Using mesoscopic tract-tracing data to guide the estimation of fiber orientation distributions in the mouse brain from diffusion MRI.

Diffusion MRI (dMRI) tractography is the only tool for non-invasive mapping of macroscopic structural connectivity over the entire brain. Although it has been successfully used to reconstruct large white matter tracts in the human and animal brains, the sensitivity and specificity of dMRI tractography remained limited. In particular, the fiber orientation distributions (FODs) estimated from dMRI signals, key to tractography, may deviate from histologically measured fiber orientation in crossing fibers and gray matter regions. In this study, we demonstrated that a deep learning network, trained using mesoscopic tract-tracing data from the Allen Mouse Brain Connectivity Atlas, was able to improve the estimation of FODs from mouse brain dMRI data. Tractography results based on the network generated FODs showed improved specificity while maintaining sensitivity comparable to results based on FOD estimated using a conventional spherical deconvolution method. Our result is a proof-of-concept of how mesoscale tract-tracing data can guide dMRI tractography and enhance our ability to characterize brain connectivity.

Towards reliable reconstruction of the mouse brain corticothalamic connectivity using diffusion MRI.

Diffusion magnetic resonance imaging (dMRI) tractography has yielded intriguing insights into brain circuits and their relationship to behavior in response to gene mutations or neurological diseases across a number of species. Still, existing tractography approaches suffer from limited sensitivity and specificity, leading to uncertain interpretation of the reconstructed connections. Hence, in this study, we aimed to optimize the imaging and computational pipeline to achieve the best possible spatial overlaps between the tractography and tracer-based axonal projection maps within the mouse brain corticothalamic network. We developed a dMRI-based atlas of the mouse forebrain with structural labels imported from the Allen Mouse Brain Atlas (AMBA). Using the atlas and dMRI tractography, we first reconstructed detailed node-to-node mouse brain corticothalamic structural connectivity matrices using different imaging and tractography parameters. We then investigated the effects of each condition for accurate reconstruction of the corticothalamic projections by quantifying the similarities between the tractography and the tracer data from the Allen Mouse Brain Connectivity Atlas (AMBCA). Our results suggest that these parameters significantly affect tractography outcomes and our atlas can be used to investigate macroscopic structural connectivity in the mouse brain. Furthermore, tractography in mouse brain gray matter still face challenges and need improved imaging and tractography methods.

Early adversity changes the economic conditions of mouse structural brain network organization.

Early adversity can change educational, cognitive, and mental health outcomes. However, the neural processes through which early adversity exerts these effects remain largely unknown. We used generative network modeling of the mouse connectome to test whether unpredictable postnatal stress shifts the constraints that govern the organization of the structural connectome. A model that trades off the wiring cost of long-distance connections with topological homophily (i.e., links between regions with shared neighbors) generated simulations that successfully replicate the rodent connectome. The imposition of early life adversity shifted the best-performing parameter combinations toward zero, heightening the stochastic nature of the generative process. Put simply, unpredictable postnatal stress changes the economic constraints that reproduce rodent connectome organization, introducing greater randomness into the development of the simulations. While this change may constrain the development of cognitive abilities, it could also reflect an adaptive mechanism that facilitates effective responses to future challenges.

Acceleration of three-dimensional diffusion magnetic resonance imaging using a kernel low-rank compressed sensing method.

