A method for evaluating the addictiveness and neurotoxicity of fentanyl-related drugs based on fish school conditional location preferences.

Abstract

This invention discloses a method for evaluating the addictiveness and neurotoxicity of fentanyl-like drugs based on conditional position preference in fish schools. The method involves administering zebrafish to adult zebrafish via intraperitoneal injection, followed by behavioral experiments such as conditional position preference, social modeling, exploration modeling, mirror modeling, diving modeling, and T-maze experiments. These experiments reveal the conditional position preference exhibited by the zebrafish school, as well as indicators of anxiety, aggression, social behavior, and memory in individuals within the school. The behavioral toxicity and impact of the drug are evaluated using the analytical parameters of these characteristics. Furthermore, the method utilizes the neurotransmitter levels in the zebrafish brains following the behavioral experiments to correlate with the results, thereby determining the mechanism by which fentanyl-like drugs affect nerve conduction. This approach effectively improves the accuracy of evaluating the addiction risk and neurotoxicity of such drugs.

Description

Technical Field

[0001] This invention relates to the field of drug behavioral toxicity evaluation technology, and in particular to a method for evaluating the addictive and neurotoxic effects of a drug on adult zebrafish. Background Technology

[0002] Fentanyl-related substances are addictive. Their strong lipophilicity allows them to quickly cross the blood-brain barrier and enter the brain, forming a peak in the bloodstream within a short period, which easily leads to tolerance and drug dependence in the body. At the same time, fentanyl also poses a very high risk.

[0003] Zebrafish are an effective vertebrate model for behavioral research, allowing for the assessment of the rewarding effects of novel psychoactive substances through self-dosing and conditioned place preference. In zebrafish models, conditioned place preference tests have been used to evaluate the rewarding effects of drugs such as morphine, methamphetamine, ethanol, and nicotine. These studies exploring the reward effects of drugs using zebrafish typically employ conditioned place preference experiments with a single subject because behavioral data from individual fish is easily tracked and quantified. However, significant individual variability leads to unstable experimental results. In these experiments, unstable location preferences or anomalous behavioral abilities exhibited by the fish can result in experimental failure and increase experimental costs. Summary of the Invention

[0004] With advancements in model animal behavior tracking devices and image tracking algorithms, it is now possible to quantify the group behavior of zebrafish, analyzing the preferences (average number over a certain time) and group behavioral characteristics of zebrafish schools in a designated area (distance between a fish and its nearest neighbor, distance between a fish and all other fish, and the magnitude of the average vector of all fish, etc.). Individual zebrafish may be compelled to specific locations by stronger preferences among their peers within the school, thus reducing the instability caused by individual behavioral abnormalities. To our knowledge, zebrafish group behavior has not yet been used in CPP trials to study the drug reward effect.

[0005] The evaluation of fentanyl addiction based on fish swarm conditional location preference is more efficient, accurate, and significantly reduces experimental instability compared to traditional individual-based experiments. Further research using multiple behavioral models to investigate the effects of fentanyl exposure on anxiety, aggression, social behavior, and memory in zebrafish, combined with changes in neurotransmitters in the zebrafish brain, can elucidate the impact of these psychoactive substances on drug reward effects and provide a basis for evaluating the neurotoxicity caused by addiction to these drugs.

[0006] This invention provides a method for evaluating the addictiveness and neurotoxicity of fentanyl-like drugs based on conditional position preference in fish groups. By studying the drug reward effect through group behavior, it optimizes the traditional individual-based conditional position preference experiment and can more conveniently evaluate the addictiveness and neurotoxicity of fentanyl-like drugs.

[0007] A method for evaluating the addictiveness and neurotoxicity of fentanyl-like drugs based on fish school conditional location preferences includes the following steps:

[0008] (1) Adult zebrafish were separated and raised individually, and then exposed to fentanyl drugs by intraperitoneal injection. After the injection, the entire group of fish was isolated in a medicated tank. The next day, physiological saline was injected and the fish were isolated in a blank tank. The above operation was repeated for two cycles.

