CN114667956A - Method for constructing zebra fish memory evaluation model and application thereof - Google Patents
Method for constructing zebra fish memory evaluation model and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of drug evaluation and screening, and discloses a construction method and application of a zebra fish memory evaluation model, wherein the construction method comprises the following steps: taking fertilized zebra fish juvenile fish, and respectively arranging a normal group and a model group, wherein a dimethyl sulfoxide solution is added into the normal group, and a cycloheximide solution is added into the model group; respectively moving the micro-porous plates into the micro-porous plates, and opening a behavior analysis system; entering a training stage, circularly stimulating and training for 130-150 times according to a stimulation mode of '15 s bright-1 s dark', and resting for 10-20 min; repeating 4 training cycles, washing to remove medicinal liquid, and continuously irradiating; entering a testing stage, and circularly stimulating and training for 10-20 times according to a 60s bright-1 s dark stimulation mode; calculating the memory intensity learning rate. The invention combines a behavior analysis system and a microplate technology which are generally applicable in the field of zebra fish behaviors, and can realize high-flux analysis; the data processing is more objective and scientific, and the experimental efficiency is improved.
Description
Technical Field
The invention relates to the technical field of drug evaluation and screening, in particular to a construction method and application of a zebra fish memory evaluation model.
Background
The zebra fish juvenile fish is the only higher vertebrate animal suitable for high-flux drug screening at present due to high homology (87%) with human genes and small individual, and can be used for rapid evaluation of memory function. The zebrafish juvenile fish can experience sudden rapid movement reaction due to sudden loss of light (dark flash), which can lead to long-term habitual performance after repeated dark flash stimulation training. Habituation is a phenomenon of habituation of an organism to repeated dangerous stimuli, and can be divided into short-term habituation and long-term habituation (over 24 h) according to the retention time, and the quality of memory is closely related to long-term habituation.
In the prior art, when the memory ability of the zebra fish is evaluated by using habitual clothes, a high-speed camera is generally adopted to capture the body form phenotype of the zebra fish under the stimulation of sound and light and the like. Since the somatic reaction lasts for a very short time (within 20 ms), it cannot be distinguished by the naked eye and must be captured by a high-speed camera and then identified manually or by software. It has the following disadvantages: firstly, the realization mode is single; secondly, the experimental efficiency is low due to small flux; thirdly, the specialization degree of related equipment and analysis software is high, the investment cost is high, the specialization degree of related equipment and the like is high, and the application is single; fourthly, the subjectivity is high during the analysis of the picture data types, and the scientificity is insufficient.
The Chinese patent with publication number CN112034113A discloses a method for detecting memory improvement effect of health food, which comprises the following steps: (1) randomly selecting a plurality of zebra fish, putting an inducer after memory training, and establishing a dysmnesia model control group; (2) after memory training, putting an inducer and a sample to be tested, and establishing a sample group; (3) after memory training, adding an inducer and edaravone, and establishing a positive control group; (4) memory training, establishing a normal control group; (5) and respectively enabling the zebra fish of the memory impairment model control group, the test sample group, the positive control group and the normal control group to enter a T-shaped maze experiment, recording the latency time (T) from the starting point to the eutrophication area of the zebra fish, and calculating the memory improvement effect (%) of the test sample to be tested. However, the memory training of the method is only effective for short-term memory, has low flux and insufficient scientificity, and is not suitable for evaluating the long-term memory efficacy.
Disclosure of Invention
In order to solve the technical problems, the invention provides a construction method and application of a zebra fish memory evaluation model, and the construction method is combined with a behavior analysis system and a microplate technology which are universally applicable in the zebra fish behavioral field to improve the existing evaluation method.
