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Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. It is limited to obtain efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs)- and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase/improve the visual restorative effect.

Neuromodulation via the intracellular second messenger cAMP is ubiquitous at presynaptic nerve terminals. This modulation of synaptic transmission allows exocytosis to adapt to stimulus levels and reliably encode information. The AII amacrine cell (AII-AC) is a central hub for signal processing in the mammalian retina. The main apical dendrite of the AII-AC is connected to several lobular appendages that release glycine onto OFF cone bipolar cells and ganglion cells. However, the influence of cAMP on glycine release is not well understood. Using membrane capacitance measurements from mouse AII-ACs to directly measure exocytosis, we observe that intracellular dialysis of 1 mmcAMP enhances exocytosis without affecting the L-type Ca2+current. Responses to depolarizing pulses of various durations show that the size of the readily releasable pool of vesicles nearly doubles with cAMP, while paired-pulse depression experiments suggest that release probability does not change. Specific agonists and antagonists for exchange protein activated by cAMP 2 (EPAC2) revealed that the cAMP-induced enhancement of exocytosis requires EPAC2 activation. Furthermore, intact Ca2+stores were also necessary for the cAMP potentiation of exocytosis. Postsynaptic recordings from OFF cone bipolar cells showed that increasing cAMP with forskolin potentiated the frequency of glycinergic spontaneous IPSCs. We propose that cAMP elevations in the AII-AC lead to a robust enhancement of glycine release through an EPAC2 and Ca2+store signaling pathway. Our results thus contribute to a better understanding of how AII-AC crossover inhibitory circuits adapt to changes in ambient luminance.SIGNIFICANCE STATEMENTThe mammalian retina operates over a wide dynamic range of light intensities and contrast levels. To optimize the signal-to-noise ratio of processed visual information, both excitatory and inhibitory synapses within the retina must modulate their gain in synaptic transmission to adapt to different levels of ambient light. Here we show that increases of cAMP concentration within AII amacrine cells produce enhanced exocytosis from these glycinergic interneurons. Therefore, we propose that light-sensitive neuromodulators may change the output of glycine release from AII amacrine cells. This novel mechanism may fine-tune the amount of tonic and phasic synaptic inhibition received by bipolar cell terminals and, consequently, the spiking patterns that ganglion cells send to the upstream visual areas of the brain.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH+) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH+ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH+ACs. Optogenetic stimulation of local CRH+ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH+ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits.SIGNIFICANCE STATEMENTMelanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons.

Neurons in the central nervous system (CNS) are distinguished by the neurotransmitter types they release, their synaptic connections, morphology, and genetic profiles. To fully understand how the CNS works, it is critical to identify all neuronal classes and reveal their synaptic connections. The retina has been extensively used to study neuronal development and circuit formation. Here, we describe a previously unidentified interneuron in mammalian retina. This interneuron shares some morphological, physiological, and molecular features with retinal bipolar cells, such as receiving input from photoreceptors and relaying visual signals to retinal ganglion cells. It also shares some features with amacrine cells (ACs), particularly Aii-ACs, such as their neurite morphology in the inner plexiform layer, the expression of some AC-specific markers, and possibly the release of the inhibitory neurotransmitter glycine. Thus, we unveil an uncommon interneuron, which may play an atypical role in vision.

The retinas of many species show regional specialisations that are evident in the differences in the processing of visual input from different parts of the visual field. Regional specialisation is thought to reflect an adaptation to the natural visual environment, optical constraints, and lifestyle of the species. Yet, little is known about regional differences in synaptic circuitry. Here, we were interested in the topographical distribution of connexin-36 (Cx36), the major constituent of electrical synapses in the retina. We compared the retinas of mice, rats, and cats to include species with different patterns of regional specialisations in the analysis. First, we used the density of Prox1-immunoreactive amacrine cells as a marker of any regional specialisation, with higher cell density signifying more central regions. Double-labelling experiments showed that Prox1 is expressed in AII amacrine cells in all three species. Interestingly, large Cx36 plaques were attached to about 8–10% of Prox1-positive amacrine cell somata, suggesting the strong electrical coupling of pairs or small clusters of cell bodies. When analysing the regional changes in the volumetric density of Cx36-immunoreactive plaques, we found a tight correlation with the density of Prox1-expressing amacrine cells in the ON, but not in the OFF sublamina in all three species. The results suggest that the relative contribution of electrical synapses to the ON- and OFF-pathways of the retina changes with retinal location, which may contribute to functional ON/OFF asymmetries across the visual field.

Purpose Primary cilia are conserved organelles found in polarized cells within the eye that regulate cell growth, migration, and differentiation. Although the role of cilia in photoreceptors is well-studied, the formation of cilia in other retinal cell types has received little attention. In this study, we examined the ciliary profile focused on the inner nuclear layer of retinas in mice and rhesus macaque primates. Methods Retinal sections or flatmounts from Arl13b-Cetn2 tg transgenic mice were immunostained for cell markers (Pax6, Sox9, Chx10, Calbindin, Calretinin, ChaT, GAD67, Prox1, TH, and vGluT3) and analyzed by confocal microscopy. Primate retinal sections were immunostained for ciliary and cell markers (Pax6 and Arl13b). Optical coherence tomography (OCT) and ERGs were used to assess visual function of Vift88 mice. Results During different stages of mouse postnatal eye development, we found that cilia are present in Pax6-positive amacrine cells, which were also observed in primate retinas. The cilia of subtypes of amacrine cells in mice were shown by immunostaining and electron microscopy. We also removed primary cilia from vGluT3 amacrine cells in mouse and found no significant vision defects. In addition, cilia were present in the outer limiting membrane, suggesting that a population of Muller glial cells forms cilia. Conclusions We report that several subpopulations of amacrine cells in inner nuclear layers of the retina form cilia during early retinal development in mice and primates.

Connexin-36 (Cx36) is the major constituent of mammalian retinal gap junctions positioned in key signal pathways. Here, we examined the laminar and large-scale topographical distribution of Cx36 punctate immunolabels in the retina of the cat, a classical model of the mammalian visual system. Calretinin-immunoreactive (CaR-IR) cell populations served to outline the nuclear and plexiform layers and to stain specific neuronal populations. CaR-IR cells included horizontal cells in the outer retina, numerous amacrine cells, and scattered cells in the ganglion cell layer. Cx36-IR plaques were found among horizontal cell dendrites albeit without systematic colocalization of the two labels. Diffuse Cx36 immunoreactivity was found in the cytoplasm of AII amacrine cells, but no colocalization of Cx36 plaques was observed with either the perikarya or the long varicose dendrites of the CaR-IR non-AII amacrine cells. Cx36 puncta were seen throughout the entire inner plexiform layer showing their highest density in the ON sublamina. The densities of AII amacrine cell bodies and Cx36 plaques in the ON sublamina were strongly correlated across a wide range of eccentricities suggesting their anatomical association. However, the high number of plaques per AII cell suggests that a considerable fraction of Cx36 gap junctions in the ON sublamina is formed by other cell types than AII amacrine cells drawing attention to extensive but less studied electrically coupled networks. Electronic supplementary material The online version of this article (10.1007/s00429-019-01876-y) contains supplementary material, which is available to authorized users.

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development., During retinal development, waves of cholinergic neural activity play a role in retinal circuit development. Here, the authors show that this activity also contributes to layer-specific angiogenesis and formation of the blood-retinal barrier.

Trimethyltin chloride (TMT), one of the most widely used organotin compounds in industrial and agricultural fields, is widespread in soil, aquatic systems, foodstuffs and household items. TMT reportedly has toxic effects on the nervous system; however, there is limited information about its effects on eye development and no clear associated mechanisms have been identified. Therefore, in the present study, we investigated eye morphology, vison-related behavior, reactive oxygen species (ROS) production, apoptosis, histopathology, and gene expression to evaluate the toxicity of TMT during ocular development in zebrafish embryos. Exposure to TMT decreased the axial length and surface area of the eye and impaired the ability of zebrafish to recognize light. 2′,7′-dichlorofluorescein diacetate and acridine orange assays revealed dose-dependent increases in ROS formation and apoptosis in the eye. Furthermore, pyknosis of retinal cells was confirmed through histopathological analysis. Antioxidative enzyme-related genes were downregulated and apoptosis-inducing genes were upregulated in TMT-treated zebrafish compared to expression in controls. Retinal cell-specific gene expression was suppressed mainly in retinal ganglion cells, bipolar cells, and photoreceptor cells, whereas amacrine cell-, horizontal cell-, and Muller cell-specific gene expression was enhanced. Our results demonstrate for the first time the toxicity of TMT during eye development, which occurs through the induction of ROS-mediated apoptosis in retinal cells during ocular formation.

Purpose The balance of neuronal excitation and inhibition is important for proper retinal signaling. A previous report showed that diabetes selectively reduces light-evoked inhibition to the retinal dim light rod pathway, changing this balance. Here, changes in mechanisms of retinal inhibitory synaptic transmission after 6 weeks of diabetes are investigated. Methods Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ, 75 mg/kg), and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell voltage-clamp recordings of electrically evoked inhibitory postsynaptic currents from rod bipolar cells and light-evoked excitatory postsynaptic currents from A17-amacrine cells were made in dark-adapted retinal slices. Results Diabetes shortened the timecourse of directly activated lateral GABAergic inhibitory amacrine cell inputs to rod bipolar cells. The timing of GABA release onto rod bipolar cells depends on a prolonged amacrine cell calcium signal that is reduced by slow calcium buffering. Therefore, the effects of calcium buffering with EGTA-acetoxymethyl ester (AM) on diabetic GABAergic signaling were tested. EGTA-AM reduced GABAergic signaling in diabetic retinas more strongly, suggesting that diabetic amacrine cells have reduced calcium signals. Additionally, the timing of release from reciprocal inhibitory inputs to diabetic rod bipolar cells was reduced, but the activation of the A17 amacrine cells responsible for this inhibition was not changed. Conclusions These results suggest that reduced light-evoked inhibitory input to rod bipolar cells is due to reduced and shortened calcium signals in presynaptic GABAergic amacrine cells. A reduction in calcium signaling may be a common mechanism limiting inhibition in the retina.

Signal processing within the retina is generally mediated by graded potentials, whereas output is conveyed by action potentials transmitted along optic nerve axons. Among retinal neurons, amacrine cells seem to be an exception to this general rule, as several types generate voltage-gated Na+ (Nav ) channel-dependent action potentials. The AII, a narrow-field, bistratified axon-less amacrine cell found in mammalian retinas, displays a unique process that resembles an axon initial segment (AIS), with expression of Nav channels colocalized with the cytoskeletal protein ankyrin-G, and generates action potentials. As the role of spiking in AIIs is uncertain, we hypothesized that the morphological properties of the AIS-like process could provide information relevant for its functional importance, including potential pre- and/or postsynaptic connectivity. For morphological analysis, we injected AII amacrine cells in slices with fluorescent dye and immunolabeled the slices for ankyrin-G. Subsequently, this enabled us to reliably identify AII-type processes among ankyrin-G labeled processes in wholemount retina. We systematically analyzed the laminar localization, spatial orientation, and distribution of the AIS-like processes as a function of retinal eccentricity. In the horizontal plane, the processes displayed no preferred orientation and terminal endings were randomly distributed. In the vertical plane, the processes displayed a horizontal preference, but also ascended and descended into the inner nuclear layer and proximal inner plexiform layer, respectively. These results suggest that the AII amacrine AIS-like process is unlikely to take part in conventional synaptic connections, but may instead be adapted to respond to volume neurotransmission by means of extrasynaptic receptors. This article is protected by copyright. All rights reserved.

Our previous research showed that increased phosphorylation of connexin (Cx)36 indicated extended coupling of AII amacrine cells (ACs) in the rod-dominant mouse myopic retina. This research will determine whether phosphorylation at serine 276 of Cx35-containing gap junctions increased in the myopic chicken, whose retina is cone-dominant. Refractive errors and ocular biometric dimensions of 7-days-old chickens were determined following 12 h and 7 days induction of myopia by a −10D lens. The expression pattern and size of Cx35-positive plaques were examined in the early (12 h) and compensated stages (7 days) of lens-induced myopia (LIM). At the same time, phosphorylation at serine 276 (functional assay) of Cx35 in strata 5 (S5) of the inner plexiform layer was investigated. The axial length of the 7 days LIM eyes was significantly longer than that of non-LIM controls (P

One of the causes of nervous system degeneration is an excess of glutamate released upon several diseases. Glutamate analogs, like N-methyl-DL-aspartate (NMDA) and kainic acid (KA), have been shown to induce experimental retinal neurotoxicity. Previous results have shown that NMDA/KA neurotoxicity induces significant changes in the full field electroretinogram response, a thinning on the inner retinal layers, and retinal ganglion cell death. However, not all types of retinal neurons experience the same degree of injury in response to the excitotoxic stimulus. The goal of the present work is to address the effect of intraocular injection of different doses of NMDA/KA on the structure and function of several types of retinal cells and their functionality. To globally analyze the effect of glutamate receptor activation in the retina after the intraocular injection of excitotoxic agents, a combination of histological, electrophysiological, and functional tools has been employed to assess the changes in the retinal structure and function. Retinal excitotoxicity caused by the intraocular injection of a mixture of NMDA/KA causes a harmful effect characterized by a great loss of bipolar, amacrine, and retinal ganglion cells, as well as the degeneration of the inner retina. This process leads to a loss of retinal cell functionality characterized by an impairment of light sensitivity and visual acuity, with a strong effect on the retinal OFF pathway. The structural and functional injury suffered by the retina suggests the importance of the glutamate receptors expressed by different types of retinal cells. The effect of glutamate agonists on the OFF pathway represents one of the main findings of the study, as the evaluation of the retinal lesions caused by excitotoxicity could be specifically explored using tests that evaluate the OFF pathway. This study was funded by the Instituto de Salud Carlos III (RETICS-FEDER RD16/0008/0016, RD16/0008/0020, and FIS/PI18-00754) and cofounded with the European Regional Development Fund (ERDF) within the “Plan Estatal de Investigación Científica y Técnica y de Innovación 2017–2020”, the Spanish Ministry of Economy and Competitiveness (RTI2018-098969-B-100 and FEDER-PID2019-106230-RB-100), Generalitat Valenciana (PROMETEO/2017/060, IDIFEDER/2017/064, and APOSTD/2020/245), and by the Bidons Egara Research Chair of the University Miguel Hernández.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer's disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access. This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

