Neuroimaging Evidence of Major Morpho-Anatomical and Functional Abnormalities in the BTBR T+TF/J Mouse Model of Autism

BTBR T+tf/J (BTBR) mice display prominent behavioural deficits analogous to the defining symptoms of autism, a feature that has prompted a widespread use of the model in preclinical autism research. Because neuro-behavioural traits are described with respect to reference populations, multiple investigators have examined and described the behaviour of BTBR mice against that exhibited by C57BL/6J (B6), a mouse line characterised by high sociability and low self-grooming. In an attempt to probe the translational relevance of this comparison for autism research, we used Magnetic Resonance Imaging (MRI) to map in both strain multiple morpho-anatomical and functional neuroimaging readouts that have been extensively used in patient populations. Diffusion tensor tractography confirmed previous reports of callosal agenesis and lack of hippocampal commissure in BTBR mice, and revealed a concomitant rostro-caudal reorganisation of major cortical white matter bundles. Intact inter-hemispheric tracts were found in the anterior commissure, ventro-medial thalamus, and in a strain-specific white matter formation located above the third ventricle. BTBR also exhibited decreased fronto-cortical, occipital and thalamic gray matter volume and widespread reductions in cortical thickness with respect to control B6 mice. Foci of increased gray matter volume and thickness were observed in the medial prefrontal and insular cortex. Mapping of resting-state brain activity using cerebral blood volume weighted fMRI revealed reduced cortico-thalamic function together with foci of increased activity in the hypothalamus and dorsal hippocampus of BTBR mice. Collectively, our results show pronounced functional and structural abnormalities in the brain of BTBR mice with respect to control B6 mice. The large and widespread white and gray matter abnormalities observed do not appear to be representative of the neuroanatomical alterations typically observed in autistic patients. The presence of reduced fronto-cortical metabolism is of potential translational relevance, as this feature recapitulates previously-reported clinical observations

Mouse Cognition-Related Behavior in the Open-Field: Emergence of Places of Attraction

Spatial memory is often studied in the Morris Water Maze, where the animal's spatial orientation has been shown to be mainly shaped by distal visual cues. Cognition-related behavior has also been described along “well-trodden paths”—spatial habits established by animals in the wild and in captivity reflecting a form of spatial memory. In the present study we combine the study of Open Field behavior with the study of behavior on well-trodden paths, revealing a form of locational memory that appears to correlate with spatial memory. The tracked path of the mouse is used to examine the dynamics of visiting behavior to locations. A visit is defined as either progressing through a location or stopping there, where progressing and stopping are computationally defined. We then estimate the probability of stopping at a location as a function of the number of previous visits to that location, i.e., we measure the effect of visiting history to a location on stopping in it. This can be regarded as an estimate of the familiarity of the mouse with locations. The recently wild-derived inbred strain CZECHII shows the highest effect of visiting history on stopping, C57 inbred mice show a lower effect, and DBA mice show no effect. We employ a rarely used, bottom-to-top computational approach, starting from simple kinematics of movement and gradually building our way up until we end with (emergent) locational memory. The effect of visiting history to a location on stopping in it can be regarded as an estimate of the familiarity of the mouse with locations, implying memory of these locations. We show that the magnitude of this estimate is strain-specific, implying a genetic influence. The dynamics of this process reveal that locations along the mouse's trodden path gradually become places of attraction, where the mouse stops habitually

Genetic Variation and Population Substructure in Outbred CD-1 Mice: Implications for Genome-Wide Association Studies

Outbred laboratory mouse populations are widely used in biomedical research. Since little is known about the degree of genetic variation present in these populations, they are not widely used for genetic studies. Commercially available outbred CD-1 mice are drawn from an extremely large breeding population that has accumulated many recombination events, which is desirable for genome-wide association studies. We therefore examined the degree of genome-wide variation within CD-1 mice to investigate their suitability for genetic studies. The CD-1 mouse genome displays patterns of linkage disequilibrium and heterogeneity similar to wild-caught mice. Population substructure and phenotypic differences were observed among CD-1 mice obtained from different breeding facilities. Differences in genetic variation among CD-1 mice from distinct facilities were similar to genetic differences detected between closely related human populations, consistent with a founder effect. This first large-scale genetic analysis of the outbred CD-1 mouse strain provides important considerations for the design and analysis of genetic studies in CD-1 mice

Cell Membrane Modification for Rapid Display of Bi-Functional Peptides: A Novel Approach to Reduce Complement Activation

