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

Abstract

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 ␣-calcium͞calmodulin-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 timelapse 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.patch-clamp recording ͉ two-photon imaging

Inhibition of Histone Methyltransferases SUV39H1 and G9a Leads to Neuroprotection in an in vitro Model of Cerebral Ischemia

“…With the BLOCK-iT Pol II miR Expression Vector Kit (Invitrogen, Darmstadt, Germany) microRNA-embedded short hairpin RNA (miR-shRNA) constructs were generated containing the respective sequences. The miR-shRNA constructs were finally cloned into a third-generation lentiviral vector based on Addgene plasmid 27232 (ref 15) with the following modifications: microRNA delivery was driven by a ubiquitin promoter and the initial reporter protein was exchanged with a myc-tagged red fluorescent protein (RFP). HEK 293FT cells (Invitrogen, Darmstadt, Germany) were used as a producer cell line.…”

Mapping somatosensory connectivity in adult mice using diffusion MRI tractography and super-resolution track density imaging

“…The structural composition and connectivity of the "whisker pathways" has been described using 2D histologic and functional activation methods. Network tracking techniques include lesion studies, (Killackey and Fleming, 1985;Killackey and Leshin, 1975); carbocyanine dyes (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine Perchlorate; DiI, DiA) (Kivrak and Erzurumlu, 2013;Seehaus et al, 2013) and myelin staining (Barrera et al, 2012); these techniques are also complemented by Lenti and Adeno-associated viral vector expression of fluorescent proteins (Aronoff et al, 2010;Dittgen et al, 2004;Wimmer et al, 2010). A range of macroscopic to microscopic functional connectivity mapping techniques include functional MRI (Kim et al, 2012;Yang et al, 1996), 2-photon imaging of electrical activity using voltage-sensitive dyes (Petersen et al, 2003a;Petersen et al, 2003b) and channelrhodopsins to map neuronal connectivity have also been conducted (Paz et al, 2011;Petreanu et al, 2007).…”

Principles of designing interpretable optogenetic behavior experiments

“…In mammals, viruses expressing opsins under cell-specific promoters in principle are useful, but many cell-specific promoters are not small enough to fit into a virus. Some commonly used promoters can target neurons (e.g., synapsin-1), or simply express pancellularly (e.g., CAG, EF1a) (Kügler et al 2003;Dittgen et al 2004;Betley and Sternson 2011), when used in adeno-associated virus (AAV) or lentivirus, two popular methods of viral gene delivery into the mammalian brain. Complicating attempts to target expression, viruses themselves can have various tropisms, i.e., they can infect some cell types preferentially over others.…”