Light-sensitive Ion Channels Research Articles

Combined-electrical optogenetic stimulation but not channelrhodopsin kinetics improves the fidelity of high rate stimulation in the auditory pathway in mice

Novel stimulation methods are needed to overcome the limitations of contemporary cochlear implants. Optogenetics is a technique that confers light sensitivity to neurons via the genetic introduction of light-sensitive ion channels. By controlling neural activity with light, auditory neurons can be activated with higher spatial precision. Understanding the behaviour of opsins at high stimulation rates is an important step towards their translation. To elucidate this, we compared the temporal characteristics of auditory nerve and inferior colliculus responses to optogenetic, electrical, and combined optogenetic-electrical stimulation in virally transduced mice expressing one of two channelrhodopsins, ChR2-H134R or ChIEF, at stimulation rates up to 400 pulses per second (pps). At 100 pps, optogenetic responses in ChIEF mice demonstrated higher fidelity, less change in latency, and greater response stability compared to responses in ChR2-H134R mice, but not at higher rates. Combined stimulation improved the response characteristics in both cohorts at 400 pps, although there was no consistent facilitation of electrical responses. Despite these results, day-long stimulation (up to 13 h) led to severe and non-recoverable deterioration of the optogenetic responses. The results of this study have significant implications for the translation of optogenetic-only and combined stimulation techniques for hearing loss.

Effect of Optogenetic Stimulation Parameters on Firing Rate of Basal Ganglia Neurons in Parkinsonian State BG and RT Networks

Purpose: Parkinson's disease is a neurodegenerative disorder that affects the basal ganglia of the brain, which plays an important role in movement. Basal Ganglia-Thalamic network model including Subthalamic nucleus, Globus Pallidus externa, Globus Pallidus interna and Thalamus neurons. Optogenetics is a combination of optical and genetic tools used to stimulate basal ganglia neurons by light-sensitive ion channels (opsins) to eliminate the pathological effects of Parkinson's disease. Materials and Methods: To analyze the effect of optogenetic stimulation on Parkinsonian nervous systems, two complete models of BG and RT (including STN, GPe, Gpi, and TH neurons) have been selected and developed for Parkinson’s disease and to apply three and four-state optogenetic stimulation. For this purpose, ChETA, ChRwt, and NpHR opsins have been selected in three-state and four-state stimulations and different stimulation conditions according to different parameters in both models have been investigated. Results: To evaluate the performance of two models for each gene in three- and four-state stimulation conditions with different values of basic parameters, the value of error index is calculated and stimulation conditions that created an error index equal to zero have been introduced as optimal conditions. Based on the results, frequencies of 20 and 200 Hz in the four-state ChRwt model and frequency of 80 Hz in the three-state ChETA model have been introduced as optimal genes, frequencies, and models. To verify the developed model, the obtained results have been compared with the results of experimental studies. Conclusion: In optimal conditions, STN provides excitatory input and GPe provide appropriate inhibitory input to GPi, and GPi can provide appropriate inhibitory input to TH, and as a result, its function improves and pathological effects of Parkinson's disease disappear. The response of GPe neurons is consistent with the experimental results and the response of other neurons is also similar to the response of GPe neurons.

AAV8 vector induced gliosis following neuronal transgene expression.

Expression of light sensitive ion channels by selected neurons has been achieved by viral mediated transduction with gene constructs, but for this to have therapeutic uses, for instance in treating epilepsy, any adverse effects of viral infection on the cerebral cortex needs to be evaluated. Here, we assessed the impact of adeno-associated virus 8 (AAV8) carrying DNA code for a soma targeting light activated chloride channel/FusionRed (FR) construct under the CKIIa promoter. Viral constructs were harvested from transfected HEK293 cells in vitro and purified. To test functionality of the opsin, cultured rodent neurons were transduced and the light response of transduced neurons was assayed using whole-cell patch-clamp recordings. In vivo expression was confirmed by immunofluorescence for FR. Unilateral intracranial injections of the viral construct were made into the mouse neocortex and non-invasive fluorescence imaging of FR expression made over 1-4 weeks post-injection using an IVIS Spectrum system. Sections were also prepared from injected mouse cortex for immunofluorescence staining of FR, alongside glial and neuronal marker proteins. In vitro, cortical neurons were successfully transduced, showing appropriate physiological responses to light stimulation. Following injections in vivo, transduction was progressively established around a focal injection site over a 4-week period with spread of transduction proportional to the concentration of virus introduced. Elevated GFAP immunoreactivity, a marker for reactive astrocytes, was detected near injection sites associated with, and proportional to, local FR expression. Similarly, we observed reactive microglia around FR expressing cells. However, we found that the numbers of NeuN+ neurons were conserved close to the injection site, indicating that there was little or no neuronal loss. In control mice, injected with saline only, astrocytosis and microgliosis was limited to the immediate vicinity of the injection site. Injections of opsin negative viral constructs resulted in comparable levels of astrocytic reaction as seen with opsin positive constructs. We conclude that introduction of an AAV8 vector transducing expression of a transgene under a neuron specific promotor evokes a mild inflammatory reaction in cortical tissue without causing extensive short-term neuronal loss. The expression of an opsin in addition to a fluorescent protein does not significantly increase neuroinflammation.

Biohybrid Photonic Platform for Subcellular Stimulation and Readout of In Vitro Neurons.

Targeted manipulation of neural activity via light has become an indispensable tool for gaining insights into the intricate processes governing single neurons and complex neural networks. To shed light onto the underlying interaction mechanisms, it is crucial to achieve precise control of individual neural activity, as well as a spatial read-out resolution on the nanoscale. Here, a versatile photonic platform with subcellular resolution for stimulation and monitoring of in-vitro neurons is demonstrated. Low-loss photonic waveguides are fabricated on glass substrates using nanoimprint lithography and featuring a loss of only -0.9±0.2dBcm-1 at 489nm and are combined with optical fiber-based waveguide-access and backside total internal reflection fluorescence microscopy. Neurons are grown on the bio-functionalized photonic chip surface and, expressing the light-sensitive ion channel Channelrhodopsin-2, are stimulated within the evanescent field penetration depth of 57nm of the biocompatible waveguides. The versatility and cost-efficiency of the platform, along with the possible subcellular resolution, enable tailor-made investigations of neural interaction dynamics with defined spatial control and high throughput.

Recent Advances in Neurotechnologies to Understand and Direct Specific Behavior of Live Animals

Neuroscience has traditionally faced difficulty in accessing and understanding the behavior of the brain at a single-cell level. Many of these challenges are due to the brain’s complexity; the brain has 100 billion neurons and well over 100 trillion connections and synapses, all operating at a millisecond timescale with functionally distinct circuits crossing over each other within microscale regions. However, starting with breakthroughs in the 2000s, such as fluorescent genetically-encoded calcium ion (Ca2+) indicators (GECIs)—where Ca2+ detection is used as a proxy of neural activity—and optogenetics—where neuron firing is controlled using light-sensitive ion channels in neurons—new advances in revolutionary neurotechnology have made it increasingly feasible for neuroscientists to understand how the brain operates on a cellular level. In the past few years, four distinct categories of neurotechnology advances have catalyzed this change in understanding, including chemogenic control, electrical recording and control, optogenetics-based control, and optogenetics recording and visualization.

1533-P: Mind over Metabolism—A Molecular Subtype of Vagal Neurons That Can Improve Glucose Tolerance

Parasympathetic neurons of the dorsal motor nucleus of the vagus (DMV) control glucose metabolism through the vagus nerve and so are potential targets for diabetes treatment. Currently, little is known about the molecular identity or organization of the glucose-regulating DMV neurons. To identify these neurons for further study, we optogenetically activated them in awake mice during a glucose tolerance test (GTT). We transgenically targeted a blue light-sensitive ion channel, CaTCh, selectively to all DMV neurons based on co-expression of Chat - and Phox2b- driven recombinases (Chat-Cre::Phox2b-Flp::CaTCh mice). We implanted optical fibers over the DMV in these adult mice (2 females, 1 male), acclimated them to the test environment and procedure, and then fasted them overnight. After recording baseline glucose levels, we administered intraperitoneal glucose (20% glucose, 10uL/g) and concurrently began photostimulation (time=0-30min; 5Hz, 1ms pulse, 2.5s off, 1s on) or sham stimulation. We performed tail blood glucometry at 5-min increments from -20-120min and compared average readings between stimulation and sham trials. Results: sham 0-30min 472.6 ± 74mg/dL, stim 0-30min 352 ± 48mg/dL (p=0.0005, paired t-test); other timepoints, p>0.05 between conditions. Our results indicate that activating DMV neurons can significantly improve glucose tolerance. To identify specific DMV neurons controlling glucose tolerance, we optogenetically activated a molecular subtype of DMV neurons defined by co-expression of Calb2 and Chat (2 female Calb2-Cre::Chat-Flp::CaTCh adult mice). We photostimulated Calb2+ DMV neurons while performing GTT as described above. Results: sham 0-30min 493 ± 90mg/dL, stim 0-30min 407 ± 69mg/dL (p=0.0390, paired t-test); other timepoints, p>0.05 between conditions. Together our data indicates that activating DMV neurons generally or Calb2+ DMV neurons specifically improves glucose tolerance. Disclosure N.J.Conley: None. L.S.Kauffman: None. J.Campbell: None. Funding American Diabetes Association (1-18-INI-14 to J.C.)

Optogenetic Control of Muscles: Potential Uses and Limitations.

Optogenetics is a technique where a cell is transduced with a light-sensitive ion channel. This technique can be used to control muscle cell contraction in conjunction with commonly used viral vectors. However, this technique has not yet become widely applied. In this study, we discuss the mechanisms and techniques involved in opsin transfer to muscle tissue, the clinical applicability of these approaches, and the major limitations facing this technique.

Evaluation of Non-invasive Optogenetic Stimulation with Transcranial Functional Ultrasound Imaging.

Optogenetics employs engineered viruses to genetically modify cells to express specific light-sensitive ion channels. The standard method for gene delivery in the brain involves invasive craniotomies that expose the brain and direct injections of viruses that invariably damage neural tissue along the syringe tract. A recently proposed alternative in which non-invasive optogenetics is performed with focused ultrasound (FUS)-mediated blood-brain barrier (BBB) openings has been found to non-invasively facilitate gene delivery for optogenetics in mice. Although gene delivery can be performed non-invasively, validating successful viral transduction and expression of encoded ion channels in target tissue typically involves similar invasive techniques, such as craniotomies in longitudinal studies and/or postmortem histology. Functional ultrasound imaging (fUSi) is an emerging neuroimaging technique that can be used to transcranially detect changes in cerebral blood volume following introduction of a stimulus. In this study, we implemented a fully non-invasive combined FUS-fUSi technique for performing optogenetics in mice. FUS successfully delivered viruses encoding the red-shifted channelrhodopsin variant ChrimsonR in all treated subjects. fUSi successfully identified stimulus-evoked cerebral blood volume changes preferentially in brain regions expressing the light-sensitive ion channels. Improvements in cell-specific targeting of viral vectors and transcranial ultrasound imaging will make the combined technique a useful tool for neuroscience research in small animals.

Role of opsins and light or heat activated transient receptor potential ion channels in the mechanisms of photobiomodulation and infrared therapy

Photobiomodulation (otherwise known as low level light therapy) is an emerging approach for treating many diseases and conditions such as pain, inflammation, wound healing, brain disorders, hair regrowth etc. The light used in this therapy generally lies in the red and near-infrared spectral regions. Despite many positive studies for treating different conditions, this therapy still faces some skepticism, which has prevented its widespread adoption in clinics. The main reasons behind this skepticism are the lack of comprehensive information about the molecular, cellular, and tissular mechanisms of action, which underpin the positive effects of photobiomodulation. Moreover, there is also another therapeutic application using longer wavelength infrared radiation, involving either infrared saunas or heat lamps which are powered by electricity, as well as infrared emitting textiles and garments which are solely powered by the wearer's own body heat. In recent years, much knowledge has been gained about the mechanism of action underlying these treatments, which will be summarized in this review. There are three broad classes of primary chromophores, which have so far been identified. One is mitochondrial cytochromes (including cytochrome c oxidase), another is opsins and light or heat-sensitive calcium ion channels, and a third is nanostructured water clusters. Light sensitive ion channels are activated by the absorption of light by the chromophore proteins, opsin-3 and opsin-4, while mitochondrial chromophores are activated by red or near-infra red (NIR) light up to about 850 nm. However NIR light at 980 nm or longer wavelengths can activate transient receptor potential (TRP) ion channels, probably after being absorbed by nanostructured water clusters. Heat-activated TRP channels undergo a conformational change triggered by only small temperature changes. Here we will discuss the role of opsins and light or heat activated TRP channels in the mechanism of photobiomodulation and infrared therapy.

A nanoscale inorganic coating strategy for stabilizing hydrogel neural probes in vivo.

Hydrogels with adaptable optical and mechanical characteristics show considerable promise for light delivery in vivo with neuroengineering applications. However, the unlinked amorphous polymer chains within hydrogels can cause volumetric swelling after water absorption under physiological conditions over time. Chemically cross-linked poly(vinyl alcohol) (PVA) hydrogels showcase fatigue-resistant attributes and promising biocompatibility for the manufacture of soft neural probes. However, possible swelling of the PVA hydrogel matrix could impact the structural stability of hydrogel-based bioelectronics and their long-term in vivo functionality. In this study, we utilized an atomic layer deposition (ALD) technique to generate an inorganic, silicon dioxide (SiO2) coating layer on chemically cross-linked PVA hydrogel fibers. To evaluate the stability of SiO2-coated PVA hydrogel fibers mimicking the in vivo environment, we conducted accelerated stability tests. SiO2-coated PVA hydrogel fibers showed improved stability over a one-week incubation period under a harsh environment, preventing swelling and preserving their mechanical and optical properties compared to uncoated fibers. These SiO2-coated PVA hydrogel fibers demonstrated nanoscale polymeric crystalline domains (6.5 ± 0.1 nm), an elastic modulus of 73.7 ± 31.7 MPa, a maximum elongation of 113.6 ± 24.2%, and minimal light transmission loss (1.9 ± 0.2 dB cm-1). Lastly, we applied these SiO2-coated PVA hydrogel fibers in vivo to optically activate the motor cortex of transgenic Thy1::ChR2 mice during locomotor behavioral tests. This mouse cohort was genetically modified to express the light-sensitive ion channel, channelrhodopsin-2 (ChR2), and implanted with hydrogel fibers to deliver light to the motor cortex area (M2). Light stimulation via hydrogel fibers resulted in optogenetically modulated mouse locomotor behaviors, including increased contralateral rotation, mobility speeds, and travel distances.

1315-P: Optogenetic Activation of Vagal Motor Neurons in Mice Modulates Heart Rate but Not Blood Glucose Levels

Parasympathetic neurons in the dorsal motor nucleus of the vagus (DMV) control glucose metabolism through the vagus nerve and so are potential targets for diabetes treatment. Yet, little is known about the molecular identity and organization of glucose-regulating DMV neurons. To identify these neurons for further investigation, we optogenetically activated them in mice while measuring blood glucose. We targeted a light-sensitive ion channel, CaTCh, selectively to DMV neurons based on their co-expression of Chat and Phox2b, which resulted in CaTCh expression in most or all DMV neurons but no surrounding neurons. To photostimulate CaTCh+ DMV neurons, we then implanted optical fibers over the DMV in 9 adult Chat::Phox2b::CaTCh mice (4 females, 5 males; mean age ± S.D., 24 ± 13 weeks) . After acclimation we fasted the mice overnight and then delivered pulses of blue laser light (20Hz, 10ms pulse, 3 sec off, 1 sec on) for 15 min to activate DMV neurons. We used a glucometer on tail blood samples drawn every 5 minutes for 1 hour, beginning 20 minutes before and ending 25 minutes after photostimulation. Before, during, and after photostimulation, blood glucose averaged 2± 35 mg/dL, 220 ± 36 mg/dL, and 215 ± 45 mg/dL and did not change significantly with photostimulation (p>0.05, ANOVA) . To rule out stress as a confound, we repeated these studies after anesthetizing 4-hour fasted mice with isoflurane but still did not detect a significant effect of photostimulation on blood glucose. Interestingly, however, heart rate decreased significantly with photostimulation (pre, 5± 23 bpm; during, 429 ± 32 bpm; post, 517 ± 24 bpm; p<0.001, ANOVA) , indicating that the photostimulation parameters were sufficient to activate vagal neurons. Further work is underway to address whether the lack of effect of DMV poststimulation on blood glucose is due to antagonistic signaling (e.g., coincident release of insulin and glucagon) or represents a limitation of the method. Disclosure J.Campbell: None. N.J.Conley: None. L.S.Kauffman: None. Funding American Diabetes Association (1-18-INI-14) ;

Astrocyte-derived adenosine excites sleep-promoting neurons in the ventrolateral preoptic nucleus: Astrocyte-neuron interactions in the regulation of sleep.

Although ATP and/or adenosine derived from astrocytes are known to regulate sleep, the precise mechanisms underlying the somnogenic effects of ATP and adenosine remain unclear. We selectively expressed channelrhodopsin-2 (ChR2), a light-sensitive ion channel, in astrocytes within the ventrolateral preoptic nucleus (VLPO), which is an essential brain nucleus involved in sleep promotion. We then examined the effects of photostimulation of astrocytic ChR2 on neuronal excitability using whole-cell patch-clamp recordings in two functionally distinct types of VLPO neurons: sleep-promoting GABAergic projection neurons and non-sleep-promoting local GABAergic neurons. Optogenetic stimulation of VLPO astrocytes demonstrated opposite outcomes in the two types of VLPO neurons. It led to the inhibition of non-sleep-promoting neurons and excitation of sleep-promoting neurons. These responses were attenuated by blocking of either adenosine A1 receptors or tissue-nonspecific alkaline phosphatase (TNAP). In contrast, exogenous adenosine decreased the excitability of both VLPO neuron populations. Moreover, TNAP was expressed in galanin-negative VLPO neurons, but not in galanin-positive sleep-promoting projection neurons. Taken together, these results suggest that astrocyte-derived ATP is converted into adenosine by TNAP in non-sleep-promoting neurons. In turn, adenosine decreases the excitability of local GABAergic neurons, thereby increasing the excitability of sleep-promoting GABAergic projection neurons. We propose a novel mechanism involving astrocyte-neuron interactions in sleep regulation, wherein endogenous adenosine derived from astrocytes excites sleep-promoting VLPO neurons, and thus decreases neuronal excitability in arousal-related areas of the brain.

In Utero Electroporation for Manipulation of Specific Neuronal Populations.

The complexity of brain functions is supported by the heterogeneity of brain tissue and millisecond-scale information processing. Understanding how complex neural circuits control animal behavior requires the precise manipulation of specific neuronal subtypes at high spatiotemporal resolution. In utero electroporation, when combined with optogenetics, is a powerful method for precisely controlling the activity of specific neurons. Optogenetics allows for the control of cellular membrane potentials through light-sensitive ion channels artificially expressed in the plasma membrane of neurons. Here, we first review the basic mechanisms and characteristics of in utero electroporation. Then, we discuss recent applications of in utero electroporation combined with optogenetics to investigate the functions and characteristics of specific regions, layers, and cell types. These techniques will pave the way for further advances in understanding the complex neuronal and circuit mechanisms that underlie behavioral outputs.

Serotype-based evaluation of an optogenetic construct in rat cortical astrocytes

Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

The Effect of Optogenetically Activating Glia on Neuronal Function

Glia, or glial cells, are considered a vital component of the nervous system, serving as an electrical insulator and a protective barrier from the interstitial (extracellular) media. Certain glial cells (i.e., astrocytes, microglia, and oligodendrocytes) within the CNS have been shown to directly affect neural functions, but these properties are challenging to study due to the difficulty involved with selectively-activating specific glia. To overcome this hurdle, we selectively expressed light-sensitive ion channels (i.e., channel rhodopsin, ChR2-XXL) in glia of larvae and adult Drosophila melanogaster. Upon activation of ChR2, both adults and larvae showed a rapid contracture of body wall muscles with the animal remaining in contracture even after the light was turned off. During ChR2-XXL activation, electrophysiological recordings of evoked excitatory junction potentials within body wall muscles of the larvae confirmed a train of motor nerve activity. Additionally, when segmental nerves were transected from the CNS and exposed to light, there were no noted differences in quantal or evoked responses. This suggests that there is not enough expression of ChR2-XXL to influence the segmental axons to detect in our paradigm. Activation of the glia within the CNS is sufficient to excite the motor neurons.

Light-controllable recellularized ventricular and atrial tissues

Abstract Introduction Construction of three-dimensional ventricular and atrial patches using decellularized extracellular matrix could allow the creation of “organ-like” structures for disease modeling, drug testing and regenerative medicine applications. To engineer functional artificial anatomical hearts several obstacles need to be overcome including generation of chamber-specific cells, prevention of immune reaction and donor/host tissue electrical coupling. Purpose In order to tackle these challenges we aimed to combine human induced pluripotent stem cell (hiPSC) technology, developmental biology inspired differentiation system to generate chamber-specific cardiomyocytes, decellularization/recellularization processes, and optogenetics utilizing light-sensitive ion channels to generate light-controllable atrial/ventricular tissue engineered patches. Material and method Atrial and ventricular patches of adult rats were decellularized using 1% SDS, 3% triton-X. The decellularized scaffolds were then recellularized with hiPSC- derived cardiomyocytes (2x107 atrial or ventricular cells). Adenoviral transduction was used to express the light-sensitive cationic channel ChR2 in the tissue engineered constructs. Results and discussion Eight days after cell seeding, we observed the development of spontaneous contraction of the atrial/ventricular tissues. Immunostaining for atrial (Cx40) or ventricular (MLC-2V) markers, gene expression, action-potential morphology, and the response to chamber-specific pharmacology confirmed the atrial/ventricular specific identity of the patches. Histological examination confirmed the preservation of the macro/micro- atrial/ventricular anatomical features in the generated chamber-specific recellularized patches. Optical mapping, using an EM-CCD camera, was used to characterize the conduction and repolarization properties of the generated tissues. The engineered patches could be paced and their electrical activity controlled by either electrical or optogenetic stimulation. Finally, arrhythmogenic reentrant arrhythmias could be induced in the tissue models and terminated by using optogenetic stimulation (“optogenetic-defibrillation”). Conclusions Three-dimensional light-sensitive chamber-specific engineered heart patches could be generated that could be controlled and manipulated through electrical and light pacing. These tissue could be used for several pathophysiological, drug testing, disease modeling and regenerative medicine applications. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): H2020-ERC-COG

Advanced Cardiac Rhythm Management by Applying Optogenetic Multi-Site Photostimulation in Murine Hearts.

Ventricular tachyarrhythmias are a major cause of mortality and morbidity worldwide. Electrical defibrillation using high-energy electric shocks is currently the only treatment for life-threatening ventricular fibrillation. However, defibrillation may have side-effects, including intolerable pain, tissue damage, and worsening of prognosis, indicating a significant medical need for the development of more gentle cardiac rhythm management strategies. Besides energy-reducing electrical approaches, cardiac optogenetics was introduced as a powerful tool to influence cardiac activity using light-sensitive membrane ion channels and light pulses. In the present study, a robust and valid method for successful photostimulation of Langendorff perfused intact murine hearts will be described based on multi-site pacing applying a 3 x 3 array of micro light-emitting diodes (micro-LED). Simultaneous optical mapping of epicardial membrane voltage waves allows the investigation of the effects of region-specific stimulation and evaluates the newly induced cardiac activity directly on-site. The obtained results show that the efficacy of defibrillation is strongly dependent on the parameters chosen for photostimulation during a cardiac arrhythmia. It will be demonstrated that the illuminated area of the heart plays a crucial role for termination success as well as how the targeted control of cardiac activity during illumination for modifying arrhythmia patterns can be achieved. In summary, this technique provides a possibility to optimize the on-site mechanism manipulation on the way to real-time feedback control of cardiac rhythm and, regarding the region specificity, new approaches in reducing the potential harm to the cardiac system compared to the usage of non-specific electrical shock applications.

An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans

BackgroundOptogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal, and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular, and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm.ResultsThe OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles, and light color. We demonstrate OptoArm’s power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit ageing.ConclusionWe have assembled an economical, reliable, and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research community.

Superpulsed 904 nm laser photobiomodulation combined with coenzyme Q10 synergistically augment burn wound healing

The management of multifaceted burn wound imparts a huge health care burden. Innumerable complexities associated with non-healing burn wounds led to the search for new therapeutic healing modalities. Earlier, our studies have shown that photobiomodulation (PBM) with superpulsed 904 nm laser increased cellular proliferation, bioenergetics activation, reduced inflammation and nitroxidative stress during burn wound healing. Coenzyme Q10 (CoQ10), a mitochondrial electron carrier acts as a potent antioxidant, maintains redox balance and mitochondrial dysfunction. Multi-target therapy aims to accelerate healing of intractable impaired wounds, which could exhibit a synergistic effect to potentially affecting different phases of the repair process. The present study investigates the efficacy of 904 nm superpulsed laser-mediated PBM (200 ns pulse-width, 100 Hz frequency, 0.24 J/cm2 total energy density, 0.40 mW/cm2 average power density and 19.77 W/cm2 peak power density for 10 min once daily for 7 days post-wounding) and CoQ10 (0.6%, w/v) topical treatments individually and in combination on full-thickness burn wound healing in rats and further explore the underlying mechanisms of action. The dual treatment showed significant (p < 0.05) synergism in enhancing pro-reparative healing markers: wound closure, mitogenesis (DNA, protein), angiogenesis (HIF-1α, VEGF), re-epithelialization, collagen deposition and bioenergetics activation (mitochondrial activity, cytochrome c oxidase and ATP), redox homeostasis (increased Nrf2, HO1, TXNRD2, catalase, SOD and decreased reactive oxygen species, lipid peroxidation levels) as compared to the standalone treatment and burn control groups. The PBM-CoQ10 combination treatment demonstrated significantly (p < 0.05) increased cellular proliferation (TGF-β2), light-sensitive ion channel regulation (TRPV3) and cytoprotection (HSP90, GRP78) during burn wound repair. Collectively, the combined therapy of PBM and CoQ10 is highly effective than either treatment alone in augmenting burn wound healing by efficiently attenuating oxidative stress and enhancing cellular proliferation, collagen deposition and bioenergetics activation. The study shows that combined treatment distinctly augments full-thickness burn repair in rats, which could pave the path for multi-target therapy approaches for non-healing impaired wounds in clinical care.

Near-infrared stimulation of the auditory nerve: A decade of progress toward an optical cochlear implant.

ObjectivesWe provide an appraisal of recent research on stimulation of the auditory system with light. In particular, we discuss direct infrared stimulation and ongoing controversies regarding the feasibility of this modality. We also discuss advancements and barriers to the development of an optical cochlear implant.MethodsThis is a review article that covers relevant animal studies.ResultsThe auditory system has been stimulated with infrared light, and in a much more spatially selective manner than with electrical stimulation. However, there are experiments from other labs that have not been able to reproduce these results. This has resulted in an ongoing controversy regarding the feasibility of infrared stimulation, and the reasons for these experimental differences still require explanation. The neural response characteristics also appear to be much different than with electrical stimulation. The electrical stimulation paradigms used for modern cochlear implants do not apply well to optical stimulation and new coding strategies are under development. Stimulation with infrared light brings the risk of heat accumulation in the tissue at high pulse repetition rates, so optimal pulse shapes and combined optical/electrical stimulation are being investigated to mitigate this. Optogenetics is another promising technique, which makes neurons more sensitive to light stimulation by inserting light sensitive ion channels via viral vectors. Challenges of optogenetics include the expression of light sensitive channels in sufficient density in the target neurons, and the risk of damaging neurons by the expression of a foreign protein.ConclusionOptical stimulation of the nervous system is a promising new field, and there has been progress toward the development of a cochlear implant that takes advantage of the benefits of optical stimulation. There are barriers, and controversies, but so far none that seem intractable.Level of evidenceNA (animal studies and basic research).