The Y-maze is an unconditioned behavioural test used to assess exploratory behaviour and spatial working memory function in both rodents and fish.

Paradigms used in the Y-maze include: the spontaneous alternation test and the recognition memory test. These tests take advantage of an animal’s natural exploration and, as they do not involve conditioned learning, provide a test that is quick with minimal training time.

Dr Matt Parker, University of Portsmouth, explains the benefits of the using the Y-maze with zebrafish, which include that the fish don't need to be individually housed beforehand and the task provides detailed information on cognitive processes quickly.


Experimental set up

Depending on your choice of animal model, both the Zantiks MWP unit or the Zantiks AD unit can be used for Y-maze testing. The Zantiks MWP can be used to test Drosophila or zebrafish larvae, while the Zantiks AD unit can test adult fish.

In the Zantiks MWP unit, a custom-made Y-maze well plate is used to create separate arenas to test up to 6 individual animals simultaneously (see image ii below).

In the Zantiks AD unit, the double Y-maze insert is used to create the testing environment for two separate adult fish (see image iv below). The test is typically conducted in 3 litres of water and without lights, to reduce stress to the fish.

Target zones are assigned to each of the three arms of the Y-maze. Time spent in each arm; the visits to each arm, and distance travelled in each arm can be calculated automatically by the Zantiks unit (see image iii).

Y Maze 2 1 Plates Mwp
i) Y-maze plates for the MWP, with one and two Y-mazes per plate
ii) Image of six Y-maze plate in the MWP, photo captured in the MWP to make an arena asset
Kastureymazes Zones
iii) Arena asset for a six Y-maze plate in the MWP, showing the different zones
Y Maze 2 Zebrafish Ad
iv) Example of a two Y-maze insert for the AD, used with adult zebrafish
Y Maze Lt Copy Annotated
v) Example of Y-maze insert for the LT unit that could be used with mice, or bigger fish, showing each arm's dimensions - 152 x 50 x 155 (L,W,H in mm).

Tracking Drosophila in a y-maze in the Zantiks MWP. The real-time video also displays the 6 different arenas, and then the four different zones in each arena. Video courtesy of Ameya Kasture, Thomas Hummel's lab, University of Vienna


Continuous spontaneous alternation task

In this paradigm, the subject is allowed to freely explore the three arms for a set time period. The arms of the maze typically do not contain any reward or internal and external cues. Subjects are placed into the maze, allowed to explore, and the sequence of arm choices is recorded.

Animals show a tendency to explore a new arm of the maze rather than returning to the arm that was most recently visited. When the animal chooses a different arm than the one it just exited, this choice is called an alternation.

The number of arm entries and the number of triads (sequence of three consecutive arm entries) are recorded in order to calculate the percentage of alternation. A triad containing all three arms is scored as spontaneous alternation.

Alternation percentage is a measure of working memory. A subject with impaired spatial working memory will show decreased spontaneous alternation as indicated by a low percentage of alternation.

Number of triads = (Total Entries - 2)

Alternation [%] = [(# of Spontaneous Alternations / # of Triads) *100]

Perseveration can be calculated using series of tetragrams (sequence of four consecutive arm entries). Sequences RRRR and LLLL represent pure repetitions and sequences RLRL, LRLR represent pure alternations. High repetitions of alternations or of repetition sequences may indicate perseveration behaviour.

One way to analyse the y-maze data can be found on github. Here you will find instructions and an R script which is designed to batch analyse raw data files created by the Zantiks unit into tetragrams.


Recognition memory task

Another use of the Y-maze test is the recognition memory test. This task consists of two trials. During the first trial, one arm of the Y-maze is blocked off and the animal is allowed to freely explore the remaining two arms. During the second trial, the animal is returned to the maze and all three arms are accessible. Memory can be analysed by varying the inter-trial interval (ITI) between the two trials.

The time spent and distance travelled within the novel and the familiar arms during trial two are used as memory indices.

Recognition Memory Index [%] = [(time spent in the novel arm / total time spent in all arms during the first minute of the second trial) *100].

Discrimination Memory Index [%] = percent of subjects which entered the novel arm first during the second trial.

Locomotor activity index = # of visits in each arm and total distance in the novel arm in the second trial.


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