RESUMEN
The zebrafish visual system has become a paradigmatic preparation for behavioral and systems neuroscience. Around 40 types of retinal ganglion cells (RGCs) serve as matched filters for stimulus features, including light, optic flow, prey, and objects on a collision course. RGCs distribute their signals via axon collaterals to 12 retinorecipient areas in forebrain and midbrain. The major visuomotor hub, the optic tectum, harbors nine RGC input layers that combine information on multiple features. The retinotopic map in the tectum is locally adapted to visual scene statistics and visual subfield-specific behavioral demands. Tectal projections to premotor centers are topographically organized according to behavioral commands. The known connectivity in more than 20 processing streams allows us to dissect the cellular basis of elementary perceptual and cognitive functions. Visually evoked responses, such as prey capture or loom avoidance, are controlled by dedicated multistation pathways that-at least in the larva-resemble labeled lines. This architecture serves the neuronal code's purpose of driving adaptive behavior.
Asunto(s)
Células Ganglionares de la Retina , Colículos Superiores , Vías Visuales , Pez Cebra , Animales , Vías Visuales/fisiología , Pez Cebra/fisiología , Células Ganglionares de la Retina/fisiología , Colículos Superiores/fisiología , Percepción Visual/fisiologíaRESUMEN
The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs1. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates2. By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.
Asunto(s)
Evolución Biológica , Neuronas , Retina , Vertebrados , Visión Ocular , Animales , Humanos , Neuronas/clasificación , Neuronas/citología , Neuronas/fisiología , Retina/citología , Retina/fisiología , Células Ganglionares de la Retina/clasificación , Análisis de Expresión Génica de una Sola Célula , Vertebrados/fisiología , Visión Ocular/fisiología , Especificidad de la Especie , Células Amacrinas/clasificación , Células Fotorreceptoras/clasificación , Células Ependimogliales/clasificación , Células Bipolares de la Retina/clasificación , Percepción VisualRESUMEN
Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1-5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6-8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12-14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.
Asunto(s)
Aglomeración , Neuronas , Conducta Social , Colículos Superiores , Tálamo , Vías Visuales , Pez Cebra , Animales , Mapeo Encefálico , Calcio/análisis , Hipotálamo/citología , Hipotálamo/fisiología , Locomoción , Microscopía Electrónica , Neuronas/citología , Neuronas/fisiología , Neuronas/ultraestructura , Reconocimiento Visual de Modelos , Estimulación Luminosa , Colículos Superiores/citología , Colículos Superiores/fisiología , Tálamo/citología , Tálamo/fisiología , Vías Visuales/citología , Vías Visuales/fisiología , Vías Visuales/ultraestructura , Pez Cebra/fisiologíaRESUMEN
Synaptic connections between neurons form the basis for perception and behavior. Synapses are often clustered in space, forming stereotyped layers. In the retina and optic tectum, multiple such synaptic laminae are stacked on top of each other, giving rise to stratified neuropil regions in which each layer combines synapses responsive to a particular sensory feature. Recently, several cellular and molecular mechanisms that underlie the development of multilaminar arrays of synapses have been discovered. These mechanisms include neurite guidance and cell-cell recognition. Molecules of the Slit, Semaphorin, Netrin, and Hedgehog families, binding to their matching receptors, bring axons and dendrites into spatial register. These guidance cues may diffuse over short distances or bind to sheets of extracellular matrix, thus conditioning the local extracellular milieu, or are presented on the surface of cells bordering the future neuropil. In addition, mutual recognition of axons and dendrites through adhesion molecules with immunoglobulin domains ensures cell type-specific connections within a given layer. Thus, an elaborate genetic program assembles the parallel processing channels that underlie visual perception.
Asunto(s)
Retina/fisiología , Sinapsis/metabolismo , Vías Visuales , Percepción Visual , Animales , Proteínas del Ojo/metabolismo , Retina/citologíaRESUMEN
The mechanisms that generate specific neuronal connections in the brain are under intense investigation. In zebrafish, retinal ganglion cells project their axons into at least six layers within the neuropil of the midbrain tectum. Each axon elaborates a single, planar arbor in one of the target layers and forms synapses onto the dendrites of tectal neurons. We show that the laminar specificity of retinotectal connections does not depend on self-sorting interactions among RGC axons. Rather, tectum-derived Slit1, signaling through axonal Robo2, guides neurites to their target layer. Genetic and biochemical studies indicate that Slit binds to Dragnet (Col4a5), a type IV Collagen, which forms the basement membrane on the surface of the tectum. We further show that radial glial endfeet are required for the basement-membrane anchoring of Slit. We propose that Slit1 signaling, perhaps in the form of a superficial-to-deep gradient, presents laminar positional cues to ingrowing retinal axons.
