RU2789575C1 - Device for testing animal behavior and its use method - Google Patents

Abstract

FIELD: experimental physiology.

SUBSTANCE: group of inventions relates to experimental physiology, and it is used in study of neurophysiological mechanisms of learning and memory, spatial navigation, motor and search activity of laboratory animals. A device for testing animal behavior includes a labyrinth made in the form of an arena and consisting of outer walls and inner partitions. At the same time, the labyrinth itself is an arena with square compartments formed using fixed white and black removable partitions used as signal sources and with floor covering with color marks applied to it and used as outer signals. There is an animal passage on at least one partition, and a final compartment contains a feeder. Options of a method for the use of the specified device are also presented.

EFFECT: obtainment of an assessment of cognitive abilities of experimental animals, when they perform a refreshing search task, at which a sequence of equivalent alternative choices in different labyrinth configurations is made.

12 cl, 5 dwg

Description

Technical field

The claimed technical solution relates to experimental physiology and is used in the study of neurophysiological mechanisms of learning and memory, spatial navigation, motor and search activity of laboratory animals in the implementation of their purposeful food-procuring behavior, as well as in testing cognitive impairment in model experiments on animals and can be applied in experimental pharmacology and experimental medicine.

State of the art

Classical mazes are known from the prior art for studying the spatial choice of an animal - this is the T-maze and its modification Υ-maze (Zhang, Q., Kobayashi, Y., Goto, Η., Itohara, S. An Automated T-maze Based Apparatus and Protocol for Analyzing Delay- and Effort-based Decision Making in Free Moving Rodents. J. Vis. Exp.(138), e57895, doi:10.3791/57895 (2018).) (Swonger AK, Rech RH (1972) Serotonergic and cholinergic involvement in habituation of activity and spontaneous alternation of rats in a Y maze J Comp Physiol Psychol 81(3):509-522). In this maze, the animal chooses one of two or one of three arms based on cues, past choices, or simply exploratory behavior.

The disadvantage of these labyrinths is a small (two or three options) number of available choices, which does not make it possible to make an unambiguous conclusion about the selection strategy used by the animal, which affects the results of the experiment. In addition, the limited route does not allow us to explore the mechanisms underlying spatial navigation.

Multipath mazes are also known (Olton, D.S.; Samuelson, R.J. (1976). "Remembrance of places passed: Spatial memory in rats". Journal of Experimental Psychology: Animal Behavior Processes. 2 (2): 97-116) or the Barnes maze ( Barnes, CA (1979) "Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat." with reinforcements or one of the many holes to avoid the annoying factor. That is, a feature of multipath mazes is the use of many alternative choices, which makes them more attractive compared to Τ or Υ mazes.

The disadvantage of all ray labyrinths is the inability to separate and exclude the coincidence of a random choice with the correct one, which affects the purity of the experiment.

Also known is the Hebb-Williams maze, which provides different routes between fixed start and finish points for animals (Hebb, D. O. and K. A. Williams. 1946. A method of rating animal intelligence. J. Gen. Psychol. 34: 59-65 Pritchett-Corning, Kathleen & Mulder, Guy (2004) Hebb-Williams mazes Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science 43. 44-5. The maze is primarily focused on egocentric navigation and memorization of the spatial location of the start and finish points, although there are modifications of this maze using external signals.

The disadvantage is that the passage options in this maze are not identical in terms of difficulty.

There is a known method for assessing spatial orientation and cognitive impairment in experimental animals (RU 2551308 C2 dated 2012.10.15, class IPC A61B 5/16, G09B 23/28), where the orienting-exploratory and motor behavior of rats is studied against the background of developing a food-procuring skill by differentiating the trajectory movement of animals in the W-shaped maze. The assessment is carried out according to the following forms of motor actions: movements to the loci of the labyrinth without food reinforcement; purposeful movements to the locus with food reinforcement; research bypasses of all sites of the labyrinth; movements to a locus that is mirror opposite to the locus with food reinforcement; repetitive movements between two loci of the labyrinth; fading at the launch pad. The method allows to increase the reliability of the study, which is achieved through a multifactorial assessment of the functions of the central nervous system.

