A method for intervening in conditioned place preference in mice using bilateral alternating visual stimuli

Abstract

The present invention specifically relates to a method for intervening in conditioned place preference of mice by bilateral alternating visual stimulation, belonging to the field of basic medicine. The present invention uses a bilateral alternating visual stimulation device that includes a light strip that can be alternately lit with variable frequencies and external support hardware, and can apply different stimulation frequencies. Step S1: Train the mice for alcohol-conditioned place preference; Step S2: Administer alcohol to the modeled mice by intraperitoneal injection to activate the brain regions related to alcohol-conditioned place preference in the mice and cause the reconsolidation of the alcohol preference memory in the mice; Step S3: Perform bilateral alternating visual stimulation interventions with different frequencies on the modeled mice; Step S4: Compare the effects of different frequency intervention methods on the alcohol-conditioned place preference of the mice. The present invention provides a non-invasive and highly efficient intervention method for mice modeled with alcohol-conditioned place preference.

Description

Technical Field

[0001] The present invention belongs to the field of basic medicine, and particularly relates to a method for intervening in conditioned place preference of mice by bilateral alternating visual stimulation. More specifically, it relates to a method for performing bilateral visual stimulation on mice using an alternately flashing light strip to intervene in the conditioned place preference of mice with conditioned place preference models. Background Art

[0002] Alcohol is the most widely used addictive substance in the world, permeating all aspects of people's lives. Once an individual forms alcohol addiction, the resulting addictive symptoms will seriously affect their normal life and social interactions. Addicts will also spend a large amount of money and time on drinking. In addition, addicts are unable to work normally, causing economic losses to society and disrupting social stability. In recent years, more and more research evidence at home and abroad has supported the view that once alcohol addiction is formed, it will cause brain damage and genetic defects in addicted individuals. The characteristics of alcohol addiction are compulsive drinking. Even though an individual knows the harm caused by drinking, they still cannot control their drinking behavior and will have withdrawal reactions after stopping drinking.

[0003] Currently, there are mainly two methods for alcohol addicts to quit drinking: the drug level and the psychological level. Using drugs is one of the very important methods for addicts to quit drinking because relevant drugs can help patients relieve alcohol withdrawal symptoms and prevent the occurrence of alcohol addiction complications. Many studies have explored the alcohol abstinence effects of relevant drugs. For example, hallucinogenic ibogaine, GABA receptor agonist Baclofen, and some benzodiazepine drugs, etc., all have the effect of helping to quit drinking. However, using drug treatment is often accompanied by great side effects. For alcohol addicts, psychological treatment is equally important.

[0004] People around alcohol addicts should continuously encourage and support them, enabling the addicts themselves to establish the confidence and determination to overcome alcohol addiction. At the same time, with the cooperation of supportive treatment for a period of time, good results will also be achieved. However, there is still a lack of effective methods to reduce craving and prevent relapse for alcohol addiction at present.

[0005] The Conditioned Place Preference (CPP) experiment is a classic experimental model for evaluating drug psychological dependence and an effective tool for finding anti-drug-seeking behaviors in animals. It is a method for determining the rewarding effect of substances based on the theory of "responsive reinforcement". "Responsive reinforcement" means that if a rewarding stimulus is repeatedly associated with a specific non-rewarding neutral stimulus (such as a specific environment), the latter can acquire rewarding properties, that is, this specific environment can induce the unconditioned behavioral effects originally associated with the unconditioned reward. However, the specific neural mechanism by which environmental stimuli induce animals to approach actively is not fully understood. Modeling conditioned place preference in mice and detecting the effects of different intervention methods on the preference for addictive drugs are of great significance for studying the circuits and molecular mechanisms behind the formation of drug conditioned place preference and finding potential therapeutic targets for addiction.

[0006] Alternating bilateral sensory stimulation (ABS) is a method used to treat post-traumatic stress disorder. It is also known as eye movement desensitization and reprocessing. Its process can be roughly divided into two parts. First, the patient is required to recall traumatic memories while receiving alternating bilateral sensory stimulation to achieve a permanent treatment effect on the traumatic memories. Currently, some studies have also applied this clinical method to the mouse model of conditioned fear and successfully extinguished the fear memories of mice. We applied this method to intervene in mice modeled with alcohol conditioned place preference, providing new ideas and methods for studying the circuits and molecular mechanisms of the formation of alcohol conditioned place preference. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for intervening in conditioned place preference in mice using alternating bilateral visual stimulation. The present invention uses a light strip that can be alternately lit with variable frequencies and external support hardware to apply alternating bilateral visual stimulation with different stimulation frequencies to intervene in conditioned place preference in mice.

