Subrata Batabyal

Broad spectral excitation of opsin for enhanced stimulation of cells

Optical stimulation of cells expressing light-sensitive proteins (opsins) has allowed targeted activation with cellular specificity. However, since narrow-band light has been used for excitation of these optogenetic probes, only active stimulation strategies are being attempted for clinical applications such as restoration of vision. Here, we report use of broad spectral excitation (white light) for optogenetic stimulation of opsin-sensitized cells. We found that ReaChR is optimally excited with white light offering significantly higher photocurrents compared to spectrally filtered narrow-band light stimulation. Our findings open up the possibility of passive stimulation strategy by use of natural sunlight for retinal stimulation, which could have benefits for ambient light stimulated vision restoration.

Restoring vision in mice with retinal degeneration using multicharacteristic opsin

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.

Ultrafast laser-assisted spatially targeted optoporation into cortical axons and retinal cells in the eye

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.

Label-free optical detection of action potential in mammalian neurons

We describe an optical technique for label-free detection of the action potential in cultured mammalian neurons. Induced morphological changes due to action potential propagation in neurons are optically interrogated with a phase sensitive interferometric technique. Optical recordings composed of signal pulses mirror the electrical spike train activity of individual neurons in a network. The optical pulses are transient nanoscale oscillatory changes in the optical path length of varying peak magnitude and temporal width. Exogenous application of glutamate to cortical neuronal cultures produced coincident increase in the electrical and optical activity; both were blocked by application of a Na-channel blocker, Tetrodotoxin. The observed transient change in optical path length in a single optical pulse is primarily due to physical fluctuations of the neuronal cell membrane mediated by a yet unknown electromechanical transduction phenomenon. Our analysis suggests a traveling surface wave in the neuronal cell membrane is responsible for the measured optical signal pulses.

Broadband activation by white-opsin lowers intensity threshold for cellular stimulation

Photoreceptors, which initiate the conversion of ambient light to action potentials via retinal circuitry, degenerate in retinal diseases such as retinitis pigmentosa and age related macular degeneration leading to loss of vision. Current prosthetic devices using arrays consisting of electrodes or LEDs (for optogenetic activation of conventional narrow-band opsins) have limited spatial resolution and can cause damage to retinal circuits by mechanical or photochemical (by absorption of intense narrow band light) means. Here, we describe a broad-band light activatable white-opsin for generating significant photocurrent at white light intensity levels close to ambient daylight conditions. White-opsin produced an order of magnitude higher photocurrent in response to white light as compared to narrow-band opsin channelrhodopsin-2, while maintaining the ms-channel kinetics. High fidelity of peak-photocurrent (both amplitude and latency) of white-opsin in response to repetitive white light stimulation of varying pulse width was observed. The significantly lower intensity stimulation required for activating white-opsin sensitized cells may facilitate ambient white light-based restoration of vision for patients with widespread photoreceptor degeneration.

Broad-band activatable white-opsin

Currently, the use of optogenetic sensitization of retinal cells combined with activation/inhibition has the potential to be an alternative to retinal implants that would require electrodes inside every single neuron for high visual resolution. However, clinical translation of optogenetic activation for restoration of vision suffers from the drawback that the narrow spectral sensitivity of an opsin requires active stimulation by a blue laser or a light emitting diode with much higher intensities than ambient light. In order to allow an ambient light-based stimulation paradigm, we report the development of a ‘white-opsin’ that has broad spectral excitability in the visible spectrum. The cells sensitized with white-opsin showed excitability at an order of magnitude higher with white light compared to using only narrow-band light components. Further, cells sensitized with white-opsin produced a photocurrent that was five times higher than Channelrhodopsin-2 under similar photo-excitation conditions. The use of fast white-opsin may allow opsin-sensitized neurons in a degenerated retina to exhibit a higher sensitivity to ambient white light. This property, therefore, significantly lowers the activation threshold in contrast to conventional approaches that use intense narrow-band opsins and light to activate cellular stimulation.

Optical stimulation and monitoring of the visual system using bioluminescent opsin (Conference Presentation)

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.

Targeted nano-enhanced Optical delivery of opsin for dry-AMD therapy (Conference Presentation)

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.

In-vivo label-free optical detection of neural activities in retina (Conference Presentation)

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.

Label-free optical detection of action potential in mammalian neurons (Conference Presentation)

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.