Early postnatal development of the MDGA2+/- mouse model of synaptic dysfunction.

Synaptic dysfunction underlies many neurodevelopmental disorders (NDDs). The membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) regulate synaptic development by modulating neurexin-neuroligin complex formation. Since understanding the neurodevelopmental profile and the sex-based differences in the manifestation of the symptoms of NDDs is important for their early diagnosis, we tested a mouse model haploinsufficient for MDGA2 (MDGA2+/-) on a neurodevelopmental test battery, containing sensory, motor, and cognitive measures, as well as ultrasonic vocalizations. When male and female MDGA2+/- and wildtype (WT) C57BL/6 J mice were examined from 2 to 23 days of age using this test battery, genotype and sex differences in body weight, sensory-motor processes, and ultrasonic vocalizations were observed. The auditory startle reflex appeared earlier in the MDGA2+/- than in WT mice and the MDGA2+/- mice produced fewer ultrasonic vocalizations. The MDGA2+/- mice showed reduced locomotion and rearing than WT mice in the open field after 17 days of age and spent less time investigating a novel object than WT mice at 21 days of age. Female MDGA2+/- mice weighed less than WT females and showed lower grip strength, indicating a delay in sensory-motor development in MDGA2+/- mice, which appears to be more pronounced in females than males. The behavioural phenotypes resulting from MDGA2 haploinsufficiency suggests that it shows delayed development of motor behaviour, grip strength and exploratory behaviour, non-social phenotypes of NDDs.

Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1-42 in Alzheimer's disease.

Reduced insulin-like growth factor 2 (IGF2) levels in Alzheimer's disease (AD) may be the mechanism relating age-related metabolic disorders to dementia. Since Igf2 is an imprinted gene, we examined age and sex differences in the relationship between amyloid-beta 1-42 (Aß42) accumulation and epigenetic regulation of the Igf2/H19 gene cluster in cerebrum, liver, and plasma of young and old male and female 5xFAD mice, in frontal cortex of male and female AD and non-AD patients, and in HEK293 cell cultures. We show IGF2 levels, Igf2 expression, histone acetylation, and H19 ICR methylation are lower in females than males. However, elevated Aß42 levels are associated with Aß42 binding to Igf2 DMR2, increased DNA and histone methylation, and a reduction in Igf2 expression and IGF2 levels in 5xFAD mice and AD patients, independent of H19 ICR methylation. Cell culture results confirmed the binding of Aß42 to Igf2 DMR2 increased DNA and histone methylation, and reduced Igf2 expression. These results indicate an age- and sex-related causal relationship among Aß42 levels, epigenomic state, and Igf2 expression in AD and provide a potential mechanism for Igf2 regulation in normal and pathological conditions, suggesting IGF2 levels may be a useful diagnostic biomarker for Aß42 targeted AD therapies.

The effect of background strain on the behavioral phenotypes of the MDGA2+/- mouse model of autism spectrum disorder.

The membrane-associated mucin (MAM) domain containing glycosylphosphatidylinositol anchor 2 protein single knock-out mice (MDGA2+/- ) are models of ASD. We examined the behavioral phenotypes of male and female MDGA2+/- and wildtype mice on C57BL6/NJ and C57BL6/N backgrounds at 2 months of age and measured MDGA2, neuroligin 1 and neuroligin 2 levels at 7 months. Mice on the C57BL6/NJ background performed better than those on the C57BL6/N background in visual ability and in learning and memory performance in the Morris water maze and differed in measures of motor behavior and anxiety. Mice with the MDGA2+/- genotype differed from WT mice in motor, social and repetitive behavior and anxiety, but most of these effects involved interactions between MDGA2+/- genotype and background strain. The background strain also influenced MDGA2 levels and NLGN2 association in MDGA2+/- mice. Our findings emphasize the importance of the background strain used in studies of genetically modified mice.

Whisker exploration behaviours in the 5xFAD mouse are affected by sex and retinal degeneration.

