Genetic background modulates behavioral impairments in R6/2 mice and suggests a role for dominant genetic modifiers in Huntington's disease pathogenesis.

Variability and modification of the symptoms of Huntington's disease (HD) are commonly observed in both patient populations and animal models of the disease. Utilizing a stable line of the R6/2 HD mouse model, the present study investigated the role of genetic background in the onset and severity of HD symptoms in a transgenic mouse. R6/2 congenic C57BL/6J and C57BL/6J×DBA/2J F1 (B6D2F1) mice were evaluated for survival and a number of behavioral phenotypes. This study reports that the presence of the DBA/2J allele results in amelioration or exacerbation of several HD-like phenotypes characteristic of the R6/2 mouse model and indicates the presence of dominant genetic modifiers of HD symptoms. This study is the first step in identifying genes that confer natural genetic variation and modify the HD symptoms. This identification may lead to novel targets for treatment and help elucidate the molecular mechanisms of HD pathogenesis.

Neuronal aggregates are associated with phenotypic onset in the R6/2 Huntington's disease transgenic mouse.

Huntington's disease (HD) is caused by the expansion of the polyglutamine tract expressed in the huntingtin protein. Data from patients show a strong negative correlation between CAG repeat size and age of disease onset. Recent studies in mixed background C57×CBA R6/2 mice suggest the inverse correlation observed in the human disease may not be replicated in some animal models of HD. To further clarify the relationship between repeat length and age of onset, congenic C57BL6/J R6/2 transgenic mice expressing 110, 260 or 310 CAG were tested in a comprehensive behavioral battery at multiple ages. Data confirmed the findings of earlier studies and indicate that on a pure C57BL6/J genetic background, R6/2 mice with larger repeats exhibit a delay in phenotypic onset with increasing polyglutamine size (6 weeks in 110 CAG and 17 weeks in 310 CAG mice). Further analysis confirmed a decrease in transgene transcript expression in 310 CAG mice as well as differential aggregated protein localization in association with repeat length. Mice expressing 110 CAG developed aggregates that localized almost exclusively to the nucleus of neuronal cells in the striatum and cortex. In contrast, tissue from 310 CAG mice exhibited predominantly extranuclear inclusions. Novel mutant protein analysis obtained using time-resolved fluorescence resonance energy transfer (FRET) revealed that soluble protein levels decreased with disease onset in R6/2 mice while aggregated protein levels increased. We believe that these data suggest a role for aggregation and inclusion localization in HD pathogenesis and propose a mechanism for the age of onset delay observed in R6/2 mice.

Genetic reduction of muscarinic M4 receptor modulates analgesic response and acoustic startle response in a mouse model of fragile X syndrome (FXS).

INTRODUCTION: The G-protein coupled muscarinic acetylcholine receptors, widely expressed in the CNS, have been implicated in fragile X syndrome (FXS). Recent studies have reported an overactive signaling through the muscarinic receptors in the Fmr1KO mouse model. Hence, it was hypothesized that reducing muscarinic signaling might modulate behavioral phenotypes in the Fmr1KO mice. Pharmacological studies from our lab have provided evidence for this hypothesis, with subtype-preferring muscarinic M1 and M4 receptor antagonists modulating select behaviors in the Fmr1KO mice. Since the pharmacological antagonists were not highly specific, we investigated the specific role of M4 receptors in the Fmr1KO mouse model, using a genetic approach. METHODS: We created a double mutant heterozygous for the M4 receptor gene and hemizygous for the Fmr1 gene and examined the mutants on various behaviors. Each animal was tested on a behavior battery comprising of open-field activity (activity), light-dark (anxiety), marble burying (perseverative behavior), prepulse inhibition (sensorimotor gating), rotarod (motor coordination), passive avoidance (learning and memory) and hotplate (analgesia). Animals were also tested on the audiogenic seizure protocol and testis weights were measured. RESULTS: Reduction of M4 receptor expression in the heterozygotes completely rescued the analgesic response and partly rescued the acoustic startle response phenotype in the Fmr1KO mice. However, no modulation was observed in a number of behaviors including learning and memory, activity, perseverative behavior and audiogenic seizures. CONCLUSION: Reducing M4 receptor signaling altered only select behavioral phenotypes in the Fmr1KO mouse model, suggesting that other targets are involved in the modulation of fragile X behaviors.

Group I metabotropic glutamate receptor antagonists alter select behaviors in a mouse model for fragile X syndrome.

