Experimenter effects on behavioral test scores of eight inbred mouse strains under the influence of ethanol.

Eight standard inbred mouse strains were evaluated for ethanol effects on a refined battery of behavioral tests in a study that was originally designed to assess the influence of rat odors in the colony on mouse behaviors. As part of the design of the study, two experimenters conducted the tests, and the study was carefully balanced so that equal numbers of mice in all groups and times of day were tested by each experimenter. A defect in airflow in the facility compromised the odor manipulation, and in fact the different odor exposure groups did not differ in their behaviors. The two experimenters, however, obtained markedly different results for three of the tests. Certain of the experimenter effects arose from the way they judged behaviors that were not automated and had to be rated by the experimenter, such as slips on the balance beam. Others were not evident prior to ethanol injection but had a major influence after the injection. For several measures, the experimenter effects were notably different for different inbred strains. Methods to evaluate and reduce the impact of experimenter effects in future research are discussed.

A contemporary view of genes and behavior: complex systems and interactions.

Several large-scale searches for genes that influence complex human traits, such as intelligence and personality, in the normal range of variation have failed to identify even one gene that makes a significant difference. All previously published claims for genetic influences of this kind now appear to have been false positives. For more serious psychiatric and medical disorders such as schizophrenia and autism, several genes have been found where a rare mutation contributes to abnormal behavior, but in many instances they are de novo mutations not obtained from a parent. Despite the many disappointments in the search for genes influencing human behavior, the field of molecular genetics has made remarkable progress to the extent that several broadly applicable principles can now be affirmed. These principles show how development is regulated by networks of interacting genes that function in an environmental context. They invalidate several key assumptions of statistical genetic analysis that are made when estimating heritability. There is now a need to reform the teaching of genetics to our students and to restrict the funding of further searches for elusive genes that account for so little variance in normal behaviors.

The hunt for gene effects pertinent to behavioral traits and psychiatric disorders: from mouse to human.

The field of behavioral genetics was reviewed in the classic 1960 text by Fuller and Thompson. Since then, there has been remarkable progress in the genetic analysis of animal behavior. Many molecular genetic methods in common use today were not even anticipated in 1960. Animal models for many human psychiatric disorders have been discovered or created. In human behavior genetics, however, powerful new methods have failed to reveal even one bona fide, replicable gene effect pertinent to the normal range of variation in intelligence and personality. There is no explanatory or predictive value in that genetic information. For several psychiatric disorders, including autism and schizophrenia, many large genetic effects arise from de novo mutations. Genetically, the disorders are heterogeneous; different cases with the same diagnosis have different causes. The promises of the molecular genetic revolution have not been fulfilled in behavioral domains of most interest to human psychology.

Reversed light-dark cycle and cage enrichment effects on ethanol-induced deficits in motor coordination assessed in inbred mouse strains with a compact battery of refined tests.

The laboratory environment existing outside the test situation itself can have a substantial influence on results of some behavioral tests with mice, and the extent of these influences sometimes depends on genotype. For alcohol research, the principal issue is whether genotype-related ethanol effects will themselves be altered by common variations in the lab environment or instead will be essentially the same across a wide range of lab environments. Data from 20 inbred strains were used to reduce an original battery of seven tests of alcohol intoxication to a compact battery of four tests: the balance beam and grip strength with a 1.25 g/kg ethanol dose and the accelerating rotarod and open-field activation tests with 1.75 g/kg. The abbreviated battery was then used to study eight inbred strains housed under a normal or reversed light-dark cycle, or a standard or enriched home cage environment. The light-dark cycle had no discernable effects on any measure of behavior or response to alcohol. Cage enrichment markedly improved motor coordination in most strains. Ethanol-induced motor coordination deficits were robust; the well-documented strain-dependent effects of ethanol were not altered by cage enrichment.

The precision of video and photocell tracking systems and the elimination of tracking errors with infrared backlighting.