[0009] (2) The reward effect of fentanyl drugs was evaluated by an optimized fish swarm-based conditional position preference experiment, and the neurotoxicity of fentanyl was evaluated by an optimized social model experiment, exploratory model experiment, mirror model experiment, diving model experiment and T-maze experiment based on individual behavioral characteristics.

[0010] (3) Study the neurotoxic mechanism of fentanyl based on changes in neurotransmitter content.

[0011] The above evaluation method, through behavioral experiments on adult zebrafish with fully developed nervous systems, can more objectively demonstrate the biological and behavioral effects of fentanyl-like drugs on zebrafish, thus improving the accuracy of toxicity evaluation of this class of drugs.

[0012] The fentanyl-class drugs may be substances whose chemical structure, compared to fentanyl (N-[1-(2-phenylethyl)-4-piperidinyl]-N-phenylpropionamide), meets one or more of the following conditions: 1. The propionyl group is replaced by another acyl group; 2. The phenyl group directly bonded to the nitrogen atom is replaced by any substituted or unsubstituted monocyclic aromatic group; 3. Alkyl, alkenyl, alkoxy, ester, ether, hydroxyl, halogen, haloalkyl, amino, and nitro substituents are present on the piperidine ring; 4. The phenylethyl group is replaced by any other group (except hydrogen atom).

[0013] The zebrafish mentioned are wild AB strain zebrafish. Zebrafish should be kept in accordance with their suitable growth environment parameters, without strict requirements, but their health and well-developed state should be ensured.

[0014] Adult zebrafish were separated from the system and placed in individual tanks before the experiment. They were then transferred to a room with a suitable temperature and quiet environment and kept individually. The indoor lights were turned on at 8:00 AM daily, and the water in the tanks was changed using the zebrafish rearing system. The lights were turned off at 10:00 PM. Feeding was stopped after two days of isolation. The water in the individual tanks was completely changed after each day's experiment.

[0015] The drug exposure method is intraperitoneal injection. The drug concentration is not strictly controlled and can be set using conventional concentration gradients or concentrations found in environments affected by fentanyl. The injection volume is 5 μL per injection.

[0016] Baseline measurements for conditional position preference experiments should be performed before drug exposure, with an observation period of 20 minutes and sampling intervals of 1 minute per interval. During preference training, adult zebrafish exposed to fentanyl-like drugs were isolated in a medicated tank for 40 minutes, and after blank exposure, they were also isolated in a blank tank for 40 minutes.

[0017] The social model experiment, exploration model experiment, mirror model experiment, and diving model experiment were all conducted after the conditional position preference experiment, with a total observation time of 5 minutes for each experiment.

[0018] When conducting the above experiments, a quiet environment and the same light intensity should be maintained. Optionally, the light intensity should be the same as that used for the daily rearing of adult zebrafish. In particular, a lower light intensity, one-sixth of the rearing light intensity, should be maintained when conducting diving tests.

[0019] The indicators of zebrafish's location preference for adult conditions include averagecount (the average number of fish in the medicated box and the empty box per unit time), nnd (the average distance between fish i and its nearest neighbor), iid (the average distance between fish i and all other fish), polarization (the average size of the vector of all fish), and average speed.

[0020] The metrics for the social model experiment, exploration model experiment, mirror model experiment, and diving model experiment include area duration, movement distance, tactile attraction, and number of behaviors.

[0021] The metrics for the T-maze experiment include duration, distance traveled, and time to first entry.

[0022] The neurotransmitter analysis targets were glutamine, γ-aminobutyric acid, dopamine, and serotonin.

