Sensitive dLight for imaging broad-spectrum dopamine events across brain regions.

Dopaminergic neurons modulate movement, motivation, and learning by dynamically regulating dopamine release across distributed neural circuits. However, existing genetically encoded dopamine sensors lack the sensitivity and resolution to capture the full amplitude and temporal complexity of in vivo dopamine signaling, limiting insight into its functions across behavioral contexts. Here, we present dLight3.

Structural basis of liver de-targeting and neuronal tropism of CNS-targeted AAV capsids.

Crossing the blood-brain barrier while minimizing liver transduction is a key challenge in developing safe adeno-associated virus (AAV) vectors for treating brain disorders. In mice, the engineered capsid PHP.eB shows enhanced brain transduction, while the further engineered CAP-B10 is also de-targeted from astrocytes and liver.

The cell-surface shared proteome of astrocytes and neurons and the molecular foundations of their multicellular interactions.

Neurons and astrocytes are predominant brain cells that extensively interact, but the molecular basis of their interactions remains largely unexplored. We identified and mapped striatal astrocytic and neuronal cell-surface proteins (CSPs) and found that many were shared, representing the cell-surface shared proteome of astrocytes and neurons (CS SPAN) bridging striatal astrocyte-neuron interaction sites. CS SPAN was replete with extracellular matrix proteins, cell adhesion molecules, transporters, ion channels, and G protein-coupled receptors.

Axially decoupled photo-stimulation and two photon readout () for mapping functional connectivity of neural circuits.

All optical physiology provides a conduit for investigating the function of neural circuits in 3-D. Here, we report a new strategy for flexible, axially-decoupled photo-stimulation and two photon readout () of neuronal activity. To achieve axially-contained widefield optogenetic patterned stimulation, we couple a digital micro-mirror device illuminated by a solid-state laser with a motorized holographic diffuser.

Spatial genomics of AAV vectors reveals mechanism of transcriptional crosstalk that enables targeted delivery of large genetic cargo.

Cell-type-specific regulatory elements such as enhancers can direct expression of recombinant adeno-associated viruses (AAVs) to specific cell types, but this approach is limited by the relatively small packaging capacity of AAVs. In this study, we used spatial genomics to show that transcriptional crosstalk between individual AAV genomes provides a general method for cell-type-specific expression of large cargo by separating distally acting regulatory elements into a second AAV genome. We identified and profiled transcriptional crosstalk in AAV genomes carrying 11 different enhancers active in mouse brain.

Molecularly-guided spatial proteomics captures single-cell identity and heterogeneity of the nervous system.

Single-cell proteomics is an emerging field with significant potential to characterize heterogeneity within biological tissues. It offers complementary insights to single-cell transcriptomics by revealing unbiased proteomic changes downstream of the transcriptome. Recent advancements have focused on enhancing proteome coverage and depth, mostly in cultured cell lines, and a few recent studies have explored the potential of analyzing tissue micro-samples but were limited to homogenous peripheral tissues.

Synthetic dosage-compensating miRNA circuits allow precision gene therapy for Rett syndrome.

A longstanding challenge in gene therapy is expressing a dosage-sensitive gene within a tight therapeutic window. For example, loss of function causes Rett syndrome, while its duplication causes duplication syndrome. Viral gene delivery methods generate variable numbers of gene copies in individual cells, creating a need for gene dosage-invariant expression systems.

Peripheral neuronal activation shapes the microbiome and alters gut physiology.

The gastrointestinal (GI) tract is innervated by intrinsic neurons of the enteric nervous system (ENS) and extrinsic neurons of the central nervous system and peripheral ganglia. The GI tract also harbors a diverse microbiome, but interactions between the ENS and the microbiome remain poorly understood. Here, we activate choline acetyltransferase (ChAT)-expressing or tyrosine hydroxylase (TH)-expressing gut-associated neurons in mice to determine effects on intestinal microbial communities and their metabolites as well as on host physiology.

Removal of a partial genomic duplication restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Background: Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies.

Immediate responses to ambient light reveal distinct subpopulations of suprachiasmatic VIP neurons.

The circadian rhythm pacemaker, the suprachiasmatic nucleus (SCN), mediates light entrainment via vasoactive intestinal peptide (VIP) neurons (SCN). Yet, how these neurons uniquely respond and connect to intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing melanopsin (Opn4) has not been determined functionally in freely behaving animals. To address this, we first used monosynaptic tracing from SCN neurons in mice and identified two SCN subpopulations.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species.

Removal of a genomic duplication by double-nicking CRISPR restores synaptic transmission and behavior in the MyosinVA mutant mouse Flailer.

Copy number variations, and particularly duplications of genomic regions, have been strongly associated with various neurodegenerative conditions including autism spectrum disorder (ASD). These genetic variations have been found to have a significant impact on brain development and function, which can lead to the emergence of neurological and behavioral symptoms. Developing strategies to target these genomic duplications has been challenging, as the presence of endogenous copies of the duplicate genes often complicates the editing strategies.

Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors.

The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis.

CRISPR-clear imaging of melanin-rich B16-derived solid tumors.

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.