SummaryUnderstanding the molecular anatomy and neural connectivity of the brain requires imaging technologies that can map the 3D nanoscale distribution of specific proteins in the context of brain ultrastructure. Light and electron microscopy (EM) enable visualization of either specific labels or anatomical ultrastructure, but combining molecular specificity with anatomical context is challenging. Here, we present pan-Expansion Microscopy of tissue (pan-ExM-t), an all-optical mouse brain imaging method that combines ∼24-fold linear expansion of biological samples with fluorescent pan-staining of protein densities (providing EM-like ultrastructural context), and immunolabeling of protein targets (for molecular imaging). We demonstrate the versatility of this approach by imaging the established synaptic markers Homer1, Bassoon, PSD-95, Synaptophysin, the astrocytic protein GFAP, myelin basic protein (MBP), and anti-GFP antibodies in dissociated neuron cultures and mouse brain tissue sections. pan-ExM-t reveals these markers in the context of ultrastructural features such as pre and postsynaptic densities, 3D nanoarchitecture of neuropil, and the fine structures of cellular organelles. pan-ExM-t is adoptable in any neurobiological laboratory with access to a confocal microscope and has therefore broad applicability in the research community.Highlightspan-ExM-t visualizes proteins in the context of synaptic ultrastructureLipid labeling in pan-ExM-t reveals organellar and cellular membranesAll-optical, easily accessible alternative to correlative light/electron microscopyHigh potential for high throughput connectomics studies

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells.

Understanding cellular organization demands the best possible spatial resolution in all three dimensions (3D). In fluorescence microscopy, this is achieved by 4Pi nanoscopy methods that combine the concepts of using two opposing objectives for optimal diffraction-limited 3D resolution with switching fluorescent molecules between bright and dark states to break the diffraction limit. However, optical aberrations have limited these nanoscopes to thin samples and prevented their application in thick specimens. Here we have developed an improved isoSTED nanoscope, which utilizes an advanced adaptive optics strategy to achieve sub-50 nm isotropic resolution of structures such as neuronal synapses and ring canals previously inaccessible in tissue. The adaptive optics scheme presented in this work is generally applicable to any microscope with a similar beam path geometry involving two opposing objectives to optimize resolution when imaging deep in aberrating specimens.

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Impulsivity is a multidimensional heritable phenotype that broadly refers to the tendency to act prematurely and is associated with multiple forms of psychopathology, including substance use disorders. We performed genome-wide association studies (GWAS) of eight impulsive personality traits from the Barratt Impulsiveness Scale and the short UPPS-P Impulsive Personality Scale (N = 123,509–133,517 23andMe research participants of European ancestry), and a measure of Drug Experimentation (N = 130,684). Because these GWAS implicated the gene CADM2, we next performed single-SNP phenome-wide studies (PheWAS) of several of the implicated variants in CADM2 in a multi-ancestral 23andMe cohort (N = 3,229,317, European; N = 579,623, Latin American; N = 199,663, African American). Finally, we produced Cadm2 mutant mice and used them to perform a Mouse-PheWAS (“MouseWAS”) by testing them with a battery of relevant behavioral tasks. In humans, impulsive personality traits showed modest chip-heritability (~6–11%), and moderate genetic correlations (rg = 0.20–0.50) with other personality traits, and various psychiatric and medical traits. We identified significant associations proximal to genes such as TCF4 and PTPRF, and also identified nominal associations proximal to DRD2 and CRHR1. PheWAS for CADM2 variants identified associations with 378 traits in European participants, and 47 traits in Latin American participants, replicating associations with risky behaviors, cognition and BMI, and revealing novel associations including allergies, anxiety, irritable bowel syndrome, and migraine. Our MouseWAS recapitulated some of the associations found in humans, including impulsivity, cognition, and BMI. Our results further delineate the role of CADM2 in impulsivity and numerous other psychiatric and somatic traits across ancestries and species.

Brain development is likely impacted by micronutrients. This is supported by the effects of the ω-3 fatty acid docosahexaenoic acid (DHA) during early neuronal differentiation, when it increases neurite growth. Aiming to delineate DHA roles in postnatal stages, we selected the visual cortex due to its stereotypic maturation. Immunohistochemistry showed that young mice that received dietary DHA from birth exhibited more abundant presynaptic and postsynaptic specializations. DHA also increased density and size of synapses in a dose-dependent manner in cultured neurons. In addition, dendritic arbors of neurons treated with DHA were more complex. In agreement with improved connectivity, DHA enhanced physiological parameters of network maturation in vitro, including bursting strength and oscillatory behavior. Aiming to analyze functional maturation of the cortex, we performed in vivo electrophysiological recordings from awake mice to measure responses to patterned visual inputs. Dietary DHA robustly promoted the developmental increase in visual acuity, without altering light sensitivity. The visual acuity of DHA-supplemented animals continued to improve even after their cortex had matured and DHA abolished the acuity plateau. Our findings show that the ω-3 fatty acid DHA promotes synaptic connectivity and cortical processing. These results provide evidence that micronutrients can support the maturation of neuronal networks.

Multiple trans-synaptic complexes organize synapse development, yet their roles in the mature brain and cooperation remain unclear. We analyzed the postsynaptic adhesion protein LRRTM1 in the prefrontal cortex (PFC), a region relevant to cognition and disorders. LRRTM1 knockout (KO) mice had fewer synapses, and we asked whether other synapse organizers counteract further loss. This determined that the immunoglobulin family member SynCAM 1 controls synapse number in PFC and was upregulated upon LRRTM1 loss. Combined LRRTM1 and SynCAM 1 deletion substantially lowered dendritic spine number in PFC, but not hippocampus, more than the sum of single KO impairments. Their cooperation extended presynaptically, and puncta of Neurexins, LRRTM1 partners, were less abundant in double KO (DKO) PFC. Electrophysiology and fMRI demonstrated aberrant neuronal activity in DKO mice. Further, DKO mice were impaired in social interactions and cognitive tasks. Our results reveal concerted roles of LRRTM1 and SynCAM 1 across synaptic, network, and behavioral domains.