Search

Microbial rhodopsin (MRs) ion channels and pumps have become invaluable optogenetic tools for neuroscience as well as biomedical applications. Recently, MR-optogenetics expanded towards subcellular organelles opening principally new opportunities in optogenetic control of intracellular metabolism and signaling via precise manipulations of organelle ion gradients using light. This new optogenetic field expands the opportunities for basic and medical studies of cancer, cardiovascular, and metabolic disorders, providing more detailed and accurate control of cell physiology. This review summarizes recent advances in studies of the cellular metabolic processes and signaling mediated by optogenetic tools targeting mitochondria, endoplasmic reticulum (ER), lysosomes, and synaptic vesicles. Finally, we discuss perspectives of such an optogenetic approach in both fundamental and applied research. [ABSTRACT FROM AUTHOR]

Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics., Introduction The electrical cochlear implant (CI) is considered the most successful neuroprosthesis. Implanted in more than 200,000 hearing-impaired subjects worldwide, CIs enable open speech comprehension in the majority of users [...]

Spatial asymmetries in neural connectivity have an important role in creating basic building blocks of neuronal processing (1,2). A key circuit module of directionally selective (DS) retinal ganglion cells is a spatially asymmetric inhibitory input from starburst amacrine cells (3-5). It is not known how and when this circuit asymmetry is established during development. Here we photostimulate mouse starburst cells targeted with channelrhodopsin-2 (refs 6-8) while recording from a single genetically labelled type of DS cell (9,10). We follow the spatial distribution of synaptic strengths between starburst and DS cells during early postnatal development before these neurons can respond to a physiological light stimulus, and confirm connectivity by monosynaptically restricted trans-synaptic rabies viral tracing. We show that asymmetry develops rapidly over a 2-day period through an intermediate state in which random or symmetric synaptic connections have been established. The development of asymmetry involves the spatially selective reorganization of inhibitory synaptic inputs. Intriguingly, the spatial distribution of excitatory synaptic inputs from starburst cells is significantly more symmetric than that of the inhibitory inputs at the end of this developmental period. Our work demonstrates a rapid developmental switch from a symmetric to asymmetric input distribution for inhibition in the neural circuit of a principal cell., DS retinal ganglion cells respond to movement in a 'preferred' direction with robust spiking, but show minimal response to movement in the opposite, or 'null', direction (2,11-13). DS cells receive [...]

Author(s): Feng Zhang [1]; Li-Ping Wang [1]; Martin Brauner [2]; Jana F. Liewald [2]; Kenneth Kay [1]; Natalie Watzke [4]; Phillip G. Wood [4]; Ernst Bamberg [3, 4]; Georg Nagel [...]

The [Na.sup.+]/[K.sup.+]-ATPase is a ubiquitous plasma membrane ion pump that utilizes ATP hydrolysis to regulate the intracellular concentration of [Na.sup.+] and [K.sup.+]. It is comprised of at least two subunits, a large catalytic subunit that mediates ATP hydrolysis and ion transport, and an ancillary [beta] subunit that is required for proper trafficking of the holoenzyme. Although processes mediated by the a subunit have been extensively studied, little is known about the participation of the [beta] subunit in conformational changes of the enzyme. To elucidate the role of the [beta] subunit during ion transport, extracellular amino acids proximal to the transmembrane region of the sheep [[beta].sub.1] subunit were individually replaced for cysteines. This enabled sulfhydryl-specific labeling with the environmentally sensitive fluorescent dye tetramethylrhodamine-6-maleimide (TMRM) upon expression in Xenopus oocytes. Investigation by voltage-clamp fluorometry identified three reporter positions on the [[beta].sub.1] subunit that responded with fluorescence changes to alterations in ionic conditions and/or membrane potential. These experiments for the first time show real-time detection of conformational rearrangements of the [Na.sub.+]/[K.sub.+]-ATPase through a fluorophore-labeled [beta] subunit. Simultaneous recording of presteady-state or stationary currents together with fluorescence signals enabled correlation of the observed environmental changes of the [beta] subunit to certain reaction steps of the [Na.sup.+]/[K.sup.+]-ATPase, which involve changes in the occupancy of the two principle conformational states, [E.sub.1]P and [E.sub.2]P. From these experiments, evidence is provided that the [[beta].sub.1]-S62C mutant can be directly used to monitor the conformational state of the enzyme, while the F64C mutant reveals a relaxation process that is triggered by sodium transport but evolves on a much slower time scale. Finally, shifts in voltage dependence and kinetics observed for mutant K65C show that this charged lysine residue, which is conserved in [[beta].sub.1] isoforms, directly influences the effective potential that determines voltage dependence of extracellular cation binding and release. KEY WORDS: voltage-clamp fluorometry * conformational change * kinetics * electrogenic steps * subunit interaction

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active [Na.sup.+]/[K.sup.+]-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep [[alpha].sub.1]-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ([approximately equal to] 5%) on addition of extracellular [K.sup.+]. When the enzyme was subjected to voltage jumps under [Na.sup.+]/[Na.sup.+]-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the [E.sub.1]P-[E.sub.2]P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of [Na.sup.+] by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating [K.sup.+] concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing [K.sup.+] concentrations. As a result, the stationary distribution between [E.sub.1] and [E.sub.2] states and voltage-dependent relaxation times can be determined at any time and membrane potential under [Na.sup.+]/[Na.sup.+] exchange as well as [Na.sup.+]/[K.sup.+] turnover conditions.