Behavioural and anatomical characterization of mutant mice with targeted deletion of D1 dopamine receptor-expressing cells: response to acute morphine.

Considerable topographic overlap exists between brain opioidergic and dopaminergic neurons. Pharmacological blockade of the dopamine D(1) receptor (Drd1a) reverses several behavioural phenomena elicited by opioids. The present study examines the effects of morphine in adult mutant (MUT) mice expressing the attenuated diphtheria toxin-176 gene in Drd1a-expressing cells, a mutant line shown previously to undergo post-natal striatal atrophy and loss of Drd1a-expression. MUT and wild-type mice were assessed behaviourally following acute administration of 10 mg/kg morphine. Treatment with morphine reduced locomotion and rearing similarly in both genotypes but reduced total grooming only in MUT mice. Morphine-induced Straub tail and stillness were heightened in MUT mice. Chewing and sifting were decreased in MUT mice and these effects were not modified by morphine. Loss of striatal Drd1-positive cells and up-regulated D(2)-expression, as reflected in down-regulated D(1)-like and up-regulated D(2)-like binding, respectively, is not uniform along the cranio-caudal extent in this model but appears to be greater in the caudal striatum. Preferential caudal loss of µ-opioid-expression, a marker for the striosomal compartment, was seen. These data indicate that Drd1a-positive cell loss modifies the exploratory behavioural response elicited by morphine, unmasking novel morphine-induced MUT-specific behaviours and generating a hypersensitivity to morphine for others.

Learning and memory deficits in Notch mutant mice.

Notch is a critical component of evolutionarily conserved signaling mechanisms that regulate development and may contribute to plasticity-related processes, including changes in neurite structure and maintenance of neural stem cells. Deficits in the Notch pathway are responsible for Alagille and Cadasil syndromes, which are associated with mental retardation and dementia. Additionally, in postmitotic neurons, Notch proteins interact with presenilins and with beta-amyloid precursor protein and could therefore have a role in the memory deficits associated with familial and sporadic Alzheimer's disease. To test if alterations in Notch signaling can lead to learning and memory deficits, we studied mice with mutations in this pathway. Here, we show that null heterozygous mutations in Notch1 result in deficits in spatial learning and memory without affecting other forms of learning, motor control, or exploratory activity. We also show that null heterozygous mutations in the downstream cofactor RBP-J result in similarly specific spatial learning and memory deficits. These data indicate that a constitutive decrease in Notch signaling can result in specific learning and memory deficits and suggest that abnormalities in Notch-dependent transcription may contribute to the cognitive deficits associated with Alzheimer's disease and Alagille and Cadasil syndromes.

Generation and analysis of GluR5(Q636R) kainate receptor mutant mice.

The physiological significance of RNA editing of transcripts that code for kainate-preferring glutamate receptor subunits is unknown, despite the fact that the functional consequences of this molecular modification have been well characterized in cloned receptor subunits. RNA editing of the codon that encodes the glutamine/arginine (Q/R) site in the second membrane domain (MD2) of glutamate receptor 5 (GluR5) and GluR6 kainate receptor subunits produces receptors with reduced calcium permeabilities and single-channel conductances. Approximately 50% of the GluR5 subunit transcripts from adult rat brain are edited at the Q/R site in MD2. To address the role of glutamate receptor mRNA editing in the brain, we have made two strains of mice with mutations at amino acid 636, the Q/R-editing site in GluR5, using embryonic stem cell-mediated transgenesis. GluR5(RloxP/RloxP) mice encode an arginine at the Q/R site of the GluR5 subunit, whereas GluR5(wt(loxP)/wt(loxP)) mice encode a glutamine at this site, similar to wild-type mice. Mutant animals do not exhibit developmental abnormalities, nor do they show deficits in the behavioral paradigms tested in this study. Kainate receptor current densities were reduced by a factor of six in acutely isolated sensory neurons of dorsal root ganglia from GluR5(RloxP/RloxP) mice compared with neurons from wild-type mice. However, the editing mutant mice did not exhibit altered responses to thermal and chemical pain stimuli. Our investigations with the GluR5-editing mutant mice have therefore defined a set of physiological processes in which editing of the GluR5 subunit is unlikely to play an important role.

MeCP2 dysfunction in humans and mice.

