Optogenetic Actuators Research Articles

All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC). The voltage-indicator QuasAr2 provides information for fast, closed-loop optical feedback to the bidirectional optogenetic actuator BiPOLES. Voltage-dependent fluorescence is held within tight margins, thus clamping the cell to distinct potentials. We established the OVC in muscles and neurons of Caenorhabditis elegans, and transferred it to rat hippocampal neurons in slice culture. Fluorescence signals were calibrated to electrically measured potentials, and wavelengths to currents, enabling to determine optical I/V-relationships. The OVC reports on homeostatically altered cellular physiology in mutants and on Ca2+-channel properties, and can dynamically clamp spiking in C. elegans. Combining non-invasive imaging with control capabilities of electrophysiology, the OVC facilitates high-throughput, contact-less electrophysiology in individual cells and paves the way for true optogenetic control in behaving animals.

Near infrared voltage sensitive dyes based on chromene electron donors.

Excellent hemicyanine voltage sensitive dyes (VSDs) are available for optical electrophysiology over a wide spectral range out to the far red. Near infrared fluorescent VSDs would be advantageous because they allow deeper light penetration into tissue because of decreased light scattering and also because they can be used in combination with other fluorescent indicators or optogenetic actuators. The usual approach to constructing long wavelength chromophores is to lengthen the π-system; however, this is generally accompanied by a significant decrease in fluorescence quantum efficiency and can also decrease aqueous solubility, making it difficult to deliver the probes to the target tissue. The VSDs developed in our lab are generally composed of an amino-naphthyl electron donor moiety linked to a pyridinium acceptor. Here we show that replacing the naphthalene with chromene induces a 60-80 nm red-shift in the absorption and emission spectra while maintaining the structure at the same size and fluorescence quantum yield at the same level. The voltage sensitivity of these chromene-based VSDs is also comparable to our older VSDs and they display opposite polarity sensitivity when excited at the short and long wavelength edges of the absorbance spectra; thus they can be used in dual wavelength ratio measurements to eliminate artifacts from motion and bleaching. We have tested one of these VSDs in voltage imaging of a whole murine heart at 730 nm, by far the longest wavelength reported in voltage imaging. It resolves action potentials in single sweep scans with 10.1% ΔF/F per action potential. Chromene-based electron donors are likely to find wide applications in generating long wavelength hemicyanines or cyanines. (Supported by NIH grant MH116830. Conflict of Interest statement: LML, CDA, and PY are founders and owners of Potentiometric Probes LLC.)

Towards next-generation optogenetic actuators of voltage-gated Ca2+ channels.

CaV1 channels (CaV1.2/1.3) are fundamentally involved in the normal function and pathophysiology of the heart and the brain. These channels convey Ca2+ influx that sculpts action potentials, triggers muscle contraction, and mediates excitation-transcription coupling. To orchestrate these diverse functions, CaV1 activity is fine-tuned by multiple regulatory processes, including Ca2+-dependent inactivation (CDI), which is mediated by the Ca2+-binding protein, calmodulin (CaM). Disruption of CaM regulation is linked to cardiac arrhythmias and neurodevelopmental disorders. However, the precise role of this modulation in various physiological settings is not fully understood. In part, this gap in understanding stems from a lack of approaches to dynamically alter CDI. Here, we engineer a novel optogenetic tool to acutely prevent CDI. To do so, we undertook a biomimetic approach by leveraging naturally occurring CaV1 regulatory mechanisms that modify CDI. Specifically, SH3 and cysteine-rich-domain (stac) protein was recently found to selectively prevent CaV1 CDI. Our previous work identified a short amino acid segment of stac called the U-domain that strongly inhibits CDI. Here, we attached the stac3 U-domain to a light-sensitive Lov2 domain to optically switch its accessibility for modulating CaV1 channels, yielding an actuator known as LovU-CaV1. Whole-cell electrophysiological recordings showed that co-expression of LovU-CaV1 resulted in low basal inhibition of CDI of CaV1.2. However, upon light activation of LovU-CaV1, we observed a rapid reduction in CDI that was then reversible. LovU-CaV1 provides a new avenue to optically manipulate endogenous Ca channels in both the heart and the brain. Future studies will employ this tool to dissect the importance of L-type channel inactivation in both cardiomyocytes and in neurons.

Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation

cAMP, a key player in many physiological processes, was classically considered to originate solely from the plasma membrane (PM). This view was recently challenged by observations showing that upon internalization GsPCRs can sustain signaling from endosomes and/or the trans-Golgi network (TGN). In this new view, after the first PM-generated cAMP wave, the internalization of GsPCRs and ACs generates a second wave that was strictly associated with nuclear transcriptional events responsible for triggering specific biological responses. Here, we report that the endogenously expressed TSHR, a canonical GsPCR, triggers an internalization-dependent, calcium-mediated nuclear sAC activation that drives PKA activation and CREB phosphorylation. Both pharmacological and genetic sAC inhibition, which did not affect the cytosolic cAMP levels, blunted nuclear cAMP accumulation, PKA activation, and cell proliferation, while an increase in nuclear sAC expression significantly enhanced cell proliferation. Furthermore, using novel nuclear-targeted optogenetic actuators, we show that light-stimulated nuclear cAMP synthesis can mimic the proliferative action of TSH by activating PKA and CREB. Therefore, based on our results, we propose a novel three-wave model in which the "third" wave of cAMP is generated by nuclear sAC. Despite being downstream of events occurring at the PM (first wave) and endosomes/TGN (second wave), the nuclear sAC-generated cAMP (third wave) is sufficient and rate-limiting for thyroid cell proliferation.

Functional integrity of visual coding following advanced photoreceptor degeneration

Photoreceptor degeneration sufficient to produce severe visual loss often spares the inner retina. This raises hope for vision restoration treatments using optogenetics or electrical stimulation, which generate a replacement light input signal in surviving neurons. The success of these approaches is dependent on the capacity of surviving circuits of the visual system to generate and propagate an appropriate visual code in the face of neuroanatomical remodeling. To determine whether retinally degenerate animals possess this capacity, we generated a transgenic mouse model expressing the optogenetic actuator ReaChR in ON bipolar cells (second-order neurons in the visual projection). After crossing this with the rd1 model of photoreceptor degeneration, we compared ReaChR-derived responses with photoreceptor-driven responses in wild-type (WT) mice at the level of retinal ganglion cells and the visual thalamus. The ReaChR-driven responses in rd1 animals showed low photosensitivity, but in other respects generated a visual code that was very similar to the WT. ReaChR rd1 responses had high trial-to-trial reproducibility and showed sensitivity normalization to code contrast across background intensities. At the single unit level, ReaChR-derived responses exhibited broadly similar variations in response polarity, contrast sensitivity, and temporal frequency tuning as the WT. Units from the WT and ReaChR rd1 mice clustered together when subjected to unsupervised community detection based on stimulus-response properties. Our data reveal an impressive ability for surviving circuitry to recreate a rich visual code following advanced retinal degeneration and are promising for regenerative medicine in the central nervous system.

Portable low-cost macroscopic mapping system for all-optical cardiac electrophysiology.

All-optical cardiac electrophysiology enables the visualization and control of key parameters relevant to the detection of cardiac arrhythmias. Mapping such responses in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is of great interest for cardiotoxicity and personalized medicine applications. We introduce and validate a very low-cost compact mapping system for macroscopic all-optical electrophysiology in layers of hiPSC-CMs. The system uses oblique transillumination, low-cost cameras, light-emitting diodes, and off-the-shelf components (total ) to capture voltage, calcium, and mechanical waves under electrical or optical stimulation. Our results corroborate the equivalency of electrical and optogenetic stimulation of hiPSC-CMs, and similarity in conduction under pacing. Green-excitable optical sensors are combinable with blue optogenetic actuators (chanelrhodopsin2) only under very low green light ( ). Measurements in warmer culture medium yield larger spread of action potential duration and higher conduction velocities compared to Tyrode's solution at room temperature. As multiple optical sensors and actuators are combined, our results can help handle the "spectral congestion" and avoid parameter distortion. We illustrate the utility of the system for uncovering the action of cellular uncoupling agents and show extensibility to an epi-illumination mode for future imaging of thicker native or engineered tissues.

