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The ability to express a gene of interest in a spatio-temporal manner using Gal4-UAS system has allowed the use of Drosophila model to study various biological phenomenon. During Drosophila eye development, a synchronous wave of differentiation called Morphogenetic furrow (MF) initiates at the posterior margin resulting in differentiation of retinal neurons. This synchronous differentiation is also observed in the differentiating retina of vertebrates. Since MF is highly dynamic, it can serve as an excellent model to study patterning and differentiation. However, there are not any Gal4 drivers available to observe the gain- of- function or loss- of- function of a gene specifically along the dynamic MF. The decapentaplegic (dpp) gene encodes a secreted protein of the transforming growth factor-beta (TGF-beta) superfamily that expresses at the posterior margin and then moves with the MF. However, unlike the MF associated pattern of dpp gene expression, the targeted dpp-Gal4 driver expression is restricted to the posterior margin of the developing eye disc. We screened GMR lines harboring regulatory regions of dpp fused with Gal4 coding region to identify MF specific enhancer of dpp using a GFP reporter gene. We employed immuno-histochemical approaches to detect gene expression. The rationale was that GFP reporter expression will correspond to the dpp expression domain in the developing eye. We identified two new dpp-Gal4 lines, viz., GMR17E04-Gal4 and GMR18D08-Gal4 that carry sequences from first intron region of dpp gene. GMR17E04-Gal4 drives expression along the MF during development and later in the entire pupal retina whereas GMR18D08-Gal4 drives expression of GFP transgene in the entire developing eye disc, which later drives expression only in the ventral half of the pupal retina. Thus, GMR18D08-Gal4 will serve as a new reagent for targeting gene expression in the ventral half of the pupal retina. We compared misexpression phenotypes of Wg, a negative regulator of eye development, using GMR17E04-Gal4, GMR18D08-Gal4 with existing dpp-Gal4 driver. The eye phenotypes generated by using our newly identified MF specific driver are not similar to the ones generated by existing dpp-Gal4 driver. It suggests that misexpression studies along MF needs revisiting using the new Gal4 drivers generated in our studies.

The ability of the retina to adapt to changes in mean light intensity and contrast is well known. Classically, however, adaptation is thought to affect gain but not to change the visual modality encoded by a given type of retinal neuron. Recent findings reveal unexpected dynamic properties in mouse retinal neurons that challenge this view. Specifically, certain cell types change the visual modality they encode with variations in ambient illumination or following repetitive visual stimulation. These discoveries demonstrate that computations performed by retinal circuits with defined architecture can change with visual input. Moreover, they pose a major challenge for central circuits that must decode properties of the dynamic visual signal from retinal outputs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In recent years, multielectrode arrays and large silicon probes have been developed to record simultaneously between hundreds and thousands of electrodes packed with a high density. However, they require novel methods to extract the spiking activity of large ensembles of neurons. Here, we developed a new toolbox to sort spikes from these large-scale extracellular data. To validate our method, we performed simultaneous extracellular and loose patch recordings in rodents to obtain 'ground truth' data, where the solution to this sorting problem is known for one cell. The performance of our algorithm was always close to the best expected performance, over a broad range of signal-to-noise ratios, in vitro and in vivo. The algorithm is entirely parallelized and has been successfully tested on recordings with up to 4225 electrodes. Our toolbox thus offers a generic solution to sort accurately spikes for up to thousands of electrodes., Competing Interests: PY, GS, EE, BL, SD, CG, MS, FJ, GZ, SP, JD, OM No competing interests declared, (© 2018, Yger et al.)

The clustered protocadherins (Pcdhs) comprise 58 cadherin-related proteins encoded by three tandemly arrayed gene clusters, Pcdh -α, Pcdh -β, and Pcdh -γ ( Pcdha , Pcdhb , and Pcdhg , respectively). Pcdh isoforms from different clusters are combinatorially expressed in neurons. They form multimers that interact homophilically and mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, axonal tiling, and dendritic self-avoidance. Most studies have analyzed clusters individually. Here, we assessed functional interactions between Pcdha and Pcdhg clusters. To circumvent neonatal lethality associated with deletion of Pcdhgs , we used Crispr-Cas9 genome editing in mice to combine a constitutive Pcdha mutant allele with a conditional Pcdhg allele. We analyzed roles of Pcdhas and Pcdhgs in the retina and cerebellum from mice (both sexes) lacking one or both clusters. In retina, Pcdhgs are essential for survival of inner retinal neurons and dendritic self-avoidance of starburst amacrine cells, whereas Pcdhas are dispensable for both processes. Deletion of both Pcdha and Pcdhg clusters led to far more dramatic defects in survival and self-avoidance than Pcdhg deletion alone. Comparisons of an allelic series of mutants support the conclusion that Pcdhas and Pcdhgs function together in a dose-dependent and cell-type-specific manner to provide a critical threshold of Pcdh activity. In the cerebellum, Pcdhas and Pcdhgs also cooperate to mediate self-avoidance of Purkinje cell dendrites, with modest but significant defects in either single mutant and dramatic defects in the double mutant. Together, our results demonstrate complex patterns of redundancy between Pcdh clusters and the importance of Pcdh cluster diversity in postnatal CNS development. SIGNIFICANCE STATEMENT The formation of neural circuits requires diversification and combinatorial actions of cell surface proteins. Prominent among them are the clustered protocadherins (Pcdhs), a family of ∼60 neuronal recognition molecules. Pcdhs are encoded by three closely linked gene clusters called Pcdh -α, Pcdh -β, and Pcdh -γ. The Pcdhs mediate a variety of developmental processes, including neuronal survival, synaptic maintenance, and spatial patterning of axons and dendrites. Most studies to date have been limited to single clusters. Here, we used genome editing to assess interactions between Pcdh -α and Pcdh -γ gene clusters. We examined two regions of the CNS, the retina and cerebellum and show that the 14 α-Pcdhs and 22 γ-Pcdhs act synergistically to mediate neuronal survival and dendrite patterning., (Copyright © 2018 the authors 0270-6474/18/382713-17$15.00/0.)

Aim To compare the relationship between white matter hyperintensities (WMH) on brain magnetic resonance imaging and retinal nerve fiber layer (RNFL), choroid, and ganglion cell layer (GCL) thicknesses in migraine patients and healthy subjects. We also assessed the role of cerebral hypoperfusion in the formation of these WMH lesions. Methods We enrolled 35 migraine patients without WMH, 37 migraine patients with WMH, and 37 healthy control subjects examined in the Neurology outpatient clinic of our tertiary center from May to December 2015. RFNL, choroid, and GCL thicknesses were measured by optic coherence tomography. Results There were no differences in the RFNL, choroid, or GCL thicknesses between migraine patients with and without WMH ( p > 0.05). Choroid layer thicknesses were significantly lower in migraine patients compared to control subjects ( p < 0.05), while there were no differences in RFNL and GCL thicknesses ( p > 0.05). Conclusions The 'only cerebral hypoperfusion' theory was insufficient to explain the pathophysiology of WMH lesions in migraine patients. In addition, the thinning of the choroid thicknesses in migraine patients suggests a potential causative role for cerebral hypoperfusion and decreased perfusion pressure of the choroid layer.

