Biology subjects

Novarino, G.

Publications and source records attributed to Novarino, G..

7 recordsLinked to original sources

bioRxiv2024

Light-microscopy based dense connectomic reconstruction of mammalian brain tissue

The information-processing capability of the brains cellular network depends on the physical wiring pattern between neurons and their molecular and functional characteristics. Mapping neurons and resolving their individual synaptic connections can be achieved by volumetric imaging at nanoscale resolution with dense cellular labeling. Light microscopy is uniquely positioned to visualize specific molecules but dense, synapse-level circuit reconstruction by light microscopy has been out of reach due to limitations in resolution, contrast, and volumetric imaging capability. Here we developed light-microscopy based connectomics (LICONN). We integrated specifically engineered hydrogel embedding and expansion with comprehensive deep-learning based segmentation and analysis of connectivity, thus directly incorporating molecular information in synapse-level brain tissue reconstructions. LICONN will allow synapse-level brain tissue phenotyping in biological experiments in a readily adoptable manner. One-Sentence SummaryHydrogel expansion enables molecularly informed reconstruction of brain tissue at synaptic resolution with light microscopy.

neuroscience↗

bioRxiv2022

Subcortical circuit dysfunctions delay perceptual decision-making in autism models

Despite the diverse genetic origins of autism spectrum disorders (ASDs), affected individuals share strikingly similar and correlated behavioural traits that include perceptual and sensory processing challenges. Notably, the severity of these sensory symptoms is often predictive of the expression of other autistic traits. However, the origin of these perceptual deficits remains largely elusive. Here, we show a recurrent impairment in visual threat perception that is similarly impaired in three independent models of ASD with different molecular aetiologies. Interestingly, this deficit is associated with reduced avoidance of threatening environments - a non-perceptual trait. Focusing on a common cause of ASDs, the Setd5 gene mutation, we define the molecular mechanism. We show that the perceptual impairment is caused by a potassium channel (Kv1) mediated hypoexcitability in a subcortical node essential for the initiation of escape responses, the dorsal periaqueductal grey (dPAG). Targeted pharmacological Kv1 blockade rescued both perceptual and place avoidance deficits, causally linking seemingly unrelated trait deficits to the dPAG. Our findings reveal a link between rapid perception controlled by subcortical pathways and appropriate learned interactions with the environment, and define a non-developmental source of such deficits in ASD.

neuroscience↗

bioRxiv2022

Uncovering brain tissue architecture across scales with super-resolution light microscopy

Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the systems molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.

neuroscience↗

bioRxiv2022

Large neutral amino acid levels tune perinatal neuronal excitability and survival

Surprisingly little is known about the critical metabolic changes that neural cells have to undergo during development and how even mild, temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically-relevant large neutral amino acids, lead to a form of autism spectrum disorder, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different stages of life. We found that the cerebral cortex undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes. But what are the consequences of interfering with this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of large neutral amino acids and lipids in the cerebral cortex are highly interconnected. Deletion of Slc7a5 in neurons perturbs specifically the postnatal metabolic state leading to a shift in lipid metabolism and a stage- and cell-type-specific alteration in neuronal activity patterns, resulting in a long-term cortical circuit dysfunction.

neuroscience↗

bioRxiv2022

Saturated reconstruction of living brain tissue

Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full in-silico reconstruction1-5, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches6-12 facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy6,13 in extracellularly labelled tissue14 for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.

neuroscience↗

bioRxiv2020

CHD8 haploinsufficiency alters the developmental trajectories of human excitatory and inhibitory neurons linking autism phenotypes with transient cellular defects

Chromodomain helicase DNA-binding 8 (CHD8) is one of the most frequently mutated genes causative of autism spectrum disorder (ASD). While its phenotypic spectrum often encompasses macrocephaly and hence implicates cortical abnormalities in this form of ASD, the neurodevelopmental impact of human CHD8 haploinsufficiency remains unexplored. Here we combined human cerebral organoids and single cell transcriptomics to define the effect of ASD-linked CHD8 mutations on human cortical development. We found that CHD8 haploinsufficiency causes a major disruption of neurodevelopmental trajectories with an accelerated generation of inhibitory neurons and a delayed production of excitatory neurons alongside the ensuing protraction of the proliferation phase. This imbalance leads to a significant enlargement of cerebral organoids aligned to the macrocephaly observed in patients with CHD8 mutations. By adopting an isogenic design of patient-specific mutations and mosaic cerebral organoids, we define genotype-phenotype relationships and uncover their cell-autonomous nature. Finally, our results assign different CHD8-dependent molecular defects to particular cell types, pointing to an abnormal and extended program of proliferation and alternative splicing specifically affected in, respectively, the radial glial and immature neuronal compartments. By identifying temporally restricted cell-type specific effects of human CHD8 mutations, our study uncovers developmental alterations as reproducible endophenotypes for neurodevelopmental disease modelling.

neuroscience↗

bioRxiv2020

Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3 mutant brain displays cortical lamination abnormalities due to defective neuronal migration and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level, employing a quantitative proteomic approach, we show that Cul3 regulates cytoskeletal and adhesion protein abundance in mouse embryos. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neuronal cells results in atypical organization of the actin mesh at the cell leading edge, likely causing the observed migration deficits. In contrast to these important functions early in development, Cul3 deficiency appears less relevant at adult stages. In fact, induction of Cul3 haploinsufficiency in adult mice does not result in the behavioral defects observed in constitutive Cul3 haploinsufficient animals. Taken together, our data indicate that Cul3 has a critical role in the regulation of cytoskeletal proteins and neuronal migration and that ASD-associated defects and behavioral abnormalities are primarily due to Cul3 functions at early developmental stages.

neuroscience↗