Virtual reality-empowered deep-learning analysis of brain cells.

Automated detection of specific cells in three-dimensional datasets such as whole-brain light-sheet image stacks is challenging. Here, we present DELiVR, a virtual reality-trained deep-learning pipeline for detecting c-Fos+ cells as markers for neuronal activity in cleared mouse brains. Virtual reality annotation substantially accelerated training data generation, enabling DELiVR to outperform state-of-the-art cell-segmenting approaches. Our pipeline is available in a user-friendly Docker container that runs with a standalone Fiji plugin. DELiVR features a comprehensive toolkit for data visualization and can be customized to other cell types of interest, as we did here for microglia somata, using Fiji for dataset-specific training. We applied DELiVR to investigate cancer-related brain activity, unveiling an activation pattern that distinguishes weight-stable cancer from cancers associated with weight loss. Overall, DELiVR is a robust deep-learning tool that does not require advanced coding skills to analyze whole-brain imaging data in health and disease.

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Defensive and offensive behaviours in a Kleefstra syndrome mouse model.

Kleefstra syndrome in humans is characterized by a general delay in development, intellectual disability and autistic features. The mouse model of this disease (Ehmt1±) expresses anxiety, autistic-like traits, and aberrant social interactions with non-cagemates. To investigate how Ehmt1± mice behave with unfamiliar conspecifics, we allowed adult, male animals to freely interact for 10 min in a neutral, novel environment within a host-visitor setting. In trials where the Ehmt1± mice were hosts, there were defensive and offensive behaviors. Our key finding was that Ehmt1± mice displayed defensive postures, attacking and biting; in contrast, wild-type (WT) interacting with other WT did not enact such behaviors. Further, if there was a fight between an Ehmt1± and a WT mouse, the Ehmt1± animal was the most aggressive and always initiated these behaviors.

Spatial proteomics in three-dimensional intact specimens.

Spatial molecular profiling of complex tissues is essential to investigate cellular function in physiological and pathological states. However, methods for molecular analysis of large biological specimens imaged in 3D are lacking. Here, we present DISCO-MS, a technology that combines whole-organ/whole-organism clearing and imaging, deep-learning-based image analysis, robotic tissue extraction, and ultra-high-sensitivity mass spectrometry. DISCO-MS yielded proteome data indistinguishable from uncleared samples in both rodent and human tissues. We used DISCO-MS to investigate microglia activation along axonal tracts after brain injury and characterized early- and late-stage individual amyloid-beta plaques in a mouse model of Alzheimer's disease. DISCO-bot robotic sample extraction enabled us to study the regional heterogeneity of immune cells in intact mouse bodies and aortic plaques in a complete human heart. DISCO-MS enables unbiased proteome analysis of preclinical and clinical tissues after unbiased imaging of entire specimens in 3D, identifying diagnostic and therapeutic opportunities for complex diseases. VIDEO ABSTRACT.

FriendlyClearMap: an optimized toolkit for mouse brain mapping and analysis.

BACKGROUND: Tissue clearing is currently revolutionizing neuroanatomy by enabling organ-level imaging with cellular resolution. However, currently available tools for data analysis require a significant time investment for training and adaptation to each laboratory's use case, which limits productivity. Here, we present FriendlyClearMap, an integrated toolset that makes ClearMap1 and ClearMap2's CellMap pipeline easier to use, extends its functions, and provides Docker Images from which it can be run with minimal time investment. We also provide detailed tutorials for each step of the pipeline. FINDINGS: For more precise alignment, we add a landmark-based atlas registration to ClearMap's functions as well as include young mouse reference atlases for developmental studies. We provide an alternative cell segmentation method besides ClearMap's threshold-based approach: Ilastik's Pixel Classification, importing segmentations from commercial image analysis packages and even manual annotations. Finally, we integrate BrainRender, a recently released visualization tool for advanced 3-dimensional visualization of the annotated cells. CONCLUSIONS: As a proof of principle, we use FriendlyClearMap to quantify the distribution of the 3 main GABAergic interneuron subclasses (parvalbumin+ [PV+], somatostatin+, and vasoactive intestinal peptide+) in the mouse forebrain and midbrain. For PV+ neurons, we provide an additional dataset with adolescent vs. adult PV+ neuron density, showcasing the use for developmental studies. When combined with the analysis pipeline outlined above, our toolkit improves on the state-of-the-art packages by extending their function and making them easier to deploy at scale.

