Retinal Ganglion Cell Dendrites Research Articles

Shootin1 Regulates Retinal Ganglion Cell Neurite Development: Insights From an RGC Direct Somatic Cell Reprogramming Model.

Retinal ganglion cells (RGCs) connect the retina to the brain. Proper development of the axons and dendrites of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of Shtn1 (which encodes shootin1) in RGC neurite development. Immunofluorescence (IF) was used to characterize the expression pattern of marker genes. An in vitro direct somatic cell reprogramming system was used to generate RGC-like neurons (iRGCs), which was subsequently used to study the function of Shtn1. Short-hairpin RNAs (shRNAs) were used to knock down Shtn1, and the coding sequence (CDS) of Shtn1 was used to overexpress the gene. Lentiviruses were used to deliver shRNAs or CDSs into iRGCs. The patch clamp technique was used to measure the electrophysiological properties of the iRGCs. RNA sequencing (RNA-seq) was used to examine transcriptome expression. Using IF, we demonstrated that shootin1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites and thus the electrophysiological maturation, of iRGCs. RNA-seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. Shtn1 promotes RGC neurite development. These findings improve our understanding of the molecular machinery governing RGC neurite development and may help to optimize future RGC regeneration methods.

Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

Neuroprotection and axon regeneration in glaucoma models by gene therapy using modified tyrosine kinase receptors

Aims/Purpose: To determine whether gene therapy using the modified tyrosine kinase receptors (insulin receptor or TrkB) can protect retinal ganglion cells (RGCs) and stimulate axon regeneration in glaucoma model mice.Methods: To examine the effects on the signalling pathways, Cos7 cells were transfected with intracellular region of insulin receptor (iIR) or TrkB (iTrkB) for 24 h, and immunoblot analysis was performed using antibodies against total and phosphorylated ERK and AKT. To evaluate their neuroprotective effects in vivo, the modified IR or TrkB was overexpressed in RGCs in GLAST knockout mice, a mouse model of normal tension glaucoma, by an intraocular injection of AAV‐iIR or AAV‐iTrkB. For optic nerve crush (ONC) model, AAV‐iIR or AAV‐iTrkB was injected into the mouse eyes 2 weeks before ONC. The number of surviving RGCs was examined by whole‐mount immunostaining of the retina with an RBPMS antibody. RGC dendrites were visualized by GFP labeling and quantified using the microscopy image analysis software IMARIS. Optic nerve regeneration was examined by the intraocular injection of Cholera Toxin Subunit B at 2 days before sacrifice.Results: iIR and iTrkB transfection in vitro both strongly induced phosphorylation of ERK and AKT, and in vivo, AAV‐iIR and AAV‐iTrkB both significantly increased the number of surviving RGCs in GLAST knockout mice and ONC mice. Moreover, AAV‐iIR and AAV‐iTrkB promoted axon regeneration in ONC mice and preserved the structure of RGC dendrites.Conclusions: Our system overcomes the small size limitation of the genome packaging in AAV and may be useful for the treatment of retinal and optic nerve degeneration.References Nishijima E, Honda S, Kitamura Y, Namekata K, Kimura A, Guo X, Azuchi Y, Harada C, Murakami A, Matsuda A, Nakano T, Parada LF, Harada T. Vision protection and robust axon regeneration in glaucoma models by membrane‐associated Trk receptors. Molecular Therapy 31(3), 810–824, 2023. doi:10.1016/j.ymthe.2022.11.018

Longitudinal in vivo evaluation of retinal ganglion cell complex layer and dendrites in mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE), induced by the immunization of myelin oligodendrocyte glycoprotein (MOG), is related to human MOG antibody-associated disease (MOGAD). Neuroinflammation and demyelination of the optic nerve can lead to retinal ganglion cell (RGC) death and axonal damage in MOGAD. Here, we aimed to evaluate the structural changes in RGCs longitudinally by in vivo imaging in mice with RGCs expressing yellow fluorescent protein along the course of EAE. Successful induction of EAE was confirmed by the neurological function scores and histology analyses. The changes in the thickness of ganglion cell complex (GCC) layer and RGC survival and dendrites were monitored longitudinally along the course of EAE. Before the onset of EAE, there were no significant changes in the number and morphology of RGCs and the thickness of the GCC layer as compared to the mice without EAE induction. After the onset of EAE, the thickness of the GCC layer and the RGC number and dendritic network all gradually decreased along the course of EAE. Notably, dendritic shrinkage could be detected earlier than the thinning of the GCC layer. In summary, this study delineated the longitudinal profile of RGC structural changes in EAE mice, providing an assessment platform for monitoring outcomes of RGC treatments.