The specific technical scheme of the invention is as follows:
in a first aspect, the invention provides a method for constructing a zebra fish memory evaluation model, which comprises the following steps:
(1) respectively arranging fertilized zebra fish juvenile fish into a normal group and a model group, adding a dimethyl sulfoxide solution into the normal group, and adding a cycloheximide solution into the model group; after adapting under the illumination condition, respectively moving the zebra fish larvae into a micropore plate, wherein each hole is provided with one zebra fish larva;
(2) opening a behavior analysis system, and balancing the zebra fish juvenile fish in the micropore plate for a period of time under the illumination condition;
(3) entering a training stage, circularly stimulating and training for 130-150 times according to a stimulation mode of '15 s bright-1 s dark', and taking the stimulation mode as 1 training cycle; under the condition of continuous illumination, resting for 10-20 min;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) after the training stage is finished, washing off a dimethyl sulfoxide solution or a cycloheximide solution on the zebra fish juvenile fish, and continuously illuminating for 4-6 h;
(6) entering a testing stage, and circularly stimulating and training for 10-20 times according to a 60s bright-1 s dark stimulation mode;
(7) calculating the memory strength learning rate of each group.
Under the stimulation of dark flash, the expression change of the fast movement response of the zebra fish is within 20ms, the maintenance time is very short, and the zebra fish cannot be distinguished by naked eyes. The rapid movement response can be identified through a behavior analysis system, the behavior analysis system is a device which is widely applied in the research of the behavioral science of the zebra fish juvenile fish, and the behavioral analysis of a plurality of zebra fish juvenile fishes can be completed by the aid of the device, such as common behavioral (track, speed, liveness), position preference and the like. And the microplate technology is matched, so that high-flux analysis can be realized, the test period is shortened, the interference of movement among individuals is reduced, and the problems of insufficient flux and low experimental efficiency in the prior art are solved.
In addition, the invention adopts a high-frequency stimulation mode of '15 s bright-1 s dark' to circularly stimulate and train for a higher frequency in the training stage of the zebra fish, under the training frequency, the habituation rate of a normal group reaches 70-80%, enough space causing memory decline can be provided for a subsequent model group in an actual experiment, a significant difference can be formed statistically, the training frequency is continuously increased, the test period is prolonged, and the initial purpose of rapid screening of the zebra fish experiment is not met. After the training cycle, the young fish is given a rest period to enhance memory, during which time the young fish is continuously exposed to the same intensity of light as during the experiment. After continuous illumination, long-term habituation memory is formed, a testing stage is used for checking the maintenance condition of a long-term habituation phenomenon, after a large number of early-stage repeated training, the exercise reaction ratio of the zebra fish at the stage starts from a lower value, and due to the existence of spontaneous exercise and other inducement, the circulation stimulation frequency set for 10-20 times is enough to be used for memory evaluation of the zebra fish.
The inducer of the cycloheximide inhibits the synthesis of biological protein by hindering the extension of polypeptide chain in the protein synthesis, thereby destroying the material basis of memory, causing the memory of a model group to decline, and being incapable of well keeping the habitual performance of dark flash in a test stage. The dimethyl sulfoxide solution in the normal groups does not influence the memory ability of the zebra fish juvenile fish, so that better habit formation can be observed, and the memory intensity can be evaluated by comparing the retention degree of the habit formation among the groups.
Preferably, in the step (1), the zebra fish juvenile fish is juvenile fish of 5-7 days after fertilization; the adaptive time under the illumination condition is 30-60 min. The zebra fish juvenile fish (5-7dpf) has better memory capacity and is beneficial to the construction and evaluation of a model.
Preferably, in the step (1), 5-8 parallel groups are respectively arranged on the normal group and the model group, and each parallel group comprises 32-48 zebra fish juvenile fishes; the volume fraction of the dimethyl sulfoxide solution in the normal group is 1-3 per mill; the concentration of the cycloheximide solution in the model group is 10-15 mu M.
Preferably, in the step (2), the zebra fish juvenile fish is balanced for 10-20 min under the illumination condition.
Preferably, the illumination conditions are illumination with the same intensity, and the illumination is set to be 8-15% of the illumination on the behavior analysis system.