Active whisking in mice and rats is one of the fastest behaviours known in mammals and is used to guide complex behaviours such as exploration and navigation. During object contact, whisker movements are actively controlled and undergo robust changes in timing, speed and position. This study quantifies whisker movements in 6- to 7-month old male and female 5xFAD mice, and their C57/SJL F1 wild-type (WT) controls. As well as genotype, we examined sex differences and the effects of retinal degeneration (rd). Mice were filmed using a high-speed video camera at 500 frames per second (fps), under infrared light while behaving freely in three tasks: object exploration, sequential object exploration and tunnel running. Measures of whisker position, amplitude, speed and asymmetry were extracted and analysed for each task. The 5xFAD mice had significantly altered whisker angular positions, amplitude and asymmetry during object contacts and female 5xFAD mice with rd had lower mean angular positions during object contact. There were no significant effects of genotype on sequential object exploration or on tunnel running but differences due to sex and rd were found in both tasks, with female mice making larger and faster whisker movements overall, and mice with rd making larger and faster whisker movements during object contact. There were sex differences in whisker movements during sequential object exploration and females with rd had higher whisker retraction speeds in tunnel running. These data show that measuring whisker movements can quantify genotype and sex differences and the effects of rd during exploratory behaviour in these mice.

Recommendations for measuring whisker movements and locomotion in mice with sensory, motor and cognitive deficits.

BACKGROUND: Previous studies have measured whisker movements and locomotion to characterise mouse models of neurodegenerative disease. However, these studies have always been completed in isolation, and do not involve standardized procedures for comparisons across multiple mouse models and background strains. NEW METHOD: We present a standard method for conducting whisker movement and locomotion studies, by carrying out qualitative scoring and quantitative measurement of whisker movements from high-speed video footage of mouse models of Amyotrophic Lateral Sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, Cerebellar Ataxia, Somatosensory Cortex Development and Ischemic stroke. RESULTS: Sex, background strain, source breeder and genotype all affected whisker movements. All mouse models, apart from Parkinson's disease, revealed differences in whisker movements during locomotion. R6/2 CAG250 Huntington's disease mice had the strongest behavioural phenotype. Robo3R3-5-CKO and RIM-DKOSert mouse models have abnormal somatosensory cortex development and revealed significant changes in whisker movements during object exploration. COMPARISON WITH EXISTING METHOD(S): Our results have good agreement with past studies, which indicates the robustness and reliability of measuring whisking. We recommend that differences in whisker movements of mice with motor deficits can be captured in open field arenas, but that mice with impairments to sensory or cognitive functioning should also be filmed investigating objects. Scoring clips qualitatively before tracking will help to structure later analyses. CONCLUSIONS: Studying whisker movements provides a quantitative measure of sensing, motor control and exploration. However, the effect of background strain, sex and age on whisker movements needs to be better understood.

Effects of the Novel IDO Inhibitor DWG-1036 on the Behavior of Male and Female 3xTg-AD Mice.

The kynurenine pathway metabolizes tryptophan into nicotinamide adenine dinucleotide, producing a number of intermediary metabolites, including 3-hydroxy kynurenine and quinolinic acid, which are involved in the neurodegenerative mechanisms that underlie Alzheimer's disease (AD). Indolamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of this pathway, is increased in AD, and it has been hypothesized that blocking this enzyme may slow the progression of AD. In this study, we treated male and female 3xTg-AD and wild-type mice with the novel IDO inhibitor DWG-1036 (80 mg/kg) or vehicle (distilled water) from 2 to 6 months of age and then tested them in a battery of behavioral tests that measured spatial learning and memory (Barnes maze), working memory (trace fear conditioning), motor coordination and learning (rotarod), anxiety (elevated plus maze), and depression (tail suspension test). The 3xTg-AD mice treated with DWG-1036 showed better memory in the trace fear conditioning task and significant improvements in learning but poorer spatial memory in the Barnes maze. DWG-1036 treatment also ameliorated the behaviors associated with increased anxiety in the elevated plus maze and depression-like behaviors in the tail suspension test in 3xTg-AD mice. However, the effects of DWG-1036 treatment on the behavioral tasks were variable, and sex differences were apparent. In addition, high doses of DWG-1036 resulted in reduced body weight, particularly in females. Taken together, our results suggest that the kynurenine pathway is a promising target for treating AD, but more work is needed to determine the effective compounds, examine sex differences, and understand the side effects of the compounds.

