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The goal of this optimized 'everyday memory' protocol in an event arena was to employ a stable home-base that encourages the use of allocentric spatial representations. This animal model provides an effective test-bed for future research into the formation and retention of event memories using behavioral and physiological techniques.
The event arena provides an optimal platform to investigate learning and memory. The appetitive everyday memory task described in this paper provides a robust protocol for the investigation of episodic and spatial memory in rodents, which specifically fosters allocentric memory representation. Rats are trained to find and dig for food during the encoding phase and, after a time delay, rats are given a choice to find the reward food pellet in the correct location. There are two key elements that promote the use of an allocentric strategy in this protocol: 1) rats start from different start locations within and between sessions, 2) a stable home-base is deployed where rats have to carry their food to eat. By means of these modifications, we effectively encourage the rodents to use allocentric spatial representations to perform the task. In addition, the task provides a good paradigm for within-subject experimental design and allows experimenters to manipulate different conditions to reduce variability. Used in conjunction with behavioral and physiological techniques, the resulting rodent model provides an effective test-bed for future research into memory formation and retention.
To investigate the neurobiology of learning and memory, invasive techniques are required, which are not generally feasible in humans. Thus, for over a century, behaviouralΒ protocols have been designed for laboratory animals to model various forms of human memory. The design and choice of both task and apparatus are central to the success of effective models of human memory. Numerous paradigms have been developed with diverse complexity, ranging from simple classical and instrumental conditioning protocols1,2,3 to mazes such as the T-maze4, radial arm maze5, Barnes maze6, watermaze7, and the cheese-board maze8. Yet, while these tasks capture facets of associative learning and spatial navigation, they cannot be used unambiguously to study the memory representation of momentary events (i.e., episodic-like memory). And, although novel object recognition9 and permutations of this spontaneous memory task, such as object-place memory10, have provided valuable insights into recognition memory, they do not test explicit recall of events. To address this demand, the event arena was specifically developed, and its use has enabled research into long-term, paired-associate memory encoding and recall11,12,13 as well as the encoding and recall of discrete events happening in a familiar space14,15,16,17,18. The latter theme is the focus of this manuscript.
The event arena is a large, square, open-field area where events occur for rodents. The size of the arena can be scaled to accommodate either rats or mice, and rodents are encouraged to enter and explore. A typical example of an event that takes place within the arena is the finding and retrieval of food from a sandwell at a specific location. The event arena is designed for such appetitive tasks, in which rats or mice are trained to search for, find, and dig up food. It capitalizes on their natural tendency to carry food back to a dark environment, which in this case is located adjacent to the arena, where they then eat it. After minimal training to dig for food, rodents take to this task naturally and perform well in the encoding trial, and in the recall choice trial, which follows the encoding trials after a short, 30-min delay. In a choice trial, several sandwells (i.e., locations for digging) are available, but only one is rewarded.
Different tasks can be performedΒ within the event arena (e.g., spatial memory, episodic-like memory, and paired-associate learning). Given the interest in developing effective models of episodic-like memory, the following protocol was developed,Β in which the location where food can be found is altered daily. In this task, rodents are required to remember where the event of digging for, and successfully retrieving, a food reward happened most recently within the event arena. The protocol outlined below entails an encoding trial in which rats search for a sandwell in a new place each day followed, after a delay, by a recallΒ choice trial, where the recently encoded sandwell location is rewarded, while the other, alternative sandwells in different locations do not contain accessible food. Remembering where the food was on a previous day is not helpful: the correct location has to be encoded and remembered, at least for a while, each day. Accordingly, we have introduced the term 'everyday memory' to capture the form of memory modeled in this task, which we, as humans, use on a daily basis. A human example of everyday memory is remembering where one has parked one's car at the shopping mall (Figure 1A) or has put one's glasses down around the house. In this protocol, allΒ intra- and extra-arena cues are all stable, just as they are in the settings of our everyday lives (i.e., homes, offices, car parks, etc.). Thus, rodents must remember where something happened most recently within a familiar environment (Figure 1B). The task is analogous to, but an improvement on, the delayed-matching-to-place (DMP) task in the watermaze19. Being an appetitive task, it exploits rodents' natural behavior to forage for food20, instead of their desire to escape from the water. However, as in the watermaze7, there are no local cues differentiating correct from incorrect locations; animals must use recall rather than recognition to locate the correct sandwell location after varying memory delay durations.
Figure 1: Everyday memory. (A) Human everyday memory. Schematic showing a green car parked in a car park. After a delay, the driver attempts to remember exactly where she parked her car. (B) Animal everyday memory. Schematic showing a rat digging and retrieving a pellet from a sandwell at a location within the event arena. After a delay, the rat is given a choice trial with multiple incorrect sandwells (gray) and one correct sandwell (green). Please click here to view a larger version of this figure.
