Microbial Status and Genetic Evaluation of Mice and Rats: Proceedings of the 1999 US/Japan Conference (2000)

Jacqueline N. Crawley

Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, Intramural Research Program, National Institute of Mental Health, Bethesda, MD

Experimental manipulation of the mouse genome provides a powerful new technology to generate animal models of human genetic disorders. A well-defined phenotype of the mouse model can serve as a quantitative, robust surrogate marker to evaluate the efficacy of potential treatments for the human disease.

Neuropsychiatric illnesses generally present as a complex set of symptoms. Multiple genes contribute to primary causes and to susceptibility factors. Symptoms are often cyclical and may vary with age and level of neurodegeneration. Biologic and environmental components interact in determining the etiology of the disease. Targeted gene mutation mouse models can be useful in parceling out each of the genetic components of the disease.

More than 100 transgenic and knockout mice with mutations in genes expressed in the nervous system have been generated to date (Picciotto 1999). Aberrant behavioral phenotypes have been documented in many lines of mice with mutations in genes expressed in the central nervous system. Mouse models of neuropsychiatric disorders mimic the human behavioral symptoms to a greater or lesser extent in targeted gene mutations relevant to Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, ataxia, epilepsy, generalized anxiety, schizophrenia, and obesity (Bauer and others 1999; Bedell and others 1997; Burright and others 1997; Campbell and Gold 1996; Contarino and others 1999; Gingrich and Roder 1998; Jucker and Ingram 1997; Klockgether and Evert 1998; Lee and others 1996; Nelson and Young 1998; Picciotto 1999; Price and Sisodia 1998; Smithies 1993; Wahlsten 1999).

To define the precise behavioral concomitants of the genetic manipulation, our laboratory has been addressing methodologic issues for the behavioral phenotyping of mutant mice. Guidelines based on strategies that have proven successful in studying a variety of new transgenic and knockout mice in our laboratory and others are extensively described in recent publications (Crawley 1999, 2000; Crawley and Paylor, 1997; Crawley and others 1997a,b; Picciotta 1999; Rogers and others 1997; Silver 1995; Wehner and Silva 1996). This discussion highlights the critical features of existing guidelines.

Two or more strains are often used to develop the set of mice for behavioral experiments. The 129/SvJ or another 129 substrain is used for the embryonic stem cells. C57BL/6J or another inbred or outbred strain is used for the blastocyst donors. C57BL/6J or an outbred strain is often used for the breeders. Varying ratios of the genetic backgrounds from each strain will be present in each offspring. Background genes from each parent may have profound effects on behavioral tests. Unknown interactions between the mutated gene and the varying background genes will compromise the interpretation of the behavioral phenotype of the mutation.

Congenic breeding of the mutation into the chosen inbred strain for seven generations will create a uniform genetic background, reduce unknown gene product interactions, and reduce variability due to random assortment of parental alleles (Crawley and others 1997a; Picciotto, 1999; Silver 1995; Wehner and Silva 1996). Suggestions for optimizing the choice of inbred strain for breeding the mutation are reviewed in Crawley and others (1997a). C57BL/6J is a strain that breeds relatively well and shows average scores on many behavioral tasks, allowing detection of both increases and decreases in the behavioral scores in a mutant line bred onto a C57BL/6J background.

One extreme individual can dramatically skew the results of a pilot experiment with small Ns. Larger numbers of mice are required for behavioral experiments than for many other phenotypic assays. To obtain statistically meaningful results, most behavioral experiments require 10 to 20 mice per treatment group. The treatment groups are the homozygous mutants (−/−), heterozygous mutant littermates (+/−), and wild-type littermates (+/+). If sex differences are detected, N = 10-20 for each sex of each genotype is required. Ages of the mice must be approximately the same across treatment groups. Adult mice at ages 3 to 8 months are relatively homogeneous on most behavioral tasks.

If large numbers of animals are not available simultaneously, experiments can be repeated with small groups as litters become available. All three geno

types must be represented within each set of experiments. Data across repeated experiments can be combined if no differences are detected between the wild-type controls across the dates of testing.

Characterization of the behavioral phenotype is best conducted in progressive stages (Crawley 1999, 2000; Crawley and Paylor 1997; Paylor and others 1998). A four-step characterization for behavioral evaluation of a new mutant mouse line is recommended based on the experiences of our laboratory in the behavioral phenotyping of over 25 transgenic and knockout mouse lines.

