The Hexagonal Tunnel Maze is used to study the exploratory behaviors of rodents. It is a complex automated maze with opaque walls, ceilings, and floors and is composed of a series of darkened tunnels resembling a natural environment for rodents to explore. Since no interior light and extra-visual cues are available, the Hexagonal Tunnel Maze takes advantage of rodents’ natural inclination to explore dark enclosed spaces.

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Hexagonal Tunnel Maze

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Introduction

The Hexagonal Tunnel Maze is used to study the exploratory behaviors of rodents. It is a complex automated maze with opaque walls, ceilings, and floors and is composed of a series of darkened tunnels resembling a natural environment for rodents to explore. Since no interior light and extra-visual cues are available, the Hexagonal Tunnel Maze takes advantage of rodents’ natural inclination to explore dark enclosed spaces.

The subject is placed in the central open field of the maze and is allowed to explore the maze for a specified time. Since no interior light is present, the rodent’s position within the maze is recorded through a computer interfaced with photocells uniformly distributed throughout the maze. Unlike several other mazes such as the T-Maze and Y-Maze, the Hexagonal Tunnel Maze does not utilize any food reward to motivate the subjects to explore; instead, the opportunity to explore presumably serves as motivation. The Hexagonal Tunnel Maze can also be easily modified according to different experimental needs. At different points of the maze, the insertion of barriers can modify the maze into a six-arm Radial Maze with each arm consisting of a blind alley and a long arm. Thus, by utilizing the modified radial maze version of the Hexagonal Tunnel Maze, learning, short-term memory, and long-term memory can also be assessed by observing repetitive arm entries during each trial. Furthermore, reversal learning can also be examined using a mirrored version of the six-arm radial maze.  

The Hexagonal Tunnel Maze can also be used to observe the effect of different diseases and disorders, and drugs on rodents’ exploratory behaviors. Other mazes used to study rodent’s exploratory behaviors include the Elevated Plus Maze, the Zero Maze, the Morris Water Maze, and the Hole board.

Apparatus and Equipment

The Hexagonal Tunnel Maze is an automated maze measuring 150 cm in diameter with opaque walls, ceiling, and floor. The tunnels of the maze measure 8 cm in width and 15 cm in height. The configuration of the Hexagonal Tunnel Maze can easily be altered by inserting barriers in different locations. Forty-two photocells are present throughout the maze mounted 4 cm above the maze’s floor, spaced 19-30 cm apart, which help monitor and record the subject’s location in the maze. 

The Hexagonal Tunnel Maze can easily be modified into a modified version of a six-arm Radial Maze with each arm consisting of a blind alley and a long arm. Since the barriers are removable, a mirror image of the six-arm Radial Maze can also be made.

Training Protocol

Clean the maze thoroughly after each trial. Wash the floor of the maze and allow it to dry before starting another trial.

Habituation 

Handle the subjects for 5 days before testing. Allow the subject to adapt to the dark experiment room 10 minutes before starting testing. 

Hexagonal Tunnel Maze Task 

Place the subject in the Hexagonal Tunnel Maze’s central open field through the ceiling’s trap door. Allow the subject to explore the maze for 6 minutes. Lift the maze and remove the subject from the maze and place it back into its home cage. Conduct test sessions once a day for three consecutive days. 

The following is a sample protocol for the modified six-arm radial version of the Hexagonal Tunnel Maze.

Modified Six-Arm Radial Maze Task 

Place the subject in the central open field of the maze. Allow the subject to explore the maze and enter any of the six arms for 6 minutes.

Literature Review

Investigation of the effect of the Neurokinin Substance P (SP) on Hexagonal Tunnel Maze performance in rats 

Nagel, Welzl, Bättig, and Huston (1993) investigated the effect of SP’s systemic injection on the activity and exploratory activity of forty male Wistar rats using the Hexagonal Tunnel Maze. The subjects were randomly assigned to five groups and received intraperitoneal injections of 0, 10, 100, 500, or 1000 µg of SP. During testing, the subjects were required to explore the maze for 6 minutes, and their locomotor activity, perimeter walking, and exploratory efficiency were measured. Trials were performed once a day for three consecutive days; however, the SP injection was only given on day 1. Results indicated no significant differences in the maze’s total activity levels between the control group and all SP-injected rats on all three testing days.  All groups, except the 1000 µg group, exhibited an increase in activity levels from day 1 to 2. Observation of perimeter walking revealed that the subjects spent 65-70% of their total activity on the perimeter on day 1. Perimeter walking decreased with each testing day where the subjects only spent 45% of their activity on the maze’s perimeter on day two and spent most of their time in the center of the maze on day 3. No differences in perimeter walking were observed between all groups on days 1 and 3. However, a significant difference in perimeter walking was observed on day 2 between the 10µg group and all other groups, where the 10µg group spent more time along the perimeter. The results from exploratory efficiency indicated no significant differences between all groups on all three days. 