The disadvantage of this method is that it is focused on the free search behavior of rats, remembering the route and its reproduction under unchanged conditions of the same modification of the maze.

It is also known that the standard for testing spatial memory is the Morris water maze, in which the animal learns to find a platform hidden under water (Morris RGM. Spatial localization does not require the presence of local cues. Learn Motiv 1981;12:239-260). In the specified labyrinth, the trajectory of the animal's movement, its time spent in the sectors of the maze and the time it takes to reach the platform are investigated. During training, the animal's starting position is varied, where orientation is carried out on the basis of signals external to the pool. It is assumed that animals use sequentially or, more often simultaneously, various strategies to achieve the goal, which include, in addition to spatial orientation, but also non-spatial (Whishaw IQ, Mittleman G. Visits to starts, routes, and places by rats (Rattus norvegicus) in swimming pool navigation tasks J Comp Psychol 1986 Dec;100(4):422-31 PMID: 3802786.). This maze allows you to explore the mechanisms of memory and learning, using both allocentric and egocentric navigation.

Another similar water maze is the Cincinnati maze, which is a multiple T-maze (Vorhees CV, Williams MT. Cincinnati water maze: A review of the development, methods, and evidence as a test of egocentric learning and memory. Neurotoxicol Teratol 2016;57:1-19. doi:10.1016/j.ntt.2016.08.002). In the stated labyrinth, the animal makes a choice of the right or left sleeve, one of which leads to a dead end, and the other to the place of the next choice. The labyrinth consists of nine interconnected T-junctions. The animals are forced to find their way from one end of the maze to the other by moving through holes in the walls rather than at the end of each passage. The walls are wide enough to prevent the animals from leaning against the wall, while the walls themselves are made of plexiglass to prevent unwanted animal behavior such as climbing over the wall or looking for seams in the walls to grab onto. A feature of this labyrinth is the ability to use it both with external signals and without them, which allows you to explore both types of navigation. The disadvantage of this labyrinth is its invariable configuration. This labyrinth is taken as a prototype.

The disadvantage of the above water labyrinths is the laboriousness of their use, due to their size, since such labyrinths occupy an entire room, and, in addition, require special equipment for the delivery and discharge of water, as well as its heating. In addition, the method implemented in the labyrinths is focused on the avoidance reaction, where the animal is looking for a platform, trying to avoid the discomfort associated with being in the water. The stress that accompanies such conditions can have an impact on cognitive function, which affects the results of the experiment.

Additionally, we note that any maze used allows us to evaluate the cognitive abilities of animals based on the trajectory of its passage, the time it takes to reach the goal, and the number of errors.

The objective of the proposed technical solution is to obtain an assessment of the cognitive abilities of experimental animals, when they perform the task of searching for reinforcement, in which a chain of equivalent alternative choices is made in various maze configurations, both using external signals and without them, through the use of a maze with a modular design.

The difference from the prototype is the possibility of multiple modification of the route, as well as the fact that the search for reinforcements by animals is carried out on the land arena, which allows them to avoid the stress associated with tasks performed in the water.

Disclosure of the essence of the technical solution

The technical result of the above task is achieved by developing a device for testing animal behavior, including a labyrinth made in the form of an arena, and consisting of external walls and internal partitions, where the labyrinth itself is an arena, with square compartments formed using fixed removable partitions , while at least one partition has a passage, and one of the compartments has a feeder. The specified technical solution provides a modular design that allows you to change the configuration of the labyrinth by changing the position of the removable partitions.

A variant of the technical solution is possible, where the partitions are fixed with the help of columns with grooves located in the inner space of the labyrinth and semi-columns with grooves located on the outer walls of the labyrinth. This technical solution allows you to create different configurations of the labyrinth, changing the position of individual partitions, without using the floor of the labyrinth, which remains homogeneous, which allows you to quickly remove it and eliminate any traces of animals that could serve as signals.

A variant of the technical solution is possible, where the partitions are fixed with the help of a perforated floor and pins along the lower edge of the partitions. This technical solution allows you to create different configurations of the labyrinth without installing additional elements in the interior of the labyrinth.

