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% volumetric accuracy at 40-km 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 the radiometer include a large 6-m 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 that the electrical and thermal characteristics of the electronics and internal calibration sources are very stable and promote excellent gain stability. Radiometer NEDT < 1 K for 17-ms samples. The gain spectrum exhibits low noise at frequencies >1 MHz and 1/f 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 that the instrument is operating as expected.

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 (GluClalphabeta), 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 GluClalphabeta-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.

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 GluClalpha1 and GluClbeta 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, GluClalpha1 subunit) and enhanced cyan fluorescent protein (EFCP, GluClbeta 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 EC(50) of 1.2 nM and a Hill coefficient of 1.9. We also confirm that the Y182F mutation in the codon-optimized beta subunit results in a heteromeric channel that retains the response to ivermectin while reducing the response to 100 microM 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 beta-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 alpha. For GluCl alphabeta(Y182F), the EC(50) 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 alpha, beta and beta(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.

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 alpha and beta) 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 alpha and beta 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 alpha and cyan fluorescent protein-tagged (ECFP) GluCl beta. The present study provides an in vitro model 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.