Dry eye disease (DED) is a common chronic condition with multifactorial etiologies that is increasing in prevalence worldwide, up to 20% in the elderly. The economic burden and impact of DED on vision, quality of life, work productivity, psychological and physical impact of pain, are considerable. Chronic ocular pain is the most common symptom of DED and there is currently no topical ocular analgesic therapy available to treat this debilitating disease. Eye pain can be perceived as itch, irritation, dryness, grittiness, burning, aching, and light sensitivity. Ocular pain is triggered by corneal nociceptors (cornea being the most sensory innervated tissue of the body). It was clearly established that repeated direct damage to ocular surface and per se corneal nerves can cause peripheral and central sensitization mechanisms explaining the ocular pain in some patients with DED. However, the brain regions and the neuronal pathways associated with ocular pain are still unclear. Thus, a better characterization of chronic ocular pain and an understanding of the peripheral and central molecular and cellular mechanisms involved are crucial issues for developing effective management and therapeutic strategy to alleviate ocular pain. In this review, we first describe the nociceptive corneal nerve pathways and the classification and the neurochemistry of primary afferents innervating the cornea. Then, an update of the fundamental and clinical studies related to the inflammatory processes linked to ocular pain is detailed. The last part of the review presents the diagnostic tools used in clinic for evaluating corneal sensitivity and corneal inflammation., (© Société de Biologie, 2018.)

The mouse retina has a layered structure that is composed of five classes of neurons supported by Müller glial and pigment epithelial cells. Recent studies have made progress in the classification of bipolar and ganglion cells, and also in the wiring of rod-driven signaling, color coding, and directional selectivity. Molecular biological techniques, such as genetic manipulation, transcriptomics, and fluorescence imaging, have contributed a lot to these advancements. The mouse retina has consistently been an important experimental system for both basic and clinical neurosciences.

Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.

In the developing retina, as in other regions of the CNS, neural progenitors give rise to individual cell types during discrete temporal windows. Pax6 is expressed in retinal progenitor cells (RPCs) throughout the course of retinogenesis, and has been shown to be required during early retinogenesis for generation of most early-born cell types. In this study, we examined the function of Pax6 in postnatal mouse retinal development. We found that Pax6 is essential for the generation of late-born interneurons, while inhibiting photoreceptor differentiation. Generation of bipolar interneurons requires Pax6 expression in RPCs, while Pax6 is required for the generation of glycinergic, but not for GABAergic or non-GABAergic-non-glycinergic (nGnG) amacrine cell subtypes. In contrast, overexpression of either full-length Pax6 or its 5a isoform in RPCs induces formation of cells with nGnG amacrine features, and suppresses generation of other inner retinal cell types. Moreover, overexpression of both Pax6 variants prevents photoreceptor differentiation, most likely by inhibiting Crx expression. Taken together, these data show that Pax6 acts in RPCs to control differentiation of multiple late-born neuronal cell types., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons., Results: We found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα -/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production., Conclusion: We identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits.

In traditional electrophysiology, spatially inefficient electronics and the need for tissue-to-electrode proximity defy non-invasive interfaces at scales of more than a thousand low noise, simultaneously recording channels. Using compressed sensing concepts and silicon complementary metal-oxide-semiconductors (CMOS), we demonstrate a platform with 65,536 simultaneously recording and stimulating electrodes in which the per-electrode electronics consume an area of 25.5 μm by 25.5 μm. Application of this platform to mouse retinal studies is achieved with a high-performance processing pipeline with a 1 GB/s data rate. The platform records from 65,536 electrodes concurrently with a ~10 µV r.m.s. noise; senses spikes from more than 34,000 electrodes when recording across the entire retina; automatically sorts and classifies greater than 1700 neurons following visual stimulation; and stimulates individual neurons using any number of the 65,536 electrodes while observing spikes over the entire retina. The approaches developed here are applicable to other electrophysiological systems and electrode configurations.

Simultaneous electrical stimulation and recording using multi-electrode arrays can provide a valuable technique for studying circuit connectivity and engineering neural interfaces. However, interpreting these measurements is challenging because the spike sorting process (identifying and segregating action potentials arising from different neurons) is greatly complicated by electrical stimulation artifacts across the array, which can exhibit complex and nonlinear waveforms, and overlap temporarily with evoked spikes. Here we develop a scalable algorithm based on a structured Gaussian Process model to estimate the artifact and identify evoked spikes. The effectiveness of our methods is demonstrated in both real and simulated 512-electrode recordings in the peripheral primate retina with single-electrode and several types of multi-electrode stimulation. We establish small error rates in the identification of evoked spikes, with a computational complexity that is compatible with real-time data analysis. This technology may be helpful in the design of future high-resolution sensory prostheses based on tailored stimulation (e.g., retinal prostheses), and for closed-loop neural stimulation at a much larger scale than currently possible.

Aims: To identify clinical characteristics and risk factors of retinal neurodegeneration represented by macular ganglion cell/inner plexiform layer (mGCIPL) thinning in patients with long-standing type 2 diabetes mellitus (T2DM)., Methods: Patients who had T2DM for >15 years were prospectively recruited from September 2014 to July 2015. Clinical data and samples were collected according to the Common Data Element and Standards of Procedure developed by the Korean Diabetes Association research council. Baseline characteristics included age, gender, family history, medical record of comorbidity, and microvascular complications. All patients underwent optical coherence tomography with automatic segmentation of the mGCIPL in six parafoveal regions. Multivariable regression analysis identified factors associated with mGCIPL thinning., Results: Of 220 registered patients, 162 were included after ophthalmologic examination. The mean (SD) age was 65.0 (9.3) years, the mean duration of T2DM was 20.5 (4.0) years; mGCIPL thickness was 76.2 (8.5) µm. Hypertension, diabetic retinopathy, statin medication, estimated glomerular filtration rate, conduction velocity of the posterior tibial, peroneal, and sural nerves, and cardiac autonomic neuropathy (CAN) score were significantly correlated with mGCIPL thickness. Multivariate regression analysis showed that the CAN score (coefficient = -1.78, p = 0.001) and sural nerve velocity (coefficient = 0.458, p = 0.035) yielded a significant high regression correlation with mGCIPL thickness (overall R 2  = 0.46)., Conclusions: This study demonstrated that various clinical features were associated with retinal neurodegeneration in T2DM. In particular, peripheral nerve conduction and autonomic nerve function were confirmed to be strong risk factors for mGCIPL thinning in patients with T2DM.

Müller glia (MG) are major retinal supporting cells that participate in retinal metabolism, function, maintenance, and protection. During the pathogenesis of diabetic retinopathy (DR), a neurovascular disease and a leading cause of blindness, MG modulate vascular function and neuronal integrity by regulating the production of angiogenic and trophic factors. In this article, I will (1) briefly summarize our work on delineating the role and mechanism of MG-modulated vascular function through the production of vascular endothelial growth factor (VEGF) and on investigating VEGF signaling-mediated MG viability and neural protection in diabetic animal models, (2) explore the relationship among VEGF and neurotrophins in protecting Müller cells in in vitro models of diabetes and hypoxia and its potential implication to neuroprotection in DR and hypoxic retinal diseases, and (3) discuss the relevance of our work to the effectiveness and safety of long-term anti-VEGF therapies, a widely used strategy to combat DR, diabetic macular edema, neovascular age-related macular degeneration, retinopathy of prematurity, and other hypoxic retinal vascular disorders., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X 2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X 2 purinoceptors acquire permeability to large cations, such as N -methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,β-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X 2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X 2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina. NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X 2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X 2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo., (Copyright © 2017 the American Physiological Society.)