Bipolar cells containing protein kinase Cα mediate attentional facilitation of the avian retinal ganglion cells by the retinopetal signal.

Birds have a well-developed retinopetal system projecting from the midbrain to the contralateral retina. The signal sent to the retina through the retinopetal system facilitates visual responses of the retinal ganglion cells (RGCs), and the retinopetal signals function as attentional signals during visual search. Thus, the retinopetal signal somehow reaches and facilitates visual responses of the RGCs. However, the tertiary neuron of the retinopetal system, the isthmo-optic target cell (IOTC), is unlikely to contact most RGCs directly, because the IOTCs form axon terminals localized in the outermost sublayer (lamina 1) of the inner plexiform layer (IPL) where few RGC dendrites terminate. Therefore, some other intrinsic retinal neurons must be involved in the centrifugal attentional enhancement of visual responses of the RGCs. We investigated connections of the target cells of the IOTCs in chicken and quail, using light and electron microscopic immunohistochemistry. We show that axon terminals of the IOTC make synaptic contacts with protein kinase Cα (PKCα)-immunoreactive (ir) bipolar cells (PKCα-BCs) in lamina 1 of the IPL. Furthermore, with prolonged electrical stimulation of the isthmo-optic nucleus (ION) on one side, whose neurons send their axons to the contralateral retina and make synaptic contacts there with IOTCs, phosphorylation of cAMP response element-binding protein was observed in the PKCα-BCs in the contralateral retina, but not in the ipsilateral retina. This suggests that electrical stimulation of ION activated PKCα-BCs through synapses from IOTCs to PKCα-BCs, thus stimulating transcription in PKCα-BCs. Thus, centrifugal attentional signals may facilitate visual responses of RGCs via the PKCα-BCs.

Brain-derived neurotrophic factor released from blood platelets prevents dendritic atrophy of lesioned adult central nervous system neurons.

In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the BDNF gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the Bdnf gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using 'humanized' mice engineered to express the Bdnf gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 ± 11.36 ng/mL for homozygous and 17.02 ± 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 ± 258, 1776 ± 435 and 1763 ± 256 versus 1406 ± 315 in the wild-type control group (P ≤ 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing ∼15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 ± 690 and 1921 ± 392, P = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing ∼50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an ex vivo and in vivo model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.

The adenosine A2A receptor antagonist KW6002 distinctly regulates retinal ganglion cell morphology during postnatal development and neonatal inflammation.

Adenosine A2A receptors (A2ARs) appear early in the retina during postnatal development, but the roles of the A2ARs in the morphogenesis of distinct types of retinal ganglion cells (RGCs) during postnatal development and neonatal inflammatory response remain undetermined. As the RGCs are rather heterogeneous in morphology and functions in the retina, here we resorted to the Thy1-YFPH transgenic mice and three-dimensional (3D) neuron reconstruction to investigate how A2ARs regulate the morphogenesis of three morphologically distinct types of RGCs (namely Type I, II, III) during postnatal development and neonatal inflammation. We found that the A2AR antagonist KW6002 did not change the proportion of the three RGC types during retinal development, but exerted a bidirectional effect on dendritic complexity of Type I and III RGCs and cell type-specifically altered their morphologies with decreased dendrite density of Type I, decreased the dendritic field area of Type II and III, increased dendrite density of Type III RGCs. Moreover, under neonatal inflammation condition, KW6002 specifically increased the proportion of Type I RGCs with enhanced the dendrite surface area and volume and the proportion of Type II RGCs with enlarged the soma area and perimeter. Thus, A2ARs exert distinct control of RGC morphologies to cell type-specifically fine-tune the RGC dendrites during normal development but to mainly suppress RGC soma and dendrite volume under neonatal inflammation.

