Reversible Silencing of Neuronal Excitability in Behaving Mice by a Genetically Targeted, Ivermectin-Gated Cl^− Channel

Several genetic strategies for inhibiting neuronal function in mice have been described, but no system that directly suppresses membrane excitability and is triggered by a systemically administered drug, has been validated in awake behaving animals. We expressed unilaterally in mouse striatum a modified heteromeric ivermectin (IVM)-gated chloride channel from C. elegans (GluClαβ), systemically administered IVM, and then assessed amphetamine-induced rotational behavior. Rotation was observed as early as 4 hr after a single intraperitoneal IVM injection (10 mg/kg), reached maximal levels by 12 hr, and was almost fully reversed by 4 days. Multiple cycles of silencing and recovery could be performed in a single animal. In striatal slice preparations from GluClαβ-expressing animals, IVM rapidly suppressed spiking. The two-subunit GluCl/IVM system permits “intersectional” strategies designed to increase the cellular specificity of silencing in transgenic animals

Selective Electrical Silencing of Mammalian Neurons In Vitro by the Use of Invertebrate Ligand-Gated Chloride Channels

Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of human neurological disorders and (2) provide insight into the global function of specific sets of neurons. We focus on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl α and β) with a Sindbis virus and then activate these channels with IVM to produce inhibition via a Cl^− conductance. We constructed a three-cistron Sindbis virus that expresses the α and β subunits of a glutamate-gated chloride channel (GluCl) along with the green fluorescent protein (EGFP) marker. Expression of the C. elegans channel does not affect the normal spike activity or GABA/glutamate postsynaptic currents of cultured embryonic day 18 hippocampal neurons. At concentrations as low as 5 nM, IVM activates a Cl^− current large enough to silence infected neurons effectively. This conductance reverses in 8 hr. These low concentrations of IVM do not potentiate GABA responses. Comparable results are observed with plasmid transfection of yellow fluorescent protein-tagged (EYFP) GluCl α and cyan fluorescent protein-tagged (ECFP) GluCl β. The present study provides an in vitromodel mimicking conditions that can be obtained in transgenic mice and in viral-mediated gene therapy. These experiments demonstrate the feasibility of using invertebrate ligand-activated Cl^− channels as an approach to modulate excitability

SMAP L-Band Microwave Radiometer: Instrument Design and First Year on Orbit

The Soil Moisture Active Passive (SMAP) L-band microwave radiometer is a conical scanning instrument designed to measure soil moisture with 4 percent volumetric accuracy at 40-kilometer spatial resolution. SMAP is NASA's first Earth Systematic Mission developed in response to its first Earth science decadal survey. Here, the design is reviewed and the results of its first year on orbit are presented. Unique features of radiometer include a large 6-meter rotating reflector, fully polarimetric radiometer receiver with internal calibration, and radio-frequency interference detection and filtering hardware. The radiometer electronics are thermally controlled to achieve good radiometric stability. Analyses of on-orbit results indicate the electrical and thermal characteristics of the electronics and internal calibration sources are very stable and promote excellent gain stability. Radiometer NEdT (Noise Equivalent differential Temperature) less than 1 degree Kelvin for 17-millisecond samples. The gain spectrum exhibits low noise at frequencies greater than 1 megahertz and 1 divided by f (pink) noise rising at longer time scales fully captured by the internal calibration scheme. Results from sky observations and global swath imagery of all four Stokes antenna temperatures indicate the instrument is operating as expected

The Evolution of the MSL Heatshield

This presentation was part of the session : Current Planetary Probe Science and TechnologySixth International Planetary Probe WorkshopThe MSL entry vehicle will be experiencing aerothermal environments that no previous Mars entry vehicle has seen, and as such, will require a heatshield that no previous Mars entry vehicle has flown. Early assessments of the environments led engineers down the path to "fly as we've always flown", with the heritage Mars heatshield material, SLA-561V. Screening tests in arc jet facilities in stagnation model configurations substantiated the claims that the SLA-561V could survive high heating rates expected on the heatshield, albeit, with a melting surface. However, due to the size and mass of the vehicle, along with the need to reenter at angle of attack, high heating and high shear in turbulent flow was predicted on the leeward side of the vehicle. Testing in shear showed that the SLA-561V exhibited catastrophic ablation which led to the abandonment of the material for this application and the fast-tracked development of a tiled PICA heatshield design, on track for a 2009 launch

Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels

Organisms use synonymous codons in a highly non-random fashion. These codon usage biases sometimes frustrate attempts to express high levels of exogenous genes in hosts of widely divergent species. The Caenorhabditis elegans GluClα1 and GluClβ genes form a functional glutamate and ivermectin-gated chloride channel when expressed in Xenopus oocytes, but expression is weak in mammalian cells. We have constructed synthetic genes that retain the amino acid sequence of the wild-type GluCl channel proteins, but use codons that are optimal for mammalian cell expression. We have tagged the native and codon-optimized GluCl cDNAs with enhanced yellow fluorescent protein (EYFP, GluClα1 subunit) and enhanced cyan fluorescent protein (EFCP, GluClβ subunit), expressed the channels in E18 rat hippocampal neurons and measured the relative expression levels of the two genes with fluorescence microscopy as well as with electrophysiology. Codon optimization provides a 6- to 9-fold increase in expression, allowing the conclusions that the ivermectin-gated channel has an EC50 of 1.2 nM and a Hill coefficient of 1.9. We also confirm that the Y182F mutation in the codon-optimized β subunit results in a heteromeric channel that retains the response to ivermectin while reducing the response to 100 μM glutamate by 7-fold. The engineered GluCl channel is the first codon-optimized membrane protein expressed in mammalian cells and may be useful for selectively silencing specific neuronal populations in vivo

Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels

Organisms use synonymous codons in a highly non-random fashion. These codon usage biases sometimes frustrate attempts to express high levels of exogenous genes in hosts of widely divergent species. The Caenorhabditis elegans GluClα1 and GluClβ genes form a functional glutamate and ivermectin-gated chloride channel when expressed in Xenopus oocytes, but expression is weak in mammalian cells. We have constructed synthetic genes that retain the amino acid sequence of the wild-type GluCl channel proteins, but use codons that are optimal for mammalian cell expression. We have tagged the native and codon-optimized GluCl cDNAs with enhanced yellow fluorescent protein (EYFP, GluClα1 subunit) and enhanced cyan fluorescent protein (EFCP, GluClβ subunit), expressed the channels in E18 rat hippocampal neurons and measured the relative expression levels of the two genes with fluorescence microscopy as well as with electrophysiology. Codon optimization provides a 6- to 9-fold increase in expression, allowing the conclusions that the ivermectin-gated channel has an EC50 of 1.2 nM and a Hill coefficient of 1.9. We also confirm that the Y182F mutation in the codon-optimized β subunit results in a heteromeric channel that retains the response to ivermectin while reducing the response to 100 μM glutamate by 7-fold. The engineered GluCl channel is the first codon-optimized membrane protein expressed in mammalian cells and may be useful for selectively silencing specific neuronal populations in vivo

Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel

Glutamate‐gated chloride (GluCl) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis elegans GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure–function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the β‐subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six‐fold, with little change to IVM responses, when coexpressed with wild‐type (WT) GluCl α. For GluCl αβ(Y182F), the EC₅₀ and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3–M4 loop of the GluCl α, β and β(Y182F). We found no significant functional difference between these XFP‐tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies