Neural Endophenotype Assessment in Zebrafish Larvae Using Optomotor and ZebraBox Locomotion Assessment.

Due to the highly conserved genetics across the central nervous system, the easily probed visual system can act as an endophenotype for assessing neurological function. Here, we describe a psychophysics approach to assess visually driven swimming behavior in the high-throughput zebrafish genetic model system. We use the optomotor response test together with general locomotion behavior to assess neural processing while excluding motor defects related to muscle function.

Her6 and Prox1a are novel regulators of photoreceptor regeneration in the zebrafish retina.

Damage to light-sensing photoreceptors (PRs) occurs in highly prevalent retinal diseases. As humans cannot regenerate new PRs, these diseases often lead to irreversible blindness. Intriguingly, animals, such as the zebrafish, can regenerate PRs efficiently and restore functional vision. Upon injury, mature Müller glia (MG) undergo reprogramming to adopt a stem cell-like state. This process is similar to cellular dedifferentiation, and results in the generation of progenitor cells, which, in turn, proliferate and differentiate to replace lost retinal neurons. In this study, we tested whether factors involved in dedifferentiation of Drosophila CNS are implicated in the regenerative response in the zebrafish retina. We found that hairy-related 6 (her6) negatively regulates of PR production by regulating the rate of cell divisions in the MG-derived progenitors. prospero homeobox 1a (prox1a) is expressed in differentiated PRs and may promote PR differentiation through phase separation. Interestingly, upon Her6 downregulation, Prox1a is precociously upregulated in the PRs, to promote PR differentiation; conversely, loss of Prox1a also induces a downregulation of Her6. Together, we identified two novel candidates of PR regeneration that cross regulate each other; these may be exploited to promote human retinal regeneration and vision recovery.

Heterogeneity in quiescent Müller glia in the uninjured zebrafish retina drive differential responses following photoreceptor ablation.

Introduction: Loss of neurons in the neural retina is a leading cause of vision loss. While humans do not possess the capacity for retinal regeneration, zebrafish can achieve this through activation of resident Müller glia. Remarkably, despite the presence of Müller glia in humans and other mammalian vertebrates, these cells lack an intrinsic ability to contribute to regeneration. Upon activation, zebrafish Müller glia can adopt a stem cell-like state, undergo proliferation and generate new neurons. However, the underlying molecular mechanisms of this activation subsequent retinal regeneration remains unclear. Methods/Results: To address this, we performed single-cell RNA sequencing (scRNA-seq) and report remarkable heterogeneity in gene expression within quiescent Müller glia across distinct dorsal, central and ventral retina pools of such cells. Next, we utilized a genetically driven, chemically inducible nitroreductase approach to study Müller glia activation following selective ablation of three distinct photoreceptor subtypes: long wavelength sensitive cones, short wavelength sensitive cones, and rods. There, our data revealed that a region-specific bias in activation of Müller glia exists in the zebrafish retina, and this is independent of the distribution of the ablated cell type across retinal regions. Notably, gene ontology analysis revealed that injury-responsive dorsal and central Müller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Through scRNA-seq analysis, we identify a shared genetic program underlying initial Müller glia activation and cell cycle entry, followed by differences that drive the fate of regenerating neurons. We observed an initial expression of AP-1 and injury-responsive transcription factors, followed by genes involved in Notch signaling, ribosome biogenesis and gliogenesis, and finally expression of cell cycle, chromatin remodeling and microtubule-associated genes. Discussion: Taken together, our findings document the regional specificity of gene expression within quiescent Müller glia and demonstrate unique Müller glia activation and regeneration features following neural ablation. These findings will improve our understanding of the molecular pathways relevant to neural regeneration in the retina.

Dedifferentiation-derived neural stem cells exhibit perturbed temporal progression.