Age and sex differences in motivation and spatial working memory in 3xTg-AD mice in the Hebb-Williams maze.

The 3xTg-AD mouse model of Alzheimer's disease (AD) has both amyloid beta plaque and tau tangle pathology. However, the results of behavioural testing with these mice have been inconsistent due to age- and sex-related differences, as well as differences in the difficulty of the tests used to measure cognitive function. In order to better understand the sex- and age-related spatial working memory deficits in the 3xTg-AD mice compared to their B6129S/F2 wildtype controls, we tested 4 and 7-month-old males and females and 13-month-old females in the Hebb-Williams maze. In the acquisition phase, the 3xTg-AD mice performed better than the WT controls, but the females of both genotypes showed motivational deficits; often returning to the start box and not eating the food reward, thus taking longer than males to meet the criterion for acquisition. The 3xTg-AD mice showed more working memory deficits than WT mice during the test phase, and the difference increased as the problems increased in difficulty. The results of this study indicate that female 3xTg-AD mice may have motivational deficits in tests using food reward and that the cognitive deficits of the 3xTg-AD mice are not apparent when the tests are too easy; the more difficult the task, the more deficits are shown in the 3xTg-AD mice compared to WT controls. Thus, the inconsistency in previous results may result from differences in motivation and in test difficulty and these must be considered when evaluating cognitive deficits in the 3xTg-AD mice.

Cognitive Decline, Cerebral-Spleen Tryptophan Metabolism, Oxidative Stress, Cytokine Production, and Regulation of the Txnip Gene in a Triple Transgenic Mouse Model of Alzheimer Disease.

Pathologic inflammation in response to injury, infection, or oxidative stress is a proposed mechanism relating cognitive decline to dementia. The kynurenine pathway and thioredoxin-interacting protein (TXNIP) activity regulate inflammation and neurotoxicity in Alzheimer disease (AD). We examined cognitive deficits, kynurenine pathway mediators, TXNIP, and oxidative damage in the cerebrum and spleen, including inflammatory cytokine production by stimulated splenocytes, from female triple transgenic (3xTg-AD) mice in early and late stages of disease progression, and characterized tissue-specific epigenetic regulation of Txnip gene expression. We show that cognitive deficits in 7-month-old 3xTg-AD mice are associated with a stable increase in cerebrum and spleen tryptophan metabolites, with a concomitant increase in amyloid ß 40 (Aß40)/Aß42 and tau/hyperphosphorylated tau pathologies and a coordinated reduction in spleen proinflammatory cytokine production in 17-month-old mice. The enhanced cerebrum TXNIP expression is associated with increased histone acetylation, transcription factor [Aß42 or CCCTC-binding factor (CTCF)] binding, and Txnip promoter hypomethylation, whereas the attenuated spleen TXNIP expression is associated with increased histone methylation, reduced CTCF binding, and Txnip promoter hypermethylation. These results suggest a causal relationship among epigenomic state, TXNIP expression, cerebral-spleen tryptophan metabolism, inflammatory cytokine production, and cognitive decline; and they provide a potential mechanism for Txnip gene regulation in normal and pathologic conditions, suggesting TXNIP levels may be a useful predictive or diagnostic biomarker for Aß40/Aß42 targeted AD therapies.

Interval timing is disrupted in female 5xFAD mice: An indication of altered memory processes.

Temporal information processing in the seconds-to-minutes range is disrupted in patients with Alzheimer's disease (AD). In this study, we investigated the timing behavior of the 5xFAD mouse model of AD in the peak interval (PI) procedure. Nine-month-old female mice were trained with sucrose solution reinforcement for their first response after a fixed-interval (FI) and tested in the inter-mixed non-reinforced PI trials that lasted longer than FI. Timing performance indices were estimated from steady-state timed anticipatory nose-poking responses in the PI trials. We found that the time of maximal reward expectancy (peak time) of the 5xFAD mice was significantly earlier than that of the wild-type (WT) controls with no differences in other indices of timing performance. These behavioral differences corroborate the findings of previous studies on the disruption of temporal associative memory abilities of 5xFAD mice and can be accounted for by the scalar timing theory based on altered long-term memory consolidation of temporal information in the 5xFAD mice. This is the first study to directly show an interval timing phenotype in a genetic mouse model of AD.