Targeted sensors for glutamatergic neurotransmission.

Optical report of neurotransmitter release allows visualisation of excitatory synaptic transmission. Sensitive genetically-encoded fluorescent glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-γ2 and SnFR-γ8, were enriched at synapses, retained function and reported spontaneous glutamate release in rat hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic mouse neurons cultured on astrocytic microislands, evoked neurotransmitter release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal neurotransmitter release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-γ2 is a linear reporter of the global quantal parameters and short-term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest (ROIs) showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-γ2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

Optogenetic stimulation reveals a latent tipping point in cortical networks during ictogenesis.

Brain-state transitions are readily apparent from changes in brain rhythms,1 but are difficult to predict, suggestive that the underlying cause is latent to passive recording methods. Among the most important transitions, clinically, are the starts of seizures. We here show that an 'active probing' approach may have several important benefits for epileptic management, including by helping predict these transitions. We used mice expressing the optogenetic actuator, channelrhodopsin, in pyramidal cells, allowing this population to be stimulated in isolation. Intermittent stimulation at frequencies as low as 0.033 Hz (period = 30 s) delayed the onset of seizure-like events in an acute brain slice model of ictogenesis, but the effect was lost if stimulation was delivered at even lower frequencies (1/min). Notably, active probing additionally provides advance indication of when seizure-like activity is imminent, revealed by monitoring the postsynaptic response to stimulation. The postsynaptic response, recorded extracellularly, showed an all-or-nothing change in both amplitude and duration, a few hundred seconds before seizure-like activity began-a sufficient length of time to provide a helpful warning of an impending seizure. The change in the postsynaptic response then persisted for the remainder of the recording, indicative of a state change from a pre-epileptic to a pro-epileptic network. This occurred in parallel with a large increase in the stimulation-triggered Ca2+ entry into pyramidal dendrites, and a step increase in the number of evoked postsynaptic action potentials, both consistent with a reduction in the threshold for dendritic action potentials. In 0 Mg2+ bathing media, the reduced threshold was not associated with changes in glutamatergic synaptic function, nor of GABAergic release from either parvalbumin or somatostatin interneurons, but simulations indicate that the step change in the optogenetic response can instead arise from incremental increases in intracellular [Cl-]. The change in the response to stimulation was replicated by artificially raising intracellular [Cl-], using the optogenetic chloride pump, halorhodopsin. By contrast, increases in extracellular [K+] cannot account for the firing patterns in the response to stimulation, although this, and other cellular changes, may contribute to ictal initiation in other circumstances. We describe how these various cellular changes form a synergistic network of positive feedback mechanisms, which may explain the precipitous nature of seizure onset. This model of seizure initiation draws together several major lines of epilepsy research as well as providing an important proof-of-principle regarding the utility of open-loop brain stimulation for clinical management of the condition.

Spatiotemporal dynamics of membrane surface charge regulates cell polarity and migration.

During cell migration and polarization, numerous signal transduction and cytoskeletal components self-organize to generate localized protrusions. Although biochemical and genetic analyses have delineated many specific interactions, how the activation and localization of so many different molecules are spatiotemporally orchestrated at the subcellular level has remained unclear. Here we show that the regulation of negative surface charge on the inner leaflet of the plasma membrane plays an integrative role in the molecular interactions. Surface charge, or zeta potential, is transiently lowered at new protrusions and within cortical waves of Ras/PI3K/TORC2/F-actin network activation. Rapid alterations of inner leaflet anionic phospholipids-such as PI(4,5)P2, PI(3,4)P2, phosphatidylserine and phosphatidic acid-collectively contribute to the surface charge changes. Abruptly reducing the surface charge by recruiting positively charged optogenetic actuators was sufficient to trigger the entire biochemical network, initiate de novo protrusions and abrogate pre-existing polarity. These effects were blocked by genetic or pharmacological inhibition of key signalling components such as AKT and PI3K/TORC2. Conversely, increasing the negative surface charge deactivated the network and locally suppressed chemoattractant-induced protrusions or subverted EGF-induced ERK activation. Computational simulations involving excitable biochemical networks demonstrated that slight changes in feedback loops, induced by recruitment of the charged actuators, could lead to outsized effects on system activation. We propose that key signalling network components act on, and are in turn acted upon, by surface charge, closing feedback loops, which bring about the global-scale molecular self-organization required for spontaneous protrusion formation, cell migration and polarity establishment.