Dedifferentiation is the reversion of mature cells to a stem cell-like fate, whereby gene expression programs are altered and genes associated with multipotency are (re)expressed. Misexpression of multipotency factors and pathways causes the formation of ectopic neural stem cells (NSCs). Whether dedifferentiated NSCs faithfully produce the correct number and types of progeny, or undergo timely terminal differentiation, has not been assessed. Here, we show that ectopic NSCs induced via bHLH transcription factor Deadpan (Dpn) expression fail to undergo appropriate temporal progression by constantly expressing mid-temporal transcription factor(tTF), Sloppy-paired 1/2 (Slp). Consequently, this resulted in impaired terminal differenation and generated an excess of Twin of eyeless (Toy)-positive neurons at the expense of Reversed polarity (Repo)-positive glial cells. Preference for a mid-temporal fate in these ectopic NSCs is concordant with an enriched binding of Dpn at mid-tTF loci and a depletion of Dpn binding at early- and late-tTF loci. Retriggering the temporal series via manipulation of the temporal series or cell cycle is sufficient to reinstate neuronal diversity and timely termination.

Exposure to an environmentally relevant concentration of 17α-ethinylestradiol disrupts craniofacial development of juvenile zebrafish.

Endocrine disrupting chemicals (EDCs) can interact with native hormone receptors to interfere with and disrupt hormone signalling that is necessary for a broad range of developmental pathways. EDCs are pervasive in our environment, in particular in our waterways, making aquatic wildlife especially vulnerable to their effects. Many of these EDCs are able to bind to and activate oestrogen receptors, causing aberrant oestrogen signalling. Craniofacial development is an oestrogen-sensitive process, with oestrogen receptors expressed in chondrocytes during critical periods of development. Previous studies have demonstrated a negative effect of high concentrations of oestrogen on early craniofacial patterning in the aquatic model organism, the zebrafish (Danio rerio). In order to determine the impacts of exposure to an oestrogenic EDC, we exposed zebrafish larvae and juveniles to either a high concentration to replicate previous studies, or a low, environmentally relevant concentration of the oestrogenic contaminant, 17α-ethinylestradiol. The prolonged / chronic exposure regimen was used to replicate that seen by many animals in natural waterways. We observed changes to craniofacial morphology in all treatments, and most strikingly in the larvae-juveniles exposed to a low concentration of EE2. In the present study, we have demonstrated that the developmental stage at which exposure occurs can greatly impact phenotypic outcomes, and these results allow us to understand the widespread impact of oestrogenic endocrine disruptors. Given the conservation of key craniofacial development pathways across vertebrates, our model can further be applied in defining the risks of EDCs on mammalian organisms.

Phytochemicals, Antioxidant Activities, and Toxicological Screening of Native Australian Fruits Using Zebrafish Embryonic Model.

Phytochemicals play a pivotal role in human health and drug discovery. The safety evaluation of plant extracts is a prerequisite to ensure that all phytochemicals are safe before translational development and human exposure. As phytochemicals are natural, they are generally considered safe, although this is not always true. The objective of this study was to investigate and compare the phytochemical composition, antioxidant potential, and safety evaluation of native Australian Muntries (Kunzea pomifera), Kakadu plum (Terminalia ferdinandiana), Davidson plum (Davidsonia) and Quandong peach (Santalum acuminatum) through the in vivo vertebrate zebrafish embryonic model. The highest total phenolic content (TPC; 793.89 ± 22.27 µg GAE/mg) was quantified in Kakadu plum, while the lowest TPC (614.44 ± 31.80 µg GAE/mg) was quantified in Muntries. Developmental alterations, mortality, and morbidity were assessed for toxicological screening of these selected native Australian fruit extracts. In this study, muntries were quantified as having the least LC50 value (169 mg/L) compared to Davidson plum (376 mg/L), Kakadu plum (>480 mg/L), and Quandong peach (>480 mg/L), which indicates that muntries extract was more toxic than other fruit extracts. Importantly, we found that adverse effects were not correlated to the total phenolic content and antioxidant potential of these native Australian fruits and cannot simply be predicted from the in vitro analysis. Conclusively, these selected native Australian fruit extracts are categorized as safe. This study could explore the use of these native Australian fruits in cosmetics, pharmaceuticals, and drug discovery.

Photoreceptor ablation following ATP induced injury triggers Müller glia driven regeneration in zebrafish.

Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina - Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.

Altered Visual Function in a Larval Zebrafish Knockout of Neurodevelopmental Risk Gene pdzk1.

