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In the vertebrate visual system, all output of the retina is carried by retinal ganglion cells. Each type encodes distinct visual features in parallel for transmission to the brain. How many such ‘output channels’ exist and what each encodes are areas of intense debate. In the mouse, anatomical estimates range from 15 to 20 channels, and only a handful are functionally understood. By combining two-photon calcium imaging to obtain dense retinal recordings and unsupervised clustering of the resulting sample of more than 11,000 cells, here we show that the mouse retina harbours substantially more than 30 functional output channels. These include all known and several new ganglion cell types, as verified by genetic and anatomical criteria. Therefore, information channels from the mouse eye to the mouse brain are considerably more diverse than shown thus far by anatomical studies, suggesting an encoding strategy resembling that used in state-of-the-art artificial vision systems. Two-photon calcium imaging reveals that the mouse retina contains more than 30 functionally distinct retinal ganglion cells, including some that have not been described before, exceeding current estimates and suggesting that the functional diversity of retinal ganglion cells may be much larger than previously thought. Multiple retinal ganglion cell types Retinal ganglion cells (RGCs) convey visual information from the retina to the brain. How many types of RGC exist and how they should be classified have been long-standing questions. Thomas Euler and colleagues used two-photon calcium imaging to record responses to stimuli in more than 11,000 cells in a patch of the mouse ganglion cell layer, and applied unsupervised clustering of the resulting data. This revealed that the mouse retina harbours more than 30 distinct functional RGC types, including several that have not been described before. This number substantially exceeds current estimates and indicates that the functional diversity of RGCs is greater than previously thought.

The melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) are a relatively recently discovered class of atypical ganglion cell photoreceptor. These ipRGCs are a morphologically and physiologically heterogeneous population that project widely throughout the brain and mediate a wide array of visual functions ranging from photoentrainment of our circadian rhythms, to driving the pupillary light reflex to improve visual function, to modulating our mood, alertness, learning, sleep/wakefulness, regulation of body temperature, and even our visual perception. The presence of melanopsin as a unique molecular signature of ipRGCs has allowed for the development of a vast array of molecular and genetic tools to study ipRGC circuits. Given the emerging complexity of this system, this review will provide an overview of the genetic tools and methods used to study ipRGCs, how these tools have been used to dissect their role in a variety of visual circuits and behaviors in mice, and identify important directions for future study.

Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes. Retinal ganglion cells (RGCs) are diverse in cellular function and physiology. This study demonstrates additional RGC heterogeneity using single cell transcriptomic analyses to classify 40 classes of RGCs in early postnatal mice before eye opening.

Optogenetics may enable mutation-independent, circuit-specific restoration of neuronal function in neurological diseases. Retinitis pigmentosa is a neurodegenerative eye disease where loss of photoreceptors can lead to complete blindness. In a blind patient, we combined intraocular injection of an adeno-associated viral vector encoding ChrimsonR with light stimulation via engineered goggles. The goggles detect local changes in light intensity and project corresponding light pulses onto the retina in real time to activate optogenetically transduced retinal ganglion cells. The patient perceived, located, counted and touched different objects using the vector-treated eye alone while wearing the goggles. During visual perception, multichannel electroencephalographic recordings revealed object-related activity above the visual cortex. The patient could not visually detect any objects before injection with or without the goggles or after injection without the goggles. This is the first reported case of partial functional recovery in a neurodegenerative disease after optogenetic therapy. Combined intraocular injection of an adeno-associated viral vector, encoding an optogenetic sensor, with light stimulation via engineered goggles enables partial recovery of visual function in a blind patient.

