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The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and function in vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving putative synaptic structures and achieving three-dimensional cellular resolution in the mouse retina in vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions with sub-capillary details in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high-resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatment in vivo.

Magnetogenetics promises remote control of neurons, but its validity is questioned due to controversies surrounding the underlying mechanisms and deficits in reproducibility. Recent studies discovered that ferritin, used in Magnetogenetics, transduces radiofrequency (RF) magnetic fields into biochemical signals (reactive oxygen species and oxidized lipids). Magnetic stimulation of ferritin-tethered TRPV channels induces Ca2+responses and modulates behavior but electrophysiological studies indicate that a particular channel, Magneto2.0, is ineffective in affecting the neuronal bioelectrical properties. We investigated this problem using the Magnetogenetic technique FeRIC. We resolved the electromagnetic interference caused by RF in patch-clamp recordings and supported the data with voltage imaging experiments. In neurons expressing TRPV4FeRIC, RF depolarizes the membrane potential and increases the spiking frequency. In neurons expressing the chloride-permeable TMEM16AFeRIC, RF hyperpolarizes the membrane potential and decreases the spiking frequency. Our study reveals the control of neuronal bioelectrical properties with Magnetogenetics that is non-instantaneous, long-lasting, and moderate, but effective and comparable to that induced by endogenous signaling molecules.

The retina, behind the transparent optics of the eye, is the only neural tissue whose physiology and pathology can be non-invasively probed by optical microscopy. The aberrations intrinsic to the mouse eye, however, prevent high-resolution investigation of retinal structure and functionin vivo. Optimizing the design of a two-photon fluorescence microscope (2PFM) and sample preparation procedure, we found that adaptive optics (AO), by measuring and correcting ocular aberrations, is essential for resolving synapses and achieving three-dimensional cellular resolution in the mouse retinain vivo. Applying AO-2PFM to longitudinal retinal imaging in transgenic models of retinal pathology, we characterized microvascular lesions and observed microglial migration in a proliferative vascular retinopathy model, and found Lidocaine to effectively suppress retinal ganglion cell hyperactivity in a retinal degeneration model. Tracking structural and functional changes at high resolution longitudinally, AO-2PFM enables microscopic investigations of retinal pathology and pharmacology for disease diagnosis and treatmentin vivo.

Rod and cone photoreceptors degenerate in retinitis pigmentosa (RP). While downstream neurons survive, they undergo physiological changes, including accelerated spontaneous firing in retinal ganglion cells (RGCs). Retinoic acid (RA) is the molecular trigger of RGC hyperactivity, but whether this interferes with visual perception is unknown. Here, we show that inhibiting RA synthesis with disulfiram, a deterrent of human alcohol abuse, improves behavioral image detection in vision-impaired mice. In vivo Ca 2+ imaging shows that disulfiram sharpens orientation tuning of visual cortical neurons and strengthens fidelity of responses to natural scenes. An RA receptor inhibitor also reduces RGC hyperactivity, sharpens cortical representations, and improves image detection. These findings suggest that photoreceptor degeneration is not the only cause of vision loss in RP. RA-induced corruption of retinal information processing also degrades vision, pointing to RA synthesis and signaling inhibitors as potential therapeutic tools for improving sight in RP and other retinal degenerative disorders.

To better understand neural circuits and behavior, microbial opsins have been developed as optogenetic tools for stimulating or inhibiting action potentials with high temporal and spatial precision. However, if we seek a more reductionist understanding of how neuronal circuits operate, we also need high-resolution tools for perturbing the function of synapses. By combining photochemical tools and molecular biology, a wide variety of light-regulated neurotransmitter receptors have been developed, enabling photo-control of excitatory, inhibitory, and modulatory synaptic transmission. Here we focus on photo-control of GABA

Author(s): Adams, Dany Spencer; Djamgoz, Mustafa; Levin, Michael; Zhao, Min; Rajnicek, Ann; Nuccitelli, Richard; Arcangeli, Annarosa; Kramer, Richard; Bates, Emily Anne

In degenerative retinal disorders, rod and cone photoreceptors die, causing vision impairment and blindness. Downstream neurons survive but undergo morphological and physiological remodeling, with some retinal ganglion cells (RGC) exhibiting heightened spontaneous firing. Retinoic acid (RA) has been implicated as the key signaling molecule that induces RGC hyperactivity, obscuring RGC light responses and reducing light avoidance behaviors triggered by residual rods and cones. However, evidence that RA-dependent remodeling corrupts image-forming vision has been lacking. Here we show that disulfiram, an FDA-approved drug that inhibits RA synthesis, and BMS 493, an RA receptor (RAR) inhibitor, reduce RGC hyperactivity and augment image detection in visually impaired mice. Functional imaging of visual cortical neurons shows that disulfiram and BMS 493 sharpen orientation-tuning and strengthen response fidelity to naturalistic scenes. These findings establish a causal link between RA-induced retinal hyperactivity and vision impairment and define molecular targets and candidate drugs for boosting image-forming vision in retinal degeneration.

