A potassium channel β-subunit couples mitochondrial electron transport to sleep

The essential but enigmatic functions of sleep; 1,2; must be reflected in molecular changes sensed by the brain's sleep-control systems. In the fruitfly Drosophila, about two dozen sleep-inducing neurons; 3; with projections to the dorsal fan-shaped body (dFB) adjust their electrical output to sleep need; 4; , via the antagonistic regulation of two potassium conductances: the leak channel Sandman imposes silence during waking, whereas increased A-type currents through Shaker support tonic firing during sleep; 5; . Here we show that oxidative byproducts of mitochondrial electron transport; 6,7; regulate the activity of dFB neurons through a nicotinamide adenine dinucleotide phosphate (NADPH) cofactor bound to the oxidoreductase domain; 8,9; of Shaker's K; V; β subunit, Hyperkinetic; 10,11; . Sleep loss elevates mitochondrial reactive oxygen species in dFB neurons, which register this rise by converting Hyperkinetic to the NADP; +; -bound form. The oxidation of the cofactor slows the inactivation of the A-type current and boosts the frequency of action potentials, thereby promoting sleep. Energy metabolism, oxidative stress, and sleep-three processes implicated independently in lifespan, ageing, and degenerative disease; 6,12-14; -are thus mechanistically connected. K; V; β substrates; 8,15,16; or inhibitors that alter the ratio of bound NADPH to NADP; +; (and hence the record of sleep debt or waking time) represent prototypes of potential sleep-regulatory drugs

Dendritic Integration of Sensory Evidence in Perceptual Decision-Making

Perceptual decisions require the accumulation of sensory information to a response criterion. Most accounts of how the brain performs this process of temporal integration have focused on evolving patterns of spiking activity. We report that subthreshold changes in membrane voltage can represent accumulating evidence before a choice. αβ core Kenyon cells (αβc KCs) in the mushroom bodies of fruit flies integrate odor-evoked synaptic inputs to action potential threshold at timescales matching the speed of olfactory discrimination. The forkhead box P transcription factor (FoxP) sets neuronal integration and behavioral decision times by controlling the abundance of the voltage-gated potassium channel Shal (KV4) in αβc KC dendrites. αβc KCs thus tailor, through a particular constellation of biophysical properties, the generic process of synaptic integration to the demands of sequential sampling

A genetically encoded reporter of synaptic activity in vivo

To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses

Dielectric Properties of the Quasi-Two-Dimensional Electron Liquid in Heterojunctions

A quasi-two-dimensional (Q2D) electron liquid (EL) is formed at the interface of a semiconductor heterojunction. For an accurate characterization of the Q2D EL, many-body effects need to be taken into account beyond the random phase approximation. In this theoretical work, the self-consistent static local-field correction known as STLS is applied for the analysis of the Q2D EL. The penetration of the charge distribution to the barrier-acting material is taken into consideration through a variational approach. The Coulomb from factor that describes the effective 2D interaction is rigorously treated. The longitudinal dielectric function and the plasmon dispersion of the Q2D EL are presented for a wide range of electron and ionized acceptor densities choosing GaAs/AlGaAs as the physical system. Analytical expressions fitted to our results are also supplied to enable a widespread use of these results.Comment: 39 pages (in LaTeX), including 8 PostScript figure

Fabrication and characterization of dual function nanoscale pH-scanning ion conductance microscopy (SICM) probes for high resolution pH mapping

The easy fabrication and use of nanoscale dual function pH-scanning ion conductance microscopy (SICM) probes is reported. These probes incorporate an iridium oxide coated carbon electrode for pH measurement and an SICM barrel for distance control, enabling simultaneous pH and topography mapping. These pH-SICM probes were fabricated rapidly from laser pulled theta quartz pipets, with the pH electrode prepared by in situ carbon filling of one of the barrels by the pyrolytic decomposition of butane, followed by electrodeposition of a thin layer of hydrous iridium oxide. The other barrel was filled with an electrolyte solution and Ag/AgCl electrode as part of a conductance cell for SICM. The fabricated probes, with pH and SICM sensing elements typically on the 100 nm scale, were characterized by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and various electrochemical measurements. They showed a linear super-Nernstian pH response over a range of pH (pH 2–10). The capability of the pH-SICM probe was demonstrated by detecting both pH and topographical changes during the dissolution of a calcite microcrystal in aqueous solution. This system illustrates the quantitative nature of pH-SICM imaging, because the dissolution process changes the crystal height and interfacial pH (compared to bulk), and each is sensitive to the rate. Both measurements reveal similar dissolution rates, which are in agreement with previously reported literature values measured by classical bulk methods

M153R Mutation in a pH-Sensitive Green Fluorescent Protein Stabilizes Its Fusion Proteins

BACKGROUND: Green fluorescent protein (GFP) and its fusion proteins have been used extensively to monitor and analyze a wide range of biological processes. However, proteolytic cleavage often removes GFP from its fusion proteins, not only causing a poor signal-to-noise ratio of the fluorescent images but also leading to wrong interpretations. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the M153R mutation in a ratiometric pH-sensitive GFP, pHluorin, significantly stabilizes its fusion products while the mutant protein still retaining a marked pH dependence of 410/470 nm excitation ratio of fluorescence intensity. The M153R mutation increases the brightness in vivo but does not affect the 410/470-nm excitation ratios at various pH values. CONCLUSIONS/SIGNIFICANCE: Since the pHluorin(M153R) probe can be directly fused to the target proteins, we suggest that it will be a potentially powerful tool for the measurement of local pH in living cells as well as for the analysis of subcellular localization of target proteins

Opto-Current-Clamp Actuation of Cortical Neurons Using a Strategically Designed Channelrhodopsin

BACKGROUND: Optogenetic manipulation of a neuronal network enables one to reveal how high-order functions emerge in the central nervous system. One of the Chlamydomonas rhodopsins, channelrhodopsin-1 (ChR1), has several advantages over channelrhodopsin-2 (ChR2) in terms of the photocurrent kinetics. Improved temporal resolution would be expected by the optogenetics using the ChR1 variants with enhanced photocurrents. METHODOLOGY/PRINCIPAL FINDINGS: The photocurrent retardation of ChR1 was overcome by exchanging the sixth helix domain with its counterpart in ChR2 producing Channelrhodopsin-green receiver (ChRGR) with further reform of the molecule. When the ChRGR photocurrent was measured from the expressing HEK293 cells under whole-cell patch clamp, it was preferentially activated by green light and has fast kinetics with minimal desensitization. With its kinetic advantages the use of ChRGR would enable one to inject a current into a neuron by the time course as predicted by the intensity of the shedding light (opto-current clamp). The ChRGR was also expressed in the motor cortical neurons of a mouse using Sindbis pseudovirion vectors. When an oscillatory LED light signal was applied sweeping through frequencies, it robustly evoked action potentials synchronized to the oscillatory light at 5-10 Hz in layer 5 pyramidal cells in the cortical slice. The ChRGR-expressing neurons were also driven in vivo with monitoring local field potentials (LFPs) and the time-frequency energy distribution of the light-evoked response was investigated using wavelet analysis. The oscillatory light enhanced both the in-phase and out-phase responses of LFP at the preferential frequencies of 5-10 Hz. The spread of activity was evidenced by the fact that there were many c-Fos-immunoreactive neurons that were negative for ChRGR in a region of the motor cortex. CONCLUSIONS/SIGNIFICANCE: The opto-current-clamp study suggests that the depolarization of a small number of neurons wakes up the motor cortical network over some critical point to the activated state