Rett syndrome is a leading cause of postnatal neurodevelopmental regression. Rett syndrome is caused by mutations in MECP2, the gene encoding methyl-CpG binding protein 2. In up to 96% of all classic cases, Rett syndrome cases are caused by mutations or deletions in MECP2. The phenotypic spectrum of MECP2 mutations is broad and includes mental retardation with or without seizures, Angelman syndrome-like phenotype, and autism. Mecp308/Y mice carry a truncating mutation and display many of the features seen in Rett syndrome. Social behavior abnormalities and impaired social interactions in Mecp308/Y mice suggest that MeCP2 plays a role in modulating the activity of genes and neurons important for social interactions. Mice that overexpress MeCP2 at twice the endogenous levels develop a progressive neurologic disorder, demonstrating that MeCP2 levels are tightly regulated and raising the possibility that duplications or gain-of-function mutations of MECP2 might underlie some cases of neurodevelopmental X-linked disorders.

Behavioral phenotyping enhanced--beyond (environmental) standardization.

It is basic biology that the phenotype of an animal is the product of a complex and dynamic interplay between nature (genotype) and nurture (environment). It is far less clear, however, how this might translate into experimental design and the interpretation of animal experiments. Animal experiments are a compromise between modelling real world phenomena with maximal validity (complexity) and designing practicable research projects (abstraction). Textbooks on laboratory animal science generally favour abstraction over complexity. Depending on the area of research, however, abstraction can seriously compromise information gain, with respect to the real world phenomena an experiment is designed to model. Behavioral phenotyping of mouse mutants often deals with particularly complex manifestations of life, such as learning, memory or anxiety, that are strongly modulated by environmental factors. A growing body of evidence indicates that current approaches to behavioral phenotyping might often produce results that are idiosyncratic to the study in which they were obtained, because the interactive nature of genotype-environment relationships underlying behavioral phenotypes was not taken into account. This paper argues that systematic variation of genetic and environmental backgrounds, instead of excessive standardization, is needed to control the robustness of the results and to detect biologically relevant interactions between the mutation and the genetic and environmental background of the animals.

The fallacy of behavioral phenotyping without standardisation.

Behavioral phenotyping of mutant mice is a new and challenging task for the behavioral neuroscientist. Therefore, standardisation of the experimental conditions is required to permit comparisons between the results of experiments within and between laboratories. Once mutation-induced behavioral changes have been identified, phenotyping of mouse mutants should be performed along a systematic trajectory, which allows for an in-depth characterisation of the mutant under investigation.

The individuality of mice.

Mutant mice simulating human CNS disorders are used as models for therapeutic drug development. Drug evaluation requires a coherent correlation between behavioral phenotype and drug status. Variations in behavioral responses could mask such correlations, a problem highlighted by the three-site studies of Crabbe et al. (1999) and Wahlsten et al. (2003a). Factors contributing to variation are considered, focusing on differences between individual animals. Genetic differences due to minisatellite variation suggest that each mouse is genetically distinct. Effects during gestation, including maternal stress, influence later life behavior; while endocrine exchanges between fetus and parent, and between male and female fetuses dependent on intrauterine position, also contribute. Pre and perinatal nutrition and maternal attention also play a role. In adults, endocrine cyclicity in females is a recognized source of behavioral diversity. Notably, there is increasing recognition that groups of wild and laboratory mice have complex social structures, illustrated through consideration of Crowcroft (1966). Dominance status can markedly modify behavior in test paradigms addressing anxiety, locomotion and aggressiveness, to an extent comparable to mutation or drug status. Understanding how such effects amplify the behavioral spectrum displayed by otherwise identical animals will improve testing.

Spontaneous circling behavior and dopamine neuron loss in a genetically hypothyroid mouse.

The genetically hypothyroid mouse, Tshr(hyt), has a single point mutation resulting in a defective thyroid-stimulating hormone receptor, and therefore a non-functional thyroid gland. This is an autosomal recessive disorder and affected mice have been reported to have a number of somatic and behavioral deficits. This study reports a pronounced, spontaneous, asymmetrical circling behavior in the Tshr(hyt) mouse. The spontaneous circling behavior appeared in about 25% of the homozygous animals, in both males and females. The circling usually appeared by postnatal day 35 and continued throughout the lifespan of the animal. The circling was in one direction only, either clockwise or counterclockwise, with the directional preference being almost absolute. A stereological analysis of tyrosine hydroxylase immunoreactive neurons in the substantia nigra and adjacent ventral tegmental area of circling homozygous mice, non-circling homozygous mice and heterozygous mice revealed that the circlers had significantly fewer (40% reduction) midbrain dopamine neurons than those animals that did not circle. There was not an association between the direction of the circling and an asymmetry in the number of dopamine neurons in the midbrains of these mice. There was no difference in the number of dopamine neurons in the midbrain of the homozygous non-circlers and the heterozygous mice. These studies indicate that about 25% of genetically hypothyroid mice demonstrated a spontaneous, perseverative, unilateral circling behavior that was associated with a significant reduction in the number of their midbrain dopamine neurons. Thus congenitally hypothyroid mice are at risk for a reduction in the number of nigral dopamine neurons and an associated repetitive movement disorder.