A variant of a technical solution is possible, where the internal partitions are not fixed in all possible positions and the compartments formed by them are not square. The specified technical solution allows you to expand the number of possible multiple modifications of routes.

A variant of the technical solution is possible, where the passage in the partition is implemented in the form of an arch or in the form of a hole. The specified technical solution allows you to create routes for the animal from the compartment selected as the start to the compartment selected as the finish with passages that are optimal for laboratory animals.

A variant of the technical solution is possible, where the partitions have a different color for each side, or have the same color for each side, or the color of all partitions is the same, or the color of at least one partition is different on at least one side, or the partitions have the same color, and the floor covering individual compartments has a different color, or partitions and flooring have the same color. This technical solution allows the use of color as external signals for the animal, where the choice of passages marked with a certain color will provide the best route to receive reinforcements.

A variant of the technical solution is possible, where the arena is made in the form of a square or rectangular shape. The specified technical solution increases the options for modifications of the labyrinth.

A variant of the technical solution is possible, where the feeder is installed on a removable partition or the inside of an external wall or installed on the floor or in a floor recess. The specified technical solution provides the variability of routes for the animal by changing the compartment in which the animal receives reinforcements.

A variant of the technical solution is possible, where the arena has no ceiling, or there is a transparent ceiling, or the arena has an opaque ceiling. The specified technical solution increases the options for modifications of the labyrinth in which the animal is isolated to varying degrees from the environment.

A variant of the technical solution is possible, where the labyrinth is provided with a video camera. The specified technical solution allows for video recording of the behavior of the animal in the maze for further analysis.

A variant of a technical solution is possible, where the labyrinth is equipped with at least one motion sensor. The specified technical solution allows to automatically determine the coordinates of the animal in the labyrinth under conditions of complete isolation of the animal from the external environment, in the case of using a labyrinth with opaque walls and ceiling.

The technical result of the above problem is achieved through the method of using the claimed device, which includes a labyrinth made in the form of an arena, and consisting of external walls and internal partitions, and characterized in that, in order to provide multiple modifications of routes, the labyrinth is made modular, by changing the configuration partitions with walkways, as well as changing the location of the start and finish compartments, in less than 5 minutes, so that each subsequent run of the animal can be implemented in new conditions. The specified technical solution allows, during testing, to present to the animal successively different tasks, represented by different routes (chains of alternative choices), both equivalent in length and level of complexity, and not equivalent.

The technical result of the above task is achieved through the method of using the claimed device, which includes a labyrinth made in the form of an arena, and consisting of external walls and internal partitions, and characterized in that, in order to provide multiple modifications of the orientation of the routes relative to the environment, the labyrinth is made mobile, due to the size and weight, which allow it to be rotated, carried and mounted in less than 30 minutes. The specified technical solution allows testing animals by varying the location of the routes relative to the external space, which leads to minimization of the influence of external signs and reduces the likelihood of using external, in relation to the maze, landmarks for navigation.

Brief list of drawings

Additionally, we note that, as shown in FIG. 1-5 show the most preferred embodiment of the technical solution and cannot be considered as limiting the content of the technical solution, which includes other embodiments.

An example of the implementation of a technical solution, namely a maze for studying the behavior of rats, is reflected in

fig. 1 - drawing of the labyrinth, top view;

fig. 2 - a good example, a view of the labyrinth from above;

fig. 3 - options for different routes of the maze;

fig. 4 - variants of different positions in the space of one route;

fig. 5 - test version of the maze

pos. 1 - labyrinth, made in the form of an arena with removable internal partitions 3;

pos. 2 - side (external) walls of labyrinth 1, not less than 20 cm high;

pos. 3 - internal removable partitions forming compartments with a side of at least 15x15 cm of labyrinth 1, while removable partitions 3 are presented in three versions: white solid partitions 3, white partitions 3 with an arch 5 for the passage of an animal, partitions are white on one side, and with the other side is black with an arch 5 for the passage of the animal;

pos. 4 - feeder, which is placed in the compartment designated in this configuration as the finishing one. Depending on the task, the feeder is located at a height of 5 cm and above from the floor on a black or white partition (the high location of the feeder is necessary in order to exclude the possibility of its visual detection from the adjacent compartment). And, depending on the compartment assigned as the finishing one, the wall with the feeder is either inserted into the grooves of the columns, or attached to the outer wall with double-sided tape.

pos. 5 - a passage made in the form of an arch or hole, located on the internal removable partitions 3, labyrinth 1.

pos. 6 - a column with grooves or semi-columns with grooves located on the outer walls of the labyrinth, providing fastening of the partitions 3 between the individual compartments of the labyrinth 1.