Automated tracking offers a number of advantages over both manual and photocell tracking methodologies, including increased reliability, validity, and flexibility of application. Despite the advantages that video offers, our experience has been that video systems cannot track a mouse consistently when its coat color is in low contrast with the background. Furthermore, the local lab lighting can influence how well results are quantified. To test the effect of lighting, we built devices that provide a known path length for any given trial duration, at a velocity close to the average speed of a mouse in the open-field and the circular water maze. We found that the validity of results from two commercial video tracking systems (ANY-maze and EthoVision XT) depends greatly on the level of contrast and the quality of the lighting. A photocell detection system was immune to lighting problems but yielded a path length that deviated from the true length. Excellent precision was achieved consistently, however, with video tracking using infrared backlighting in both the open field and water maze. A high correlation (r=0.98) between the two software systems was observed when infrared backlighting was used with live mice.

Digit ratio (2Dratio4D) differences between 20 strains of inbred mice.

The second to fourth digit ratio (2Dratio4D) is sexually differentiated in a variety of species, including humans, rats, birds, and lizards. In humans, this ratio tends to be lower in males than in females. Lower digit ratios are believed to indicate increased prenatal testosterone exposure, and are associated with more masculinized behavior across a range of traits. The story seems more complicated in laboratory mice. We have previously shown that there is no sex difference in the digit ratios of inbred mice, but found behavioral evidence to suggest that higher 2Dratio4D is associated with more masculinized behaviors. Work examining intrauterine position effects show that neighbouring males raise pup digit ratio, suggesting again that higher digit ratios are associated with increased developmental androgens. Other work has suggested that masculinization is associated with lower digit ratios in lab mice. Here, we examine the fore- and hindlimb digit ratios of 20 inbred mouse strains. We find large inter-strain differences, but no sexual dimorphism. Digit ratios also did not correlate with mice behavioral traits. This result calls into question the use of this trait as a broadly applicable indicator for prenatal androgen exposure. We suggest that the inbred mice model presents an opportunity for researchers to investigate the genetic, and gene-environmental influence on the development of digit ratios.

Calibration of rotational acceleration for the rotarod test of rodent motor coordination.

The latency of mice and rats to fall from the accelerating rotarod can differ markedly between laboratories using the same brand of rod as well as between studies using different kinds of rods. These discrepancies can arise from different rod diameters, surface textures, test protocols, or laboratory environmental factors beyond the test itself, but it is also possible that the actual acceleration rates of the different rods do not correspond to the nominal rates set on the devices. This paper describes a simple method to measure acceleration rate of the rotarod and to set the rate to a desired value for any brand of rod.

Overview of mouse assays of ethanol intoxication.

There are many behavioral assays to assess sensitivity to ethanol intoxication in mice. Most are simple to implement, and are sensitive to a particular dose range of ethanol. Most reflect genetic influences, and each test appears to reflect the contribution of a relatively distinct collection of genes. This genetic heterogeneity implies that no single test can claim to capture the construct "ethanol intoxication" completely. Depending on the test, and when measurements are made, acute functional tolerance to even a single dose of ethanol must be considered as a contributing factor. Whether or not a test is conducted in naïve mice or as part of a test battery can influence sensitivity, and do so in a strain-dependent manner. This unit reviews existing tests and recommends several.

Impaired fear memory, altered object memory and modified hippocampal synaptic plasticity in split-brain mice.

The hippocampus is critical for memory formation. However, the contributions of the hippocampal commissure (HC) and the corpus callosum (CC) are less clear. To elucidate the role of the forebrain commissures in learning and memory, we performed a behavioural and electrophysiological characterization of an inbred mouse strain that displays agenesis of the CC and congenitally reduced HC (BTBR T+ tf/J; 'BTBR'). Compared to a control strain, BTBR mice have severely impaired contextual fear memory, with normal object recognition memory. Interestingly, continuous environmental "enrichment" significantly increased object recognition in BTBR, but not in control C57BL/6 ('BL/6') mice. In area CA1 of hippocampal slices, BTBR displayed intact expression of long-term potentiation (LTP), paired-pulse facilitation (PPF) and basal synaptic transmission, compared to BL/6 mice. However, BTBR hippocampal slices show an increased susceptibility to depotentiation (DPT), an activity-induced reversal of LTP. We conclude that the HC and CC are critical for some forms of hippocampal memory and for synaptic resistance to DPT. Agenesis of the CC and HC may unmask some latent ability to encode, store or retrieve certain forms of recognition memory. We suggest that the increased susceptibility to DPT in BTBR may underlie the memory phenotype reported here.