Purpose: To identify and characterise numerically and topographically the population of alpha retinal ganglion cells (αRGCs) and their subtypes, the sustained-response ON-centre αRGCs (ONs-αRGCs), which correspond to the type 4 intrinsically photosensitive RGCs (M4-ipRGCs), the transient-response ON-centre αRGCs (ONt-αRGCs), the sustained-response OFF-centre αRGCs (OFFs-αRGCs) and the transient-response OFF-centre αRGCs (OFFt-αRGCs) in the adult pigmented mouse retina. Methods: The αRGC population and its subtypes were studied in flat-mounted retinas and radial sections immunodetected against: non-phosphorylated high molecular weight neurofilament subunit (SMI-32) or osteopontin (OPN), two αRGCs pan-markers; Calbindin, expressed in ONs-αRGCs, and amacrines; T-box transcription factor T-brain 2 (Tbr2), a key transcriptional regulator for ipRGC development and maintenance, expressed in ipRGCs and GABA-displaced amacrine cells; OPN4, an anti-melanopsin antibody; or Brn3a and Brn3c, markers of RGCs. The total population of RGCs was counted automatically and αRGCs and its subtypes were counted manually, and colour-coded neighbourhood maps were used for their topographical representation. Results: The total mean number of αRGCs per retina is 2252±306 SMI32+αRGCs and 2315±175 OPN+αRGCs (n=10), representing 5.08% and 5.22% of the total number of RGCs traced from the optic nerve, respectively. αRGCs are distributed throughout the retina, showing a higher density in the temporal hemiretina. ONs-αRGCs represent ≈36% (841110 cells (n=10)) of all αRGCs and are located throughout the retina, with the highest density in the temporal region. ONt-αRGCs represent ≈34% (797±146 cells (n=10)) of all αRGCs and are mainly located in the central retinal region. OFF-αRGCs represent the remaining 32% of total αRGCs and are divided equally between OFFs-αRGCs and OFFt-αRGCs (363±50 cells (n=10) and 376±36 cells (n=10), respectively). OFFs-αRGCs are mainly located in the supero-temporal peripheral region of the retina and OFFt-αRGCs in the mid-peripheral region of the retina, especially in the infero-temporal region. Conclusions: The combination of specific antibodies is a useful tool to identify and study αRGCs and their subtypes. αRGCs are distributed throughout the retina with a higher density in the temporal area. The sustained ON and OFF response subtypes are mainly located in the periphery while the transient ON and OFF response subtypes are found in the central regions of the retina.

Astrocytes regulate synaptic connectivity in the CNS through secreted signals. Here we identified two astrocyte-secreted proteins, hevin and SPARC, as regulators of excitatory synaptogenesis in vitro and in vivo. Hevin induces the formation of synapses between cultured rat retinal ganglion cells. SPARC is not synaptogenic, but specifically antagonizes synaptogenic function of hevin. Hevin and SPARC are expressed by astrocytes in the superior colliculus, the synaptic target of retinal ganglion cells, concurrent with the excitatory synaptogenesis. Hevin-null mice had fewer excitatory synapses; conversely, SPARC-null mice had increased synaptic connections in the superior colliculus. Furthermore, we found that hevin is required for the structural maturation of the retinocollicular synapses. These results identify hevin as a positive and SPARC as a negative regulator of synapse formation and signify that, through regulation of relative levels of hevin and SPARC, astrocytes might control the formation, maturation, and plasticity of synapses in vivo.

Glaucoma is the most common cause of age-related blindness in the United States. There is currently no cure, and once vision is lost, the condition is irreversible. Williams et al. now report that vitamin B 3 (also known as niacin) prevents eye degeneration in glaucoma-prone mice (see the Perspective by Crowston and Trounce). Supplementing the diets of young mice with vitamin B 3 averted early signs of glaucoma. Vitamin B 3 also halted further glaucoma development in aged mice that already showed signs of the disease. Thus, healthy intake of vitamin B 3 may protect eyesight. Science , this issue p. 756 ; see also p. 688 Oral supplementation with vitamin B 3 protects against glaucoma development in mice. Glaucomas are neurodegenerative diseases that cause vision loss, especially in the elderly. The mechanisms initiating glaucoma and driving neuronal vulnerability during normal aging are unknown. Studying glaucoma-prone mice, we show that mitochondrial abnormalities are an early driver of neuronal dysfunction, occurring before detectable degeneration. Retinal levels of nicotinamide adenine dinucleotide (NAD + , a key molecule in energy and redox metabolism) decrease with age and render aging neurons vulnerable to disease-related insults. Oral administration of the NAD + precursor nicotinamide (vitamin B 3 ), and/or gene therapy (driving expression of Nmnat1 , a key NAD + -producing enzyme), was protective both prophylactically and as an intervention. At the highest dose tested, 93% of eyes did not develop glaucoma. This supports therapeutic use of vitamin B 3 in glaucoma and potentially other age-related neurodegenerations.

