Behavioral Phenotyping of Transgenic and Knockout Mice: Experimental Design and Evaluation of General Health, Sensory Functions, Motor Abilities, and Specific Behavioral Tests

Jacqueline N. Crawley, Ph.D., is Chief of the Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, Bethesda, Maryland. The following article is reprinted from Brain Research 1999, 835:18–26, with permission from Elsevier Science. Targeted gene mutation technology represents a powerful new tool for biomedical research. When the targeted gene is expressed in the brain, the behavioral phenotype of the mutant mice may reveal genetic mechanisms underlying normal behaviors and may increase our knowledge of genetic factors in neuropsychiatric disorders. Transgenic mice have a new gene, or an additional copy of an existing gene, added to the genome. Knockout mice have a targeted gene deletion, such that no product of the mutated gene is synthesized in the null mutants. The methods for developing the mutation, and breeding strategies to generate null mutants in the F2 and subsequent generations, are extensively described in [ Brain Research Vol 835] and elsewhere ( Campbell and Gold 1996 ; Nelson and Young 1998 ; Wehner and Silva 1996 ). Approximately 100 different genes expressed in the central nervous system have been targeted and phenotyped in transgenic and knockout mice to date ( Campbell and Gold 1996 ; Bedell et al. 1997 ; Silva et al. 1997 ; Murphy et al. 1998 ; Nelson and Young 1998 ). Reported behavioral phenotypes include aberrant social, reproductive, and parental behaviors, aggression, feeding disorders, learning and memory impairments, anxiety-like behaviors, and altered responses to antidepressants, antipsychotics, ethanol, and psychostimulant drugs of abuse. Experimental design is presently being optimized for thorough evaluation of behavioral phenotyping in mutant mice. This review is designed to suggest general methods that have been validated in our laboratory and others. Specific protocols for individual behavioral tasks can be found in the original publications referenced throughout the text, and in several recent reviews ( Wehner et al. 1996 ; Lee et al. 1996 ; Campbell and Gold 1996 ; Crawley et al. 1997, 1998 ; Crawley and Paylor 1997 ; Jucker and Ingram 1997 ; Nelson and Young 1998 ) [see also Crawley 2000 1 ]. After the mutation has been successfully introduced, and the gene product shown to be correctly overexpressed or absent, the first chimeras are mated with wildtype mice. A Mendelian distribution of F2 offspring is predicted. The strain of mouse used for the embryonic stem cells, for the donor blastulas, and for the breeding can greatly affect the behavioral phenotype. For example, some 129 substrains (e.g., 129/J and 129/SvJ) have an incomplete or missing corpus callosum ( Livy and Wahlsten 1991 ), and perform poorly on learning and memory tasks ( Wehner and Silva 1996 ; Crawley et al. 1997 ). Background genes that are randomly contributed by each parental strain often interact with the mutated gene of interest, creating false positives ( Wehner and Silva 1996 ; Gerlai 1996 ; Crawley 1996 ; Banbury Conference 1997 ; Crawley et al. 1997 ). For example, human Alzheimer’s mutant β-amyloid precursor protein overexpressed in C57B6 breeder mice produces amyloid plaques in the brain and poor learning and memory on the Morris water task ( Hsiao et al. 1996 ). The same transgenic insertion in FVB/N mice did not show plaque formation and was lethal at too young an age for learning and memory testing. No single strain can solve all of the potential breeding problems. For some experiments, breeding into the 129/Sv strain is a good solution, to unify the background genes by using the same strain for the embryonic stem cells and the parental breeders ( Lijam et al. 1997 ). Other experiments benefit from a congenic breeding strategy into C57BL/6J, a standard, commercially available inbred strain that shows intermediate values on most behavioral phenotypes, has been characterized on a large number of behavioral tasks, and is a reasonably prolific breeder ( Silver 1995 ; Crawley et al. 1997 ; Banbury Conference 1997 ). Numbers of animals for standardized experimental designs and appropriate statistical tests are a minimum of N = 10 −/− null mutants, N = 10 +/− heterozygotes, and N = 10 +/+ wildtype littermate controls. If a gender effect is detected, N = 10 of each gender and each genotype is required. Ages of the animals are approximately equivalent across genotypes, in accordance with the goals of the experiment. for example, adult mice are best tested between ages 3 and 8 months; aged mice between 12 and 18 months; juvenile mice between 2 and 6 weeks. A series of carefully conducted preliminary observations of general health, home cage behaviors, sensory abilities, and motor functions is first conducted for each mouse to avoid spurious false positives. If an animal has a major health problem or a gross motor defect, it will be unable to perform many behavioral