The development of fluorescent dyes that emit and absorb light at wavelengths greater than 700 nm and that respond to biochemical and biophysical events in living systems remains an outstanding challenge for noninvasive optical imaging. Here, we report the design, synthesis, and application of near-infrared (NIR)-absorbing and -emitting optical voltmeter based on a sulfonated, phosphine-oxide (po) rhodamine for voltage imaging in intact retinas. We find that po-rhodamine based voltage reporters, or poRhoVRs, display NIR excitation and emission profiles at greater than 700 nm, show a range of voltage sensitivities (13 to 43% ΔF/F per 100 mV in HEK cells), and can be combined with existing optical sensors, like Ca2+-sensitive fluorescent proteins (GCaMP), and actuators, like light-activated opsins ChannelRhodopsin-2 (ChR2). Simultaneous voltage and Ca2+ imaging reveals differences in activity dynamics in rat hippocampal neurons, and pairing poRhoVR with blue-light based ChR2 affords all-optical electrophysiology. In ex vivo retinas isolated from a mouse model of retinal degeneration, poRhoVR, together with GCaMP-based Ca2+ imaging and traditional multielectrode array (MEA) recording, can provide a comprehensive physiological activity profile of neuronal activity, revealing differences in voltage and Ca2+ dynamics within hyperactive networks of the mouse retina. Taken together, these experiments establish that poRhoVR will open new horizons in optical interrogation of cellular and neuronal physiology in intact systems.

Vision impairment and blindness in humans are most frequently caused by the degeneration and loss of photoreceptor cells in the outer retina, as is the case for age-related macular degeneration, retinitis pigmentosa, retinal detachment and many other diseases. While inner retinal neurons survive degeneration, they undergo fundamental pathophysiological changes, collectively known as “remodeling.” Inner retinal remodeling downstream to photoreceptor death occurs across mammalian retinas from mice to humans, independently of the cause of degeneration. It results in pervasive spontaneous hyperactivity and membrane hyperpermeability in retinal ganglion cells, which funnel all retinal signals to the brain. Remodeling reduces light detection in vision-impaired patients and precludes meaningful vision restoration in blind individuals. In this review, we summarize current hypotheses proposed to explain remodeling and their potential medical significance highlighting the important role played by retinoic acid and its receptor.

Horizontal cells (HCs) form reciprocal synapses with rod and cone photoreceptors, an arrangement that underlies lateral inhibition in the retina. HCs send negative and positive feedback signals to photoreceptors, but how HCs initiate these signals remains unclear. Unfortunately, because HCs have no unique neurotransmitter receptors, there are no pharmacological treatments for perturbing membrane potential specifically in HCs. Here we use transgenic zebrafish whose HCs express alien receptors, enabling cell-type-specific control by cognate alien agonists. To depolarize HCs, we used the Phe-Met-Arg-Phe-amide (FMRFamide)-gated Na+channel (FaNaC) activated by the invertebrate neuropeptide FMRFamide. To hyperpolarize HCs we used a pharmacologically selective actuator module (PSAM)-glycine receptor (GlyR), an engineered Cl–selective channel activated by a synthetic agonist. Expression of FaNaC or PSAM-GlyR was restricted to HCs with the cell-type selective promoter for connexin-55.5. We assessed HC-feedback control of photoreceptor synapses in three ways. First, we measured presynaptic exocytosis from photoreceptor terminals using the fluorescent dye FM1-43. Second, we measured the electroretinogram (ERG) b-wave, a signal generated by postsynaptic responses. Third, we used Ca2+imaging in retinal ganglion cells (RGCs) expressing the Ca2+indicator GCaMP6. Addition of FMRFamide significantly decreased FM1-43 destaining in darkness, whereas the addition of PSAM-GlyR significantly increased it. However, both agonists decreased the light-elicited ERG b-wave and eliminated surround inhibition of the Ca2+response of RGCs. Taken together, our findings show that chemogenetic tools can selectively manipulate negative feedback from HCs, providing a platform for understanding its mechanism and helping to elucidate its functional roles in visual information processing at a succession of downstream stages.

