Implementation of a manual for working with wobbler mice and criteria for discontinuation of the experiment.

Mouse breeding is of importance to a whole range of medical and biological research. There are many known mouse models for motor neuron diseases. However, it must be kept in mind that especially mouse models for amyotrophic lateral sclerosis develop severe symptoms causing intense stress. This article is designed to summarize conscientious work with the wobbler mouse, a model for the sporadic form of amyotrophic lateral sclerosis. This mouse model is characterized by a degeneration of α-motor-neurons leading to head tremor, loss of body weight and rapidly progressive paralysis. Although this mouse model has been known since 1956, there are no guidelines for breeding wobbler mice. Due to the lack of such guidelines the present study tries to close this gap and implements a manual for further studies. It includes the whole workflow in regard to wobbler mice from breeding and animal care taking, genotyping and phenotype analysis, but also gives some examples for the use of various neuronal tissues for histological investigation. Beside the progress in research a second aim should always be the enhancement of mouse welfare and reduction of stress for the laboratory animals.

Neurobehavioral performances and brain regional metabolism in Dab1(scm) (scrambler) mutant mice.

As disabled-1 (DAB1) protein acts downstream in the reelin signaling pathway modulating neuronal migration, glutamate neurotransmission, and cytoskeletal function, the disabled-1 gene mutation (scrambler or Dab1(scm) mutation) results in ataxic mice displaying dramatic neuroanatomical defects similar to those observed in the reeler gene (Reln) mutation. By comparison to non-ataxic controls, Dab1(scm) mutants showed severe motor coordination impairments on stationary beam, coat-hanger, and rotorod tests but were more active in the open-field. Dab1(scm) mutants were also less anxious in the elevated plus-maze but with higher latencies in the emergence test. In mutants versus controls, changes in regional brain metabolism as measured by cytochrome oxidase (COX) activity occurred mainly in structures intimately connected with the cerebellum, in basal ganglia, in limbic regions, particularly hippocampus, as well as in visual and parietal sensory cortices. Although behavioral results characterized a major cerebellar disorder in the Dab1(scm) mutants, motor activity impairments in the open-field were associated with COX activity changes in efferent basal ganglia structures such as the substantia nigra, pars reticulata. Metabolic changes in this structure were also associated with the anxiety changes observed in the elevated plus-maze and emergence test. These results indicate a crucial participation of the basal ganglia in the functional phenotype of ataxic Dab1(scm) mutants.

Paranodal permeability in "myelin mutants".

Fluorescent dextran tracers of varying sizes have been used to assess paranodal permeability in myelinated sciatic nerve fibers from control and three "myelin mutant" mice, Caspr-null, cst-null, and shaking. We demonstrate that in all of these the paranode is permeable to small tracers (3 kDa and 10 kDa), which penetrate most fibers, and to larger tracers (40 kDa and 70 kDa), which penetrate far fewer fibers and move shorter distances over longer periods of time. Despite gross diminution in transverse bands (TBs) in the Caspr-null and cst-null mice, the permeability of their paranodal junctions is equivalent to that in controls. Thus, deficiency of TBs in these mutants does not increase the permeability of their paranodal junctions to the dextrans we used, moving from the perinodal space through the paranode to the internodal periaxonal space. In addition, we show that the shaking mice, which have thinner myelin and shorter paranodes, show increased permeability to the same tracers despite the presence of TBs. We conclude that the extent of penetration of these tracers does not depend on the presence or absence of TBs but does depend on the length of the paranode and, in turn, on the length of "pathway 3," the helical extracellular pathway that passes through the paranode parallel to the lateral edge of the myelin sheath.

Unraveling the complexity of amyotrophic lateral sclerosis: recent advances from the transgenic mutant SOD1 mice.