[0023] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0024] The use of fish swarm-based conditional location preference in fentanyl addiction assessment is more efficient, accurate, and significantly reduces experimental instability compared to traditional individual-based experiments. Furthermore, multiple behavioral testing models comprehensively reveal social, anxiety, aggressive, and memory changes following fentanyl addiction. In addition, the study of neurotransmitter levels in the brains of adult zebrafish was conducted to investigate the toxicity mechanism, further improving the accuracy of toxicity assessment for this class of drugs. Attached Figure Description

[0025] Figure 1 Diagram of the CPP test cylinder; Figure 2 As a social testing ground;

[0026] Figure 3 Explore the tank for novel objects;

[0027] Figure 4 It is a mirror cylinder;

[0028] Figure 5 For diving test cylinders;

[0029] Figure 6 For the T-maze;

[0030] Figure 7 The dwell and activity parameters of fish groups in the conditional location preference experiment;

[0031] Figure 8 The differences in average distance traveled, time spent in the two zones, number of times of crossing, and latency to enter the social zone among adult zebrafish in a social experiment;

[0032] Figure 9 Differences in average distance traveled, time spent in two zones, number of times of crossing, and latency to enter the new object zone among adult zebrafish in the novel object exploration model.

[0033] Figure 10 The differences in average moving distance, time spent in the mirror zone, number of shuttles, and rapid movement time in the mirror zone for adult zebrafish in the mirror model;

[0034] Figure 11 The differences in average distance traveled, time spent in the top zone, number of shuttles, and latency in the top zone for adult zebrafish in the diving model;

[0035] Figure 12 The duration and distance traveled by adult zebrafish in the two zones during the T-maze experiment, and the time difference in the time it takes for an individual to first reach the food memory zone (red zone);

[0036] Figure 13 The levels of glutamine, GABA, dopamine hydrochloride, hydrocortisone, and serotonin in the brain of adult zebrafish; Detailed Implementation

[0037] To more clearly illustrate the Conditional Place Preferred Experiment, Social Model Experiment, Exploration Model Experiment, Mirror Model Experiment, Diving Model Experiment, and T-Maze Experiment, a more detailed explanation will be provided below with reference to the accompanying drawings and specific embodiments.

[0038] Figures 1-6 This is the experimental apparatus used in this invention. Figure 1The experimental tank was selected based on the preferred location of the test chamber, specifically a 9L (30cm×20cm×15cm, length×width×height) transparent acrylic tank. The tank was divided into two areas: a blank area and a dot matrix area (medication kit). The blank area was empty, while the dot matrix area had four 2cm diameter black dots affixed to the bottom. A removable transparent partition separated the two areas.

[0039] Figure 2 This is a social testing tank, a transparent acrylic tank with a volume of 3L (30cm×10cm×10cm, length×width×height). The tank has a removable partition at one-third of its long side; the isolated 10cm×10cm×10cm area is the social gathering area, and the other part is the testing area.

[0040] Figure 3 The "Novelty Exploration Tank" is a transparent acrylic water tank with a volume of 2L (20cm×10cm×10cm, length×width×height). Inside the tank is an opaque cylinder with a diameter of 6cm. There is a gap of more than 1cm between the object and each tank wall.

[0041] Figure 4 This is a mirror tank, a 2L transparent acrylic water tank (20cm×10cm×10cm, length×width×height), with a mirror attached to one side of the tank wall. The area 5cm away from the mirror is the attack zone.

[0042] Figure 6 It is a diving test tank, specifically an irregularly shaped transparent tank with a volume of 2L, a trapezoidal cross-section, and a height of 20cm.

[0043] Figure 7 The structure is a T-shaped fish tank with a volume of 9L. One end of the shorter end of the T is covered with a red opaque plastic film, and the other end is covered with a green opaque plastic film. The longer end is left untreated.

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. This embodiment is a method for evaluating the effects of fentanyl on the social behavior and addiction of zebrafish.