The event arena has already been successfully utilized in investigations of 'everyday memory'. These are memories that are automatically encoded each day, retained in long-term memory, but often forgotten after relatively short time periods. Bast et al.14 showed monotonic delay-dependent event memory, which varies from excellent memory after short intervals to chance level after 24 h. The retention of memory can, however, be successfully enhanced by post-encoding novelty or, with multiple encoding trials, with extended trial spacing15,17.
The event arena is versatile and relatively non-stressful; no aversive stimuli are used. The size of the arena, and the tasks it accommodates, can be adapted for both rats14,15 and mice16. Also, as a land-based task, it is amenable to physiological recording and calcium imaging studies, unlike the watermaze21. Moreover, in accordance with the principles of the 3Rs (reduction, refinement, replacement), studies employing the event arena require fewer animals to obtain statistical power, as within-subject experimental designs are feasible (in which each animal serves as its own control for pharmacological interventions, optogenetic stimulations, etc.) and no aversive stimulation is required for motivation. Although initial training demands more time and occurs over more sessions than in, for example, novelty recognition tasks, once animals achieve a stable, asymptotic level of task performance, manipulations such as drug, vehicle-control, or optogenetic stimulation may be interspersed with a relatively small number of additional training sessions17. In addition, distinct facets of representation come under direct experimental control in the event arena, such as the nature of the spatial representation employed when solving the task.
The issue of representation concerns the mental framework employed by rats when remembering where recent events happen18. Do they remember where the food is located, orΒ do they only remember how to get to the food? Rats can use allocentric (map-like) or egocentric (body-centered) spatial representations to solve an appetitive task within the arena18. However, to control and identify the spatial strategy employed by each experimental subject when performing the task, there are distinct training protocols that are able to selectively promote the use of only one spatial representation. Usually, an egocentric-based representation is employed when rats take their food reward back to the same location from which they started the day's trial, which allows several opportunities to remember the reward location during runs back and forth. This spatial strategy can be employed regardless of whether the start location is changed from day to day or kept constant. In contrast, an allocentric representation is favored when rats are required to carry food reward to a fixed home-base location at the side of the arena, which is different from the changing starting locations. There are numerous advantages of allocentric representations with respect to the brain's storage capacity.
In this paper, we have outlined the home-base protocol, which encourages the employment of only an allocentric representation. WeΒ have provided representative results for this task, which clearly illustrate the advantages of using this rodent model of 'everyday memory' in the investigation of learning and memory and highlights how allocentric representations of episodic-like spatial memory can be promoted.
The methods described in this paper have been approved by the University of Edinburgh Ethical Review Committee; they are compliant with the UK Animals (Scientific Procedures) Act 1986 and the European Communities Council Directive of 24 November 1986 (86/609/EEC) legislation governing the maintenance of laboratory animals and their use in scientific experiments.
NOTE: The experimental subject of the protocol outlined below is Lister-hooded rats, but it can be adapted for other rodent strains.
1. Animal handling, housing, and food control
2. Setting up the apparatus
Figure 2: The event arena and cues. (A) Schematic showing the event arena (Abbreviations: N= North, E= East, S= South, W= West). (B) The event arena with intra- and extra-arena cues. (C) The two 3D intra-arena cues (left to right): golf ball stack and cylindrical black bottle. (D) Several 3D extra-arena cues (from left to right): patterned spherical lantern; red star lantern; blue lantern. (E) One of four black boxes is positioned midway along each event arena wall. Three of these black boxes serve as start-boxes, which provide a starting position for the rats at the start of each trial. The fourth black box is a home-base where rats consume the food reward that they retrieve from the arena. Please click here to view a larger version of this figure.
Figure 3: Sandwells. (A) Schematic showing an empty sandwell with the accessible and inaccessible sections labeled. (B) An empty sandwell with an accessible section and inaccessible section. (C) Schematic illustrating the pellet arrangement in a rewarded (left) and non-rewarded (right) sandwell. Both the rewarded and non-rewarded sandwells contain a total of 12 pellets and are filled with specially prepared sand, which conceals the pellets in the sandwells'. (D) Series of photographs showing the preparation of a rewarded sandwell, including the correct placement of the pellets in the accessible section (step 1-4). Please click here to view a larger version of this figure.
Figure 4: The experimental setup of the event arena. (A) Schematic showing the experimental setup of the experimental and control rooms. (B) Screenshot showing a live feed of the experimental room viewed through the custom computer software. The custom computer software allows the experimenters to control the startbox doors remotely and provides other measurements. Please click here to view a larger version of this figure.
3. Habituation protocol
NOTE: During habituation, the rats are trained to search for sandwells, dig for a food reward, and explore the event arena.
