Ganglion Cell Activity Research Articles

Regulatory mechanism of melatonin on the retinal ganglion cell photoreaction in mice.

The study aimed to analyze the regulatory mechanism of melatonin (MLT) on the retinal ganglion cell photoreaction in mice. Forty-eight, 3-week-old healthy ICR mice, regardless of gender, were randomly divided into 4 groups. Group A was exposed to an illumination/dark time of 0 h/24 h, 6 h/18 h in group B, 12 h/12 h in group C and 18 h/6 h in group D, for up to 6 weeks. Four mice in each group were sacrificed at week 1, 3 and 6, respectively, for harvesting of retinal ganglion cells. ELISA was used to detect nocturnal plasma MLT levels at midnight. Immunohistochemistry was used to detect the expression of the retinal MLT receptor and the expression levels of inducible nitric oxide synthase (iNOS) and c-fos protein. The plasma MLT levels, MLT receptor levels and c-fos protein expression levels of group C, after 1, 3 and 6 weeks of light application, were the highest, followed by groups B and D, while group A had the lowest levels. For each illumination time, the iNOS levels of group C were the lowest and group A was the highest. Differences were all statistically significant (P<0.05). In conclusion, appropriate illumination regimens can increase c-fos protein, decrease iNOS activity and regulate the physiological activities of the retinal ganglion cells by regulating the expressions of MLT and its receptor.

Rabbit Retinal Ganglion Cell Activation Thresholds in Response to Various Electrical Stimulation Waveforms using a Different Spatial Resolution Electrode Array

Rabbit Retinal Ganglion Cell Activation Thresholds in Response to Various Electrical Stimulation Waveforms using a Different Spatial Resolution Electrode Array

Stimulation Strategies for Selective Activation of Retinal Ganglion Cells.

Retinal prosthetic implants have shown potential to restore partial vision to patients blinded by retinitis pigmentosa or dry age-related macular degeneration, via a camera-driven multielectrode array that electrically stimulates surviving retinal neurons. Commercial epi-retinal prostheses mostly use charge-balanced symmetric cathodic-first biphasic pulses to depolarize retinal ganglion cells (RGCs) and bipolar cells (BCs), resulting in the perception of light in blind patients. However, previous clinical study for patients with Argus II epiretinal implants reported most percepts evoked by single electrode stimulation were elongated and aligned with estimated axon path of retinal ganglion cells, suggesting the activation of axon bundles. In this project, using an established genetically encoded calcium indicator (GECI), we performed in vitro calcium imaging for different stimulation paradigms, focusing primarily on short duration pulse that can avoid axonal stimulation and selective activate targeted RGC soma. The findings support the possibility to manipulate the responses of RGCs through varying the stimulation waveform, thus potentially forming more ideal shape perception with higher spatial resolution in future retinal prosthesis design.

IPSC-Derived Retina Transplants Improve Vision in rd1 End-Stage Retinal-Degeneration Mice.

iPSC-Derived Retina Transplants Improve Vision in rd1 End-Stage Retinal-Degeneration Mice.

Present Molecular Limitations of ON-Bipolar Cell Targeted Gene Therapy.

Recent studies have demonstrated the safety and efficacy of ocular gene therapy based on adeno-associated viral vectors (AAVs). Accordingly, a surge in promising new gene therapies is entering clinical trials, including the first optogenetic therapy for vision restoration. To date, optogenetic therapies for vision restoration target either the retinal ganglion cells (GCs) or presynaptic ON-bipolar cells (OBCs). Initiating light responses at the level of the OBCs has significant advantages over optogenetic activation of GCs. For example, important neural circuitries in the inner retina, which shape the receptive fields of GCs, remain intact when activating the OBCs. Current drawbacks of AAV-mediated gene therapies targeting OBCs include (1) a low transduction efficiency, (2) off-target expression in unwanted cell populations, and (3) a poor performance in human tissue compared to the murine retina. Here, we examined side-by-side the performance of three state-of-the art AAV capsid variants, AAV7m8, AAVBP2, and AAV7m8(Y444F) in combination with the 4xGRM6-SV40 promoter construct in the healthy and degenerated mouse retina and in human post-mortem retinal explants. We find that (1) the 4xGRM6-SV40 promoter is not OBC specific, (2) that all AAV variants possess broad cellular transduction patterns, with differences between the transduction patterns of capsid variants AAVBP2 and AAV7m8 and, most importantly, (3) that all vectors target OBCs in healthy tissue but not in the degenerated rd1 mouse model, potentially limiting the possibilities for an OBC-targeted optogenetic therapy for vision restoration in the blind.