A kinetic-optimized CoChR variant with enhanced high-frequency spiking fidelity

Channelrhodopsins are a promising toolset for noninvasive optical manipulation of genetically identifiable neuron populations. Existing channelrhodopsins have generally suffered from a trade-off between two desired properties: fast channel kinetics and large photocurrent. Such a trade-off hinders spatiotemporally precise optogenetic activation during both one-photon and two-photon photostimulation. Furthermore, the simultaneous use of spectrally separated genetically encoded indicators and channelrhodopsins has generally suffered from non-negligible crosstalk in photocurrent or fluorescence. These limitations have hindered crosstalk-free dual-channel experiments needed to establish relationships between multiple neural populations. Recent large-scale transcriptome sequencing revealed one potent optogenetic actuator, the channelrhodopsin from species Chloromonas oogama (CoChR), which possessed high cyan light-driven photocurrent but slow channel kinetics. We rationally designed and engineered a kinetic-optimized CoChR variant that was faster than native CoChR while maintaining large photocurrent amplitude. When expressed in cultured hippocampal pyramidal neurons, our CoChR variant improved high-frequency spiking fidelity under one-photon illumination. Our CoChR variant’s blue-shifted excitation spectrum enabled simultaneous cyan photostimulation and red calcium imaging with negligible photocurrent crosstalk.

Ascl1 phospho-site mutations enhance neuronal conversion of adult cortical astrocytes in vivo.

Direct neuronal reprogramming, the process whereby a terminally differentiated cell is converted into an induced neuron without traversing a pluripotent state, has tremendous therapeutic potential for a host of neurodegenerative diseases. While there is strong evidence for astrocyte-to-neuron conversion in vitro, in vivo studies in the adult brain are less supportive or controversial. Here, we set out to enhance the efficacy of neuronal conversion of adult astrocytes in vivo by optimizing the neurogenic capacity of a driver transcription factor encoded by the proneural gene Ascl1. Specifically, we mutated six serine phospho-acceptor sites in Ascl1 to alanines (Ascl1 SA 6) to prevent phosphorylation by proline-directed serine/threonine kinases. Native Ascl1 or Ascl1 SA 6 were expressed in adult, murine cortical astrocytes under the control of a glial fibrillary acidic protein (GFAP) promoter using adeno-associated viruses (AAVs). When targeted to the cerebral cortex in vivo, mCherry+ cells transduced with AAV8-GFAP-Ascl1 SA 6-mCherry or AAV8-GFAP-Ascl1-mCherry expressed neuronal markers within 14 days post-transduction, with Ascl1 SA 6 promoting the formation of more mature dendritic arbors compared to Ascl1. However, mCherry expression disappeared by 2-months post-transduction of the AAV8-GFAP-mCherry control-vector. To circumvent reporter issues, AAV-GFAP-iCre (control) and AAV-GFAP-Ascl1 (or Ascl1 SA 6)-iCre constructs were generated and injected into the cerebral cortex of Rosa reporter mice. In all comparisons of AAV capsids (AAV5 and AAV8), GFAP promoters (long and short), and reporter mice (Rosa-zsGreen and Rosa-tdtomato), Ascl1 SA 6 transduced cells more frequently expressed early- (Dcx) and late- (NeuN) neuronal markers. Furthermore, Ascl1 SA 6 repressed the expression of astrocytic markers Sox9 and GFAP more efficiently than Ascl1. Finally, we co-transduced an AAV expressing ChR2-(H134R)-YFP, an optogenetic actuator. After channelrhodopsin photostimulation, we found that Ascl1 SA 6 co-transduced astrocytes exhibited a significantly faster decay of evoked potentials to baseline, a neuronal feature, when compared to iCre control cells. Taken together, our findings support an enhanced neuronal conversion efficiency of Ascl1 SA 6 vs. Ascl1, and position Ascl1 SA 6 as a critical transcription factor for future studies aimed at converting adult brain astrocytes to mature neurons to treat disease.