[0008] According to the purpose of the present invention, there is provided a method for intervening in conditioned place preference in mice using alternating bilateral visual stimulation, including the following steps:

[0009] (1) First, conduct conditioned place preference training on mice to complete the modeling of conditioned place preference in mice;

[0010] (2) Administer alcohol to the modeled mice by intraperitoneal injection to activate the brain regions related to alcohol conditioned place preference in mice and cause the reconsolidation of the preference memory for alcohol in mice;

[0011] (3) Conduct visual stimulation on mice with a light strip that is alternately lit in sequence, that is, complete the intervention of alternating bilateral visual stimulation.

[0012] Preferably, the frequency of the alternately lit light strip is 1HZ - 8HZ.

[0013] Preferably, the frequency of the alternately lit light strip is 4HZ - 8HZ.

[0014] Preferably, the alternately lit light strip is placed on the inner wall of the black cylinder and surrounds the inner wall of the black cylinder in a circle; the diameter of the cylinder is 20cm - 23cm, and the height of the cylinder is 20cm - 23 cm.

[0015] Preferably, the height of the alternately lit light strip from the bottom of the black cylinder is 25cm - 27 cm.

[0016] Preferably, the alternately lit light strip is an LED light strip, including 30 - 35 LED lights, and the LED lights are alternately lit in sequence from one end to the other end during the visual stimulation process.

[0017] Preferably, the time of the bilateral alternating visual stimulation intervention is 30min - 40min.

[0018] Preferably, the conditional place preference training for mice specifically includes the following steps:

[0019] (1) Place the mice in the conditional place preference box for adaptation; the conditional place preference box is divided into three compartments, the walls of the two side compartments are white and black respectively, the two side compartments have different floor hollow shapes, and there is a passage with a black wall between the two side compartments; the adaptation is to open the doors of the two side compartments to allow the mice to move in the entire conditional place preference box; the side compartment where the mice stay for a longer time is taken as the preferred side.

[0020] (2) Inject alcohol intraperitoneally into the mice and place them on the non - preferred side, and inject the same amount of normal saline intraperitoneally into the mice the next day and place them on the preferred side to complete one training; complete 4 - 5 times of training according to this method to obtain the modeled mice.

[0021] Preferably, after the bilateral alternating visual stimulation intervention, the conditional place preference of the mice is detected; that is, open the doors of the two side compartments of the conditional place preference box to allow the mice to move in the entire conditional place preference box. If the staying time of the mice on the alcohol - paired side becomes shorter compared with the staying time of the mice without bilateral alternating visual stimulation intervention on the alcohol - paired side, that is, the alcohol - conditional place preference of the mice is reduced.

[0022] Generally speaking, compared with the prior art, the above - mentioned technical solution conceived by the present invention mainly has the following technical advantages:

[0023] (1) The method provided by the present invention for intervening in the conditioned place preference of mice using bilateral alternating visual stimuli has a significant effect in the intervention of mice modeled with conditioned place preference, can effectively reduce the alcohol preference of mice, provides new ideas and methods for the long-term and efficient intervention of mice with alcohol-induced conditioned place preference in scientific research, and provides a new animal model for exploring the neurobiological mechanism of alcohol-induced conditioned place preference.

[0024] (2) By intervening in the alcohol-induced conditioned place preference of mice using bilateral alternating visual stimuli, on the one hand, alcohol is given before the intervention, causing the alcohol memory of mice to be in an unstable stage and entering the memory reconsolidation process. During this process, bilateral alternating visual stimuli can accelerate the forgetting of their alcohol-related memories; on the other hand, bilateral alternating visual stimuli cause eye movements in mice, which can activate the brain regions related to eye movements in the brain. This brain region has an inhibitory input to the brain regions related to addiction, thereby affecting the neuronal activity of the brain regions related to addiction, and thus is a possible mechanism for intervening in the alcohol-induced conditioned place preference of mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is an overall physical diagram of the conditioned place preference box.

[0026] Figure 2 It is an overall physical diagram of the device for applying bilateral alternating visual stimuli.

[0027] Figure 3 It is a schematic diagram of the alcohol-induced conditioned place preference of mice and the intervention process.