The first stage is a set of preliminary observations to evaluate overall health (Crawley and Paylor 1997; Paylor and others 1998). A general examination of the mice is conducted in the home cages. Any gross abnormalities in overall health, home cage nesting, sleeping, feeding, grooming, and condition of the fur are noted. Body weight and body temperature are measured. Any unusual patterns of locomotion, hyperreactivity to handling, or fighting in the home cage are recorded. Abnormal appearances and home cage behaviors provide important clues for subsequent experiments to define the behavioral phenotype. Ataxias and seizures are often first detected in the home cage (Brennan and others 1997). Aggressive behaviors in nitric oxide synthase knockout mice were first detected by animal caretakers who reported fighting in the home cage (Nelson and others 1995). Absence of normal huddled sleeping patterns in the home cages led to the discovery of social interaction abnormalities in dishevelled-1 knockout mice (Lijam and others 1997).

Several quick tests reveal debilitating neurologic and physiologic problems (Crawley and Paylor 1997; Paylor and others 1998). The righting reflex is a simple test in which the mouse is turned onto its back; the time it takes for the mouse to right itself onto all four paws is measured. Eye blink reflex occurs when the cornea is approached with a cotton tip swab. Ear twitch reflex occurs when the ear is touched with a cotton tip swab, resulting in immediate movement of the pinna. The whisker-orienting reflex is observed by touching the vibrissae on one side; the whiskers stop moving and the head turns to the side on which the whiskers were touched.

Quantitative measures of sensory functions and motor skills are obtained with several tasks. Many are of short (such as 5-minute) duration. Most require

specialized equipment. The better tests for acuity in evaluating vision, hearing, and smell require sophisticated neurophysiologic recording equipment or complex operant discrimination tasks.

Gross hearing ability is assessed by the acoustic startle response. A mouse will flinch in response to a sudden loud sound. Acoustic startle to a loud tone is quantitated by an automated startle system that measures amplitude of whole body flinch (Davis and others 1982). Sensitive measures of hearing acuity are conducted with neurophysiologic recording from the auditory nerve using the auditory brainstem response (Erway and others 1993).

A visual cliff detects blindness. The visual cliff response is quantitated in a box with a horizontal surface and a vertical wall drop-off that represents a ledge (Fox 1965). The inner horizontal surface of the box and vertical drop-off are covered with black and white checkerboard contact paper, which emphasizes the cliff-like drop-off. A piece of clear Plexiglas spans the ledge so that there is no actual drop-off but only the appearance of a cliff. The mouse is placed on a platform at the border between the horizontal surface and the vertical drop-off. Normal mice will step down mostly onto the horizontal surface to avoid the cliff they see on the other side of the platform. Blind mice, not seeing the apparent cliff, will step down an equal number of times onto the horizontal surface and the cliff-like drop-off. This test is compromised by the ability of normal mice to use sensory feedback from the whiskers and feet for edge detection. Another simple test of gross visual ability is the latency for a mouse to enter a dark area when the mouse is placed in a brightly lit area. Because mice are nocturnal and prefer the dark, a mouse with normal light/dark perception will quickly enter the darkened chamber. A blind mouse will have a much longer latency to enter the darkened chamber.

More sensitive measures of visual acuity are obtained with tasks that require training, using visual stimuli in a conditioned reward paradigm. Neurophysiologic recording from the optic nerve or the visual cortex during presentation of visual stimuli will yield the most precise measures of visual acuity.

A simple test for olfactory anosmia is failure to retrieve a buried food source. A simple test for taste insensitivity is failure to avoid water flavored with quinine. However, the quick versions of these sensory tests have not been well characterized in mice and are influenced by motivational factors. Sensitive measures of

smell or taste require training on operant discrimination tasks with graded olfactory or gustatory stimuli (Ackroff and Sclafani 1998). Components of learning and memory may confound a purely sensory interpretation of operant discrimination tasks.

Accurate measures of olfactory acuity are obtained with neurophysiologic recording from the olfactory epithelium or olfactory cortex in response to odorants (Belluscio and others 1998). Accurate measures of gustation are obtained by neurophysiologic recording from the chorda tympani branch of the facial nerve in response to lingual application of taste stimuli (Wong and others 1996).

