Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions. The specialized downstream circuit for rod signalling, called the primary rod pathway, is well characterized in mammals, but circuitry for rod signalling in non-mammals is largely unknown. Here we demonstrate that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA sequencing, we identified two bipolar cell types in zebrafish that are related to mammalian rod bipolar cell (RBCs), the only bipolar type that directly carries rod signals from the outer to the inner retina in the primary rod pathway. By combining electrophysiology, histology and ultrastructural reconstruction of the zebrafish RBCs, we found that, similar to mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells postsynaptic to one RBC type is strikingly similar to that of mammalian RBCs and their amacrine partners, suggesting that the cell types and circuit design of the primary rod pathway emerged before the divergence of teleost fish and mammals. The second RBC type, which forms separate pathways, was either lost in mammals or emerged in fish.

New twists in the evolution of retinal direction selectivity.

In mammals, starburst amacrine cells are centrally involved in motion vision and a new study in PLOS Biology, by Yan and colleagues finds that zebrafish have them, too. They coexist with a second pair of starburst-like neurons, but neither appears to be strongly motion selective.

Setting up a state-of-the-art laboratory in resource limited settings: A case study of the biomedical science research and training centre in Northeast Nigeria.

African science has substantial potential, yet it grapples with significant challenges. Here we describe the establishment of the Biomedical Science Research and Training Centre (BioRTC) in Yobe State, Northeast Nigeria, as a case study of a hub fostering on-continent research and describe strategies to overcome current barriers. We detail the steps taken to establish BioRTC, emphasising the critical importance of stakeholder engagement, community involvement, resource optimisation and collaborations. With its state-of-the-art facilities and commitment to training African scientists, BioRTC is poised to significantly advance neuroscience research and training in the region. Although we are in the early stages of our journey, our model, emphasizing open access and inclusivity, offers a replicable blueprint for neuroscience research development in similar resource-limited settings, promising to enrich the global neuroscience community. We invite the support and collaboration of those who share our vision and believe in our potential.

Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage. Thus, it has been long assumed that the primary rod pathway evolved in mammals. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs, suggesting that the cell types and circuit design of the primary rod pathway have emerged before the divergence of teleost fish and amniotes. The second RBC type, which forms separate pathways, is either lost in mammals or emerged in fish.

Ancient origin of the rod bipolar cell pathway in the vertebrate retina.

Vertebrates rely on rod photoreceptors for vision in low-light conditions1. Mammals have a specialized downstream circuit for rod signaling called the primary rod pathway, which comprises specific cell types and wiring patterns that are thought to be unique to this lineage2-6. Thus, it has been long assumed that the primary rod pathway evolved in mammals3,5-7. Here, we challenge this view by demonstrating that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA-sequencing, we identified two bipolar cell (BC) types in zebrafish that are related to mammalian rod BCs (RBCs) of the primary rod pathway. By combining electrophysiology, histology, and ultrastructural reconstruction of the zebrafish RBCs, we found that, like mammalian RBCs8, both zebrafish RBC types connect with all rods and red-cones in their dendritic territory, and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells post-synaptic to one RBC type is strikingly similar to that of mammalian RBCs. This suggests that the cell types and circuit design of the primary rod pathway may have emerged before the divergence of teleost fish and amniotes (mammals, bird, reptiles). The second RBC type in zebrafish, which forms separate pathways from the first RBC type, is either lost in mammals or emerged in fish to serve yet unknown roles.

Amacrine cells differentially balance zebrafish color circuits in the central and peripheral retina.

The vertebrate inner retina is driven by photoreceptors whose outputs are already pre-processed; in zebrafish, outer retinal circuits split "color" from "grayscale" information across four cone-photoreceptor types. It remains unclear how the inner retina processes incoming spectral information while also combining cone signals to shape grayscale functions. We address this question by imaging the light-driven responses of amacrine cells (ACs) and bipolar cells (BCs) in larval zebrafish in the presence and pharmacological absence of inner retinal inhibition. We find that ACs enhance opponency in some bipolar cells while at the same time suppressing pre-existing opponency in others, so that, depending on the retinal region, the net change in the number of color-opponent units is essentially zero. To achieve this "dynamic balance," ACs counteract intrinsic color opponency of BCs via the On channel. Consistent with these observations, Off-stratifying ACs are exclusively achromatic, while all color-opponent ACs stratify in the On sublamina.

