The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Summary

The goal of this protocol is to investigate spatial cognition in rodents. The double-H water maze is a novel test, which is particularly useful to elucidate the different components of learning, consolidation and memory, as well as the interplay of memory systems.

Abstract

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their behavioral flexibility and required memory duration, the number of goals and pathways, and also the overall task complexity. A confounding feature in many of these tasks is the lack of control over the strategy employed by the rodents to reach the goal, e.g., allocentric (declarative-like) or egocentric (procedural) based strategies. The double-H maze is a novel water-escape memory task that addresses this issue, by allowing the experimenter to direct the type of strategy learned during the training period. The double-H maze is a transparent device, which consists of a central alleyway with three arms protruding on both sides, along with an escape platform submerged at the extremity of one of these arms.

Rats can be trained using an allocentric strategy by alternating the start position in the maze in an unpredictable manner (see protocol 1; §4.7), thus requiring them to learn the location of the platform based on the available allothetic cues. Alternatively, an egocentric learning strategy (protocol 2; §4.8) can be employed by releasing the rats from the same position during each trial, until they learn the procedural pattern required to reach the goal. This task has been proven to allow for the formation of stable memory traces.

Memory can be probed following the training period in a misleading probe trial, in which the starting position for the rats alternates. Following an egocentric learning paradigm, rats typically resort to an allocentric-based strategy, but only when their initial view on the extra-maze cues differs markedly from their original position. This task is ideally suited to explore the effects of drugs/perturbations on allocentric/egocentric memory performance, as well as the interactions between these two memory systems.

Introduction

In animals, learning is principally mediated by the hippocampal- and striatal-based memory systems^1,2, which play central roles regarding place- and procedural-memory, respectively. The relationship between these two systems is complex, and they are known to interact with each other in cooperative or competitive manners^1,3. In addition, studies have shown that the influence of either of these memory systems on animal behavior can increase following the absence or damage of the other system^4-7. Both of these systems are connected to the prefrontal cortex via the thalamus.

Numerous neurological disorders and neurodegenerative diseases can affect spatial cognition in humans, which rely on the interplay between procedural and declarative memory systems. Examples include Parkinson’s disease (PD), Huntington’s disease (HD)^8-10, Alzheimer’s disease (AD)^11-14, as well as amyotrophic lateral sclerosis (ALS)¹⁵. Animal models, which are relevant to these disorders can be induced through various drug treatments which block certain receptors¹⁶, as well as through targeted lesions. When such animals are used with spatial memory tasks, a valuable insight can be gained into the underlying mechanisms related to these disorders, as well as to various treatment options.

There are many different types of spatial memory tasks in rodents, which collectively are designed to assess specific aspects of learning and memory, as well as the effects of potential treatments for various disorders^17,18. These tasks can be distinguished by the number of goals and pathways, the degree of behavioral flexibility in solving the task, the memory duration or delay, as well as the choice of strategy used in solving the task. A good performance may be acquired based on external cues or landmarks which are used to orientate the animal towards the goal (an allocentric or place strategy). Alternatively, a rodent may develop a strategy which is based on bodily direction and cues with regards to the direction to move in (an egocentric or procedural strategy), e.g., if a rat knows that the goal is one left turn followed by one right turn, then there is little need for an allocentric or place strategy. Maze tasks often differ based on the degree of flexibility offered to the rodent in solving them. For instance, in the Morris Water Maze, a dry version of the latter (e.g., ¹⁹) or the Barnes maze (e.g., ²⁰), there are potentially infinite routes the rat can take to reach the goal. In the Morris Water Maze, for example, the location of the goal may be learned based on external landmarks or cues (allocentric strategy), or by simply swimming in circles towards the center until the platform is found (egocentric strategy)²¹. Certain tasks have multiple goals and a high degree of flexibility, such as the cone-field task²² or Olton’s radial maze²³. At the other end of the scale are tasks, which offer limited flexibility in reaching the goal, e.g., the Stone maze, or the alternating version of the T-maze. These tasks provide only one correct way of reaching the goal and facilitate the emergence of cognitive routines that are principally governed by the striatal-based procedural memory system.