Purpose: The human PDZK1 gene is located in a genomic susceptibility region for neurodevelopmental disorders. A genome-wide association study identified links between PDZK1 polymorphisms and altered visual contrast sensitivity, an endophenotype for schizophrenia and autism spectrum disorder. The PDZK1 protein is implicated in neurological functioning, interacting with synaptic molecules including postsynaptic density 95 (PSD-95), N-methyl-d-aspartate receptors (NMDARs), corticotropin-releasing factor receptor 1 (CRFR1), and serotonin 2A receptors. The purpose of the present study was to elucidate the role of PDZK1. Methods: We generated pdzk1-knockout (pdzk1-KO) zebrafish using CRISPR/Cas-9 genome editing. Visual function of 7-day-old fish was assessed at behavioral and functional levels using the optomotor response and scotopic electroretinogram (ERG). We also quantified retinal morphology and densities of PSD-95, NMDAR1, CRFR1, and serotonin in the synaptic inner plexiform layer at 7 days, 4 weeks, and 8 weeks of age. Standard RT-PCR and nonsense-mediated decay interference treatment were also performed to assess genetic compensation in mutants. Results: Relative to wild-type, pdzk1-KO larvae showed spatial frequency tuning functions with increased amplitude (likely due to abnormal gain control) and reduced ERG b-waves (suggestive of inner retinal dysfunction). No synaptic phenotypes, but possible morphological retinal phenotypes, were identified. We confirmed that the absence of major histological phenotypes was not attributable to genetic compensatory mechanisms. Conclusions: Our findings point to a role for pdzk1 in zebrafish visual function, and our model system provides a platform for investigating other genes associated with abnormal visual behavior.

Experience-dependent development of visual sensitivity in larval zebrafish.

The zebrafish (Danio rerio) is a popular vertebrate model for studying visual development, especially at the larval stage. For many vertebrates, post-natal visual experience is essential to fine-tune visual development, but it is unknown how experience shapes larval zebrafish vision. Zebrafish swim with a moving texture; in the wild, this innate optomotor response (OMR) stabilises larvae in moving water, but it can be exploited in the laboratory to assess zebrafish visual function. Here, we compared spatial-frequency tuning inferred from OMR between visually naïve and experienced larvae from 5 to 7 days post-fertilisation. We also examined development of synaptic connections between neurons by quantifying post-synaptic density 95 (PSD-95) in larval retinae. PSD-95 is closely associated with N-methyl-D-aspartate (NMDA) receptors, the neurotransmitter-receptor proteins underlying experience-dependent visual development. We found that rather than following an experience-independent genetic programme, developmental changes in visual spatial-frequency tuning at the larval stage required visual experience. Exposure to motion evoking OMR yielded no greater improvement than exposure to static form, suggesting that increased sensitivity as indexed by OMR was driven not by motor practice but by visual experience itself. PSD-95 density varied with visual sensitivity, suggesting that experience may have up-regulated clustering of PSD-95 for synaptic maturation in visual development.

Correspondence Between Behavioral, Physiological, and Anatomical Measurements of Visual Function in Inhibitory Neuron-Ablated Zebrafish.

Purpose: To compare the effects of reduced inhibitory neuron function in the retina across behavioral, physiological, and anatomical levels. Methods: Inhibitory neurons were ablated in larval zebrafish retina. The Ptf1a gene, which determines inhibitory neuron fate in developing vertebrates, was used to express nitroreductase. By exposing larvae to the prodrug metronidazole, cytotoxicity was selectively induced in inhibitory neurons. Visual phenotypes were characterized at behavioral, physiological, and anatomical levels using an optomotor response (OMR) assay, electroretinography (ERG), and routine histology, respectively. Nonvisual locomotion was also assessed to reveal any general behavioral effects due to ablation of other nonvisual neurons that also express Ptf1a. Results: Injured larvae showed severely reduced OMR relative to controls. Locomotor assessment showed unaltered swimming ability, indicating that reduced OMR was due to visual deficits. For ERG, injured larvae manifested either reduced (type-I) or absent (type-II) b-wave signals originating from bipolar interneurons in the retina. Histologic analysis showed altered retinal morphology in injured larvae, with reductions in synaptic inner plexiform layer (IPL) thickness and synaptic density more pronounced in type-II than type-I larvae; type-II larvae also had smaller retinae overall. Conclusions: The consequences of inhibitory neuron ablation corresponded closely across behavioral, physiological, and anatomical levels. Inhibitory neuron loss likely increases the ratio of neural excitation to inhibition, leading to hyperexcitability. In addition to modulating visual signals, inhibitory neurons may be critical for maintaining retinal structure and organization. This study highlights the utility of a multidisciplinary approach and provides a template for characterizing other zebrafish models of neurological disease.