Cadherin-13 is a critical regulator of GABAergic modulation in human stem-cell-derived neuronal networks.

Activity in the healthy brain relies on a concerted interplay of excitation (E) and inhibition (I) via balanced synaptic communication between glutamatergic and GABAergic neurons. A growing number of studies imply that disruption of this E/I balance is a commonality in many brain disorders; however, obtaining mechanistic insight into these disruptions, with translational value for the patient, has typically been hampered by methodological limitations. Cadherin-13 (CDH13) has been associated with autism and attention-deficit/hyperactivity disorder. CDH13 localizes at inhibitory presynapses, specifically of parvalbumin (PV) and somatostatin (SST) expressing GABAergic neurons. However, the mechanism by which CDH13 regulates the function of inhibitory synapses in human neurons remains unknown. Starting from human-induced pluripotent stem cells, we established a robust method to generate a homogenous population of SST and MEF2C (PV-precursor marker protein) expressing GABAergic neurons (iGABA) in vitro, and co-cultured these with glutamatergic neurons at defined E/I ratios on micro-electrode arrays. We identified functional network parameters that are most reliably affected by GABAergic modulation as such, and through alterations of E/I balance by reduced expression of CDH13 in iGABAs. We found that CDH13 deficiency in iGABAs decreased E/I balance by means of increased inhibition. Moreover, CDH13 interacts with Integrin-ß1 and Integrin-ß3, which play opposite roles in the regulation of inhibitory synaptic strength via this interaction. Taken together, this model allows for standardized investigation of the E/I balance in a human neuronal background and can be deployed to dissect the cell-type-specific contribution of disease genes to the E/I balance.

The emerging role of chromatin remodelers in neurodevelopmental disorders: a developmental perspective.

Neurodevelopmental disorders (NDDs), including intellectual disability (ID) and autism spectrum disorders (ASD), are a large group of disorders in which early insults during brain development result in a wide and heterogeneous spectrum of clinical diagnoses. Mutations in genes coding for chromatin remodelers are overrepresented in NDD cohorts, pointing towards epigenetics as a convergent pathogenic pathway between these disorders. In this review we detail the role of NDD-associated chromatin remodelers during the developmental continuum of progenitor expansion, differentiation, cell-type specification, migration and maturation. We discuss how defects in chromatin remodelling during these early developmental time points compound over time and result in impaired brain circuit establishment. In particular, we focus on their role in the three largest cell populations: glutamatergic neurons, GABAergic neurons, and glia cells. An in-depth understanding of the spatiotemporal role of chromatin remodelers during neurodevelopment can contribute to the identification of molecular targets for treatment strategies.

EHMT1 regulates Parvalbumin-positive interneuron development and GABAergic input in sensory cortical areas.

Mutations in the Euchromatic Histone Methyltransferase 1 (EHMT1) gene cause Kleefstra syndrome, a rare form of intellectual disability (ID) with strong autistic traits and sensory processing deficits. Proper development of inhibitory interneurons is crucial for sensory function. Here we report a timeline of Parvalbumin-positive (PV+) interneuron development in the three most important sensory cortical areas in the Ehmt1+/- mouse. We find a hitherto unreported delay of PV+ neuron maturation early in sensory development, with layer- and region-specific variability later in development. The delayed PV+ maturation is also reflected in a delayed maturation of GABAergic transmission in Ehmt1+/- auditory cortex, where we find a reduced GABA release probability specifically in putative PV+ synapses. Together with earlier reports of excitatory impairments in Ehmt1+/- neurons, we propose a shift in excitatory-inhibitory balance towards overexcitability in Ehmt1+/- sensory cortices as a consequence of early deficits in inhibitory maturation.