[0045] Zebrafish farming

[0046] Zebrafish broodstock of the WT-AB ​​strain were used and raised in a photocycle system with a constant temperature of 28.5℃ and a daily light cycle of 14 hours and 10 hours of darkness (Shanghai Haisheng Aquarium Equipment Co., Ltd.). The pH of the water was maintained at 7.0-8.0, the salinity at 0.25‰-0.50‰, the conductivity at 500-800μS / cm, and the total ammonia nitrogen at less than 0.02mg / L. They were fed twice a day with hatched and cleaned brine shrimp.

[0047] Preparation of experimental subjects

[0048] Sixty-four healthy, age-appropriate adult zebrafish of uniform sex and with well-proportioned body shape were selected. The selected adult fish were randomly divided into four groups: 0 mg / L (control group), 10 mg / L, 100 mg / L, and 1000 mg / L, with 16 fish in each group. The 0 mg / L group served as the control group, and the other three groups were the experimental groups. Each group was placed in an independent aquarium, separated from the system, and transferred to a room with a suitable temperature and quiet environment for individual rearing. The indoor lights were turned on at 8:00 AM daily, and the water in the tanks was changed using water from the zebrafish rearing system. The lights were turned off at 10:00 PM. Feeding was stopped after two days of isolation.

[0049] Video data collection and analysis

[0050] Baseline and preference determinations were performed using the Viewpoint zebrafish behavior tracking software in Multicount mode, which collected the average counts of experimental subjects in the analysis area in real time. The video sampling frequency was 5 times / s, and the counting frequency was 1 minute / time. Videos from the social model, exploration model, mirror model, diving model, and T-maze experiments were analyzed using Noldus' EthoVisionXT 14 software, with the object tracking method being dynamic silhouette tracking, and the video sampling frequency was 5 times / s.

[0051] Data Statistics and Analysis

[0052] All results in this study are expressed as mean ± SEM (standard error of the mean). All data were analyzed using GraphPad Prism 8 software with two-way ANOVA followed by multiple comparisons or one-way ANOVA and t-tests. Significance levels were set as *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

[0053] Behavioral experiments

[0054] Example 1: Conditional Place Preference Experiment (CPP) of Fish Schools:

[0055] Baseline determination: After rinsing the CPP tank multiple times with purified water, it was then rinsed once with the system's circulating water from the zebrafish rearing system. System water was added to the CPP tank, with the water level at approximately 6-8 cm. All zebrafish from each group were placed in the CPP tank, and the tank was then placed in an adult fish observation box. The activity of the experimental subjects in the tank was recorded. The lighting parameters in the observation box were set to top-emitting light with a fixed intensity. The observation duration was 20 minutes, and sampling was performed every 1 minute. After the observation period, the fish in the CPP tank were transferred back to the rearing tank. After changing groups, the box door was closed, and observations continued for subsequent groups.

[0056] Preference Training: The experiment consisted of two-day cycles, repeated for two periods. DAY 1: Drug Injection and Training: The CPP tank was placed in the observation box, with environmental settings such as light intensity identical to the baseline measurement. A transparent partition separated the blank tank from the medicated tank, preventing zebrafish from crossing between the two areas. Using a micro-injection needle, 5 μL of the target solution was drawn and inserted approximately 3 mm into the abdominal cavity from around the pelvic fin. The solution was then gently injected into the adult fish. The injected adult fish were then placed in the medicated tank, and the observation box door was closed for 40 minutes of training. The control group was injected with fish saline solution, while the experimental groups were injected with a diluted solution of fentanyl hydrochloride in fish saline solution at concentration gradients of 10 mg / L, 100 mg / L, and 1000 mg / L. DAY 2 Blank Injection and Reverse Training: After repeating the baseline measurement of the CPP tank cleaning operation, the fish tank was placed. The placement of the CPP tank and the environmental settings of the observation box remained unchanged. Both the control group and the experimental group were injected with 5 μL of fish physiological saline and then placed in the blank box with the observation box door closed for training for 40 minutes.