Wenlong Sheng,1 Shijun Weng,2 Fei Li,1 Yun Zhang,1 Qiuxia He,1 Wenxiang Sheng,1 Ying Fu,3 Haiyue Yan,4 Kechun Liu1 1Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan, People’s Republic of China; 2State Key Laboratory of Medical Neurobiology, Institutes of Brain Science, Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China; 3Shandong Science and Technology Exchange Center, Jinan, People’s Republic of China; 4Shandong Institute of Scientific and Technical Information, Jinan, People’s Republic of ChinaCorrespondence: Wenlong Sheng; Kechun Liu Email shengwenlong1121@163.com; liukechun2000@163.comBackground: Melatonin (N-acetyl-5-methoxytryptamine), a significant indoleamine neuromodulator implicated in circadian rhythms and sleep patterns, regulates diverse rhythmic functions via activating its high-affinity G-protein-coupled receptors. However, the detailed cellular expression of the Mel1a receptor in the retina is still a research gap.Methods: The expression of the Mel1a receptor in pigeon retina was assessed using Western blot analysis and immunofluorescent staining. The cellular localization of the Mel1a receptor was studied using double immunofluorescent staining and laser-scanning confocal microscopy.Results: Our data suggested that the Mel1a receptor was extensively expressed in the outer segment of Rho4D2-labeled rod and L/M-opsin-labeled red/green cone and in the somata of the CB-labeled horizontal cell, TH-labeled dopaminergic amacrine cell, ChAT-labeled cholinergic amacrine cell, PV-labeled AII amacrine cell, Brn3a-labeled conventional ganglion cell, melanopsin-containing ganglion celland CRALBP-labeled Müller glial cell. In addition, the Mel1a receptor was diffusely distributed throughout the full thickness of the inner plexiform layer. However, the outer segment of S-opsin-labeled blue cone, the somata of ChX-10-labeled bipolar cell and outer plexiform layer seemed to lack immunoreactivity of the Mel1a receptor.Conclusion: The finding that multiple types of retinal cells express the Mel1a receptor provides a new neurobiological basis for the participation of melatonin in the regulation of retinal functions through activating the Mel1a receptor.Keywords: circadian rhythm, retinal Mel1a receptor, cellular localization, diurnal animal

The mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 can partially reverse the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells.

Transcription factor Sox2 is widely recognized for its critical roles in the nervous system, including the neural retina. Here, we aimed to reveal the function of Sox2 in the process of mouse postnatal development. After the suppression of Sox2 at P0, there was an increase number in bipolar cells but a decrease in amacrine cells. Inhibited Sox2 expression also led to decreased visual function. Furthermore, we found a distinctive type of retinal cells expressing the characteristic proteins of both bipolar cells and amacrine cells at P6, which may be an intermediate state in which amacrine cells were transforming into bipolar cells. Transcription factors associated with the development of bipolar cells and amacrine cells also support those changes. Our work indicated that inhibition of Sox2 could change cell fate by affecting transcription factors in the development of bipolar cells and amacrine cells, may provide new directions for the study and treatment of retinal genetic diseases and retinal dysplasia.

The presence of α-synuclein aggregates in the retina of Parkinson’s disease patients has been associated with vision impairment. In this study we sought to determine the effects of α-synuclein overexpression on the survival and function of dopaminergic amacrine cells (DACs) in the retina. Adult mice were intravitreally injected with an adeno-associated viral (AAV) vector to overexpress human wild-type α-synuclein in the inner retina. Before and after systemic injections of levodopa (L-DOPA), retinal responses and visual acuity-driven behavior were measured by electroretinography (ERG) and a water maze task, respectively. Amacrine cells and ganglion cells were counted at different time points after the injection. α-synuclein overexpression led to an early loss of DACs associated with a decrease of light-adapted ERG responses and visual acuity that could be rescued by systemic injections of L-DOPA. The data show that α-synuclein overexpression affects dopamine neurons in the retina. The approach provides a novel accessible method to model the underlying mechanisms implicated in the pathogenesis of synucleinopathies and for testing novel treatments.

Cell therapy approaches hold great potential for treating retinopathies, which are currently incurable. This study addresses the problem of inadequate migration and integration of transplanted cells into the host retina. To this end, we have identified the chemokines that were most upregulated during retinal degeneration and that could chemoattract mesenchymal stem cells (MSCs). The results were observed using a pharmacological model of ganglion/amacrine cell degeneration and a genetic model of retinitis pigmentosa, from both mice and human retinae. Remarkably, MSCs overexpressing Ccr5 and Cxcr6, which are receptors bound by a subset of the identified chemokines, displayed improved migration after transplantation in the degenerating retina. They also led to enhanced rescue of cell death and to preservation of electrophysiological function. Overall, we show that chemokines released from the degenerating retinae can drive migration of transplanted stem cells, and that overexpression of chemokine receptors can improve cell therapy-based regenerative approaches. This work was supported by Velux Stiftung (976 a to M.P.C.), La Caixa Health (HR17-00231), the Ministerio de Ciencia e Innovación, (BFU2017-86760-P to M.P.C.) (AEI/FEDER, UE), the AGAUR grant from Secretaria d’Universitats i Recerca del Departament d’Empresa i Coneixement de la Generalitat de Catalunya, (2017 SGR 689 to M.P.C.), the subprograma estatal de Formación del Ministerio de Economía y Competitividad ref. BES-2015-075802 (to M.P.) and ref. BES-2015-075805 (to S.A.B.-P.), the Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement de la Generalitat de Catalunya ref. 2018FI_B_00637 (to R.S.-P.), and the co‐finance of Fondo Social Europeo (FSE to R.S.-P.). We acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme / Generalitat de Catalunya

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N -ethyl- N -nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3 −/− mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3 −/− mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Adapting between scotopic and photopic illumination involves switching the routing of retinal signals between rod and cone-dominated circuits. In the daytime, cone signals pass through parallel On and Off cone bipolar cells, that are sensitive to increments and decrements in luminance, respectively. At night, rod signals are routed into these cone-pathways via a key glycinergic interneuron, the AII amacrine cell (AII-AC). In primates, it is not known whether AII-ACs contact all Off-bipolar cell types indiscriminately, or whether their outputs are biased towards specific Off-bipolar cell types. Here, we show that the rod-driven glycinergic output of AII-ACs is strongly biased towards a subset of macaque Off-cone bipolar cells. The Off-bipolar types that receive this glycinergic input have sustained physiological properties and include the Off-midget bipolar cells, which provide excitatory input to the Off-midget ganglion cells (parvocellular pathway). The kinetics of the glycinergic events are consistent with the involvement of the α1 glycine receptor subunit. Taken together with results in mouse retina, our findings point towards a conserved motif whereby rod signals are preferentially routed into sustained Off signaling pathways.Significance StatementVisual signals pass through different retinal neurons depending on the prevailing level of illumination. Under night-time light levels, signals from rods pass through the AII amacrine cell, an inhibitory interneuron that routes rod signals into On and Off bipolar cells to detect increments and decrements in light intensity, respectively. Here, we show in primate retina that the output of AII amacrine cells is strongly biased towards specific Off bipolar cell types, which suggests that rod signals reach the brain via specific neural channels. Our results further our understanding of how visual signals are routed through visual circuits during night-time vision.