The Object Space Task reveals increased expression of cumulative memory in a mouse model of Kleefstra syndrome.

Kleefstra syndrome is a disorder caused by a mutation in the EHMT1 gene characterized in humans by general developmental delay, mild to severe intellectual disability and autism. Here, we characterized cumulative memory in the Ehmt1+/- mouse model using the Object Space Task. We combined conventional behavioral analysis with automated analysis by deep-learning networks, a session-based computational learning model, and a trial-based classifier. Ehmt1+/- mice showed more anxiety-like features and generally explored objects less, but the difference decreased over time. Interestingly, when analyzing memory-specific exploration, Ehmt1+/- show increased expression of cumulative memory, but a deficit in a more simple, control memory condition. Using our automatic classifier to differentiate between genotypes, we found that cumulative memory features are better suited for classification than general exploration differences. Thus, detailed behavioral classification with the Object Space Task produced a more detailed behavioral phenotype of the Ehmt1+/- mouse model.

Neuronal network dysfunction in a model for Kleefstra syndrome mediated by enhanced NMDAR signaling.

Kleefstra syndrome (KS) is a neurodevelopmental disorder caused by mutations in the histone methyltransferase EHMT1. To study the impact of decreased EHMT1 function in human cells, we generated excitatory cortical neurons from induced pluripotent stem (iPS) cells derived from KS patients. Neuronal networks of patient-derived cells exhibit network bursting with a reduced rate, longer duration, and increased temporal irregularity compared to control networks. We show that these changes are mediated by upregulation of NMDA receptor (NMDAR) subunit 1 correlating with reduced deposition of the repressive H3K9me2 mark, the catalytic product of EHMT1, at the GRIN1 promoter. In mice EHMT1 deficiency leads to similar neuronal network impairments with increased NMDAR function. Finally, we rescue the KS patient-derived neuronal network phenotypes by pharmacological inhibition of NMDARs. Summarized, we demonstrate a direct link between EHMT1 deficiency and NMDAR hyperfunction in human neurons, providing a potential basis for more targeted therapeutic approaches for KS.

V1 connections reveal a series of elongated higher visual areas in the California ground squirrel, Otospermophilus beecheyi.

For studies of visual cortex organization, mouse is becoming an increasingly more often used model. In addition to its genetic tractability, the relatively small area of cortical surface devoted to visual processing simplifies efforts in relating the structure of visual cortex to visual function. However, the nature of this compact organization can make some comparisons to the much larger non-human primate visual cortex difficult. The squirrel, as a highly visual rodent offers a useful means for better understanding how mouse and monkey cortical organization compares. More in line with primates than their nocturnal rodent cousin, squirrels rely much more on sight and have evolved a larger expanse of cortex devoted to visual processing. To reveal the detailed organization of visual cortex in squirrels, we injected a highly sensitive monosynaptic retrograde tracer (glycoprotein deleted rabies virus) into several locations of primary visual cortex (V1) in California ground squirrels. The resulting pattern of connectivity revealed an organizational scheme in the squirrel that retains some of the basic features of the mouse visual cortex along the medial and posterior borders of V1, but unlike mouse has an elaborate and extensive pattern laterally that is more similar to the early visual cortex organization found in monkeys. In this way, we show that the squirrel can serve as a useful model for comparison to both mouse and primate visual systems, and may help facilitate comparisons between these two very different yet widely used animal models of visual processing.

Increased GABAB receptor signaling in a rat model for schizophrenia.