Amyotrophic Lateral Sclerosis (ALS), which accounts for the majority of motor neuron disorders, is a progressive and fatal neurodegenerative disease leading to complete paralysis of skeletal muscles and premature death usually from respiratory failure. About 10% of all ALS cases are inherited, with the responsible gene having been identified in approximately 25% of these individuals. Mutations in the copper-zinc superoxide dismutase (SOD1) gene were the first to be recognized nearly twenty years ago, and since then different animal models, in particular transgenic rodents, have been developed. They replicate many of the clinical, neuropathological and molecular features of ALS patients and have contributed significantly to our understanding of the pathogenic mechanisms of this disease. Although results obtained so far with mutant SOD1 mice have not translated into effective therapies in ALS patients, these models still represent the only experimentally accessible system to study multiple aspects of disease pathogenesis and to provide proof-of-principle for the development of new therapeutic strategies. This review will examine the most recent discoveries obtained from these animal models in an attempt to elucidate the complex mechanisms of the disease. In particular it will focus on the contribution of multiple cell types in governing the disease development and progression.

Developmental anatomy of reeler mutant mouse.

The reeler mouse is one of the most famous spontaneously occurring mutants in the research field of neuroscience, and this mutant has been used as a model animal to understand mammalian brain development. The classical observations emphasized that laminar structures of the reeler brain are highly disrupted. Molecular cloning of Reelin, the gene responsible for reeler mutant provided insights into biochemistry of Reelin signal, and some models had been proposed to explain the function of Reelin signal in brain development. However, recent reports of reeler found that non-laminated structures in the central nervous system are also affected by the mutation, making function of Reelin signal more controversial. In this review, we summarized reported morphological and histological abnormalities throughout the central nervous system of the reeler comparing to those of the normal mouse. Based on this overview of the reeler abnormalities, we discuss possible function of Reelin signal in the neuronal migration and other morphological events in mouse development.

Mouse forward genetics in the study of the peripheral nervous system and human peripheral neuropathy.

Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics. Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as a human disease gene causing multiple forms of peripheral neuropathy, to the more recent identification of the claw paw and sprawling mutations, forward genetics has long been a tool for probing the physiology, pathogenesis, and genetics of the PNS. Even as spontaneous and mutagenized mice continue to enable the identification of novel genes, provide allelic series for detailed functional studies, and generate models useful for clinical research, new methods, such as the piggyBac transposon, are being developed to further harness the power of forward genetics.

Gene-environment interaction during early development in the heterozygous reeler mouse: clues for modelling of major neurobehavioral syndromes.

Autism and schizophrenia are multifactorial disorders with increasing prevalence in the young population. Among candidate molecules, reelin (RELN) is a protein of the extracellular matrix playing a key role in brain development and synaptic plasticity. The heterozygous (HZ) reeler mouse provides a model for studying the role of reelin deficiency for the onset of these syndromes. We investigated whether early indices of neurobehavioral disorders can be identified in the infant reeler, and whether the consequences of ontogenetic adverse experiences may question or support the suitability of this model. A first study focused on the link between early exposure to Chlorpyryfos and its enduring neurobehavioral consequences. Our data are interesting in view of recently discovered cholinergic abnormalities in autism and schizophrenia, and may suggest new avenues for early pharmacological intervention. In a second study, we analyzed the consequences of repeated maternal separation early in ontogeny. The results provide evidence of how unusual stress early in development are converted into altered behavior in some, but not all, individuals depending on gender and genetic background. A third study aimed to verify the reliability of the model at critical age windows. Data suggest reduced anxiety, increased impulsivity and disinhibition, and altered pain threshold in response to morphine for HZ, supporting a differential organization of brain dopaminergic, serotonergic and opioid systems in this genotype. In conclusion, HZ exhibited a complex behavioral and psycho-pharmacological phenotype, and differential responsivity to ontogenetic adverse conditions. HZ may be used to disentangle interactions between genetic vulnerability and environmental factors. Such an approach could help to model the pathogenesis of neurodevelopmental psychiatric diseases.

Neurobehavioral mutants identified in an ENU-mutagenesis project.