[0057] Preference observation: The procedures and parameter settings for the baseline measurement were repeated. Observations were recorded for each group of fish after preference training, and the results were compared with the baseline performance. The results are shown in [the table below]. Figure 7 .

[0058] As fentanyl concentration increased, the percentage of zebrafish in the drug-paired zone (black spots) showed an increasing trend. Figure 7 B). The 100 mg / L and 1000 mg / L concentration groups showed a significantly higher percentage of zebrafish on the drug-pairing side compared to the control group (p<0.001 and p<0.05, respectively). Compared to the control group, the 10 mg / L fentanyl concentration did not significantly increase zebrafish position preference (p>0.05). Compared to the control group, the 1000 mg / L fentanyl-treated zebrafish showed a significantly increased nearest neighbor distance (NND) (p<0.05). Furthermore, compared to the control group, the inter-individual distance (IID) of zebrafish injected with 100 mg / L and 1000 mg / L fentanyl was significantly increased (p<0.01 and p<0.0001, respectively). No significant change in the average speed of zebrafish was observed after fentanyl treatment compared to the control group. Figure 7 D). These results indicate that fentanyl at concentrations of 100 mg / L and 1000 mg / L produced a significant preference for the drug-treated area without reducing swimming ability, demonstrating a clear drug reward effect. At the same time, the overall cohesion of the fish school decreased, reflecting the influence of fentanyl on group behavior.

[0059] Example 3: Social Model

[0060] Four healthy, non-experimental adult zebrafish were added to the social isolation zone of the socialization test tank as social subjects for the experimental zebrafish. Each zebrafish (16 zebrafish per concentration) was then placed in the test zone. The zebrafish were observed in an observation box for 5 minutes, and their activity was recorded under a light intensity of 100 lux. Behavioral data for each zebrafish were recorded. Data from the first two minutes were used for adaptation analysis, and data from the last three minutes were used for behavioral analysis, including the total distance the zebrafish moved in the tank, the time spent in the social zone, the latency period for entering the social zone, and the number of times the zebrafish entered the social zone. The results are shown below. Figure 8 .

[0061] We examined the neurotoxicity of fentanyl by observing changes in their social preferences. Compared to the control, there was no significant difference in swimming distance among fentanyl-treated zebrafish. Figure 8 B)(all concentrations p>0.05). Zebrafish in the 100 mg / L and 1000 mg / L concentration groups spent significantly less time moving in the social zone than control zebrafish. Figure 8 C) (both concentrations p<0.05), but there was no significant difference in the number of trips to the social area. Figure 8 D)(all concentrations p>0.05). Furthermore, the latency period for zebrafish to enter the social zone after fentanyl treatment tended to increase with increasing concentration, but the difference was not significant. Figure 8 E) (all concentrations p>0.05). The results showed that 100 mg / L and 1000 mg / L fentanyl reduced the social preferences of zebrafish individuals.

[0062] Example 4 Exploration Model

[0063] Adult zebrafish from each group were individually placed into a novelty exploration tank. A 5-minute observation test was conducted in a Viewpoint observation chamber, and the exploration behavior of each individual within each group was recorded (16 zebrafish were selected for each concentration). The light intensity was 30 lux. The first 2 minutes were used for acclimatization, and the following 3 minutes were used for behavioral data analysis, including the total distance the zebrafish moved in the tank, the time spent in the novelty area, the delay time before entering the novelty area, and the number of times the zebrafish entered the novelty area. The results are shown below. Figure 9 .

[0064] The effect of fentanyl on the ability of zebrafish to recognize new objects was evaluated by introducing environmentally friendly green novel objects. Within 5 minutes of the test, the behavioral ability of fentanyl-treated zebrafish tended to decrease with increasing fentanyl concentration, but the difference was not significant compared to the control group. Figure 9 B)(all concentrations p>0.05). Zebrafish treated with high doses of fentanyl (1000 mg / L) showed a significant reduction in migration time to new target areas. Figure 9C)(p<0.05), and showed a significant reduction in the number of shuttles entering the new object region (C)(p<0.05). Figure 9 D)(p<0.05) indicates that fentanyl leads to a decrease in exploratory behavior preference. With increasing fentanyl concentration, the latency period for entering new target areas tends to increase, but the difference is not significant compared to the control fish. Figure 9 E)(all concentrations p>0.05). The results showed that 1000 mg / L fentanyl reduced the zebrafish's preference for new objects without significantly affecting their activity level.