The double-H maze is a novel spatial memory testing device, which was designed to allow the experimenter to direct the type of strategy that is learned by rodents in solving the task²⁴. Consisting of three parallel run arms intersected by a perpendicular central alleyway, the double-H maze is a water-escape task in which rodents learn to reach an escape platform that is immersed in one of the maze locations. During training, a procedural strategy can be developed by maintaining the same start and goal locations throughout. Alternatively, an allocentric strategy may be developed by alternating the starting location in a random order, thus requiring the rat to learn the location of the hidden platform based on environmental cues as it has to do in a water maze. This overcomes an obstacle present in many different maze tasks, in which the experimenter otherwise has little control over the type of strategy that rodents utilize. This is important when considering that the effects of certain cognition-enhancing drug candidates rely on the hippocampal-based place-memory system, thus the emergence of cognitive routines or procedures may confound the interpretation of the behavioral observations when animals, for example switch from allocentric to procedural memory during the course of training. Similarly, it may be desirable to assess the effects of drugs and treatments on procedural memory, without the influence of allocentric place-based memory. Finally, this device can be utilized to study the cooperative or competitive interactions between these memory systems, and the conditions under which rodents may switch from one system to another.

Protocol

1. General Considerations

This protocol is approved by the Animal Care and Use Committee of University Hospital Freiburg (same for Strasbourg). Visual acuity is necessary for performance in tests of spatial learning. Rodents with impaired visual systems are thus not suitable. Also, lighting must be sufficient in order for the rats to see the different cues located on the surrounding walls. It is useful to utilize basic-shaped (square, circle, triangle) but well-contrasted cues (e.g., black-painted cues on a white-painted background). Likewise, severe motoric deficits are exclusion criteria because swimming is required for this test and drowning may occur. Finally, hyper-anxious rodents can display a strongly biased search behavior, which impacts on performance.

2. Apparatus Set Up

1. Construct a double-H maze consisting of a 160 cm central alleyway, which is intersected at both ends and in the center by three 160 cm parallel run arms (see Figure 1). Ensure that the central alleyway and its perpendicular arms are 20 cm wide, and are surrounded by 35 cm high transparent Plexiglas walls. Thickness of the Plexiglas is 6 mm for all parts.
2. Secure the walls in place using glue and screws, and waterproof the maze using silicone joints at all internal angles. These joints can be replaced easily should they lose their waterproofness. Place a drain outlet at the extremity of one of the corner arms (or in the middle of the maze) for emptying.
3. Place the maze on an 80 cm high table, with adequate space around it for a) walking around the maze, and b) placement of well-contrasted cues. Designate each arm in the maze by its position, i.e., northwest (NW), north (N), northeast (NE), southwest (SW), south (S), and southeast (SE).
4. Place a ceiling-mounted camera above the maze for post-test analysis of animal behavior, using either manual or automatic (video-tracking) methods (see §5).

3. General Comments

1. Prior to use, fill the maze with water to an approximate height of 18 cm (200 L). This is high enough to prevent rats from touching the bottom of the maze with their feet, but shallow enough to prevent escape.
2. After pre-training, render the water opaque by mixing 250 g skim milk powder. Change the water on a daily basis to prevent the milk-water from becoming rancid. Maintain the water temperature between 21 - 23 °C to provide incentive for the rats to seek the escape platform.
   NOTE: As rats become familiar with the position of the platform during pre-training, its position is moved to a different arm during training.
3. Prior to use, immerse a 17 cm high, 10 cm diameter platform at the extremity of one of the corner arms (NE, NW, SE or SW). Ensure the height to be submerged is 1 cm below the surface of the water surface. Train the rats to reach the target platform using either an allocentric or egocentric learning strategy, which is dependent on the type of paradigm used (see §4).

4. Basic Training Protocols

NOTE: Rats are typically provided with an initial day of pre-training, which allows them to become familiar with the maze.