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode.

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Different Fgfs have distinct roles in regulating neurogenesis after spinal cord injury in zebrafish.

BACKGROUND: Despite conserved developmental processes and organization of the vertebrate central nervous system, only some vertebrates including zebrafish can efficiently regenerate neural damage including after spinal cord injury. The mammalian spinal cord shows very limited regeneration and neurogenesis, resulting in permanent life-long functional impairment. Therefore, there is an urgent need to identify the cellular and molecular mechanisms that can drive efficient vertebrate neurogenesis following injury. A key pathway implicated in zebrafish neurogenesis is fibroblast growth factor signaling. METHODS: In the present study we investigated the roles of distinct fibroblast growth factor members and their receptors in facilitating different aspects of neural development and regeneration at different timepoints following spinal cord injury. After spinal cord injury in adults and during larval development, loss and/or gain of Fgf signaling was combined with immunohistochemistry, in situ hybridization and transgenes marking motor neuron populations in in vivo zebrafish and in vitro mammalian PC12 cell culture models. RESULTS: Fgf3 drives neurogenesis of Islet1 expressing motor neuron subtypes and mediate axonogenesis in cMet expressing motor neuron subtypes. We also demonstrate that the role of Fgf members are not necessarily simple recapitulating development. During development Fgf2, Fgf3 and Fgf8 mediate neurogenesis of Islet1 expressing neurons and neuronal sprouting of both, Islet1 and cMet expressing motor neurons. Strikingly in mammalian PC12 cells, all three Fgfs increased cell proliferation, however, only Fgf2 and to some extent Fgf8, but not Fgf3 facilitated neurite outgrowth. CONCLUSIONS: This study demonstrates differential Fgf member roles during neural development and adult regeneration, including in driving neural proliferation and neurite outgrowth of distinct spinal cord neuron populations, suggesting that factors including Fgf type, age of the organism, timing of expression, requirements for different neuronal populations could be tailored to best drive all of the required regenerative processes.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In vivo expression of Nurr1/Nr4a2a in developing retinal amacrine subtypes in zebrafish Tg(nr4a2a:eGFP) transgenics.

The Nuclear receptor subfamily 4 group A member 2 (Nr4a2) is crucial for the formation or maintenance of dopaminergic neurons in the central nervous system including the retina, where dopaminergic amacrine cells contribute to visual function. Little is known about which cells express Nr4a2 at which developmental stage. Furthermore, whether Nr4a2 functions in combination with other genes is poorly understood. Thus, we generated a novel transgenic to visualize Nr4a2 expression in vivo during zebrafish retinogenesis. A 4.1 kb fragment of the nr4a2a promoter was used to drive green fluorescent protein expression in this Tg(nr4a2a:eGFP) line. In situ hybridization showed that transgene expression follows endogenous RNA expression at a cellular level. Temporal expression and lineages were quantified using in vivo time-lapse imaging in embryos. Nr4a2 expressing retinal subtypes were characterized immunohistochemically. Nr4a2a:eGFP labeled multiple neuron subtypes including 24.5% of all amacrine interneurons. Nr4a2a:eGFP labels all tyrosine hydroxylase labeled dopaminergic amacrine cells, and other nondopaminergic GABAergic amacrine populations. Nr4a2a:eGFP is confined to a specific progenitor lineage identified by sequential expression of the bhlh transcription factor Atonal7 (Atoh7) and Pancreas transcription factor 1a (Ptf1a), and labels postmitotic postmigratory amacrine cells. Thus, developmental Nr4a2a expression indicates a role during late differentiation of specific amacrine interneurons. Tg(nr4a2a:eGFP) is an early marker of distinct neurons including dopaminergic amacrine cells. It can be utilized to assess consequences of gene manipulations and understand whether Nr4a2 only carries out its role in the presence of specific coexpressed genes. This will allow Nr4a2 use to be refined for regenerative approaches.