Diffusion Magnetic Resonance Imaging (dMRI) has shown great potential in probing tissue microstructure and structural connectivity in the brain but is often limited by the lengthy scan time needed to sample the diffusion profile by acquiring multiple diffusion weighted images (DWIs). Although parallel imaging technique has improved the speed of dMRI acquisition, attaining high resolution three dimensional (3D) dMRI on preclinical MRI systems remained still time consuming. In this paper, kernel principal component analysis, a machine learning approach, was employed to estimate the correlation among DWIs. We demonstrated the feasibility of such correlation estimation from low-resolution training DWIs and used the correlation as a constraint to reconstruct high-resolution DWIs from highly under-sampled k-space data, which significantly reduced the scan time. Using full k-space 3D dMRI data of post-mortem mouse brains, we retrospectively compared the performance of the so-called kernel low rank (KLR) method with a conventional compressed sensing (CS) method in terms of image quality and ability to resolve complex fiber orientations and connectivity. The results demonstrated that the KLR-CS method outperformed the conventional CS method for acceleration factors up to 8 and was likely to enhance our ability to investigate brain microstructure and connectivity using high-resolution 3D dMRI.

Deletion of the mu opioid receptor gene in mice reshapes the reward-aversion connectome.

Connectome genetics seeks to uncover how genetic factors shape brain functional connectivity; however, the causal impact of a single gene's activity on whole-brain networks remains unknown. We tested whether the sole targeted deletion of the mu opioid receptor gene (Oprm1) alters the brain connectome in living mice. Hypothesis-free analysis of combined resting-state fMRI diffusion tractography showed pronounced modifications of functional connectivity with only minor changes in structural pathways. Fine-grained resting-state fMRI mapping, graph theory, and intergroup comparison revealed Oprm1-specific hubs and captured a unique Oprm1 gene-to-network signature. Strongest perturbations occurred in connectional patterns of pain/aversion-related nodes, including the mu receptor-enriched habenula node. Our data demonstrate that the main receptor for morphine predominantly shapes the so-called reward/aversion circuitry, with major influence on negative affect centers.

Open-source versatile 3D-print animal conditioning platform design for in vivo preclinical brain imaging in awake mice and anesthetized mice and rats.

Proper animal conditioning is a key factor in the quality and success of preclinical neuroimaging applications. Here, we introduce an open-source easy-to-modify multimodal 3D printable design for rodent conditioning for magnetic resonance imaging (MRI) or other imaging modalities. Our design can be used for brain imaging in anesthetized or awake mice, and in anesthetized rats. We show ease of use and reproducibility of subject conditioning with anatomical T2-weighted imaging for both mice and rats. We also demonstrate the application of our design for awake functional MRI in mice using both visual evoked potential and olfactory stimulation paradigms. In addition, using a combined MRI, positron emission tomography and X-ray computed tomography experiment, we demonstrate that our proposed cradle design can be utilized for multiple imaging modalities.

Early adversity causes sex-specific deficits in perforant pathway connectivity and contextual memory in adolescent mice.

BACKGROUND: Early life adversity impairs hippocampal development and function across diverse species. While initial evidence indicated potential variations between males and females, further research is required to validate these observations and better understand the underlying mechanisms contributing to these sex differences. Furthermore, most of the preclinical work in rodents was performed in adult males, with only few studies examining sex differences during adolescence when such differences appear more pronounced. To address these concerns, we investigated the impact of limited bedding (LB), a mouse model of early adversity, on hippocampal development in prepubescent and adolescent male and female mice. METHODS: RNA sequencing, confocal microscopy, and electron microscopy were used to evaluate the impact of LB and sex on hippocampal development in prepubescent postnatal day 17 (P17) mice. Additional studies were conducted on adolescent mice aged P29-36, which included contextual fear conditioning, retrograde tracing, and ex vivo diffusion magnetic resonance imaging (dMRI). RESULTS: More severe deficits in axonal innervation and myelination were found in the perforant pathway of prepubescent and adolescent LB males compared to LB female littermates. These sex differences were due to a failure of reelin-positive neurons located in the lateral entorhinal cortex (LEC) to innervate the dorsal hippocampus via the perforant pathway in males, but not LB females, and were strongly correlated with deficits in contextual fear conditioning. CONCLUSIONS: LB impairs the capacity of reelin-positive cells located in the LEC to project and innervate the dorsal hippocampus in LB males but not female LB littermates. Given the critical role that these projections play in supporting normal hippocampal function, a failure to establish proper connectivity between the LEC and the dorsal hippocampus provides a compelling and novel mechanism to explain the more severe deficits in myelination and contextual freezing found in adolescent LB males.