Preferably, in the step (7), the memory strength learning rate is calculated by the following formula:
the A is the average value of the individual percentage of the motor response of the zebra fish juvenile fish in the previous 10 cycles in the step (6); and B is the average value of the individual percentage of the moving response of the zebra fish juvenile fish in the previous 10 times of the training cycle in the 1 st training cycle in the step (4).
The zebra fish juvenile fish can not make quick motion response to the dark flash when keeping the habit of taking clothes, also can not be detected by the instrument to this calculates the rate of taking clothes. In the testing stage, because of a large amount of training, the exercise response of the normal group is reduced to the minimum in about 10 cycles, the data after the previous 10 cycles are not greatly different, correspondingly, the data of the previous 10 cycles in the 1 st training cycle are also taken in the training stage, and the accuracy in the calculation of the exercise rate is improved.
Preferably, the step (7) further includes calculating learning ability learning rate by the following formula:
c is the average value of the individual percentage of the zebra fish juvenile fish exercise reaction of the previous 10 times of training cycles in the 4 th training cycle in the step (4); and D is the average value of the individual percentage of the moving response of the zebra fish juvenile fish in the previous 10 times of the training cycle in the 1 st training cycle in the step (4).
Preferably, the movement reaction is that the movement speed exceeds a speed threshold set by a behavior analysis system; the speed threshold value is 13-15 mm/s.
By setting a speed threshold value of the behavior analysis system, when the motion speed of the zebra fish is larger than the speed threshold value after the dark flashes are applied, the zebra fish is recorded, and therefore the proportion of individuals with reactions in each dark flash is counted. In addition, zebrafish can move due to various reasons, such as spontaneous swimming, influence of sound and disturbance of vibration, and in order to filter out irrelevant reasons as much as possible, especially spontaneous movement, and improve the high correlation between quick movement response and dark flashing, the speed threshold is most suitable in practical operation.
On the other hand, the invention also provides application of the zebra fish memory evaluation model construction method in evaluating drugs or health-care food for assisting in improving memory functions.
Preferably, a group of positive control groups are arranged besides the experimental group and the model group, medicine liquid of medicine or health-care food and cycloheximide solution are added into the positive control groups, and the habitual administration rate of each group is calculated according to the construction method from the step (1) to the step (6); statistically processing the results of the individual groups of uniform rates to
Showing that the comparison between groups was performed by one-factor ANOVA, and the comparison between groups was statistically processed by Dunnett's T-test, p<0.05 was significantly different.
Compared with the prior art, the invention has the beneficial effects that:
(1) the zebra fish behavior analysis system is mature in technology, stable in equipment performance, applicable to almost all behavioral experiments of zebra fish, and has application and expansion space, and equipment investment is relatively reasonable and flexible;
(2) by means of the micropore plate technology, high-flux analysis can be realized, the test period is shortened, the interference of movement among zebra fish individuals is reduced, and the problems of insufficient flux and low test efficiency in the prior art are solved;
(3) the digitalized data type analysis and processing is more objective and scientific, and the efficiency of experimental result analysis is improved.