[0028] Figure 4 Detection result diagrams of the control group of mice, the conditioned place preference modeling group, and different frequency ABS intervention groups after ABS intervention on alcohol-induced conditioned place preference according to the method provided by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] A method of the present invention for intervening in the alcohol-induced conditioned place preference of mice using bilateral alternating visual stimuli, using a bilateral alternating visual stimulus device that can apply different stimulation frequencies, which includes an alternately lit light strip with variable brightness and frequency and external support hardware, and the technical solution implemented includes the following steps:

[0031] Step Sl: The mice are subjected to alcohol conditioned place preference training, that is, the mice are modeled for alcohol conditioned place preference: The mice are alternately intraperitoneally injected with 20% alcohol or an equal amount of physiological saline daily and placed on different sides of the conditioned place preference box respectively;

[0032] Step S2: The mice after modeling are intervened with bilateral alternating visual stimuli at different frequencies. Before the intervention, the mice are intraperitoneally injected with the same amount of alcohol as that during training;

[0033] Preferably, the time of ABS intervention in Step S2 is 30 min;

[0034] Preferably, after Step S2, that is, 24 h after the end of the intervention, the mice are subjected to conditioned place preference detection, that is, Step S3 for detecting the intervention effect;

[0035] Step S3: The mice are placed in the conditioned preference box, and the doors of the preference boxes on both sides are opened so that the mice can freely explore therein, and the preference of the modeled and intervened mice for the position boxes on both sides is detected.

[0036] In some embodiments, an external device for bilateral alternating visual stimulus intervention is constituted by a black plastic cylinder, the height and diameter of which are both 20 cm, and the mice can move freely therein.

[0037] In some embodiments, an LED light strip is used for bilateral alternating stimulation of the mice. It is located on the inner wall of the cylinder 5 cm above the ground inside the cylindrical external device, and about 35 LED lights are included in one circle. During the whole visual stimulus process, the LED lights are turned on and off in sequence from one end to the other end, and vice versa.

[0038] In some embodiments, before the intervention of the mice with alcohol conditioned place preference by bilateral alternating visual stimulus, an appropriate amount of alcohol is given to the mice by intraperitoneal injection to activate the brain regions related to alcohol conditioned place preference of the mice and cause the reconsolidation of the alcohol preference memory of the mice.

[0039] The following are specific embodiments

[0040] Example 1

[0041] This example is a method for intervening in the alcohol conditioned place preference of mice by bilateral alternating visual stimulus. The general flow chart is as Figure 3 shown. Adult male C57BL / 6J mice (8 weeks old) are used. Ethanol is administered by intraperitoneal injection at a dose of 2 g / kg (20% dissolved in physiological saline). As Figure 1The mouse conditioned place preference box of Shanghai Xinruan Information Technology Co., Ltd. shown in the figure. The device is divided into three rooms. The preference boxes on both sides have different wall colors (black and white) and floor hollow shapes (striped and circular). The black wall is paired with the striped cut, and the white wall is paired with the circular cut. There is a narrow black compartment between the two rooms. Before each experiment, the box is cleaned with 70% ethanol and dried. The simple alcohol conditioned place preference group and the 1Hz, 4Hz, and 8Hz ABS intervention groups are subjected to conditioned place preference training. The behavioral paradigm includes 1 day of adaptation (open the compartment door to allow the mouse to explore in the entire conditioned place box for 20 minutes, record the residence time of the mouse in the black box and the white box within 20 minutes respectively to evaluate the natural preference effect of the mouse for the two boxes on both sides, select the side with the shorter residence time as the alcohol-paired side during training, and the side with the longer residence time as the normal saline-paired side during training), 8 days of training days (close the compartment doors on both sides, the mouse undergoes one training per day, with alcohol and normal saline alternating. Specifically described as: on the first day of training, the mouse is intraperitoneally injected with 20% alcohol and then placed on the alcohol-paired side for 5 minutes. On the second day, it is injected with an equal amount of normal saline and then placed on the other side. The process is repeated on the third to eighth days of training, and a total of four rounds of training are carried out), and 1 post-training detection (free entry into the two rooms for 20 minutes). The control group is injected with normal saline throughout the process without modeling. One day is added between the training and detection days to intervene in the mice in the alcohol conditioned place preference intervention group: after intraperitoneal injection of the same amount of alcohol as during training, bilateral alternating visual stimulation is carried out for 30 minutes. The intervention uses the device as shown in Figure 2 . The device is externally composed of a black plastic cylinder with a height and diameter of 20 cm. The mouse can move freely inside it. There is a circle of LED light strips 5 cm above the ground inside the cylinder. There are approximately 35 LED lights in a circle. During the entire visual stimulation process, the LED lights are turned on and off in sequence from one end to the other, and then return from the other end in the same way. Among them, the 1Hz stimulation is one round trip within 1 s, and 4Hz and 8Hz are 4 or 8 round trips in 1 s respectively. The LED light strip is controlled by an Arduino program to enable different frequencies of visual stimulation.

