Merged magnetic resonance and light sheet microscopy of the whole mouse brain.

We have developed workflows to align 3D magnetic resonance histology (MRH) of the mouse brain with light sheet microscopy (LSM) and 3D delineations of the same specimen. We start with MRH of the brain in the skull with gradient echo and diffusion tensor imaging (DTI) at 15 µm isotropic resolution which is ~ 1,000 times higher than that of most preclinical MRI. Connectomes are generated with superresolution tract density images of ~5 µm. Brains are cleared, stained for selected proteins, and imaged by LSM at 1.8 µm/pixel. LSM data are registered into the reference MRH space with labels derived from the ABA common coordinate framework. The result is a high-dimensional integrated volume with registration (HiDiver) with alignment precision better than 50 µm. Throughput is sufficiently high that HiDiver is being used in quantitative studies of the impact of gene variants and aging on mouse brain cytoarchitecture and connectomics.

Transcriptome analysis identifies an ASD-Like phenotype in oligodendrocytes and microglia from C58/J amygdala that is dependent on sex and sociability.

BACKGROUND: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. METHODS: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. RESULTS: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using Bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. LIMITATIONS: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its' potential as an ASD therapeutic. CONCLUSIONS: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

Prenatal heroin exposure alters brain morphology and connectivity in adolescent mice.

The United States is experiencing a dramatic increase in maternal opioid misuse and, consequently, the number of individuals exposed to opioids in utero. Prenatal opioid exposure has both acute and long-lasting effects on health and wellbeing. Effects on the brain, often identified at school age, manifest as cognitive impairment, attention deficit, and reduced scholastic achievement. The neurobiological basis for these effects is poorly understood. Here, we examine how in utero exposure to heroin affects brain development into early adolescence in a mouse model. Pregnant C57BL/6J mice received escalating doses of heroin twice daily on gestational days 4-18. The brains of offspring were assessed on postnatal day 28 using 9.4 T diffusion MRI of postmortem specimens at 36 µm resolution. Whole-brain volumes and the volumes of 166 bilateral regions were compared between heroin-exposed and control offspring. We identified a reduction in whole-brain volume in heroin-exposed offspring and heroin-associated volume changes in 29 regions after standardizing for whole-brain volume. Regions with bilaterally reduced standardized volumes in heroin-exposed offspring relative to controls include the ectorhinal and insular cortices. Regions with bilaterally increased standardized volumes in heroin-exposed offspring relative to controls include the periaqueductal gray, septal region, striatum, and hypothalamus. Leveraging microscopic resolution diffusion tensor imaging and precise regional parcellation, we generated whole-brain structural MRI diffusion connectomes. Using a dimension reduction approach with multivariate analysis of variance to assess group differences in the connectome, we found that in utero heroin exposure altered structure-based connectivity of the left septal region and the region that acts as a hub for limbic regulatory actions. Consistent with clinical evidence, our findings suggest that prenatal opioid exposure may have effects on brain morphology, connectivity, and, consequently, function that persist into adolescence. This work expands our understanding of the risks associated with opioid misuse during pregnancy and identifies biomarkers that may facilitate diagnosis and treatment.

A time-course study of actively stained mouse brains: Diffusion tensor imaging parameters and connectomic stability over 1 year.

While the application of diffusion tensor imaging (DTI), tractography, and connectomics to fixed tissue is a common practice today, there have been limited studies examining the effects of fixation on brain microstructure over extended periods. This mouse model time-course study reports the changes of regional brain volumes and diffusion scalar parameters, such as fractional anisotropy, across 12 representative brain regions as measures of brain structural stability. The scalar DTI parameters and regional volumes were highly variable over the first 2 weeks after fixation. The same parameters were consistent over a 2-8-week window after fixation, which means confounds from tissue stability over that scanning window were minimal. Quantitative connectomes were analyzed over the same time with extension out to 1 year. While there was some change in the scalar metrics at 1 year after fixation, these changes were sufficiently small, particularly in white matter, to support reproducible connectomes over a period ranging from 2-weeks to 1-year post-fixation. These findings delineate a scanning period, during which brain volumes, diffusion scalar metrics, and connectomes are remarkably consistent.

Transcriptome Analysis Identifies An ASD-Like Phenotype In Oligodendrocytes And Microglia From C58/J Amygdala That Is Dependent On Sex and Sociability.

Background: Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with higher incidence in males and is characterized by atypical verbal/nonverbal communication, restricted interests that can be accompanied by repetitive behavior, and disturbances in social behavior. This study investigated brain mechanisms that contribute to sociability deficits and sex differences in an ASD animal model. Methods: Sociability was measured in C58/J and C57BL/6J mice using the 3-chamber social choice test. Bulk RNA-Seq and snRNA-Seq identified transcriptional changes in C58/J and C57BL/6J amygdala within which DMRseq was used to measure differentially methylated regions in amygdala. Results: C58/J mice displayed divergent social strata in the 3-chamber test. Transcriptional and pathway signatures revealed immune-related biological processes differ between C58/J and C57BL/6J amygdala. Hypermethylated and hypomethylated genes were identified in C58/J versus C57BL/6J amygdala. snRNA-Seq data in C58/J amygdala identified differential transcriptional signatures within oligodendrocytes and microglia characterized by increased ASD risk gene expression and predicted impaired myelination that was dependent on sex and sociability. RNA velocity, gene regulatory network, and cell communication analysis showed diminished oligodendrocyte/microglia differentiation. Findings were verified using bulk RNA-Seq and demonstrated oxytocin's beneficial effects on myelin gene expression. Limitations: Our findings are significant. However, limitations can be noted. The cellular mechanisms linking reduced oligodendrocyte differentiation and reduced myelination to an ASD phenotype in C58/J mice need further investigation. Additional snRNA-Seq and spatial studies would determine if effects in oligodendrocytes/microglia are unique to amygdala or if this occurs in other brain regions. Oxytocin's effects need further examination to understand its potential as an ASD therapeutic. Conclusions: Our work demonstrates the C58/J mouse model's utility in evaluating the influence of sex and sociability on the transcriptome in concomitant brain regions involved in ASD. Our single-nucleus transcriptome analysis elucidates potential pathological roles of oligodendrocytes and microglia in ASD. This investigation provides details regarding regulatory features disrupted in these cell types, including transcriptional gene dysregulation, aberrant cell differentiation, altered gene regulatory networks, and changes to key pathways that promote microglia/oligodendrocyte differentiation. Our studies provide insight into interactions between genetic risk and epigenetic processes associated with divergent affiliative behavior and lack of positive sociability.