tasks, for reasons not necessarily specific to the mutation. If an animal is blind or deaf, specific behavioral tests can be designed around the sensory deficit, such as olfactory learning tasks for blind mice or tactile startle tasks for deaf mice. Our laboratory developed a set of preliminary observations and neurological reflexes which we use to assess gross defects in mutant mice ( Crawley and Paylor 1997 ). This first step is likened to “giving your mouse a physical exam.” The mouse is weighed, its body temperature is taken, and the appearance of its fur and whiskers is noted. Home cage locomotion, grooming, nesting, sleeping, and fighting patterns are recorded. Neurological reflexes are tested in each mouse. These include eye blink, ear twitch, whisker twitch, and righting reflex. We next evaluate motor functions. Each mouse is tested for normal exploratory locomotion on the Digiscan open field ( Norflus et al. 1998 ). The mouse is placed in the photocellequipped automated open field box for a 5-min test session, during which the Accuscan software calculates total distance traversed, number of movements, horizontal activity, vertical activity, and center/perimeter time. Each mouse is next tested for motor coordination on the Basile automated accelerating rotarod ( Jones and Roberts 1968 ; Sango et al. 1995 ). The mouse is placed on the cylinder and the speed of the cylinder rotation is gradually accelerated from 4 to 40 revolutions per minute over a 5-min period. Latency to fall from the rotarod is recorded. The fall is approximately 6 inches, a height that mice can easily fall and land on their feet without injury. Neuromuscular strength is tested by the wire hang test ( Paylor et al. 1998 ). The mouse is placed on a wire cage lid and the lid is gently waved in the air so the mouse grips the wire. The lid is then turned upside down, approximately 6 inches above the surface of soft bedding material. Latency to fall onto the bedding is recorded, with a 60-sec cut-off time. Gait is measured with the footprint test ( Barlow et al. 1996 ). The two hindpaws are dipped into black ink. The mouse is then immediately placed onto white paper in a dark, narrow tunnel, approximately 12 inches long. The ink is then wiped off the feet, and the pattern and pathway of the black footprints on the white paper are calculated. Sensory functions are then assessed. Visual ability is measured in the visual cliff test conducted in a box with a ledge. The inner surface of the box and ledge are covered with black and white checkerboard contact paper, which emphasizes the ledge drop-off. A piece of clear plexiglas spans the ledge so that there is no actual drop-off, just the visual appearance of a cliff. Normal mice will stop at the “edge” and explore the Plexiglas floor before walking forward. Blind mice will not see the appearance of the edge and will walk forward across the plexiglas immediately. Hearing is assessed with the acoustic startle test ( Paylor and Crawley 1997 ). The reflexive flinch and eyeblink to a sudden loud noise comprise the standard acoustic startle response. An automated startle system is used to deliver the startle stimulus and measure the response of the mouse. The mouse is placed in a small cylindrical restraining tube within a soundattenuating chamber. Stimulus tones are varied randomly from 70 dB to 120 dB, 40 msec duration, in the presence of background white noise at 70 dB. A standard battery of tones begins at threshold sound levels of 72 and 75 dB, includes moderately loud tones of 80, 90, and 100 dB, and ends at the loudest sound levels, 110, 115, and 120. Whole body flinch amplitude is automatically recorded by the pressure transducer. Tactile startle is measured by whole body flinch amplitude to a brief puff of air delivered by the system to an area near the face. 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Behavioral phenotypes in the mutant mouse can as to test the of potential major of the transgenic and knockout technology its to the general of the of genes in normal and The first is the presence of the mutation in all A gene expressed in the brain may also be expressed in many is to a behavioral in the mutant mouse to a specific brain or or to the nervous are being to solve problem ( et al. 1996 ; et al. 1996 ; et al. 1996 ; et al. 1997 ; and see reviews in [ Brain Research Vol The is the presence of the mutation from the of Other genes may during for the of the gene, or for the of the of behavioral phenotype may be to by a gene which over the of the missing An behavioral phenotype to that may be to by or An behavioral phenotype may in the of the gene, not the targeted mutation. are being to solve problem ( et al. 1996 ; et al. 1996 ; and see reviews in [ Brain Research Vol If and it is that the transgenic and knockout technology will the tool to our of the genetic of may such as drugs that as at may also of brain or to the functions of and Behavioral phenotyping methods to to our to the of the mutant mouse