Parvalbumin-expressing interneurons (PVs) in the dentate gyrus provide activity-dependent regulation of adult neurogenesis as well as maintain inhibitory control of mature neurons. In mature neurons, PVs evoke GABAA postsynaptic currents (GPSCs) with fast rise and decay phases that allow precise control of spike timing, yet synaptic currents with fast kinetics do not appear in adult-born neurons until several weeks after cell birth. Here we used mouse hippocampal slices to address how PVs signal to newborn neurons prior to the appearance of fast GPSCs. Whereas PV-evoked currents in mature neurons exhibit hallmark fast rise and decay phases, newborn neurons display slow GPSCs with characteristics of spillover signaling. We also unmasked slow spillover currents in mature neurons in the absence of fast GPSCs. Our results suggest that PVs mediate slow spillover signaling in addition to conventional fast synaptic signaling, and that spillover transmission mediates activity-dependent regulation of early events in adult neurogenesis.

The excitatory synapse between hippocampal CA3 and CA1 pyramidal neurons exhibits long-term potentiation (LTP), a positive feedback process implicated in learning and memory in which postsynaptic depolarization strengthens synapses, promoting further depolarization. Without mechanisms for interrupting positive feedback, excitatory synapses could strengthen inexorably, corrupting memory storage. Here, we reveal a hidden form of inhibitory synaptic plasticity that prevents accumulation of excitatory LTP. We developed a knockin mouse that allows optical control of endogenous α5-subunit-containing γ-aminobutyric acid (GABA)

Degenerative retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) affect millions of people around the world and lead to irreversible vision loss if left untreated. A number of therapeutic strategies have been developed over the years to treat these diseases or restore vision to already blind patients. In this Review, we describe the development and translational application of light-sensitive chemical photoswitches to restore visual function to the blind retina and compare the translational potential of photoswitches with other vision-restoring therapies. This therapeutic strategy is enabled by an efficient fusion of chemical synthesis, chemical biology, and molecular biology and is broadly applicable to other biological systems. We hope this Review will be of interest to chemists as well as neuroscientists and clinicians.

A discrete De Rham complex enables compatible, structure-preserving discretizations for a broad range of partial differential equations problems. Such discretizations can correctly reproduce the physics of interface problems, provided the grid conforms to the interface. However, large deformations, complex geometries, and evolving interfaces makes generation of such grids difficult. We develop and demonstrate two formally equivalent approaches that, for a given background mesh, dynamically construct an interface-conforming discrete De Rham complex. Both approaches start by dividing cut elements into interface-conforming subelements but differ in how they build the finite element basis on these subelements. The first approach discards the existing non-conforming basis of the parent element and replaces it by a dynamic set of degrees of freedom of the same kind. The second approach defines the interface-conforming degrees of freedom on the subelements as superpositions of the basis functions of the parent element. These approaches generalize the Conformal Decomposition Finite Element Method (CDFEM) and the extended finite element method with algebraic constraints (XFEM-AC), respectively, across the De Rham complex.

BACKGROUND AND PURPOSE The photo-isomerizable local anaesthetic, quaternary ammonium-azobenzene-quaternary ammonium (QAQ), provides rapid, optical control over pain signalling without involving genetic modification. In darkness or in green light, trans-QAQ blocks voltage-gated K+ and Na+ channels and silences action potentials in pain-sensing neurons. Upon photo-isomerization to cis with near UV light, QAQ blockade is rapidly relieved, restoring neuronal activity. However, the molecular mechanism of cis and trans QAQ blockade is not known. Moreover, the absorption spectrum of QAQ requires UV light for photo-control, precluding use deep inside neural tissue. EXPERIMENTAL APPROACH Electrophysiology and molecular modelling were used to characterize the binding of cis and trans QAQ to voltage-gated K+ channels and to develop quaternary ammonium-ethylamine-azobenzene-quaternary ammonium (QENAQ), a red-shifted QAQ derivative controlled with visible light. KEY RESULTS trans QAQ was sixfold more potent than cis QAQ, in blocking current through Shaker K+ channels. Both isomers were use-dependent, open channel blockers, binding from the cytoplasmic side, but only trans QAQ block was slightly voltage dependent. QENAQ also blocked native K+ and Na+ channels preferentially in the trans state. QENAQ was photo-isomerized to cis with blue light and spontaneously reverted to trans within seconds in darkness, enabling rapid photo-control of action potentials in sensory neurons. CONCLUSIONS AND IMPLICATIONS Light-switchable local anaesthetics provide a means to non-invasively photo-control pain signalling with high selectivity and fast kinetics. Understanding the mode of action of QAQ and related compounds will help to design of drugs with improved photo-pharmacological properties. LINKED ARTICLES This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Photopharmacological control of neuronal activity using synthetic photochromic ligands, or photoswitches, is a promising approach for restoring visual function in patients suffering from degenerative retinal diseases. Azobenzene photoswitches, such as AAQ and DENAQ, have been shown to restore the responses of retinal ganglion cells to light in mouse models of retinal degeneration but do not recapitulate native retinal signal processing. Here, we describe diethylamino-azo-diethylamino (DAD), a third-generation photoswitch that is capable of restoring retinal ganglion cell light responses to blue or white light. In acute brain slices of murine layer 2/3 cortical neurons, we determined that the photoswitch quickly relaxes to its inactive form in the dark. DAD is not permanently charged, and the uncharged form enables the photoswitch to rapidly and effectively cross biological barriers and thereby access and photosensitize retinal neurons. Intravitreal injection of DAD restored retinal light responses and light-driven behavior to blind mice. Unlike DENAQ, DAD acts upstream of retinal ganglion cells, primarily conferring light sensitivity to bipolar cells. Moreover, DAD was capable of generating ON and OFF visual responses in the blind retina by utilizing intrinsic retinal circuitry, which may be advantageous for restoring visual function.