Stability of inbred mouse strain differences in behavior and brain size between laboratories and across decades.

If we conduct the same experiment in two laboratories or repeat a classical study many years later, will we obtain the same results? Recent research with mice in neural and behavioral genetics yielded different results in different laboratories for certain phenotypes, and these findings suggested to some researchers that behavior may be too unstable for fine-scale genetic analysis. Here we expand the range of data on this question to additional laboratories and phenotypes, and, for the first time in this field, we formally compare recent data with experiments conducted 30-50 years ago. For ethanol preference and locomotor activity, strain differences have been highly stable over a period of 40-50 years, and most strain correlations are in the range of r = 0.85-0.98, as high as or higher than for brain weight. For anxiety-related behavior on the elevated plus maze, on the other hand, strain means often differ dramatically across laboratories or even when the same laboratory is moved to another site within a university. When a wide range of phenotypes is considered, no inbred strain appears to be exceptionally stable or labile across laboratories in any general sense, and there is no tendency to observe higher correlations among studies done more recently. Phenotypic drift over decades for most of the behaviors examined appears to be minimal.

Effects of genetic and procedural variation on measurement of alcohol sensitivity in mouse inbred strains.

Mice from eight inbred strains were studied for their acute sensitivity to ethanol as indexed by the degree of hypothermia (HT), indexed as the reduction from pre-injection baseline of their body temperature. Two weeks later, mice were tested for their loss of righting reflex (LRR) after a higher dose of ethanol. The LRR was tested using the "classical" method of watching for recovery in animals placed on their backs in a V-shaped trough and recording duration of LRR. In a separate test, naive animals of the same strains were tested for HT repeatedly to assess the development of rapid (RTOL) and chronic tolerance (CTOL). We have recently developed a new method for testing LRR that leads to a substantial increase in the sensitivity of the test. Strains also have been found to differ in the new LRR test, as well as in the development of acute functional tolerance (AFT) to this response. In addition, our laboratory has periodically published strain difference data on the older versions of the HT and LRR responses. The earlier tests used some of the exact substrains tested currently, while for some strains, different substrains (usually, Nih versus Jax) were tested. We examined correlations of strain means to see whether patterns of strain differences were stable across time and across different test variants assessing the same behavioral construct. HT strain sensitivity scores were generally highly correlated across a 10-23 years period and test variants. The CTOL to HT was well-correlated across studies, and was also genetically similar to RTOL. The AFT, however, was related to neither RTOL nor CTOL, although this may be because different phenotypic end points were compared. The LRR data, which included a variant of the classical test, were not as stable. Measures of LRR onset were reasonably well correlated, as were those taken at recovery (e.g., duration). However, the two types of measures of LRR sensitivity to ethanol appear to be tapping traits that differ genetically. Also, the pattern of genetic correlation between HT and LRR initially reported in 1983 was not seen in current and contemporaneous studies. In certain instances, substrain seems to matter little, while in others, substrains differed a great deal. These data are generally encouraging about the stability of genetic differences.

Different rankings of inbred mouse strains on the Morris maze and a refined 4-arm water escape task.