Drawings
FIG. 1 is a scattergram of the learning process of zebra fish juvenile fish in example 1;
FIG. 2 is a graph showing the memory strength of the positive drug 1 in examples 2 and 3;
FIG. 3 is a graph showing the memory strength of the positive drug 2 in examples 4 and 5;
FIG. 4 is a graph showing the memory strength of the positive drug 3 in example 6, example 7 and example 8;
FIG. 5 is a graph showing the learning ability of the positive drug 1 in examples 2 and 3;
FIG. 6 is a graph showing the learning ability of the positive drug 2 in examples 4 and 5;
FIG. 7 is a graph showing the learning ability of the positive drug 3 in examples 6, 7 and 8.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
Constructing a zebra fish memory evaluation model:
(1) taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; respectively setting a normal group and a model group, wherein each group comprises 5 parallel groups, each parallel group comprises 32 zebra fish juvenile fishes, after deducting the sample adding volume of each hole of a six-hole plate, 12 mu L of 100% dimethyl sulfoxide solution is added into each hole of the normal group, and 12 mu L of 10mM cycloheximide solution is added into the model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-well plate, wherein each hole is filled with 300 mu L of zebra fish fries;
(2) Turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training 150 times according to a stimulation mode of '15 s light-1 s dark' as 1 training cycle, and consuming 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate;
the memory strength learning rate is calculated by the following formula:
a is the average value of the individual percentage of the motor response of the zebra fish juvenile fish in the previous 10 cycles in the step (6); b is the average value of the individual percentage of the juvenile zebra fish exercise reaction of the previous 10 times of training cycles in the 1 st training cycle in the step (4);
the formula for calculating learning ability learning rate is as follows:
c is the average value of the individual percentage of the zebra fish juvenile fish exercise reaction of the previous 10 times of training cycles in the 4 th training cycle in the step (4);
D is the average value of the individual percentages of the zebra fish juvenile fish movement responses in the first 10 training cycles in the step (4).
Wherein the illumination conditions are 8% illumination on the behavior analysis system; the motor response is that the motor speed exceeds the 13mm/s speed threshold set by the behavior analysis system.
Example 2
Meclofenoxate hydrochloride (positive drug 1, low concentration) was evaluated using the zebrafish memory evaluation model in example 1:
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is fixed to 12mL by standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 5mg/mL meclofenoxate hydrochloride solution, and carrying out overnight pre-exposure on zebra fish juvenile fish with 6 dpf; on the experimental day, 6dpf of zebra fish juvenile fish is adapted for 30min under the illumination condition, and 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after the solution is adapted for 30min under the illumination condition again, the solution is respectively moved into a 96-hole plate, one zebra fish juvenile fish is added into each hole, and 300uL of the solution is added into each hole;
(2) Turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training 150 times according to a stimulation mode of '15 s light-1 s dark' as 1 training cycle, and consuming 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
Example 3
Meclofenoxate hydrochloride (positive drug 1, high concentration) was evaluated using the zebrafish memory evaluation model in example 1:
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
Positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is determined to 12mL by standard dilution water; after the sample adding volume is deducted from each hole of a six-hole plate, 12 mu L of 10mg/mL meclofenoxate hydrochloride solution is added, and overnight pre-exposure is carried out on zebra fish juvenile fishes with 6 dpf; on the experimental day, 6dpf of zebra fish juvenile fish is adapted for 30min under the illumination condition, and 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after adapting for 30min under the illumination condition again, respectively moving the zebra fish into 96-pore plates, wherein each pore is filled with one zebra fish juvenile fish, and the volume of liquid in each pore is 300 uL;
(2) turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training for 150 times according to a stimulation mode of '15 s bright-1 s dark', wherein 1 training cycle takes 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) Entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
Example 4
Oxiracetam was evaluated using the zebrafish memory evaluation model of example 1 (positive 2, low concentration):
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is fixed to 12mL by standard dilution water; after the sample adding volume of each hole of the six-hole plate is deducted, 12 mu L of 40mg/mL oxiracetam solution is added, and overnight pre-exposure is carried out on zebra fish juvenile fish with 6 dpf; on the experimental day, 6dpf of zebra fish juvenile fish is adapted for 30min under the illumination condition, and 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after the solution is adapted for 30min under the illumination condition again, the solution is respectively moved into a 96-hole plate, one zebra fish juvenile fish is added into each hole, and 300uL of the solution is added into each hole;