Ischemia and reperfusion of organs is an unavoidable consequence of transplantation. Inflammatory events associated with reperfusion injury are in part attributed to excessive complement activation. Systemic administration of complement inhibitors reduces reperfusion injury but leaves patients vulnerable to infection. Here, we report a novel therapeutic strategy that decorates cells with an anti-complement peptide. An analog of the C3 convertase inhibitor Compstatin (C) was synthesized with a hexahistidine (His6) tag to create C-His6. To decorate cell membranes with C-His6, fusogenic lipid vesicles (FLVs) were used to incorporate lipids with nickel (Ni2+) tethers into cell membranes, and these could then couple with C-His6. Ni2+ tether levels to display C-His6 were modulated by changing FLV formulation, FLV incubation time and FLV levels. SKOV-3 cells decorated with C-His6 effectively reduced complement deposition in a classical complement activation assay. We conclude that our therapeutic approach appears promising for local ex vivo treatment of transplanted organs to reduce complement-mediated reperfusion injury

Genetic Variations Strongly Influence Phenotypic Outcome in the Mouse Retina

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2-nb1 at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases

Expression and purification of recombinant toxicshock‐syndrome toxin I

Toxic‐shock‐syndrome toxin I (TSSTI), an exotoxin produced by certain strains of Staphylococcus aureus, has been closely associated with the pathogenesis of toxic shock syndrome. Outside the context of its staphylococcal host, TSSTI may offer therapeutic uses. We report here a strategy for high‐level expression and simplified purification of TSSTI. We have subcloned the coding region for TSSTI into a vector containing an inducible T7 promoter sequence and expressed the protein in an Escherichia coli host strain. The recombinant TSSTI protein contained ten sequential histidine residues (Histag) at its N‐terminus, which enabled its efficient purification using nickel‐agarose‐affinity resin. Histag‐TSSTI (H‐TSSTI) was further purified to homogeneity using a size‐exclusion column. By this system, 80 mg of highly purified H‐TSSTI can be consistently obtained per litre of culture in under 3 days. H‐TSSTI retained biological activity and was unaffected by the presence of the Histag, as measured in lymphocyte proliferation assays

Gene Expression in Brain and Liver Produced by Three Different Regimens of Alcohol Consumption in Mice: Comparison with Immune Activation

Chronically available alcohol escalates drinking in mice and a single injection of the immune activator lipopolysaccharide can mimic this effect and result in a persistent increase in alcohol consumption. We hypothesized that chronic alcohol drinking and lipopolysaccharide injections will produce some similar molecular changes that play a role in regulation of alcohol intake. We investigated the molecular mechanisms of chronic alcohol consumption or lipopolysaccharide insult by gene expression profiling in prefrontal cortex and liver of C57BL/6J mice. We identified similar patterns of transcriptional changes among four groups of animals, three consuming alcohol (vs water) in different consumption tests and one injected with lipopolysaccharide (vs. vehicle). The three tests of alcohol consumption are the continuous chronic two bottle choice (Chronic), two bottle choice available every other day (Chronic Intermittent) and limited access to one bottle of ethanol (Drinking in the Dark). Gene expression changes were more numerous and marked in liver than in prefrontal cortex for the alcohol treatments and similar in the two tissues for lipopolysaccharide. Many of the changes were unique to each treatment, but there was significant overlap in prefrontal cortex for Chronic-Chronic Intermittent and for Chronic Intermittent-lipopolysaccharide and in liver all pairs showed overlap. In silico cell-type analysis indicated that lipopolysaccharide had strongest effects on brain microglia and liver Kupffer cells. Pathway analysis detected a prefrontal cortex-based dopamine-related (PPP1R1B, DRD1, DRD2, FOSB, PDNY) network that was highly over-represented in the Chronic Intermittent group, with several genes from the network being also regulated in the Chronic and lipopolysaccharide (but not Drinking in the Dark) groups. Liver showed a CYP and GST centered metabolic network shared in part by all four treatments. We demonstrate common consequences of chronic alcohol consumption and immune activation in both liver and brain and show distinct genomic consequences of different types of alcohol consumption.This work was supported by grants from the National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA) Integrated Neuroscience Initiative on Alcoholism (INIA-West, http://www.scripps.edu/california/resear​ch/inia/; AA13520), NIH K award to IP (AA017234), and NIH grant AA013518 to RAH (NIH, http://www.nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.Pharmac