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Environmental Factor, October 2012

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Integrating learning and memory tests into developmental neurotox studies

By Sheetal Thakur

Charles Vorhees, Ph.D.

Vorhees stressed that researchers should be clear about their objectives when they design their studies. They need to understand what aspect of learning and memory is being studied, and whether the study is for screening or in-depth analysis, as well as the pros and cons of the tests being considered. (Photo courtesy of Charles Vorhees)

Cincinnati Water Maze (CWM) shown above

Shown from above, the CWM is partially filled with water. The length of time it takes a rodent to negotiate the labyrinth, and the number of its errors, can reflect the extent to which learning and memory are affected by the drug or chemical administered. (Photo courtesy of Charles Vorhees)

Drawing of Cincinnati Water Maze (CWM)

To eliminate distal cues, the CWM is conducted in complete darkness, and monitored with an infrared light emitter and camera. According to the Animal Behavioral Core website, the test provides a method for evaluating an animal’s entorhinal cortex, where grid cells integrate information from the hippocampus and the pre- and post-subiculum, where head direction cells are located. (Photo courtesy of Charles Vorhees)

An enthusiastic audience of toxicologists and neuroscientists was on hand Sept. 6 to hear a presentation by animal behavioral testing expert Charles Vorhees, Ph.D.

Vorhees’ seminar, “Assessment of Learning and Memory in Developmental Neurotoxicology Studies,” was hosted by his longtime research associate, NTP neuroscientist Jean Harry, Ph.D., who had invited him to share insights from his extensive experience in using rodent models for evaluating effects of neonatal exposures to toxicants that later in life lead to behavioral and cognitive impairment.

The seminar was organized as a part of NTP’s efforts to collaborate with basic researchers, in the course of developing and validating improved testing strategies and strengthening scientific knowledge on potential developmental toxicants.

Vorhees is a professor of pediatric neurology and director of the Animal Behavioral Core in the Cincinnati Children’s Hospital Medical Center at the University of Cincinnati (UC). He is a leading authority in the field of behavioral toxicology and has published numerous articles on chemical-induced and drug-induced alteration of learning and memory in laboratory animals.

Advantages of mazes for testing learning and memory

Vorhees began his talk by highlighting the distinction between learning and memory. He added that both learning and memory can be classified into different types and pointed to subtle differences between different types of memory. He emphasized that it is crucial for researchers to know what kind of memory they are testing and to select a testing paradigm that addresses the specific scientific question. Different types of memory are stored in different parts of the brain and memory circuits involved can change dramatically between types.

Learning and memory tests can be broadly classified into the operant-type and mazes, and according to the type of reinforcement used. In the context of regulatory toxicology studies, Vorhees recommended maze testing, with escape from water serving as the motivator, but offered caveats about the inherent limitations of any single model of testing.

“Swimming mazes are basically immune to appetite differences,” said Vorhees. This could be a major advantage for testing toxicants that affect appetite and growth. From the screening perspective, these mazes require little or no training, are unaffected by body mass differences, and the motivation is intrinsic. In addition, they have adequate learning curves, no dropouts, and moderate inter-individual differences, and can be integrated and expanded to more complicated types of assessment. He cautioned on the use of certain types of tests, such as passive avoidance tests and simple T, Y, and M mazes, which have certain confounding factors associated with them, including aversive motivation and strain differences in response.

Things to consider while choosing the right test

Vorhees stressed that learning and memory involve a complex set of functions, and there is no single test that can address all aspects of learning and memory. He emphasized the importance of a standardized and validated testing approach. “It worries me that in contract laboratory settings the emphasis is on efficiency rather than validity,” he added.

Vorhees encouraged researchers to use a robust negative control, and choose tests that have low false positives and negatives. He explained that before starting a testing paradigm, it is important to understand the pros and cons of each test, and to consult external experts when necessary.

According to Vorhees, some of the commonly used tests in laboratory settings include the Morris Water Maze (MWM), an open-pool analysis of spatial navigation and reference memory involved in finding a platform, and the Cincinnati Water Maze (CWM), a swimming labyrinth test of an animal’s ability to navigate in darkness using route-based internal cues.

Because of widespread use since its development in 1981, Vorhees said, “MWM is the most validated test of hippocampal-dependent spatial learning that exists.” He pointed out that there are variations of MWM and CWM that could be used to conduct further evaluation of memory deficits. He also cautioned the audience about potential sex and strain differences in response.

(Sheetal Thakur, Ph.D., is an Intramural Research Training Award fellow in the NTP Toxicology Branch.)

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