Optic nerve injury-induced regeneration in the adult zebrafish is accompanied by spatiotemporal changes in mitochondrial dynamics

Axonal regeneration in the central nervous system is an energy-intensive process. In contrast to mammals, adult zebrafish can functionally recover from neuronal injury. This raises the question of how zebrafish can cope with this high energy demand. We previously showed that in adult zebrafish, subjected to an optic nerve crush, an antagonistic axon-dendrite interplay exists wherein the retraction of retinal ganglion cell dendrites is a prerequisite for effective axonal repair. We postulate a 'dendrites for regeneration' paradigm that might be linked to intraneuronal mitochondrial reshuffling, as ganglion cells likely have insufficient resources to maintain dendrites and restore axons simultaneously. Here, we characterized both mitochondrial distribution and mitochondrial dynamics within the different ganglion cell compartments (dendrites, somas, and axons) during the regenerative process. Optic nerve crush resulted in a reduction of mitochondria in the dendrites during dendritic retraction, whereafter enlarged mitochondria appeared in the optic nerve/tract during axonal regrowth. Upon dendritic regrowth in the retina, mitochondrial density inside the retinal dendrites returned to baseline levels. Moreover, a transient increase in mitochondrial fission and biogenesis was observed in retinal ganglion cell somas after optic nerve damage. Taken together, these findings suggest that during optic nerve injury-induced regeneration, mitochondria shift from the dendrites to the axons and back again and that temporary changes in mitochondrial dynamics support axonal and dendritic regrowth after optic nerve crush.

Influences of Glaucoma on the Structure and Function of Synapses in the Visual System.

Significance: Glaucoma is an age-related neurodegenerative disorder of the visual system associated with sensitivity to intraocular pressure (IOP). It is the leading irreversible cause of vision loss worldwide, and vision loss results from damage and dysfunction of the retinal output neurons known as retinal ganglion cells (RGCs). Recent Advances: Elevated IOP and optic nerve injury triggers pruning of RGC dendrites, altered morphology of excitatory inputs from presynaptic bipolar cells, and disrupted RGC synaptic function. Less is known about RGC outputs, although evidence to date indicates that glaucoma is associated with altered mitochondrial and synaptic structure and function in RGC-projection targets in the brain. These early functional changes likely contribute to vision loss and might be a window into early diagnosis and treatment. Critical Issues: Glaucoma affects different RGC populations to varying extents and along distinct time courses. The influence of glaucoma on RGC synaptic function as well as the mechanisms underlying these effects remain to be determined. Since RGCs are an especially energetically demanding population of neurons, altered intracellular axon transport of mitochondria and mitochondrial function might contribute to RGC synaptic dysfunction in the retina and brain as well as RGC vulnerability in glaucoma. Future Directions: The mechanisms underlying differential RGC vulnerability remain to be determined. Moreover, the timing and mechanisms of RGCs synaptic dysfunction and degeneration will provide valuable insight into the disease process in glaucoma. Future work will be able to capitalize on these findings to better design diagnostic and therapeutic approaches to detect disease and prevent vision loss. Antioxid. Redox Signal. 37, 842-861.

The m6A reader YTHDF2 is a negative regulator for dendrite development and maintenance of retinal ganglion cells.

The precise control of growth and maintenance of the retinal ganglion cell (RGC) dendrite arborization is critical for normal visual functions in mammals. However, the underlying mechanisms remain elusive. Here, we find that the N6-methyladenosine (m6A) reader YTHDF2 is highly expressed in the mouse RGCs. Conditional knockout (cKO) of Ythdf2 in the retina leads to increased RGC dendrite branching, resulting in more synapses in the inner plexiform layer. Interestingly, the Ythdf2 cKO mice show improved visual acuity compared with control mice. We further demonstrate that Ythdf2 cKO in the retina protects RGCs from dendrite degeneration caused by the experimental acute glaucoma model. We identify the m6A-modified YTHDF2 target transcripts which mediate these effects. This study reveals mechanisms by which YTHDF2 restricts RGC dendrite development and maintenance. YTHDF2 and its target mRNAs might be valuable in developing new treatment approaches for glaucomatous eyes.

Immunohistochemical Characterisation of the Whale Retina.

