Maze Basics

Maze Basics: Lashley III

By February 6, 2019 No Comments


In searching for a localized storage point in the brain for learned information, termed an ‘‘engram,’’ Karl Lashley developed numerous rodent mazes in the early 20th century. He believed that if rats could learn to find a maze exit, he could then systematically lesion various brain regions until that particular piece of information disappeared, thus proving that it had been stored in a particular area of the brain.[1] While he was unsuccessful in this pursuit (we now know that memories are widely distributed throughout cortical regions), his various rodent mazes continue to be used for studies of learning and memory in many rodent models.

One such maze, the Lashley III, has been widely used for low-stress assays of spatial learning and memory. The apparatus consists of a box in which the animal starts in, followed by four interconnected alleyways of maze space and a finishing box which may be modified to mimic the animal’s home-cage. Animals are motivated to find the finishing box either by the presence of a food-based reward placed therein or by the use of a simulated home-cage in place of the goal box which uses bedding from their actual home cage. Previously, researchers have found these two stimuli to be equally efficacious in driving the subject’s motivation to solve the maze.[2][3]

Outcome measures in the Lashley III maze

Over multiple trials, the animals progressively learn to navigate the maze with increased efficiency. Outcome measures for quantifying this learning process include the time required to arrive at the goal as well as the number of errors (i.e. wrong turns) made in the alleyways leading there. Finally, total distance traveled may be measured as a means of quantifying the animal’s efficiency in solving the maze. Since the speed of travel across all animals may not be equal, this measure may provide a more accurate representation of maze-solving efficiency than the total time spent in the maze. In naive or untreated animals (i.e. those without learning deficits), all of these measures decrease over repeated trials.

What is the Lashley III maze used for?

The Lashley III maze has been used for a wide variety of testing, from the effects of pharmacological agents to the impact of disease models on learning and memory.

Disease/disorder states

Researchers have used the Lashley III maze to model learning deficits in Alzheimer’s disease and Fragile X mental retardation by using a transgenic mouse model which lacks the Kv4.2 potassium channel (Kv4.2 KO mice). Mice of this strain were found to make more errors while navigating the maze, suggesting a deficit in learning acquisition.[4]

Additionally, the Lashley III maze has been used to identify learning differences between animals of differing social status. Social domination is a common form of inducing chronic stress in laboratory animals, which has been found to be associated with poor memory performance. For instance, mice which have been socially dominated and therefore considered socially subordinate, exhibit deficiencies in the Lashley III maze. Specifically, subordinate mice are slower to learn the maze, showing increased error-rates and higher latencies to finding the goal-box as compared to mice of non-subordinate social status.[5] Thus, the Lashley III maze may be used to assess memory deficits associated with stress in addition to those induced by disease states.

Finally, drugs which either enhance or diminish memory functions have been evaluated using the Lashley III maze. Cognitive-enhancers such as nicotine have been shown to increase performance in the maze, exhibited by accelerated acquisition in young mice given the drug.[6] Conversely, the offspring of mice treated with the psychomotor stimulant amphetamine during pregnancy, which is known to have deleterious effects on cognition, show slower rates of maze acquisition.[7]


Performance in the Lashley III maze is likely to be highly dependent on the function of the hippocampus. By comparing multiple mouse strains, researchers have found that cortical ectopias caused by autoimmune disorders did not impact performance in the maze when they tested the commonly used RF mouse strain (which displays no abnormal cortical development) as compared to the BXSB mouse strain (an autoimmune disease model which exhibits cortical ectopias). However, when testing NZB mice who exhibit similar cortical ectopias in addition to developmental malformation of the hippocampus, they observed significantly worse performance in the maze. Thus, despite Karl Lashley’s initial assertion that cortical function was the primary mediator of learning and memory in this spatial task,[1] it appears that this behavior is more dependent on the hippocampus than the cortex.[8]

The Lashley III maze is normally comprised of plexiglass walls of opaque color in order to eliminate external visual cues and therefore restrict learning to egocentric motivators such as finding food or finding the home cage. However, the maze may also be used with transparent walls for the assessment of maze-learning with allocentric cues. In this latter condition, researchers have found increased reliance on a brain region known as the anteromedial extrastriate complex than in the egocentric condition (i.e. without external visual cues).[10] Thus, the Lashley III maze may be modified for the assessment of function in multiple types of memory with variable neuroanatomical localizations.