Childhood adversity, such as severe deprivation and neglect, leads to structural changes in human brain development that are associated with learning deficits and behavioral difficulties. Some of the most consistent findings in individuals exposed to childhood adversity are reduced hippocampal volume and abnormal hippocampal function. This is important because the hippocampus is necessary for learning and memory, and it plays a crucial role in depression and anxiety. Although initial studies suggested more pronounced hippocampal deficits in men, additional research is needed to confirm these findings and to elucidate the mechanisms responsible for these sex differences. We found that male and female mice exposed to early impoverishment and deprivation exhibit similar structural changes to those observed in deprived children. Interestingly, adolescent male mice, but not females, display severe deficits in their ability to freeze when placed back in a box where they were previously shocked. The ability to associate "shock/danger" with a "box/place" is referred to as contextual fear conditioning and requires normal connections between the entorhinal cortex and the hippocampus. We found that these connections did not form properly in male mice exposed to impoverished conditions, but they were only minimally affected in females. These findings appear to explain why exposure to impoverished conditions impairs contextual fear conditioning in male mice but not in female mice. Additional work is needed to determine whether similar sex-specific changes in these connections are also observed in adolescents exposed to neglect and deprivation.

Cerebellar output neurons impair non-motor behaviors by altering development of extracerebellar connectivity.

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the minimum requirement for these neurons is for motor coordination and not learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations.

Virtual mouse brain histology from multi-contrast MRI via deep learning.

1H MRI maps brain structure and function non-invasively through versatile contrasts that exploit inhomogeneity in tissue micro-environments. Inferring histopathological information from magnetic resonance imaging (MRI) findings, however, remains challenging due to absence of direct links between MRI signals and cellular structures. Here, we show that deep convolutional neural networks, developed using co-registered multi-contrast MRI and histological data of the mouse brain, can estimate histological staining intensity directly from MRI signals at each voxel. The results provide three-dimensional maps of axons and myelin with tissue contrasts that closely mimic target histology and enhanced sensitivity and specificity compared to conventional MRI markers. Furthermore, the relative contribution of each MRI contrast within the networks can be used to optimize multi-contrast MRI acquisition. We anticipate our method to be a starting point for translation of MRI results into easy-to-understand virtual histology for neurobiologists and provide resources for validating novel MRI techniques.

Translational Structural and Functional Signatures of Chronic Alcohol Effects in Mice.

BACKGROUND: Alcohol acts as an addictive substance that may lead to alcohol use disorder. In humans, magnetic resonance imaging showed diverse structural and functional brain alterations associated with this complex pathology. Single magnetic resonance imaging modalities are used mostly but are insufficient to portray and understand the broad neuroadaptations to alcohol. Here, we combined structural and functional magnetic resonance imaging and connectome mapping in mice to establish brain-wide fingerprints of alcohol effects with translatable potential. METHODS: Mice underwent a chronic intermittent alcohol drinking protocol for 6 weeks before being imaged under medetomidine anesthesia. We performed open-ended multivariate analysis of structural data and functional connectivity mapping on the same subjects. RESULTS: Structural analysis showed alcohol effects for the prefrontal cortex/anterior insula, hippocampus, and somatosensory cortex. Integration with microglia histology revealed distinct alcohol signatures, suggestive of advanced (prefrontal cortex/anterior insula, somatosensory cortex) and early (hippocampus) inflammation. Functional analysis showed major alterations of insula, ventral tegmental area, and retrosplenial cortex connectivity, impacting communication patterns for salience (insula), reward (ventral tegmental area), and default mode (retrosplenial cortex) networks. The insula appeared as a most sensitive brain center across structural and functional analyses. CONCLUSIONS: This study demonstrates alcohol effects in mice, which possibly underlie lower top-down control and impaired hedonic balance documented at the behavioral level, and aligns with neuroimaging findings in humans despite the potential limitation induced by medetomidine sedation. This study paves the way to identify further biomarkers and to probe neurobiological mechanisms of alcohol effects using genetic and pharmacological manipulations in mouse models of alcohol drinking and dependence.

Macroscopic Structural and Connectome Mapping of the Mouse Brain Using Diffusion Magnetic Resonance Imaging.

Translational work in rodents elucidates basic mechanisms that drive complex behaviors relevant to psychiatric and neurological conditions. Nonetheless, numerous promising studies in rodents later fail in clinical trials, highlighting the need for improving the translational utility of preclinical studies in rodents. Imaging of small rodents provides an important strategy to address this challenge, as it enables a whole-brain unbiased search for structural and dynamic changes that can be directly compared to human imaging. The functional significance of structural changes identified using imaging can then be further investigated using molecular and genetic tools available for the mouse. Here, we describe a pipeline for unbiased search and characterization of structural changes and network properties, based on diffusion MRI data covering the entire mouse brain at an isotropic resolution of 100 µm. We first used unbiased whole-brain voxel-based analyses to identify volumetric and microstructural alterations in the brain of adult mice exposed to unpredictable postnatal stress (UPS), which is a mouse model of complex early life stress (ELS). Brain regions showing structural abnormalities were used as nodes to generate a grid for assessing structural connectivity and network properties based on graph theory. The technique described here can be broadly applied to understand brain connectivity in other mouse models of human disorders, as well as in genetically modified mouse strains. Graphic abstract: Pipeline for characterizing structural connectome in the mouse brain using diffusion magnetic resonance imaging. Scale bar = 1 mm.

Early life stress causes sex-specific changes in adult fronto-limbic connectivity that differentially drive learning.

It is currently unclear whether early life stress (ELS) affects males and females differently. However, a growing body of work has shown that sex moderates responses to stress and injury, with important insights into sex-specific mechanisms provided by work in rodents. Unfortunately, most of the ELS studies in rodents were conducted only in males, a bias that is particularly notable in translational work that has used human imaging. Here we examine the effects of unpredictable postnatal stress (UPS), a mouse model of complex ELS, using high resolution diffusion magnetic resonance imaging. We show that UPS induces several neuroanatomical alterations that were seen in both sexes and resemble those reported in humans. In contrast, exposure to UPS induced fronto-limbic hyper-connectivity in males, but either no change or hypoconnectivity in females. Moderated-mediation analysis found that these sex-specific changes are likely to alter contextual freezing behavior in males but not in females.

Mapping GPR88-Venus illuminates a novel role for GPR88 in sensory processing.

GPR88 is an orphan G-protein coupled receptor originally characterized as a striatal-enriched transcript and is a potential target for neuropsychiatric disorders. At present, gene knockout studies in the mouse have essentially focused on striatal-related functions and a comprehensive knowledge of GPR88 protein distribution and function in the brain is still lacking. Here, we first created Gpr88-Venus knock-in mice expressing a functional fluorescent receptor to fine-map GPR88 localization in the brain. The receptor protein was detected in neuronal soma, fibers and primary cilia depending on the brain region, and remarkably, whole-brain mapping revealed a yet unreported layer-4 cortical lamination pattern specifically in sensory processing areas. The unique GPR88 barrel pattern in L4 of the somatosensory cortex appeared 3 days after birth and persisted into adulthood, suggesting a potential function for GPR88 in sensory integration. We next examined Gpr88 knockout mice for cortical structure and behavioral responses in sensory tasks. Magnetic resonance imaging of live mice revealed abnormally high fractional anisotropy, predominant in somatosensory cortex and caudate putamen, indicating significant microstructural alterations in these GPR88-enriched areas. Further, behavioral analysis showed delayed responses in somatosensory-, visual- and olfactory-dependent tasks, demonstrating a role for GPR88 in the integration rather than perception of sensory stimuli. In conclusion, our data show for the first time a prominent role for GPR88 in multisensory processing. Because sensory integration is disrupted in many psychiatric diseases, our study definitely positions GPR88 as a target to treat mental disorders perhaps via activity on cortical sensory networks.