The eye of the largest adult mammal in the world, the whale, offers a unique opportunity to study the evolution of the visual system and its adaptation to aquatic environments. However, the difficulties in obtaining cetacean samples mean these animals have been poorly studied. Thus, the aim of this study was to characterise the different neurons and glial cells in the whale retina by immunohistochemistry using a range of molecular markers. The whale retinal neurons were analysed using different antibodies, labelling retinal ganglion cells (RGCs), photoreceptors, bipolar and amacrine cells. Finally, glial cells were also labelled, including astrocytes, Müller cells and microglia. Thioflavin S was also used to label oligomers and plaques of misfolded proteins. Molecular markers were used to label the specific structures in the whale retinas, as in terrestrial mammalian retinas. However, unlike the retina of most land mammals, whale cones do not express the cone markers used. It is important to highlight the large size of whale RGCs. All the neurofilament (NF) antibodies used labelled whale RGCs, but not all RGCs were labelled by all the NF antibodies used, as it occurs in the porcine and human retina. It is also noteworthy that intrinsically photosensitive RGCs, labelled with melanopsin, form an extraordinary network in the whale retina. The M1, M2, and M3 subtypes of melanopsin positive-cells were detected. Degenerative neurite beading was observed on RGC axons and dendrites when the retina was analysed 48 h post-mortem. In addition, there was a weak Thioflavin S labelling at the edges of some RGCs in a punctuate pattern that possibly reflects an early sign of neurodegeneration. In conclusion, the whale retina differs from that of terrestrial mammals. Their monochromatic rod vision due to the evolutionary loss of cone photoreceptors and the well-developed melanopsin-positive RGC network could, in part, explain the visual perception of these mammals in the deep sea.

Exposure to Blue Light Reduces Melanopsin Expression in Intrinsically Photoreceptive Retinal Ganglion Cells and Damages the Inner Retina in Rats.

PurposeThe purpose of this study was to investigative the effects of blue light on intrinsically photoreceptive retinal ganglion cells (ipRGCs).MethodsBrown Norway rats were used. Nine rats were continuously exposed to blue light (light emitting diodes [LEDs]: 463 nm; 1000 lx) for 2 days (acute exposure [AE]); 9 rats were exposed to 12 hours of blue light and 12 hours of darkness for 10 days (long-term exposure [LTE]); 6 control rats were exposed to 12 hours of white fluorescent light (1000 lx) and 12 hours of darkness for 10 days. Whole-mount retinas were immunolabelled with melanopsin antibodies; melanopsin-positive (MP) ipRGC somas and processes were counted and measured with Neuron J. To detect apoptosis, retinal cryo-sections were stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling. Ultra-thin sections were visualized with transmission electron microscopy.ResultsThe number of MP ipRGC somas was significantly lower in retinas from AE and LTE rats than in those from control rats (P < 0.001 and = 0.002, respectively). The mean length of MP areas of processes was significantly lower in AE rats (P < 0.001). AE rats had severe retinal damage and massive apoptosis in the outer nuclear layer; their mitochondria were damaged in the axons and dendrites of the nerve fiber layer and the inner plexiform layer. Retinal ganglion cells (RGCs) in AE rats appeared to have reduced amounts of free ribosomes and rough endoplasmic reticulum.ConclusionsAE to blue light reduces melanopsin expression and damages RGCs, likely including ipRGCs. Changes in the axons and dendrites of RGCs suggest possible disruption of intraretinal and extraretinal signal transmission.

The cell adhesion molecule Sdk1 shapes assembly of a retinal circuit that detects localized edges.

Nearly 50 different mouse retinal ganglion cell (RGC) types sample the visual scene for distinct features. RGC feature selectivity arises from their synapses with a specific subset of amacrine (AC) and bipolar cell (BC) types, but how RGC dendrites arborize and collect input from these specific subsets remains poorly understood. Here we examine the hypothesis that RGCs employ molecular recognition systems to meet this challenge. By combining calcium imaging and type-specific histological stains, we define a family of circuits that express the recognition molecule Sidekick-1 (Sdk1), which include a novel RGC type (S1-RGC) that responds to local edges. Genetic and physiological studies revealed that Sdk1 loss selectively disrupts S1-RGC visual responses, which result from a loss of excitatory and inhibitory inputs and selective dendritic deficits on this neuron. We conclude that Sdk1 shapes dendrite growth and wiring to help S1-RGCs become feature selective.

Progression of optic atrophy in traumatic optic neuropathy: retrograde neuronal degeneration in humans.