The Developmental Progression of Eight Opsin Spectral Signals Recorded from the Zebrafish Retinal Cone Layer Is Altered by the Timing and Cell Type Expression of Thyroxin Receptor ß2 (trß2) Gain-Of-Function Transgenes.

Zebrafish retinal cone signals shift in spectral shape through larval, juvenile, and adult development as expression patterns of eight cone-opsin genes change. An algorithm extracting signal amplitudes for the component cone spectral types is developed and tested on two thyroxin receptor ß2 (trß2) gain-of-function lines crx:mYFP-2A-trß2 and gnat2:mYFP-2A-trß2, allowing correlation between opsin signaling and opsin immunoreactivity in lines with different developmental timing and cell-type expression of this red-opsin-promoting transgene. Both adult transgenics became complete, or nearly complete, "red-cone dichromats," with disproportionately large long-wavelength-sensitive (LWS)1 opsin amplitudes as compared with controls, where LWS1 and LWS2 amplitudes were about equal, and significant signals from SWS1, SWS2, and Rh2 opsins were detected. But in transgenic larvae and juveniles of both lines it was LWS2 amplitudes that increased, with LWS1 cone signals rarely encountered. In gnat2:mYFP-2A-trß2 embryos at 5 d postfertilization (dpf), red-opsin immunoreactive cone density doubled, but red-opsin amplitudes (LWS2) increased <10%, and green-opsin, blue-opsin, and UV-opsin signals were unchanged, despite co-expressed red opsins, and the finding that an sws1 UV-opsin reporter gene was shut down by the gnat2:mYFP-2A-trß2 transgene. By contrast both LWS2 red-cone amplitudes and the density of red-cone immunoreactivity more than doubled in 5-dpf crx:mYFP-2A-trß2 embryos, while UV-cone amplitudes were reduced 90%. Embryonic cones with trß2 gain-of-function transgenes were morphologically distinct from control red, blue or UV cones, with wider inner segments and shorter axons than red cones, suggesting cone spectral specification, opsin immunoreactivity and shape are influenced by the abundance and developmental timing of trß2 expression.

Ancestral circuits for vertebrate color vision emerge at the first retinal synapse.

For color vision, retinal circuits separate information about intensity and wavelength. In vertebrates that use the full complement of four "ancestral" cone types, the nature and implementation of this computation remain poorly understood. Here, we establish the complete circuit architecture of outer retinal circuits underlying color processing in larval zebrafish. We find that the synaptic outputs of red and green cones efficiently rotate the encoding of natural daylight in a principal components analysis­like manner to yield primary achromatic and spectrally opponent axes, respectively. Blue cones are tuned to capture most remaining variance when opposed to green cones, while UV cone present a UV achromatic axis for prey capture. We note that fruitflies use essentially the same strategy. Therefore, rotating color space into primary achromatic and chromatic axes at the eye's first synapse may thus be a fundamental principle of color vision when using more than two spectrally well-separated photoreceptor types.

Spectral inference reveals principal cone-integration rules of the zebrafish inner retina.

Retinal bipolar cells integrate cone signals at dendritic and axonal sites. The axonal route, involving amacrine cells, remains largely uncharted. However, because cone types differ in their spectral sensitivities, insights into bipolar cells' cone integration might be gained based on their spectral tunings. We therefore recorded in vivo responses of bipolar cell presynaptic terminals in larval zebrafish to widefield but spectrally resolved flashes of light and mapped the results onto spectral responses of the four cones. This "spectral circuit mapping" allowed explaining ∼95% of the spectral and temporal variance of bipolar cell responses in a simple linear model, thereby revealing several notable integration rules of the inner retina. Bipolar cells were dominated by red-cone inputs, often alongside equal sign inputs from blue and green cones. In contrast, UV-cone inputs were uncorrelated with those of the remaining cones. This led to a new axis of spectral opponency where red-, green-, and blue-cone "Off" circuits connect to "natively-On" UV-cone circuits in the outermost fraction of the inner plexiform layer-much as how key color opponent circuits are established in mammals. Beyond this, and despite substantial temporal diversity that was not present in the cones, bipolar cell spectral tunings were surprisingly simple. They either approximately resembled both opponent and non-opponent spectral motifs already present in the cones or exhibited a stereotyped non-opponent broadband response. In this way, bipolar cells not only preserved the efficient spectral representations in the cones but also diversified them to set up a total of six dominant spectral motifs, which included three axes of spectral opponency.