Behavioral phenotyping of rodents.

Established methods for analyzing behavioral traits in mutant lines of mice allow researchers to understand the outcomes of genetic manipulations in the nervous system. A rigorous six-tiered behavioral phenotyping strategy is described. Recommendations are offered for the design of mouse behavioral testing suites in animal housing facilities.

Phenotype assessment: are you missing something?

BACKGROUND AND PURPOSE: Phenotype assessment of genetically modified rodents is an essential component of animal model development and their eventual use. Described here is a paradigm to consider as we collectively pursue phenotype assessment of the constantly increasing number of genetically modified rodent models, as well as those with spontaneously occurring mutations. METHODS: Review of past experiences and the literature provides useful examples to illustrate the principles described. CONCLUSION: A practical approach to phenotype assessment can be divided into a primary level of assessment to find abnormalities, and a secondary level of more specialized assessment to quantify and evaluate the abnormalities detected. There are many subtle, but important phenotypic characteristics that can be markedly affected by the background genetics and environment of the animal being assessed.

Establishment of an immunodeficient alcohol mouse model to study the effects of alcohol on human cells in vivo.

OBJECTIVE: The effects of alcohol (ethanol) are well documented and contribute to significant health problems and financial burden on the health care system. Several mouse models have been described that facilitate studies of the effects of alcohol on the mouse immune system. Our goal was to establish a chronic alcohol mouse model using the immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse. This severely immunodeficient model has previously been shown to allow efficient engraftment of human hematopoietic repopulating cells and cancer cells, thereby facilitating diverse studies on human hematopoiesis, immune cell function, and oncogenesis in vivo. METHOD: NSG mice were provided ethanol in their drinking water as the only available fluid, starting at 5% weight/volume (w/v) and subsequently were increased to 10%, 15%, and 20% w/v. Mice were then maintained at 20% w/v, a level that models chronic alcohol use in humans. Alcohol consumption and weight were monitored. RESULTS: NSG mice readily consumed alcohol throughout the study and showed no adverse effects. No significant difference between group mean weights was identified the day before increasing the ethanol dose or at the end of 5 weeks at 20% w/v (p > .28). While the mice were maintained at 20% w/v ethanol, the mean daily ethanol intake was 27.2 g/kg of body weight, 32% of caloric intake. CONCLUSIONS: Here we have established a chronic alcohol mouse model using the powerful immunodeficient NSG mouse. This model should allow for novel studies on the effects of alcohol on engrafted human cells, including studies on the effects of alcohol on hematopoiesis, immunity, and cancer.

Neurobehavioral deficits in db/db diabetic mice.

Recent clinical studies indicate neurobehavioral disturbances in type-2 diabetics. However, there is paucity of preclinical research to support this concept. The validity of db/db mouse as an animal model to study type-2 diabetes and related complications is known. The present study was designed to investigate comprehensively the db/db mouse behavior as preclinical evidence of type-2 diabetes related major neurobehavioral complications. We tested juvenile (5-6weeks) and adult (10-11weeks) db/db mice for behavioral depression in forced swim test (FST), psychosis-like symptoms using pre-pulse inhibition (PPI) test, anxiety behavior employing elevated plus maze (EPM) test, locomotor behavior and thigmotaxis using open field test and working memory deficits in Y-maze test. Both juvenile and adult group db/db mice displayed behavioral despair with increased immobility time in FST. There was an age-dependent progression of psychosis-like symptoms with disrupted PPI in adult db/db mice. In the EPM test, db/db mice were less anxious as observed by increased percent open arms time and entries. They were also hypo-locomotive as evident by a decrease in their basic and fine movements. There was no impairment of working memory in the Y-maze test in db/db mice. This is the first report of depression, psychosis-like symptoms and anxiolytic behavior of db/db mouse strain. It is tempting to speculate that this mouse strain can serve as useful preclinical model to study type-2 diabetes related neurobehavioral complications.

Varying intertrial interval reveals temporally defined memory deficits and enhancements in NTAN1-deficient mice.

