Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo

It is becoming increasingly clear that single cortical neurons encode complex and behaviorally relevant signals, but efficient means to study gene functions in small networks and single neurons in vivo are still lacking. Here, we establish a method for genetic manipulation and subsequent phenotypic analysis of individual cortical neurons in vivo. First, lentiviral vectors are used for neuron-specific gene delivery from α-calcium/calmodulin-dependent protein kinase II or Synapsin I promoters, optionally in combination with gene knockdown by means of U6 promoter-driven expression of short-interfering RNAs. Second, the phenotypic analysis at the level of single cortical cells is carried out by using two-photon microscopy-based techniques: high-resolution two-photon time-lapse imaging is used to monitor structural dynamics of dendritic spines and axonal projections, whereas cellular response properties are analyzed electrophysiologically by two-photon microscopydirected whole-cell recordings. This approach is ideally suited for analysis of gene functions in individual neurons in the intact brain.

[1]  D. Hubel,et al.  Receptive fields, binocular interaction and functional architecture in the cat's visual cortex , 1962, The Journal of physiology.

[2]  M. Wong-Riley.  Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry , 1979, Brain Research.

[3]  K. Horikawa,et al.  A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates , 1988, Journal of Neuroscience Methods.

[4]  M. Waxham,et al.  In situ hybridization histochemistry of Ca2+/calmodulin-dependent protein kinase in developing rat brain , 1990, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[5]  Yamamura Ken-ichi,et al.  Efficient selection for high-expression transfectants with a novel eukaryotic vector , 1991 .

[6]  T Friedmann,et al.  Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B. Krippl,et al.  The 5'-flanking region of the rat synapsin I gene directs neuron-specific and developmentally regulated reporter gene expression in transgenic mice. , 1993, The Journal of biological chemistry.

[8]  G. Brewer,et al.  Optimized survival of hippocampal neurons in B27‐supplemented neurobasal™, a new serum‐free medium combination , 1993, Journal of neuroscience research.

[9]  F. Gage,et al.  In Vivo Gene Delivery and Stable Transduction of Nondividing Cells by a Lentiviral Vector , 1996, Science.

[10]  E. Kandel,et al.  Control of Memory Formation Through Regulated Expression of a CaMKII Transgene , 1996, Science.

[11]  Pico Caroni,et al.  Overexpression of growth-associated proteins in the neurons of adult transgenic mice , 1997, Journal of Neuroscience Methods.

[12]  Robert U Muller,et al.  Memory and behavior: a second generation of genetically modified mice , 1997, Current Biology.

[13]  Luigi Naldini,et al.  Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo , 1997, Nature Biotechnology.

[14]  Fred H. Gage,et al.  Development of a Self-Inactivating Lentivirus Vector , 1998, Journal of Virology.

[15]  M. Cramer,et al.  Biological Activity and Modular Structure of RE-1-silencing Transcription Factor (REST), a Repressor of Neuronal Genes* , 1998, The Journal of Biological Chemistry.

[16]  B. Sauer.  Inducible gene targeting in mice using the Cre/lox system. , 1998, Methods.

[17]  Rafael Yuste,et al.  Imaging neurons : a laboratory manual , 1999 .

[18]  J. Bloch,et al.  Lentiviral Gene Transfer to the Nonhuman Primate Brain , 1999, Experimental Neurology.

[19]  K. Svoboda,et al.  Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo , 2000, Nature.

[20]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[21]  D. Trono.  Lentiviral vectors: turning a deadly foe into a therapeutic agent , 2000, Gene Therapy.

[22]  C. Lois,et al.  Retroviruses as tools to study the immune system. , 2001, Current opinion in immunology.

[23]  D. Wilkin,et al.  Neuron , 2001, Brain Research.

[24]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[25]  B. Sakmann,et al.  In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain , 2002, Pflügers Archiv.

[26]  Stacy L DeRuiter,et al.  RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  David Baltimore,et al.  Germline Transmission and Tissue-Specific Expression of Transgenes Delivered by Lentiviral Vectors , 2002, Science.

[28]  Hermann Bujard,et al.  Studying gene function in eukaryotes by conditional gene inactivation. , 2002, Annual review of genetics.

[29]  K. Svoboda,et al.  Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex , 2002, Nature.

[30]  D. Baltimore,et al.  Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[31]  R. Brady,et al.  Gene transfer into the central nervous system in vivo using a recombinanat lentivirus vector , 2002, Journal of neuroscience research.

[32]  Haibin Xia,et al.  siRNA-mediated gene silencing in vitro and in vivo , 2002, Nature Biotechnology.

[33]  L. Ailles,et al.  HIV-1-derived lentiviral vectors. , 2002, Current topics in microbiology and immunology.

[34]  N. Kasthuri,et al.  Long-term dendritic spine stability in the adult cortex , 2002, Nature.

[35]  Winfried Denk,et al.  Targeted Whole-Cell Recordings in the Mammalian Brain In Vivo , 2003, Neuron.

[36]  Emilio Salinas,et al.  Cognitive neuroscience: Flutter Discrimination: neural codes, perception, memory and decision making , 2003, Nature Reviews Neuroscience.

[37]  B. Sakmann,et al.  Dynamic Receptive Fields of Reconstructed Pyramidal Cells in Layers 3 and 2 of Rat Somatosensory Barrel Cortex , 2003, The Journal of physiology.

[38]  Michael T. McManus,et al.  A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference , 2003, Nature Genetics.

[39]  B. Nuttin,et al.  Lentiviral vector-mediated delivery of short hairpin RNA results in persistent knockdown of gene expression in mouse brain. , 2003, Human gene therapy.

[40]  Robert M Sears,et al.  Local gene knockdown in the brain using viral-mediated RNA interference , 2003, Nature Medicine.

[41]  Inder M Verma,et al.  A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[42]  I. Verma,et al.  Gene therapy with viral vectors. , 2003, Annual review of pharmacology and toxicology.

[43]  B. Sakmann,et al.  Whisker movements evoked by stimulation of single pyramidal cells in rat motor cortex , 2004, Nature.

[44]  W. Newsome,et al.  What electrical microstimulation has revealed about the neural basis of cognition , 2004, Current Opinion in Neurobiology.

[45]  C. Glover,et al.  Gene expression profiling for hematopoietic cell culture , 2006 .