Notch-mediated re-specification of neuronal identity during central nervous system development.

Neuronal identity has long been thought of as immutable, so that once a cell acquires a specific fate, it is maintained for life.1 Studies using the overexpression of potent transcription factors to experimentally reprogram neuronal fate in the mouse neocortex2,3 and retina4,5 have challenged this notion by revealing that post-mitotic neurons can switch their identity. Whether fate reprogramming is part of normal development in the central nervous system (CNS) is unclear. While there are some reports of physiological cell fate reprogramming in invertebrates,6,7 and in the vertebrate peripheral nervous system,8 endogenous fate reprogramming in the vertebrate CNS has not been documented. Here, we demonstrate spontaneous fate re-specification in an interneuron lineage in the zebrafish retina. We show that the visual system homeobox 1 (vsx1)-expressing lineage, which has been associated exclusively with excitatory bipolar cell (BC) interneurons,9-12 also generates inhibitory amacrine cells (ACs). We identify a role for Notch signaling in conferring plasticity to nascent vsx1 BCs, allowing suitable transcription factor programs to re-specify them to an AC fate. Overstimulating Notch signaling enhances this physiological phenotype so that both daughters of a vsx1 progenitor differentiate into ACs and partially differentiated vsx1 BCs can be converted into ACs. Furthermore, this physiological re-specification can be mimicked to allow experimental induction of an entirely distinct fate, that of retinal projection neurons, from the vsx1 lineage. Our observations reveal unanticipated plasticity of cell fate during retinal development.

Distinct synaptic transfer functions in same-type photoreceptors.

Many sensory systems use ribbon-type synapses to transmit their signals to downstream circuits. The properties of this synaptic transfer fundamentally dictate which aspects in the original stimulus will be accentuated or suppressed, thereby partially defining the detection limits of the circuit. Accordingly, sensory neurons have evolved a wide variety of ribbon geometries and vesicle pool properties to best support their diverse functional requirements. However, the need for diverse synaptic functions does not only arise across neuron types, but also within. Here we show that UV-cones, a single type of photoreceptor of the larval zebrafish eye, exhibit striking differences in their synaptic ultrastructure and consequent calcium to glutamate transfer function depending on their location in the eye. We arrive at this conclusion by combining serial section electron microscopy and simultaneous 'dual-colour' two-photon imaging of calcium and glutamate signals from the same synapse in vivo. We further use the functional dataset to fit a cascade-like model of the ribbon synapse with different vesicle pool sizes, transfer rates, and other synaptic properties. Exploiting recent developments in simulation-based inference, we obtain full posterior estimates for the parameters and compare these across different retinal regions. The model enables us to extrapolate to new stimuli and to systematically investigate different response behaviours of various ribbon configurations. We also provide an interactive, easy-to-use version of this model as an online tool. Overall, we show that already on the synaptic level of single-neuron types there exist highly specialised mechanisms which are advantageous for the encoding of different visual features.

Spherical arena reveals optokinetic response tuning to stimulus location, size, and frequency across entire visual field of larval zebrafish.

Many animals have large visual fields, and sensory circuits may sample those regions of visual space most relevant to behaviours such as gaze stabilisation and hunting. Despite this, relatively small displays are often used in vision neuroscience. To sample stimulus locations across most of the visual field, we built a spherical stimulus arena with 14,848 independently controllable LEDs. We measured the optokinetic response gain of immobilised zebrafish larvae to stimuli of different steradian size and visual field locations. We find that the two eyes are less yoked than previously thought and that spatial frequency tuning is similar across visual field positions. However, zebrafish react most strongly to lateral, nearly equatorial stimuli, consistent with previously reported spatial densities of red, green, and blue photoreceptors. Upside-down experiments suggest further extra-retinal processing. Our results demonstrate that motion vision circuits in zebrafish are anisotropic, and preferentially monitor areas with putative behavioural relevance.