Sense of touch is evaluated by the reflexive twitch response to Von Frey hairs, fine wires of graded thickness touched to the paw (Pitcher and others 1999). Pain sensitivity is measured by the latency to lick or lift a hindpaw in the hot plate test, or to move the tail out of the path of an intense light beam in the tail-flick test (Matthes and others 1996; Sora and others 1997).

Open field exploratory locomotion is the most common measure of general motor abilities. Open field activity is measured with a photocell-equipped automated apparatus that quantitates locomotion and rearings in an empty open field (Pierce and Kalivas 1997). Coordination and balance are quantitated on the rotarod, consisting of a precisely accelerating rotating cylinder (Carter and others 1999; Lalonde and others 1996). The ability of the mouse to climb up or down a pole, and to walk along a narrow beam, represent additional measures of balance and coordination (Carter and others 1999; Paylor and others 1998). Measurement of the ability of the mouse to hang from a wire by its paws provides an index of neuromuscular strength (Paylor and others 1998). Footprint pathway analysis to quantitate abnormal gait is conducted by videotaping locomotion in a Plexiglas tunnel, or by dipping the hindpaws in black ink and allowing the mouse to walk across white paper through a tunnel (Barlow and others 1996; Carter and others 1999; Clarke and Still 1999). These several tests detect major abnormalities in spinal motor neurons and cerebellum.

Specific behavioral tasks are then designed to test hypotheses about the function of the gene and to model the symptoms of the human genetic disease. Relevant behavioral phenotypes are often discovered during sensory and motor analyses. The auditory brainstem response detects impaired acoustic acuity in mice (Erway and others 1993) and can be used to analyze hearing in mice with

deafness candidate genes (Robertson and others 1997). A mutant mouse model of Tay-Sachs and Sandhoff diseases, deficient in the hexosaminidase enzyme that degrades gangliosides, shows neuronal ganglioside accumulation and concomitant progressive decline in performance on the rotarod task (Sango and others 1995), analogous to the motor deficits that characterize this human syndrome. Atm knockout mice, a model of ataxia telangiectasia, are impaired on the open field and rotarod tests and show unusual footprint patterns (Barlow and others 1996), analogous to the ataxia seen in the clinical syndrome.

To investigate genes with unknown functions, the experimental design often requires several hypotheses and a thorough knowledge of the tests available in the existing behavioral neuroscience literature. Genes expressed primarily in the cerebellum would be investigated in tasks that measure motor coordination and motor learning. Genes expressed primarily in the hypothalamus would be investigated in tasks including feeding, stress responses, and reproductive behaviors. Genes expressed in the hippocampus and cortex would be tested in learning, memory, and attentional and habituation tasks. Genes expressed in the mesocorticolimbic dopamine pathway would be investigated in motivational, appetitively rewarded, stressor, and drug abuse paradigms. Genes expressed in the periaqueductal grey and dorsal horn of the spinal cord would be investigated in pain threshold tests and for responses to analgesics.

Many good behavioral tests are available for each of the behavioral domains of interest. Reviews cited above describe specific tests and reference the source literature for methodologic details.

Learning and memory tests for mice include spatial navigation learning tasks such as the Morris water task, Barnes maze, radial maze, T-maze, and Y-maze; rewarded tasks such as nose-poke for a food reward in an operant chamber or a five-hole chamber on various schedules; and aversive tasks such as passive avoidance, cued and contextual conditioning, and taste aversion. These tasks have been applied to the behavioral phenotyping of a variety of transgenic mouse models of Alzheimer 's disease (Hsiao and others 1996) and mutations in signaling genes (Silva and others 1997). Knockouts of genes expressed in the hippocampus and regulating neuronal calcium-related signaling show deficits in learning and memory tasks (Cho and others 1998; Impey and others, 1998; Mayford and others 1996). Feeding tests include 24-hour consumption, limited daily access, macronutrient sources, taste discrimination, and sham feeding. Some of these tasks have been applied to study genes regulating feeding and obesity (Huszar and others 1997; Pelleymounter and others 1995). Reproductive behaviors are quantitated by standardized scoring of sexual activity in male mice, lordosis response in female mice, and parental latency to retrieve pups to the nest and to nurse, groom, and nest with the pups. Estrogen receptor knockout mice are impaired on sexual behaviors (Rissman and others, 1997). Oxytocin-deficient mice fail to lactate (Nishimori and others 1996; Young and others 1996). Good models of anxiety-related behaviors include the elevated plus maze, the

elevated zero maze, light↔dark exploratory transitions, and the Vogel conflict test. Corticotropin-releasing factor transgenics and knockouts for genes expressed in the amygdala show unusual anxiety-related behaviors on stress-related tasks (Contarino and others 1999; Heinrichs and others 1997). Drug abuse tendencies can be measured with conditioned place preference, two-bottle choices, and intravenous self-administration. Opiate receptor knockout mice are aberrant on tests for pain responsivity, analgesic effects of morphine, morphine withdrawal responses, and conditioned place preference (Matthes and others 1996; Sora and others 1997).

Our laboratory recommends an order of testing that begins with the home cage observations, continues with observations of general health and neurologic reflexes, then addresses sensory and motor abilities, and finally focuses on the behavioral domains relevant to the specific hypotheses. This approach allows the investigator to detect underlying physiologic abnormalities in the mutant mice that might limit their ability to perform the procedures necessary for complex behavioral tasks. False positives are prevented, which would have been caused by artifacts such as blindness limiting performance in a visual discrimination learning task, hearing and olfactory deficits responsible for poor parental pup retrieval, or ataxias impeding elevated plus maze arm entries. Instead, the hypothesis-driven tests are designed to accommodate the physical limitations of the mice. For example, an auditory tone cue instead of a visual light cue is used in the automated operant chamber in a learning task for blind mice.

To avoid false negatives, our laboratory recommends choosing three or more tasks within the behavioral domain of interest. Different types of memory, different types of anxiety, different components of feeding, different types of parental care, different symptoms relevant to schizophrenia and so forth may be differentially regulated by the gene of interest. Spreading a wider net allows the investigator to catch the particular type of phenotype relevant to the mutated gene. Choice of multiple tasks is further based on differing sensory modalities and motor requirements. For example, three complimentary memory tasks would include cued and contextual conditioning (employing auditory and olfactory cues, with minimal motor requirements), the Morris water task (spatial navigation with visual cues, swimming, and stress components), and taste aversion (gustatory cues, long retention time). If deficits in learning and memory are detected in all three tasks, the findings are likely to be biologically meaningful and highly replicable. If deficits in learning and memory are detected in only one or two tasks, that type of cognitive function is further explored. For example, a deficit only on the Morris water task would be further explored with other spatial navigation tasks such as the Barnes maze and the radial arm maze.

In some cases, combinations of tests cannot be conducted in the same mouse. Interference between tasks often becomes an issue when two tasks are very

similar. Passive avoidance is similar to cued and contextual fear conditioning and to light dark exploration. These tasks require the mouse to remember sensory associations with a dark chamber or a location where a footshock was previously received. These three tests are best conducted with different sets of mice. Alternatively, it may be possible to conduct related tests in the same mice with sufficient intervention between the tests.

Carryover effects limit some combinations of tests in the same individuals. Repeated testing in the Digiscan open field induces habituation to the novelty of the open field environment. Stressful tasks such as the Morris water task will affect performance on sensitive anxiety tests and are therefore best administered as the last behavioral assay. Similarly, drug treatments should be administered at the end of the behavioral phenotyping series. Some drugs are slowly metabolized, such that residual drug remains in the mouse for several days. Other drugs induce sensitization or tolerance to repeated doses and to doses of other drugs in the same class. Past treatments with amphetamine and cocaine induce sensitization to the effects of an acute dose of these psychostimulants on hyperlocomotion and dopamine release (White and Kalivas 1999). Repeated treatments with neuroleptics and D1 antagonists result in increased catalepsy scores (Chinen and Frussa-Filho 1999).

Molecular geneticists planning to begin behavioral phenotyping experiments are encouraged to develop collaborations with reputable behavioral neuroscientists. Correct choices and implementation of behavioral tasks in mice require knowledge of more than 50 years of scientific literature in behavioral neuro-science, understanding of the standard methods for the basic behavioral paradigms, and familiarity with the technical tricks that make any method work well. Experience with proper testing and handling of mice, to minimize stress factors and to meet the international guidelines for the care and use of laboratory rodents, can best be gained by spending some time working in an established behavioral neuroscience laboratory. Entering into a scientific collaboration with a recognized behavioral laboratory will help to avoid artifacts, generate statistically and biologically meaningful data, and complete behavioral phenotyping experiments with the maximum speed and precision.

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