The submerged platform or Morris water escape task is widely used to study genetic variation in spatial learning and memory, but interpretation is sometimes difficult because of wall hugging, jumping off the platform, floating or non-spatial swim strategies. We modified the task by introducing four wide arms into the circular tank and adding features that reduced, eliminated, or compensated for several competing behaviors. Three versions of the 4-arm task were evaluated in detail, and the third version yielded good results for six of eight inbred strains. Furthermore, the 4-arm task could be scored adequately without computerized video tracking. Although performance on the 4-arm task was generally superior to the Morris maze, the extent of the improvement was strain dependent. Two strains with retinal degeneration (C3H/HeJ, FVB/NJ) performed poorly on both the Morris and 4-arm mazes, whereas C57BL/6J and DBA/2J did well on both mazes. A/J performed poorly on the Morris task but became very proficient on the 4-arm maze, despite its strong tendency to hug the walls of the tank. The BALB/cByJ strain, on the other hand, exhibited the best probe trial performance on the Morris maze but was very slow in acquiring the 4-arm task. We conclude that no single task can reveal the full richness of spatially guided behavior in a wide range of mouse genotypes.

An analysis of the genetics of alcohol intoxication in inbred mice.

We compared the behaviors of eight inbred mouse strains across 18 variables, using 11 behavioral assays, and gave ethanol (EtOH) as an intoxicant. Genetic influences on behavior and sensitivity to EtOH were pronounced, but strain sensitivities were generally only modestly correlated across tasks. Certain well-correlated clusters of responses suggested that some genes affect similar neurobiological substrates. No strains of mice were generally sensitive or resistant to intoxication across tasks. Anthropomorphically appealing concepts like 'muscle strength' had little explanatory power across tasks. A battery of selected tests was proposed for future studies. Overall, the results show that each mouse behavioral assay captures only a portion of ataxia, a genetically complex behavioral domain. Conversely, multiple behavioral capacities are apparently required for performance in each specific assay. Thus, if only one or two tests are used to evaluate motor function in genetically engineered mutant mice, only a small portion of the domain will be assessed and results may be misleading. This caveat likely extends to many behavioral domains (e.g. learning and memory, anxiety).

Observer-rated ataxia: rating scales for assessment of genetic differences in ethanol-induced intoxication in mice.

Identification of genetic and physiological mechanisms underlying a drug's or mutation's effects on motor performance could be aided by the existence of a simple observation-based rating scale of ataxia for mice. Rating scales were developed to assess ataxia after ethanol (2.75, 3.0, and 3.25 g/kg) in nine inbred mouse strains. Each scale independently rates a single behavior. Raters, blinded to dose, scored four behaviors (splay of hind legs, wobbling, nose down, and belly drag) at each of four time points after injection. The severities of hind leg splaying and wobbling were quantifiable, whereas nose down and belly dragging were expressed in all-or-none fashion. Interrater reliabilities were substantial (0.75 0 at some time), but all doses were equally effective. Incidence of nose down and belly dragging behaviors increased strain dependently after ethanol, but strains did not differentially respond to dose. Ethanol-induced splaying was modestly, and negatively, genetically correlated with wobbling. Nose down and belly dragging tended to be associated with splaying and wobbling at later times. Four distinct ataxia-related behaviors were sensitive to ethanol. Strains differed in ethanol sensitivity for all measures. Modest strain mean correlations among behaviors indicate that these behaviors are probably under control of largely different genes and that ataxia rating scales should rate separate behaviors on discrete scales.

Influence of task parameters on rotarod performance and sensitivity to ethanol in mice.