LITOS: a versatile LED illumination tool for optogenetic stimulation

Optogenetics has become a key tool to manipulate biological processes with high spatio-temporal resolution. Recently, a number of commercial and open-source multi-well illumination devices have been developed to provide throughput in optogenetics experiments. However, available commercial devices remain expensive and lack flexibility, while open-source solutions require programming knowledge and/or include complex assembly processes. We present a LED Illumination Tool for Optogenetic Stimulation (LITOS) based on an assembled printed circuit board controlling a commercially available 32 × 64 LED matrix as illumination source. LITOS can be quickly assembled without any soldering, and includes an easy-to-use interface, accessible via a website hosted on the device itself. Complex light stimulation patterns can easily be programmed without coding expertise. LITOS can be used with different formats of multi-well plates, petri dishes, and flasks. We validated LITOS by measuring the activity of the MAPK/ERK signaling pathway in response to different dynamic light stimulation regimes using FGFR1 and Raf optogenetic actuators. LITOS can uniformly stimulate all the cells in a well and allows for flexible temporal stimulation schemes. LITOS’s affordability and ease of use aims at democratizing optogenetics in any laboratory.

Optogenetics at the presynapse.

Optogenetic actuators enable highly precise spatiotemporal interrogation of biological processes at levels ranging from the subcellular to cells, circuits and behaving organisms. Although their application in neuroscience has traditionally focused on the control of spiking activity at the somatodendritic level, the scope of optogenetic modulators for direct manipulation of presynaptic functions is growing. Presynaptically localized opsins combined with light stimulation at the terminals allow light-mediated neurotransmitter release, presynaptic inhibition, induction of synaptic plasticity and specific manipulation of individual components of the presynaptic machinery. Here, we describe presynaptic applications of optogenetic tools in the context of the unique cell biology of axonal terminals, discuss their potential shortcomings and outline future directions for this rapidly developing research area.

Advantages, Pitfalls, and Developments of All Optical Interrogation Strategies of Microcircuits in vivo.

The holy grail for every neurophysiologist is to conclude a causal relationship between an elementary behaviour and the function of a specific brain area or circuit. Our effort to map elementary behaviours to specific brain loci and to further manipulate neural activity while observing the alterations in behaviour is in essence the goal for neuroscientists. Recent advancements in the area of experimental brain imaging in the form of longer wavelength near infrared (NIR) pulsed lasers with the development of highly efficient optogenetic actuators and reporters of neural activity, has endowed us with unprecedented resolution in spatiotemporal precision both in imaging neural activity as well as manipulating it with multiphoton microscopy. This readily available toolbox has introduced a so called all-optical physiology and interrogation of circuits and has opened new horizons when it comes to precisely, fast and non-invasively map and manipulate anatomically, molecularly or functionally identified mesoscopic brain circuits. The purpose of this review is to describe the advantages and possible pitfalls of all-optical approaches in system neuroscience, where by all-optical we mean use of multiphoton microscopy to image the functional response of neuron(s) in the network so to attain flexible choice of the cells to be also optogenetically photostimulated by holography, in absence of electrophysiology. Spatio-temporal constraints will be compared toward the classical reference of electrophysiology methods. When appropriate, in relation to current limitations of current optical approaches, we will make reference to latest works aimed to overcome these limitations, in order to highlight the most recent developments. We will also provide examples of types of experiments uniquely approachable all-optically. Finally, although mechanically non-invasive, all-optical electrophysiology exhibits potential off-target effects which can ambiguate and complicate the interpretation of the results. In summary, this review is an effort to exemplify how an all-optical experiment can be designed, conducted and interpreted from the point of view of the integrative neurophysiologist.

Insights on gastrointestinal motility through the use of optogenetic sensors and actuators.