[0065] Example 5 Mirror Model

[0066] Sixteen zebrafish were selected for each concentration and placed in a mirror tank. The total distance the zebrafish moved, the time spent in the mirror area, the latency period before entering the mirror area, and the duration of rapid swimming in the mirror area (rapid movement was defined as a speed greater than 60% of the average speed) were analyzed for 5 minutes.

[0067] In our study, there was no significant difference or trend in the behavioral abilities of fentanyl-treated zebrafish compared to the control group. Figure 10 B)(all concentrations p>0.05). In the aggression assessment, zebrafish in the fentanyl group spent more time in the mirror area than zebrafish in the control group, but the difference was not statistically significant. Figure 10 C) (all concentrations p>0.5). Fentanyl did not cause a significant change in the number of times zebrafish shuttled to the mirror area. Figure 10 D)(all concentrations p>0.05). However, compared with the control, zebrafish treated with 100 mg / L and 1000 mg / L fentanyl showed a significant increase in the duration of rapid swimming in the mirror zone (D). Figure 10 E)(p<0.01 and p<0.05). The results showed that 100 mg / L and 1000 mg / L fentanyl increased the aggression of zebrafish against mirrors without significantly affecting their activity.

[0068] Example 6: Diving Model

[0069] Each group of adult zebrafish was placed into the underwater test tank one by one. All lights in the adult fish observation room were turned off, while maintaining the light intensity. For ie W30 lux. Zebrafish activity within the tank was recorded over 3 minutes using a horizontal observation camera in an animal behavior observation device. The tank was divided into two equal-height areas, the top and the bottom. The total distance traveled by the zebrafish in the tank, the time spent in the top area, the delay in entering the top area, and the number of times the zebrafish entered the top area were analyzed. The results are shown in Figure 11.

[0070] Diving tests in zebrafish have proven to be an effective method for assessing anxiety. Increased preference for the top zone in zebrafish was associated with decreased anxiety. We evaluated the distance individual zebrafish swam in the top and bottom zones at each concentration and found that zebrafish in the 0, 10, 100, and 1000 concentration groups generally preferred the bottom zone, exhibiting anxiety-like behavior. Figure 11 A). There was no significant difference in behavioral abilities between the fentanyl-treated groups and the control group. Figure 11 B) (all concentrations p>0.05), but the zebrafish in the 1000 mg / L fentanyl group had a significantly increased time in the top region (Fig. 11C) (p<0.01), which reduced the latency to enter the top region. Figure 11 D)(p<0.01). All fentanyl treatment groups showed a significant increase in the number of shuttles to the top region (D)(p<0.01). Figure 11 E)(p<0.05, p<0.01 and p<0.05). The results showed that 1000 mg / L fentanyl reduced the anxiety level of zebrafish individuals without significantly affecting their activity level.

[0071] Example 7: T-maze experiment

[0072] (1) Food location memory training: Slow-release fish food patches were placed in the red area of ​​the T-maze, and water was added to the aquaculture system until the water level was 2cm. After the fish food began to be released, the adult zebrafish from each group were placed in the empty area of ​​the T-maze. After the zebrafish explored the T-maze for 5 minutes, they were driven to the red area and isolated with a transparent partition for 20 minutes to allow them to feed. This operation was repeated twice for each group. The ambient light intensity was maintained at 30 lux. (2) Memory ability test: The day after the training, the T-maze was placed in a Viewpoint observation box, and the light intensity was set to 30 units. The adult zebrafish from each group were placed one by one into the empty area of ​​the T-maze. The swimming time and distance of the zebrafish in the red, green, and white arms within 5 minutes, as well as their first arrival time in the red arm, were tested. The results are shown in […]. Figure 12 .