Schizophrenia is a complex disorder that affects cognitive function and has been linked, both in patients and animal models, to dysfunction of the GABAergic system. However, the pathophysiological consequences of this dysfunction are not well understood. Here, we examined the GABAergic system in an animal model displaying schizophrenia-relevant features, the apomorphine-susceptible (APO-SUS) rat and its phenotypic counterpart, the apomorphine-unsusceptible (APO-UNSUS) rat at postnatal day 20-22. We found changes in the expression of the GABA-synthesizing enzyme GAD67 specifically in the prelimbic- but not the infralimbic region of the medial prefrontal cortex (mPFC), indicative of reduced inhibitory function in this region in APO-SUS rats. While we did not observe changes in basal synaptic transmission onto LII/III pyramidal cells in the mPFC of APO-SUS compared to APO-UNSUS rats, we report reduced paired-pulse ratios at longer inter-stimulus intervals. The GABAB receptor antagonist CGP 55845 abolished this reduction, indicating that the decreased paired-pulse ratio was caused by increased GABAB signaling. Consistently, we find an increased expression of the GABAB1 receptor subunit in APO-SUS rats. Our data provide physiological evidence for increased presynaptic GABAB signaling in the mPFC of APO-SUS rats, further supporting an important role for the GABAergic system in the pathophysiology of schizophrenia.

Tracing inputs to inhibitory or excitatory neurons of mouse and cat visual cortex with a targeted rabies virus.

BACKGROUND: Cortical inhibition plays a critical role in controlling and modulating cortical excitation, and a more detailed understanding of the neuronal circuits contributing to each will provide more insight into their roles in complex cortical computations. Traditional neuronal tracers lack a means for easily distinguishing between circuits of inhibitory and excitatory neurons. To overcome this limitation, we have developed a technique for retrogradely labeling inputs to local clusters of inhibitory or excitatory neurons, but not both, using neurotropic adenoassociated and lentiviral vectors, cell-type-specific promoters, and a modified rabies virus. RESULTS: Applied to primary visual cortex (V1) in mouse, the cell-type-specific tracing technique labeled thousands of presynaptically connected neurons and revealed that the dominant source of input to inhibitory and excitatory neurons is local in origin. Neurons in other visual areas are also labeled; the percentage of these intercortical inputs to excitatory neurons is somewhat higher (~20%) than to inhibitory neurons (<10%), suggesting that intercortical connections have less direct control over inhibition. The inputs to inhibitory neurons were also traced in cat V1, and when aligned with the orientation preference map revealed for the first time that long-range inputs to inhibitory neurons are well tuned to orientation. CONCLUSIONS: These novel findings for inhibitory and excitatory circuits in the visual cortex demonstrate the efficacy of our new technique and its ability to work across species, including larger-brained mammals such as the cat. This paves the way for a better understanding of the roles of specific cell types in higher-order perceptual and cognitive processes.

High serotonin levels during brain development alter the structural input-output connectivity of neural networks in the rat somatosensory layer IV.

Homeostatic regulation of serotonin (5-HT) concentration is critical for "normal" topographical organization and development of thalamocortical (TC) afferent circuits. Down-regulation of the serotonin transporter (SERT) and the consequent impaired reuptake of 5-HT at the synapse, results in a reduced terminal branching of developing TC afferents within the primary somatosensory cortex (S1). Despite the presence of multiple genetic models, the effect of high extracellular 5-HT levels on the structure and function of developing intracortical neural networks is far from being understood. Here, using juvenile SERT knockout (SERT(-/-)) rats we investigated, in vitro, the effect of increased 5-HT levels on the structural organization of (i) the TC projections of the ventroposteromedial thalamic nucleus toward S1, (ii) the general barrel-field pattern, and (iii) the electrophysiological and morphological properties of the excitatory cell population in layer IV of S1 [spiny stellate (SpSt) and pyramidal cells]. Our results confirmed previous findings that high levels of 5-HT during development lead to a reduction of the topographical precision of TCA projections toward the barrel cortex. Also, the barrel pattern was altered but not abolished in SERT(-/-) rats. In layer IV, both excitatory SpSt and pyramidal cells showed a significantly reduced intracolumnar organization of their axonal projections. In addition, the layer IV SpSt cells gave rise to a prominent projection toward the infragranular layer Vb. Our findings point to a structural and functional reorganization of TCAs, as well as early stage intracortical microcircuitry, following the disruption of 5-HT reuptake during critical developmental periods. The increased projection pattern of the layer IV neurons suggests that the intracortical network changes are not limited to the main entry layer IV but may also affect the subsequent stages of the canonical circuits of the barrel cortex.