Vision loss in age-related macular degeneration (AMD) stems from disruption of photoreceptor cells in the macula, the central retinal area required for high-acuity vision. Mice and rats have no macula, but surgical insertion of a subretinal implant can induce localized photoreceptor degeneration due to chronic separation from retinal pigment epithelium, simulating a key aspect of AMD. We find that the implant-induced loss of photoreceptors in rat retina leads to local changes in the physiology of downstream retinal ganglion cells (RGCs), similar to changes in RGCs of rodent models of retinitis pigmentosa (RP), an inherited disease causing retina-wide photoreceptor degeneration. The local implant-induced changes in RGCs include enhanced intrinsic excitability leading to accelerated spontaneous firing, increased membrane permeability to fluorescent dyes, and enhanced photosensitization by azobenzene photoswitches. The local physiological changes are correlated with an increase in retinoic acid receptor-induced (RAR-induced) gene transcription, the key process underlying retinal remodeling in mouse models of RP. Hence the loss of photoreceptors, whether by local physical perturbation or by inherited mutation, leads to a stereotypical set of pathophysiological consequences in RGCs. These findings implicate RAR as a possible common therapeutic target for reversing the signal-corrupting effects of retinal remodeling in both RP and AMD.

The excitatory synapse between hippocampal CA3 and CA1 pyramidal neurons exhibits long-term potentiation (LTP), a positive feedback process implicated in learning and memory in which postsynaptic depolarization strengthens synapses, promoting further depolarization. Without mechanisms for interrupting positive feedback, excitatory synapses could strengthen inexorably, corrupting memory storage. Here, we reveal a hidden form of inhibitory synaptic plasticity that prevents accumulation of excitatory LTP. We developed a knockin mouse that allows optical control of endogenous α5-subunit-containing γ-aminobutyric acid (GABA)A receptors (α5-GABARs). Induction of excitatory LTP relocates α5-GABARs, which are ordinarily extrasynaptic, to inhibitory synapses, quashing further NMDA receptor activation necessary for inducing more excitatory LTP. Blockade of α5-GABARs accelerates reversal learning, a behavioral test for cognitive flexibility dependent on repeated LTP. Hence, inhibitory synaptic plasticity occurs in parallel with excitatory synaptic plasticity, with the ensuing interruption of the positive feedback cycle of LTP serving as a possible critical early step in preserving memory.

Algebras introduced by, or attributed to, Sugihara, Belnap, Meyer, and Church are representable as algebras of binary relations with set-theoretically defined operations. They are definitional reducts or subreducts of proper relation algebras. The representability of Sugihara matrices yields sound and complete set-theoretical semantics for R-mingle., 39 pages, 6 tables, 5 figures, submitted to Journal of Logical and Algebraic Methods in Programming, third version, math-LO