Neurons in the CNS are distinguished from each other by their morphology, the types of the neurotransmitter they release, their synaptic connections, and their genetic profiles. While attempting to characterize the retinal bipolar cell (BC) input to retinal ganglion cells (RGCs), we discovered a previously undescribed type of interneuron in mice and primates. This interneuron shares some morphological, physiological, and molecular features with traditional BCs, such as having dendrites that ramify in the outer plexiform layer (OPL) and axons that ramify in the inner plexiform layer (IPL) to relay visual signals from photoreceptors to inner retinal neurons. It also shares some features with amacrine cells, particularly Aii amacrine cells, such as their axonal morphology and possibly the release of the inhibitory neurotransmitter glycine, along with the expression of some amacrine cell specific markers. Thus, we unveil an unrecognized type of interneuron, which may play unique roles in vision.Significance StatementCell types are the building blocks upon which neural circuitry is based. In the retina, it is widely believed that all neuronal types have been identified. We describe a cell type, which we call the Campana cell, that does not fit into the conventional neuronal retina categories but is evolutionarily conserved. Unlike retinal bipolar cells, the Campana cell receives synaptic input from both rods and cones, has broad axonal ramifications, and may release an inhibitory neurotransmitter. Unlike retinal amacrine cells, the Campana cell receives direct photoreceptor input has bipolar-like ribbon synapses. With this discovery, we open the possibility for new forms of visual processing in the retina.

Objective: Considering the demonstrated implication of the retina in Parkinson disease (PD) pathology and the importance of dopaminergic cells in this tissue, we aimed to analyze the state of the dopaminergic amacrine cells and some of their main postsynaptic neurons in the retina of PD. Methods: Using immunohistochemistry and confocal microscopy, we evaluated morphology, number, and synaptic connections of dopaminergic cells and their postsynaptic cells, AII amacrine and melanopsin‐containing retinal ganglion cells, in control and PD eyes from human donors. Results: In PD, dopaminergic amacrine cell number was reduced between 58% and 26% in different retinal regions, involving a decline in the number of synaptic contacts with AII amacrine cells (by 60%) and melanopsin cells (by 35%). Despite losing their main synaptic input, AII cells were not reduced in number, but they showed cellular alterations compromising their adequate function: (1) a loss of mitochondria inside their lobular appendages, which may indicate an energetic failure; and (2) a loss of connexin 36, suggesting alterations in the AII coupling and in visual signal transmission from the rod pathway. Interpretation: The dopaminergic system impairment and the affection of the rod pathway through the AII cells may explain and be partially responsible for the reduced contrast sensitivity or electroretinographic response described in PD. Also, dopamine reduction and the loss of synaptic contacts with melanopsin cells may contribute to the melanopsin retinal ganglion cell loss previously described and to the disturbances in circadian rhythm and sleep reported in PD patients. These data support the idea that the retina reproduces brain neurodegeneration and is highly involved in PD pathology. This work was supported by the Michael J. Fox Foundation for Parkinson’s Research. I.O.-L. and X.S.-S. acknowledge financial support from the Ministry of Education, Spain (FPU 14/03166; FPU 16/04114). N.C. acknowledges financial support from the Ministry of Economy and Competitiveness, Spain (MINECO-FEDER-BFU2015-67139-R), Carlos III Institute of Health (RETICS-FEDER RD16/0008/0016), Retina Asturias Association, and Generalitat Valenciana-European Regional Development Fund (IDIFEDER/2017/064). The Brain and Body Donation Program has been supported by the NIH National Institute of Neurological Disorders and Stroke (U24 NS072026), the NIH National Institute on Aging (P30 AG19610), the Arizona Department of Health Services, the Arizona Biomedical Research Commission, and the Michael J. Fox Foundation for Parkinson’s Research.

The fragile X mental retardation protein (FMRP) is a regulator of local translation through its mRNA targets in the neurons. Previous studies have demonstrated that FMRP may function in distinct ways during the development of different visual subcircuits. However, the localization of the FMRP in different types of retinal cells is unclear. In this work, the FMRP expression in rat retina was detected by Western blot and immunofluorescence double labeling. Results showed that the FMRP expression could be detected in rat retina and that the FMRP had a strong immunoreaction (IR) in the ganglion cell (GC) layer, inner nucleus layer (INL), and outer plexiform layer (OPL) of rat retina. In the outer retina, the bipolar cells (BCs) labeled by homeobox protein ChX10 (ChX10) and the horizontal cells (HCs) labeled by calbindin (CB) were FMRP-positive. In the inner retina, GABAergic amacrine cells (ACs) labeled by glutamate decarbonylase colocalized with the FMRP. The dopaminergic ACs (tyrosine hydroxylase marker) and cholinergic ACs (choline acetyltransferase (ChAT) marker) were co-labeled with the FMRP. In most GCs (labeled by Brn3a) and melanopsin-positive intrinsically photosensitive retinal GCs (ipRGCs) were also FMRP-positive. The FMRP expression was observed in the cellular retinal binding protein-positive Müller cells. These results suggest that the FMRP could be involved in the visual pathway transmission.

Night vision in mammals depends fundamentally on rod photoreceptors and the well-studied rod bipolar (RB) cell pathway. The central neuron in this pathway, the AII amacrine cell (AC), exhibits a spatially tuned receptive field, composed of an excitatory center and an inhibitory surround, that propagates to ganglion cells, the retina’s projection neurons. The circuitry underlying the surround of the AII, however, remains unresolved. Here, we combined structural, functional and optogenetic analyses of the mouse retina to discover that surround inhibition of the AII depends primarily on a single interneuron type, the NOS-1 AC: a multistratified, axon-bearing GABAergic cell, with dendrites in both ON and OFF synaptic layers, but with a pure ON (depolarizing) response to light. Our study demonstrates generally that novel neural circuits can be identified from targeted connectomic analyses and specifically that the NOS-1 AC mediates long-range inhibition during night vision and is a major element of the RB pathway.

