Monthly Archives: June 2012

SypHTomato: a red indicator of presynaptic activity

As already anticipated by many, the next big step of optogenetics will consist in multiplexing optogenetic tools in the same experiment (see the page Combining Optogenetic Tools). Tools used in parallel will need to have well-separated spectral characteristics, a requirement that FRET sensors will have a hard time meeting. The problems of FRET sensors might run deeper than their bandwidth of operation, with issues including differential donor/acceptor photobleaching, worse 2P performance &  more scattering of the excitation wavelength (usually blue-shifted), the difficulty to fuse them with other proteins and their signal-to-noise ratio being lower than for 1-FP sensors. The race for diversifying 1-FP sensors has already begun, with new hue-variants of the GCaMP scaffold (the GECOs series and RCaMP), of kinase activity sensors (Cyan Sinphos) and of voltage-sensitive fluorescent proteins (VSFP3s).

The lab of Richard Tsien has just added a new member to this emerging family of hue-shifted single-FP sensors: a novel red pH-sensitive red fluorescent protein called pHTomato. In a nicely-done 2-author paper published in Nature Neuroscience, Li and Tsien demonstrate the usefulness and efficiency of pHTomato by fusing it to the vesicular membrane protein synaptophysin. The resulting protein, SypHTomato, can report vesicle fusion and recycling as well as its … Continue reading

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The GCaMP3 reporter strain published

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

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New Cre-dependent optogenetic reporter lines

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

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