We report on a battery of behavioral screening tests that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU-mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and used a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open-field mutants (one displaying hyperlocomotion, the other hypolocomotion), four tail-suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning-and-memory mutant (displaying reduced response to the conditioned stimulus). These findings highlight the utility of a set of behavioral tasks used in a high-throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

The functions of UCH-L1 and its relation to neurodegenerative diseases.

Parkinson's disease (PD) and Alzheimer's disease (AD), the most common neurodegenerative diseases, are caused by both genetic and environmental factors. Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is involved in the pathogenesis of both of these neurodegenerative diseases. Several functions of UCH-L1, other than as an ubiquitin hydrolase, have been proposed; these include acting as an ubiquitin ligase and stabilizing mono-ubiquitin. This review focuses on recent findings on the functions and the regulation of UCH-L1, in particular those that relate to PD and AD.

Retrograde infection of precerebellar nuclei neurons by injection of a recombinant adenovirus into the cerebellar cortex of normal and reeler mice.

The reeler mouse is an autosomal recessive mutant mouse caused by mutation of the reelin gene and characterized by cerebellar ataxia. To determine whether the distribution pattern of precerebellar nuclei neurons in the brainstem of the reeler mouse changes, we injected a small volume of a replication-defective recombinant adenovirus carrying E. coli beta-galactosidase (lacZ) into the cerebellar cortex of normal and reeler mice. Five days later, the mice were transcardially perfused by a fixative solution. X-gal staining of coronal or sagittal sections of the brainstem revealed that many origins for reticulocerebellar, cuneocerebellar, trigeminocerebellar, and pontocerebellar projections were retrogradely labeled, but only a few olivocerebellar neurons were labeled. Retrogradely labeled neurons in the lateral reticular nucleus tended to locate more laterally and be more condensed into a small compartment in the reeler compared with their normal counterparts. Retrogradely labeled neurons in the external cuneate nucleus were more dorsally shifted in the reeler mice compared with their normal counterparts. We could not find any differences between the normal and reeler mice in the distribution patterns of their trigeminocerebellar projection neurons. Retrogradely labeled pontocerebellar neurons in the basilar pons of the reeler mouse were reduced in number compared with their normal counterparts in addition to being more ventrally and laterally shifted. These findings strongly suggest that the migration of some precerebellar nuclei neurons from the rhombic lip to their final loci may be obstructed in the reeler mice.

Hormonal and morphological study of the pituitaries in reeler mice.

Reelin is a neuronal glycoprotein that plays a crucial role in brain layer formation during prenatal development. The reeler mutant mouse lacks Reelin, leading to abnormalities in the neuronal layering of cerebral cortex and cerebellum, producing ataxia, tremor and abnormal locomotion. Reeler mice are reported to have growth retardation and most of them are sterile or unable to bring up their newborns. Since the brain is one of the main regulator of pituitary hormone secretion and no information was reported regarding pituitary function and structure in these mutant mice, we studied pituitary endocrine activity and morphology in reeler mice. Mice were classified in three groups as reeler homozygote (RHM), reeler heterozygote (RHT) or control (CO). Pituitary hormone blood levels were assessed by enzyme immunoassay (EIA) and immunoradiometric assay (IRMA). Animals and their pituitaries were weighted and pituitaries were studied by histology, immunohistochemistry and electron microscopy. Results showed statistically significant differences in body weight and in adrenocorticotropic hormone (ACTH) and luteinizing hormone (LH) blood levels between the three groups. In contrast, growth hormone (GH) blood levels showed a high individual variation and no decrease in reeler groups compared with CO. Morphological studies revealed no differences in pituitary cell types except that somatotrophs appeared to be slightly smaller in RHM and RHT. Although it seems that pituitary hypofunction is not responsible for growth retardation, more studies are needed to obtain a deeper insight into the endocrine status of these mutant mice to elucidate the cause of their low body weight and reproductive behaviour.

The natural history and treatment of epilepsy in a murine model of tuberous sclerosis.