A second experiment was also performed on the same subjects using the modified six-arm radial version of the Hexagonal Tunnel Maze. During trials, the subject’s locomotor activity, number of maze arms entered, and exploratory efficiency was assessed for 6 minutes. In addition, the subject’s short-term memory and long-term memory were also examined. Trials were performed for five days without any injection, and on the sixth day, the subjects were tested on a mirror version of the six-arm radial maze and were injected with SP or the vehicle (0,10, 100, 500, 1000 µg/kg SP). The mirror maze configuration trials were performed as a form of reversal learning for the subjects. The subjects underwent further testing on the mirror image of the maze for two more days without injection. The results indicated that no differences in maze performance were observed between all groups on the first five days of testing. However, when the maze was changed to the mirror configuration, all groups except the 1000µg group increased their activity levels. Moreover, all groups except the 500µg group showed an increase in the amount of activity required to visit all six maze arms on day 6. On day 7, a decrease in exploratory efficiency was observed in all SP-injected groups. The 100µg and 500µg groups explored more efficiently than controls; however, the groups receiving lower SP doses did not differ from controls in exploratory performance. Observation of short-term memory indicated that when the maze was changed to the mirror configuration, the 0, 10, and 100µg SP groups increased their number of repetitive arm entries than the 500µg and 1000µg groups. No group differences were observed with respect to blind alley choices on day 6. However, on day 7, only the 100µg group chose the blind alley less often than controls compared to all the other SP-injected groups. Overall results indicated that SP produced facilitation of measures of exploratory efficiency, short-term memory, and long-term memory without affecting activity. 

A third experiment was performed on 66 male Wistar rats in which the subjects were injected with either 500µg SP or 50µg SP pretrial or post-trial on the six-arm radial maze and the mirrored configuration. The results indicated that pretrial injections of 500µg SP facilitated a stronger performance concerning short-term memory, long-term memory, and exploratory efficiency compared to the 50µg SP. In contrast, no significant effect on performance was observed with post-trial injections. 

Investigation of the behavioral performance of rats on the Hexagonal Tunnel Maze 

Martin, Ottenga, and Bättig (1986) investigated behavioral parameters such as habituation, and locomotor activity of portacaval shunt (PCS) operated and sham-operated male Sprague Dawley rats using the Hexagonal Tunnel Maze and its modified six-arm radial maze version. Fifty-four rats were used in the study and were required to explore each version of the maze for 6 minutes during trials. Testing was first conducted on the Hexagonal Tunnel Maze for six consecutive days, followed by six days of testing on the radial maze. Results from the Hexagonal Tunnel Maze indicated a significant interaction of surgical conditions with repeated testing. The PCS group showed increased activity levels on day 1 of testing, and the sham-operated group showed greater activity levels on the later days of testing. Further results revealed that intrasession habituation was more rapid from the PCS group than the sham-operated group, which rapidly increased each day as trials progressed. In the radial configuration of the Hexagonal Tunnel Maze, the number of blind alley entries was evaluated. Results indicated a decrease in blind alley entries over the six-day testing period. However, no significant effect of surgical condition and repeated testing on blind alley entries was observed. Overall results indicated that depending on the maze configuration, the PCS rats showed a decline in activity and spontaneous locomotor activity.

Data Analysis

The following parameters can be observed using the Hexagonal Tunnel Maze:

  • Locomotor activity of the subject measured by the total number of photocell interruptions 
  • Perimeter walking of the subject
  • Exploratory efficiency of the subject measured by the total number of photocells activated 
  • Time spent in the central area of the maze 
  • Total number of entries into the central area of the maze
  • Total number of photocells activated during each trial 

The following parameters can be observed using the modified six-arm radial version of the Hexagonal Tunnel Maze:

  • Number of blind alley entries 
  • Number of long arm entries 
  • Total number of arms entered 
  • Number of repetitive arm entries 

Strengths and Limitations

Strengths 

The Hexagonal Tunnel Maze contains multiple darkened tunnels to study rodents’ exploratory behaviors in a laboratory setting that resembles their natural environment. Trials are easy to perform on the Hexagonal Tunnel Maze and takes a short amount of time. The maze does not utilize any internal maze cues or food rewards for motivation and solely relies on rodents’ natural behavior to explore during trials. The Hexagonal Tunnel Maze can easily be modified by inserting barriers at different maze locations to make a modified six-arm radial maze to assess learning and memory, in addition to exploratory behaviors. Moreover, using a mirrored version of the radial maze, reversal learning can also be assessed. 

Limitations 

Exploratory behaviors of the subjects are highly essential for task performance. The age, gender, weight, or strain of the subject can affect their exploratory performance. 

Summary

  • The Hexagonal Tunnel Maze is used to study the exploratory behaviors of rodents.
  • It is a complex automated maze with opaque walls, ceilings, and floors and consists of several darkened tunnels. 
  • The Hexagonal Tunnel Maze can be modified into a six-arm radial maze by inserting barriers at different maze locations. 
  • The Hexagonal Tunnel Maze can be used to test the effect of diseases and drugs on the exploratory behaviors of rodents. 

References

  1. Martin, J. R., Oettinger, R., & Bättig, K. (1986). Behavioral effects of experimental portacaval anastomosis measured in Dashiell and radial tunnel maze configurationsPhysiology & behavior38(1), 21–24. https://doi.org/10.1016/0031-9384(86)90127-7
  2. Nagel, J. A., Welzl, H., Bättig, K., & Huston, J. P. (1993). Facilitation of tunnel maze performance by systemic injection of the neurokinin substance PPeptides14(1), 85–95. https://doi.org/10.1016/0196-9781(93)90014-8

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