Asunto(s)
Encéfalo/embriología , Colágeno Tipo IV/metabolismo , Techo del Mesencéfalo/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Axones/metabolismo , Encéfalo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Células Ganglionares de la Retina/metabolismo , Transducción de Señal , Pez Cebra/metabolismoRESUMEN
Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm3. We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system.
Asunto(s)
Sinapsis , Pez Cebra , Animales , Larva , Sinapsis/ultraestructura , Encéfalo/ultraestructura , Microscopía ElectrónicaRESUMEN
Vertebrate behavior is strongly influenced by light. Light receptors, encoded by functional opsin proteins, are present inside the vertebrate brain and peripheral tissues. This expression feature is present from fishes to human and appears to be particularly prominent in diurnal vertebrates. Despite their conserved widespread occurrence, the nonvisual functions of opsins are still largely enigmatic. This is even more apparent when considering the high number of opsins. Teleosts possess around 40 opsin genes, present from young developmental stages to adulthood. Many of these opsins have been shown to function as light receptors. This raises the question of whether this large number might mainly reflect functional redundancy or rather maximally enables teleosts to optimally use the complex light information present under water. We focus on tmt-opsin1b and tmt-opsin2, c-opsins with ancestral-type sequence features, conserved across several vertebrate phyla, expressed with partly similar expression in non-rod, non-cone, non-retinal-ganglion-cell brain tissues and with a similar spectral sensitivity. The characterization of the single mutants revealed age- and light-dependent behavioral changes, as well as an impact on the levels of the preprohormone sst1b and the voltage-gated sodium channel subunit scn12aa. The amount of daytime rest is affected independently of the eyes, pineal organ, and circadian clock in tmt-opsin1b mutants. We further focused on daytime behavior and the molecular changes in tmt-opsin1b/2 double mutants, and found that-despite their similar expression and spectral features-these opsins interact in part nonadditively. Specifically, double mutants complement molecular and behavioral phenotypes observed in single mutants in a partly age-dependent fashion. Our work provides a starting point to disentangle the highly complex interactions of vertebrate nonvisual opsins, suggesting that tmt-opsin-expressing cells together with other visual and nonvisual opsins provide detailed light information to the organism for behavioral fine-tuning. This work also provides a stepping stone to unravel how vertebrate species with conserved opsins, but living in different ecological niches, respond to similar light cues and how human-generated artificial light might impact on behavioral processes in natural environments.
Asunto(s)
Encéfalo/fisiología , Ecosistema , Opsinas/fisiología , Oryzias , Animales , Animales Modificados Genéticamente , Conducta Animal/fisiología , Encéfalo/embriología , Embrión no Mamífero , Interacción Gen-Ambiente , Opsinas/genética , Oryzias/embriología , Oryzias/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
The different cell types in the central nervous system develop from a common pool of progenitor cells. The nuclei of progenitors move between the apical and basal surfaces of the neuroepithelium in phase with their cell cycle, a process termed interkinetic nuclear migration (INM). In the retina of zebrafish mikre oko (mok) mutants, in which the motor protein Dynactin-1 is disrupted, interkinetic nuclei migrate more rapidly and deeply to the basal side and more slowly to the apical side. We found that Notch signaling is predominantly activated on the apical side in both mutants and wild-type. Mutant progenitors are, thus, less exposed to Notch and exit the cell cycle prematurely. This leads to an overproduction of early-born retinal ganglion cells (RGCs) at the expense of later-born interneurons and glia. Our data indicate that the function of INM is to balance the exposure of progenitor nuclei to neurogenic versus proliferative signals.
Asunto(s)
Núcleo Celular/metabolismo , Células Neuroepiteliales/citología , Organogénesis , Retina/embriología , Animales , Tipificación del Cuerpo , Ciclo Celular , Diferenciación Celular , Complejo Dinactina , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genética , Mutación , Células Neuroepiteliales/metabolismo , Receptores Notch/metabolismo , Retina/citología , Células Ganglionares de la Retina/metabolismo , Células Madre/citología , Células Madre/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.
Asunto(s)
Microscopía/métodos , Pez Cebra/anatomía & histología , Animales , Encéfalo/ultraestructura , Núcleo Celular/ultraestructura , Biología Evolutiva/métodos , Larva/anatomía & histología , Neurociencias/métodos , Sinapsis/ultraestructura , Pez Cebra/embriologíaRESUMEN
All-optical methods enable the control and monitoring of neuronal activity with minimal perturbation of the system. Although imaging and optogenetic manipulations can be performed at cellular resolution, the morphology of single cells in a dense neuronal population has often remained unresolvable. Here we describe in detail two recently established optogenetic protocols for systematic description of function and morphology of single neurons in zebrafish. First, the Optobow toolbox allows unbiased mapping of excitatory functional connectivity. Second, the FuGIMA technique enables selective labeling and anatomical tracing of neurons that are responsive to a given sensory stimulus or correlated with a specific behavior. Both strategies can be genetically targeted to a neuronal population of choice using the Gal4/UAS system. As these in vivo approaches are non-invasive, we envision useful applications for the study of neuronal structure, function and connectivity during development and behavior.
