Nina Milosavljevic

Restoration of Vision with Ectopic Expression of Human Rod Opsin

Summary Many retinal dystrophies result in photoreceptor loss, but the inner retinal neurons can survive, making them potentially amenable to emerging optogenetic therapies. Here, we show that ectopically expressed human rod opsin, driven by either a non-selective or ON-bipolar cell-specific promoter, can function outside native photoreceptors and restore visual function in a mouse model of advanced retinal degeneration. Electrophysiological recordings from retinal explants and the visual thalamus revealed changes in firing (increases and decreases) induced by simple light pulses, luminance increases, and naturalistic movies in treated mice. These responses could be elicited at light intensities within the physiological range and substantially below those required by other optogenetic strategies. Mice with rod opsin expression driven by the ON-bipolar specific promoter displayed behavioral responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at levels of contrast and illuminance (≈50–100 lux) typical of natural indoor environments. These data reveal that virally mediated ectopic expression of human rod opsin can restore vision under natural viewing conditions and at moderate light intensities. Given the inherent advantages in employing a human protein, the simplicity of this intervention, and the quality of vision restored, we suggest that rod opsin merits consideration as an optogenetic actuator for treating patients with advanced retinal degeneration.

Chemogenetic Activation of Melanopsin Retinal Ganglion Cells Induces Signatures of Arousal and/or Anxiety in Mice

Summary Functional imaging and psychometric assessments indicate that bright light can enhance mood, attention, and cognitive performance in humans. Indirect evidence links these events to light detection by intrinsically photosensitive melanopsin-expressing retinal ganglion cells (mRGCs) [1, 2, 3, 4, 5, 6, 7, 8, 9]. However, there is currently no direct demonstration that mRGCs can have such an immediate effect on mood or behavioral state in any species. We addressed this deficit by using chemogenetics to selectively activate mRGCs, simulating the excitatory effects of bright light on this cell type in dark-housed mice. This specific manipulation evoked circadian phase resetting and pupil constriction (known consequences of mRGC activation). It also induced c-Fos (a marker of neuronal activation) in multiple nuclei in the hypothalamus (paraventricular, dorsomedial, and lateral hypothalamus), thalamus (paraventricular and centromedian thalamus), and limbic system (amygdala and nucleus accumbens). These regions influence numerous aspects of autonomic and neuroendocrine activity and are typically active during periods of wakefulness or arousal. By contrast, c-Fos was absent from the ventrolateral preoptic area (active during sleep). In standard behavioral tests (open field and elevated plus maze), mRGC activation induced behaviors commonly interpreted as anxiety like or as signs of increased alertness. Similar changes in behavior could be induced by bright light in wild-type and rodless and coneless mice, but not melanopsin knockout mice. These data demonstrate that mRGCs drive a light-dependent switch in behavioral motivation toward a more alert, risk-averse state. They also highlight the ability of this small fraction of retinal ganglion cells to realign activity in brain regions defining widespread aspects of physiology and behavior.

Melanopsin-driven increases in maintained activity enhance thalamic visual response reliability across a simulated dawn

Significance Irradiance-dependent (“luxotonic”) changes in baseline firing were first described in neurones of the early visual system decades ago. However, the origin and function (if any) of this visual response is still poorly understood. Here we address both questions by recording electrophysiological activity in mouse dorsal lateral geniculate nucleus over a simulated dawn. First, we show that in the photopic regime luxotonic activity becomes increasingly driven by inner-retinal melanopsin photoreceptors as irradiance increases. Then, that irradiance-dependent increases in activity apply not only to baseline firing but also to the amplitude of fast visual responses, producing increases in signal:noise across the simulated dawn, revealing a function for luxotonic activity and a new way in which inner retinal photoreceptors support conventional vision. Twice a day, at dawn and dusk, we experience gradual but very high amplitude changes in background light intensity (irradiance). Although we perceive the associated change in environmental brightness, the representation of such very slow alterations in irradiance by the early visual system has been little studied. Here, we addressed this deficit by recording electrophysiological activity in the mouse dorsal lateral geniculate nucleus under exposure to a simulated dawn. As irradiance increased we found a widespread enhancement in baseline firing that extended to units with ON as well as OFF responses to fast luminance increments. This change in baseline firing was equally apparent when the slow irradiance ramp appeared alone or when a variety of higher-frequency artificial or natural visual stimuli were superimposed upon it. Using a combination of conventional knockout, chemogenetic, and receptor-silent substitution manipulations, we continued to show that, over higher irradiances, this increase in firing originates with inner-retinal melanopsin photoreception. At the single-unit level, irradiance-dependent increases in baseline firing were strongly correlated with improvements in the amplitude of responses to higher-frequency visual stimuli. This in turn results in an up to threefold increase in single-trial reliability of fast visual responses. In this way, our data indicate that melanopsin drives a generalized increase in dorsal lateral geniculate nucleus excitability as dawn progresses that both conveys information about changing background light intensity and increases the signal:noise for fast visual responses.