A high-density narrow-field inhibitory retinal interneuron with direct coupling to 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 labelling 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 (dLGN) showed selective connections between MACs and a subpopulation of 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 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.

Thyroid hormone receptor beta mutations alter photoreceptor development and function in Danio rerio (zebrafish).

We investigate mutations in trß2, a splice variant of thrb, identifying changes in function, structure, and behavior in larval and adult zebrafish retinas. Two N-terminus CRISPR mutants were identified. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings of isolated cone signals showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Both larval and adult 6BP+1 mutants exhibit a loss of red-cone contribution to the ERG and an increase in UV-cone contribution. Transgenic reporters show loss of cone trß2 activation in the 6BP+1 mutant but increase in the density of cones with active blue, green, and UV opsin genes. Antibody reactivity for red-cone LWS1 and LWS2 opsin was absent in the 6BP+1 mutant, as was reactivity for arrestin3a. Our results confirm a critical role for trß2 in long-wavelength cone development.

Zebrafish Retinal Ganglion Cells Asymmetrically Encode Spectral and Temporal Information across Visual Space.

In vertebrate vision, the tetrachromatic larval zebrafish permits non-invasive monitoring and manipulating of neural activity across the nervous system in vivo during ongoing behavior. However, despite a perhaps unparalleled understanding of links between zebrafish brain circuits and visual behaviors, comparatively little is known about what their eyes send to the brain via retinal ganglion cells (RGCs). Major gaps in knowledge include any information on spectral coding and information on potentially critical variations in RGC properties across the retinal surface corresponding with asymmetries in the statistics of natural visual space and behavioral demands. Here, we use in vivo two-photon imaging during hyperspectral visual stimulation as well as photolabeling of RGCs to provide a functional and anatomical census of RGCs in larval zebrafish. We find that RGCs' functional and structural properties differ across the eye and include a notable population of UV-responsive On-sustained RGCs that are only found in the acute zone, likely to support visual prey capture of UV-bright zooplankton. Next, approximately half of RGCs display diverse forms of color opponency, including many that are driven by a pervasive and slow blue-Off system-far in excess of what would be required to satisfy traditional models of color vision. In addition, most information on spectral contrast was intermixed with temporal information. Taken together, our results suggest that zebrafish RGCs send a diverse and highly regionalized time-color code to the brain.

Fovea-like Photoreceptor Specializations Underlie Single UV Cone Driven Prey-Capture Behavior in Zebrafish.

In the eye, the function of same-type photoreceptors must be regionally adjusted to process a highly asymmetrical natural visual world. Here, we show that UV cones in the larval zebrafish area temporalis are specifically tuned for UV-bright prey capture in their upper frontal visual field, which may use the signal from a single cone at a time. For this, UV-photon detection probability is regionally boosted more than 10-fold. Next, in vivo two-photon imaging, transcriptomics, and computational modeling reveal that these cones use an elevated baseline of synaptic calcium to facilitate the encoding of bright objects, which in turn results from expressional tuning of phototransduction genes. Moreover, the light-driven synaptic calcium signal is regionally slowed by interactions with horizontal cells and later accentuated at the level of glutamate release driving retinal networks. These regional differences tally with variations between peripheral and foveal cones in primates and hint at a common mechanistic origin.

Conditional and biased regeneration of cone photoreceptor types in the zebrafish retina.

A major challenge in regenerative medicine is replacing cells lost through injury or disease. While significant progress has been made, much remains unknown about the accuracy of native regenerative programs in cell replacement. Here, we capitalized on the regenerative capacity and stereotypic retinal organization of zebrafish to determine the specificity with which retinal Müller glial cells replace lost neuronal cell types. By utilizing a targeted genetic ablation technique, we restricted death to all or to distinct cone photoreceptor types (red, blue, or UV-sensitive cones), enabling us to compare the composition of cones that are regenerated. We found that Müller glia produce cones of all types upon nondiscriminate ablation of these photoreceptors, or upon selective ablation of red or UV cones. Pan-ablation of cones led to regeneration of the various cone types in relative abundances that resembled those of nonablated controls, that is, red > green > UV ~ blue cones. Moreover, selective loss of red or UV cones biased production toward the cone type that was ablated. In contrast, ablation of blue cones alone largely failed to induce cone production at all, although it did induce cell division in Müller glia. The failure to produce cones upon selective elimination of blue cones may be due to their low abundance compared to other cone types. Alternatively, it may be that blue cone death alone does not trigger a change in progenitor competency to support cone genesis. Our findings add to the growing notion that cell replacement during regeneration does not perfectly mimic programs of cell generation during development.

Zebrafish Differentially Process Color across Visual Space to Match Natural Scenes.

Animal eyes have evolved to process behaviorally important visual information, but how retinas deal with statistical asymmetries in visual space remains poorly understood. Using hyperspectral imaging in the field, in vivo 2-photon imaging of retinal neurons, and anatomy, here we show that larval zebrafish use a highly anisotropic retina to asymmetrically survey their natural visual world. First, different neurons dominate different parts of the eye and are linked to a systematic shift in inner retinal function: above the animal, there is little color in nature, and retinal circuits are largely achromatic. Conversely, the lower visual field and horizon are color rich and are predominately surveyed by chromatic and color-opponent circuits that are spectrally matched to the dominant chromatic axes in nature. Second, in the horizontal and lower visual field, bipolar cell terminals encoding achromatic and color-opponent visual features are systematically arranged into distinct layers of the inner retina. Third, above the frontal horizon, a high-gain UV system piggybacks onto retinal circuits, likely to support prey capture.

α2δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses.

α2δ-4 is an auxiliary subunit of voltage-gated Cav1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the CACNA2D4 gene encoding α2δ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of α2δ-4, we used genome editing to generate an α2δ-4 knock-out (α2δ-4 KO) mouse. In male and female α2δ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in α2δ-4 KO retina. In reconstructions of α2δ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in α2δ-4 KO mice correlates with a progressive loss of Cav1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in α2δ-4 KO mice and better under photopic than scotopic conditions. We conclude that α2δ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with CACNA2D4 mutations.SIGNIFICANCE STATEMENT In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for α2δ-4, a subunit of voltage-gated Ca2+ channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca2+ channels are progressively lost and that rod and cone synapses are disrupted in mice that lack α2δ-4. Our results suggest that alterations in presynaptic Ca2+ signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the CACNA2D4 gene encoding α2δ-4.

Stimulation of functional neuronal regeneration from Müller glia in adult mice.

Many retinal diseases lead to the loss of retinal neurons and cause visual impairment. The adult mammalian retina has little capacity for regeneration. By contrast, teleost fish functionally regenerate their retina following injury, and Müller glia (MG) are the source of regenerated neurons. The proneural transcription factor Ascl1 is upregulated in MG after retinal damage in zebrafish and is necessary for regeneration. Although Ascl1 is not expressed in mammalian MG after injury, forced expression of Ascl1 in mouse MG induces a neurogenic state in vitro and in vivo after NMDA (N-methyl-d-aspartate) damage in young mice. However, by postnatal day 16, mouse MG lose neurogenic capacity, despite Ascl1 overexpression. Loss of neurogenic capacity in mature MG is accompanied by reduced chromatin accessibility, suggesting that epigenetic factors limit regeneration. Here we show that MG-specific overexpression of Ascl1, together with a histone deacetylase inhibitor, enables adult mice to generate neurons from MG after retinal injury. The MG-derived neurons express markers of inner retinal neurons, synapse with host retinal neurons, and respond to light. Using an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we show that the histone deacetylase inhibitor promotes accessibility at key gene loci in the MG, and allows more effective reprogramming. Our results thus provide a new approach for the treatment of blinding retinal diseases.