Asunto(s)
Red Nerviosa/fisiología , Neuronas/fisiología , Optogenética/métodos , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Técnicas Biosensibles/métodos , Encéfalo/citología , Encéfalo/fisiología , Calcio/química , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/efectos de la radiación , Red Nerviosa/citología , Estimulación Luminosa/métodos , Factores de Transcripción/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Enfermedades Hereditarias del Ojo/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Miopía/metabolismo , Ceguera Nocturna/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Sinapsis/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Adulto , Animales , Canales de Calcio Tipo L/genética , Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/embriología , Enfermedades Hereditarias del Ojo/genética , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/embriología , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Humanos , Masculino , Miopía/embriología , Miopía/genética , Ceguera Nocturna/embriología , Ceguera Nocturna/genética , Retina/embriología , Retina/metabolismo , Sinapsis/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
G-protein coupled inwardly rectifying potassium (GIRK) channels are an integral part of inhibitory signal transduction pathways, reducing the activity of excitable cells via hyperpolarization. They play crucial roles in processes such as cardiac output, cognition and the coordination of movement. Therefore, the precision control of GIRK channels is of critical importance. Here, we describe the development of the azobenzene containing molecule VLOGO (Visible Light Operated GIRK channel Opener), which activates GIRK channels in the dark and is promptly deactivated when illuminated with green light. VLOGO is a valuable addition to the existing tools for the optical control of GIRK channels as it circumvents the need to use potentially harmful UV irradiation. We therefore believe that VLOGO will be a useful research tool for studying GIRK channels in biological systems.
Asunto(s)
Compuestos Azo/química , Compuestos Azo/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/agonistas , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Células HEK293 , Humanos , Luz , Técnicas de Placa-Clamp , Procesos Fotoquímicos/efectos de los fármacosRESUMEN
The major active retinoid, all-trans retinoic acid, has long been recognized as critical for the development of several organs, including the eye. Mutations in STRA6, the gene encoding the cellular receptor for vitamin A, in patients with Matthew-Wood syndrome and anophthalmia/microphthalmia (A/M), have previously demonstrated the importance of retinol metabolism in human eye disease. We used homozygosity mapping combined with next-generation sequencing to interrogate patients with anophthalmia and microphthalmia for new causative genes. We used whole-exome and whole-genome sequencing to study a family with two affected brothers with bilateral A/M and a simplex case with bilateral anophthalmia and hypoplasia of the optic nerve and optic chiasm. Analysis of novel sequence variants revealed homozygosity for two nonsense mutations in ALDH1A3, c.568A>G, predicting p.Lys190*, in the familial cases, and c.1165A>T, predicting p.Lys389*, in the simplex case. Both mutations predict nonsense-mediated decay and complete loss of function. We performed antisense morpholino (MO) studies in Danio rerio to characterize the developmental effects of loss of Aldh1a3 function. MO-injected larvae showed a significant reduction in eye size, and aberrant axonal projections to the tectum were noted. We conclude that ALDH1A3 loss of function causes anophthalmia and aberrant eye development in humans and in animal model systems.
Asunto(s)
Aldehído Oxidorreductasas/genética , Anoftalmos/genética , Codón sin Sentido/genética , Microftalmía/genética , Quiasma Óptico/anomalías , Nervio Óptico/anomalías , Aldehído Oxidorreductasas/metabolismo , Animales , Anoftalmos/metabolismo , Niño , Preescolar , Exoma , Ojo/crecimiento & desarrollo , Ojo/patología , Femenino , Genoma , Homocigoto , Humanos , Lactante , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Microftalmía/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.
