TagsBooks chloride ChRs Cryptochrome Dimerizers Electroporation Epilepsy Expression fiberoptics FRET GECI GEVI Highlighters Holography hVoS Indicators LED Lines LOV Opsins Optics Optrodes Phototropin prosthetics protons Pumps Recordings Reporter Retina Reviews Scattering Therapeutics Thoughts Toxicity Viruses Voltage waveguides
Tag Archives: Lines
Genetically encoded optical voltage sensors expand the optogenetic toolkit to enable the imaging of electrical activity from genetically defined populations of neurons. In a recent paper that appeared in the Journal of Neurophysiology, Wang et al reported the imaging of electrical activity in hippocampal slices from transgenic mice expressing hybrid voltage sensors (hVoS). hVoS probes are membrane targeted fluorescent proteins that have been optimized for a FRET interaction with dipicrylamine, a lipophilic molecule that partitions into lipid bilayers. A change in voltage alters the FRET interaction between the fluorescent protein and dipicrylamine to produce an optical signal that can be imaged.
Among the various genetically encoded voltage sensors currently under development in various labs, hVOS probes have a signal amplitude comparable to other probes (20-30% for 100 mV), but a very rapid response time (~0.5 msec). Thus, these probes are rapid enough to detect action potentials. Wang et al generated transgenic mice with two different high-performance hVoS probes under control of a neuron-specific thy-1 promoter. Hippocampal slices from these animals present distinct spatial patterns of expression, and electrical stimulation evoked fluorescence changes as high as 3%.
In some instances, clear responses were recorded in a single trial without averaging. One … Continue reading
Optogenetics has proven to be a powerful tool capable of manipulating the activity of a specific population of cells in a complex multicellular organism. This approach is enthusiastically pursued in recent neuroscience field and the causal relationship between neural activity and behavior is finally starting to become unveiled. However, most studies utilize virus mediated gene transfer for the induction of light-sensitive proteins, such as channelrhodopsin-2 (ChR2), and such method inevitably introduces surgical injuries and variability of expression between trials. Therefore, transgenic approach has long been sought, however, satisfying the demands of the specificity as well as the abundance of expression were difficult.
In a recent paper published in the Cell Reports, Tanaka and Matsui and their colleagues at the National Institute for Physiological Sciences (Okazaki, Japan) established Knockin-mediated ENhanced Gene Expression by improved tetracycline-controlled gene induction system (KENGE-tet). The authors found that high levels of tTA-mediated transcription can be achieved by knocking in tetO-ChR2 cassette into a locus at a housekeeping gene, beta-actin. The authors crossed this tetO-ChR2 knockin mouse with 7 different tTA lines and achieved ChR2 expression in specific cell-types including sub-populations of neurons, astrocytes, oligodendrocytes, and microglial cells. In all cases, the level of ChR2 expression was … Continue reading
A couple months ago the latest Cre-dependent optogenetic reporter mouse lines were published in a Nature Neuroscience paper. But one particular line, the GCaMP3 reporter strain, was missing at roll call. This line was published almost at the same time in the Journal of Neuroscience. In the February 29 issue of J. Neurosci, Zariwala et al. show that when crossed with Cre lines the Ai38 line yields stable GCaMP3 expression without the typical toxicity observed with AAV infections (which correlates with GCaMP3 diffusing into the nucleus and is observed as early as 4-6 weeks after infection). For a side-by-side comparison of GCaMP3 and Oregon Green BAPTA-1 (OGB-1), the authors looked at the visual cortex of anesthetized mice. GCaMP3-expressing pyramidal cells of the visual cortex (obtained using a Wfs1-Tg2-Cre line) showed preserved orientation selectivity to moving oriented gratings. Interestingly, the maximum ΔF/F achieved in GCaMP3-expressing neurons was substantially higher than with OGB-1. On the opposite, OGB-1 gave higher ΔF/F for low responder cells. One downside is that imaging from the Ai38 line required about 3 x more laser power than when using AAVs or OGB-1. But overall it looks like GcaMP reporter strains are on the right track and already experiment-ready. Now the … Continue reading
Cre-dependent mouse strains that would express optogenetic control tools have been seen as the holy grail for the past 6 years. Not only such strains would simplify experimental procedures by eliminating the need for gene delivery (in utero electroporation or viral infection) but they would potentially yield more reproducible experiments by providing more homogeneous and predictable expression levels in a given neuronal population. Many labs gave it a try and failed. The challenge resided in achieving high expression using a ubiquitous promoter and a Cre-activated cassette (principle below).
Some time ago it seemed like the folks at the Allen Institute were on a promising track and their first ChR2 reporter strain called Ai27 leaked out to several labs. But many were discouraged by the insufficient expression level of this line and the small light-evoked responses obtained with it. But the Ai27 mouse was just the beginning. Last year the Allen Institute released a new line, codename Ai32, with significantly improved ChR2 expression. Apparently the same ones which were disappointed by the Ai27 line were pretty unanimous about the good results of this Ai32 mouse. The word spread quickly and the mouse was made available through the Jackson Laboratory together with … Continue reading