Age-related changes in social behaviours in the 5xFAD mouse model of Alzheimer's disease.

In addition to memory impairments, patients with Alzheimer's disease (AD) exhibit a number of behavioural and psychological symptoms that can affect social interactions over the course of the disease. While altered social interactions have been demonstrated in a number of mouse models of AD, many models only recapitulate the initial stages of the disease, and these behavioural changes have yet to be examined over the course of disease progression. By performing a longitudinal study using the 5xFAD mouse model, we have demonstrated that transgenic females exhibit progressive alterations in social investigation compared to wild-type controls. Transgenic females exhibited an age-related reduction in interest for social odours, as well as reduced investigative behaviours towards novel conspecifics in a novel environment. However, transgenic mice exhibited no obvious olfactory deficits, nor any changes in scent-marking behaviour compared to wild-type controls, indicating that changes in investigative behaviour were due to motivation to engage with a social stimulus. This evidence suggests that transgenic 5xFAD females exhibit increased social anxiety in novel environments compared to wild-type controls. Overall, transgenic 5xFAD female mice mimic some features of social withdrawal observed in human AD patients suggesting this strain may be suitable for modelling aspects of the social dysfunction observed in human patients.

Sex differences in the timing behavior performance of 3xTg-AD and wild-type mice in the peak interval procedure.

We investigated interval timing behavior of 10-month-old male and female 3xTg-AD mice compared with their B6129F2/J wild type controls using the peak interval procedure with a 15 s target interval. Multiple parameters reflecting different aspects of timing performance were extracted from steady-state anticipatory nose-poking behavior using two different approaches: single trial analyses and average response curve analyses. These measures can dissociate the differences in performance due to distortions in the interval timing ability or to motivational decline (i.e. apathy); both of which have been reported in Alzheimer patients. We found that the interval timing ability of male and female 3xTg-AD mice did not differ from wild-type controls. However, in measures reflecting motivational state, we found significant sex differences regardless of genotype. Specifically, female mice initiated anticipatory responding later in the trial and had lower response amplitudes than males. Although our findings can also be interpreted in terms of differences in temporal control for response initiation, they more strongly suggest the effect of differential incentive motivation between sexes on timing behavior in these mice.

Sex Differences in Healthspan Predict Lifespan in the 3xTg-AD Mouse Model of Alzheimer's Disease.

Mouse models of Alzheimer's disease (AD) exhibit marked differences in life expectancy depending on their genotype and sex. The assessment of frailty could provide a measure of healthspan to facilitate comparisons between different AD models. We used a validated mouse frailty index (FI) assessment tool to explore genotype and sex differences in lifespan and healthspan of 3xTg-AD mice and their B6129F2 wild-type (WT) controls. This tool is based on an approach commonly used in people and quantifies frailty by counting the accumulation of age-related health deficits. The number of deficits in an individual divided by the total number measured yields an FI score theoretically between 0 and 1, with higher scores denoting more frailty. Male 3xTg-AD mice aged 300-600 days had higher FI scores (Mean FI = 0.21 ± 0.03) than either male WT (Mean FI = 0.15 ± 0.01) or female 3xTg-AD mice (Mean FI = 0.10 ± 0.01), and the elevated frailty scores were accompanied by parallel increases in mortality. Frailty increased exponentially with age, and higher rates of deficit accumulation elevated mortality risk in all groups of mice. When mice were stratified by FI score, frailty predicted mortality, at least in females. Therefore, the mouse clinical FI provides a valuable tool for evaluating healthspan in mouse models of AD with different lifespans.

Age-Related Changes in the Spatial Frequency Threshold of Male and Female 3xTg-AD Mice Using OptoMotry.

Visual impairments and retinal abnormalities occur in patients with Alzheimer's disease (AD) and in mouse models of AD. It is important to know the visual ability of mouse models of AD to ensure that age-related cognitive deficits are not confounded by visual impairments. Using OptoMotry, the spatial frequency thresholds of male and female 3xTg-AD mice did not differ from their B6129SF2 wildtype controls between 1-18 months of age, but females had higher spatial frequency thresholds than males. However, the differences were quite small, and the visual ability of all mice was comparable to that of C57BL/6 mice.

