Reconstruction of neocortex: Organelles, compartments, cells, circuits, and activity.

We assembled a semi-automated reconstruction of L2/3 mouse primary visual cortex from ∼250 × 140 × 90 µm3 of electron microscopic images, including pyramidal and non-pyramidal neurons, astrocytes, microglia, oligodendrocytes and precursors, pericytes, vasculature, nuclei, mitochondria, and synapses. Visual responses of a subset of pyramidal cells are included. The data are publicly available, along with tools for programmatic and three-dimensional interactive access. Brief vignettes illustrate the breadth of potential applications relating structure to function in cortical circuits and neuronal cell biology. Mitochondria and synapse organization are characterized as a function of path length from the soma. Pyramidal connectivity motif frequencies are predicted accurately using a configuration model of random graphs. Pyramidal cells receiving more connections from nearby cells exhibit stronger and more reliable visual responses. Sample code shows data access and analysis.

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

The Allen Mouse Brain Common Coordinate Framework: A 3D Reference Atlas.

Recent large-scale collaborations are generating major surveys of cell types and connections in the mouse brain, collecting large amounts of data across modalities, spatial scales, and brain areas. Successful integration of these data requires a standard 3D reference atlas. Here, we present the Allen Mouse Brain Common Coordinate Framework (CCFv3) as such a resource. We constructed an average template brain at 10 µm voxel resolution by interpolating high resolution in-plane serial two-photon tomography images with 100 µm z-sampling from 1,675 young adult C57BL/6J mice. Then, using multimodal reference data, we parcellated the entire brain directly in 3D, labeling every voxel with a brain structure spanning 43 isocortical areas and their layers, 329 subcortical gray matter structures, 81 fiber tracts, and 8 ventricular structures. CCFv3 can be used to analyze, visualize, and integrate multimodal and multiscale datasets in 3D and is openly accessible (https://atlas.brain-map.org/).

Gradients of functional connectivity in the mouse cortex reflect neocortical evolution.

Understanding cortical organization is a fundamental goal of neuroscience that requires comparisons across species and modalities. Large-scale connectivity gradients have recently been introduced as a data-driven representation of the intrinsic organization of the cortex. We studied resting-state functional connectivity gradients in the mouse cortex and found robust spatial patterns across four data sets. The principal gradient of functional connectivity shows a striking overlap with an axis of neocortical evolution from two primordial origins. Additional gradients reflect sensory specialization and aspects of a sensory-to-transmodal hierarchy, and are associated with transcriptomic features. While some of these gradients strongly resemble observations in the human cortex, the overall pattern in the mouse cortex emphasizes the specialization of sensory areas over a global functional hierarchy.

Compensatory molecular and functional mechanisms in nervous system of the Grm1(crv4) mouse lacking the mGlu1 receptor: a model for motor coordination deficits.

The metabotropic glutamate type 1 (mGlu1) and type 5 (mGlu5) receptors, the only members of group I mGlu receptors, are implicated in synaptic plasticity and mechanisms of feedback control of glutamate release. They exhibit nearly complementary distributions throughout the central nervous system, well evident in the cerebellum, where mGlu1 receptor is most intensely expressed while mGlu5 receptor is not. Despite their different distribution, they show a similar subcellular localization and use common transducing pathways. We recently described the Grm1(crv4) mouse with motor coordination deficits and renal anomalies caused by a spontaneous mutation inactivating the mGlu1 receptor. To define the neuropathological mechanisms in these mice, we evaluated expression and function of the mGlu5 receptor in cerebral and cerebellar cortices. Western blot and immunofluorescence analyses showed mGlu5 receptor overexpression. Quantitative reverse transcriptase-polymerase chain reaction results indicated that the up-regulation is already evident at RNA level. Functional studies confirmed an enhanced glutamate release from cortical cerebral and cerebellar synaptosomes when compared with wild-type that is abolished by the mGlu5 receptor-specific inhibitor, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP). Finally, acute MPEP treatment of Grm1(crv4/crv4) mice induced an evident although incomplete improvement of motor coordination, suggesting that mGlu5 receptors enhanced activity worsens, instead of improving, the motor-coordination defects in the Grm1(crv4/crv4) mice.