Increased Alcohol Seeking in Mice Lacking Gpr88 Involves Dysfunctional Mesocorticolimbic Networks.

BACKGOUND: Alcohol use disorder (AUD) is devastating and poorly treated, and innovative targets are actively sought for prevention and treatment. The orphan G protein-coupled receptor GPR88 is enriched in mesocorticolimbic pathways, and Gpr88 knockout mice show hyperactivity and risk-taking behavior, but a potential role for this receptor in drug abuse has not been examined. METHODS: We tested Gpr88 knockout mice for alcohol-drinking and -seeking behaviors. To gain system-level understanding of their alcohol endophenotype, we also analyzed whole-brain functional connectivity in naïve mice using resting-state functional magnetic resonance imaging. RESULTS: Gpr88 knockout mice showed increased voluntary alcohol drinking at both moderate and excessive levels, with intact alcohol sedation and metabolism. Mutant mice also showed increased operant responding and motivation for alcohol, while food and chocolate operant self-administration were unchanged. Alcohol place conditioning and alcohol-induced dopamine release in the nucleus accumbens were decreased, suggesting reduced alcohol reward in mutant mice that may partly explain enhanced alcohol drinking. Seed-based voxelwise functional connectivity analysis revealed significant remodeling of mesocorticolimbic centers, whose hallmark was predominant weakening of prefrontal cortex, ventral tegmental area, and amygdala connectional patterns. Also, effective connectivity from the ventral tegmental area to the nucleus accumbens and amygdala was reduced. CONCLUSIONS: Gpr88 deletion disrupts executive, reward, and emotional networks in a configuration that reduces alcohol reward and promotes alcohol seeking and drinking. The functional connectivity signature is reminiscent of alterations observed in individuals at risk for AUD. The Gpr88 gene, therefore, may represent a vulnerability/resilience factor for AUD, and a potential drug target for AUD treatment.

Remodeling of Sensorimotor Brain Connectivity in Gpr88-Deficient Mice.

Recent studies have demonstrated that orchestrated gene activity and expression support synchronous activity of brain networks. However, there is a paucity of information on the consequences of single gene function on overall brain functional organization and connectivity and how this translates at the behavioral level. In this study, we combined mouse mutagenesis with functional and structural magnetic resonance imaging (MRI) to determine whether targeted inactivation of a single gene would modify whole-brain connectivity in live animals. The targeted gene encodes GPR88 (G protein-coupled receptor 88), an orphan G protein-coupled receptor enriched in the striatum and previously linked to behavioral traits relevant to neuropsychiatric disorders. Connectivity analysis of Gpr88-deficient mice revealed extensive remodeling of intracortical and cortico-subcortical networks. Most prominent modifications were observed at the level of retrosplenial cortex connectivity, central to the default mode network (DMN) whose alteration is considered a hallmark of many psychiatric conditions. Next, somatosensory and motor cortical networks were most affected. These modifications directly relate to sensorimotor gating deficiency reported in mutant animals and also likely underlie their hyperactivity phenotype. Finally, we identified alterations within hippocampal and dorsal striatum functional connectivity, most relevant to a specific learning deficit that we previously reported in Gpr88-/- animals. In addition, amygdala connectivity with cortex and striatum was weakened, perhaps underlying the risk-taking behavior of these animals. This is the first evidence demonstrating that GPR88 activity shapes the mouse brain functional and structural connectome. The concordance between connectivity alterations and behavior deficits observed in Gpr88-deficient mice suggests a role for GPR88 in brain communication.