[0073] We used changes in color preference to assess food-induced spatial memory. In our study, compared to red-armed zebrafish, control zebrafish showed no significant preference for green-armed zebrafish at the end of training in terms of distance traveled and time spent, indicating a change in the original green preference due to food induction. Figure 12 A-12C). However, zebrafish treated with fentanyl at concentrations of 10 mg / L, 100 mg / L, and 1000 mg / L moved significantly greater distances in the green area than in the red area (A-12C). Figure 12B)(p < 0.05, p < 0.01, and p < 0.05). Furthermore, zebrafish treated with fentanyl at concentrations of 100 mg / L and 1000 mg / L spent significantly more time in the green area than in the red area (B). Figure 12 C)(p<0.05, p<0.01). Importantly, the time to first entry into the red arm was much longer in the fentanyl group than in the control group (C). Figure 12 (D) (all concentrations p<0.05). These results indicate that conditioned food stimulation altered the color preferences of zebrafish in the control group, while it did not change them in the fentanyl group, suggesting that fentanyl may impair learning and memory in zebrafish.

[0074] Example 8: Detection of changes in related neurotransmitters after fentanyl administration

[0075] (1) Extraction of neurotransmitters

[0076] Following the initial behavioral experiments, zebrafish in each group were euthanized by freezing at -80°C. Brain tissue was removed using forceps and scissors and weighed, then placed into 2mL centrifuge tubes. The centrifuge tubes containing brain tissue were numbered according to their group, and two sets of 15mL centrifuge tubes were prepared and labeled accordingly. An appropriate amount of 1.89% formic acid solution was added to the isolated brain tissue, with a brain tissue to formic acid solution ratio of 10mg / g. A grinding bead was then added to each centrifuge tube, and the mixture was ground for 700s using a pre-chilled (4°C) cryogenic grinder. After grinding, the sample was centrifuged for 40 minutes at 14000 rpm at a constant temperature of 4°C to ensure sufficient precipitation of impurities. After centrifugation, the supernatant was pipetted into the first set of 15mL centrifuge tubes, and 1% formic acid acetonitrile was added at a ratio of V:V supernatant = 4:1, and the mixture was shaken well to allow sufficient protein precipitation. All samples were then centrifuged a second time for 40 minutes at 5000 rpm and 4°C. During the above procedures, the extracted brain tissue, undissected zebrafish, relevant solutions, and samples to be tested were all preserved in an ice-water mixture.

[0077] (2) Determination of neurotransmitter content by LC-MS

[0078] The target compounds were glutamine, GABA, dopamine hydrochloride, serotonin, and hydrocortisone. The supernatant after two centrifugations was concentrated to 2 mL and injected.

[0079] Chromatographic conditions: The parameters of the liquid chromatography column were ACQUITY UPLC HSS T3 (2.1 mm × 100 mm × 1.8 μm); the column temperature was 40 ℃; the mobile phase A was 0.1% formic acid aqueous solution, the mobile phase B was methanol, the flow rate was 0.25 mL / min, and the program was set to gradient system (Table 2-1).

[0080] Mass spectrometry conditions: Electrospray ionization (ESI) source (positive ion mode); Nebulizer gas: Nitrogen, flow rate 3 L / min; Drying gas: Nitrogen, flow rate 10 L / min; Collision gas: Argon, DL temperature 250℃; Interface temperature 300℃, heating block temperature 400℃; Scanning mode: Multiple reaction detection (MRM). Mass spectrometry parameters were optimized based on the above conditions, and the linear range of neurotransmitters is shown in Table 2-2.

[0081] Table 2-1 Liquid Chromatography Column Gradient System

[0082]

[0083] Table 2-2 Linear range of each neurotransmitter

[0084]

[0085] The results are shown in Figure 13 .