Isoflurane preconditioning induced genomic changes in mouse cortex.

Background: Altered patterns of genetic expression induced by isoflurane preconditioning in mouse brain have not yet been investigated. The aim of our pilot study is to examine the temporal sequence of changes in the transcriptome of mouse brain cortex produced by isoflurane preconditioning. Methods: Twelve-wk-old wild-type (C57BL/6J) male mice were randomly assigned for the experiments. Mice were exposed to isoflurane 2% in air for 1 h and brains were harvested at the following time points-immediately (0 h), and at 6, 12, 24, 36, 48, and 72 h after isoflurane exposure. A separate cohort of mice were exposed to three doses of isoflurane on days 1, 2, and 3 and brains were harvested after the third exposure. The NanoString mouse neuropathology panel was used to analyse isoflurane-induced gene expression in the cortex. The neuropathology panel included 760 genes covering pathways involved in neurodegeneration and other nervous system diseases, and 10 internal reference genes for data normalisation. Results: Genes involving several pathways were upregulated and downregulated by isoflurane preconditioning. Interestingly, a biphasic response was noted, meaning, an early expression of genes (until 6 h), followed by a transient pause (until 24 h), and a second wave of genomic response beginning at 36 h of isoflurane exposure was noted. Conclusions: Isoflurane preconditioning induces significant alterations in the genes involved in neurodegeneration and other nervous system disorders in a temporal sequence. These data could aid in the identification of molecular mechanisms behind isoflurane preconditioning-induced neuroprotection in various central nervous system diseases.

TET1 displays catalytic and non-catalytic functions in the adult mouse cortex.

TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.

Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

Glucocorticoids are important for proper organ maturation, and their levels are tightly regulated during development. Here, we use human cerebral organoids and mice to study the cell-type-specific effects of glucocorticoids on neurogenesis. We show that glucocorticoids increase a specific type of basal progenitors (co-expressing PAX6 and EOMES) that has been shown to contribute to cortical expansion in gyrified species. This effect is mediated via the transcription factor ZBTB16 and leads to increased production of neurons. A phenome-wide Mendelian randomization analysis of an enhancer variant that moderates glucocorticoid-induced ZBTB16 levels reveals causal relationships with higher educational attainment and altered brain structure. The relationship with postnatal cognition is also supported by data from a prospective pregnancy cohort study. This work provides a cellular and molecular pathway for the effects of glucocorticoids on human neurogenesis that relates to lasting postnatal phenotypes.

TEMPO enables sequential genetic labeling and manipulation of vertebrate cell lineages.

During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

Extracting Dynamical Understanding From Neural-Mass Models of Mouse Cortex.

New brain atlases with high spatial resolution and whole-brain coverage have rapidly advanced our knowledge of the brain's neural architecture, including the systematic variation of excitatory and inhibitory cell densities across the mammalian cortex. But understanding how the brain's microscale physiology shapes brain dynamics at the macroscale has remained a challenge. While physiologically based mathematical models of brain dynamics are well placed to bridge this explanatory gap, their complexity can form a barrier to providing clear mechanistic interpretation of the dynamics they generate. In this work, we develop a neural-mass model of the mouse cortex and show how bifurcation diagrams, which capture local dynamical responses to inputs and their variation across brain regions, can be used to understand the resulting whole-brain dynamics. We show that strong fits to resting-state functional magnetic resonance imaging (fMRI) data can be found in surprisingly simple dynamical regimes-including where all brain regions are confined to a stable fixed point-in which regions are able to respond strongly to variations in their inputs, consistent with direct structural connections providing a strong constraint on functional connectivity in the anesthetized mouse. We also use bifurcation diagrams to show how perturbations to local excitatory and inhibitory coupling strengths across the cortex, constrained by cell-density data, provide spatially dependent constraints on resulting cortical activity, and support a greater diversity of coincident dynamical regimes. Our work illustrates methods for visualizing and interpreting model performance in terms of underlying dynamical mechanisms, an approach that is crucial for building explanatory and physiologically grounded models of the dynamical principles that underpin large-scale brain activity.