Feedback from each retinal neuron population drives expression of subsequent fate determinant genes without influencing the cell cycle exit timing.

During neurogenesis, progenitors balance proliferation and cell cycle exit together with expression of fate determinant genes to ensure that the correct number of each of these neuron types is generated. Although intrinsic gene expression acting cell autonomously within each progenitor drives these processes, the final number of neurons generated is also influenced by extrinsic cues, representing a potential avenue to direct neurogenesis in developmental disorders or regenerative settings without the requirement to change intrinsic gene expression. Thus, it is important to understand which of these stages of neurogenesis are amenable to such extrinsic influences. Additionally, all types of neurons are specified in a highly conserved histogenic order, although its significance is unknown. This study makes use of conserved patterns of neurogenesis in the relatively simple yet highly organized zebrafish retina model, in which such histogenic birth order is well characterized. We directly visualize and quantify birth dates and cell fate determinant expression in WT vs. environments lacking different neuronal populations. This study shows that extrinsic feedback from developing retinal neurons is important for the temporal expression of intrinsic fate determinants but not for the timing of birth dates. We found no changes in cell cycle exit timing but did find a significant delay in the expression of genes driving the generation only of later- but not earlier-born cells, suggesting that the robustness of this process depends on continuous feedback from earlier-formed cell types. Thus, extrinsic cues selectively influence cell fate determinant progression, which may explain the function of the retinal histogenic order observed. J. Comp. Neurol. 524:2553-2566, 2016. © 2016 Wiley Periodicals, Inc.

Biasing amacrine subtypes in the Atoh7 lineage through expression of Barhl2.

Within the developing vertebrate retina, particular subtypes of amacrine cells (ACs) tend to arise from progenitors expressing the basic helix-loop-helix (bHLH) transcription factor, Atoh7, which is necessary for the early generation of retinal ganglion cells (RGCs). All ACs require the postmitotic expression of the bHLH pancreas transcription factor Ptf1a; however, Ptf1a alone is not sufficient to give subtype identities. Here we use functional and in vivo time-lapse studies in the zebrafish retina to investigate on the developmental programs leading to ACs specification within the subsequent divisions of Atoh7-positive progenitors. We find evidences that the homeobox transcription factor Barhl2 is an AC subtype identity-biasing factor that turns on within Atoh7-positive descendants. In vivo lineage tracing reveals that particular modes of cell division tend to generate Barhl2-positive precursors from sisters of RGCs. Additionally, Atoh7 indirectly impacts these division modes to regulate the right number of barhl2-expressing cells. We finally find that Atoh7 itself influences the subtypes of Barhl2-dependent ACs. Together, the results from our study uncover lineage-related and molecular logic of subtype specification in the vertebrate retina, by showing that specific AC subtypes arise via a particular mode of cell division and a transcriptional network cascade involving the sequential expression of first atoh7 followed by ptf1a and then barhl2.

Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish.

Adult zebrafish show a remarkable capacity to regenerate their spinal column after injury, an ability that stands in stark contrast to the limited repair that occurs within the mammalian CNS post-injury. The reasons for this interspecies difference in regenerative capacity remain unclear. Here we demonstrate a novel role for Fgf signaling during glial cell morphogenesis in promoting axonal regeneration after spinal cord injury. Zebrafish glia are induced by Fgf signaling, to form an elongated bipolar morphology that forms a bridge between the two sides of the resected spinal cord, over which regenerating axons actively migrate. Loss of Fgf function inhibits formation of this "glial bridge" and prevents axon regeneration. Despite the poor potential for mammalian axonal regeneration, primate astrocytes activated by Fgf signaling adopt a similar morphology to that induced in zebrafish glia. This suggests that differential Fgf regulation, rather than intrinsic cell differences, underlie the distinct responses of mammalian and zebrafish glia to injury.

Origin and determination of inhibitory cell lineages in the vertebrate retina.