(2) Turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training 150 times according to a stimulation mode of '15 s light-1 s dark' as 1 training cycle, and consuming 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
Example 5
Oxiracetam was evaluated using the zebrafish memory evaluation model of example 1 (positive 2, high concentration):
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group;
After adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is fixed to 12mL by standard dilution water; after the sample adding volume of each hole of the six-hole plate is deducted, 12 mu L of oxiracetam solution with the concentration of 80mg/mL is added, and overnight pre-exposure is carried out on zebra fish juvenile fish with the concentration of 6 dpf; on the experimental day, 6dpf of zebra fish juvenile fish is adapted for 30min under the illumination condition, and 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after the solution is adapted for 30min under the illumination condition again, the solution is respectively moved into a 96-hole plate, one zebra fish juvenile fish is added into each hole, and 300uL of the solution is added into each hole;
(2) turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training for 150 times according to a stimulation mode of '15 s bright-1 s dark', wherein 1 training cycle takes 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) The training phase is ended. Immediately washing young fishes in a 96-pore plate to remove dimethyl sulfoxide solution or cycloheximide solution, and then continuously illuminating and exposing for 4 hours under the same illumination intensity in the training process;
(6) entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
Example 6
Evaluation of ginkgo leaf-desert cistanche tablet (positive drug 3, low concentration) using zebrafish memory evaluation model in example 1:
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
Positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is determined to 12mL by standard dilution water; adding 12 mu L of 125mg/mL ginkgo leaf and cistanche slice solution after deducting the sample adding volume of each hole of a six-hole plate, and carrying out overnight pre-exposure to zebra fish juvenile fish with 6 dpf; on the day of experiment, 6dpf of zebra fish juvenile fish is adapted for 30min under the condition of illumination, and then 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after the solution is adapted for 30min under the illumination condition again, the solution is respectively moved into a 96-hole plate, one zebra fish juvenile fish is added into each hole, and 300uL of the solution is added into each hole;
(2) turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training 150 times according to a stimulation mode of '15 s light-1 s dark' as 1 training cycle, and consuming 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) Entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
Example 7
Evaluation of ginkgo leaf and cistanche deserticola tablets (positive drug 3, medium concentration) by using the zebra fish memory evaluation model in example 1:
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is fixed to 12mL by standard dilution water; adding 12 mu L of 250mg/mL ginkgo leaf and cistanche slice solution after deducting the sample adding volume of each hole of a six-hole plate, and carrying out overnight pre-exposure to 6dpf zebra fish juvenile fish; on the experimental day, 6dpf of zebra fish juvenile fish is adapted for 30min under the illumination condition, and 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after the solution is adapted for 30min under the illumination condition again, the solution is respectively moved into a 96-hole plate, one zebra fish juvenile fish is added into each hole, and 300uL of the solution is added into each hole;
(2) Turning on a behavior analysis system, after parameters are correctly set and preheated, turning on a background lamp to balance the zebra fish juvenile fishes in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training for 150 times according to a stimulation mode of '15 s bright-1 s dark', wherein 1 training cycle takes 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
Example 8
Evaluation of ginkgo leaf-desert cistanche tablet (positive drug 3, high concentration) using zebrafish memory evaluation model in example 1:
(1) respectively setting a normal group, a model group and a positive control group, wherein each group comprises 5 parallel groups, and each parallel group comprises 32 zebra fish juvenile fishes;
normal group, model group: taking 6dpf of zebra fish juvenile fish to adapt for 30min under the illumination condition, selecting active individuals, placing the active individuals in six-hole plates, wherein 35-40 individuals are placed in each hole, and the volume of each hole is fixed to 12mL by using standard dilution water; after deducting the sample adding volume of each hole of the six-hole plate, adding 12 mu L of 100% dimethyl sulfoxide solution into each hole of a normal group, and adding 12 mu L of 10mM cycloheximide solution into a model group; after adapting for 30min under the illumination condition, respectively moving the zebra fish fries into a 96-hole plate, wherein each hole is filled with one zebra fish fry, and the volume of liquid in each hole is 300 uL;