Spatial receptive fields in the retina and dorsal lateral geniculate nucleus of mice lacking rods and cones

In advanced retinal degeneration loss of rods and cones leaves melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) as the only source of visual information. ipRGCs drive non-image-forming responses (e.g., circadian photoentrainment) under such conditions but, despite projecting to the primary visual thalamus [dorsal lateral geniculate nucleus (dLGN)], do not support form vision. We wished to determine what precludes ipRGCs supporting spatial discrimination after photoreceptor loss, using a mouse model (rd/rd cl) lacking rods and cones. Using multielectrode arrays, we found that both RGCs and neurons in the dLGN of this animal have clearly delineated spatial receptive fields. In the retina, they are typically symmetrical, lack inhibitory surrounds, and have diameters in the range of 10-30° of visual space. Receptive fields in the dLGN were larger (diameters typically 30-70°) but matched the retinotopic map of the mouse dLGN. Injections of a neuroanatomical tracer (cholera toxin β-subunit) into the dLGN confirmed that retinotopic order of ganglion cell projections to the dLGN and thalamic projections to the cortex is at least superficially intact in rd/rd cl mice. However, as previously reported for deafferented ipRGCs, onset and offset of light responses have long latencies in the rd/rd cl retina and dLGN. Accordingly, dLGN neurons failed to track dynamic changes in light intensity in this animal. Our data reveal that ipRGCs can convey spatial information in advanced retinal degeneration and identify their poor temporal fidelity as the major limitation in their ability to provide information about spatial patterns under natural viewing conditions.

Chemogenetic Activation of ipRGCs Drives Changes in Dark-Adapted (Scotopic) Electroretinogram.

Purpose The purpose of this study was to investigate the impact of activating melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) on dark-adapted (scotopic) electroretinograms (ERG). Methods We used mice (Opn4Cre/+) expressing cre recombinase in melanopsin-expressing cells for a targeted gene delivery of a chemogenetic Gq-coupled receptor, hM3Dq, to ipRGCs. Intraperitoneal injection of clozapine N-oxide (CNO) at 5 mg/kg was used for acute activation of hM3Dq and thus excitation of ipRGCs in darkness. Dark-adapted flash ERGs were recorded across a 9-fold range of irradiances from hM3Dq Opn4Cre/+ and control Opn4Cre/+ mice before and after intraperitoneal injection of CNO. A- and b-wave amplitudes and implicit times and oscillatory potentials (OPs) were analyzed. Paired-flash stimuli were used to isolate cone-driven responses. Results Clozapine N-oxide application suppressed a- and b-wave amplitudes of the dark-adapted ERG across the flash intensity range in hM3Dq Opn4Cre/+ mice compared to control mice. Examination of the normalized irradiance-response functions revealed a shift in b-wave but not a-wave sensitivity. No changes in a- and b-wave implicit times were detected. Total OP amplitudes were also reduced in hM3Dq Opn4Cre/+ mice compared to controls following CNO administration. The paired-flash method revealed reduction in both the first (rods and cones) and second (cones only) flash response. Conclusions Acute and selective activation of ipRGCs modulates the amplitude of both a- and b-waves of the scotopic ERG, indicating that the influence of this ganglion cell class on the retinal physiology extends to the photoreceptors as well as their downstream pathways.