How to use the Lashley III maze

Apparatus/environmental habituation

If using a food reward as the motivating factor in the goal-box, researchers should acclimate the animal to the food pellet prior to testing. On the day prior to the first trial, animals should first be given sample food pellets while in their home cage in order to familiarize them with the reward. Next, animals should be placed in each of the four alleyways of the testing apparatus for a duration of 4 minutes with the openings in each wall blocked. Then, the animal should be placed in the goal-box along with additional reward pellets for a duration of 6 minutes. In this way, the animal becomes familiar with the apparatus environment and the presence of a reward without learning to solve the maze, thereby reducing novelty stress on the following day when testing begins.[2]

If using a home-cage simulation box as the end-goal of the maze, researchers will see an improved behavioral performance by habituating the animal to the simulated home-cage box for two weeks prior to the commencement of the experimental protocol.[3] Additionally, researchers should follow the above apparatus habituation protocol on the day prior to testing to minimize the stress associated with environmental novelty.


Levels of motivation differ between murine models, including between species (mice versus rats) and strains within species. In some cases, food deprivation may be used to increase motivation to solve the maze in the case of a food-based reward in the goal box.[2] In this case, ad libitum food sources are removed at the end of the light cycle on the day prior to testing. However, the use of food deprivation for motivational acceleration is not required for positive behavioral performance in the Lashley III maze, which makes it an advantageous apparatus for low-stress learning assessment.[10]


The Lashley III maze is a valuable tool for researchers interested in multiple forms of memory. In its basic form, the Lashley III maze is a low-stress test of learning which can be used to evaluate the rate of learning across multiple animal models and conditions which impact memory acquisition, such as stress, neurodegenerative disorders, and pharmacological interventions. Simple modifications can render the maze as an apparatus for the assessment of visuospatial memory by allowing for the inclusion of extra-maze visual cues.


  1. Lashley, K. S. (1933). INTEGRATIVE FUNCTIONS OF THE CEREBRAL CORTEX. Physiological Reviews, 13(1), 1–42.doi:10.1152/physrev.1933.13.1.1
  2. Matzel, L. D., Han, Y. R., Grossman, H., Karnik, M. S., Patel, D., Scott, N., … Gandhi, C. C. (2003). Individual Differences in the Expression of a “General” Learning Ability in Mice. Journal of Neuroscience, 23(16), 6423–6433.
  3. Blizard, D. A., Weinheimer, V. K., Klein, L. C., Petrill, S. A., Cohen, R., & McClearn, G. E. (2006). “Return to Home Cage” as a Reward for Maze Learning in Young and Old Genetically Heterogeneous Mice. Comparative Medicine, 56(3), 196-201.
  4. Smith, G. D., Gao, N., & Lugo, J. N. (2017). Kv4.2 knockout mice display learning and memory deficits in the Lashley maze. F1000Research, 5, 2456.
  5. Colas-Zelin, D., Light, K. R., Kolata, S., Wass, C., Denman-Brice, A., Rios, C., Szalk, K., … Matzel, L. D. (2012). The imposition of, but not the propensity for, social subordination impairs exploratory behaviors and general cognitive abilities. Behavioural brain research, 232(1), 294-305.
  6. Fisher, A., Hanin, I., Poewe, W., & Windisch, M. (2007). Alzheimer’s and Parkinson’s Diseases. Karger Medical and Scientific Publishers.
  7. Nasello, A. G., & Ramirez, O. A. (1978). Open-field and Lashley III maze behaviour of the offspring of amphetamine-treated rats. Psychopharmacology, 58(2), 171–173.
  8. Schrott, L. M., Denenberg, V. H., Sherman, G. F., Rosen, G. D., & Galaburda, A. M. (1992). Lashley maze learning deficits in NZB mice. Physiology & Behavior, 52(6), 1085–1089
  9. Espinoza-Cifuentes, S., & Leander Zeise, M. (2008). The anteromedial extrastriate complex is critical for the use of allocentric visual cues and in the retention of the Lashley III maze task in rats. Biological Research, 41(4).
  10. Bressler, A., Blizard, D., & Andrews, A. (2010). Low-stress route learning using the Lashley III maze in mice. Journal of visualized experiments : JoVE, (39), 1786.

About Andrew Scheyer

Andrew Scheyer is a post-doctoral fellow at the Institut de Neurobiologie de la Méditerranée. He completed his PhD in Neuroscience in 2015 at Rosalind Franklin University of Medicine and Science, studying the synaptic underpinnings of protracted withdrawal from cocaine self-administration. Currently, his research focuses on the impact of marijuana exposure on neurological development. He specializes in electrophysiological studies of synaptic plasticity and in vivo deep-brain calcium imaging in freely moving rats.