Photoreceptor deficits appear at eye opening in Rs1 mutant mouse models of X-linked retinoschisis.

X-linked retinoschisis (XLRS) is an early onset degenerative retinal disease characterized by cystic lesions in the middle layers of the retina. These structural changes are accompanied by a loss of visual acuity and decreased contrast sensitivity. XLRS is caused by mutations in the gene Rs1 which encodes the secreted protein Retinoschisin 1. Young Rs1-mutant mouse models develop key hallmarks of XLRS including intraretinal schisis and abnormal electroretinograms. The electroretinogram (ERG) comprises activity of multiple cellular generators, and it is not known how and when each of these is impacted in Rs1 mutant mice. Here we use an ex vivo ERG system and pharmacological blockade to determine how ERG components generated by photoreceptors, ON-bipolar, and Müller glial cells are impacted in Rs1 mutants and to determine the time course of these changes. We report that ERG abnormalities begin near eye-opening and that all ERG components are involved.

From 2D slices to a 3D model: Training students in digital microanatomy analysis techniques through a 3D printed neuron project.

The reconstruction of two-dimensional (2D) slices to three-dimensional (3D) digital anatomical models requires technical skills and software that are becoming increasingly important to the modern anatomist, but these skills are rarely taught in undergraduate science classrooms. Furthermore, learning opportunities that allow students to simultaneously explore anatomy in both 2D and 3D space are increasingly valuable. This report describes a novel learning activity that trains students to digitally trace a serially imaged neuron from a confocal stack and to model that neuron in 3D space for 3D printing. By engaging students in the production of a 3D digital model, this learning activity is designed to provide students a novel way to enhance their understanding of the content, including didactic knowledge of neuron morphology, technical research skills in image analysis, and career exploration of neuroanatomy research. Moreover, students engage with microanatomy in a way that starts in 2D but results in a 3D object they can see, touch, and keep. This discursive article presents the learning activity, including videos, instructional guides, and learning objectives designed to engage students on all six levels of Bloom's Taxonomy. Furthermore, this work is a proof of principle modeling workflow that is approachable, inexpensive, achievable, and adaptable to cell types in other organ systems. This work is designed to motivate the expansion of 3D printing technology into microanatomy and neuroanatomy education.

Light drives the developmental progression of outer retinal function.

The complex nature of rod and cone photoreceptors and the light-evoked responsivity of bipolar cells in the mature rodent retina have been well characterized. However, little is known about the emergent light-evoked response properties of the mouse retina and the role light plays in shaping these emergent responses. We have previously demonstrated that the outer retina is responsive to green light as early as postnatal day 8 (P8). Here, we characterize the progression of both photoreceptors (rods and cones) and bipolar cell responses during development and into adulthood using ex vivo electroretinogram recordings. Our data show that the majority of photoreceptor response at P8 originates from cones and that these outputs drive second-order bipolar cell responses as early as P9. We find that the magnitude of the photoresponse increases concurrently with each passing day of postnatal development and that many functional properties of these responses, as well as the relative rod/cone contributions to the total light-evoked response, are age dependent. We compare these responses at eye opening and maturity to age-matched animals raised in darkness and found that the absence of light diminishes emergent and mature cone-to-bipolar cell signaling. Furthermore, we found cone-evoked responses to be significantly slower in dark-reared retinas. Together, this work characterizes the developmental photoresponsivity of the mouse retina while highlighting the importance of properly timed sensory input for the maturation of the first visual system synapse.

Modeling cholinergic retinal waves: starburst amacrine cells shape wave generation, propagation, and direction bias.

Stage II cholinergic retinal waves are one of the first instances of neural activity in the visual system as they are present at a developmental timepoint in which light-evoked activity remains largely undetectable. These waves of spontaneous neural activity sweeping across the developing retina are generated by starburst amacrine cells, depolarize retinal ganglion cells, and drive the refinement of retinofugal projections to numerous visual centers in the brain. Building from several well-established models, we assemble a spatial computational model of starburst amacrine cell-mediated wave generation and wave propagation that includes three significant advancements. First, we model the intrinsic spontaneous bursting of the starburst amacrine cells, including the slow afterhyperpolarization, which shapes the stochastic process of wave generation. Second, we establish a mechanism of wave propagation using reciprocal acetylcholine release, synchronizing the bursting activity of neighboring starburst amacrine cells. Third, we model the additional starburst amacrine cell release of GABA, changing the spatial propagation of retinal waves and in certain instances, the directional bias of the retinal wave front. In total, these advancements comprise a now more comprehensive model of wave generation, propagation, and direction bias.