Choroideremia: melanopsin-mediated postillumination pupil relaxation is abnormally slow.

To investigate the rod-cone and melanopsin pupillary light response (PLR) pathways in choroideremia. Eight patients with choroideremia and 18 healthy age-matched controls underwent chromatic pupillometry by applying blue (463nm) and red light (643nm) at 100lux intensity to the right eye while recording pupil diameters. Absolute baseline pupil size (mm), normalized maximal pupil constriction and the early and late postillumination pupillary dilation, from 0 to 10seconds and 10 to 30seconds after the end of illumination, respectively, were determined. Postillumination responses to blue light were considered to be primarily driven by melanopsin activation of the intrinsic photosensitive retinal ganglion cells. Baseline pupil diameters were comparable in patients with choroideremia and control subjects (p=0.48). The maximum pupil constriction in patients with choroideremia was severely weakened in red light but only mildly weakened in blue light (p<0.05). Postillumination dilation of the pupil was normal after red illumination but extremely protracted after blue illumination. Also, in contrast to healthy subjects, no abrupt change in the dilation curve was seen in the patients after the end of blue illumination, the early-phase dilation being completely abolished (p<0.01). Rod-cone-driven pupil responses were decreased as expected in an outer retinal degeneration, and near-normal pupil constriction in blue light supports that the melanopsin system is normal. In contrast, the lack of brisk early-phase dilation after blue illumination in choroideremia is remarkable and may be interpreted to mean that the absence of photoreceptor inhibition promotes a tonic contraction of the pupil.

Pupillary responses to short-wavelength light are preserved in aging

With aging, less blue light reaches the retina due to gradual yellowing of the lens. This could result in reduced activation of blue light-sensitive melanopsin-containing retinal ganglion cells, which mediate non-visual light responses (e.g., the pupillary light reflex, melatonin suppression, and circadian resetting). Herein, we tested the hypothesis that older individuals show greater impairment of pupillary responses to blue light relative to red light. Dose-response curves for pupillary constriction to 469-nm blue light and 631-nm red light were compared between young normal adults aged 21–30 years (n = 60) and older adults aged ≥50 years (normal, n = 54; mild cataract, n = 107; severe cataract, n = 18). Irrespective of wavelength, pupillary responses were reduced in older individuals and further attenuated by severe, but not mild, cataract. The reduction in pupillary responses was comparable in response to blue light and red light, suggesting that lens yellowing did not selectively reduce melanopsin-dependent light responses. Compensatory mechanisms likely occur in aging that ensure relative constancy of pupillary responses to blue light despite changes in lens transmission.

The Spatial Extent of Epiretinal Electrical Stimulation in the Healthy Mouse Retina

Background/Aims: Retinal prostheses use electrical stimulation to restore functional vision to patients blinded by retinitis pigmentosa. A key detail is the spatial pattern of ganglion cells activated by stimulation. Therefore, we characterized the spatial extent of network-mediated electrical activation of retinal ganglion cells (RGCs) in the epiretinal monopolar electrode configuration. Methods: Healthy mouse RGC activities were recorded with a micro-electrode array (MEA). The stimuli consisted of monophasic rectangular cathodic voltage pulses and cycling full-field light flashes. Results: Voltage tuning curves exhibited significant hysteresis, reflecting adaptation to electrical stimulation on the time scale of seconds. Responses decreased from 0 to 300 µm, and were also dependent on the strength of stimulation. Applying the Rayleigh criterion to the half-width at half-maximum of the electrical point spread function suggests a visual acuity limit of no better than 20/946. Threshold voltage showed only a modest increase across these distances. Conclusion: The existence of significant hysteresis requires that future investigations of electrical retinal stimulation control for such long-memory adaptation. The spread of electrical activation beyond 200 µm suggests that neighbouring electrodes in epiretinal implants based on indirect stimulation of RGCs may be indiscriminable at interelectrode spacings as large as 400 µm.

Amplitude Modulation-based Electrical Stimulation for Encoding Multipixel Spatiotemporal Visual Information in Retinal Neural Activities.