The stereospecific interaction sites and target specificity of cGMP analogs in mouse cortex.

cGMP interactors play a role in several pathologies and may be targets for cGMP analog-based drugs, but the success of targeting depends on the biochemical stereospecificity between the cGMP-analog and the interactor. The stereospecificity between general cGMP analogs-or such that are selectivity-modified to obtain, for example, inhibitory actions on a specific target, like the cGMP-dependent protein kinase-have previously been investigated. However, the importance of stereospecificity for cGMP-analog binding to interactors is not known. We, therefore, applied affinity chromatography on mouse cortex proteins utilizing analogs with cyclic phosphate (8-AET-cGMP, 2-AH-cGMP, 2'-AHC-cGMP) and selectivity-modified analogs with sulfur-containing cyclic phosphorothioates (Rp/Sp-8-AET-cGMPS, Rp/Sp-2'-AHC-cGMPS) immobilized to agaroses. The results illustrate the cGMP analogs' stereospecific binding for PKG, PKA regulatory subunits and PKA catalytic subunits, PDEs, and EPAC2 and the involvement of these in various KEGG pathways. For the seven agaroses, PKG, PKA regulatory subunits, and PKA catalytic subunits were more prone to be enriched by 2-AH-, 8-AET-, Rp-8-AET-, and Sp-8-AET-cGMP, whereas PDEs and EPAC2 were more likely to be enriched by 2-AH-, Rp-2'-AHC-, and Rp-8-AET-cGMP. Our findings help elucidate the stereospecific-binding sites essential for the interaction between individual cGMP analogs and cGMP-binding proteins, as well as the cGMP analogs' target specificity, which are two crucial parameters in drug design.

Optogenetic strategies for high-efficiency all-optical interrogation using blue-light-sensitive opsins.

All-optical methods for imaging and manipulating brain networks with high spatial resolution are fundamental to study how neuronal ensembles drive behavior. Stimulation of neuronal ensembles using two-photon holographic techniques requires high-sensitivity actuators to avoid photodamage and heating. Moreover, two-photon-excitable opsins should be insensitive to light at wavelengths used for imaging. To achieve this goal, we developed a novel soma-targeted variant of the large-conductance blue-light-sensitive opsin CoChR (stCoChR). In the mouse cortex in vivo, we combined holographic two-photon stimulation of stCoChR with an amplified laser tuned at the opsin absorption peak and two-photon imaging of the red-shifted indicator jRCaMP1a. Compared to previously characterized blue-light-sensitive soma-targeted opsins in vivo, stCoChR allowed neuronal stimulation with more than 10-fold lower average power and no spectral crosstalk. The combination of stCoChR, tuned amplified laser stimulation, and red-shifted functional indicators promises to be a powerful tool for large-scale interrogation of neural networks in the intact brain.

The Spatial Structure of Neural Encoding in Mouse Posterior Cortex during Navigation.

Navigation engages many cortical areas, including visual, parietal, and retrosplenial cortices. These regions have been mapped anatomically and with sensory stimuli and studied individually during behavior. Here, we investigated how behaviorally driven neural activity is distributed and combined across these regions. We performed dense sampling of single-neuron activity across the mouse posterior cortex and developed unbiased methods to relate neural activity to behavior and anatomical space. Most parts of the posterior cortex encoded most behavior-related features. However, the relative strength with which features were encoded varied across space. Therefore, the posterior cortex could be divided into discriminable areas based solely on behaviorally relevant neural activity, revealing functional structure in association regions. Multimodal representations combining sensory and movement signals were strongest in posterior parietal cortex, where gradients of single-feature representations spatially overlapped. We propose that encoding of behavioral features is not constrained by retinotopic borders and instead varies smoothly over space within association regions.

Two-Photon STED Microscopy for Nanoscale Imaging of Neural Morphology In Vivo.