[0086] Following behavioral tests, the levels of Glu, GABA, DA, 5-HT, and Cortisol in the zebrafish brain were measured using LC-MS / MS. The results showed that, compared to the control group, the levels of Glu, GABA, DA, and 5-HT in the zebrafish brain of the 1000 mg / L group were significantly increased. Figure 13 A-13D). Compared with the control group, the average level of cortisol in the brains of zebrafish in the fentanyl group was reduced, but the difference was not statistically significant. Figure 13 E). The neurotransmitter disruption caused by high concentrations of fentanyl is consistent with the concentration results that lead to behavioral changes.

Claims

1. A method for evaluating the addictiveness and neurotoxicity of fentanyl-related drugs based on fish school conditional location preferences, characterized in that, Includes the following steps: (1) Adult zebrafish were separated and raised individually, and then exposed to fentanyl drugs by intraperitoneal injection. After the injection, the entire group of fish was isolated in a medicated tank. The next day, physiological saline was injected and the fish were isolated in a blank tank. The above operation was repeated for two cycles. (2) The reward effect of fentanyl drugs was evaluated by an optimized fish swarm-based conditional position preference experiment, and the neurotoxicity of fentanyl was evaluated by an optimized social model experiment, exploration model experiment, mirror model experiment, diving model experiment and T-maze experiment based on individual behavioral characteristics. (3) Study the neurotoxic mechanism of fentanyl based on the changes in neurotransmitter content.

2. The method for assessing addictive and neurotoxicity risks according to claim 1, characterized in that, In step (1), the adult zebrafish is a 5-6 month old wild AB strain zebrafish with a body length of 3cm±0.5cm and a weight of 0.25g±0.1g.

3. The method for evaluating addictiveness and neurotoxicity according to claim 1, characterized in that, In step (1), before the experiment, the zebrafish were moved out of the system and raised independently. The rearing environment was a constant temperature of 28.5℃, 14 hours of light and 10 hours of darkness per day. The pH of the water was maintained at 7.0-8.0, the salinity was maintained at 0.25‰-0.50‰, the conductivity was maintained at 500-800 μS / cm, and the total ammonia nitrogen was below 0.02 mg / L. The water in the rearing tank was changed after the experiment was completed each day.

4. The method for evaluating addictiveness and neurotoxicity according to claim 1, characterized in that, In step (2), the conditional position preference index of the zebrafish adult fish group includes the average count of the fish group consisting of 16 zebrafish in the medicated box and the blank box per unit time, the average distance between fish i and its nearest neighbor, the average distance between fish i and all other fish, and the magnitude or average speed of the average vector of all fish. The metrics for the social model experiment, exploration model experiment, mirror model experiment, and diving model experiment include the duration of the area, the distance traveled, and the number of tactile responses or behaviors. The metrics for the T-maze experiment include duration, distance traveled, or time to first entry; The neurotransmitter analysis targets are glutamine, γ-aminobutyric acid, dopamine, or serotonin.

5. The method for evaluating addictiveness and neurotoxicity according to claim 1, characterized in that, The differences in behavioral parameters of adult zebrafish were determined by the following method: All parameters are expressed as mean ± SEM (standard error of the mean) or median ± quartiles; Data that conform to a normal distribution are subjected to two-way ANOVA followed by multiple comparisons, or one-way ANOVA followed by Turkish post-hoc tests. Data that do not conform to a normal distribution are subjected to nonparametric tests. The significance level is set to , , , .

6. The method for evaluating addictiveness and neurotoxicity according to claim 1, characterized in that, The detection of neurotransmitters in the brains of adult zebrafish was performed using high performance liquid chromatography coupled with mass spectrometry. Tissue samples were separated by treatment with formic acid water and formic acid acetonitrile, and then concentrated and redissolved to the volume before protein precipitation.

Images