The complex pathology of diabetic retinopathy (DR) affects both vascular and neural tissue. The characteristics of neurodegeneration are well-described in animal models but have more recently been confirmed in the clinical setting, mostly by using non-invasive imaging approaches such as spectral domain optical coherence tomography (SD-OCT). The most frequent observations report loss of tissue in the nerve fiber layer and inner plexiform layer, confirming earlier findings from animal models. In several cases the reduction in inner retinal layers is reported in patients with little evidence of vascular lesions or macular edema, suggesting that degenerative loss of neural tissue in the inner retina can occur after relatively short durations of diabetes. Animal studies also suggest that neurodegeneration leading to retinal thinning is not limited to cell death and tissue loss but also includes changes in neuronal morphology, reduced synaptic protein expression and alterations in neurotransmission, including changes in expression of neurotransmitter receptors as well as neurotransmitter release, reuptake and metabolism. The concept of neurodegeneration as an early component of DR introduces the possibility to explore alternative therapies to prevent the onset of vision loss, including neuroprotective therapies and drugs targeting individual neurotransmitter systems, as well as more general neuroprotective approaches to preserve the integrity of the neural retina. In this review we consider some of the evidence for progressive retinal neurodegeneration in diabetes, and explore potential neuroprotective therapies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: We determined the effects of a remodeled inner retina on the flicker electroretinograms (ERGs) in a rabbit eye at an advanced stage of inherited retinal degeneration., Methods: Six wild-type (WT) and four rhodopsin P347L transgenic (Tg) rabbits were studied at 18 months of age. Flicker ERGs were elicited by sinusoidal stimuli at frequencies of 3.906 to 50.781 Hz. To block the ON and OFF retinal pathways, 2-amino-4-phosphonobutyric acid (APB), and 6-cyano-7-nitroquinoxaline-2, 3(1H, 4H)-dione (CNQX), respectively, were injected intravitreally. The amplitudes and phases of the fundamental components of the pre- and postdrug ERGs were analyzed. The postsynaptic APB (ON-) and CNQX (OFF-) sensitive components were determined by examining the phases and amplitude vectors., Results: The temporal properties of the Tg rabbits were different from those of the WT rabbits and had unique features; at 3.906 Hz, the amplitude was depressed but it increased by more than 3.5-fold at 15.625 Hz. The reduction of the amplitude at 3.906 Hz in Tg rabbits was caused by a cancelation of the ON and OFF components by a phase difference of 180°. On the other hand, an increase in the amplitude at 15.625 Hz in Tg rabbits was caused by the summation of the ON and OFF components, which had an approximate 120° phase difference., Conclusions: The temporal properties of the flicker ERGs of Tg rabbits were affected markedly by the remodeling of the retinal neurons. Evaluations of the flicker ERGs in RP eyes must be done with careful considerations of the current findings.

Advances in multi-unit recordings pave the way for statistical modeling of activity patterns in large neural populations. Recent studies have shown that the summed activity of all neurons strongly shapes the population response. A separate recent finding has been that neural populations also exhibit criticality, an anomalously large dynamic range for the probabilities of different population activity patterns. Motivated by these two observations, we introduce a class of probabilistic models which takes into account the prior knowledge that the neural population could be globally coupled and close to critical. These models consist of an energy function which parametrizes interactions between small groups of neurons, and an arbitrary positive, strictly increasing, and twice differentiable function which maps the energy of a population pattern to its probability. We show that: 1) augmenting a pairwise Ising model with a nonlinearity yields an accurate description of the activity of retinal ganglion cells which outperforms previous models based on the summed activity of neurons; 2) prior knowledge that the population is critical translates to prior expectations about the shape of the nonlinearity; 3) the nonlinearity admits an interpretation in terms of a continuous latent variable globally coupling the system whose distribution we can infer from data. Our method is independent of the underlying system's state space; hence, it can be applied to other systems such as natural scenes or amino acid sequences of proteins which are also known to exhibit criticality.

Retinal progenitor cell is a promising candidate in the treatment of retinal pigmentosa diseases. The limiting factors of stem cell transplantation are the proliferation and differentiation capacities of hRPCs, which may be governed by culture conditions. Previous studies have proved that the secretome of human Umbilical Cord Mesenchymal stem cells (hUCMSCs) and human Adipose derived stem cells (hADSCs), including more active cytokines and neurotrophic factors, have the paracrine potential of enhancing proliferation and differentiation in several cell types. The aim of this study was to investigate whether hRPCs could effectively proliferate, adhere and differentiate towards specific retinal cell types by treating with the condition medium (CM) of hUCMSCs (hUCMSCCM) or hADSCs (hADSCCM). Here, we show that hUCMSCCM or hADSCCM enhances the proliferation rate of the S and G2 phase cells, with an upregulation of Ki67 expression. Moreover, the upregulation expression of NF, Recoverin and Rhodopsin indicates that specialized retinal cells including ganglion cells and photoreceptors are favored over hRPCs differentiation due to hUCMSCCM or hADSCCM. Under FBS induced differentiation conditions, hRPCs treated with hUCMSCCM or hADSCCM increase the expression of retinal neuron and photoreceptor specific markers. These results suggest that hUCMSCCM and hADSCCM can stimulate the hRPC proliferation, promote its adherence and support hRPC neuronal and photoreceptor differentiation. These findings may provide a new strategy to improve the viability of hRPCs and photoreceptor differentiation capacities., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The zebrafish has become a commonly used vertebrate model for toxicity assessment, of particular relevance to the study of toxic effects on the visual system because of the structural similarities shared by zebrafish and human retinae. In this article we present a colour preference-based technique that, by assessing the functionality of photoreceptors, can be used to evaluate the effects of toxicity on behaviour. A digital camera was used to record the locomotor behaviour of individual zebrafish swimming in a water tank consisting of two compartments separated by an opaque perforated wall through which the fish could pass. The colour of the lighting in each compartment could be altered independently (producing distinct but connected environments of white, red or blue) to allow association of the zebrafish's swimming behaviour with its colour preference. The functionality of the photoreceptors was evaluated based on the ability of the zebrafish to sense the different colours and to swim between the compartments. The zebrafish tracking was carried out using our algorithm developed with MATLAB. We found that zebrafish preferred blue illumination to white, and white illumination to red. Acute treatment with acrylamide (2mM for 36h) resulted in a marked reduction in locomotion and a concomitant loss of colour-preferential swimming behaviour. Histopathological examination of acrylamide-treated zebrafish eyes showed that acrylamide exposure had caused retinal damage. The colour preference tracking technique has applications in the assessment of neurodegenerative disorders, as a method for preclinical appraisal of drug efficacy and for behavioural evaluation of toxicity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The structure of the physical world projects images onto our eyes. However, those images are often poorly representative of environmental structure: well-defined boundaries within the eye may correspond to irrelevant features of the physical world, while critical features of the physical world may be nearly invisible at the retinal projection. The challenge for the visual cortex is to sort these two types of features according to their utility in ultimately reconstructing percepts and interpreting the constituents of the scene. We describe a novel paradigm that enabled us to selectively evaluate the relative role played by these two feature classes in signal reconstruction from corrupted images. Our measurements demonstrate that this process is quickly dominated by the inferred structure of the environment, and only minimally controlled by variations of raw image content. The inferential mechanism is spatially global and its impact on early visual cortex is fast. Furthermore, it retunes local visual processing for more efficient feature extraction without altering the intrinsic transduction noise. The basic properties of this process can be partially captured by a combination of small-scale circuit models and large-scale network architectures. Taken together, our results challenge compartmentalized notions of bottom-up/top-down perception and suggest instead that these two modes are best viewed as an integrated perceptual mechanism.