Restoration of vision with ectopic expression of human rod opsin

Abstract Background Inherited retinal degenerations that lead to irreversible blindness due to progressive loss of rods and cones in the outer retina affect 1 in 2500 people worldwide. However, despite the substantial photoreceptor loss, the inner retinal neurons can survive, making them potentially amenable to emerging optogenetic therapies. The aim of this study was to determine whether it is possible to recreate vision in blind mice using ectopic expression of human rod opsin. Methods The rod opsin expressing adeno-associated virus serotype 2 vector, driven by either a non-selective or ON-bipolar cell-specific promoter, was injected intravitreally into adult rd1 mice, a mouse model of advanced retinal degeneration. Retinal function was assessed in vitro with multielectrode array recordings, and in vivo with electrophysiological recordings from the visual thalamus. Behavioural studies were developed to test the visual responses in treated mice under a range of light conditions. Findings Ectopically expressed human rod opsin, driven by either a non-selective or ON-bipolar cell-specific promoter, functioned outside native photoreceptors and restored visual function in rd1 mice. Electrophysiological recordings from retinal explants and the visual thalamus revealed changes in firing induced by simple light pulses, luminance increases, and naturalistic movies in treated mice. These responses could be elicited at light intensities within the physiological range and substantially below those required by other optogenetic strategies. Mice with rod opsin expression driven by the ON-bipolar specific promoter displayed behavioural responses to increases in luminance, flicker, coarse spatial patterns, and elements of a natural movie at natural levels of contrast and illuminance. Interpretation These data reveal that virally mediated ectopic expression of human rod opsin can restore vision under natural viewing conditions and at moderate light intensities. Given the inherent advantages of using a human protein, the simplicity of this intervention, and the quality of vision restored, we suggest that rod opsin merits consideration as an optogenetic actuator for treating patients with advanced retinal degeneration. Funding Medical Research Council (clinical research training fellowship to JCK), Medical Research Council Confidence in Concept Award, European Research Council.

Optogenetic interrogation reveals separable G-protein-dependent and -independent signalling linking G-protein-coupled receptors to the circadian oscillator

BackgroundEndogenous circadian oscillators distributed across the mammalian body are synchronised among themselves and with external time via a variety of signalling molecules, some of which interact with G-protein-coupled receptors (GPCRs). GPCRs can regulate cell physiology via pathways originating with heterotrimeric G-proteins or β-arrestins. We applied an optogenetic approach to determine the contribution of these two signalling modes on circadian phase.ResultsWe employed a photopigment (JellyOp) that activates Gαs signalling with better selectivity and higher sensitivity than available alternatives, and a point mutant of this pigment (F112A) biased towards β-arrestin signalling. When expressed in fibroblasts, both native JellyOp and the F112A arrestin-biased mutant drove light-dependent phase resetting in the circadian clock. Shifts induced by the two opsins differed in their circadian phase dependence and the degree to which they were associated with clock gene induction.ConclusionsOur data imply separable G-protein and arrestin inputs to the mammalian circadian clock and establish a pair of optogenetic tools suitable for manipulating Gαs- and β-arrestin-biased signalling in live cells.

Functional Characterization of Na + /H + Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Efficacy and Safety of Glycosidic Enzymes for Improved Gene Delivery to the Retina following Intravitreal Injection in Mice

Viral gene delivery is showing great promise for treating retinal disease. Although subretinal vector delivery has mainly been used to date, intravitreal delivery has potential advantages if low retinal transduction efficiency can be overcome. To this end, we investigated the effects of co-injection of glycosaminoglycan-degrading enzymes, singly or in combination, with AAV2 as a method of increasing retinal transduction. Experiments using healthy mice demonstrated that these enzymes enhance retinal transduction. We found that heparinase III produced the greatest individual effect, and this was enhanced further by combination with hyaluronan lyase. In addition, this optimized AAV2-enzyme combination led to a marked improvement in transduction in retinas with advanced retinal degeneration compared with AAV2 alone. Safety studies measuring retinal function by flash electroretinography indicated that retinal function was unaffected in the acute period and at least 12 months after enzyme treatment, whereas pupillometry confirmed that retinal ganglion cell activity was unaffected. Retinal morphology was not altered by the enzyme injection. Collectively these data confirm the efficacy and safety of this intravitreal approach in enhancing retinal transduction efficiency by AAV in rodents. Translating this method into other species, such as non-human primates, or for clinical applications will have challenges and require further studies.