Adaptive Processes in Thalamus and Cortex Revealed by Silencing of Primary Visual Cortex during Contrast Adaptation.

Visual adaptation illusions indicate that our perception is influenced not only by the current stimulus but also by what we have seen in the recent past. Adaptation to stimulus contrast (the relative luminance created by edges or contours in a scene) induces the perception of the stimulus fading away and increases the contrast detection threshold in psychophysical tests [1, 2]. Neural correlates of contrast adaptation have been described throughout the visual system including the retina [3], dorsal lateral geniculate nucleus (dLGN) [4, 5], primary visual cortex (V1) [6], and parietal cortex [7]. The apparent ubiquity of adaptation at all stages raises the question of how this process cascades across brain regions [8]. Focusing on V1, adaptation could be inherited from pre-cortical stages, arise from synaptic depression at the thalamo-cortical synapse [9], or develop locally, but what is the weighting of these contributions? Because contrast adaptation in mouse V1 is similar to classical animal models [10, 11], we took advantage of the optogenetic tools available in mice to disentangle the processes contributing to adaptation in V1. We disrupted cortical adaptation by optogenetically silencing V1 and found that adaptation measured in V1 now resembled that observed in dLGN. Thus, the majority of adaptation seen in V1 neurons arises through local activity-dependent processes, with smaller contributions from dLGN inheritance and synaptic depression at the thalamo-cortical synapse. Furthermore, modeling indicates that divisive scaling of the weakly adapted dLGN input can predict some of the emerging features of V1 adaptation.

A mitochondrial therapeutic reverses visual decline in mouse models of diabetes.

Diabetic retinopathy is characterized by progressive vision loss and the advancement of retinal micoraneurysms, edema and angiogenesis. Unfortunately, managing glycemia or targeting vascular complications with anti-vascular endothelial growth factor agents has shown only limited efficacy in treating the deterioration of vision in diabetic retinopathy. In light of growing evidence that mitochondrial dysfunction is an independent pathophysiology of diabetes and diabetic retinopathy, we investigated whether selectively targeting and improving mitochondrial dysfunction is a viable treatment for visual decline in diabetes. Measures of spatial visual behavior, blood glucose, bodyweight and optical clarity were made in mouse models of diabetes. Treatment groups were administered MTP-131, a water-soluble tetrapeptide that selectively targets mitochondrial cardiolipin and promotes efficient electron transfer, either systemically or in eye drops. Progressive visual decline emerged in untreated animals before the overt symptoms of metabolic and ophthalmic abnormalities were manifest, but with time, visual dysfunction was accompanied by compromised glucose clearance, and elevated blood glucose and bodyweight. MTP-131 treatment reversed the visual decline without improving glycemic control or reducing bodyweight. These data provide evidence that visuomotor decline is an early complication of diabetes. They also indicate that selectively treating mitochondrial dysfunction with MTP-131 has the potential to remediate the visual dysfunction and to complement existing treatments for diabetic retinopathy.

Prevention of vision loss protects against age-related impairment in learning and memory performance in DBA/2J mice.

The DBA/2J mouse is a model of pigmentary glaucoma in humans as it shows age-related increases in intraocular pressure (IOP), retinal ganglion cell death and visual impairment. Previously, we showed that visual ability declines from 9 to 12 months of age and visual impairment is correlated with poor learning and memory performance in visuo-spatial tasks but not in tasks that do not depend on visual cues. To test the "sensory impairment" hypothesis of aging, which postulates that sensory impaired individuals are disadvantaged in their performance on psychometric tests as a direct result of difficulties in sensory perception, we treated DBA/2J mice with a conventional glaucoma medication used in humans (Timoptic-XE, 0.00, 0.25, or 0.50%) daily from 9 weeks to 12 months of age to determine whether prevention of vision loss prevented the decline in visuo-spatial learning and memory performance. At all ages tested (3, 6, 9, and 12 months of age), mice treated with Timoptic-XE (0.25 and 0.50%) maintained a high level of performance, while 12 month old control mice (0.00%) exhibited impaired performance in visually-dependent, but not non-visual tasks. These results demonstrate that when sensory function is preserved, cognitive performance is normalized. Thus, as in many aging humans, DBA/2J mice show age-related decrements in performance on visually presented cognitive tests, not because of cognitive impairment but as a direct consequence of poor visual ability. Our results demonstrate that age-related impairment in performance in visuo-spatial tasks in DBA/2J mice can be prevented by the preservation of visual ability.