PURPOSE: Patients with tuberous sclerosis complex (TSC) often have severe epilepsy that is intractable to available therapies. The development of novel treatments for epilepsy in TSC would benefit greatly from a suitable animal model, but most animal models of TSC to date have few reported neurological abnormalities, such as epilepsy. We previously described a novel model of TSC, due to conditional inactivation of the Tsc1 gene in glia (Tsc1(GFAP)CKO mice), in which mice develop epilepsy and premature death. Here, we characterize the natural history of the epilepsy in Tsc1(GFAP)CKO mice in more detail and report acute effects of treatment with standard antiepileptic drugs on seizures in these mice. METHODS: Video-EEG recordings were obtained from Tsc1(GFAP)CKO mice on a weekly basis, starting at 4 weeks of age until death, to monitor progression of interictal EEG abnormalities and seizures. In separate experiments, Tsc1(GFAP)CKO mice were monitored for interictal EEG abnormalities and seizures before and during treatment with phenobarbital, phenytoin, or saline. RESULTS: Tsc1(GFAP)CKO mice developed seizures around 4-6 weeks of age and subsequently had progressive worsening of the interictal EEG background and seizure frequency over a month, culminating in death. Treatment with phenobarbital or phenytoin caused a reduction in seizure frequency, but did not improve EEG background or prevent death. CONCLUSIONS: Tsc1(GFAP)CKO mice develop progressive epilepsy. Acute treatment with standard antiepileptic drugs suppresses seizures in these mice, but does not affect long-term prognosis. Tsc1(GFAP)CKO mice represent a good model to test other drugs that may have true antiepileptogenic actions in TSC.

Cochlear pathology of the circling mouse: a new mouse model of DFNB6.

CONCLUSION: The circling mouse (cir/cir) has phenotypes which follow the pattern of neuroepithelial defects of deafness from 10 days after birth. The cir mouse is defective in Tmie gene, the function of which should be further elucidated. OBJECTIVES: We previously reported a recessive mutation of deafness called circling mice (cir/cir). The present study focused on investigating phenotypes and histological findings of the cochlea in circling mice with respect to age. MATERIALS AND METHODS: In order to analyze cochlear pathology over time, five different age groups of circling mice were examined (10, 18, 21, 35, and 90 days old). The organs of Corti and spiral ganglion neurons in basal and middle turns were evaluated. RESULTS: The pathology of the organ of Corti followed the pattern of neuroepithelial defects. Hair cells in organs of Corti had degenerated in circling mice at 10 days old, in a time-dependent manner. Scanning electron microscopy (SEM) showed that stereociliary bundles were irregular in size and had shortened at 10 days, and that this degeneration was complete at 21 days. The number of spiral ganglion neurons significantly reduced with age. RT-PCR analysis indicated that the transmembrane inner ear gene (Tmie) was absent in various organs in circling mice.

Alteration of cerebellar neurotropin messenger ribonucleic acids and the lack of thyroid hormone receptor augmentation by staggerer-type retinoic acid receptor-related orphan receptor-alpha mutation.

The mutant mouse staggerer (sg) harbors a deletion within the gene encoding the retinoic acid receptor-related orphan receptor-alpha (RORalpha). Homozygotes show aberrant cerebellar development. However, the mechanisms responsible for the cerebellar defect are still poorly understood. In the present study, the involvement of neurotropins (NTs), including nerve growth factor, brain-derived neurotropic factor, NT-3 and NT-4/5, and their receptors, which play a crucial role in brain development, on the cerebellar defects of sg mice was studied by semiquantitative RT-PCR and in situ hybridization histochemistry. An evident alteration of these mRNA levels was observed in both heterozygotes and homozygotes. Such difference was most evident in the internal granule cell layer. Because the changes in NT expression as well as morphological alterations in sg cerebellum are similar to those in hypothyroid animals, the effect of mutant RORalpha (RORsg) on transcriptional regulation through the thyroid hormone (TH) response element or the ROR response element (RORE) was then studied. RORsg neither activated the transcription through RORE nor suppressed RORalpha-induced transcription, indicating that it does not function as a dominant negative inhibitor. On the other hand, although wild-type RORalpha augmented TH receptor (TR)alpha1/beta1-mediated transcription through various TH response elements, RORsg was not effective in augmenting TR action. These results suggest that the cerebellar defect of the sg mouse is partly caused by the altered expression of NTs and the lack of augmentation of TR-mediated transcription by RORalpha as well as the absence of RORalpha action through RORE.