Retinal implants have been developed as a promising way to restore partial vision for the blind. The observation and analysis of neural activities can offer valuable insights for successful prosthetic electrical stimulation. Retinal ganglion cell (RGC) activities have been investigated to provide knowledge on the requirements for electrical stimulation, such as threshold current and the effect of stimulation waveforms. To develop a detailed ‘stimulation strategy’ for faithful delivery of spatiotemporal visual information to the brain, it is essential to examine both the temporal and spatial characteristics of RGC responses, whereas previous studies were mainly focused on one or the other. In this study, we investigate whether the spatiotemporal visual information can be decoded from the RGC network activity evoked by patterned electrical stimulation. Along with a thorough characterization of spatial spreading of stimulation current and temporal information encoding, we demonstrated that multipixel spatiotemporal visual information can be accurately decoded from the population activities of RGCs stimulated by amplitude-modulated pulse trains. We also found that the details of stimulation, such as pulse amplitude range and pulse rate, were crucial for accurate decoding. Overall, the results suggest that useful visual function may be restored by amplitude modulation-based retinal stimulation.

Are long stimulus pulse durations the answer to improving spatial resolution in retinal prostheses?

Retinal prostheses can provide artificial vision to patients with degenerate retinae by electrically stimulating the remaining inner retinal neurons. The evoked perception is generally adequate for light localization, but of limited spatial resolution owing to the indiscriminate activation of multiple retinal cell types, leading to distortions in the perceived image. Here we present a perspective on a recent work by Weitz and colleagues who demonstrate a focal confinement of retinal ganglion cell (RGC) activation when using extended pulse durations in the stimulation waveform. Using real-time calcium imaging, they provide evidence that long pulse durations selectively stimulate inner retinal neurons, whilst avoiding unwanted axonal activations. The application of this stimulation technique may provide enhanced spatial resolution for retinal prosthesis users. These experiments provide a robust analysis of the effects of increasing pulse duration and introduce the potential for alternative stimulation paradigms in retinal prostheses.

An Evolutionarily Conserved Mechanism for Activity-Dependent Visual Circuit Development.

Neural circuit development is an activity-dependent process. This activity can be spontaneous, such as the retinal waves that course across the mammalian embryonic retina, or it can be sensory-driven, such as the activation of retinal ganglion cells (RGCs) by visual stimuli. Whichever the source, neural activity provides essential instruction to the developing circuit. Indeed, experimentally altering activity has been shown to impact circuit development and function in many different ways and in many different model systems. In this review, we contemplate the idea that retinal waves in amniotes, the animals that develop either in ovo or utero (namely reptiles, birds and mammals) could be an evolutionary adaptation to life on land, and that the anamniotes, animals whose development is entirely external (namely the aquatic amphibians and fish), do not display retinal waves, most likely because they simply don’t need them. We then review what is known about the function of both retinal waves and visual stimuli on their respective downstream targets, and predict that the experience-dependent development of the tadpole visual system is a blueprint of what will be found in future studies of the effects of spontaneous retinal waves on instructing development of retinorecipient targets such as the superior colliculus (SC) and the lateral geniculate nucleus.

Two light-activated neuroendocrine circuits arising in the eye trigger physiological and morphological pigmentation.

Two biological processes regulate light-induced skin colour change. A fast 'physiological pigmentation change' (i.e. circadian variations or camouflage) involves alterations in the distribution of pigment containing granules in the cytoplasm of chromatophores, while a slower 'morphological pigmentation change' (i.e. seasonal variations) entails changes in the number of pigment cells or pigment type. Although linked processes, the neuroendocrine coordination triggering each response remains largely obscure. By evaluating both events in Xenopus laevis embryos, we show that morphological pigmentation initiates by inhibiting the activity of the classical retinal ganglion cells. Morphological pigmentation is always accompanied by physiological pigmentation, and a melatonin receptor antagonist prevents both responses. Physiological pigmentation also initiates in the eye, but with repression of melanopsin-expressing retinal ganglion cell activity that leads to secretion of alpha-melanocyte-stimulating hormone (α-MSH). Our findings suggest a model in which eye photoperception links physiological and morphological pigmentation by altering α-MSH and melatonin production, respectively.

