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
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
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
Plants have developed elaborate light-sensing pathways to be able to adequately react on the quality and quantity of light. These signal transduction pathways regulate among others growth directionality (phototropism), flowering, stomatal opening, and chloroplast movement. Light-signalling pathways in plants converge on the ubiquitin-protein ligase COP1 that regulates stability of many transcription factors involved in light-dependent responses. This puts regulated proteolysis by the ubiquitin proteasome system at the heart of light-depending signaling. Recently, Renicke et al., reported in Chemistry & Biology on the engineering of a synthetic tool that concentrates light-regulated proteolysis into a single construct. The module developed by the authors comprises the light-sensing LOV2 domain of Arabidopsis thaliana phot1 and the ornithine decarboxylase (ODC) like degradation sequence (degron) cODC1. Activity of this so-called photo-sensitive degron (psd) module depends on the presence of blue light: the conformational changes within the LOV2 domain expose the degron, which induces proteasomal degradation. In contrast to the majority of proteasomal substrates, the cODC1 degron is directly recognized by the proteasome, without polyubiquitylation by an ubiquitin-protein ligase. Thus, the psd module is a minimized plant light-signalling pathway that comprises a light-responsive domain fused with a degradation-inducing sequence. The module can be fused to target proteins … Continue reading
Sensory information is typically represented in distributed patterns of activity across large populations of neurons. Therefore, many emerging neuro-photonic applications, such as optogenetic retinal prostheses require systems capable of delivering intense, parallel and dynamic light patterns. Such a system will ideally allow photo-control with single-cell selectivity of large neural populations expressing optogenetic probes, rather than nonspecific flashed illumination of the whole population (as provided by many current optogenetic light delivery systems). In a recent Nature Communications report, Reutsky-Gefen, Shoham and colleagues demonstrate holographic optogenetic control of retinal neural activity which is shown to provide rapid cellular-resolution, massively parallel excitation across macroscopic (millimeter-scale) coverage areas. The study illustrates that diffractive wavefront shaping (holographic) tools offer a powerful modality for dynamic patterned photo-stimulation as they naturally combine the high intensity, efficiency and resolution that are characteristic of sequential laser deflectors (like acousto-optical deflectors) with the simultaneous scan-less parallel illumination of multiple locations of microdisplay array projectors, but without their respective limitations. Holographic tools were previously shown to allow structured excitation of dendritic arbors and neurons using neurotransmitter photolysis, as well as two-photon optogenetic stimulation of proximal neurons in brain slices.
The study’s main goal was to develop a prosthetic system that would … Continue reading
Microelectrodes are powerful tools for in vivo functional studies. However they are limited in the number of information they provide. In January 2011, LeChasseur and colleagues (Nature Methods 8(4), 319-325, 2011) developed a glass microelectrode which was integrating an optical micro-channel for light delivery and fluorescence collection. The probe serves for specific cellular fluorescence optical detection and activation/inhibition. In a recent issue of PLOS ONE, Dufour et al. extended the multimodal aspect of this micro-optrode. They introduce a, aluminum-coated, fibre optic-based glass microprobe (diameter ≤ 10 μm) with multiple electrical and optical detection capabilities. The probe enables optical separation from individual cells in transgenic mice expressing multiple fluorescent proteins in distinct populations of neurons within the same deep brain nucleus. It also enables color conversion of photoswitchable fluorescent proteins, which can be used for post-hoc identification of the recorded cells and finally it enables dual electrical recordings. Figure 1 shows a representation of the microp-optrode and the modalities described in this paper.
These modalities are in addition to the calcium monitoring and optogenetic cellular activation previously reported (Nature Methods 8(4), 319-325, 2011). In this study, two different excitation sources and detection pathways were used simultaneously to differentiate two different populations … Continue reading
In optogenetic experiments aiming at controlling neuronal activity light-sensitive molecules such as channelrhodopsins or proton pumps are activated through application of light from a physical light source, such as arc lamps, lasers or LEDs. An interesting extension of such experiments could be achieved by controlling light-sensing molecules with a biological light source. Luciferases are such light producing proteins; they are enzymes, used by a variety of bioluminescent organisms, which produce light by oxidizing a substrate molecule. Replacing the physical light source with a biological one, i.e. a light-emitting protein, should allow non-invasive activation of light-sensitive molecules. In addition, the advantage of genetically encoding both light production and light sensing introduces unique combinatorial possibilities.