Primates have specialized domains in inferior temporal (IT) cortex that are responsive to particular image categories. Though IT traditionally has been regarded as lacking retinotopy, several recent studies in monkeys have shown that retinotopic maps extend to face patches along the lower bank of the superior temporal sulcus (STS) and neighboring regions of IT cortex. Here, we used fMRI to map the retinotopic organization of medial ventral temporal cortex in four monkeys (2 male and 2 female). We confirm the presence of visual field maps within and around the lower bank of the STS and extend these prior findings to scene-selective cortex in the ventral-most regions of IT. Within the occipitotemporal sulcus (OTS), we identified two retinotopic areas, OTS1 and OTS2. The polar angle representation of OTS2 was a mirror reversal of the OTS1 representation. These regions contained representations of the contralateral periphery and were selectively active for scene versus face, body, or object images. The extent of this retinotopy parallels that in humans and shows that the organization of the scene network is preserved across primate species. In addition retinotopic maps were identified in dorsal extrastriate, posterior parietal, and frontal cortex as well as the thalamus, including both the lateral geniculate nucleus and pulvinar. Together, it appears that most, if not all, of the macaque visual system contains organized representations of visual space. SIGNIFICANCE STATEMENT Primates have specialized domains in inferior temporal (IT) cortex that are responsive to particular image categories. Though retinotopic maps are considered a fundamental organizing principle of posterior visual cortex, IT traditionally has been regarded as lacking retinotopy. Recent imaging studies have demonstrated the presence of several visual field maps within the lateral IT. Using neuroimaging, we found multiple representations of visual space within ventral IT cortex of macaques that included scene-selective cortex. Scene domains were biased toward the peripheral visual field. These data demonstrate the prevalence of visual field maps throughout the primate visual system, including late stages in the ventral visual hierarchy, and support the idea that domains representing different categories are biased toward different parts of the visual field., (Copyright © 2017 Arcaro and Livingstone.)

Purpose: Rod photoreceptor terminals respond to retinal injury with retraction and sprouting. Since the guidance cue Semaphorin3A (Sema3A) is observed in the retina after injury, we asked whether Sema3A contributes to structural plasticity in rod photoreceptors., Methods: We used Western blots and alkaline phosphatase (AP)-tagged neuropilin-1 (NPN-1) to detect the expression of Sema3A in an organotypic model of porcine retinal detachment. We then examined Sema3A binding to cultured salamander rod photoreceptors using AP-tagged Sema3A. For functional analysis, we used a microspritzer to apply a gradient of Sema3A-Fc to isolated salamander rod photoreceptors over 24 hours., Results: Sema3A protein was biochemically detected in porcine retinal explants in the retina 7, 24, and 72 hours after detachment. In sections, NPN-1 receptor was bound to the inner and outer retina. For isolated rod photoreceptors, Sema3A localized to synaptic terminals and to neuritic processes after 1 week in vitro. In microspritzed rod photoreceptors, process initiation occurred away from high concentrations of Sema3A. Sema3A significantly decreased the number of processes formed by rod photoreceptors although the average length of processes was not affected. The cellular orientation of rod photoreceptors relative to the microspritzer also significantly changed over time; this effect was reduced with the Sema3A inhibitor, xanthofulvin., Conclusion: Sema3A is expressed in the retina after detachment, binds to rod photoreceptors, affects cell orientation, and reduces photoreceptor process initiation in vitro. Our results suggest that Sema3A contributes to axonal retraction in retinal injury, whereas rod neuritic sprouting and regenerative synaptogenesis may require a reduction in semaphorin signaling.

The prospect of replacing damaged body parts with artificial implants is being transformed from science fiction to science fact through the increasing application of electronics to interface with human neurons in the limbs, the brain, and the retina. We propose bio-inspired electronics which adopt the fractal geometry of the neurons they interface with. Our focus is on retinal implants, although performance improvements will be generic to many neuronal types. The key component is a multifunctional electrode; light passes through this electrode into a photodiode which charges the electrode. Its electric field then stimulates the neurons. A fractal electrode might increase both light transmission and neuron proximity compared to conventional Euclidean electrodes. These advantages are negated if the fractal's field is less effective at stimulating neurons. We present simulations demonstrating how an interplay of fractal properties generates enhanced stimulation; the electrode voltage necessary to stimulate all neighboring neurons is over 50% less for fractal than Euclidean electrodes. This smaller voltage can be achieved by a single diode compared to three diodes required for the Euclidean electrode's higher voltage. This will allow patients, for the first time, to see with the visual acuity necessary for navigating rooms and streets.

Key Points: Large conductance, Ca 2+ -activated K + (BK Ca ) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells. BK Ca channel blockers paxilline and iberiotoxin, as well as Ca 2+ free solutions and divalent cation Ca v channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150 mm K + , single channels had a conductance close to 250 pS, within the range of all known BK Ca channels. In current clamped horizontal cells, BK Ca channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BK Ca channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells., Abstract: Large conductance, calcium-activated potassium (BK Ca ) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca 2+ ] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between -30 and +60 mV is carried primarily in BK Ca channels in isolated horizontal cells of rats and mice. Whole-cell outward currents were maximal at +50 mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BK Ca channel blocker paxilline (100 nm), iberiotoxin (10 μm), Ca 2+ free solutions and divalent cation Ca v channel blockers. It was activated by the BK Ca channel activator NS1619 (30 μm). Single channel recordings revealed the conductance of the channels to be 244 ± 11 pS (n = 17; symmetrical 150 mm K + ) with open probability being both voltage- and Ca 2+ -dependent. The channels showed fast activation kinetics in response to Ca 2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BK Ca current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BK Ca current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BK Ca current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: Visual input to the suprachiasmatic nucleus circadian clock is critical for animals to adapt their physiology and behaviour in line with the solar day. In addition to direct retinal projections, the clock receives input from the visual thalamus, although the role of this geniculohypothalamic pathway in circadian photoreception is poorly understood. In the present study, we develop a novel brain slice preparation that preserves the geniculohypothalamic pathway to show that GABAergic thalamic neurons inhibit retinally-driven activity in the central clock in a circadian time-dependent manner. We also show that in vivo manipulation of thalamic signalling adjusts specific features of the hypothalamic light response, indicating that the geniculohypothalamic pathway is primarily activated by crossed retinal inputs. Our data provide a mechanism by which geniculohypothalamic signals can adjust the magnitude of circadian and more acute hypothalamic light responses according to time-of-day and establish an important new model for future investigations of the circadian visual system., Abstract: Sensory input to the master mammalian circadian clock, the suprachiasmatic nucleus (SCN), is vital in allowing animals to optimize physiology and behaviour alongside daily changes in the environment. Retinal inputs encoding changes in external illumination provide the principle source of such information. The SCN also receives input from other retinorecipient brain regions, primarily via the geniculohypothalamic tract (GHT), although the contribution of these indirect projections to circadian photoreception is currently poorly understood. To address this deficit, in the present study, we established an in vitro mouse brain slice preparation that retains connectivity across the extended circadian system. Using multi-electrode recordings, we first confirm that this preparation retains intact optic projections to the SCN, thalamus and pretectum and a functional GHT. We next show that optogenetic activation of GHT neurons selectively suppresses SCN responses to retinal input, and also that this effect exhibits a pronounced day/night variation and involves a GABAergic mechanism. This inhibitory action was not associated with overt circadian rhythmicity in GHT output, indicating modulation at the SCN level. Finally, we use in vivo electrophysiological recordings alongside pharmacological inactivation or optogenetic excitation to show that GHT signalling actively modulates specific features of the SCN light response, indicating that GHT cells are primarily activated by crossed retinal projections. Taken together, our data establish a new model for studying network communication in the extended circadian system and provide novel insight into the roles of GHT-signalling, revealing a mechanism by which thalamic activity can help gate retinal input to the SCN according to time of day., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Emotional experience changes visual perception, leading to the prioritization of sensory information associated with threats and opportunities. These emotional biases have been extensively studied by basic and clinical scientists, but their underlying mechanism is not known. The present study combined measures of brain-electric activity and autonomic physiology to establish how threat biases emerge in human observers. Participants viewed stimuli designed to differentially challenge known properties of different neuronal populations along the visual pathway: location, eye, and orientation specificity. Biases were induced using aversive conditioning with only 1 combination of eye, orientation, and location predicting a noxious loud noise and replicated in a separate group of participants. Selective heart rate-orienting responses for the conditioned threat stimulus indicated bias formation. Retinotopic visual brain responses were persistently and selectively enhanced after massive aversive learning for only the threat stimulus and dissipated after extinction training. These changes were location-, eye-, and orientation-specific, supporting the hypothesis that short-term plasticity in primary visual neurons mediates the formation of perceptual biases to threat. (PsycINFO Database Record, ((c) 2017 APA, all rights reserved).)