Liberal Representationalism: A Deflationist Defense

The idea that only complex brains can possess genuine representations is an important element in mainstream philosophical thinking. An alternative view, which I label ‘liberal representationalism’, holds that we should accept the existence of many more full-blown representations, from activity in retinal ganglion cells to the neural states produced by innate releasing mechanisms in cognitively unsophisticated organisms. A promising way of supporting liberal representationalism is to show it to be a consequence of our best naturalistic theories of representation. However, several philosophers and scientists have recently argued against this strategy. In the paper I counter these objections in defense of liberal representationalism.

Probing the functional impact of sub-retinal prosthesis.

Retinal prostheses are promising tools for recovering visual functions in blind patients but, unfortunately, with still poor gains in visual acuity. Improving their resolution is thus a key challenge that warrants understanding its origin through appropriate animal models. Here, we provide a systematic comparison between visual and prosthetic activations of the rat primary visual cortex (V1). We established a precise V1 mapping as a functional benchmark to demonstrate that sub-retinal implants activate V1 at the appropriate position, scalable to a wide range of visual luminance, but with an aspect-ratio and an extent much larger than expected. Such distorted activation profile can be accounted for by the existence of two sources of diffusion, passive diffusion and activation of ganglion cells' axons en passant. Reverse-engineered electrical pulses based on impedance spectroscopy is the only solution we tested that decreases the extent and aspect-ratio, providing a promising solution for clinical applications.

A computational model of orientation-dependent activation of retinal ganglion cells.

Currently, a challenge in electrical stimulation for epiretinal prostheses is the avoidance of stimulation of axons of passage in the nerve fiber layer that originate from distant regions of the ganglion cell layer. A computational model of extracellular stimulation that captures the effect of neurite orientation in anisotropic tissue is developed using a modified version of the standard volume conductor model, known as the cellular composite model, embedded in a four layer model of the retina. Simulations are conducted to investigate the interaction of neural tissue orientation, electrode placement, and stimulation pulse duration and amplitude. Using appropriate multiple electrode configurations and higher frequency stimulation, preferential activation of the axon initial segment is shown to be possible for a range of realistic electrode-retina separation distances. These results establish a quantitative relationship between the time-course of stimulation and physical properties of the tissue, such as fiber orientation.

Neural activity promotes long-distance, target-specific regeneration of adult retinal axons.

Axons in the mammalian central nervous system (CNS) fail to regenerate after injury. Here we show that if retinal ganglion cell (RGC) activity is increased by visual stimulation or using chemogenetics, their axons regenerate. We also show that if enhancement of neural activity is combined with elevation of the cell growth-promoting pathway involving mammalian target of rapamycin (mTOR), RGC axons regenerate the long distances necessary to re-innervate the brain. Analysis of genetically-labeled RGCs revealed this regrowth can be target specific: RGC axons navigated back to their correct visual targets and avoided targets incorrect for their function. Moreover, these regenerated connections were successful in partially rescuing a subset of visual behaviors. Our findings indicate that combining neural activity with activation of mTOR can serve as powerful tool for enhancing axon regeneration and they highlight the remarkable capacity of CNS neurons to re-establish accurate circuit connections in the adult brain.

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

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

Brimonidine Enhances the Electrophysiological Response of Retinal Ganglion Cells through the Trk-MAPK/ERK and PI3K Pathways in Axotomized Eyes

ABSTRACTPurpose: To investigate changes in retinal ganglion cell (RGC) activity by measuring the positive scotopic threshold response (pSTR) of the electroretinogram (ERG) in axotomized eyes after brimonidine injection.Methods: In 50 adult Sprague-Dawley rats, the left eye was axotomized and injected with phosphate buffered saline (PBS) or brimonidine and the contralateral right eye was left untreated. Scotopic ERGs were recorded simultaneously from both eyes on days 1, 2, 3, 7, and 10 after the intravitreal injection, and the amplitude of the a- and b-waves and the pSTR were measured. Surviving RGCs in the flat-mounted retinas were counted 10 days after axotomy. In addition to brimonidine, K252a (an inhibitor of tyrosine kinase phosphorylation of the Trk receptors), U0126 (a MAPK/ERK kinase inhibitor), and LY294002 (phosphoinositide 3-kinases [PI3Ks]) were also injected intravitreally into the left eye, and ERGs were recorded using the same protocol.Results: The pSTR amplitude increased significantly in the axotomized eyes with brimonidine, to 122.9 ± 5.0%, 161.8 ± 8.3%, and 133.6 ± 8.1% on days 1, 2, and 3 (P < 0.01), respectively, compared to the axotomized eyes treated with PBS (control). The increased pSTR amplitude returned to normal (103.6 ± 6.7%) on day 7, although there were a greater number of surviving RGCs in the treatment groups than in the controls. The intravitreal injection of K252a, U0126, or LY294002 significantly attenuated the increase in pSTR induced by intravitreal brimonidine (P < 0.01).Conclusion: Intravitreal brimonidine enhanced the survival and electrophysiological activity of the RGCs in rats. The mechanism of this electrophysiological change may involve activation of the Trk-MAPK/ERK and Trk-PI3K signals.