The N-end rule is one ubiquitin-proteolytic pathway that relates the in vivo half-life of a protein to the identity of its N-terminal residue. NTAN1 deamidates N-terminal asparagine to aspartate, which is conjugated to arginine by ATE1. An N-terminal arginine-bearing substrate protein is recognized, ubiquitylated by UBR1/E3alpha, and subsequently degraded by 26S proteasomes. Previous research showed that NTAN1-deficient mice exhibited impaired long-term memory in the Lashley III maze. Therefore, a series of studies, designed to assess the role of NTAN1 in short- and intermediate-term memory processes, was undertaken. Two hundred sixty mice (126 -/-; 134 +/ +) received Lashley III maze training with intertrial intervals ranging from 2-180 min. Results indicated that inactivation of NTAN1 amidase differentially affects short-, intermediate-, and long-term memory.

A behavior profile of the MRL/Mp lpr/lpr mouse and its association with hydrocephalus.

Since the NZB and BXSB autoimmune mouse strains have cortical ectopias and associated behavioral changes, mice from the MRL/l (MRL/Mp lpr/lpr) autoimmune strain were given a series of behavioral tests followed by anatomical analyses of their brains and immune assays of plasma. No cortical ectopias were found, but this strain was discovered to have a high incidence of hydrocephalus: 46% had severe dilatation of the lateral and third ventricles, 30% had moderate dilatation, and 24% had minimal or no enlargement of the ventricles (total N = 57). Those mice in the severe hydrocephalus group were poorer in a nonspatial discrimination learning task, had less paw asymmetry, and were less active in a swimming task. No association was found between degree of hydrocephalus and any immune parameter.

A behavioral profile of autoimmune lupus-prone MRL mice.

Manifestations of the human autoimmune disease systemic lupus erythematosus (SLE) include a number of behavioral and cognitive deficits. The present study asks whether neurobehavioral dysfunction is present also in MRL mice that spontaneously develop most of the fundamental immunological aberrations of SLE. There are two congenic substrains of MRL mice that differ in the time of disease onset: MRL-lpr mice develop lupus early and MRL(-)+/+ develop the typical signs of disease relatively late in life. The behavior of these substrains was assessed at 7 to 11 weeks of age, a time that coincides with the onset of disease in MRL-lpr mice and the absence of known lupus symptoms in the MRL(-)+/+ group. When compared to the congenic MRL(-)+/+ control substrain, MRL-lpr mice were spontaneously less active, traversed a crossbeam slower, and ceased responding to the novelty of a new environment sooner. They were also more reluctant to leave their home base or travel far away from it and perseverated in their response bias during extinction and reversal learning. Immunological status was characterized by moderate proteinuria in both substrains and high titers of antinuclear antibodies in MRL-lpr but not MRL(-)+/+ mice. Histological analysis revealed minimal or no signs of joint pathology in MRL-lpr mice. Thus, this study shows the presence of behavioral dysfunction in mice with early stages of autoimmune disease and gives support for the idea that MRL mice may provide a useful model of neurobehavioral dysfunction in SLE. It is suggested that the behavioral profile of MRL-lpr mice may indicate increased "timidity," related to genetics, autoimmunity, or both.

Behavior and immune status of MRL mice in the postweaning period.

The notion that the MRL-lpr substrain is a useful model of behavioral and cognitive deficits found in systemic autoimmune diseases is supported by the recent findings of behavioral dysfunction in autoimmune MRL-lpr mice compared to their congenic control MRL +/+ mice. However, it has not been established whether the altered behavioral profile in MRL-lpr mice is the result of the autoimmune process itself or reflects a subtle difference in genetic background or both. To address the question whether MRL-lpr mice are born with behavioral dysfunction the present study compares the behavior of the two MRL substrains in the early postweaning period, when their immune status does not show detectable difference. Results show that the prediseased (4- to 6-week-old) MRL-lpr mice are not distinguishable from the congenic MRL +/+ controls on most behavioral measures except for speed of locomotion and novelty-induced hyperactivity in activity monitors. The immune status of the two substrains is also similar except for a lower hematocrit in the MRL-lpr group. Surprisingly, low amounts of antinuclear and brain-reactive antibodies (possibly transferred from diseased mothers) were detected in the serum of about 50% of the mice in both groups. The lack of major differences in behavior in the premorbid period suggests that appearance of previously reported behavioral dysfunction in the disease state reflects the presence of autoimmunity, time-determined genetic activation, or both.