Implementation of the technical solution

In the claimed technical solution, the following terms and definitions are used:

Passages are 5 arches or openings in the interchangeable walls of maze 1. Animals are primarily laboratory rodents: rats or mice, but any laboratory animals in maze 1 of the appropriate size, depending on the purpose of use, can also be used, for example, hamsters or marine pigs.

Compartment - a separate room, made in the form of a square compartment inside the labyrinth 1.

The claimed technical solution is implemented in Fig. 1-5, where the device itself, made in the form of a labyrinth 1, is an arena divided into square compartments due to internal removable partitions 3.

The total weight and dimensions of labyrinth 1 make it easy to turn it to any angle relative to its original position, move it in space and use it in any room.

While the arena of the labyrinth 1 can be made square or rectangular.

The square shape of labyrinth 1 is an arena, which is divided by partitions into an equal number of square floor compartments along both axes of the coordinate grid and is described as (n x n) in the general case, where η can be anything. A version of the maze (3 x 3) is shown in Fig. 1-5.

The rectangular shape of labyrinth 1 is an arena, which is divided by partitions into a different number of square floor compartments along both axes of the coordinate grid and is described as (n x m) in the general case, where n and m can be any.

Labyrinth 1 is made of white opaque plastic or Plexiglas. The choice of material is due to the optimal properties of plastic for cleaning the labyrinth, opacity provides isolation of the animal from extraneous visual signals.

Internal partitions 3 separating square compartments, replaceable. The removable partitions 3 are fixed with the help of grooves (not shown in the figure) located on the semi-columns installed on the inner surface of the side walls 2 of the labyrinth 1 and on the columns 6 located in the inner space of the labyrinth, in the corners of the central compartment.

One of the partitions 3 is equipped with a feeder located at a height of 10 cm from the floor. Depending on the task, the partition 3 with the attached feeder is placed either in one of the grooves (not shown in the figure) that separates the compartments or, if the feeder is placed on the side wall 2, it is fixed with double-sided tape. In maze 1, both food and drink reinforcements can be used.

The starting compartment in this maze 1 can be any compartment assigned by the researcher, the finishing compartment is the compartment with a feeder, the location of which can also be varied. After reaching the finish line and receiving reinforcements, the animal is removed from maze 1.

Maze 1 allows learning of food-procuring behavior in accordance with the signals. The color of interchangeable partitions 3 is used as signals. The task in maze 1 is to choose a passage through white or black partitions 3. So, if the choice of passages through black partitions 3 is reinforced, then passages 5 through white partitions lead to dead ends, that is, compartments with a single entrance, and passages 5 through black partitions lead to the next compartment, where the animal again has a choice between a black or white passage. In FIG. 2, the gray arrow shows the route from start to finish through the passages located on the black partitions.

The use of passages 5 in the form of arches allows the animal to pass from one compartment to another without overcoming the threshold, which makes it possible to explore the behavior of the animal in a single floor space.

The use of passages 5 in the form of holes allows the animal to pass from one compartment to another overcoming the threshold, which makes it possible to explore the behavior of the animal in conditions that limit individual compartments from all sides, including the floor.

In FIG. Figure 3 shows 4 main options for combinations of passages for a 3x3 maze, in which each square compartment of labyrinth 1 is either a dead end, when choosing a white passage, or a passage to the next compartment, when choosing a black passage. The letters Τ denote dead-end compartments, the letters B - compartments with the possibility of choosing a passage, C - the starting compartment, which is also a selection compartment, and Φ - the finishing compartment with a feeder. The gray line on the diagrams describes the route.

All 4 options are equivalent in complexity for the animal, since in all cases an equal number of internal partitions are used - (12) which is divided by an equal number of white and black partitions 3 with passages and solid partitions (4 each). At the same time, 4 configurations of the labyrinth provide an equal number of dead-end compartments (4) and selection compartments (4).