Irreversible vision loss due to disease or age is responsible for a reduced quality of life. The experiments in this study test the hypothesis that the α7 nicotinic acetylcholine receptor agonist, PNU-282987, leads to the generation of retinal neurons in an adult mammalian retina in the absence of retinal injury or exogenous growth factors. Using antibodies against BrdU, retinal ganglion cells, progenitor cells and Müller glia, the results of this study demonstrate that multiple types of retinal cells and neurons are generated after eye drop application of PNU-282987 in adult Long Evans rats in a dose-dependent manner. The results of this study provide evidence that progenitor cells, derived from Müller glia after treatment with PNU-282987, differentiate and migrate to the photoreceptor and retinal ganglion cell layers. If retinas were treated with the alpha7 nAChR antagonist, methyllycaconitine, before agonist treatment, BrdU-positive cells were significantly reduced. As adult mammalian neurons do not typically regenerate or proliferate, these results have implications for reversing vision loss due to neurodegenerative disease or the aging process to improve the quality of life for millions of patients., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

World-wide, two degenerative retinal diseases, glaucoma and age-related macular degeneration, are estimated to affect more than 12% of individuals over the age of 40 (Tham et al., 2014; Wong et al., 2014). Current therapies can slow progression, but cannot restore lost neurons or vision. Thus, there is increasing interest in developing strategies for therapeutic retinal regeneration. Nearly 50years of research on retinal neurogenesis and regeneration has identified Müller glia as intrinsic retinal stem cells in teleost fish. In the mammalian retina, there is no de novo neurogenesis in adults and only very limited injury-induced regeneration has been induced using exogenous growth factors. The study by (Webster et al., 2017) (Evidence of BrdU Positive Retinal Neurons after Application of an Alpha7 Nicotinic Acetylcholine Receptor Agonist, this issue) is the first to show robust, retinal neurogenesis in an adult, mammalian retina in the absence of overt injury and provides evidence that the source of the new neurons is likely to be the Müller glia. This exciting finding has the potential to be a game-changer in the field of retinal regeneration., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Purpose: To determine the contribution of second- and third-order retinal neurons to the photopic electroretinograms (ERGs) after the degeneration of the rods in rhodopsin P347L transgenic rabbits (Tg)., Methods: Four wild-type (WT) rabbits and four Tg rabbits were studied at 18 months of age. The photopic ERGs elicited at stimulus onset and offset were analyzed. To block different retinal pathways, 2-amino-4-phosphonobutyric acid (APB), 6-cyano-7-nitroquinoxaline-2, 3 (1H,4H)-dione (CNQX), tetrodotoxin (TTX), and N-methyl-DL-aspartic acid (NMDA) were injected intravitreally. Digital subtraction of the postdrug ERGs from the predrug ERGs was used to determine the contributions of the ON-components blocked by APB, the OFF-components blocked by CNQX, and the third-order neurons blocked by TTX+NMDA., Results: Contribution of the cone photoreceptors to the photopic ERGs in Tg rabbits was approximately 10% of that in WT rabbits. The amplitudes of the positive waves of the ON-components at stimulus onset in Tg rabbits were approximately one-half as large as those in WT. On the other hand, the amplitudes of the positive waves of the OFF-components at stimulus offset in Tg rabbits were approximately 1.4 to 2.3 times larger than those in WT. Transgenic rabbits had a positive wave at stimulus offset, which was reduced after the TTX+NMDA injection., Conclusions: A reduced ON-component and an augmented OFF-component with abnormal responses of the third-order neurons contributed to the cone ERGs after the loss of rod function in Tg rabbits. Our results suggest a complex synaptic remodeling of the residual retinal cells in the advanced stage in Tg rabbits.