The muscularis of the gastrointestinal (GI) tract consists of smooth muscle cells (SMCs) and various populations of interstitial cells of Cajal (ICC), platelet-derived growth factor receptor α+ (PDGFRα+ ) cells, as well as excitatory and inhibitory enteric motor nerves. SMCs, ICC and PDGFRα+ cells form an electrically coupled syncytium, which together with inputs from the enteric nervous system (ENS) regulates GI motility. Early studies evaluating Ca2+ signalling behaviours in the GI tract relied upon indiscriminate loading of tissues with Ca2+ dyes. These methods lacked the means to study activity in specific cells of interest without encountering contamination from other cells within the preparation. Development of mice expressing optogenetic sensors (green calmodulin fusion protein (GCaMP), red calmodulin fusion protein (RCaMP)) has allowed visualization of Ca2+ signalling behaviours in a cell specific manner. Additionally, availability of mice expressing optogenetic modulators (channelrhodopsins or halorhodospins) has allowed manipulation of specific signalling pathways using light. GCaMP-expressing animals have been used to characterize Ca2+ signalling behaviours of distinct classes of ICC and SMCs throughout the GI musculature. These findings illustrate how Ca2+ signalling in ICC is fundamental in GI muscles, contributing to tone in sphincters, pacemaker activity in rhythmic muscles and relaying enteric signals to SMCs. Animals that express channelrhodopsin in specific neuronal populations have been used to map neural circuitry and to examine post junctional neural effects on GI motility. Thus, optogenetic approaches provide a novel means to examine the contribution of specific cell types to the regulation of motility patterns within complex multi-cellular systems.

Optogenetic actuator – ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics

Combining single‐cell measurements of ERK activity dynamics with perturbations provides insights into the MAPK network topology. We built circuits consisting of an optogenetic actuator to activate MAPK signaling and an ERK biosensor to measure single‐cell ERK dynamics. This allowed us to conduct RNAi screens to investigate the role of 50 MAPK proteins in ERK dynamics. We found that the MAPK network is robust against most node perturbations. We observed that the ERK‐RAF and the ERK‐RSK2‐SOS negative feedback operate simultaneously to regulate ERK dynamics. Bypassing the RSK2‐mediated feedback, either by direct optogenetic activation of RAS, or by RSK2 perturbation, sensitized ERK dynamics to further perturbations. Similarly, targeting this feedback in a human ErbB2‐dependent oncogenic signaling model increased the efficiency of a MEK inhibitor. The RSK2‐mediated feedback is thus important for the ability of the MAPK network to produce consistent ERK outputs, and its perturbation can enhance the efficiency of MAPK inhibitors.

Nociceptive and Opioidergic Cell-types, Circuitry, and Function in the Cingulate Cortex

Pain is an unpleasant emotional experience driven by the transformation of sensory neural signals into affective-cognitive information in cortical regions, including the anterior cingulate cortex (ACC). Opioid action in the ACC ameliorates aspects of the aversive quality of pain through mu opioid receptors (MOR). We hypothesized that MOR-expressing and functionally nociceptive ACC neurons represents a crucial neural circuit cell-type to pain affect and pathological attention to chronic pain. Using single nuclei RNA sequencing of murine ACC, we found that three cell-subtypes of Slc17a7 glutamatergic neurons were the most transcriptionally active to noxious stimuli (identified by weighted expression of 139 immediate early genes (IEGs)) and differential gene expression analysis between nuclei from uninjured and chronic neuropathic pain mice. These cell-subtypes all co-expressed Oprm1 with single genetic identifiers—Otof, Figf, Npr3—that we spatially resolved to label layer-specific ensembles in L2/3 IT, L5 ET, and L5 PT neurons, respectively. To gain genetic access to ACC nociceptive-Oprm1 ensembles, we developed an intersectional approach combining genetic elements of IEGs and Oprm1 with retrograde viral recombinases to drive expression of circuit mapping tools and optogenetic actuators with activity-, molecular-, and projection-dependency. We are beginning to mimic opioid analgesia with optical inhibition of ACC MOR+ nociceptive cell-types with an iC++ opsin, while capturing pain-related behaviors with a novel deep-learning system for unbiased pose-estimation of nocifensive behaviors. Our unpublished work will provide additional insight into the input connectivity from subcortical structures and the output projection map of the nociceptive ACC cell-types. Key among these is a possible reciprocal circuit between a subpopulation of medial basolateral amygdalar nociceptive cells and ACC L2 Otof+ cells. Identifying the local cortical and brain-wide structure of specific cell-type networks underlying opioid analgesia can aid the development of circuit targeted treatments with improved selectivity, safety, and lowered addiction liabilities. Grant support from 1DP2GM140923-01 - Harnessing cortical neuromodulation to disrupt pain perception Whitehall Foundation Award in Neurobiology - Integrative cortical circuits encoding attention to pain 1 F32 DA053099-01 (Nora McCall, Corder Lab postdoc) - Modulating pain through cortical endogenous opioid circuits.