Retinal organoid light responsivity: current status and future opportunities.

The ability to generate human retinas in vitro from pluripotent stem cells opened unprecedented opportunities for basic science and for the development of therapeutic approaches for retinal degenerative diseases. Retinal organoid models not only mimic the histoarchitecture and cellular composition of the native retina, but they can achieve a remarkable level of maturation that allows them to respond to light stimulation. However, studies evaluating the nature, magnitude, and properties of light-evoked responsivity from each cell type, in each retinal organoid layer, have been sparse. In this review we discuss the current understanding of retinal organoid function, the technologies used for functional assessment in human retinal organoids, and the challenges and opportunities that lie ahead.

Ex vivo electroretinograms made easy: performing ERGs using 3D printed components.

KEY POINTS: Rod and cone photoreceptors convert light into electrochemical signals that are transferred to second order cells, initiating image-forming visual processing. Electroretinograms (ERGs) can detect the associated light-induced extracellular transretinal events, allowing for physiological assessment of cellular activity from morphologically intact retinas. We outline a method for economically configuring a traditional patch-clamp rig for performing high signal-to-noise ex vivo ERGs. We accomplish this by incorporating various 3D printed components and by modifying existing light pathways in a typical patch-clamp rig. This methodology provides an additional set of tools to labs interested in studying the physiological function of neuronal populations in isolated retinal tissue. ABSTRACT: Rod and cone photoreceptors of the retina are responsible for the initial stages in vision and convey sensory information regarding our visual world across a wide range of lighting conditions. These photoreceptors hyperpolarize in the presence of light and subsequently transmit signals to second-order bipolar and horizontal cells. The electrical components of these events are experimentally detectable, and in conjunction with pharmacological agents, can be further separated into their respective cellular contributions using electroretinograms (ERGs). Extracellular activity from populations of rods and cones generate the negative-going a-wave, while ON-bipolar cells generate positive-going b-waves. ERGs can be performed in vivo or alternatively using an ex vivo configuration, where retinas are isolated and transretinal photovoltages are recorded at high signal-to-noise ratios. However, most ERG set-ups require their own unique set of tools. We demonstrate how, at low cost, to reconfigure a typical patch-clamp rig for ERG recordings. The bulk of these modifications require implementation of various 3D printed components, which can alternatively aid in generating a stand-alone ERG set-up without a patch-rig. Further, we discuss how to configure an ERG system without a patch-clamp rig. Compared to in vivo ERGs, these are superior when measuring small responses, such as those that are cone-evoked or those from immature mouse retinae. This recording configuration provides high signal-to-noise detection of a-waves (300-600 µV) and b-waves (1-3 mV), and is ultimately capable of discerning small (1-2 µV) photovoltages from noise. These quick and economical modifications allow researchers to equip their technical arsenal with an interchangeable patch-clamp/ERG system.

Crosstalk: The diversity of melanopsin ganglion cell types has begun to challenge the canonical divide between image-forming and non-image-forming vision.

Melanopsin ganglion cells have defied convention since their discovery almost 20 years ago. In the years following, many types of these intrinsically photosensitive retinal ganglion cells (ipRGCs) have emerged. In the mouse retina, there are currently six known types (M1-M6) of melanopsin ganglion cells, each with unique morphology, mosaics, connections, physiology, projections, and functions. While melanopsin-expressing cells are usually associated with behaviors like circadian photoentrainment and the pupillary light reflex, the characterization of multiple types has demonstrated a reach that may extend far beyond non-image-forming vision. In fact, studies have shown that individual types of melanopsin ganglion cells have the potential to impact image-forming functions like contrast sensitivity and color opponency. Thus, the goal of this review is to summarize the morphological and functional aspects of the six known types of melanopsin ganglion cells in the mouse retina and to highlight their respective roles in non-image-forming and image-forming vision. Although many melanopsin ganglion cell types do project to image-forming brain targets, it is important to note that this is only the first step in determining their influence on image-forming vision. Even so, the visual system has canonically been divided into these two functional realms and melanopsin ganglion cells have begun to challenge the boundary between them, providing an overlap of visual information that is complementary rather than redundant. Further studies on these ganglion cell photoreceptors will no doubt continue to illustrate an ever-expanding role for melanopsin ganglion cells in image-forming vision.