We used optical coherence tomography (OCT) to document the time course of retrograde neuronal degeneration following indirect optic nerve injury. We retrospectively studied patients diagnosed with unilateral indirect traumatic optic neuropathy (TON). Patients with total or near-total optic atrophy were included. All patients underwent complete ophthalmological examinations, including OCT imaging, within 1day and at 1, 2, 3, 4, 6, 8, 12, 24, and 48weeks after trauma. The mean thicknesses of the circumpapillary retinal nerve fiber layer (cpRNFL) and macular retinal ganglion cell-inner plexiform layer (mGCIPL) decreased significantly at 2weeks after trauma (p = 0.027 and p = 0.043). Changes in mGCIPL thickness preceded changes in cpRNFL thickness. The rates of reduction in mGCIPL and cpRNFL thicknesses were greatest between 2 to 4weeks and 4 to 6weeks after trauma. The reduction in mGCIPL thickness then slowed, and stabilized at 12weeks after trauma. The proportions of cpRNFL and mGCIPL losses at 2, 4, 6, 8, and 12weeks compared to 24weeks were 17.1, 33.7, 59.8, 77.9, and 87.9% and 30.0, 73.3, 76.1, 88.3, and 97.9%, respectively. OCT revealed optic atrophy progression 2weeks after trauma, which was most rapid from 2 to 6weeks, and then gradually stabilized. Loss of retinal ganglion cell bodies and dendrites seemed to precede the axonal degeneration. Observations of morphological changes in retinal layers using OCT in TON patients improve our understanding of retrograde neuronal degeneration of the central nervous system.

AMPK hyperactivation promotes dendrite retraction, synaptic loss, and neuronal dysfunction in glaucoma

BackgroundThe maintenance of complex dendritic arbors and synaptic transmission are processes that require a substantial amount of energy. Bioenergetic decline is a prominent feature of chronic neurodegenerative diseases, yet the signaling mechanisms that link energy stress with neuronal dysfunction are poorly understood. Recent work has implicated energy deficits in glaucoma, and retinal ganglion cell (RGC) dendritic pathology and synapse disassembly are key features of ocular hypertension damage.ResultsWe show that adenosine monophosphate-activated protein kinase (AMPK), a conserved energy biosensor, is strongly activated in RGC from mice with ocular hypertension and patients with primary open angle glaucoma. Our data demonstrate that AMPK triggers RGC dendrite retraction and synapse elimination. We show that the harmful effect of AMPK is exerted through inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Attenuation of AMPK activity restores mTORC1 function and rescues dendrites and synaptic contacts. Strikingly, AMPK depletion promotes recovery of light-evoked retinal responses, improves axonal transport, and extends RGC survival.ConclusionsThis study identifies AMPK as a critical nexus between bioenergetic decline and RGC dysfunction during pressure-induced stress, and highlights the importance of targeting energy homeostasis in glaucoma and other neurodegenerative diseases.

Neuroprotection by WldS depends on retinal ganglion cell type and age in glaucoma

BackgroundEarly challenges to axonal physiology, active transport, and ultrastructure are endemic to age-related neurodegenerative disorders, including those affecting the optic nerve. Chief among these, glaucoma causes irreversible vision loss through sensitivity to intraocular pressure (IOP) that challenges retinal ganglion cell (RGC) axons, which comprise the optic nerve. Early RGC axonopathy includes distal to proximal progression that implicates a slow form of Wallerian degeneration. In multiple disease models, including inducible glaucoma, expression of the slow Wallerian degeneration (WldS) allele slows axon degeneration and confers protection to cell bodies.MethodsUsing an inducible model of glaucoma along with whole-cell patch clamp electrophysiology and morphological analysis, we tested if WldS also protects RGC light responses and dendrites and, if so, whether this protection depends upon RGC type. We induced glaucoma in young and aged mice to determine if neuroprotection by WldS on anterograde axonal transport and spatial contrast acuity depends on age.ResultsWe found WldS protects dendritic morphology and light-evoked responses of RGCs that signal light onset (αON-Sustained) during IOP elevation. However, IOP elevation significantly reduces dendritic complexity and light responses of RGCs that respond to light offset (αOFF-Sustained) regardless of WldS. As expected, WldS preserves anterograde axon transport and spatial acuity in young adult mice, but its protection is significantly limited in aged mice.ConclusionThe efficacy of WldS in conferring protection to neurons and their axons varies by cell type and diminishes with age.

Organization and emergence of a mixed GABA-glycine retinal circuit that provides inhibition to mouse ON-sustained alpha retinal ganglion cells

SUMMARYIn the retina, amacrine interneurons inhibit retinal ganglion cell (RGC) dendrites to shape retinal output. Amacrine cells typically use either GABA or glycine to exert synaptic inhibition. Here, we combined transgenic tools with immunohistochemistry, electrophysiology, and 3D electron microscopy to determine the composition and organization of inhibitory synapses across the dendritic arbor of a well-characterized RGC type in the mouse retina: the ON-sustained alpha RGC. We find mixed GABA-glycine receptor synapses across this RGC type, unveiling the existence of “mixed” inhibitory synapses in the retinal circuit. Presynaptic amacrine boutons with dual release sites are apposed to ON-sustained alpha RGC postsynapses. We further reveal the sequence of postsynaptic assembly for these mixed synapses: GABA receptors precede glycine receptors, and a lack of early GABA receptor expression impedes the recruitment of glycine receptors. Together our findings uncover the organization and developmental profile of an additional motif of inhibition in the mammalian retina.