A neurobehavioral analysis of the prevention of visual impairment in the DBA/2J mouse model of glaucoma.

PURPOSE: Timoptic-XE treatment was used to examine the relationship between age-related changes in intraocular pressure (IOP), retinal cell loss, visual ability, and neuronal labeling in the superior colliculus in the DBA/2J mouse model of pigmentary glaucoma. METHODS: Mice were administered Timoptic-XE (0.0%, 0.25%, or 0.50%) daily from 9 weeks to 12 months of age. Visual ability and IOP were evaluated at 3, 6, 9, and 12 months of age. Mice from each group were then given intraocular injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), and estimates of the number of cells in the ganglion cell layer of the retina, WGA-HRP transneural labeling of cells, cell count, and cross-sectional area of Nissl-stained cells in the superior colliculus were obtained. RESULTS: Mice treated with 0.50% and 0.25% Timoptic-XE maintained a high level of performance in behavioral vision tasks, while 12-month-old untreated mice (0.0% Timoptic-XE) exhibited impaired visual performance. Timoptic-XE therapy reduced IOP and cell loss in the ganglion cell layer of the retina and prevented somal shrinkage and the decrease in WGA-HRP transneural labeling in the superior colliculus that occurred in untreated mice at 12 months of age. CONCLUSIONS: This study provides a comprehensive assessment of the efficacy of Timoptic-XE in DBA/2J mice by correlating age-related visual system changes in the retina and brain with changes in IOP and visual ability. These results showed that reducing IOP not only rescued retinal ganglion cell atrophy but also restored visual function and altered patterns of neurodegeneration that occur with blindness.

The influence of visual ability on learning and memory performance in 13 strains of mice.

We calculated visual ability in 13 strains of mice (129SI/Sv1mJ, A/J, AKR/J, BALB/cByJ, C3H/HeJ, C57BL/6J, CAST/EiJ, DBA/2J, FVB/NJ, MOLF/EiJ, SJL/J, SM/J, and SPRET/EiJ) on visual detection, pattern discrimination, and visual acuity and tested these and other mice of the same strains in a behavioral test battery that evaluated visuo-spatial learning and memory, conditioned odor preference, and motor learning. Strain differences in visual acuity accounted for a significant proportion of the variance between strains in measures of learning and memory in the Morris water maze. Strain differences in motor learning performance were not influenced by visual ability. Conditioned odor preference was enhanced in mice with visual defects. These results indicate that visual ability must be accounted for when testing for strain differences in learning and memory in mice because differences in performance in many tasks may be due to visual deficits rather than differences in higher order cognitive functions. These results have significant implications for the search for the neural and genetic basis of learning and memory in mice.

Age-related changes in visual acuity, learning and memory in C57BL/6J and DBA/2J mice.

The DBA/2J mouse is a model of age-related pigmentary glaucoma in humans. Visual detection, pattern discrimination and visual acuity were evaluated in DBA/2J, C57BL/6J, B6.mpc1d (a C57 congenic strain) and D2.mpc1b (a D2 congenic strain) mice at 6, 12, 18 and 24 months of age. Mice were also tested in the Morris Water Maze and olfactory discrimination learning task. At 6 months, DBA/2J and D2.mpc1b mice outperformed C57BL/6J and B6.mpc1d mice in the visual detection task and there were no strain differences in performance on the water maze. At 12, 18 and 24 months, C57BL/6J and B6.mpc1d mice outperformed DBA/2J and D2.mpc1b mice in the vision tasks and in the water maze. Strains did not differ in the olfactory learning task. Therefore, loss of visual function occurs between 6 and 12 months of age in DBA/2J mice. Strain differences in visual task performance accounted for a significant proportion of the variance in measures of learning and memory in the water maze at 12, 18 and 24 months of age.