Positive control group: one day before the experiment, 5dpf of zebra fish juvenile fish is placed in a six-hole plate, 35-40 zebra fish juvenile fish are placed in each hole, and the volume of each hole is fixed to 12mL by standard dilution water; adding 12 mu L of 500mg/mL ginkgo leaf and cistanche slice solution after deducting the sample adding volume of each hole of a six-hole plate, and carrying out overnight pre-exposure to zebra fish juvenile fish with 6 dpf; on the experimental day, 6dpf of zebra fish juvenile fish is adapted for 30min under the illumination condition, and 12 μ L of 10mM cycloheximide solution is added for inducing and molding; after the solution is adapted for 30min under the illumination condition again, the solution is respectively moved into a 96-hole plate, one zebra fish juvenile fish is added into each hole, and 300uL of the solution is added into each hole;
(2) turning on a behavior analysis system, correctly setting parameters and preheating, and then turning on a background lamp to balance the zebra fish juvenile fish in the micropore plate for 10min under the illumination condition;
(3) entering a training stage, circularly stimulating and training 150 times according to a stimulation mode of '15 s light-1 s dark' as 1 training cycle, and consuming 40min10 s; resting for 10min under continuous illumination;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) the training phase is ended. Immediately washing the juvenile fish in the 96-well plate to remove a dimethyl sulfoxide solution or a cycloheximide solution, and then continuously exposing for 4 hours under the same illumination intensity in the training process;
(6) Entering a testing stage, circularly stimulating and training for 20 times according to a stimulating mode of '60 s bright-1 s dark', and consuming 20min10s in total;
(7) calculating the memory strength learning rate and the learning ability learning rate.
As can be seen from the graph 1, with the increase of the number of training cycles, the percentage of individuals of zebra fish juvenile fish with high-speed movement is gradually reduced, and the habit maintenance performance in the testing stage is better. The memory-improving efficacy of the positive drugs 1 to 3 was evaluated using the zebrafish memory evaluation model of example 1, and the results of statistical treatment of the habituation rates of each group were evaluated to determine
Showing that the comparison between groups was performed by one-factor ANOVA, and the comparison between groups was statistically processed by Dunnett's T-test, p<0.05 was significantly different.
As shown in fig. 2 and 5, the memory strength learning rate of the positive drug 1 at different concentrations was # p <0.05 compared to the normal control group, which was # p <0.05 compared to the model control group; the learning ability learning rate of the positive drug 1 at different concentrations was # p <0.05 compared to the normal control group, and # p <0.05 compared to the model control group.
As shown in fig. 3 and 6, the memory strength learning rate of the positive drug 2 at different concentrations was # p <0.05 compared to the normal control group, which was # p <0.05 compared to the model control group; the learning ability learning rate of the positive drug 2 at different concentrations was # p <0.05 compared to the normal control group, and # p <0.05 compared to the model control group.
As shown in fig. 4 and 7, the memory strength exercise rate of the positive drug 3 at different concentrations was # p <0.05, # p <0.01 compared to the normal control group, which was # p <0.05, # p <0.01 compared to the model control group; the learning ability learning rate of the positive drug 3 at different concentrations was # p <0.05, # p <0.01 as compared to the normal control group, and # p <0.05 as compared to the model control group.
The results show that the positive drug 1 (meclofenoxate hydrochloride) can obviously improve the decline of the memory strength and the learning ability of a model group under the experimental concentration and has a certain dose-effect relationship; the positive drug 2 (oxiracetam) can obviously improve the memory strength and learning ability of a model group under the experimental concentration; the positive medicine 3 (ginkgo leaf and cistanche tablet) can obviously improve the memory strength and learning ability of the model group under various experimental concentrations except the medium concentration, and the verification of the positive medicine proves that the model has the function of evaluating the memory strength and the learning ability.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. A construction method of a zebra fish memory evaluation model is characterized by comprising the following steps:
(1) respectively arranging fertilized zebra fish juvenile fish into a normal group and a model group, adding a dimethyl sulfoxide solution into the normal group, and adding a cycloheximide solution into the model group; after adapting under the illumination condition, respectively moving the zebra fish larvae into a micropore plate, wherein each hole is provided with one zebra fish larva;
(2) opening a behavior analysis system, and balancing the zebra fish juvenile fish in the micropore plate for a period of time under the illumination condition;
(3) entering a training stage, circularly stimulating and training for 130-150 times according to a stimulation mode of '15 s bright-1 s dark', and taking the stimulation mode as 1 training cycle; under the condition of continuous illumination, resting for 10-20 min;
(4) repeating the step (3) for 4 times, wherein the total number of training cycles is 4;
(5) after the training stage is finished, washing off a dimethyl sulfoxide solution or a cycloheximide solution on the zebra fish juvenile fish, and continuously illuminating for 4-6 h;
(6) entering a testing stage, and circularly stimulating and training for 10-20 times according to a 60s bright-1 s dark stimulation mode;
(7) calculating the memory strength learning rate of each group.