Amacrine cells are interneurons comprising the most diverse cell type in the mammalian retina. They help encode visual features such as edges or directed motion by mediating excitatory and inhibitory interactions between input (i.e. bipolar) and output (i.e. ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, neurovascular control and metabolic regulation. Here, we report that a previously poorly characterized, but relatively abundant, inhibitory amacrine cell type in the mouse retina is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed extensive associations with Müller glia, whose processes often completely ensheathe the neurites of this amacrine cell type. Microinjections of small tracer molecules into the somas of these amacrine cells led to selective labelling of nearby Müller glia, leading us to suggest the name “Müller glia-coupled amacrine cell” or MAC. Our electrophysiological data also indicate that MACs release glycine at conventional chemical synapses with amacrine, bipolar and retinal ganglion cells (RGCs), and viral transsynaptic tracing showed connections to several known RGC types. Visually-evoked responses revealed a strong preference for light increments; these “ON” responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling to other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function.Significance StatementGap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system, and play a myriad of roles including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells are rare and poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia.

Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features, such as edges or directed motion, by mediating excitatory and inhibitory interactions between input (i.e., bipolar) and output (i.e., ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation, and neurovascular control. Here, we report that, in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labeling of nearby Müller glia, leading us to suggest the name “Müller glia-coupled amacrine cell,” or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus showed selective connections between MACs and a subpopulation of retinal ganglion cell types. Visually evoked responses revealed a strong preference for light increments; these “ON” responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function. SIGNIFICANCE STATEMENT Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics, and serial electron microscopy provide new information about the neuron's synaptic partners and physiological responses.

Myopia is a major public health problem, affecting one third of the population over 12 years old in the United States and more than 80% of people in Hong Kong. Myopia is attributable to elongation of the eyeball in response to defocused images that alter eye growth and refraction. It is known that the retina can sense the focus of an image, but the effects of defocused images on signaling of population of retinal ganglion cells (RGCs) that account either for emmetropization or refractive errors has still to be elucidated. Thorough knowledge of the underlying mechanisms could provide insight to understanding myopia. In this study, we found that focused and defocused images can change both excitatory and inhibitory conductance of ON alpha, OFF alpha and ON&ndash, OFF retinal ganglion cells in the mouse retina. The firing patterns of population of RGCs vary under the different powers of defocused images and can be affected by dopamine receptor agonists/antagonists&rsquo, application. OFF-delayed RGCs or displaced amacrine cells (dACs) with time latency of more than 0.3 s had synchrony firing with other RGCs and/or dACs. These spatial synchrony firing patterns between OFF-delayed cell and other RGCs/dACs were significantly changed by defocused image, which may relate to edge detection. The results suggested that defocused images induced changes in the multineuronal firing patterns and whole cell conductance in the mouse retina. The multineuronal firing patterns can be affected by dopamine receptors&rsquo, agonists and antagonists. Synchronous firing of OFF-delayed cells is possibly related to edge detection, and understanding of this process may reveal a potential therapeutic target for myopia patients.

We recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCα-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKCα knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

Diabetic retinopathy is now well understood as a neurovascular disease. Significant deficits early in diabetes are found in the inner retina that consists of bipolar cells that receive inputs from rod and cone photoreceptors, ganglion cells that receive inputs from bipolar cells, and amacrine cells that modulate these connections. These functional deficits can be measuredin vivoin diabetic humans and animal models using the electroretinogram (ERG) and behavioral visual testing. Early effects of diabetes on both the human and animal model ERGs are changes to the oscillatory potentials that suggest dysfunctional communication between amacrine cells and bipolar cells as well as ERG measures that suggest ganglion cell dysfunction. These are coupled with changes in contrast sensitivity that suggest inner retinal changes. Mechanisticin vitroneuronal studies have suggested that these inner retinal changes are due to decreased inhibition in the retina, potentially due to decreased gamma aminobutyric acid (GABA) release, increased glutamate release, and increased excitation of retinal ganglion cells. Inner retinal deficits in dopamine levels have also been observed that can be reversed to limit inner retinal damage. Inner retinal targets present a promising new avenue for therapies for early-stage diabetic eye disease.

Myopia is a substantial public health problem worldwide. In the myopic retina, distant images are focused in front of the photoreceptors. The cells and mechanisms for retinal signaling that account either for emmetropization (i.e., normal refraction) or for refractive errors have remained elusive. Gap junctions play a key component in enhancement of signal transmission in visual pathways. AII amacrine cells (ACs), coupled by connexin36, segregate signals into ON and OFF pathways. Coupling between AII ACs is actively modulated through phosphorylation at serine 293 via dopamine in the mouse retina. In this study, form deprivation mouse myopia models were used to evaluate the expression patterns of connexin36-positive plaques (structural assay) and the state of connexin36 phosphorylation (functional assay) in AII ACs, which was green fluorescent protein-expressing in the Fam81a mouse line. Single-cell RNA sequencing showed dopaminergic synapse and gap junction pathways of AII ACs were downregulated in the myopic retina, although Gjd2 mRNA expression remained the same. Compared with the normal refractive eye, phosphorylation of connexin36 was increased in the myopic retina, but expression of connexin36 remained unchanged. This increased phosphorylation of Cx36 could indicate increased functional gap junction coupling of AII ACs in the myopic retina, a possible adaptation to adjust to the altered noisy signaling status.

The transcription factor Otx2 is required for photoreceptor and bipolar cell formation in the retina. It directly activates the transcription factors Prdm1 and Vsx2 through cell type-specific enhancers. Prdm1 and Vsx2 work in opposition, such that Prdm1 promotes photoreceptor fate and Vsx2 bipolar cell fate. To determine how OTX2+ cell fates are regulated, we deleted Prdm1 and Vsx2 or their cell type-specific enhancers simultaneously using a CRISPR/Cas9 in vivo retina electroporation strategy. Double gene or enhancer targeting effectively removed PRDM1 and VSX2 protein expression. However, double enhancer targeting favored bipolar fate outcomes whereas double gene targeting favored photoreceptor fates. Both conditions generated excess amacrine cells. Combined, these fate changes suggest that photoreceptors are a default fate outcome in OTX2+ cells and that VSX2 must be present in a narrow temporal window to drive bipolar cell formation. Prdm1 and Vsx2 also appear to redundantly restrict the competence of OTX2+ cells, preventing amacrine cell formation. By taking a combinatorial deletion approach of both coding sequences and enhancers, our work provides new insights into the complex regulatory mechanisms that control cell fate choice.

Dopamine modulation of retinal signaling has been shown to be an important part of retinal adaptation to increased background light levels, but the role of dopamine modulation of retinal inhibition is not clear. We previously showed that light adaptation causes a large reduction in inhibition to rod bipolar cells, potentially to match the decrease in excitation after rod saturation. In this study, we determined how dopamine D1 receptors in the inner retina contribute to this modulation. We found that D1 receptor activation significantly decreased the magnitude of inhibitory light responses from rod bipolar cells, whereas D1 receptor blockade during light adaptation partially prevented this decline. To determine what mechanisms were involved in the modulation of inhibitory light responses, we measured the effect of D1 receptor activation on spontaneous currents and currents evoked from electrically stimulating amacrine cell inputs to rod bipolar cells. D1 receptor activation decreased the frequency of spontaneous inhibition with no change in event amplitudes, suggesting a presynaptic change in amacrine cell activity in agreement with previous reports that rod bipolar cells lack D1 receptors. Additionally, we found that D1 receptor activation reduced the amplitude of electrically evoked responses, showing that D1 receptors can modulate amacrine cells directly. Our results suggest that D1 receptor activation can replicate a large portion but not all of the effects of light adaptation, likely by modulating release from amacrine cells onto rod bipolar cells. NEW & NOTEWORTHY We demonstrated a new aspect of dopaminergic signaling that is involved in mediating light adaptation of retinal inhibition. This D1 receptor-dependent mechanism likely acts through receptors located directly on amacrine cells, in addition to its potential role in modulating the strength of serial inhibition between amacrine cells. Our results also suggest that another D2/D4 receptor-dependent or dopamine-independent mechanism must also be involved in light adaptation of inhibition to rod bipolar cells.