µ-Opioid Receptor Activation Directly Modulates Intrinsically Photosensitive Retinal Ganglion Cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) encode light intensity and trigger reflexive responses to changes in environmental illumination. In addition to functioning as photoreceptors, ipRGCs are post-synaptic neurons in the inner retina, and there is increasing evidence that their output can be influenced by retinal neuromodulators. Here we show that opioids can modulate light-evoked ipRGC signaling, and we demonstrate that the M1, M2 and M3 types of ipRGCs are immunoreactive for µ-opioid receptors (MORs) in both mouse and rat. In the rat retina, application of the MOR-selective agonist DAMGO attenuated light-evoked firing ipRGCs in a dose-dependent manner (IC50 < 40 nM), and this effect was reversed or prevented by co-application of the MOR-selective antagonists CTOP or CTAP. Recordings from solitary ipRGCs, enzymatically dissociated from retinas obtained from melanopsin-driven fluorescent reporter mice, confirmed that DAMGO exerts its effect directly through MORs expressed by ipRGCs. Reduced ipRGC excitability occurred via modulation of voltage-gated potassium and calcium currents. These findings suggest a potential new role for endogenous opioids in the mammalian retina and identify a novel site of action-MORs on ipRGCs-through which opioids might exert effects on reflexive responses to environmental light.

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks.

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

Melanopsin Retinal Ganglion Cells Regulate Cone Photoreceptor Lamination in the Mouse Retina.

Newborn neurons follow molecular cues to reach their final destination, but whether early life experience influences lamination remains largely unexplored. As light is among the first stimuli to reach the developing nervous system via intrinsically photosensitive retinal ganglion cells (ipRGCs), we asked whether ipRGCs could affect lamination in the developing mouse retina. We show here that ablation of ipRGCs causes cone photoreceptors to mislocalize at different apicobasal positions in the retina. This effect is partly mediated by light-evoked activity in ipRGCs, as dark rearing or silencing of ipRGCs leads a subset of cones to mislocalize. Furthermore, ablation of ipRGCs alters the cone transcriptome and decreases expression of the dopamine receptor D4, while injection of L-DOPA or D4 receptor agonist rescues the displaced cone phenotype observed in dark-reared animals. These results show that early light-mediated activity in ipRGCs influences neuronal lamination and identify ipRGC-elicited dopamine release as a mechanism influencing cone position.

A novel map of the mouse eye for orienting retinal topography in anatomical space.

Functionally distinct retinal ganglion cells have density and size gradients across the mouse retina, and some degenerative eye diseases follow topographic-specific gradients of cell death. Hence, the anatomical orientation of the retina with respect to the orbit and head is important for understanding the functional anatomy of the retina in both health and disease. However, different research groups use different anatomical landmarks to determine retinal orientation (dorsal, ventral, temporal, nasal poles). Variations in the accuracy and reliability in marking these landmarks during dissection may lead to discrepancies in the identification and reporting of retinal topography. The goal of this study was to compare the accuracy and reliability of the canthus, rectus muscle, and choroid fissure landmarks in reporting retinal orientation. The retinal relieving cut angle made from each landmark during dissection was calculated based on its relationship to the opsin transition zone (OTZ), determined via a custom MATLAB script that aligns retinas from immunostained s-opsin. The choroid fissure and rectus muscle landmarks were the most accurate and reliable, while burn marks using the canthus as a reference were the least. These values were used to build an anatomical map that plots various ocular landmarks in relationship to one another, to the horizontal semicircular canals, to lambda-bregma, and to the earth's horizon. Surprisingly, during normal locomotion, the mouse's opsin gradient and the horizontal semicircular canals make equivalent 6° angles aligning the OTZ near the earth's horizon, a feature which may enhance the mouse's ability to visually navigate through its environment.

Functional Assessment of Melanopsin-Driven Light Responses in the Mouse: Multielectrode Array Recordings.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a special subset of retinal output neurons capable of detecting and responding to light via a unique photopigment called melanopsin. Melanopsin activation is essential to a wide array of physiological functions, especially to those related to non-image-forming vision. Since ipRGCs only constitute a very small proportion of retinal ganglion cells, targeted recording of melanopsin-driven responses used to be a big challenge to vision researchers. Multielectrode array (MEA) recording provides a noninvasive, high throughput method to monitor melanopsin-driven responses. When synaptic inputs from rod/cone photoreceptors are silenced with glutamatergic blockers, extracellular electric signals derived from melanopsin activation can be recorded from multiple ipRGCs simultaneously by tens of microelectrodes aligned in an array. In this chapter we describe how our labs have approached MEA recording of melanopsin-driven light responses in adult mouse retinas. Instruments, tools and chemical reagents routinely used for setting up a successful MEA recording are listed, and a standard experimental procedure is provided. The implementation of this technique offers a useful paradigm that can be used to conduct functional assessments of ipRGCs and NIF vision.