Motor performance in mice can be assessed with multiple apparatus and protocols. Use of the rotarod (a.k.a. rotorod, rota-rod, roto-rod, or accelerod) is very common, and it is often used with the apparent assumption by the experimenters that it is a straightforward and simple assay of coordination. The rotarod is sensitive to drugs that affect motor coordination, including ethanol. However, there are few systematic data assessing the range of "normal" performance in mice. There are also few data exploring optimal task parameters (e.g. the influence of different speeds of rotation). In these experiments, we show that both accelerating and fixed-speed rotarod (FSRR) performance vary under different test protocols and conditions, and that moderate to high doses of ethanol disrupt performance. Under certain conditions, low doses of ethanol were found to enhance performance on the accelerating rotarod (ARR). Therefore, it is not possible to characterize individual differences fully using a single set of test parameters. For example, because of the biphasic effect of ethanol on performance, at least two doses of the drug are necessary to explore individual sensitivity differences. We offer recommendations of parameters we believe to be generally suitable for exploring the performance of new genotypes using the rotarod. We suggest that other putative tests of "ataxia" are similarly complex, and that characterizing the contribution of genetic differences will require similar attention to the details of task apparatus and protocols. These data also underscore the need to employ multiple behavioral assays in order to model a complex domain such as "ataxia" or "coordination."

Survey of 21 inbred mouse strains in two laboratories reveals that BTBR T/+ tf/tf has severely reduced hippocampal commissure and absent corpus callosum.

A morphometric survey of brain size and forebrain commissures of 21 inbred mouse strains from the Jackson Laboratory was done with animals tested in two laboratories as part of the Mouse Phenome Project. Strain BTBR T/+ tf/tf was found to have 100% total absence of the corpus callosum as well as severe reduction of the hippocampal commissure in almost every animal, the most severe commissure defect observed to date in any commercially available mouse strain. The strain 129S1/SvImJ had a milder defect with incomplete penetrance. Crosses of BTBR mice with inbred strains BALB/cWah1, 129P1/ReJ, and the recombinant strain 9XCA/Wah having a severe commissure defect supported a two-locus model of the genetic defect in these strains. Brain size varied greatly among strains but for any one strain was almost identical in mice housed for 5 weeks in the two laboratories. Sex differences in brain weight and forebrain commissure sizes were not statistically significant.

Genotypic differences in ethanol sensitivity in two tests of motor incoordination.

Motor incoordination is frequently used as a behavioral index of intoxication by drugs that depress the central nervous system. Two tasks that have been used to assay incoordination in mice, the balance beam and the grid test, were evaluated to optimize aspects of apparatus and testing procedures for studying genetic differences. Mice of eight inbred strains were given one of several doses of ethanol or saline and tested for intoxication. Strains differed in sensitivity to ethanol in both tests, indicating a significant influence of genotype on ethanol sensitivity. For the balance beam, the width of the beam affected the strain sensitivity pattern, and only the widest beam worked well at all doses. For the grid test, both ethanol dose and the time after drug injection affected strains differentially. Although the behavioral sign of intoxication recorded for both tests was a foot-slip error, the correlations of strain means for ethanol sensitivity across the two tasks were generally not significant. This suggests that the genes influencing ethanol sensitivity in the two tasks are mostly different. These results make clear that a single set of task parameters is insufficient to characterize genetic influences on behavior. Several other issues affect the interpretation of data using these tests.

Assessment of genetic susceptibility to ethanol intoxication in mice.

Increased use of gene manipulation in mice (e.g., targeted or random mutagenesis) has been accompanied by increased reliance on a very few rapid and simple behavioral assays, each of which aspires to model a human behavioral domain. Yet, each assay comprises multiple traits, influenced by multiple genetic factors. Motor incoordination (ataxia), a common characteristic of many neurological disorders, may reflect disordered balance, muscle strength, proprioception, and/or patterned gait. Impaired motor performance can confound interpretation of behavioral assays of learning and memory, exploration, motivation, and sensory competence. The rotarod is one of the most commonly used tests to measure coordination in mice. We show here that exactly how the rotarod test is performed can markedly alter the apparent patterns of genetic influence both in undrugged performance and sensitivity to ethanol intoxication. However, when tested with well chosen parameters, the accelerating rotarod test showed very high inter- and intralaboratory reliability. Depending on test conditions, ethanol can either disrupt or enhance performance in some strains. Genetic contribution to performance on the accelerating versus the fixed-speed rotarod assay can be completely dissociated under some test conditions, and multiple test parameters are needed to assess the range of genetic influence adequately.