[0042] Example 2

[0043] Use the SuperMaze ethology video analysis software configured with the device to analyze the behavior of the mice. The statistical results of the ethology of each group are as shown in Figure 4 .

[0044] Figure 4Among all the results of the behavioral experiments shown. Among them, paired t-tests were used for statistical analysis from A to J. ns stands for no significant, indicating that P is greater than or equal to 0.05; * represents P less than 0.05; ** means P less than 0.01; *** shows P less than 0.001. Among them, one-way ANOVA was used for statistical analysis from K to L, and post hoc multiple comparison analysis tests were performed. In addition, the number of mice in each group was 20. The results of the conditioned place preference test found ( Figure 4 ), compared with the control group, there was no statistical difference in the cpp score (the time when the mouse was on the preferred side minus the non-preferred side) before and after modeling. There were statistical significances in both the cpp score and the average time on the preferred side in the simple alcohol conditioned place preference group before and after modeling, proving that the alcohol conditioned place preference modeling in mice was successful. When the mice were intervened with 1 Hz, 4 Hz, and 8 Hz ABS respectively, especially when intervened with 4 Hz ABS, compared with the mice with conditioned place preference without intervention, the time of the mice on the alcohol-paired side was effectively reduced ( Figure 4 D, I, K-L in), that is, the alcohol preference of the mice was significantly reduced, and it had an intervention effect on the alcohol conditioned place preference of the mice.

[0045] It is easy for those skilled in the art to understand that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation, characterized in that, it includes the following steps: (1) First, conduct conditioned place preference training on the mice to complete the modeling of conditioned place preference in mice; (2) Administer alcohol to the modeled mice by intraperitoneal injection to activate the brain regions related to alcohol conditioned place preference in mice and cause the reconsolidation of alcohol preference memory in mice; (3) Conduct visual stimulation on the mice with a light belt that is alternately lit in sequence, that is, complete the intervention of bilateral alternating visual stimulation.

2. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 1, characterized in that, the frequency of the alternately lit light belt is 1HZ - 8HZ.

3. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 1, characterized in that, the frequency of the alternately lit light belt is 4HZ - 8HZ.

4. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 1, characterized in that, the alternately lit light belt is placed on the inner wall of a black cylinder and surrounds the inner wall of the black cylinder in a circle; the diameter of the cylinder is 20cm - 23cm, and the height of the cylinder is 20cm - 23cm.

5. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 1, characterized in that, the alternately lit light belt is an LED light belt, which contains 30 - 35 LED lights, and the LED lights are alternately lit in sequence from one end to the other end during the visual stimulation process.

6. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 1, characterized in that, the time of the bilateral alternating visual stimulation intervention is 30min - 40min.

7. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 1, characterized in that, the specific steps for conducting conditioned place preference training on the mice include the following: (1) Place the mice in a conditioned place preference box for adaptation; the conditioned place preference box is divided into three compartments, the walls of the two side compartments are white and black respectively, the two side compartments have different floor hollow shapes, and there is a passage with a black wall between the two side compartments; the adaptation is to open the doors of the two side compartments to allow the mice to move in the entire conditioned place preference box; use the side compartment where the mice stay longer as the preference side; (2) After intraperitoneally injecting alcohol into the mice, place them on the non - preference side, and the next day, intraperitoneally inject the same amount of physiological saline into the mice and place them on the preference side to complete one training; complete 4 - 5 times of training according to this method to obtain the modeled mice.

8. The method for intervening in conditioned place preference of mice using bilateral alternating visual stimulation according to claim 7, characterized in that, after the bilateral alternating visual stimulation intervention, conduct conditioned place preference detection on the mice; that is, open the doors of the two side compartments of the conditioned place preference box to allow the mice to move in the entire conditioned place preference box. If the staying time of the mice on the alcohol - paired side becomes shorter compared to the staying time of the mice without bilateral alternating visual stimulation intervention on the alcohol - paired side, that is, the alcohol - conditioned place preference of the mice decreases.

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