In the presence of optical blur at the fovea, blur adaptation can improve visual acuity (VA) and perceived image quality over time. However, little is known regarding blur adaptation in the peripheral retina. Here, we examined neural adaptation to myopic defocus at the fovea and parafovea (10° temporal retina) in both emmetropes and myopes. During a 60-min adaptation period, subjects (3 emmetropes and 3 myopes) watched movies with +2 diopters of defocus blur through a 6-mm artificial pupil in two separate, counter-balanced sessions for each retinal location. VA was measured at 10-min intervals under full aberration-corrected viewing using an adaptive optics (AO) vision simulator. By correcting subjects' native optical aberrations with AO, we bypassed the influence of the individual subjects' optical aberrations on visual performance. Overall, exhibited a small but significant improvement after the 60-min of adaptation at both the fovea (mean±SE VA improvement: -0.06±0.04 logMAR) and parafovea (mean±SE VA improvement: -0.07±0.04 logMAR). Myopic subjects exhibited significantly greater improvement in parafoveal VA (mean±SE VA improvement: 0.10±0.02 logMAR), than that of emmetropic subjects (mean±SE VA improvement: 0.04±0.03 logMAR). In contrast, there was no significant difference in foveal VA between the two refractive-error groups. In conclusion, our results reveal differences in peripheral blur adaptation between refractive-error groups, with myopes displaying a greater degree of adaptation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: To compare the relationship of Octopus perimeter cluster mean-defect (cluster MD) values with the spatially corresponding optical coherence tomography (OCT) sector peripapillary angioflow vessel-density (PAFD) and sector retinal nerve fiber layer thickness (RNFLT) values., Methods: High quality PAFD and RNFLT images acquired on the same day with the Angiovue/RTVue-XR Avanti OCT (Optovue Inc., Fremont, USA) on 1 eye of 27 stable early-to-moderate glaucoma, 22 medically controlled ocular hypertensive and 13 healthy participants were analyzed. Octopus G2 normal visual field test was made within 3 months from the imaging., Results: Total peripapillary PAFD and RNFLT showed similar strong positive correlation with global mean sensitivity (r-values: 0.6710 and 0.6088, P<0.0001), and similar (P = 0.9614) strong negative correlation (r-values: -0.4462 and -0.4412, P≤0.004) with global MD. Both inferotemporal and superotemporal sector PAFD were significantly (≤0.039) lower in glaucoma than in the other groups. No significant difference between the corresponding inferotemporal and superotemporal parameters was seen. The coefficient of determination (R2) calculated for the relationship between inferotemporal sector PAFD and superotemporal cluster MD (0.5141, P<0.0001) was significantly greater than that between inferotemporal sector RNFLT and superotemporal cluster MD (0.2546, P = 0.0001). The R2 values calculated for the relationships between superotemporal sector PAFD and RNFLT, and inferotemporal cluster MD were similar (0.3747 and 0.4037, respectively, P<0.0001)., Conclusion: In the current population the relationship between inferotemporal sector PAFD and superotemporal cluster MD was strong. It was stronger than that between inferotemporal sector RNFLT and superotemporal cluster MD. Further investigations are necessary to clarify if our results are valid for other populations and can be usefully applied for glaucoma research., Competing Interests: Gabor Hollo is an unpaid consultant of Optovue and Zeiss. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Fibroblast growth factor (FGF) signaling is an essential regulator of lens epithelial cell proliferation and survival, as well as lens fiber cell differentiation. However, the identities of these FGF factors, their source tissue and the genes that regulate their synthesis are unknown. We have found that Chx10-Cre;Lhx2 lox/lox mice, which selectively lack Lhx2 expression in neuroretina from E10.5, showed an early arrest in lens fiber development along with severe microphthalmia. These mutant animals showed reduced expression of multiple neuroretina-expressed FGFs and canonical FGF-regulated genes in neuroretina. When FGF expression was genetically restored in Lhx2-deficient neuroretina of Chx10-Cre;Lhx2 lox/lox mice, we observed a partial but nonetheless substantial rescue of the defects in lens cell proliferation, survival and fiber differentiation. These data demonstrate that neuroretinal expression of Lhx2 and neuroretina-derived FGF factors are crucial for lens fiber development in vivo., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Retinal regeneration from resident Müller glia cells is robust in teleost fish, but is severely limited in birds and mammals. After neurotoxic injury, chick Müller glia can proliferate, and activate neurogenic genes, but they display limited capacity to differentiate into neurons. Developmental signaling molecules enhance this process. Regeneration of retinal neurons in rodents is even more limited. However, studies show evidence of proliferation and neurogenic gene expression after injury, with stronger effects in rats than mice, and differences between mouse strains. Mitogenic growth factors and Wnt signaling potentiate the proliferative response, while misexpression of the proneural transcription factor, Ascl1, reprograms to generate neurons from Müller glial in vitro, and stimulates neuronal regeneration in young mice, in vivo., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: Zebrafish neurons regenerate from Müller glia following retinal lesions. Genes and signaling pathways important for retinal regeneration in zebrafish have been described, but our understanding of how Müller glial stem cell properties are regulated is incomplete. Mammalian Müller glia possess a latent neurogenic capacity that might be enhanced in regenerative therapies to treat degenerative retinal diseases., Methods: To identify transcriptional changes associated with stem cell properties in zebrafish Müller glia, we performed a comparative transcriptome analysis from isolated cells at 8 and 16 hours following an acute photic lesion, prior to the asymmetric division that produces retinal progenitors., Results: We report a rapid, dynamic response of zebrafish Müller glia, characterized by activation of pathways related to stress, nuclear factor-κB (NF-κB) signaling, cytokine signaling, immunity, prostaglandin metabolism, circadian rhythm, and pluripotency, and an initial repression of Wnt signaling. When we compared publicly available transcriptomes of isolated mouse Müller glia from two retinal degeneration models, we found that mouse Müller glia showed evidence of oxidative stress, variable responses associated with immune regulation, and repression of pathways associated with pluripotency, development, and proliferation., Conclusions: Categories of biological processes/pathways activated following photoreceptor loss in regeneration-competent zebrafish Müller glia, which distinguished them from mouse Müller glia in retinal degeneration models, included cytokine signaling (notably NF-κB), prostaglandin E2 synthesis, expression of core clock genes, and pathways/metabolic states associated with pluripotency. These regulatory mechanisms are relatively unexplored as potential mediators of stem cell properties likely to be important in Müller glial cells for successful retinal regeneration.

During neurogenesis, progenitors balance proliferation and cell cycle exit together with expression of fate determinant genes to ensure that the correct number of each of these neuron types is generated. Although intrinsic gene expression acting cell autonomously within each progenitor drives these processes, the final number of neurons generated is also influenced by extrinsic cues, representing a potential avenue to direct neurogenesis in developmental disorders or regenerative settings without the requirement to change intrinsic gene expression. Thus, it is important to understand which of these stages of neurogenesis are amenable to such extrinsic influences. Additionally, all types of neurons are specified in a highly conserved histogenic order, although its significance is unknown. This study makes use of conserved patterns of neurogenesis in the relatively simple yet highly organized zebrafish retina model, in which such histogenic birth order is well characterized. We directly visualize and quantify birth dates and cell fate determinant expression in WT vs. environments lacking different neuronal populations. This study shows that extrinsic feedback from developing retinal neurons is important for the temporal expression of intrinsic fate determinants but not for the timing of birth dates. We found no changes in cell cycle exit timing but did find a significant delay in the expression of genes driving the generation only of later- but not earlier-born cells, suggesting that the robustness of this process depends on continuous feedback from earlier-formed cell types. Thus, extrinsic cues selectively influence cell fate determinant progression, which may explain the function of the retinal histogenic order observed. J. Comp. Neurol. 524:2553-2566, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Changes of primary visual center evoked potentials in response to white light and optic nerve electric stimulation were investigated during retinal GABAb-receptors activation with baclofen in dark-adapted carp. It was found, that baclofen - induced b-wave ERG decreasing, was accompanied by a significant amplitude growing as in the evoked potential to light as in the evoked potential to electric nerve stimulation: It is proposed, that light evoked potential changes reflect the increasing of the third retinal neuron responses to light and/or tectal neuron responsiveness enhancement.

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in developed countries, and its prevalence will increase as the global incidence of diabetes grows exponentially. DR begins with an early nonproliferative stage in which retinal blood vessels and neurons degenerate as a consequence of chronic hyperglycemia, resulting in vasoregression and persistent retinal ischemia, metabolic disequilibrium, and inflammation. This is conducive to overcompensatory pathological neovascularization associated with advanced proliferative DR. Although DR is considered a microvascular complication, the retinal microvasculature is intimately associated with and governed by neurons and glia; neurodegeneration, neuroinflammation, and dysregulation of neurovascular cross talk are responsible in part for vascular abnormalities in both early nonproliferative DR and advanced proliferative DR. Neuronal activity directly regulates microvascular dilation and blood flow in the process of neurovascular coupling. Retinal neurons also secrete guidance cues in response to injury, ischemia, or metabolic stress that may either promote or suppress vascular outgrowth, either alleviating or exacerbating DR, contingent on the stage of disease and retinal microenvironment. Neurodegeneration, impaired neurovascular coupling, and dysregulation of neuronal guidance cues are key events in the pathogenesis of DR, and correcting these events may prevent or delay development of advanced DR. The review discusses the mechanisms of neurovascular cross talk and its dysregulation in DR, and their potential therapeutic implications., (Copyright © 2016 the American Physiological Society.)

