3D waveguide array for optogenetic neuronal stimulation

Numerous applications enabled by optogenetic tools require the delivery of different light profiles within tissue. In some instances, wide-field illumination is needed to blanket a region of a specific brain layer, while cell body-size beams are necessary in other cases to achieve highly-selective localized stimulation; in addition, spatiotemporal patterning or multi-level access of light may be incorporated. In a recent paper published in Biomedical Optics Express, Abaya, et al. presented a novel device structure that facilitates three-dimensional deep-tissue light penetration with capabilities for simultaneous spatiotemporal modulation with different wavelengths. This device consists of > 1 mm length needle-shaped waveguides that circumvent intrinsic tissue absorption and scattering. This array can be adapted to a wide variety of illumination systems (e.g., microscope, μLED arrays, optical fibers, collimated beams, spatial light modulators) and can potentially facilitate the advancement of a broad range of applications utilizing optical neural stimulation.

The waveguide array is micromachined from fused silica/quartz wafers, which are transparent to visible and near-infrared wavelengths. First, bevel blades are used with a dicing saw to create arrays of pyramids as tips for the array. Next, the shanks are formed by dicing down material in between the pyramids to produce waveguides having a pyramidal … Continue reading

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New genetically-encoded voltage-sensitive probes: ArcLight and ElectricPk

There has been almost two decades of research in the field of genetically-encoded voltage indicators (GEVIs). Early probes carried channel-based voltage sensors (e.g. derived from potassium and sodium channels) but showed poor membrane localization. Membrane localization of GEVIs was greatly improved by replacing channel-based sensors with the voltage-sensing domain of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSD). Despite this major improvement, the goal to record single action potentials and subthreshold electrical events in mammalian neurons with adequate temporal and spatial resolution still presents a challenge. The latest step toward this goal was taken by the Pieribone lab which came up with two Ci-VSD-based monochromatic GEVIs.

The first one, called ArcLight and published in Neuron, was obtained by combining the Ci-VSD and a super ecliptic pHluorin that carries a critical point mutation (A227D). Based on ArcLight, five probes were engineered with super ecliptic pHluorin A227D relocated closer to the S4 domain of the Ci-VSD, after amino acids Q239, M240, K241, A242, or S243. Out of these 5 variants, ArcLight A242 emerged as the best, exhibiting -35% change in ΔF/F in response to 100mV depolarizing steps in HEK293 cells and up to -5% change in ΔF/F for single action potentials in neurons. ArcLight A242 … Continue reading

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R-CaMP1.07, an improved genetically encoded red fluorescent Ca2+ indicator

In a recent paper published in PLoS One, Ohkura et al. introduce R-CaMP1.07, an improved variant of a recently developed red fluorescent Ca2+ indicator protein called R-GECO1. While the sensitivity of R-CaMP1.07 is similar to that of R-GECO1 (Kd for Ca2+ is around 200 nM), R-CaMP1.07 shows 1.5–2-fold greater signals than R-GECO1 due to its enhanced dynamic range (Fmax/Fmin is near 30). The greatest advantage of R-CaMP1.07 is that its excitation wavelength ranges from 500 to 580 nm, which rarely overlaps with the photo-stimulation range of channelrhodopsin-2 (ChR2). Taking this merit of R-CaMP1.07, the authors demonstrate an application example of this indicator in hippocampal pyramidal neurons expressing ChR2: the successful detection of Ca2+ signals in response to action potentials evoked by the photo-stimulation of ChR2. Needless to say, this red fluorescent R-CaMP1.07 can be used for Ca2+ imaging of cells expressing blue, cyan or green fluorescent proteins.

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Transgenic mouse lines expressing hybrid voltage sensors

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

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KENGE-tet System for Expanding the Repertoire of Optogenetically Targeted Cells

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

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Optogenetic voltage imaging in slices and living mice using VSFP-Butterfly

In a recent paper published in the Journal of Neurophysiology, the Knöpfel lab (Riken, Japan) introduced a new design variant of FRET-based voltage sensitive fluorescent proteins, termed VSFP-Butterflies, along with an extensive series of application examples in brain slices and living mice. VSFP-Butterfly 1.2 uses red shifted FP, allowing excitation at 488 to 510 nm with acceptor emission > 600 nm. While the sensitivity of VSFP-Butterfly 1.2 is similar to that of VSFP2.3 (around 22% ΔF/F at half maximal activation, V1/2), its fluorescence to voltage relationship is left shifted, resulting in a more sensitive detection of subthreshold potentials as well as of action potentials (fig. 1). VSFP-Butterfly imaging of voltage signals over the cortex of living mice revealed traveling waves generated by the activity of layer 2/3 pyramidal cells (fig 2). The application examples in this report demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterflies. The authors discuss how VSFP-based voltage imaging will opening new avenues towards a better understanding of the neuronal computations reflected in the dynamics of cortical circuits.

