The submerged plus maze as an assay for studying anxiety-like behaviour in fish

Graphical abstract


Value of the Protocol
Fish are increasingly more prevalent in anxiety research. The submerged plus maze is an aquatic adaptation of the rodent elevated plus maze. The submerged plus maze can be used to identify anxiolytic or anxiogenic drugs in fish.

Description of protocol
The elevated plus maze is used to study anxiety-like behaviour in rodents [1,2] by examining aversion to open spaces (i.e., open arms relative to closed arms) [2]. Studying anxiety using fish is becoming more popular and, as such, an equivalent test should be available. To date, there have been two aquatic mazes using a plus maze structure with four arms [3][4][5]. Walsh-Monteiro and colleagues [3] constructed a maze with two arms containing ramps that decreased the height of the water column with increasing distance from the center of the maze. In a second maze, Sackerman et al. [4] alternated the arms with white and dark walls, similar to a light/dark preference test [4,5]. Although both of these mazes use a 'plus' format, neither are analogous to an elevated plus maze in rodents because of its dependence on aversion to open spaces [2]. Most fish, like rodents, display a preference for dark areas vs. light [6], either for cover [7] or to have a dark background to hide against [8], likely for its potential to provide more protection from predators [9]. The submerged plus maze, as described here, uses aversion to open spaces as an indication of anxiety-like behaviour in fish [3]. This apparatus uses a plus maze format, which includes alternating arms constructed of black or transparent plexiglass walls (each arm 12 cm Â 4.5 cm Â 13 cm, see Figs. 1 and 2). This arrangement results in two visually closed arms, two visually open arms, and a center area similar to that of the elevated plus maze. The arms are marked in 1.5 cm increments to quantify travel in each area. A transparent plastic cylinder 4.5 cm Â 4.5 cm Â 13 cm) serves as an acclimation chamber and is placed in the centre area at the start of each trial. A video recording device views the maze from above to record the movements of the fish in the maze for later behavioural scoring. The present study used convict cichlids (Amatitlania nigrofasciata) to validate the test, however, the submerged plus maze is also amenable to testing other fish species. Individual fish of the same species may display strong biases for making turns to either the right or left depending on the individual, and these biases can be sensitive to a fish's shy-vs-bold personality and to the fish's perceived probability of impending positive or negative outcomes [10,11]. This apparatus has the advantage of being symmetrical, an arm of the opposing type to that currently occupied is immediately to the right and left when exiting the current arm. This should minimize cerebral lateralization biases, but researchers may benefit by recording whether turns are biased to either the right or left when exiting each type of arm.

Behaviour testing
1 Set up plus maze apparatus with a video recorder mounted above the maze so that all areas can be seen clearly. Ensure all areas of the maze are well-lit while reducing glare. a Experimenter should be occluded from view. 2 Fill maze with aerated stock tank water or clean water from the fish habitat to a depth of 10 cm and place clear acclimation chamber in the centre area of the maze. Ensure the water matches the physical-chemical properties (temperature, pH, etc.) of habitat water to reduce the risk of climate shock. Specifically, for convict cichlids, we recommend that the water is as follows: pH 7-8, temperature 25 AE 1 C. 3 Remove fish from home tank using a dip net and deposit in acclimation chamber of the plus maze.
Record handling time. 4 Leave fish in the acclimation chamber for two minutes.
a Start the video recording before the two minutes are fully elapsed (i.e., at 1.5 min). 5 Remove the acclimation chamber, allowing the fish to move freely within the maze. Record behaviour for 5 min. a Do not make any loud noises that may disturb the fish. Fish were placed in an acclimation chamber in the centre area for two minutes before they were released to explore the maze for five minutes.
b Allow the recording to run slightly past 5 min in case water movement from the removal of the chamber prevents immediate detection of the fish. 6 Return the fish to its home tank. 7 If possible, water should be changed between fish. Otherwise, the temperature, pH, etc. of the water in the arena should be maintained and monitored throughout testing and aerated in between trials. If the temperature drops too low (D~3 C) or water chemistry changes too drastically, then the arena should be filled with new water or adjusted. a The arena should also be rotated after every trial to eliminate any spatial biases or any other uncontrolled auditory or visual stimuli.

