is connected to an air filter 12, an integrated air humidifier / flow meter 13, an odor source chamber 14, and an odor release tube 15 in sequence through a hose; the conditioned training device is composed of an isolation chamber 16 and an insect body fixer 17; the The isolation chamber 16 is a hexahedral structure, with an opening on the right side, and the opening surface is an air outlet. The odor release tube 15 runs through the left side of the isolation chamber 16 and forms an angle of 15-45 degrees with the bottom surface of the isolation chamber 16 (the isolation chamber must be kept horizontal to facilitate the operation of the insect body and antennae). The left, front, back and top of the isolation chamber 16 are composed of a lightweight transparent thin plate, the bottom surface is wrapped with a film, and a micro hole 18 is set in the middle of the bottom film. The insect body fixer 17 is located below the micro hole of the isolation chamber, and an exhaust fan 19 is set on the right side wall of the box. The insect body fixer 17 is a fixed small tube 171 with a backrest, and a hole bracket 172 for stabilizing and fixing the small tube. The odor release tube 15 is a hard tube with a diameter of ≤10 mm. A white light 21 and an infrared light 22 are also set at the top of the box. The isolation chamber is 40*30*30 mm, and the box size is 60*40*40 cm. The top of the box is also provided with an adjustable fixing frame 20 with a clamp for fixing the odor release tube. A universal exhaust hood is also provided outside the box to prevent odor from gathering in the room.

[0022] When in use, put the insect into the insect body holder, carefully guide the insect's tiny antennae into the micro-holes at the bottom of the isolation chamber, keep the antennae vertical and extended, and seal the gap with high vacuum silicone grease; after 1 hour of adaptation, gently touch the front feet of the fruit fly in the device with cotton wool soaked in pure water to observe whether it has a proboscis reflex, remove the insect individuals that still extend their proboscis after contacting pure water, and use the remaining individuals for subsequent experiments. All conditioned training processes are carried out under infrared light; absorb the conditioned stimulus odor source substance and place it on filter paper (4×1cm), and put it into the odor source chamber; turn on the air pump, and the odor in the filter paper in the odor source chamber is blown to the antennae of the fruit fly by the airflow (1L / min). At the same time, turn on the Multiple exhaust fans are set on the side walls of the box to immediately blow away various odors and keep the odors away from the insects' bodies to ensure the success of the experiment. After each training, the filter paper is replaced; 3 seconds after the conditioned stimulus odor source substance is released, a fruit fly appetite stimulating substance solution is given as a reward, and the beak of the fruit fly is gently touched with soft absorbent cotton soaked in the fruit fly appetite stimulating substance solution for 3 seconds; the odor release is maintained for 6 seconds; in the training of the CS-US paired group, the fruit flies are subjected to a total of 5 conditioned trainings, each with an interval of 10 minutes, and the beak extension reflex to the odor source substance is recorded in each experiment; the appetite stimulating substance solution includes volatile food and non-volatile food.

[0023] After the training, the fruit flies were taken out of the isolation room and placed in a room under an infrared lamp, maintained at a temperature of 25-26°C and a relative humidity of 50-60%, and fed a small amount of sucrose solution every 1 hour to prevent the fruit flies from starving and dying. Then, memory retrieval experiments were conducted 10 minutes, 24 hours, and 48 hours later. The fruit flies that showed a proboscis reflex within 3 seconds of the odor provision were considered to have remembered the relationship between the conditioned stimulus (CS: odor source substance) and the unconditioned appetitive stimulus, that is, the CS became a signal predicting the reward and was recorded as "learned", while the other fruit flies were recorded as "not learned".

[0024] When working in an indoor laboratory, you also need to open the universal exhaust hood in the room to prevent the smell from spreading indoors and causing the experiment to fail. When working in an outdoor laboratory, you only need to turn on the exhaust fan on the box.

[0025] The film has good air-impermeability and hydrophobicity, and is used to install and fix the insect antennae upward. The size of the isolation chamber can be determined according to the size of the insect antennae and the convenience of operation. It is composed of a lightweight transparent plate and a wrapped film. A circular hole is opened at one end of the rectangular structure, which is just put on the circular pipe wall of the odor releaser and fixed. The other end is not equipped with a transparent plate, which serves as an air outlet to keep the air path unobstructed. The bottom of the rectangular structure is not installed with a lightweight transparent plate, but is wrapped with a special film to form the bottom surface.

[0026] The micro-holes support the antennae to be lifted up and fixed, and the isolation chamber isolates the space where the tiny antennae (smell) of the insect's head are located from the space of other head organs (such as the tiny beak), which can not only ensure that the antennae can only receive the stimulation of conditioned odors, but also block the chemical odors that serve as unconditioned appetite stimulants from diffusing to the antennae. The gap between the micro-holes and the antennae is sealed with high vacuum silicone grease. During operation, special attention should be paid to avoid damaging the insect antennae, otherwise it will affect subsequent experiments; because the antennae and beak are both on the head and the distance between the two is very close, the space for hand operation is very narrow.

[0027] Compared with the prior art, the utility model has the following technical effects:

[0028] The utility model has a system for simultaneously testing the insect olfactory conditioned proboscis reflex driven by volatile or non-volatile appetitive substances, and further uses this system to establish a training paradigm for conditional PER driven by volatile unconditioned appetitive stimuli. This provides a new tool for scientists and technicians around the world to evaluate the joint learning and memory status of insects' CS (conditioned odor)-US (volatile unconditioned stimulus), thereby leading and opening up new research directions around the world.

Claims

1. An experimental device for detecting insect learning and memory, characterized in that: The device comprises a box body (1) made of an opaque material, a drawer door is arranged on the front of the box body, and an odor delivery device and a conditioning training device are arranged inside the box body; the odor delivery device is an air pump (11) located outside the box body connected by a hose, and the air pump (11) is connected to an air filter (12), an integrated air humidifier / flow meter (13), an odor source chamber (14), and an odor release tube (15) in sequence through the hose; the conditioning training device consists of an isolation chamber (16) and an insect body fixer (17). The isolation chamber (16) is a hexahedral structure with an opening on the right side, the opening side being an air outlet, the odor release tube (15) passes through the left side of the isolation chamber (16) and forms an angle of 15-45 degrees with the bottom surface of the isolation chamber (16), the left, front, back and top of the isolation chamber (16) are composed of a lightweight transparent thin plate, the bottom surface is wrapped with a film, and a micro hole (18) is arranged in the middle of the bottom film, the insect body fixer (17) is located below the micro hole (18) of the isolation chamber, and an exhaust fan (19) is arranged on the right side wall of the box.

2. The experimental device for detecting insect learning and memory according to claim 1, characterized in that: The insect body fixer (17) is a small fixing tube (171) with a backrest and a bracket (172) with holes for stabilizing the small fixing tube.

3. The experimental device for detecting insect learning and memory according to claim 1, characterized in that: The odor release tube (15) is a hard tube with a diameter of ≤10 mm.

4. The experimental device for detecting insect learning and memory according to claim 1, characterized in that: A white light (21) and an infrared light (22) are also provided on the top of the box (1).

5. The experimental device for detecting insect learning and memory according to claim 1, characterized in that: The size of the box (1) is 60*40*40 cm, and the size of the isolation chamber is 40*30*30 mm.

6. The experimental device for detecting insect learning and memory according to claim 1, characterized in that: An adjustable fixing frame (20) with a clamp for fixing the odor release tube (15) is also provided on the top of the box body (1).

7. The experimental device for detecting insect learning and memory according to claim 1, characterized in that: A universal exhaust hood is also arranged outside the box.

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