Objective: The field of retinal prosthetics has made major progress over the last decade, restoring visual percepts to people suffering from retinitis pigmentosa. The stimulation pulses used by present implants are suprathreshold, meaning individual pulses are designed to activate the retina. In this paper we explore subthreshold pulse sequences as an alternate stimulation paradigm. Subthreshold pulses have the potential to address important open problems such as fading of visual percepts when patients are stimulated at moderate pulse repetition rates and the difficulty in preferentially stimulating different retinal pathways., Approach: As a first step in addressing these issues we used Gaussian white noise electrical stimulation combined with spike-triggered averaging to interrogate whether a subthreshold sequence of pulses can be used to activate the mouse retina., Main Results: We demonstrate that the retinal network can integrate multiple subthreshold electrical stimuli under an experimental paradigm immediately relevant to retinal prostheses. Furthermore, these characteristic stimulus sequences varied in their shape and integration window length across the population of retinal ganglion cells., Significance: Because the subthreshold sequences activate the retina at stimulation rates that would typically induce strong fading (25 Hz), such retinal 'tickling' has the potential to minimize the fading problem. Furthermore, the diversity found across the cell population in characteristic pulse sequences suggests that these sequences could be used to selectively address the different retinal pathways (e.g. ON versus OFF). Both of these outcomes may significantly improve visual perception in retinal implant patients.

Understanding the molecular basis by which distinct cell types are specified is a central issue in retinogenesis and retinal disease development. Here we examined the role of LIM domain only 4 (Lmo4) in retinal development using both gain-of-function and loss-of-function approaches. By immunostaining, Lmo4 was found to be expressed in mouse retina from E10.5 to mature stages. Retroviral delivery of Lmo4 into retinal progenitor cells could promote the amacrine, bipolar and Müller cell fates at the expense of photoreceptors. It also inhibited the fate of early-born retinal ganglion cells. Using a dominant-negative form of Lmo4 which suppresses transcriptional activities of all LIM domain only factors, we demonstrated that LIM domain only factors are both necessary and sufficient for promoting amacrine and bipolar cell development, but not for the differentiation of ganglion, horizontal, Müller, or photoreceptor cells. Taken together, our study uncovers multiple roles of Lmo4 during retinal development and demonstrates the importance of LIM domain only factors in ensuring proper retinal cell specification and differentiation. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 900-915, 2016., (© 2015 Wiley Periodicals, Inc.)

The largest available cellular level connectivity map, of a 0.1 mm sample of the mouse retina Inner Plexiform Layer, was analysed using network models and visualized using spectral graph layouts and observed cell coordinates. This allows key nodes in the network to be identified with retinal neurons. Their strongest synaptic links can trace pathways in the network, elucidating possible circuits. Modular decomposition of the network, by sampling signal flows over nodes and links using the InfoMap method, shows discrete modules of cone bipolar cells that form a tiled mosaic in the retinal plane. The highest flow nodes, calculated by InfoMap, proved to be the most useful landmarks for elucidating possible circuits. Their dominant links to high flow amacrine cells reveal possible circuits linking bipolar through to ganglion cells and show an Off-On discrimination between the Left-Right sections of the sample. Circuits suggested by this analysis confirm known roles for some cells and point to roles for others.

The dynamic nature of the brain is critical for the success of treatments aimed at restoring vision at the retinal level. The success of these treatments relies highly on the functionality of the surviving neurons along the entire visual pathway. Electrophysiological properties at the retina level have been investigated during the progression of retinal degeneration; however, little is known about the changes in electrophysiological properties that occur in the primary visual cortex (V1) during the course of retinal degeneration. By conducting extracellular recording, we examined the electrophysiological properties of V1 in S334ter-line-3 rats (a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa patients). We measured the orientation tuning, spatial and temporal frequency tunings and the receptive field (RF) size for 127 V1 neurons from 11 S334ter-3 rats and 10 Long-Evans (LE) rats. V1 neurons in the S334ter-3 rats showed weaker orientation selectivity, lower optimal spatial and temporal frequency values and a smaller receptive field size compared to the LE rats. These results suggest that the visual cognitive ability significantly changes during retinal degeneration.

Unlabelled: Feedforward (FF) inhibition is a common motif in many neural networks. Typically, excitatory inputs drive both principal neurons and interneurons; the interneurons then inhibit the principal neurons, thereby regulating the strength and timing of the FF signal. The interneurons introduce a likely nonlinear processing step that could distort the excitation/inhibition (E/I) ratio in the principal neuron, potentially degrading the reliability of computation in the circuit. In the retina, FF inhibition is an essential feature of the circuitry underlying direction selectivity (DS): glutamatergic bipolar cells (BCs) provide excitatory input to direction-selective ganglion cells (DSGCs) and GABAergic starburst amacrine cells (SACs), and the SACs then provide FF inhibition onto DSGCs. Robust DS computation requires a consistent synaptic E/I ratio in the DSGC in various visual conditions. Here, we show in mouse retina that the E/I ratio is maintained in DSGCs over a wide stimulus contrast range due to compensatory mechanisms in the diverse population of presynaptic BCs. BC inputs to SACs exhibit higher contrast sensitivity, so that the subsequent nonlinear transformation in SACs reduces the contrast sensitivity of FF inhibition to match the sensitivity of direct excitatory inputs onto DSGCs. Measurements of light-evoked responses from individual BC synaptic terminals suggest that the distinct sensitivity of BC inputs reflects different contrast sensitivity between BC subtypes. Numerical simulations suggest that this network arrangement is crucial for reliable DS computation., Significance Statement: Properly balanced excitation and inhibition are essential for many neuronal computations across brain regions. Feedforward inhibition circuitry, in which a common excitatory source drives both the principal cell and an interneuron, is a typical mechanism by which neural networks maintain this balance. Feedforward circuits may become imbalanced at low stimulation levels, however, if the excitatory drive is too weak to overcome the activation threshold in the interneuron. Here we reveal how excitation and inhibition remain balanced in direction selective ganglion cells in the mouse retina over a wide visual stimulus range., (Copyright © 2016 the authors 0270-6474/16/365861-16$15.00/0.)

To gain insights into microgravity-induced ophthalmic changes (microgravity ocular syndrome), and as part of a project investigating effects of future planetary habitats, we investigated the effect of acute hypercapnia following 10-day bed rest and hypoxia on posterior eye structures. Female subjects (N = 7) completed three 10-day experimental interventions: 1) normoxic bed rest [NBR; partial pressure of inspired O2 (PiO2 ) = 132.9 ± 0.3 Torr]; 2) hypoxic ambulatory confinement (HAMB; PiO2 = 90.4 ± 0.3 Torr); and 3) hypoxic bed rest (HBR; n = 12; PiO2 = 90.4 ± 0.3 Torr). Before and on the last day of each intervention, optical coherence tomography (OCT) of the optic disk was performed, and the thicknesses of the retinal nerve fiber layer (RNFL), retina, and choroid were measured. OCT examinations were conducted with the subjects breathing the prevailing normocapnic breathing mixture (either normoxic or hypoxic) and then following a 10-min period of breathing the same gas mixture, but with the addition of 1% CO2 Choroidal thickness was greater during both bed-rest conditions (NBR and HBR) compared with the ambulatory (HAMB) condition (ANOVA, P < 0.001). Increases in RNFL thickness compared with baseline were observed in the hypoxic trials (HBR, P < 0.001; and HAMB, P = 0.021), but not the normoxic trial (NBR). A further increase in RNFL thickness (P = 0.019) was observed after the 10-min hypercapnic trial in the NBR condition only. The fact that choroidal thickness was not affected by Po2 or Pco2, but increased by bed rest, suggests a hydrostatic rather than a vasoactive effect. The increments in RNFL thickness were most likely associated with local hypoxia and hypercapnia-induced dilatation of the retinal blood vessels., (Copyright © 2016 the American Physiological Society.)