270. Optogenetic Activation of Remnant Cone Cells Can Restore Ganglion Cell Light Responses in a Canine Model of Retinitis Pigmentosa

In retinitis pigmentosa, the rod photoreceptor cells are generally lost first followed by a slow loss of cone cells. Remnant cone cell bodies have been observed even after many years of vision loss in retinitis pigmentosa patients. Proof-of-principle studies in retinal degeneration mice have demonstrated vision restoration upon expression of the optogene halorhodopsin in remnant cone cells. However, whether this will work in a large animal model of retinal degeneration is unclear. Here we look at light responses and histology post-treatment with halorhodopsin in a dog model of autosomal recessive retinitis pigmentosa. Halorhodopsin was expressed in the cone cells unilaterally in rcd1 dogs (PDE6β mutation) with mid- to late stage retinal degeneration using recombinant adeno-associated viral vectors. The retinas were imaged using OCT and function was evaluated using ERG and pupillometry. The spiking activity of retinal ganglion cells (RGCs) in retinal patches adjacent to and distant from the point of injection was tested using multi electrode array recordings. Retinal patches were stimulated with 589 nm light of different durations/intensities. OCT revealed significant thinning of the photoreceptor layer in the retinas of the dogs. While the ERG and pupillometry data did not show a significant difference between the treated and untreated eyes, multi-electrode array recordings revealed a restoration of ganglion cell light responses (ON, OFF and ON/OFF-responses to discrete flashes and responses to 4Hz flicker, 589nm light) in the treated retinal patch compared to an untreated control patch of retina. The lowest intensities where responses become clearly detectable for two animals were 0.1 mW/cm2 and 2.8 mW/cm2 probably reflecting different levels of NpHR expression. We observed halorhodopsin expression by histology in the remnant cone cell bodies of the treated retinas. Restoration of ganglion cell responses in the treated retinal patches suggests that the retina can be sensitized to light even after major photoreceptor cell loss. Ongoing work will determine if this restoration of ganglion cell light responses in the dogs translate to being able to navigate in the light.

Distal retinal ganglion cell axon transport loss and activation of p38 MAPK stress pathway following VEGF-A antagonism.

There is increasing evidence that VEGF-A antagonists may be detrimental to neuronal health following ocular administration. Here we investigated firstly the effects of VEGF-A neutralization on retinal neuronal survival in the Ins2Akita diabetic and JR5558 spontaneous choroidal neovascularization (CNV) mice, and then looked at potential mechanisms contributing to cell death. We detected elevated apoptosis in the ganglion cell layer in both these models following VEGF-A antagonism, indicating that even when vascular pathologies respond to treatment, neurons are still vulnerable to reduced VEGF-A levels. We observed that retinal ganglion cells (RGCs) seemed to be the cells most susceptible to VEGF-A antagonism, so we looked at anterograde transport in these cells, due to their long axons requiring optimal protein and organelle trafficking. Using cholera toxin B-subunit tracer studies, we found a distal reduction in transport in the superior colliculus following VEGF-A neutralization, which occurred prior to net RGC loss. This phenomenon of distal transport loss has been described as a feature of early pathological changes in glaucoma, Alzheimer's and Parkinson's disease models. Furthermore, we observed increased phosphorylation of p38 MAPK and downstream Hsp27 stress pathway signaling in the retinas from these experiments, potentially providing a mechanistic explanation for our findings. These experiments further highlight the possible risks of using VEGF-A antagonists to treat ocular neovascular disease, and suggest that VEGF-A may contribute to the maintenance and function of axonal transport in neurons of the retina.