In FIG. 3 shows possible routes from one launch compartment. In the case of using a labyrinth without a ceiling, it is difficult to create identical conditions of space external to the labyrinth, and in this case the animal can use external landmarks for navigation.

In FIG. Figure 4 shows an example where, in order to minimize the influence of external features, one of the possible routes - the route (a1), is turned by 90 (a2), 180 (a3) or 270 (a4) degrees, which provides a variety of combinations of start, finish and trajectories passage to the feeder relative to the external situation.

There are options for turning the labyrinth itself to any angle.

The options shown in Fig. 2-5 include the color of the walls as external signals. It is possible to apply color marks to the floor covering, using them as external signals. In this case, you can use a set of replacement floor coverings with pre-applied labels.

It is possible to mount internal partitions using pins installed in a perforated floor.

Various combinations of internal partitions 3 form different options for the passages available to the animal, depending on the tasks set by the researcher, that is, the modular organization of the labyrinth allows you to quickly, usually in a few minutes, change the configuration of the labyrinth during the experiment, so that each subsequent run of the animal can be implemented under new conditions.

The labyrinth allows you to create both different and identical route configurations in length and complexity. The compartments formed by internal partitions can be either the same or different in shape and size.

For the convenience of analyzing the behavior under study, labyrinth 1 is equipped with a video camera located on top. Software recognition of an animal provides data on its location at each moment of the experiment. The result of the analysis of behavior in maze 1 is the trajectory of the animal and the time to reach the feeder. Entry into dead end compartments are treated as errors. A trained animal is an animal that unmistakably chooses passages 5 leading to the feeder without going into dead ends.

It is possible to use a labyrinth with a ceiling that isolates the animal from above. In the case of an opaque ceiling, the animal is deprived of the opportunity to navigate by external signals relative to the entire maze. In this case, to obtain information about the coordinate of the animal at each moment of time, it is possible to use motion sensors installed inside the maze.

The measured and comparative parameters of behavior are: the success of the task, the time that the animal spent on the route from start to finish, the number of compartments of different types visited, the number of returns, the time spent in each compartment, the length of the path traveled, the number of experimental tests necessary to achieve learning criterion.

To study the navigation strategy used in an already trained animal, you can also use a test variant in which there are no solid partitions 3 in the maze 1 and the nearest routes to the feeder are achieved by passing through passages in white partitions 3, and black partitions 3 provide the longest possible path (Fig.5 ). An animal ignoring the shortest route to the feeder check and oriented only towards the back passages can be considered as using the path selection strategy solely on signals.

The learning procedure.

24 hours before the start of training, the animal is subjected to food or drink (depending on the type of reinforcement used) deprivation. On the first experimental day, the animal is placed in one section of labyrinth 1 closed on all sides by solid walls with a feeder (or drinker) placed there. After emptying the feeder, the animal is taken out and placed in a separate cage. After filling the feeder, the animal is again placed in this compartment. This procedure is carried out until the animals are confidently trained to receive reinforcement in the feeder. On average, 5 to 10 tests are sufficient. On the second experimental day, the animal is placed in the starting compartment of one of the variants of maze 1, for example (a). When placing, at the start, they try to orient the animal with its nose into a corner or, if a non-corner compartment is chosen as a start, then with its nose to the wall so that there is no pronounced orientation to one or another passage. For this, a white partition 3 is used, which covers the passages from the animal and is installed diagonally into the starting compartment, after placing the animal, the partition is removed. Previously, reinforcements are placed in the finishing compartment. After finding reinforcements, the animal is removed and placed in a separate cage. The labyrinth is rotated by 90 degrees and the animal again sits in maze 1. Thus, 5 tests are carried out, each time turning labyrinth 1 by 90 degrees, ending with the initial position (a, a90, a180, a270, a). The position of the corners of the square arena is preserved in the same coordinates. Such variability in the placement of the same route in one place is carried out in order to minimize the influence of external features on navigation. Between tests, the floor should be cleaned, and the arches through which the animals pass should also be wiped and, if possible, varied in order to minimize the ability of the animals to use the tracks left as landmarks. It should be noted that it is also possible to obtain the same options for placing the route in space without turning the labyrinth, but by changing the internal partitions and the position of the start and finish. This takes longer, but in this case, you can be sure that when passing the route, the animal did not use the marks left on the floor, since the same route actually passes through different cells of the floor.