The fovea is an anatomical specialization of the central retina containing closely packed cone-photoreceptors providing an area of high acuity vision in humans and primates. Despite its key role in the clarity of vision, little is known about the molecular and cellular basis of foveal development, due to the absence of a foveal structure in commonly used laboratory animal models. Of the amniotes the retina in birds of prey and some reptiles do exhibit a typical foveal structure, but they have not been studied in the context of foveal development due to lack of availability of embryonic tissue, lack of captive breeding programs, and limited genomic information. However, the genome for the diurnal bifoveate reptile species Anolis carolinensis (green anole) was recently published and it is possible to collect embryos from this species in captivity. Here, we tested the feasibility of using the anole as a model to study foveal development. Eyes were collected at various stages of development for histological analysis, immunofluorescence, and apoptosis. We show that at embryonic stage (ES) 10 there is peak ganglion cell density at the incipient central foveal region and a single row of cone photoreceptor nuclei. At ES17 the foveal pit begins to form and at this stage there are 3-4 rows of cone nuclei. Post-hatching a further increase in cone density and lengthening of inner and outer segments is observed. A yellowish pigment was seen in the adult central foveal region, but not in the temporal fovea. At ES14 Pax6 was localized across the entire retina, but was more prominent in the ganglion cell layer (GCL) and the part of the inner nuclear layer (INL) containing amacrine cell bodies. However, at ES17 Pax6 expression in the ganglion cells of the central retina was markedly reduced. Bioinformatic analysis revealed that 86% of human candidate foveal hypoplasia genes had an orthologous gene or DNA sequence in the green anole. These findings provide the first insight into foveal morphogenesis in the green anole and suggest that it could be a very useful model for investigating the molecular signals driving foveal development, and thus inform on human foveal development and disease.

The retina is a highly specialised part of the brain responsible for visual processing. It is well-laminated; three layers containing five different types of neurons are compartmentalised by two synaptic layers. Among the retinal layers, the inner nuclear layer (INL) is composed of horizontal, bipolar, and amacrine cell types. Bipolar cells form one sublayer in the distal half of the IPL, while amacrine cells form another sublayer in the proximal half, without any border-like structure. Here, we report that a plexiform layer-like structure exists temporarily in the border between the bipolar and amacrine sublayers in the INL in the rat retina during retinal development. This transient intermediate plexiform layer (TIPL) appeared at postnatal day (PD) 7 and then disappeared around PD 12. Most apoptotic cells in the INL were found near the TIPL. These results suggest that the TIPL may contribute to the formation of sublayers and the cell number limit in the INL., Graphical Abstract

Purpose The combined action of the activating protein-2 (AP-2) transcription factors, AP-2α and AP-2β, is important in early retinal development, specifically in the formation of horizontal cells. However, in previous studies, it was not possible to analyze postnatal development and function of additional retinal subtypes. Methods We used a double conditional deletion of AP-2α and AP-2β from the retina to further examine the combinatory role of these genes in retinal cell patterning and function in postnatal adult mice as measured by Voronoi domain area and nearest-neighbor distance spatial analyses and ERGs, respectively. Results Conditional deletion of both AP-2α and AP-2β from the retina resulted in a variety of abnormalities, including the absence of horizontal cells, defects in the photoreceptor ribbons in which synapses failed to form, along with evidence of aberrant amacrine cell arrangement. Although no significant changes in amacrine cell population numbers were observed in the double mutants, significant irregularities in the mosaic patterning of amacrine cells was observed as demonstrated by both Voronoi domain areas and nearest-neighbor distances analyses. These changes were further accompanied by an alteration in the retinal response to light as recorded by ERGs. In particular, in the double-mutant mice lacking AP-2α and AP-2β, the b-wave amplitude, representative of interneuron signal processing, was significantly reduced compared with control littermates. Conclusions Together these findings demonstrate the requirement for both AP-2α and AP-2β in proper amacrine mosaic patterning and a normal functional light response in the retina.

SUMMARY Dendrites in many neurons actively compute information. In retinal starburst amacrine cells, transformations from synaptic input to output occur within individual dendrites and mediate direction selectivity, but directional signal fidelity at individual synaptic outputs and correlated activity among neighboring outputs on starburst dendrites have not been examined systematically. Here, we record visually evoked calcium signals simultaneously at many individual synaptic outputs within single starburst amacrine cells in mouse retina. We measure visual receptive fields of individual output synapses and show that small groups of outputs are functionally compartmentalized within starburst dendrites, creating distinct computational units. Inhibition enhances compartmentalization and directional tuning of individual outputs but also decreases the signal-to-noise ratio. Simulations suggest, however, that the noise underlying output signal variability is well tolerated by postsynaptic direction-selective ganglion cells, which integrate convergent inputs to acquire reliable directional information., In Brief Poleg-Polsky et al. examine the directional signaling fidelity of individual synapses on starburst amacrine cell dendrites. They identify functionally and morphologically distinct signaling compartments within SAC dendrites and show that inhibition enhances reliable decoding by postsynaptic directionselective ganglion cells.

Dopaminergic amacrine cells (DACs) are among the most well-characterized neurons in the mammalian retina, and their connections to AII amacrine cells have been described in detail. However, the stratification of DAC dendrites differs based on their location in the inner plexiform layer (IPL), raising the question of whether all AII lobules are modulated by dopamine release from DACs. The present study aimed to clarify the relationship between DACs and AII amacrine cells, and to further elucidate the role of dopamine at synapses with AII amacrine cell. In the rabbit retina, DAC dendrites were observed in strata 1, 3, and 5 of the IPL. In stratum 1, most DAC dendritic varicosities—the presumed sites of neurotransmitter release—made contact with the somata and lobular appendages of AII amacrine cells. However, most lobular appendages of AII amacrine cells localized within stratum 2 of the IPL exhibited little contact with DAC varicosities. In addition, double- or triple-labeling experiments revealed that DACs did not express the GABAergic neuronal markers anti-GABA, vesicular GABA transporter, or glutamic acid decarboxylase. These findings suggest that the lobular appendages of AII amacrine cells are involved in at least two different circuits. We speculate that the circuit associated with stratum 1 of the IPL is modulated by DACs, while that associated with stratum 2 is modulated by unknown amacrine cells expressing a different neuroactive substance. Our findings further indicate that DACs in the rabbit retina do not use GABA as a neurotransmitter, in contrast to those in other mammals., Graphical Abstract

