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1.
Nat Biotechnol ; 36(1): 81-88, 2018 01.
Article in English | MEDLINE | ID: mdl-29251729

ABSTRACT

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle-a functionalized glass pipette tip or magnetic nanoparticles in a pipette-that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.


Subject(s)
Brain/virology , Magnetite Nanoparticles/administration & dosage , Single-Cell Analysis/methods , Viruses/genetics , Animals , Genetic Engineering/trends , Humans , Magnetite Nanoparticles/chemistry , Mice , Organoids/metabolism , Organoids/virology , Retina/metabolism , Retina/virology , Tissue Distribution , Virus Diseases/genetics , Virus Diseases/metabolism , Virus Replication/genetics
2.
Neuron ; 89(1): 177-93, 2016 Jan 06.
Article in English | MEDLINE | ID: mdl-26711119

ABSTRACT

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.


Subject(s)
Amacrine Cells/cytology , Cytoskeletal Proteins/metabolism , Nerve Net/physiology , Neural Inhibition/physiology , Nystagmus, Congenital/metabolism , Visual Pathways/physiology , Action Potentials/physiology , Animals , Mice, Transgenic , Motion Perception/physiology , Photic Stimulation/methods , Retina/physiology , Retinal Ganglion Cells/cytology , Synapses/metabolism
3.
Neuron ; 83(3): 586-600, 2014 Aug 06.
Article in English | MEDLINE | ID: mdl-25002228

ABSTRACT

The outer segments of cones serve as light detectors for daylight color vision, and their dysfunction leads to human blindness conditions. We show that the cone-specific disruption of DGCR8 in adult mice led to the loss of miRNAs and the loss of outer segments, resulting in photoreceptors with significantly reduced light responses. However, the number of cones remained unchanged. The loss of the outer segments occurred gradually over 1 month, and during this time the genetic signature of cones decreased. Reexpression of the sensory-cell-specific miR-182 and miR-183 prevented outer segment loss. These miRNAs were also necessary and sufficient for the formation of inner segments, connecting cilia and short outer segments, as well as light responses in stem-cell-derived retinal cultures. Our results show that miR-182- and miR-183-regulated pathways are necessary for cone outer segment maintenance in vivo and functional outer segment formation in vitro.


Subject(s)
MicroRNAs/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Rod Photoreceptor Cells/metabolism , Vision, Ocular/genetics , Aging , Animals , Gene Knockout Techniques , Humans , Light , Mice , Mice, Transgenic , Retina/metabolism
4.
Nat Neurosci ; 15(3): 487-95, S1-2, 2012 Jan 22.
Article in English | MEDLINE | ID: mdl-22267162

ABSTRACT

Brain circuits are assembled from a large variety of morphologically and functionally diverse cell types. It is not known how the intermingled cell types of an individual adult brain region differ in their expressed genomes. Here we describe an atlas of cell type transcriptomes in one brain region, the mouse retina. We found that each adult cell type expressed a specific set of genes, including a unique set of transcription factors, forming a 'barcode' for cell identity. Cell type transcriptomes carried enough information to categorize cells into morphological classes and types. Several genes that were specifically expressed in particular retinal circuit elements, such as inhibitory neuron types, are associated with eye diseases. The resource described here allows gene expression to be compared across adult retinal cell types, experimenting with specific transcription factors to differentiate stem or somatic cells to retinal cell types, and predicting cellular targets of newly discovered disease-associated genes.


Subject(s)
Nerve Tissue Proteins/metabolism , Neurons/classification , Neurons/physiology , Retina/cytology , Retinal Diseases/genetics , Transcription Factors/metabolism , Animals , Cluster Analysis , Connexins/genetics , Eye Proteins/genetics , Eye Proteins/metabolism , Flow Cytometry , Gene Expression/genetics , Gene Library , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microarray Analysis/methods , Mutation/genetics , Nerve Tissue Proteins/genetics , Patch-Clamp Techniques , Potassium Channels, Voltage-Gated/genetics , Receptors, Kainic Acid/genetics , Transcription Factors/genetics , Visual Pathways/metabolism , GluK2 Kainate Receptor
5.
Nat Neurosci ; 12(9): 1197-204, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19648912

ABSTRACT

The mammalian brain is assembled from thousands of neuronal cell types that are organized in distinct circuits to perform behaviorally relevant computations. Transgenic mouse lines with selectively marked cell types would facilitate our ability to dissect functional components of complex circuits. We carried out a screen for cell type-specific green fluorescent protein expression in the retina using BAC transgenic mice from the GENSAT project. Among others, we identified mouse lines in which the inhibitory cell types of the night vision and directional selective circuit were selectively labeled. We quantified the stratification patterns to predict potential synaptic connectivity between marked cells of different lines and found that some of the lines enabled targeted recordings and imaging of cell types from developing or mature retinal circuits. Our results suggest the potential use of a stratification-based screening approach for characterizing neuronal circuitry in other layered brain structures, such as the neocortex.


Subject(s)
Mice, Transgenic/anatomy & histology , Mice, Transgenic/physiology , Retina/anatomy & histology , Retina/physiology , Amacrine Cells/cytology , Amacrine Cells/physiology , Animals , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunohistochemistry , In Vitro Techniques , Mice , Microscopy, Fluorescence , Neural Pathways/anatomy & histology , Neural Pathways/cytology , Neural Pathways/physiology , Patch-Clamp Techniques , Photoreceptor Cells, Vertebrate/cytology , Photoreceptor Cells, Vertebrate/physiology , Retina/cytology , Retinal Bipolar Cells/cytology , Retinal Bipolar Cells/physiology , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/physiology , Retinal Horizontal Cells/cytology , Retinal Horizontal Cells/physiology , Species Specificity , Synapses/physiology , Vision, Ocular/physiology
6.
Nat Methods ; 6(2): 127-30, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19122667

ABSTRACT

We developed retrograde, transsynaptic pseudorabies viruses (PRVs) with genetically encoded activity sensors that optically report the activity of connected neurons among spatially intermingled neurons in the brain. Next we engineered PRVs to express two differentially colored fluorescent proteins in a time-shifted manner to define a time period early after infection to investigate neural activity. Finally we used multiple-colored PRVs to differentiate and dissect the complex architecture of brain regions.


Subject(s)
Green Fluorescent Proteins/analysis , Herpesvirus 1, Suid/metabolism , Luminescent Proteins/analysis , Synaptic Transmission/physiology , Visual Pathways/virology , Animals , Biosensing Techniques/methods , Brain/cytology , Brain/physiology , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Herpesvirus 1, Suid/genetics , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , Mice , Neurons/physiology , Neurons/virology , Time Factors , Visual Pathways/physiology , Red Fluorescent Protein
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