In the next experimental series on the same day, the animal is presented with a different version of maze 1, for example, maze b, and 5 trials are also performed, each time with a rotation of 90 degrees (b, b90, b80, b270, b). On the third, fourth and fifth experimental days, two series of 5 trials are also carried out, while in each trial a new route of maze 1 is proposed, again ending with the first route (a, b, c, d, a). When the animal unmistakably performs running in all variants of maze 1, then verification testing is carried out in maze 1 (e).

If the animal ignores the passages in the white partitions and follows a long route through the passages in the black partitions, then it can be assumed that in this task it orients itself in accordance with external signals. If the animal repeats one of the routes that were used during training, ignoring the color of the partitions, then this may indicate that the animal was using a different strategy.

The maze can be used to study both allocentric navigation when animals are trained using cues such as color marking of passages or floors, and egocentric navigation when animals are taught to follow a route in conditions without external cues.

The claimed technical solution makes it possible to create routes identical in length and equivalent in complexity in the labyrinth (where the complexity is determined by the number of turns, the number of dead-end compartments and compartments where the animal makes a choice), due to the modular system, which is based on interchangeable internal partitions with passages or without, and with or without color indication.

Such a device is intended for testing laboratory animals in order to assess their cognitive abilities and the navigation strategies used in learning and implementing goal-directed foraging behavior based on the learning rate in the maze, the analysis of the trajectory passed, the decision time at each choice point, the total time to reach the goal and the number of errors. .

Claims (12)

1. A device for testing animal behavior, which includes a labyrinth made in the form of an arena, and consisting of external walls and internal partitions, characterized in that the labyrinth itself is an arena with square compartments formed using fixed white signals used as signal sources. and black removable partitions, and a floor covering with color marks printed on it and used as external signals, while at least one partition has a passage for the animal, and the finishing compartment contains a feeder. 2. The device for testing the behavior of animals according to claim 1, characterized in that the fixation of the partitions is carried out using columns with grooves located in the inner space of the labyrinth, and semi-columns with grooves located on the outer walls of the labyrinth. 3. A device for testing animal behavior according to claim 1, characterized in that the partitions are fixed using a perforated floor and pins along the lower edge of the partitions. 4. Device for testing the behavior of animals according to claim 1, characterized in that the passage in the partition is implemented in the form of an arch or in the form of a hole. 5. Device for testing animal behavior according to claim 1, characterized in that the partitions have a different color for each side, or have the same color for each side, or the color of all partitions is the same, or the color of at least one partition is different on at least one side , or partitions have the same color, and the flooring of individual compartments has a different color, or partitions and flooring have the same color. 6. Device for testing animal behavior according to claim 1, characterized in that the arena is made in the form of a square or rectangular shape. 7. Device for testing the behavior of animals according to claim 1, characterized in that the feeder is installed on a removable partition or the inside of an external wall or installed on the floor or in a recess in the floor. 8. Device for testing animal behavior according to claim 1, characterized in that the arena has no ceiling, or there is a transparent ceiling, or the arena has an opaque ceiling. 9. Device for testing animal behavior according to claim 1, characterized in that the labyrinth is provided with a video camera. 10. Device for testing animal behavior according to claim 1, characterized in that the labyrinth is equipped with at least one motion sensor. 11. The method of using the device according to paragraphs. 1-10, which includes placing reinforcements in the finishing section of the modular maze, testing when placing the animal in the starting section, removing the animal after it finds reinforcements, turning the maze by 90 degrees, then placing the animal in it at each test and bringing it to its original position at completion of tests. 12. The method of using the device according to paragraphs. 1-10, which includes carrying out tests with a rotation of the modular maze by 90 degrees during each of them, while changing the route of the test animal, bringing the maze to the first route at the end of the tests and conducting verification testing with the animal correctly performing movements in all variants of the maze.

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