The Development of Mid-Wavelength Photoresponsivity in the Mouse Retina.

PURPOSE: Photoreceptors in the mouse retina express much of the molecular machinery necessary for phototransduction and glutamatergic transmission prior to eye opening at postnatal day 13 (P13). Light responses have been observed collectively from rod and cone photoreceptors via electroretinogram recordings as early as P13 in mouse, and the responses are known to become more robust with maturation, reaching a mature state by P30. Photocurrents from single rod outer segments have been recorded at P12, but no earlier, and similar studies on cone photoreceptors have been done, but only in the adult mouse retina. In this study, we wanted to document the earliest time point in which outer retinal photoreceptors in the mouse retina begin to respond to mid-wavelength light. METHODS: Ex-vivo electroretinogram recordings were made from isolated mouse retinae at P7, P8, P9, P10, and P30 at seven different flash energies (561 nm). The a-wave was pharmacologically isolated and measured at each developmental time point across all flash energies. RESULTS: Outer-retinal photoreceptors generated a detectable response to mid-wavelength light as early as P8, but only at photopic flash energies. a-wave intensity response curves and kinetic response properties are similar to the mature retina as early as P10. CONCLUSION: These data represent the earliest recorded outer retinal light responses in the rodent. Photoreceptors are electrically functional and photoresponsive prior to eye opening, and much earlier than previously thought. Prior to eye opening, critical developmental processes occur that have been thought to be independent of outer retinal photic modulation. However, these data suggest light acting through outer-retinal photoreceptors has the potential to shape these critical developmental processes.

The M5 Cell: A Color-Opponent Intrinsically Photosensitive Retinal Ganglion Cell.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) combine direct photosensitivity through melanopsin with synaptically mediated drive from classical photoreceptors through bipolar-cell input. Here, we sought to provide a fuller description of the least understood ipRGC type, the M5 cell, and discovered a distinctive functional characteristic-chromatic opponency (ultraviolet excitatory, green inhibitory). Serial electron microscopic reconstructions revealed that M5 cells receive selective UV-opsin drive from Type 9 cone bipolar cells but also mixed cone signals from bipolar Types 6, 7, and 8. Recordings suggest that both excitation and inhibition are driven by the ON channel and that chromatic opponency results from M-cone-driven surround inhibition mediated by wide-field spiking GABAergic amacrine cells. We show that M5 cells send axons to the dLGN and are thus positioned to provide chromatic signals to visual cortex. These findings underscore that melanopsin's influence extends beyond unconscious reflex functions to encompass cortical vision, perhaps including the perception of color.

Melanopsin ganglion cell outer retinal dendrites: Morphologically distinct and asymmetrically distributed in the mouse retina.

A small population of retinal ganglion cells expresses the photopigment melanopsin and function as autonomous photoreceptors. They encode global luminance levels critical for light-mediated non-image forming visual processes including circadian rhythms and the pupillary light reflex. There are five melanopsin ganglion cell subtypes (M1-M5). M1 and displaced M1 (M1d) cells have dendrites that ramify within the outermost layer of the inner plexiform layer. It was recently discovered that some melanopsin ganglion cells extend dendrites into the outer retina. Outer Retinal Dendrites (ORDs) either ramify within the outer plexiform layer (OPL) or the inner nuclear layer, and while present in the mature retina, are most abundant postnatally. Anatomical evidence for synaptic transmission between cone photoreceptor terminals and ORDs suggests a novel photoreceptor to ganglion cell connection in the mammalian retina. While it is known that the number of ORDs in the retina is developmentally regulated, little is known about the morphology, the cells from which they originate, or their spatial distribution throughout the retina. We analyzed the morphology of melanopsin-immunopositive ORDs in the OPL at different developmental time points in the mouse retina and identified five types of ORDs originating from either M1 or M1d cells. However, a pattern emerges within these: ORDs from M1d cells are generally longer and more highly branched than ORDs from conventional M1 cells. Additionally, we found ORDs asymmetrically distributed to the dorsal retina. This morphological analysis provides the first step in identifying a potential role for biplexiform melanopsin ganglion cell ORDs.