1. For pre-training release the rat from the extremity of one of the center arms (e.g., S arm) and place the goal platform at the extremity of one of the corner arms (e.g., NE), then give the rats 4 consecutive 60 sec trials in which to reach the target platform.
2. Upon reaching the escape platform, allow the rat to wait there for 15 sec, so that they can rest and observe their surroundings. Regardless of the start position, always block the opposite arm with a transparent guillotine door, which prevents entry.
   NOTE: During pre-training, leave the water transparent, and adjust the platform height such that it protrudes 1 cm above the water’s surface, thus making it visible to the rat.
3. Perform daily training session consisting of up to 4 consecutive trials, separated by a 10 sec gap at least (discrete training, i.e., with intervals of several minutes between trials, is an alternative).
4. For training, relocate the platform from its pre-training position to the chosen arm (e.g., NW), and submerge it at its extremity 1 cm below the water surface. Now render the water opaque by addition of milk power and perform the following training (section 4).
5. For rats that do not reach the target platform within 60 sec, return them to the starting position, and gently guide them to the platform by the experimenter.
6. Measure several variables during the training and probe sessions, such as, distance swam, latency to goal arm / platform, time spent in each arm, as well as the number of initial/repetitive errors (see Figure 2). Keep in mind that latencies may be influenced by motor difficulties. Should the case arise, distance and errors appear as more reliable variables regarding cognitive performance.
7. Allocentric Strategy Training:
   1. Day 1 - Pre-Training:
      1. Do not add milk powder for this step. Place escape platform protruding 1 cm above the water’s surface in a fixed location. Train rats in 4 consecutive trials to reach the platform.
   2. Days 2 - 5 – Training:
      1. Add 250 g of skim milk powder to the water to render it opaque. Move the platform to a different arm (e.g., from NE to NW) and add water so that the platform is 1 cm below the water surface. Release rats from either the N or S arm in unpredictable sequences for each session, such that both arms are used twice as trial start in a single session (4 trials/day, e.g., SNNS, NSNS; see Figure 3).
   3. Day 6 – Probe Session:
      1. Remove platform for the probe trial. Release rats from a different arm to those used during training (e.g., SW), and allow them to swim for 60 sec. See Figure 4 for representative swim tracks. Analysis of the time spent in the target arm (former location of the platform) gives an indication about whether rats use a spatial strategy, another type of strategy, a sequential combination of different strategies (see below), or a disorganized search pattern.
8. Egocentric Strategy Training:
   1. Day 1 – Pre-Training:
      1. This is the same as first step in the allocentric strategy training (step 4.1). Do not add milk powder for this step. Place escape platform protruding 1 cm above the water’s surface in a fixed location. Train rats in 4 consecutive trials to reach the platform.
   2. Days 2 - 5 – Training:
      1. Add 250 g of skim milk powder to the water to render it opaque. Move the platform to a different arm and add water so that the platform is 1 cm below the water surface. Release rats from the same start arm (S or N; see Figure 5) for every trial (4 trials/day).
   3. Day 6 – Probe Session:
      1. Remove platform for the probe trial. Rats are released from an arm different from where they were released during the training. Allow the rats to swim for 60 sec. Block the opposite arm with a transparent guillotine door.
         NOTE: Modify the above training strategies for according to the requirements of the particular experiment, e.g., Testing Drug Effects etc.
9. Dry the rat off with absorbent towels after each session in the water.

5. Analysis

1. Perform measurements of latencies, initial and repetitive errors, first choices and response type manually by recording these variables from the videos taken from the overhead camera.
2. Alternatively if available, utilize commercially available video-tracking software and configured to record these variables automatically.
3. Statistical Analysis:
   NOTE: The specific implementation of statistical analysis depends on the study, which is taking place.
   1. Perform a one, two or three-way ANOVAs regarding the initial/repetitive errors, latency to goal arm/platform, and time spent in the target arm; with factors that include the test day, and treatment group(s).
   2. Where necessary, follow these ANOVAs using post-hoc Newman-Keuls multiple comparisons tests. To compare performance, use a reference value (e.g., the time spent in the target arm during a probe trial vs. the chance level), and perform t-test.
   3. If required, use non parametric statistics in addition (e.g., χ², see below) or instead when conditions for parametric ones are not fulfilled.

Results

Egocentric Learning Strategy

A study was carried out to determine whether the chosen memory strategy in rats changes based on alterations of their perspective of external environmental cues, following an egocentric-learning paradigm²⁵. Rats were trained over 4 days (4 trials/day) to reach a goal arm located at NE, and were subsequently tested on the fifth day using a misleading probe trial, in which the start arm was either moved 60 cm to the left (i.e., NE start for animals r...

Discussion

Comments on Study Design and Analysis

Since its conception, the double-H maze has been utilized in a number of behavioral experiments in rats, which collectively were designed to study egocentric and/or allocentric responses in rats under normal^24,25 and altered^26-29 brain states. The latter studies include striatal deep-brain stimulation (DBS)²⁶, animal models of neurological disorders^27,28, as well as bilateral deactivations of various cortico-hippoca...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rats or Mice	Charles River
Towels for drying	University Hospital		1 / animal
Water			~200 L / day
Skim milk powder	Grocery store		250 g / 200 L water
Garden Hose	Hardware store
Drying rack for towels	Hardware store
Kinect camera	Kinect
PC computer	any
[header]
Double H Maze, (plexiglass) (Custom-Built)
External lateral walls, 1600 × 350 × 6 mm			2
Internal lateral walls, 706 × 350 × 6 mm			8
Central corridor panels, 500 × 350 × 6 mm			4
Arm extremities, 188 × 350 × 6 mm			6
Guillotine doors, 187 × 350 × 6 mm			3
Extremity covers, 200 × 250 mm			6
Crossbars, 200 × 40 mm			6
[header]
Double-H Maze Platform (to be ballasted) (Custom-Built)
Metal platform, 100 mm diameter × 150 mm			2
Platform cover, 100 mm diameter × 6 mm			2