Magnetically guided virus stamping for the targeted infection of single cells or groups of cells

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette—loaded with magnetic nanoparticles to which viral particles are bound—is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo

Efficient transduction and optogenetic stimulation of retinal bipolar cells by a synthetic adeno-associated virus capsid and promoter

In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (∼100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use

Magnetically guided virus stamping for the targeted infection of single cells or groups of cells

To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette—loaded with magnetic nanoparticles to which viral particles are bound—is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo

The transcriptome of retinal Müller glial cells

Müller glial cells are the major type of glia in the mammalian retina. To identify the molecular machinery that defines Müller glial cell identity and function, single cell gene expression profiling was performed on Affymetrix microarrays. Identification of a cluster of genes expressed at high levels suggests a Müller glia core transcriptome, which likely underlies many of the functions of Müller glia. Expression of components of the cell cycle machinery and the Notch pathway, as well as of growth factors, chemokines, and lipoproteins might allow communication between Müller glial cells and the neurons that they support, including modulation of neuronal activity. This approach revealed a set of transcripts that were not previously characterized in (Müller) glia; validation of the expression of some of these genes was performed by in situ hybridization. Genes expressed exclusively by Müller glia were identified as novel markers. In addition, a novel BAC transgenic mouse that expresses Cre in Müller glia cells was generated. The molecular fingerprint of Müller glia provides a foundation for further studies of Müller glia development and function in normal and diseased states

Noninvasive optical inhibition with a red-shifted microbial rhodopsin

Optogenetic inhibition of the electrical activity of neurons enables the causal assessment of their contributions to brain functions. Red light penetrates deeper into tissue than other visible wavelengths. We present a red-shifted cruxhalorhodopsin, Jaws, derived from Haloarcula (Halobacterium) salinarum (strain Shark) and engineered to result in red light–induced photocurrents three times those of earlier silencers. Jaws exhibits robust inhibition of sensory-evoked neural activity in the cortex and results in strong light responses when used in retinas of retinitis pigmentosa model mice. We also demonstrate that Jaws can noninvasively mediate transcranial optical inhibition of neurons deep in the brains of awake mice. The noninvasive optogenetic inhibition opened up by Jaws enables a variety of important neuroscience experiments and offers a powerful general-use chloride pump for basic and applied neuroscience.McGovern Institute for Brain Research at MIT (Razin Fellowship)United States. Defense Advanced Research Projects Agency. Living Foundries Program (HR0011-12-C-0068)Harvard-MIT Joint Research Grants Program in Basic NeuroscienceHuman Frontier Science Program (Strasbourg, France)Institution of Engineering and Technology (A. F. Harvey Prize)McGovern Institute for Brain Research at MIT. Neurotechnology (MINT) ProgramNew York Stem Cell Foundation (Robertson Investigator Award)National Institutes of Health (U.S.) (New Innovator Award 1DP2OD002002)National Institute of General Medical Sciences (U.S.) (EUREKA Award 1R01NS075421)National Institutes of Health (U.S.) (Grant 1R01DA029639)National Institutes of Health (U.S.) (Grant 1RC1MH088182)National Institutes of Health (U.S.) (Grant 1R01NS067199)National Science Foundation (U.S.) (Career Award CBET 1053233)National Science Foundation (U.S.) (Grant EFRI0835878)National Science Foundation (U.S.) (Grant DMS0848804)Society for Neuroscience (Research Award for Innovation in Neuroscience)Wallace H. Coulter FoundationNational Institutes of Health (U.S.) (RO1 MH091220-01)Whitehall FoundationEsther A. & Joseph Klingenstein Fund, Inc.JPB FoundationPIIF FundingNational Institute of Mental Health (U.S.) (R01-MH102441-01)National Institutes of Health (U.S.) (DP2-OD-017366-01)Massachusetts Institute of Technology. Simons Center for the Social Brai

Sharp-Wave Ripple Doublets Induce Complex Dendritic Spikes in Parvalbumin Interneurons in vivo

Neuronal plasticity has been shown to be causally linked to coincidence detection through dendritic spikes (dSpikes). We demonstrate the existence of SPW-R-associated, branch-specific, local dSpikes and their computational role in basal dendrites of hippocampal PV+ interneurons in awake animals. To measure the entire dendritic arbor of long thin dendrites during SPW-Rs, we used fast 3D acousto-optical imaging through an eccentric deep-brain adapter and ipsilateral local field potential recording. The regenerative calcium spike started at variable, NMDA-AMPA-dependent, hot spots and propagated in both direction with a high amplitude beyond a critical distance threshold (~150 µm) involving voltage-gated calcium channels. A supralinear dendritic summation emerged during SPW-R doublets when two successive SPW-R events coincide within a short temporal window (~150 ms), e.g., during more complex association tasks, and generated large dSpikes with an about 2.5-3-fold amplitude increase which propagated down to the soma. Our results suggest that these doublet-associated dSpikes can work as a dendritic-level temporal and spatial coincidence detector during SPW-R-related network computation in awake mice

The first steps in vision: cell types, circuits, and repair

Abstract Dysfunction of the key sense of vision, leading to visual handicap or blindness, has a crucial effect on day‐to‐day life. In this commentary, I will summarize the work in my laboratory that is focused on a basic understanding of visual processing and the use of this information to understand disease mechanism and to develop correcting therapies. We are beginning to understand how cell types of the visual system interact in local circuits and compute visual information. This has brought insight into mechanisms of cell‐type‐specific diseases and has allowed us to design new therapies for restoring vision in genetic forms of blindness