The restoration of light response with complex spatiotemporal features in retinal degenerative diseases towards retinal prosthesis has proven to be a considerable challenge over the past decades. Herein, inspired by the structure and function of photoreceptors in retinas, we develop artificial photoreceptors based on gold nanoparticle-decorated titania nanowire arrays, for restoration of visual responses in the blind mice with degenerated photoreceptors. Green, blue and near UV light responses in the retinal ganglion cells (RGCs) are restored with a spatial resolution better than 100 µm. ON responses in RGCs are blocked by glutamatergic antagonists, suggesting functional preservation of the remaining retinal circuits. Moreover, neurons in the primary visual cortex respond to light after subretinal implant of nanowire arrays. Improvement in pupillary light reflex suggests the behavioral recovery of light sensitivity. Our study will shed light on the development of a new generation of optoelectronic toolkits for subretinal prosthetic devices. The restoration of light response using retinal prosthesis could be a way to restore vision following retinal degenerative disease. Here the authors develop gold-titania nanowire arrays that restore visual response in blind mice.

Age and elevated intraocular pressure (IOP) are the two major risk factors for developing glaucoma, a leading cause of blindness worldwide that is characterized by the loss of retinal ganglion cells (RGCs). Although vision loss is irreversible over the long term, accumulating evidence points to short‐term improvement of vision in glaucoma patients in response to certain interventions, suggesting that RGCs have the capacity to recover function. In the present study, we sought to investigate the mechanisms underlying loss and recovery of RGC function in response to aging and IOP injury, with a focus on synaptic connectivity. Using electroretinography, we found that advancing age was associated with a substantial reduction in function across all retinal layers in the absence of significant cell loss. A superimposed injury induced by IOP elevation led to the selective loss of RGC function in young and middle‐aged mice that was associated with a decrease in paired excitatory synapses. RGC functional recovery after injury was significantly delayed in middle‐aged mice and was mediated through different cellular mechanisms than in young mice. Whereas young mice regained excitatory synaptic inputs from bipolar cells, functional recovery in older mice was instead mediated through an increase in intrinsic RGC excitability, associated with modulation of the action potential threshold and axon initial segment length. Our findings provide new insights into the impact of advancing age on RGC resilience to IOP injury. Boosting the capacity for RGC recovery by reversing the effect of advancing age offers a new therapeutic approach for glaucoma management. The effect of advancing age and intraocular pressure injury on retinal ganglion cell function and synaptic connectivity. While young mice modulate their synaptic input to recover retinal ganglion cell function following injury, older mice compensate by altering their action potential threshold and axon initial segment (AIS) length to increase intrinsic excitability.

The lack of effective therapies for visual restoration in Retinitis pigmentosa and macular degeneration has led to the development of new strategies, such as optogenetics and retinal prostheses. However, visual restoration is poor due to the massive light-evoked activation of retinal neurons, regardless of the segregation of visual information in ON and OFF channels, which is essential for contrast sensitivity and spatial resolution. Here, we show that Ziapin2, a membrane photoswitch that modulates neuronal capacitance and excitability in a light-dependent manner, is capable of reinstating, in mouse and rat genetic models of photoreceptor degeneration, brisk and sluggish ON, OFF, and ON-OFF responses in retinal ganglion cells evoked by full-field stimuli, with reactivation of their excitatory and inhibitory conductances. Intravitreally injected Ziapin2 in fully blind rd10 mice restores light-driven behavior and optomotor reflexes. The results indicate that Ziapin2 is a promising molecule for reinstating physiological visual responses in the late stages of retinal degeneration. Here authors demonstrate Ziapin2, a membrane-targeted photoswitch, reinstates physiological ON/OFF light responses in retinal ganglion cells from degenerate retinas, and restores light-driven behavior and optomotor reflexes in fully blind rd10 mice.