An Open Resource: MR and light sheet microscopy stereotaxic atlas of the mouse brain.

We have combined MR histology and light sheet microscopy (LSM) of five postmortem C57BL/6J mouse brains in a stereotaxic space based on micro-CT yielding a multimodal 3D atlas with the highest spatial and contrast resolution yet reported. Brains were imaged in situ with multi gradient echo (mGRE) and diffusion tensor imaging (DTI) at 15 µm resolution (∼ 2.4 million times that of clinical MRI). Scalar images derived from the average DTI and mGRE provide unprecedented contrast in 14 complementary 3D volumes, each highlighting distinct histologic features. The same tissues scanned with LSM and registered into the stereotaxic space provide 17 different molecular cell type stains. The common coordinate framework labels (CCFv3) complete the multimodal atlas. The atlas has been used to correct distortions in the Allen Brain Atlas and harmonize it with Franklin Paxinos. It provides a unique resource for stereotaxic labeling of mouse brain images from many sources.

Volumetric analysis of the aging auditory pathway using high resolution magnetic resonance histology.

Numerous shown consequences of age-related hearing loss have been unveiled; however, the relationship of the cortical and subcortical structures of the auditory pathway with aging is not well known. Investigations into neural structure analysis remain sparse due to difficulties of doing so in animal models; however, recent technological advances have been able to achieve a resolution adequate to perform such studies even in the small mouse. We utilize 12 members of the BXD family of recombinant inbred mice and aged separate cohorts. Utilizing novel magnetic resonance histology imaging techniques, we imaged these mice and generated high spatial resolution three dimensional images which were then comprehensively labeled. We completed volumetric analysis of 12 separate regions of interest specific to the auditory pathway brainstem nuclei and cortical areas with focus on the effect of aging upon said structures. Our results showed significant interstrain variation in the age-related effect on structure volume supporting a genetic influence in this interaction. Through multivariable modeling, we observed heterogenous effects of aging between different structures. Six of the 12 regions of interests demonstrated a significant age-related effect. The auditory cortex and ventral cochlear nucleus were found to decrease in volume with age, while the medial division of the medial geniculate nucleus, lateral lemniscus and its nucleus, and the inferior colliculus increased in size with age. Additionally, no sex-based differences were noted, and we observed a negative relationship between auditory cortex volume and mouse weight. This study is one of the first to perform comprehensive magnetic resonance imaging and quantitative analysis in the mouse brain auditory pathway cytoarchitecture, offering both novel insights into the neuroanatomical basis of age-related changes in hearing as well as evidence toward a genetic influence in this interaction. High resonance magnetic resonance imaging provides a promising efficacious avenue in future mouse model hearing loss investigations.

Ex Vivo MR Histology and Cytometric Feature Mapping Connect Three-dimensional in Vivo MR Images to Two-dimensional Histopathologic Images of Murine Sarcomas.

Purpose To establish a platform for quantitative tissue-based interpretation of cytoarchitecture features from tumor MRI measurements. Materials and Methods In a pilot preclinical study, multicontrast in vivo MRI of murine soft-tissue sarcomas in 10 mice, followed by ex vivo MRI of fixed tissues (termed MR histology), was performed. Paraffin-embedded limb cross-sections were stained with hematoxylin-eosin, digitized, and registered with MRI. Registration was assessed by using binarized tumor maps and Dice similarity coefficients (DSCs). Quantitative cytometric feature maps from histologic slides were derived by using nuclear segmentation and compared with registered MRI, including apparent diffusion coefficients and transverse relaxation times as affected by magnetic field heterogeneity (T2* maps). Cytometric features were compared with each MR image individually by using simple linear regression analysis to identify the features of interest, and the goodness of fit was assessed on the basis of R2 values. Results Registration of MR images to histopathologic slide images resulted in mean DSCs of 0.912 for ex vivo MR histology and 0.881 for in vivo MRI. Triplicate repeats showed high registration repeatability (mean DSC, >0.9). Whole-slide nuclear segmentations were automated to detect nuclei on histopathologic slides (DSC = 0.8), and feature maps were generated for correlative analysis with MR images. Notable trends were observed between cell density and in vivo apparent diffusion coefficients (best line fit: R2 = 0.96, P < .001). Multiple cytoarchitectural features exhibited linear relationships with in vivo T2* maps, including nuclear circularity (best line fit: R2 = 0.99, P < .001) and variance in nuclear circularity (best line fit: R2 = 0.98, P < .001). Conclusion An infrastructure for registering and quantitatively comparing in vivo tumor MRI with traditional histologic analysis was successfully implemented in a preclinical pilot study of soft-tissue sarcomas. Keywords: MRI, Pathology, Animal Studies, Tissue Characterization Supplemental material is available for this article. © RSNA, 2021.