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Tag Archives: Pumps
Unlike electrical stimulation, optogenetics allows neuronal manipulation with great cell-type specificity, with light directly affecting only those cells expressing opsins. In a recent report in Nature Communications, Krook-Magnuson et al harnessed this specificity to stop seizures in vivo in a mouse model of temporal lobe epilepsy. Mice were implanted with electrodes to record brain activity and 200µm thick optical fibers to deliver light to the brain. A closed-loop, on-demand responsive system detected seizures in real time, allowing temporal specificity, in addition to the cell-type specificity achieved through selective opsin expression. Specifically, the authors either selectively inhibited excitatory principal cells or, alternatively, excited a subpopulation of GABAergic inhibitory neurons in the hippocampus by delivering light at the time of a seizure. Both approaches proved successful, despite the less than 5% of illuminated neurons expressing opsins in the latter approach. Light arrested ongoing electrical seizure activity and reduced the incidence of events progressing to overt behavioral seizures.
Epilepsy, a condition of recurrent, spontaneous seizures, is a prevalent disorder, with 1 out of 26 people developing epilepsy during their lifetime. Unfortunately, for over 40% of patients, seizures cannot be controlled with current treatment options. Temporal lobe epilepsy, the most common form of epilepsy … Continue reading
In the last few years, several strategies for silencing neurons optogenetically have emerged. The most popular approaches make use of two types of microbial light-driven ion pumps. One type is an optimized version of halorhodopsin, an inward chloride pump isolated from the halophilic bacterium Natronomonas pharaonis. Over the past couple years, halorhodopsin was tweaked by adding a series of trafficking signals to improve its addressing to the cytoplasmic membrane (Gradinaru et al, 2010). The latest version is eNpHR3.0. The other type corresponds to archeorhodopsins, which are outward proton pumps isolated from halophilic archaebacteria of the genus Halorubrum. The two popular archeorhodopsins are Arch from Halorubrum sodomense and ArchT from Halorubrum genus (Chow et al., 2010, Han et al., 2011). The same optimization tricks used for halorhodopsin also worked for Arch and ArchT. Both types of pumps generate a hyperpolarizing current in mammalian neurons in response to 500-600 nm light and are effective optogenetic silencers of neuronal activity.
A recent head-to-head comparison performed in the lab of K. Deisseroth has shown that, under matched experiments conditions, archeorhodopsins seem to perform slightly better than the best available halorhodopsin mutant (Mattis et al., 2012). In particular eArchT3.0 (an enhanced version of ArchT) generates about … Continue reading
The team of Karl Deisseroth conducted a series of experiments under matched conditions in order to draw a systematic comparison of several microbial opsins. Experiments aimed at comparing action spectra, peak photocurrents, steady-state/peak ratios, time-to-peak, off kinetics, desensitization kinetics, kinetics of recovery from desensitization in darkness and 50% effective light power density. The report is published in an upcoming issue of Nature Methods.
11 ChR variants were tested: ChR2, ChR2(H134R), ChR2(E123A), ChR2(T159C), ChR2(E123T/T159C), ChR2(L132C), ChIEF, channelrhodopsin-fast receiver, channelrhodopsin-green receiver, C1V1(E162T) and C1V1(E122T/E162T). 4 ultrafast control tools were compared: ChR2(E123A), ChR2(E123A/H134R), ChR2(E123T) and ChR2(E123T/H134R). 7 light-driven pumps were compared: eNpHR3.0, Arch1.0, eArch3.0, ArchT1.0, eArchT3.0, Mac1.0 and eMac3.0.
all opsin genes were packaged identically in a lentiviral backbone under the control of the mouse excitatory neuron–specific CaMKIIα promoter. all opsin coding sequences were fused in frame with the gene encoding enhanced YFP (eYFP). electrophysiological measurements were performed on transfected cultured hippocampal pyramidal neurons with matched light power densities across experiments (5 mW/mm2).
A Nature Methods article from the cohen lab shows how a microbial light-driven proton pump, Archeorhodopsin-3 (Arch), usually used to silence neuronal activity, can be used to efficiently image the membrane potential of individual neurons. Like many other microbial rhodopsins, Arch is weakly fluorescent. When excited using green light (550 nm), Arch emits light in the far red (680 nm). This fluorescence process is poorly efficient (quantum yield < 0.001) but sensitive to the membrane potential. When expressed in mammalian neurons in vitro, the fluorescence change of Arch in response to steps of potential was extremely fast (< 0.5 ms) with a high signal-to-noise ratio, allowing the detection of single action potentials in single trials. However, the fact that Arch generates an outward proton current when illuminated seriously challenges the relevance of its use as a voltage sensor. A mutated form of Arch (D95N) which does not generate any current was also tested. Arch(D95N) retains the ability to report single action potentials but with a significantly slower response time (40 ms).
The labs of Ernst Bamberg and Ed Boyden introduced a gene-fusion approach to achieve co-localized and stoichiometric expression of synergistic or antagonistic opsin pairs. The method relies on a genetic tandem cassette which intercalates the coding sequence of a transmembrane helix (from the β subunit of the rat gastric H+,K+-ATPase) between two opsin genes. The resulting fusion proteins allow strict ratiometric activation of rhodopsin pairs. Fusing ChR2 and NphR or Arch can be used for example to activate or silence all neurons in a field of view to a similar extent or to precisely simulate subthreshold events across the somatodendritic tree of a neuron (as each patch of membrane will preserve the stoichiometry of expression).