Monthly Archives: May 2013

Genetically encoded sensors for simultaneous monitoring of Cl− and H+

The simultaneous optogenetic sensing of the intracellular concentrations of Cl− and H+ is a challenging task, which requires probes with high sensitivity allowing reliable quantitative measurements without perturbation of cell functioning. The recent paper in Frontiers of Molecular Neurosciences by Mukhtarov et al. (2013) describes the intracellular calibration and functional characterization of three genetically encoded probes developed for these purposes. The first is recently proposed combined Cl−/pH sensor (ClopHensor), which was obtained by fusion of a red fluorescent protein (RFP) with a GFP variant, E2GFP, which contains a specific Cl−-binding site (Arosio et al., 2010). The second is PalmPalm-ClopHensor, a variant that is preferentially expressed at the plasma membrane thanks to the addition of two palmitoylation sites at the N-terminus. The third ClopHensor variant contains a two additional mutations (H148G/V224L) in the GFP moiety conferring improved Cl− affinity and reduced pH dependence.

For functional analysis, constructs were expressed in CHO cells and neurons. In CHO cells ClopHensor and the H148G/V224L mutant exhibits cytoplasmic intracellular distribution while the PalmPalm-ClopHensor construct, as expected, is preferentially localized in the vicinity of membranes (FIG.1).

In order to evaluate the dynamic range and sensitivity of constructs to ions, CHO cells were co-transfected with Cl−-selective glycine … Continue reading

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A new set of hue variants enriches the palette of available GECOs

In a recent study published in in ACS Chemical Neuroscience, Campbell, Li, Nagai and coworkers report the development of a new series of orange and red genetically encoded Ca2+ indicators with improved sensitivity. To expand the color palette of genetically encoded Ca2+ indicators, semi-rational design and directed evolution were used to explore different chromophore structures and to modulate the environment adjacent to the chromophore of a previously reported red Ca2+ indicator, R-GECO1. These efforts lead to the identification of O-GECO (blue shifted), R-GECO1.2 and CAR-GECO1 (red shifted emission) with Ca2+ dependent intensiometric signal changes of 14600%, 3300% and 2700%, respectively (see figure 1 below). The authors go on to describe a troublesome photoactivation phenomenon that was discovered when these new indicators were used in conjunction with ChR2. Specifically, the fluorescence signals of these orange and red Ca2+ sensors exhibit a reversible increase with the intense blue light illumination used for ChR2 activation, even when there is no change in the Ca2+ concentration (see figure 2 below). By carrying out extensive in vitro and tissue-based characterizations, the authors showed that using an appropriate intensity of blue light could minimize this photoactivation problem and allowed these new orange and red Ca2+ indicators … Continue reading

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Thermal and optical characterization of a micro-LED probe

Over the last few years, there has been a significant drive to improve light sources for in vivo optogenetic control of neuronal activity. In particular, recent work has focused on the design and microfabrication of compact devices that exhibit multiple optical stimulation sites in order to gain control over closely spaced regions within the brain (see here a list of posts related to this). In a recent Optics Letters paper, McAlinden, Massoubre and colleagues presented a novel microprobe device with integrated light sources. The probe produces sufficient light for optogenetic stimulation without causing significant heating in local brain tissue. The device (see figure below) consists of a 100 µm wide, 50-100 µm thick probe with 5 individually addressable microLEDs. Each LED has a diameter of 40 µm, but can be reduced to 10 µm if higher density probes are required. The LED probes are p-n diodes made from quantum well structures using GaN on Sapphire material. The spacing between LEDs is fixed to allow 1mm of neural tissue to be addressed.

The light output from the LEDs was measured, with scattering and absorption accounted for by experimentally measuring the light transmitted through varying thicknesses of brain tissue. The recorded light … Continue reading

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