2. The method for constructing a zebra fish memory evaluation model according to claim 1, wherein in the step (1), the zebra fish juvenile fish is juvenile fish after fertilization for 5-7 days; the adaptive time under the illumination condition is 30-60 min.
3. The method for constructing the zebra fish memory evaluation model according to claim 2, wherein in the step (1), the normal group and the model group are respectively provided with 5-8 parallel groups, and each parallel group comprises 32-48 zebra fish juvenile fishes; the volume fraction of the dimethyl sulfoxide solution in the normal group is 1-3 per mill; the concentration of the cycloheximide solution in the model group is 10-15 mu M.
4. The method for constructing the zebrafish memory evaluation model according to claim 1, wherein in the step (2), the zebrafish juvenile fish is balanced for 10-20 min under the illumination condition.
5. The method for constructing the zebra fish memory evaluation model according to claim 1, wherein the illumination conditions are illumination with the same intensity, and are set to be 8-15% illumination on a behavior analysis system.
6. The method for constructing the zebra fish memory evaluation model according to claim 1, wherein in the step (7), the memory intensity learning rate is calculated according to the following formula:
the A is the average value of the individual percentage of the motor response of the zebra fish juvenile fish in the previous 10 cycles in the step (6); and B is the average value of the individual percentage of the moving response of the zebra fish juvenile fish in the previous 10 times of the training cycle in the 1 st training cycle in the step (4).
7. The method for constructing the zebra fish memory evaluation model according to claim 1 or 6, wherein in the step (7), the learning ability learning rate is calculated according to the following formula:
c is the average value of the individual percentage of the zebra fish juvenile fish exercise reaction of the previous 10 times of training cycles in the 4 th training cycle in the step (4); and D is the average value of the individual percentage of the moving response of the zebra fish juvenile fish in the previous 10 times of the training cycle in the 1 st training cycle in the step (4).
8. The method for constructing the zebra fish memory evaluation model as claimed in claim 7, wherein the movement response is that the movement speed exceeds a speed threshold set by a behavior analysis system; the speed threshold value is 13-15 mm/s.
9. Use of the method for constructing a zebra fish memory evaluation model according to any one of claims 1 to 8 in evaluating a medicine or health food for assisting in improving memory function.
10. The use of claim 9, wherein the experimental group and the model group are excludedIn addition, a group of positive control groups is arranged, medicine liquid of medicines or health-care food and cycloheximide solution are added into the positive control groups, and the habituation rate of each group is calculated according to the construction method from the step (1) to the step (7); statistically processing the results of the individual groups of uniform rates to 
Showing that the comparison between groups was performed by one-factor ANOVA, and the comparison between groups was statistically processed by Dunnett's T-test, p<0.05 was significantly different.
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Denomination of invention: Construction method and application of a zebrafish memory evaluation model Effective date of registration: 20230725 Granted publication date: 20221206 Pledgee: Hangzhou branch of Bank of Nanjing Co.,Ltd. Pledgor: HANGZHOU HUNTER BIOTECHNOLOGY Co.,Ltd. Registration number: Y2023330001562 |