The vertebrate retina has the remarkable ability to support visual function under conditions of limited illumination, including the processing of signals evoked by single photons. Dim-light vision is regulated by several adaptive mechanisms. The mechanism explored in this study is responsible for increasing the light sensitivity and operational range of rod bipolar cells, the retinal neurons operating immediately downstream of rod photoreceptors. This sensitization is achieved through the sustained dopamine-dependent GABA release from other retinal neurons. Our goals were to identify the cell type responsible for the GABA release and the site of its modulation by dopamine. Previous studies have suggested the involvement of amacrine and/or horizontal cells. We now demonstrate, using mice of both sexes, that horizontal cells do not participate in this mechanism. Instead, sustained GABA input is provided by a subpopulation of wide-field amacrine cells, which stimulate the GABACreceptors at rod bipolar cell axons. We also found that dopamine does not act directly on either of these cells. Rather, it suppresses inhibition imposed on these wide-field cells by another subpopulation of upstream GABAergic amacrine cells, thereby sustaining the GABACreceptor activation required for rod bipolar cell sensitization.SIGNIFICANCE STATEMENTThe vertebrate retina has an exquisite ability to adjust information processing to ever-changing conditions of ambient illumination, from bright sunlight to single-photon counting under dim starlight. Operation under each of these functional regimes requires an engagement of specific adaptation mechanisms. Here, we describe a mechanism optimizing the performance of the dim-light channel of vision, which consists of sensitizing rod bipolar cells by a sustained GABAergic input originating from a population of wide-field amacrine cells. Wide-field amacrine cells span large segments of the retina, making them uniquely equipped to normalize and optimize response sensitivity across distant receptive fields and preclude any bias toward local light-intensity fluctuations.

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.

Ectopic transgene expression in the retina has been reported in various transgenic mice, indicating the importance of characterizing retinal phenotypes. We examined transgene expression in the VGAT-ChR2-EYFP mouse retina by fluorescent immunohistochemistry and electrophysiology, with special emphasis on enhanced yellow fluorescent protein (EYFP) localization in retinal neuronal subtypes identified by specific markers. Strong EYFP signals were detected in both the inner and outer plexiform layers. In addition, the ChR2-EYFP fusion protein was also expressed in somata of the great majority of inhibitory interneurons, including horizontal cells and GABAergic and glycinergic amacrine cells. However, a small population of amacrine cells residing in the ganglion cell layer were not labeled by EYFP, and a part of them were cholinergic ones. In contrast, no EYFP signal was detected in the somata of retinal excitatory neurons: photoreceptors, bipolar and ganglion cells, as well as Müller glial cells. When glutamatergic transmission was blocked, bright blue light stimulation elicited inward photocurrents from amacrine cells, as well as post-synaptic inhibitory currents from ganglion cells, suggesting a functional ChR2 expression. The VGAT-ChR2-EYFP mouse therefore could be a useful animal model for dissecting retinal microcircuits when targeted labeling and/or optogenetic manipulation of retinal inhibitory neurons are required.

Previous studies have revealed a loss of retinal ganglion cells (RGCs) and optic nerve fibers after immunization with the S100B protein. Addition of heat shock protein 27 (HSP27) also leads to a decrease of RGCs. Our present aim has been to analyze various retinal cell types after immunization with S100B or S100B + HSP27 (S100 + HSP). After 28 days, retinas were processed for immunohistology and Western blot. RGCs, immunostained for NeuN, were significantly decreased in the S100 and the S100 + HSP groups. Significantly fewer ChAT+ cells were noted in both groups, whereas parvalbumin+ cells were only affected in the S100 + HSP group. Western blot results also revealed fewer ChAT signals in both immunized groups. No changes were noted with regard to PKCα+ rod bipolar cells, whereas a significant loss of recoverin+ cone bipolar cells was observed in both groups via immunohistology and Western blot. The presynaptic marker Bassoon and the postsynaptic marker PSD95 were significantly reduced in the S100 + HSP group. Opsin+ and rhodopsin+ photoreceptors revealed no changes in either group. Thus, the inner retinal layers are affected by immunization. However, the combination of S100 and HSP27 has a stronger additive effect on the retinal synapses and AII amacrine cells.

In the vertebrate retina, dopamine is synthesized and released by a specialized type of amacrine cell, the dopaminergic amacrine cell (DAC). DAC activity is stimulated by rods, cones, and melanopsin-expressing intrinsically photosensitive retinal ganglion cells upon illumination. However, the relative contributions of these three photoreceptor systems to the DAC light-induced response are unknown. Here we found that rods excite dark-adapted DACs across a wide range of stimulation intensities, primarily through connexin-36-dependent rod pathways. Similar rod-driven responses were observed in both ventral and dorsal DACs. We further found that in the dorsal retina, M-cones and melanopsin contribute to dark-adapted DAC responses with a similar threshold intensity. In the ventral retina, however, the threshold intensity for M-cone-driven responses was two log units greater than that observed in dorsal DACs, and melanopsin-driven responses were almost undetectable. We also examined the DAC response to prolonged adapting light and found such responses to be mediated by rods under dim lighting conditions, rods/M-cones/melanopsin under intermediate lighting conditions, and cones and melanopsin under bright lighting conditions. Our results elucidate the relative contributions of the three photoreceptor systems to DACs under different lighting conditions, furthering our understanding of the role these cells play in the visual system.

The goals of this study were to describe the morphology, neurotransmitter content and synaptic connections of neurons in primate retinas that contain the neuropeptide secretoneurin. Amacrine cells were labeled with antibodies to secretoneurin in macaque and baboon retinas. Their processes formed three distinct plexuses in the inner plexiform layer: one in the outermost stratum, one in the center and one in the innermost stratum. In light microscopic double immunolabeling experiments, GABA was colocalized with secretoneurin in these cells, but glycine transporter 1 and Substance P were not. ON bipolar cell axon terminals labeled with antibody to the cholecystokinin precursor, G6-gly, have ON responses to stimulation of short wavelength sensitive (S) cones. Axons of these bipolar cells made contacts with amacrine cell dendrites containing secretoneurin. Secretoneurin-IR amacrine cells also made contacts with retinal ganglion cell dendrites labeled with antibody to the photopigment melanopsin, which have OFF responses to stimulation of S cones. Using electron microscopic immunolabeling, 436 synapses from macaque retina were analyzed. Axons from bipolar cells were identified by their characteristic synaptic ribbons; their synaptic densities were asymmetric like those of excitatory synapses in the brain. Amacrine cells made and received conventional synapses with symmetric synaptic densities, like those of inhibitory synapses in the brain. Ganglion cell dendrites were identified by their absence of presynaptic specializations; they received inputs from both amacrine cells and bipolar cells. The majority of inputs to the secretoneurin-IR amacrine cells were from other amacrine cells, but they also received 21% of their input from bipolar cells. They directed most of their output, 54%, to amacrine cells, but there were many synapses onto bipolar cell axons and ganglion cell dendrites, as well. The synaptic connections were very similar in the three plexuses with one notable exception; output synapses to bipolar cells were significantly less common in the innermost one, where the S-ON bipolar cells terminate. Taken together, these findings suggest that the secretoneurin-IR amacrine cells in primates receive excitatory input from S-ON bipolar cells and, in turn, inhibit intrinsically photosensitive retinal ganglion cells.