Ho15J: a new hotfoot allele in a hot spot in the gene encoding the delta2 glutamate receptor.

Hotfoot, a recessive mouse mutation characterized by ataxia and jerky movements of the hindlimbs, is caused by various mutations in the gene (Grid2) encoding the delta2 glutamate receptor (GluRdelta2). So far, at least 20 alleles, arising either spontaneously or through the random insertion of transgenes, have been described. Interestingly, most hotfoot mutants have deletions of one or more exons coding for portions of the most amino-terminal domain of GluRdelta2. However, because live mice colonies are no longer available for most hotfoot mutants, the possibility that the loss of a part of an intron might affect the splicing of other exons or the general efficiency of transcription could not be ruled out. Here, we report that a newly identified hotfoot mutant, ho15J, was caused by an intragenic deletion of the Grid2 gene, which indeed resulted in a new type of 52-amino-acid deletion in the most amino-terminal domain of GluRdelta2. Like GluRdelta2 proteins in ho4J mutants, GluRdelta2 proteins in ho15J mice were retained in the soma of Purkinje cells, where they were degraded. Long-term depression, a form of synaptic plasticity underlying information storage in the cerebellum, was abrogated, and ho15J mice showed severe motor discoordination on rotarod tests. The agreement between the PCR results for genomic DNA and the RT-PCR results for the ho15J allele supports the view that PCR analyses of grid2 genomic DNA can predict alterations in mRNA and protein. In addition, the present findings underscore the importance of the most amino-terminal domain in GluRdelta2 signaling and cerebellar functions.

The Lurcher mouse: fresh insights from an old mutant.

The Lurcher mouse was first discovered in 1954 as a spontaneously occurring autosomal dominant mutation that caused the degeneration of virtually all cerebellar Purkinje cells and most olivary neurons and granule cells. More recent molecular studies revealed that Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that converts an alanine to threonine in the highly conserved third hydrophobic segment of GluRdelta2. The mutation converts the receptor into a constitutively leaky cation channel. The GluRdelta2 receptor is predominantly expressed in cerebellar Purkinje cells and in the heterozygous Lurcher mutant (+/Lc). Purkinje cells die due to the mutation in the GluRdelta2 receptor, while olivary neurons and granule cells degenerate due to the loss of their Purkinje cell targets. The purpose of the review is to provide highlights from 5 decades of research on the Lurcher mutant that have provided insights into the developmental mechanisms that regulate cell number during development, cerebellar pattern formation, cerebellar physiology, and the role of the cerebellum in CNS function.

ROR alpha in genetic control of cerebellum development: 50 staggering years.

The staggerer mutation was first identified at the Jackson Laboratory in 1955. In the ensuing half-century, studies of staggerer mice have provided new insights into developmental neurobiology, gene regulatory networks, and circadian behavior. Recent work has expanded the role of RORalpha, the transcription factor mutated in staggerer, to peripheral tissues, including cholesterol and lipid metabolism, immune function, and bone development. This review focuses on the role of RORalpha in neural development and behavior revealed by the staggerer mutation and subsequent molecular studies.

Tissue, cellular and sub-cellular localization of the Vangl2 protein during embryonic development: effect of the Lp mutation.

Loop-tail (Lp) mice show a very severe neural tube defect, craniorachischisis, which is caused by mis-sense mutations in the Vangl2 gene. The membrane protein Vangl2 belongs to a highly conserved group of proteins that regulate planar polarity in certain epithelia, and that are also important for convergent extension movements during gastrulation and neurulation. A specific anti-Vangl2 antiserum was produced and used to examine the tissue, cell type, and sub-cellular localization of Vangl2 during embryogenesis. Vangl2 protein is expressed at high levels in the neural tube and shows a dynamic expression profile during neurulation. After neural tube closure, robust Vangl2 staining is detected in several neural and neurosensory tissues, including cerebral cortex, dorsal root ganglia, olfactory epithelium, retina, mechanosensory hair cells of the cochlea, and optic nerve. Vangl2 is also expressed during organogenesis in a number of tubular epithelia, including the bronchial tree, intestinal crypt/villus axis, and renal tubular segments derived from ureteric bud and from metanephric mesenchyme. Examination of Vangl2 localization in the neural tubes and cochleas of the normal and Lp/Lp embryos shows disruption of normal membrane localization of Vangl2 in independent alleles at Lp (Lp, Lp(m1Jus)) as well as overall decrease in the expression level.

Neuroanatomical phenotypes in the reeler mouse.

The reeler mouse (Reln) has been proposed as a neurodevelopmental model for certain neurological and psychiatric conditions and has been studied by qualitative histochemistry and electron microscopy. Using magnetic resonance microscopy (MRM), we have quantitated for the first time the neuromorphology of Reln mice at a resolution of 21.5 microm. The neuroanatomical phenotypes of heterozygous and homozygous mutant Reln mice were compared to those of wild type (WT) littermates using morphometry and texture analysis. The cortical, hippocampal, and cerebellar phenotypes of the heterozygous and homozygous mutant Reln mice were confirmed, and new features were revealed. The Reln(rl/rl) mice possessed a smaller brain, and both Reln(rl/+) and Reln(rl/rl) mice had increased ventricles compared to WT controls. Shape differences were found between WT and Reln(rl/rl) brains, specifically in cerebellum, olfactory bulbs, dorsomedial frontal and parietal cortex, certain regions of temporal and occipital lobes, as well as in the lateral ventricles and ventral hippocampus. These findings suggest that certain brain regions may be more severely impacted by the Reln mutation than others. Gadolinium-based active staining demonstrated that layers of the hippocampus were disorganized in Reln(rl/rl) mice and differences in thickness of these layers were identified between WT and Reln(rl/rl) mice. The intensity distributions characteristic to the dorsal, middle, and ventral hippocampus were altered in the Reln(rl/rl), especially in the ventral hippocampus. These differences were quantified using skewness and modeling the intensity distributions with a Gaussian mixture. Our results suggest that structural features of Reln(rl/rl) brain most closely phenocopy those of patients with Norman-Roberts lissencephaly.

Routine tail suspension husbandry facilitates onset of seizure susceptibility in EL mice.

PURPOSE: Tail suspension can elicit seizures in susceptible EL mice, a model of idiopathic, multifactorial epilepsy. Further, repeated tail suspension hastens the lifetime onset of seizure susceptibility in these mice. The present study tested the hypothesis that curtailing human handling during development would delay the onset of seizure susceptibility relative to EL mice handled regularly by using tail suspension for standard laboratory husbandry. METHODS: Control mice were handled by the tail for bedding changes, whereas unhandled mice bedding was changed by using specially designed connector cages that allowed mice to transfer without handling to a cage containing clean bedding. Seizure susceptibility was tested beginning at 70, 80, 90, 100, or 140 days of age by using a handling-induced seizure-susceptibility paradigm. RESULTS: Among handled mice, more than half of the sample exhibited seizures by age 80 days relative to fewer than one fourth of unhandled mice. In addition, each group was tested a second time 10 days after the initial seizure-susceptibility test to detect potential experience-induced increases in seizure susceptibility. Once again, a higher frequency of handled mice expressed seizures at significantly younger ages relative to unhandled mice. CONCLUSIONS: Although it was already known that repeated tail suspension could speed the onset of seizure susceptibility in EL mice, the present results are the first to demonstrate the converse finding that decreasing routine human handling can delay significantly the onset of seizure susceptibility. This suggests that removal of nonconsensual aspects of human-animal contact may delay or prevent the onset of seizure susceptibility.