Photoinduced bleaching in an efficient singlet oxygen photosensitizing protein: Identifying a culprit in the flavin-binding LOV-based protein SOPP3

Light-oxygen-voltage (LOV) proteins that bind flavin mononucleotide (FMN) have received attention as optogenetic systems that can photosensitize the production of singlet molecular oxygen, O2(a1Δg). Although the seminal compound in this family, mini singlet oxygen generator (miniSOG), does not make O2(a1Δg) in appreciable yield, it is a convenient standard against which other singlet oxygen photosensitizing proteins (SOPPs) can be judged. SOPP3 is a re-engineered version of miniSOG that selectively produces O2(a1Δg) in high yield but, when localized in a cell, the protein-encased FMN readily bleaches upon irradiation. In contrast, the bleaching of FMN in miniSOG is slow under the same conditions. The available evidence indicates that O2(a1Δg) is not directly involved in the bleaching reaction. Rather, electron-transfer reactions between FMN and molecules in the surrounding medium, likely mediated by residues in the LOV protein, accelerate FMN bleaching. In this regard, and identifying a culprit, replacing a glutamine that H-bonds to FMN in miniSOG with either leucine (Q103L) or valine (Q103V) increases the 3FMN lifetime and, thereby, increases both the O2(a1Δg) yield and the bleaching rate. Although the same electron transfer reactions likely influence these respective observations, the O2(a1Δg) yield principally reflects the kinetic competition between two processes that remove/deactivate 3FMN: (1) quenching by ground state oxygen, O2(X3Σg-), to produce O2(a1Δg), and (2) electron transfer to produce the FMN radical anion. Photobleaching principally reflects the accumulated electron-transfer-mediated depletion of ground state FMN, which, over time in the dark, is partly regenerated by compensating redox reactions. Despite the bleaching, irradiation of SOPP3 induces morphological change in a cell. This result, which contradicts conclusions from previous experiments with a lower light dose, indicates that SOPP3 may indeed be a viable optogenetic actuator.

Expression of Channelrhodopsin‐2 in Rod Bipolar Cells Restores ON and OFF Responses at High Spatial Resolution in Blind Mouse Retina

AbstractRod bipolar cells (rBCs) represent a promising target for vision restoration as they relay information to the major image processing pathways in the retina. However, the diversity of the retinal output and the resolution obtained by optogenetic actuation of rBCs is unclear. Herein, the photostimulation of rBCs expressing channelrhodopsin‐2 is studied in a transgenic, photoreceptor‐degeneration mouse (rd10) while simultaneously recording the retinal output using a high‐density microelectrode array. After analyzing several hundred retinal output neurons, both optogenetic ON and OFF type responses are identified at similar ratio at stimulation thresholds well below photodamage intensity. The temporal latency of both response types is in the same range ( 50 ms) as reported for healthy mouse retinal ganglion cells. The spatial resolution ranges between 0.1 and 0.2 cycles deg‐1, which is close to that found in healthy mice. Photostimulation over an extended area (29 deg) but not localized stimuli induces strong bursting activity, which may originate from large‐scale activation of a coupled network of retinal cells. The distinct ON and OFF optogenetically induced activity patterns in retinal ganglion cells and the high temporal and spatial resolution demonstrate that near‐physiological vision restoration may be achieved under optimal conditions in late stage retinal degeneration.

Power-effective scanning with AODs for 3D optogenetic applications.

Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks.