Degeneration of photoreceptors, retinal cells including retinal pigment epithelium, and retinal ganglion cells characterizes visual disorders involving retinal dysfunction. The efficient and targeted gene delivery into retinal cells is critical for treatment of a variety of visual disorders. Here, we report use of an OCT guided, spatially targeted, near-infrared laser microirradiation platform to successfully deliver genes encoding ambient-light activatable multi-characteristic opsin (MCO) to retinal cells in non-human primates. The efficacy of gene delivery was evaluated by fundus imaging of the reporter (mCherry) fluorescence, and immunohistochemistry. Functional improvement was measured by red-ERG which was attributed to MCO’s broadband activation spectrum.
Stargardt Disease is an inherited macular degeneration caused by mutation of genes, primarily ABCA4 in the photoreceptor cells leading to their dysfunction and degeneration in the macula. Here, we report results of ambient-light vision restoration in Stargardt mice (Abca4tm1Ght/J) upon intravitreal delivery of AAV-carried MCO (vMCO) as evaluated by electrophysiology, Optical Coherence Tomography (OCT) and visually guided behavior. Further, safety of MCO-enabled vision restoration therapy was evaluated by measurement of Intraocular Pressure, OCT and Immunohistochemistry. The vMCO based ambient light activatable optogenetic therapy has potential to be a disease-agnostic therapy to address the unmet need of inherited retinal degenerative diseases.
We combine the real-time structural imaging capability of OCT with laser microirradiation for causing layer-specific dystrophies to mimic Retinitis Pigmentosa and dry-AMD model. In addition, we integrated an ERG-module for functional characterization after laser-injury to monitor the disease progression as well as to evaluate therapeutic efficacy. Here, we show creation of local atrophies with our combined OCT-Laser-ERG system in animal models and measurement of different cone and rod responses to focal stimulation light of different wavelengths. By varying the different mode of laser microirradiation and focal adjustment to the targeted depth, we demonstrate layer specific primary RPE injury.
We hypothesized that PEDF gene transduction in retina can provide single-dose treatment to prevent ganglion cell damage. Here, we present OCT guided ultrafast laser based non-viral targeted delivery PEDF-encoding genes to retina for neuroprotection. The ultrafast laser gene delivery showed layer-specific reliable expression of PEDF gene in retina without any detectable damage. Monitoring of IOP and electroretinogram after ultrafast laser transfection showed no adverse changes. The ultrafast laser transfection of large PEDF genes in retina exhibited significant therapeutic benefit in an injury model. Absence of any immune response in retina subsequent to ultrafast-laser transfection provides unique opportunity for repeated dosing.
Herein, we report use of near-infrared low-coherent light for non-contact, label-free in-vivo detection of retinal activity in response to visual illumination. Our multifractal phase-OCT employ phase/multifractal analysis to decipher layer specific cellular activity during visual stimulation to assess the functional state of retina. Our OCT-based interferometric technique coupled with in-depth multifractal analysis differentiated retinal activities between wild-type and mice with retinal dystrophy. Our findings open up possibility of clinical translation of multifractal phase-OCT for non-contact label free evaluation of retina health, progression of retinal dystrophies, and as well as for monitoring functional recovery after therapy.
The ability to both induce and monitor neural activity at cellular resolution is necessary to comprehend the activation paradigm in sensory processing and for the development of effective neuromodulation treatments. Our lab has produced an actuator-sensor construct via the hybridization of an optogenetic actuator (bMCOII) and bioluminescent Ca2+ sensor (GeNL) which allows continuous monitoring of neural activity with high spatiotemporal resolution. Modeling revealed that the construct is bound to the cell membrane through 14 transmembrane helices with the Ca2+ -bioluminescence indicator domain 20-40 Å inside the cytoplasm (membrane localization confirmed by imaging). The construct can be used to stimulate neural activity with very low intensity (10 μW/mm2 ) light but unlike fluorescent methods, requires no excitation light. Activation of the opsin causes influx of Ca2+ by opening MCOII-channels via trans-cis isomerization of all-trans-retinal. When these ions bind the indicator domain there is an increase in bioluminescence intensity. We observed significant correlation between the magnitude and kinetics of induced electrical activities and Ca2+ -bioluminescence. Longterm (>14 hours) recording of evoked neural activity in the visual cortices of murine models allowed the quantification of the strength of sensory activation. Additionally, through Artificial Intelligence-based Neural Activation Parameters, the Ca2+ -bioluminescence signals were used to map network activity patterns. We also observed delayed, secondary Ca2+ - bioluminescence responses murine visual cortices. These may be astrocyte-mediated responses to direct optogenetic and indirect visual stimulation. Our technique will enable the development of a scalable, and modular interface system which can be expanded to monitor and modulate a variety of neurological activities.
The efficient and targeted delivery of genes and other impermeable therapeutic molecules into retinal cells is of immense importance for therapy of various visual disorders. Traditional methods for gene delivery require viral transfection, or use of physical and chemical methods which suffer from one or many drawbacks such as invasiveness, low efficiency, lack of spatially-targeted delivery, and can generally have deleterious effects such as unexpected inflammatory responses and immunological reactions. Further, for effective dry-age related macular degeneration (dry-AMD) therapy involving geographic atrophies of the retina, it requires to localize the delivery of the targeted opsin-encoding genes to specific retinal cells in atrophied-regions. Here, we report near-infrared laser based Nano-enhanced Optical Delivery (NOD) of opsin-encoding genes into retina of mouse models of retina degeneration in-vivo. In this method, the field enhancement by gold nanorods is utilized to transiently perforate retinal cell membrane to deliver exogenous molecules to cells in the targeted area of retina. SDOCT was used to monitor if there is any damage to retina and other ocular structures. The expression and functioning of opsin in targeted retina after in-vivo NOD in the mice models of retinal degeneration opens new vista for re-photosensitizing retinas with geographic atrophies in dry-AMD.
Though primary visual cortex is known to maintain its retinotopy in subjects with retinal degeneration despite prolonged visual loss, detailed knowledge of how optogenetic sensitization of higher order neurons manifests in restoration of visual cortical activity is currently lacking. Here, we report development and characterization of bioluminescent opsin for simultaneous optical modulation and imaging of retinal and cortical activities using spectrally separated activation and detection bands. This new bioluminescent technique does not require an additional phototoxic external excitation source (as used for fluorescence). We quantified changes in bioluminescence activities in visual cortex of mice upon visual stimulation of the retina. The observed increased neural activities were found to correlate with the visual stimulation patterns. This method will be useful for monitoring changes in visual cortical activities during progression and repair of retinal degenerative diseases. Further, with integration of stimulation source, we envision development of a modular and scalable interface system with the capability to serve a multiplicity of applications to modulate and monitor large-scale activity in the nervous system.
Monitoring of visual functioning of the retina is significant for characterizing retinal degenerative diseases. Electroretinogram is the current method for measuring the electrical responses of the retina to light. However, it requires placement of electrodes on cornea, leading to contact related uncomfortable feeling. Here, we report use of near-infrared low-coherent light for non-contact, label-free in-vivo detection of retinal activities in response to visual stimulation. We utilized phase sensitive optical coherence tomography for measuring fluctuations of light reflected from retina of wild type and retinal degenerated mice. With visual stimulation, fluctuations in optical path length difference were found to be higher than that without visual stimulation in wild type mice. However, no such changes observed in mice with photoreceptor degeneration. Our findings open up possibility for clinical use of this method for non-contact label free characterization of retinal functioning and identification of dystrophies.
Retinal degenerative diseases, such as retinitis pigmentosa (RP) and dry age-related macular degeneration, have led to loss of vision in millions of individuals. Currently, no surgical or medical treatment is available, although optogenetic therapies are in clinical development. We demonstrate vision restoration using multicharacteristics opsin (MCO1) in animal models with degenerated retina. MCO1 is reliably delivered to specific retinal cells via intravitreal injection of adeno-associated virus (vMCO1), leading to significant improvement in visually guided behavior conducted using a radial arm water maze. The time to reach the platform and the number of error arms decreased significantly after delivery of MCO1. Notably, the improvement in visually guided behavior was observed even at light intensity levels orders of magnitude lower than that required for channelrhodopsin-2 opsin. Viability of vMCO1-treated retina is not compromised by chronic light exposure. Safe virus-mediated MCO1 delivery has potential for effective gene therapy of diverse retinal degenerations in patients.
Visualization and assessment of the cellular structure and function require localized delivery of the molecules into specific cells in restricted spatial regions of the tissue and may necessitate subcellular delivery and localization. Earlier, we have shown ultrafast near-infrared laser beam-assisted optoporation of actin-staining molecules into cortical neurons with single-cell resolution and high efficiency. However, diffusion of optoporated molecules in soma degrades toward the growth cone, leading to difficulties in visualization of the actin network in the growth cone in cases of long axons. Here, we demonstrate optoporation of impermeable molecules to functional cortical neurons by precise laser subaxotomy near the growth cone, leading to visualization of the actin network in the growth cone. Further, we demonstrate patterned delivery of impermeable molecules into targeted retinal cells in the rat eye. The development of optoporation as a minimally invasive approach to reliably deliver exogenous molecules into targeted axons and soma of retinal neurons in vivo will enable enhanced visualization of the structure and function of the retina.
Electrophysiology techniques are the gold standard in neuroscience for studying functionality of a single neuron to a complex neuronal network. However, electrophysiology techniques are not flawless, they are invasive nature, procedures are cumbersome to implement with limited capability of being used as a high-throughput recording system. Also, long term studies of neuronal functionality with aid of electrophysiology is not feasible. Non-invasive stimulation and detection of neuronal electrical activity has been a long standing goal in neuroscience. Introduction of optogenetics has ushered in the era of non-invasive optical stimulation of neurons, which is revolutionizing neuroscience research. Optical detection of neuronal activity that is comparable to electro-physiology is still elusive. A number of optical techniques have been reported recording of neuronal electrical activity but none is capable of reliably measuring action potential spikes that is comparable to electro-physiology. Optical detection of action potential with voltage sensitive fluorescent reporters are potential alternatives to electrophysiology techniques. The heavily rely on secondary reporters, which are often toxic in nature with background fluorescence, with slow response and low SNR making them far from ideal. The detection of one shot (without averaging)-single action potential in a true label-free way has been elusive so far. In this report, we demonstrate the optical detection of single neuronal spike in a cultured mammalian neuronal network without using any exogenous labels. To the best of our knowledge, this is the first demonstration of label free optical detection of single action potentials in a mammalian neuronal network, which was achieved using a high-speed phase sensitive interferometer. We have carried out stimulation and inhibition of neuronal firing using Glutamate and Tetrodotoxin respectively to demonstrate the different outcome (stimulation and inhibition) revealed in optical signal. We hypothesize that the interrogating optical beam is modulated during neuronal firing by electro-motility driven membrane fluctuation in conjunction with electrical wave propagation in cellular system.
Detecting cellular activity in sub-millisecond timescale and micrometer resolution without using invasive means has been a long standing goal in the study of cellular networks. Here, we have employed phase sensitive low coherence interferometry for detecting optogenetically stimulated activity of cells. Nanoscale changes in optical path length (due to change in refractive index and changes in cell thickness) occur when cells are activated, which we aim to detect by phase sensitive low coherence interferometry. A low coherence interferometry and patch-clamp electrophysiology systems were integrated with an inverted fluorescence microscope. Blue laser beam was coupled to the electrophysiology-interferometric detection system for optogenetic stimulation. The phase-sensitive measurements were carried out on Channelrhodopsin-2 sensitized cells (identified by YFP fluorescence) as well as control cells in reflection mode for different intensities and exposures of optogenetic stimulation beam. This method offers good temporal and spatial resolution without using exogenous labeling. Results of studies on all optical stimulation and detection of cellular activity will be presented. Interpretation of the optical activity signals will be discussed in context with changes in cell physiology during stimulation. We will also discuss the potential sources of various artifacts in optical/electrical detection of cellular activity during optical stimulation.
Study of communication in cellular systems requires precise activation of targeted cell(s) in the network. In contrast to chemical, electrical, thermal, mechanical stimulation, optical stimulation is non-invasive and is better suited for stimulation of targeted cells. As compared to visible lasers, the near infrared (NIR) microsecond/nanosecond pulsed laser beams are being used as preferred stimulation tool as they provide higher penetration depth in tissues. Femotosecond (FS) laser beams in NIR are also being used for direct and indirect (i.e. via two-photon optogenetics) stimulation of cells. Here, we present a comparative evaluation of efficacy of NIR FS laser beam for direct (no optogenetic sensitization) and 2ph optogenetic stimulation of cells. Further, for the first time, we demonstrate the use of blue (~450 nm, obtained by second harmonic generation) FS laser beam for stimulation of cells with and without Channelrhodopisn-2 (ChR2) expression. Comparative analysis of photocurrent generated by blue FS laser beam and continuous wave blue light for optogenetics stimulation of ChR2 transfected HEK cells will be presented. The use of ultrafast laser micro-beam for focal, non-contact, and repeated stimulation of single cells in a cellular circuitry allowed us to study the communication between different cell types.
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