RATIONALE: Studies in the Fmr1 knockout (KO) mouse, a model of fragile X syndrome (FXS), suggest that excessive signaling through group I metabotropic glutamate receptors (mGluRs), comprised of subtypes mGluR1 and mGluR5, may play a role in the pathogenesis of FXS. Currently, no studies have assessed the effect of mGluR1 modulation on Fmr1 KO behavior, and there has not been an extensive behavioral analysis of mGluR5 manipulation in Fmr1 KO mice. OBJECTIVES: The goals for this study were to determine if pharmacologic blockade of mGluR1 may affect Fmr1 KO behavior as well as to expand on the current literature regarding pharmacologic blockade of mGluR5 on Fmr1 KO behavior. METHODS: Reduction of mGluR1 or mGluR5 activity was evaluated on a variety of behavioral assays in wild-type (WT) and Fmr1 KO mice through the use of antagonists: JNJ16259685 (JNJ, mGluR1 antagonist) and MPEP (mGluR5 antagonist). RESULTS: JNJ and MPEP decreased marble burying in both WT and Fmr1 KO mice without reductions in activity. Neither JNJ nor MPEP affected the prepulse inhibition in either WT or Fmr1 KO mice. JNJ did not affect Fmr1 KO motor coordination but did impair WT performance. MPEP improved a measure of motor learning in Fmr1 KO but not WT mice. While both JNJ and MPEP decreased the audiogenic seizures in the Fmr1 KO, MPEP completely abolished the manifestation of seizures. CONCLUSION: These data illustrate that, while the manipulation of either mGluR1 or mGluR5 can affect select behaviors in the Fmr1 KO, we observe greater effects upon mGluR5 reduction.

Onset and progression of behavioral and molecular phenotypes in a novel congenic R6/2 line exhibiting intergenerational CAG repeat stability.

In the present study we report on the use of speed congenics to generate a C57BL/6J congenic line of HD-model R6/2 mice carrying 110 CAG repeats, which uniquely exhibits minimal intergenerational instability. We also report the first identification of the R6/2 transgene insertion site. The relatively stable line of 110 CAG R6/2 mice was characterized for the onset of behavioral impairments in motor, cognitive and psychiatric-related phenotypes as well as the progression of disease-related impairments from 4 to 10 weeks of age. 110Q mice exhibited many of the phenotypes commonly associated with the R6/2 model including reduced activity and impairments in rotarod performance. The onset of many of the phenotypes occurred around 6 weeks and was progressive across age. In addition, some phenotypes were observed in mice as early as 4 weeks of age. The present study also reports the onset and progression of changes in several molecular phenotypes in the novel R6/2 mice and the association of these changes with behavioral symptom onset and progression. Data from TR-FRET suggest an association of mutant protein state changes (soluble versus aggregated) in disease onset and progression.

The modulation of fragile X behaviors by the muscarinic M4 antagonist, tropicamide.

Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors (M1-M5), grouped together into two functional classes, based on their G protein interaction. Although ubiquitously expressed in the CNS, the M4 protein shows highest expression in the neostriatum, cortex, and hippocampus. Electrophysiological and biochemical studies have provided evidence for overactive mAChR signaling in the fragile X knock-out (Fmr1KO) mouse model, and this has been hypothesized to contribute to the phenotypes seen in Fmr1KO mice. To address this hypothesis we used an M4 antagonist, tropicamide, to reduce the activity through the M4 mAChR and investigated the behavioral response in the Fmr1KO animals. Data from the marble-burying assay have shown that tropicamide treatment resulted in a decreased number of marbles buried in the wild-type (WT) and in the knockout (KO) animals. Results from the open field assay indicated that tropicamide increases activity in both the WT and KO mice. In the passive avoidance assay, tropicamide treatment resulted in the improvement of performance in both the WT and the KO animals at the lower doses (2 and 5 mg/kg), and the drug was shown to be important for the acquisition and not the consolidation process. Lastly, we observed that tropicamide causes a significant decrease in the percentage of audiogenic seizures in the Fmr1KO animals. These results suggest that pharmacological antagonism of the M4 receptor modulates select behavioral responses in the Fmr1KO mice.

Genetic reduction of group 1 metabotropic glutamate receptors alters select behaviors in a mouse model for fragile X syndrome.

INTRODUCTION: Genetic heterogeneity likely contributes to variability in the symptoms among individuals with fragile X syndrome (FXS). Studies in the Fmr1 knockout (KO) mouse model for FXS suggest that excessive signaling through group I metabotropic glutamate receptors (Gp1 mGluRs), comprised of subtypes mGluR1 and mGluR5, may play a role. Hence, Gp1 mGluRs may act as modifiers of FXS. Currently no studies have addressed whether manipulation of mGluR1 activity may alter Fmr1 KO behavioral responses, and only a few have reported the effects of mGluR5 manipulation. Therefore, the goals for this study were to extend our understanding of the effects of modulating Gp1 mGluR activity on Fmr1 KO behavioral responses. METHODS: The present study determined if genetically reducing mGluR1 or mGluR5 by 50% affects an extensive array of behaviors in the Fmr1 KO. RESULTS: Reduction of mGluR1 moderately decreased Fmr1 KO activity. Reduction of mGluR5 caused an analgesic response in the Fmr1 KO and decreased active social behavior. Modulation of either mGluR1 or mGluR5 did not significantly alter audiogenic seizures, anxiety- and perseverative-related responses, sensorimotor gating, memory, or motor responses. CONCLUSIONS: Genetic reduction of mGluR1 or mGluR5 modified a few select Fmr1 KO behaviors, although these modifications appeared to be subtle in nature and/or limited to select behaviors. This may indicate that 50% reduction of either mGluR1 or mGluR5 is insufficient to produce behavioral changes, and therefore, these receptors may not be dominant modifiers of a number of Fmr1 KO behavioral phenotypes.

Modulation of behavioral phenotypes by a muscarinic M1 antagonist in a mouse model of fragile X syndrome.

RATIONALE: Muscarinic acetylcholine receptors (mAChR) are G protein-coupled receptors, widely expressed in the CNS. Electrophysiological and molecular studies have provided evidence for overactive M1 receptor signaling in the fragile X knockout (Fmr1 KO) mouse model, suggesting the involvement of the M1 receptors in fragile X syndrome. Overactive signaling through the M1 receptor has been hypothesized to contribute to the phenotypes seen in fragile X mice. OBJECTIVE: We investigated the modulation of behavioral responses in the Fmr1 KO animals by reducing the activity through the muscarinic M1 receptor using the pharmacological agent dicyclomine, an M1 antagonist. METHODS: The behavioral assays used to investigate the pharmacological effects include marble burying (perseverative behavior), open-field exploration (activity), passive avoidance (learning and memory), prepulse inhibition (sensorimotor gating), and audiogenic seizures. RESULTS: Data from the marble-burying assay suggests that treatment with dicyclomine results in a decrease in the number of marbles buried in the wild-type and in the KO animals. To examine the possibility of drug-induced sedation, overall activity was measured in an open-field chamber. Dicyclomine only increases activity at a dose of 20 mg/kg in the wild-type mice but did not affect exploration in the KO animals. Lastly, we observed that dicyclomine causes a significant decrease in the percentage of audiogenic seizures in the Fmr1 KO animals. CONCLUSION: Our findings suggest that pharmacologically reducing the activity through the mAChR M1 alters select behavioral responses in the Fmr1 KO mice.

Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety.

RATIONALE: An increasing number of investigators utilize the marble-burying assay despite the paucity of information available regarding what underlies the behavior. OBJECTIVES: We tested the possibility that a genetic component underlies marble burying in mice and if there is a genetic correlation with other anxiety-like traits. Since findings reported in the literature indicate that marble-burying behavior reflects an anxiety-like response, we explored the assumption that the novel nature of a marble induces this anxiety. Finally, we investigated how the natural response of a mouse to dig relates to the marble-burying phenomenon. METHODS: We examined ten different inbred mouse strains to determine if marble-burying behavior is genetically regulated and correlated with anxiety-like traits in two other assays. We employed multiple variants of the "traditional" marble-burying assay to address how issues such as the novelty of marbles and digging behavior contribute to marble burying. RESULTS: Marble-burying behavior varied across strain and did not correlate with anxiety measures in other assays. Multiple tests conducted to reduce the novelty of marbles failed to alter burying behavior. Additionally, digging behavior correlated with marble burying, and the presence of marbles did not significantly impact the digging response. CONCLUSIONS: Our results indicate that mouse marble burying is genetically regulated, not correlated with other anxiety-like traits, not stimulated by novelty, and is a repetitive behavior that persists/perseveres with little change across multiple exposures. Marble burying is related to digging behavior and may in fact be more appropriately considered as an indicative measure of repetitive digging.