Multipotent progenitors in the vertebrate retina often generate clonally related mixtures of excitatory and inhibitory neurons. The postmitotically expressed transcription factor, Ptf1a, is essential for all inhibitory fates in the zebrafish retina, including three types of horizontal and 28 types of amacrine cell. Here, we show that specific types of inhibitory neurons arise from the cell-autonomous influence of Ptf1a in the daughters of fate-restricted progenitors, such as Ath5 or Vsx1/2-expressing progenitors, and that in the absence of Ptf1a, cells that would have become these specific inhibitory subtypes revert to the histogenetically appropriate excitatory subtypes of the same lineage. Altered proportions of amacrine subtypes respecified by the misexpression of Ptf1a in the Ath5 lineage suggest that Ath5-expressing progenitors are biased, favoring the generation of some subtypes more than others. Yet the full array of inhibitory cell subtypes in Ath5 mutants implies the existence of Ath5-independent factors involved in inhibitory cell specification. We also show that an extrinsic negative feedback on the expression of Ptf1a provides a control mechanism by which the number of any and all types of inhibitory cells in the retina can be regulated in this lineage-dependent way.

Bipolar input to melanopsin containing ganglion cells in primate retina.

Two morphological types of melanopsin-expressing ganglion cells have been described in primate retina. Both types show intrinsic light responses as well as rod- and cone-driven ON-type responses. Outer stratifying cells have their dendrites close to the inner nuclear layer (OFF sublamina); inner stratifying cells have their dendrites close to the ganglion cell layer (ON sublamina). Both inner and outer stratifying cells receive synaptic input via ribbon synapses, but the bipolar cell types providing this input have not been identified. Here, we addressed the question whether the diffuse (ON) cone bipolar type DB6 and/or rod bipolar cells contact melanopsin-expressing ganglion cells. Melanopsin containing ganglion cells in marmoset (Callithrix jacchus) and macaque (Macaca fascicularis) retinas were identified immunohistochemically; DB6 cells were labeled with antibodies against the carbohydrate epitope CD15, rod bipolar cells were labeled with antibodies against protein kinase C, and putative synapses between the two cells types were identified with antibodies against piccolo. For one inner cell, nearly all of the DB6 axon terminals that overlap with its dendrites in the two-dimensional space show areas of close contact. In vertical sections, the large majority of the areas of close contact also contain a synaptic punctum, suggesting that DB6 cells contact inner melanopsin cells. The output from DB6 cells accounts for about 30% of synapses onto inner melanopsin cells. Synaptic contacts between rod bipolar axons and inner dendrites were not observed. In the OFF sublamina, about 10% of the DB6 axons are closely associated with dendrites of outer cells, and in about a third of these areas, axonal en passant synapses are detected. This result suggests that DB6 cells may also provide input to outer melanopsin cells.

Synaptic inputs onto small bistratified (blue-ON/yellow-OFF) ganglion cells in marmoset retina.

The inner plexiform layer of the retina contains functional subdivisions, which segregate ON and OFF type light responses. Here, we studied quantitatively the ON and OFF synaptic input to small bistratified (blue-ON/yellow-OFF) ganglion cells in marmosets (Callithrix jacchus). Small bistratified cells display an extensive inner dendritic tier that receives blue-ON input from short-wavelength-sensitive (S) cones via blue cone bipolar cells. The outer dendritic tier is sparse and is thought to receive yellow-OFF input from medium (M)- and long (L)-wavelength-sensitive cones via OFF diffuse bipolar cells. In total, 14 small bistratified cells from different eccentricities were analyzed. The cells were retrogradely labeled from the koniocellular layers of the lateral geniculate nucleus and subsequently photofilled. Retinal preparations were processed with antibodies against the C-terminal binding protein 2, the AMPA receptor subunit GluR4, and/or gephyrin to identify bipolar and/or amacrine input. The results show that the synaptic input is evenly distributed across the dendritic tree, with a density similar to that reported previously for other ganglion cell types. The population of cells showed a consistent pattern, where bipolar input to the inner tier is about fourfold greater than bipolar input to the outer tier. This structural asymmetry of bipolar input may help to balance the weight of cone signals from the sparse S cone array against inputs from the much denser M/L cone array.