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Transcriptional amplification with two rAAVs using tetracycline-controlled gene induction

Cell-type specific expression of genetically encoded indicators or optogenetic probes is often hampered by the use of promoters that are specific but drive expression only weakly. In a recent paper of the Wang Lab at Princeton University, the authors describe a means of introducing a calcium indicator using the TET promoter system in the cerebellar cortex to boost expression by about ten-fold, as determined by quantitative determination of intracellar concentration. Kuhn et al. show specific labeling of Purkinje cells and all interneuron types. Together with a previous paper of the Wang Lab where glial cells were targeted, now nearly all cell types of the cerebellar cortex can be selectively labeled. Additionally, Kuhn et al. overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by systemic pre-injection of hyperosmotic D-mannitol, doubling the time window for functional imaging.

Original article by Kuhn et al.

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Novel AAVs for Cre-dependent control of transgene expression

Recombinant adeno-associated viruses (rAAVs) designed to activate transgene expression in only those cells expressing Cre-recombinase (Cre-On) are widely used to introduce optogenetics constructs into specific cell types and brain regions. In many experiments, what functionally distinguishes Cre-expressing cells from their non-Cre expressing neighbors is not fully understood. A recent paper by Saunders et al. (Sabatini lab, Harvard Medical School) published in Frontiers in Neural Circuits describes two rAAV strategies that allow for simultaneous Cre-On and Cre-Off transgene expression. One strategy (Cre-Switch) achieves differential transgene expression with a single rAAV. The second strategy introduces a Cre-Off vector (FAS), built with lox sites that do not efficiently recombine with loxP or lox2272 sites, which allow FAS rAAVs to be used simultaneously with popular Cre-On DIO (double-floxed inverted ORF) a.k.a FLEX (flip-excision) rAAVs. All Cre-On, Cre-Off, and Cre-Switch rAAV vectors in Saunders et al. are freely available from Addgene.

Original article by Saunders et al.

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Expanding the color palette of channelrhodopsins

In a recent paper published in the Journal of Biological Chemistry, the Hegemann and Deisseroth labs introduced several color-shifted Channelrhodopsins (ChRs) with different absorption and kinetic properties. Prigge et al. mutated several key amino acids in ChR2 and C1V1 to further separate action spectra of those two existing ChRs. The resulting colour-variants are separated by 30 nm from each other and show peak absorption at 460 ,490, 520 and 550 nm respectively (Fig. 1). All color-variants exhibit two times larger photocurrents in HEK-cells then the wild type ChR2. Further engineering yielded off-kinetics spanning the range from ms to s for each colour variant. The two most spectrally separated variants (ChR2 T159C and C1V1-triple) were used to show the feasibility of a separate, wavelength-dependent activation of a HEK cell population expressing on those variants (Fig. 2). In addition the blue absorbing mutant ChR2 T159C-L132C exhibits 3 times larger photocurrents then ChR2 H134R, has a small inactivation and a reduced proton permeation making this variant the most efficient ChR for blue activation so far.

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An “electro-endoscope” for combined imaging, photocontrol and electrical recording

Sending light and recording electrical activity in deep brain structures using optrodes

When optogenetic tools for controlling neural activity came into play in the late 2000s, it became obvious that hybrid probes for delivering light into the brain and recording photoevoked activity simultaneously would become a basic component of the optogenetic toolkit. And this for at least two reasons. First, microelectrode recordings are still the golden standard for measuring neuronal firing, surpassing by far optical sensors at least at the single cell resolution and millisecond time scale. And second, electrical recordings are and will probably remain the ultimate readout of the efficiency of optogenetic activation and inactivation protocols. In other words, the only way to show that your optogenetic activator or silencer does the job is to directly record its effects on neuronal firing.

Various types of optical⁄electrical probes for deep brain photostimulation and recording. Top left: An optical⁄electrical probe having single optical fiber and single electrode. Bottom: Three types of single optical fiber–multiple electrode combination. Bottom left – wire-wound tetrodes are combined with an optical fiber. Bottom center – “Michigan probe” integrated with an optical fiber. Bottom right – “Utah” multi-electrode array combined with a tapered optical … Continue reading

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