Background Retinal ischemia-related eye diseases result in visual dysfunction. This study investigates the protective effects and mechanisms of Xue-Fu-Zhu-Yu decoction (XFZYD) with respect to retinal ischemia. Methods Retinal ischemia (I) was induced in Wistar rats by a high intraocular pressure (HIOP) of 120 mmHg for 1 h, which was followed by reperfusion of the ischemic eye; the fellow untreated eye acted as a control. Electroretinogram (ERG), biochemistry and histopathology investigations were performed. Results Significant ischemic changes occurred after ischemia including decreased ERG b-wave ratios, less numerous retinal ganglion cells (RGCs), reduced inner retinal thickness, fewer choline acetyltransferase (ChAT) labeled amacrine cell bodies, increased glial fibrillary acidic protein (GFAP) immunoreactivity and increased vimentin Müller immunolabeling. These were accompanied by significant increases in the mRNA/protein concentrations of vascular endothelium growth factor, hypoxia-inducible factor-1α, pyruvate kinase M2 and retinoblastoma-binding protein 2. The ischemic changes were concentration-dependently and significantly altered when XFZYD was given for seven consecutive days before or after retina ischemia, compared to vehicle. These alterations included enhanced ERG b-wave amplitudes, more numerous RGCs, enhanced inner retinal thickness, a greater number of ChAT immunolabeled amacrine cell bodies and decreased GFAP/vimentin immunoreactivity. Furthermore, decreased mRNA levels of VEGF, HIF-1α, PKM2, and RBP2 were also found. Reduced protein concentrations of VEGF, HIF-1α, PKM2, and RBP2 were also demonstrated. Furthermore, there was an inhibition of the ischemia-associated increased ratios (target protein/β-actin) in the protein levels of VEGF, HIF-1α, PKM2, and RBP2, which were induced by Shikonin, JIB-04 or Avastin. Conclusion XFZYD would seem to protect against well-known retinal ischemic changes via a synergistic inhibition of RBP2 and PKM2, as well as down-regulation of HIF-1α and a reduction in VEGF secretion. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1857-2) contains supplementary material, which is available to authorized users.

Amacrine cells are a heterogeneous group of interneurons that form microcircuits with bipolar, amacrine and ganglion cells to process visual information in the inner retina. This study has characterized the morphology, neurochemistry and major cell types of a VIP-ires-Cre amacrine cell population. VIP-tdTomato and -Confetti (Brainbow2.1) mouse lines were generated by crossing a VIP-ires-Cre line with either a Cre-dependent tdTomato or Brainbow2.1 reporter line. Retinal sections and whole-mounts were evaluated by quantitative, immunohistochemical, and intracellular labeling approaches. The majority of tdTomato and Confetti fluorescent cell bodies were in the inner nuclear layer (INL) and a few cell bodies were in the ganglion cell layer (GCL). Fluorescent processes ramified in strata 1, 3, 4, and 5 of the inner plexiform layer (IPL). All tdTomato fluorescent cells expressed syntaxin 1A and GABA-immunoreactivity indicating they were amacrine cells. The average VIP-tdTomato fluorescent cell density in the INL and GCL was 535 and 24 cells/mm2 , respectively. TdTomato fluorescent cells in the INL and GCL contained VIP-immunoreactivity. The VIP-ires-Cre amacrine cell types were identified in VIP-Brainbow2.1 retinas or by intracellular labeling in VIP-tdTomato retinas. VIP-1 amacrine cells are bistratified, wide-field cells that ramify in strata 1, 4, and 5, VIP-2A and 2B amacrine cells are medium-field cells that mainly ramify in strata 3 and 4, and VIP-3 displaced amacrine cells are medium-field cells that ramify in strata 4 and 5 of the IPL. VIP-ires-Cre amacrine cells form a neuropeptide-expressing cell population with multiple cell types, which are likely to have distinct roles in visual processing.

Summary Object motion sensitive (OMS) W3-retinal ganglion cells (W3-RGCs) in mice respond to local movements in a visual scene but remain silent during self-generated global image motion. The excitatory inputs that drive responses of W3-RGCs to local motion were recently characterized, but which inhibitory neurons suppress W3-RGCs' responses to global motion, how these neurons encode motion information, and how their connections are organized along the excitatory circuit axis remains unknown. Here, we find that a genetically identified amacrine cell (AC) type, TH2-AC, exhibits fast responses to global motion and slow responses to local motion. Optogenetic stimulation shows that TH2-ACs provide strong GABA A receptor-mediated input to W3-RGCs but only weak input to upstream excitatory neurons. Cell-type-specific silencing reveals that temporally coded inhibition from TH2-ACs cancels W3-RGC spike responses to global but not local motion stimuli and, thus, controls the feature selectivity of OMS signals sent to the brain.

Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Muller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100s, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP+ astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc.

Visual stimuli of different frequencies are encoded in the retina using transient and sustained responses. Zhao et al. describe the different strategies that are used by four types of retinal ganglion cells to shape photoresponse kinetics., Retinal neurons use sustained and transient light responses to encode visual stimuli of different frequency ranges, but the underlying mechanisms remain poorly understood. In particular, although earlier studies in retinal ganglion cells (RGCs) proposed seven potential mechanisms, all seven have since been disputed, and it remains unknown whether different RGC types use different mechanisms or how many mechanisms are used by each type. Here, we conduct a comprehensive survey in mice and rats of 12 candidate mechanisms that could conceivably produce tonic rod/cone-driven ON responses in intrinsically photosensitive RGCs (ipRGCs) and transient ON responses in three types of direction-selective RGCs (TRHR+, Hoxd10+ ON, and Hoxd10+ ON-OFF cells). We find that the tonic kinetics of ipRGCs arises from their substantially above-threshold resting potentials, input from sustained ON bipolar cells, absence of amacrine cell inhibition of presynaptic ON bipolar cells, and mGluR7-mediated maintenance of light-evoked glutamatergic input. All three types of direction-selective RGCs receive input from transient ON bipolar cells, and each type uses additional strategies to promote photoresponse transience: presynaptic inhibition and dopaminergic modulation for TRHR+ cells, center/surround antagonism and relatively negative resting potentials for Hoxd10+ ON cells, and presynaptic inhibition for Hoxd10+ ON-OFF cells. We find that the sustained nature of ipRGCs’ rod/cone-driven responses depends neither on melanopsin nor on N-methyl-d-aspartate (NMDA) receptors, whereas the transience of the direction-selective cells’ responses is influenced neither by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor desensitization nor by glutamate uptake. For all cells, we further rule out spike frequency adaptation and intracellular Ca2+ as determinants of photoresponse kinetics. In conclusion, different RGC types use diverse mechanisms to produce sustained or transient light responses. Parenthetically, we find evidence in both mice and rats that the kinetics of light-induced mGluR6 deactivation determines whether an ON bipolar cell responds tonically or transiently to light.