Figure 5: The design of habituation sessions. From left column to right column: the habituation session (H1-H7); the startbox used for each session (e.g., H1: South startbox (SB)); the location where rats are required to eat their food reward (i.e., North home-base); the position of the accessible pellets in the rewarded sandwell (in both written and illustrated form; p = pellet), which will be placed in each session's designated sandwell location; the position of the pellets in the flat-based sandwell in the single cage (in both written and illustrated form), which aims to promote digging behavior and strengthen the rats' association between digging in a sandwell and receiving a food reward. The last two columns refer to sandwells in the single cages (outside the arena). Abbreviation: N/A= not applicable Please click here to view a larger version of this figure.
4. Main training protocol
NOTE: Each main training session consists of two memory encoding trials (E1, E2) followed, after a short time delay (~30 min), by one recall choice trial (C1). During all trials, rats are required to successively retrieve two pellets from the rewarded sandwell. After locating each pellet, the rats should locate and enter the home-base to eat this food reward. The location of the correct (i.e., rewarded) sandwell is counterbalanced across sessions for all rats (Figure 5).
Figure 6: Representative counterbalancing. (A) Schematic illustrating how the sandwell location map and correct sandwell location encountered by the rats (e.g., Rat 1) changes across the sessions. (B) Example of a counterbalance table for one session (e.g., Session 1). A different startbox is used for each trial within a single session (i.e., encoding trial 1 (E1) started from the South startbox (SB)), but their order of use was the same for each animal (e.g., Rat 1-3). The sandwells used for the correct location (e.g., location 2, 4, 3) and their associated sets, used in full during the recall choice trial, were counterbalanced across each session's trials (e.g., encoding 1, encoding 2, recall choice) and the animals performing the task (e.g., Rat 1-3). (C) Table outlining the sandwell sets counterbalanced within and across sessions. There are 15 sandwells in total and three sets (set 1-3) of sandwells, each containing five wells (A-E). Each rat uses different wells in each encoding and recall choice trial. For example, as mentioned in Figure 6B, Rat 1 will use Sandwell 1A in encoding trial 1, Sandwell 1B in encoding trial 2, and Sandwell 2C in the recall choice trial. Please click here to view a larger version of this figure.
5. Recall probe test
6. Non-encoding control test
NOTE: A non-encoding trial is a control measure used to determine whether the rats are using olfactory artifacts, rather than their memory of the correct sandwell location, to perform the task. As the name suggests, the 'non-encoding control test' means that there are no encoding trials performed prior to the recall choice trial; only the recall choice trial is conducted. The expectation is that without being permitted to encode the location of the everyday memory event, the rats' performance in the choice trial will be at chance level. If this is not the case, and the rats perform well in the non-encoding control test, a re-design of the sandwells, and their accessible and inaccessible compartments may be required.
7. Performance measurement
NOTE: Several parameters are measured and Supplementary Figure 1 shows an example datasheet.
8. Avoidance of unintended bias
NOTE: The following control measures are implemented throughout the protocol to ensure the reproducibility and reliability of this everyday memory task.
This stable home-base protocol has been used to successfully train rats to learn this everyday memory task using allocentric representations. There are two important elements in this protocol. First, animals start from different black boxes (e.g., East, South, and West) within and between sessions (Figure 7A). There are two encoding trials and one recall choice trial per session (or probe trial instead of the choice trial in some cases), all starting from an alternate startbox. This encourag...
Humans automatically encode single events in everyday life. We readily recall some events and forget others. The episodic-like everyday memory protocol described above provides a robust method for researchers wishing to investigate this type of memory (episodic memory) in rodents. Because the task involves the daily act of finding and retrieving food pellets from a defined location, the natural instinct of rodents to forage for food is exploited. The task rests on the reasonable assumption that the act of finding and dig...
The authors have no conflict of interest to disclose.
This work was supported by Medical Research Council Programme Grants, the European Research Council (ERC-2010-AdG-268800-NEUROSCHEMA), Wellcome Trust Advanced Investigator Grant (207481/Z/17/Z).
Name | Company | Catalog Number | Comments |
Camera | CCTVFirst | N/A | |
Event Arena | University of Edinburgh (designed and built in house) | University of Edinburgh (designed and built in house) | Event arena for everyday memory task |
Lister-hooded rats | Charles River UK | 603 | |
Multitimer Labview | University of Edinburgh (designed and built in house) | University of Edinburgh (designed and built in house) | |
Pneumatics, frames, screws of event arena | RS Components Ltd. | University of Edinburgh (P. Spooner) | Tools for building event arena |
Sandwells | Adam Plastics (http://www.adamplastics.co.uk) | University of Edinburgh (P. Spooner) | Sandwells for arena |
Startboxes | Adam Plastics (http://www.adamplastics.co.uk) | University of Edinburgh (P. Spooner) | |
Video recording | Windows 10 computers with OBS software, Blackmagic Decklink Mini Recorder cards | N/A |
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