Lateral inhibition in the vertebrate retina depends on a negative feedback synapse between horizontal cells (HCs) and rod and cone photoreceptors. A change in pH is thought to be the signal for negative feedback, but its spatial profile in the synaptic cleft is unknown. Here we use three different membrane proteins, each fused to the same genetically-encoded pH-sensitive Green Fluorescent Protein (GFP) (pHluorin), to probe synaptic pH in retina from transgenic zebrafish (Danio rerio) of either sex. We used the cone transducin promoter to express SynaptopHluorin (pHluorin on vesicle-associated membrane protein (VAMP2)) or CalipHluorin (pHluorin on an L-type Ca2+channel) and the HC-specific connexin-55.5 promoter to express AMPApHluorin (pHluorin on an AMPA receptor). Stimulus light led to increased fluorescence of all three probes, consistent with alkalinization of the synaptic cleft. The receptive field size, sensitivity to surround illumination, and response to activation of an alien receptor expressed exclusively in HCs, are consistent with lateral inhibition as the trigger for alkalinization. However, SynaptopHluorin and AMPApHluorin, which are displaced farther from cone synaptic ribbons than CalipHluorin, reported a smaller pH change. Hence, unlike feedforward glutamatergic transmission, which spills over to allow cross talk between terminals in the cone network, the pH change underlying HC feedback is compartmentalized to individual synaptic invaginations within a cone terminal, consistent with private line communication.SIGNIFICANCE STATEMENTLateral inhibition (LI) is a fundamental feature of information processing in sensory systems, enhancing contrast sensitivity and enabling edge discrimination. Horizontal cells (HCs) are the first cellular substrate of LI in the vertebrate retina, but the synaptic mechanisms underlying LI are not completely understood, despite decades of study. This paper makes a significant contribution to our understanding of LI, by showing that each HC–cone synapse is a “private-line” that operates independently from other HC–cone connections. Using transgenic zebrafish expressing pHluorin, a pH-sensitive GFP variant spliced onto three different protein platforms expressed either in cones or HCs we show that the feedback pH signal is constrained to individual cone terminals, and more stringently, to individual synaptic contact sites within each terminal.

Photoswitchable neurotransmitter receptors are powerful tools for precise manipulation of neural signaling. Through the trans-cis isomerization of azobenzene photoswitches tethered to these receptors, their activities can be rapidly and reversibly controlled by light. However, their applications for slow or long-lasting biological events are constrained by thermal relaxation of cis-azobenzene, a process that renders the receptor state unsustainable in darkness after the light stimulus. We here present a simple approach to slow down this process and thereby enhance the bi-stability (i.e. the ability to stably reside in either of the two states) of photoswitchable receptors. Introducing methyl groups to the ortho positions of azobenzene strongly retards photoswitch thermal relaxation both in solution and on cell surface. In cultured cells and intact brain tissue, modifying inhibitory neurotransmitter receptors with one of the derivatives, dMPC1, allows bi-directional receptor control with 380-nm and 500-nm light. Benefited by the bi-stability of dMPC1, the modified receptors can be locked in either an active or an inactive state in darkness after a brief pulse of light. This strategy thus enables both rapid and sustained manipulation of neurotransmission, allowing optogenetic interrogation of neural functions over a broad range of time scales.

An error in a proof of a correct theorem in the classic paper, Boolean Algebras with Operators, Part I, by Jonsson and Tarski is discussed.

Kevin J. Cao and Richard H. Kramer, who developed extended release with beta cyclodextrin, were inadvertently omitted from the author list and author contributions section of this Article. These errors have now been corrected in both the PDF and HTML versions of the Article.

QAQ (quaternary ammonium-azobenzene-quaternary ammonium) and DENAQ (diethylamine-azobenzene-quaternary ammonium) are synthetic photoswitch compounds that change conformation in response to light, altering current flow through voltage-gated ion channels in neurons. These compounds are drug candidates for restoring light sensitivity in degenerative blinding diseases, such as age-related macular degeneration (AMD).However, these photoswitch compounds are cleared from the eye within several days, they must be administered through repeated intravitreal injections. Therefore, we are investigating local, sustained delivery formulations to constantly replenish these molecules and have the potential to restore sight.Here, we encapsulate QAQ and DENAQ into several molecular weights of poly(lactic-co-glycolic) acid (PLGA) through an emulsion technique to assess the viability of delivering the compounds in their therapeutic window over many weeks. We characterize the loading efficiency, release profile and bioactivity of the compounds after encapsulation.A very small burst release was observed for all of the formulations with the majority being delivered over the following two months. The lowest molecular weight PLGA led to the highest loading and most linear delivery for both QAQ and DENAQ. Bioactivity was retained for both compounds across the polymers.These results present encapsulation into polymers by emulsion as a viable option for controlled release of QAQ and DENAQ.