Differential Distribution of RBPMS in Pig, Rat, and Human Retina after Damage.

RNA binding protein with multiple splicing (RBPMS) is expressed exclusively in retinal ganglion cells (RGCs) in the retina and can label all RGCs in normal retinas of mice, rats, guinea pigs, rabbits, cats, and monkeys, but its function in these cells is not known. As a result of the limited knowledge regarding RBPMS, we analyzed the expression of RBPMS in the retina of different mammalian species (humans, pigs, and rats), in various stages of development (neonatal and adult) and with different levels of injury (control, hypoxia, and organotypic culture or explants). In control conditions, RBPMS was localized in the RGCs somas in the ganglion cell layer, whereas in hypoxic conditions, it was localized in the RGCs dendrites in the inner plexiform layer. Such differential distributions of RBPMS occurred in all analyzed species, and in adult and neonatal retinas. Furthermore, we demonstrate RBPMS localization in the degenerating RGCs axons in the nerve fiber layer of retinal explants. This is the first evidence regarding the possible transport of RBPMS in response to physiological damage in a mammalian retina. Therefore, RBPMS should be further investigated in relation to its role in axonal and dendritic degeneration.

Tyro3 Contributes to Retinal Ganglion Cell Function, Survival and Dendritic Density in the Mouse Retina.

Retinal ganglion cells (RGCs) are the only output neurons of the vertebrate retina, integrating signals from other retinal neurons and transmitting information to the visual centers of the brain. The death of RGCs is a common outcome in many optic neuropathies, such as glaucoma, demyelinating optic neuritis and ischemic optic neuropathy, resulting in visual defects and blindness. There are currently no therapies in clinical use which can prevent RGC death in optic neuropathies; therefore, the identification of new targets for supporting RGC survival is crucial in the development of novel treatments for eye diseases. In this study we identify that the receptor tyrosine kinase, Tyro3, is critical for normal neuronal function in the adult mouse retina. The loss of Tyro3 results in a reduction in photoreceptor and RGC function as measured using electroretinography. The reduction in RGC function was associated with a thinner retinal nerve fiber layer and fewer RGCs. In the central retina, independent of the loss of RGCs, Tyro3 deficiency resulted in a dramatic reduction in the number of RGC dendrites in the inner plexiform layer. Our results show that Tyro3 has a novel, previously unidentified role in retinal function, RGC survival and RGC morphology. The Tyro3 pathway could therefore provide an alternative, targetable pathway for RGC protective therapeutics.

Distinct Developmental Mechanisms Act Independently to Shape Biased Synaptic Divergence from an Inhibitory Neuron.

Neurons often contact more than one postsynaptic partner type and display stereotypic patterns of synaptic divergence. Such synaptic patterns usually involve some partners receiving more synapses than others. The developmental strategies generating "biased" synaptic distributions remain largely unknown. To gain insight, we took advantage of a compact circuit in the vertebrate retina, whereby the AII amacrine cell (AII AC) provides inhibition onto cone bipolar cell (BC) axons and retinal ganglion cell (RGC) dendrites, but makes the majority of its synapses with the BCs. Using light and electron microscopy, we reconstructed the morphology and connectivity of mouse retinal AII ACs across postnatal development. We found that AII ACs do not elaborate their presynaptic structures, the lobular appendages, until BCs differentiate about a week after RGCs are present. Lobular appendages are present in mutant mice lacking BCs, implying that although synchronized with BC axonal differentiation, presynaptic differentiation of the AII ACs is not dependent on cues from BCs. With maturation, AII ACs maintain a constant number of synapses with RGCs, preferentially increase synaptogenesis with BCs, and eliminate synapses with wide-field amacrine cells. Thus, AII ACs undergo partner type-specific changes in connectivity to attain their mature pattern of synaptic divergence. Moreover, AII ACs contact non-BCs to the same extent in bipolarless retinas, indicating that AII ACs establish partner-type-specific connectivity using diverse mechanisms that operate in parallel but independently.