Validation
This apparatus was validated using the anxiety-reducing (anxiolytic) benzodiazepine diazepam administered by immersion (Fig. 3) [12][13][14][15]. Immersion is the method of choice because injection of substances requires anesthesia with MS-222 or cold water, which can impair behavioural responses. Fig. 2. Submerged plus maze apparatus photograph. This photo was taken during the acclimation period, when the fish is placed in a transparent cylinder for two minutes before being released to explore the maze.
Immersion can quickly and reliably be used to validate anxiety-like behavioural tests with other anxiolytic substances (e.g., ethanol) [16], as well as anxiogenic substances (e.g., GABA A receptor antagonist, gabazine) [17,18]. Note that time of immersion in a drug solution can vary from 3 min (e.g., Diazepam) [12,15] up to thirty minutes [16]. If a longer duration of immersion is used, the dosing beaker should be kept under a heating mat to maintain water temperature [16]. To reduce the amount of handling the fish experienced, we used a slotted plastic cup to transport fish between each step. This likely minimizes netting stress and damage to the slime coat of the fish. Fish were housed individually in aquaria partitioned by transparent dividers to control for aggression and social isolation effects and to ensure handling stress was restricted to the fish of interest.
Then, dilute this solution with aerated tank water to a final volume of 500 mL while maintaining the desired concentration (2.5 mg/L was used in the current study, see Table 1) in the drug administration beaker. Agitate slightly to resuspend the solution. a Note: 2.5 mg/L was the concentration used in the current study, determined after an initial pilot test to obtain a dose-response curve. Different sizes and species of fish may respond optimally to different drug concentrations and therefore any new studies should obtain an appropriate doseresponse curve. 2 Prepare an additional holding container with aerated tank water. 3 Remove the fish from its home tank with a dip net and deposit fish into the slotted plastic cup. 4 Expose the fish to the drug by placing the cup holding the fish in the drug administration beaker for 3 min.
a Note: the drug solution remains in this beaker and is not transferred to any subsequent steps. 5 Lift the cup and fish and move them into the holding container for 5 min to allow the drug to take effect. 6 Lift the cup and fish and transfer fish into the acclimation chamber of the submerged plus maze by pouring it out of the cup and begin the test.
This validation method was used in a within-subjects design, so fish were exposed to the submerged plus maze with and without prior diazepam administration. In vehicle trials, fish underwent the same steps but with only DMSO and tank water in the drug administration beaker. Drug and vehicle trial order were randomized to ensure behavioural differences between trials was not due to testing order, and we imposed a 48-h inter-trial interval to ensure drug elimination between trials. Note that we used a within-subjects design because of low numbers of fish and high individual variability, however, a between-subjects design can also be used to validate this test with other fish species.  Fig. 3. Anxiolytic validation procedure. A fish is first placed into a tank containing the drug treatment (diazepam + vehicle or vehicle only) for 3 min. Next, the fish was moved to a delay tank (tank water) for 5 min to allow the drug to take effect. Finally, the fish was moved to the submerged plus maze test, where it acclimated for 2 min before the 5 min testing period.

Additional information
The dimensions of this apparatus were designed to accommodate fish ranging between 1.0-4.5 cm in standard length. An alternate apparatus for fish ranging between 3.0-7.0 cm in standard length was constructed where each arm measured 24 cm Â 9 cm Â 16.5 cm and lines were marked in 3 cm increments. The maze dimensions should be adjusted accordingly to match target fish size.
A basin should be used to surround the maze and water should flow between the basin and the maze to alleviate pressure against the walls of the plus maze. The present maze employed small gaps at the far end of each arm to exchange water with the surrounding basin. A previous iteration of the maze used gaps too large for some of the smallest fish in our experiments and gauze had to be used to prevent the fish from wedging themselves in the gaps. Smaller gaps were used in a subsequent version, eliminating the issue.