A subset of ipRGCs regulates both maturation of the circadian clock and segregation of retinogeniculate projections in mice.

The visual system consists of two major subsystems, image-forming circuits that drive conscious vision and non-image-forming circuits for behaviors such as circadian photoentrainment. While historically considered non-overlapping, recent evidence has uncovered crosstalk between these subsystems. Here, we investigated shared developmental mechanisms. We revealed an unprecedented role for light in the maturation of the circadian clock and discovered that intrinsically photosensitive retinal ganglion cells (ipRGCs) are critical for this refinement process. In addition, ipRGCs regulate retinal waves independent of light, and developmental ablation of a subset of ipRGCs disrupts eye-specific segregation of retinogeniculate projections. Specifically, a subset of ipRGCs, comprising ~200 cells and which project intraretinally and to circadian centers in the brain, are sufficient to mediate both of these developmental processes. Thus, this subset of ipRGCs constitute a shared node in the neural networks that mediate light-dependent maturation of the circadian clock and light-independent refinement of retinogeniculate projections.

Dorsal root ganglia neurite outgrowth measured as a function of changes in microelectrode array resistance.

Current research in prosthetic device design aims to mimic natural movements using a feedback system that connects to the patient's own nerves to control the device. The first step in using neurons to control motion is to make and maintain contact between neurons and the feedback sensors. Therefore, the goal of this project was to determine if changes in electrode resistance could be detected when a neuron extended a neurite to contact a sensor. Dorsal root ganglia (DRG) were harvested from chick embryos and cultured on a collagen-coated carbon nanotube microelectrode array for two days. The DRG were seeded along one side of the array so the processes extended across the array, contacting about half of the electrodes. Electrode resistance was measured both prior to culture and after the two day culture period. Phase contrast images of the microelectrode array were taken after two days to visually determine which electrodes were in contact with one or more DRG neurite or tissue. Electrodes in contact with DRG neurites had an average change in resistance of 0.15 MΩ compared with the electrodes without DRG neurites. Using this method, we determined that resistance values can be used as a criterion for identifying electrodes in contact with a DRG neurite. These data are the foundation for future development of an autonomous feedback resistance measurement system to continuously monitor DRG neurite outgrowth at specific spatial locations.

Melanopsin ganglion cells extend dendrites into the outer retina during early postnatal development.

Melanopsin ganglion cells express the photopigment melanopsin and are the first functional photoreceptors to develop in the mammalian retina. They have been shown to play a variety of important roles in visual development and behavior in the early postnatal period (Johnson et al., 2010; Kirkby and Feller, 2013; Rao et al., 2013; Renna et al., 2011). Here, we probed the maturation of the dendritic arbors of melanopsin ganglion cells during this developmental period in mice. We found that some melanopsin ganglion cells (mainly the M1-subtype) transiently extend their dendrites not only into the inner plexiform layer (where they receive synaptic inputs from bipolar and amacrine cells) but also into the outer plexiform layer, where in mature retina, rod and cone photoreceptors are thought to contact only bipolar and horizontal cells. Thus, some immature melanopsin ganglion cells are biplexiform. This feature is much less common although still present in the mature retina. It reaches peak incidence 8-12 days after birth, before the eyes open and bipolar cells are sufficiently mature to link rods and cones to ganglion cells. At this age, some outer dendrites of melanopsin ganglion cells lie in close apposition to the axon terminals of cone photoreceptors and express a postsynaptic marker of glutamatergic transmission, postsynaptic density-95 protein (PSD-95). These findings raise the possibility of direct, monosynaptic connections between cones and melanopsin ganglion cells in the early postnatal retina. We provide a detailed description of the developmental profile of these processes and consider their possible functional and evolutionary significance.

Light acts through melanopsin to alter retinal waves and segregation of retinogeniculate afferents.

Waves of correlated activity sweeping across the early postnatal mouse retina promote the segregation and refinement of retinofugal projections. This process has been thought to be spontaneous and unaffected by visual experience. We found, however, that light prolongs spiking during the waves and enhances the segregation of retinogeniculate afferents, and that it did so by activating melanopsin-expressing, intrinsically photosensitive retinal ganglion cells.