The advent of super-resolution microscopy offers to bridge the gap between electron and light microscopy. It has opened up the possibility of visualizing cellular structures and dynamic signaling events on the "mesoscale" well below the classic diffraction barrier of light microscopy (10-200 nm), while essentially retaining the advantages of fluorescence microscopy concerning multicolor labeling, detection sensitivity, signal contrast, live-cell imaging, and temporal resolution.From among the new super-resolution techniques, STED microscopy stands out as a laser-scanning imaging modality, which enables nanoscale volume-metric imaging of cellular morphology. In combination with two-photon (2P) excitation, STED microscopy facilitates the visualization of the highly complex and dynamic morphology of neurons and glia cells deep inside living brain slices and in the intact brain in vivo.Here, we present an overview of the principles and implementation of 2P-STED microscopy in vivo, providing the neurobiological context and motivation for this technique, and illustrating its capacity by showing images of dendritic spines and microglial processes obtained from living brain tissue.

Antibody/receptor protein immunocomplex in human and mouse cortical nerve endings amplifies complement-induced glutamate release.

Previous studies have demonstrated that complement alone releases glutamate from human and mouse cortical terminals in an antibody-independent manner. In order to expand our knowledge on complement-mediated effects, we investigated whether the presence of an antigen-antibody complex in synaptosomal plasmamembranes could also trigger complement-induced functional responses that might affect neurotransmitter release. To this aim, we focused on the chemokine 5 receptor (CCR5) expressed in human and mouse cortical glutamate terminals, whose activation by CCL5 elicits [(3)H]D-aspartate ([(3)H]D-ASP) release. Preincubating synaptosomes with an antibody recognizing the NH2 terminus of the CCR5 protein (anti-NH2-CCR5 antibody) abolished the CCL5-induced [(3)H]D-ASP release. Similarly, enriching synaptosomes with an antibody recognizing the COOH terminus of CCR5 (anti-COOH-CCR5 antibody) prevented the CCL5-induced [(3)H]D-ASP release. The antagonist-like activity of the anti-NH2-CCR5 antibody turned to facilitation when anti-NH2-CCR5-treated synaptosomes were exposed to complement. In these terminals, the releasing effect was significantly higher than that elicited by complement in untreated synaptosomes. On the contrary, the complement-induced [(3)H]D-ASP release from anti-COOH-CCR5 antibody-entrapped synaptosomes did not differ from that from untreated synaptosomes. Preincubating synaptosomes with anti-beta tubulin III antibody, used as negative control, neither prevented the CCL5-induced releasing effect nor it amplified the complement-induced [(3)H]D-ASP release. Finally, serum lacking the C1q protein, i.e. the protein essential to promote the antibody-mediated activation of complement, elicited a comparable [(3)H]D-ASP release from both untreated and anti-NH2-CCR5 antibody-treated synaptosomes. Thus, we propose that antibodies raised against the outer sequence of a receptor protein can trigger the activation of the complement through the classic, C1q-mediated antibody-dependent pathway, which results in an abnormal release of glutamate that could be deleterious to central nervous system.

Directing mouse embryonic neurosphere differentiation toward an enriched neuronal population.

Neural stem cells (NSC) are self-renewing multipotent cells that have emerged as a powerful tool to repair the injured brain. These cells can be cultured as neurospheres, which are floating aggregates of neural stem/progenitor cells (NSPCs). Despite their high clonal expansion capacity, it has been suggested that in neurospheres, only a small percentage of cells are capable of proliferation and that this system is not efficient in terms of neurogenic competence. Thus, our aim was to develop a neurosphere culture method with a highly proliferative stem/progenitor cell population and particularly with a prominent neurogenic potential, surpassing some of the claimed weaknesses of the neurosphere assay. In our model, mouse neurospheres were harvested from neural tissue at E15 and after only 4 days in vitro (DIV), we have achieved highly proliferative primary neurospheres (81% Sox2 and 76% Ki67 positive cells) and a rather low number of cells expressing glial and neuronal markers (∼10%). After inducing differentiation, we have attained an enriched neuronal population (45% ß-III-tubulin positive cells at 15 DIV). Using a simple methodology, we have developed a NSPC model that can provide a valuable source of neuronal precursors, thus offering a potential starting point for cell replacement therapies following CNS injury.