Using bioluminescence in combination with optogenetic actuators hinges on the ability of luciferase to activate these actuators. In a recent paper published in PLoS ONE, Berglund and colleagues demonstrate that this is indeed possible. In this study the authors engineered fusion proteins of a luciferase from the marine copepod Gaussia princeps (GLuc) and channelrhodopsins, creating luminescent opsins, or “luminopsins”. Their study brings the proof-of-concept that light produced upon supplying the luciferase with its substrate, coelenterazine (CTZ), can activate the fused channelrhodopsin, thereby modulating neuronal activity.
Because luminopsins … Continue reading
The study of cell signaling has revealed that cells can be particularly sensitive to spatiotemporal properties of the signaling dynamics. For instance, a cell can choose divergent fates (proliferation vs. differentiation, senescence vs. apoptosis, etc.) depending on the duration and strength of a protein signal. The development of fluorescent reporters has enabled us to observe these dynamics, but researchers still lack appropriate, general tools to perturb cells on the second- and minute-timescales on which signaling dynamics can occur.
Coupling light to protein activation may offer a powerful solution due to its fast, reversible, and highly tunable nature. In the March 2013 issue of Nature Methods, Bugaj, Kane, Schaffer and colleagues accomplish this by developing a method for light inducible homo-oligomerization of proteins. In this study, the authors report a genetically encodable, generalizable system that can activate multiple signaling proteins and networks in mammalian cells.
To do this, the authors leveraged the interesting ability of the Arabidopsis protein Cryptochrome 2 (Cry2) to form oligomers in response to blue light. To date, this clustering ability had only been shown in plant cells, but upon transfection into HEK 293Ts, the authors demonstrated that Cry2 can be clustered in mammalian cells as well (fig. … Continue reading
When using optogenetics to study circuit function or animal behavior, a critical prerequisite is that optogenetic protein expression does not, in itself, perturb the circuit being studied. While short-term expression is very commonly used without observable circuit disruption, whether this is also true for long-term expression is less clear. A recent paper by Miyashita et al. in Frontiers in Neural Circuits shows that long-term, high-level expression of ChR2 can induce abnormal axonal morphology and targeting in cerebral cortex. This underscores the importance of using the lowest expression possible, particularly for long-term studies.
Miyashita et al. expressed a common construct, CAG::hChR2 (H134R)-EYFP-WPRE, in L2/3 pyramidal neurons in rat somatosensory cortex via in utero electroporation (IUE). This same strategy was used in several prior studies of S1 circuit function, with one important difference: Miyashita et al. expressed hChR2 that was codon-optimized for mammalian expression, while prior studies expressed native ChR2 (discussed below). This strategy successfully conferred light-evoked spiking in vivo and in in vitro brain slices. However, long-term expression (> 40 d) also caused major abnormalities in axonal morphology, which included cylinders of axonal membrane that enveloped pyramidal cell proximal dendrites, and spherical, calyx-like axonal swellings that surrounded neuron cell bodies. These … Continue reading
In order to perform simultaneous intracellular recording and light stimulation of a single neuron, two separate positioning systems are often needed (one to position the recording electrode, one to position a waveguide near the recorded neuron). More sophisticated solutions for single neuron photostimulation involve light patterning techniques which are not suited for deep in vivo recordings. Katz et al. came up with a simple and affordable solution for this problem, by designing a patch pipette holder containing an additional port for the insertion of an optical fiber into the pipette.
This device, which they called “OptoPatcher” allows whole cell patch-clamp recording simultaneously with direct projection of light from the recording pipette. The holder spares the use of an additional manipulator and, importantly, enables accurate, stable and reproducible illumination. Moreover, the presence of the bare fiber within an aqueous solution instead of the brain can prevent tissue damage due to heating of the brain. In addition, replacement of standard pipettes is done as easily as with the available commercial holders.
The OptoPatcher was used successfully in vivo for intracellular recordings from different cortical layers in the motor cortex of transgenic mice expressing channelrhodopsin-2 under the Thy1 promoter and it was also … Continue reading