Purpose: To investigate the safety and efficacy of a newly-developed wide-field dual-array suprachoroidal-transretinal stimulation (STS) prosthesis in middle-sized animals., Methods: The prosthesis consisted of two arrays with 50 to 74 electrodes. To test the feasibility of implanting the prosthesis and its efficacy, the prosthesis was implanted for 14 days into two rabbits. Optical coherence tomography (OCT) and ophthalmoscopy were performed 7 and 14 days after the implantation. Then the rabbits were euthanized, eyes were enucleated, and the posterior segment of the eye was examined histologically. In a second experiment, the arrays were implanted into two cats, and their ability to elicit neural responses was determined by electrically evoked potentials (EEPs) at the chiasm and by optical imaging of the retina., Results: All arrays were successfully implanted, and no major complications occurred during the surgery or during the 2-week postoperative period. Neither OCT nor ophthalmoscopy showed any major complications or instability of the arrays. Histological evaluations showed only mild cellular infiltration and overall good retinal preservation. Stimulation of the retina by the arrays evoked EEPs recorded from the chiasm. Retinal imaging showed that the electrical pulses from the arrays altered the retinal images indicating an activation of retinal neurons. The thresholds were as low as 100 μA for a chiasm response and 300 μA for the retinal imaging., Conclusion: Implantation of a newly-developed dual-array STS prosthesis for 2 weeks in rabbits was feasible surgically, and safe. The results of retinal imaging showed that the dual-array system was able to activate retinal neurons. We conclude that the dual-array design can be implanted without complication and is able to activate retinal neurons and optic nerve axons.

Purpose: We examined the relationship between retinal nerve fiber layer (RNFL) thickness and cognitive function in a population of older British adults., Methods: Participants of the European Prospective Investigation of Cancer (EPIC) Norfolk cohort study underwent ophthalmic and cognitive assessment. Measurements of RNFL thickness were made using the Heidelberg Retina Tomograph (HRT). Cognitive testing included a short form of the Mini-Mental State Examination (SF-MMSE), an animal naming task, a letter cancellation task, the Hopkins Verbal Learning Test (HVLT), the National Adult Reading Test (NART), and the Paired Associates Learning Test. Multivariable linear regression models were used to assess associations of RNFL thickness with cognitive test scores, adjusted for age, sex, education level, social class, visual acuity, axial length, and history of cataract surgery., Results: Data were available from 5563 participants with a mean age of 67 years. A thicker HRT-derived RNFL thickness was associated with better scores for the SF-MMSE (0.06; 95% confidence interval [CI], [0.02, 0.10], P = 0.005), HVLT (0.16, 95% CI [0.03, 0.29]; P = 0.014), and NART (-0.24, 95% CI [-0.46, -0.02], P = 0.035). The associations of RNFL thickness with SF-MMSE and HVLT remained significant following further adjustment for NART., Conclusions: We found a significant association between HRT-derived RNFL thickness and scores from cognitive tests assessing global function, recognition, learning, episodic memory, and premorbid intelligence. However, the associations were weak and not currently of predictive value. Further research is required to confirm and clarify the nature of these associations, and identify biological mechanisms.

Purpose: Recent studies suggest that the neural retinal response to light is compromised in diabetes. Electroretinogram studies suggest that the dim light retinal rod pathway is especially susceptible to diabetic damage. The purpose of this study was to determine whether diabetes alters rod pathway signaling., Methods: Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ; 75 mg/kg), and confirmed by blood glucose levels > 200 mg/dL. Six weeks after the first injection, whole-cell voltage clamp recordings of spontaneous and light-evoked inhibitory postsynaptic currents from rod bipolar cells were made in dark-adapted retinal slices. Light-evoked excitatory currents from rod bipolar and AII amacrine cells, and spontaneous excitatory currents from AII amacrine cells were also measured. Receptor inputs were pharmacologically isolated. Immunohistochemistry was performed on whole mounted retinas., Results: Rod bipolar cells had reduced light-evoked inhibitory input from amacrine cells but no change in excitatory input from rod photoreceptors. Reduced light-evoked inhibition, mediated by both GABAA and GABAC receptors, increased rod bipolar cell output onto AII amacrine cells. Spontaneous release of GABA onto rod bipolar cells was increased, which may limit GABA availability for light-evoked release. These physiological changes occurred in the absence of retinal cell loss or changes in GABAA receptor expression levels., Conclusions: Our results indicate that early diabetes causes deficits in the rod pathway leading to decreased light-evoked rod bipolar cell inhibition and increased rod pathway output that provide a basis for the development of early diabetic visual deficits.

During early vertebrate eye development, a regulatory network of transcription factors regulates retinal cell differentiation and survival into adulthood. Among those factors, Krüppel-like factor 4 (KLF4) plays the dual role of maintaining the stem cell status of retinal progenitors cells and repressing the intrinsic axon regeneration ability in retinal ganglion cells (RGCs) after injury. This study further investigated whether KLF4 plays a role in early retinal cell differentiation or survival into adulthood. We examined different types of retinal neurons, including RGCs, amacrine cells, bipolar cells, Müller cells, and photoreceptor cells, in adult mice in which KLF4 was conditionally deleted in early retinal development using Chx10-promoted Cre by immunohistochemistry. We compared the numbers of retinal neurons and the thickness of photoreceptor and nerve fiber layers between Chx10-Cre-driven KLF4 deletion mice and wild-type mice. There was no significant difference in cell number among any of the retinal cell types or in photoreceptor layer thickness with KLF4 deletion during early development. The thickness of axon bundles in the nerve fiber layer in the Chx10 conditional KLF4 knock-out mice was greater than that in wild-type mice. These results suggest that KLF4 is not required for retinal cell differentiation or survival, but does normally limit retinal ganglion cell axon bundle thickness. These data support a hypothesis that KLF4 suppresses axon growth during development.

After a saccade, most MST neurons respond to moving visual stimuli that had existed in their post-saccadic receptive fields and turned off before the saccade ("trans-saccadic memory remapping"). Neuronal responses in higher visual processing areas are known to be modulated in relation to gaze angle to represent image location in spatiotopic coordinates. In the present study, we investigated the eye position effects after saccades and found that the gaze angle modulated the visual sensitivity of MST neurons after saccades both to the actually existing visual stimuli and to the visual memory traces remapped by the saccades. We suggest that two mechanisms, trans-saccadic memory remapping and gaze modulation, work cooperatively in individual MST neurons to represent a continuous visual world.

Whether neurons can restore their original connectivity patterns during circuit repair is unclear. Taking advantage of the regenerative capacity of zebrafish retina, we show here the remarkable specificity by which surviving neurons reassemble their connectivity upon regeneration of their major input. H3 horizontal cells (HCs) normally avoid red and green cones, and prefer ultraviolet over blue cones. Upon ablation of the major (ultraviolet) input, H3 HCs do not immediately increase connectivity with other cone types. Instead, H3 dendrites retract and re-extend to contact new ultraviolet cones. But, if regeneration is delayed or absent, blue-cone synaptogenesis increases and ectopic synapses are made with red and green cones. Thus, cues directing synapse specificity can be maintained following input loss, but only within a limited time period. Further, we postulate that signals from the major input that shape the H3 HC's wiring pattern during development persist to restrict miswiring after damage.