Asunto(s)
Luz , Locomoción/fisiología , Médula Espinal/fisiología , Pez Cebra/genética , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Axones/fisiología , Cilios/fisiología , Femenino , Larva/genética , Larva/fisiología , Larva/efectos de la radiación , Locomoción/genética , Locomoción/efectos de la radiación , Masculino , Modelos Neurológicos , Neuronas/fisiología , Neuronas/efectos de la radiación , Médula Espinal/citología , Médula Espinal/efectos de la radiación , Natación/fisiología , Cola (estructura animal)/fisiología , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrolloRESUMEN
The axons of retinal ganglion cells (RGCs) form topographic connections in the optic tectum, recreating a two-dimensional map of the visual field in the midbrain. RGC axons are also targeted to specific positions along the laminar axis of the tectum. Understanding the sensory transformations performed by the tectum requires identification of the rules that control the formation of synaptic laminae by RGC axons. However, there is little information regarding the spatial relationships between multiple axons as they establish laminar and retinotopic arborization fields within the same region of neuropil. Moreover, the contribution of RGC axon lamination to the processing of visual information is unknown. We used Brainbow genetic labeling to visualize groups of individually identifiable axons during the assembly of a precise laminar map in the larval zebrafish tectum. Live imaging of multiple RGCs revealed that axons target specific sublaminar positions during initial innervation and maintain their relative laminar positions throughout early larval development, ruling out a model for lamina selection based on iterative refinements. During this period of laminar stability, RGC arbors undergo structural rearrangements that shift their relative retinotopic positions. Analysis of cell-type-specific lamination patterns revealed that distinct combinations of RGCs converge to form each sublamina, and this input heterogeneity correlates with different functional responses to visual stimuli. These findings suggest that lamina-specific sorting of retinal inputs provides an anatomical blueprint for the integration of visual features in the tectum.
Asunto(s)
Axones/fisiología , Retina/citología , Células Ganglionares de la Retina/citología , Colículos Superiores/fisiología , Vías Visuales/fisiología , Animales , Animales Modificados Genéticamente , Calcio/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas con Homeodominio LIM/metabolismo , Larva , Método de Montecarlo , Dinámicas no Lineales , Estimulación Luminosa , Retina/anatomía & histología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The specific partnering of synaptically connected neurons is central to nervous system function. Proper wiring requires the interchange of signals between a postmitotic neuron and its environment, a distinct pattern of transcription in the nucleus, and deployment of guidance and adhesion cues to the cell surface. To identify genes involved in neurite targeting by retinal ganglion cells (GCs), their presynaptic partners in the retina, and their postsynaptic targets in the optic tectum, we undertook a forward genetic screen for mutations disrupting visual responses in zebrafish. This rapid primary screen was subsequently refined by immunohistochemical labeling of retinal and tectal neurites to detect patterning errors. From this unbiased screen, the notorious (noto) mutant exhibited the most specific phenotypes: intact retinal and tectal differentiation but multiple neurite targeting defects in the retinal inner plexiform layer (IPL) and tectal neuropil. Positional cloning and morpholino phenocopy revealed that the mutation disrupts Topoisomerase IIß (Top2b), a broadly distributed nuclear protein involved in chromatin modifications during postmitotic differentiation. Top2b-DNA interactions are known to regulate transcription of developmentally important genes, including axon guidance factors and cell adhesion molecules, but a specific role in local synaptic targeting has not been previously described. The neurite targeting defects among GC axons are largely restricted to crossovers between sublaminae of a specific layer, SFGS, and were shown by mosaic analysis to be autonomous to the GC axons. The noto mutant provides the first example of the importance of an epigenetic regulator, Top2b, in the intricate series of events that lead to a properly wired visual system.
Asunto(s)
Axones/fisiología , ADN-Topoisomerasas de Tipo II/fisiología , Proteínas de Unión al ADN/fisiología , Dendritas/fisiología , Células Ganglionares de la Retina/fisiología , Pez Cebra/metabolismo , Animales , Epigenómica , Visión Ocular/fisiología , Vías VisualesRESUMEN
The ability to process temporal information is fundamental to sensory perception, cognitive processing and motor behaviour of all living organisms, from amoebae to humans. Neural circuit mechanisms based on neuronal and synaptic properties have been shown to process temporal information over the range of tens of microseconds to hundreds of milliseconds. How neural circuits process temporal information in the range of seconds to minutes is much less understood. Studies of working memory in monkeys and rats have shown that neurons in the prefrontal cortex, the parietal cortex and the thalamus exhibit ramping activities that linearly correlate with the lapse of time until the end of a specific time interval of several seconds that the animal is trained to memorize. Many organisms can also memorize the time interval of rhythmic sensory stimuli in the timescale of seconds and can coordinate motor behaviour accordingly, for example, by keeping the rhythm after exposure to the beat of music. Here we report a form of rhythmic activity among specific neuronal ensembles in the zebrafish optic tectum, which retains the memory of the time interval (in the order of seconds) of repetitive sensory stimuli for a duration of up to approximately 20 s. After repetitive visual conditioning stimulation (CS) of zebrafish larvae, we observed rhythmic post-CS activities among specific tectal neuronal ensembles, with a regular interval that closely matched the CS. Visuomotor behaviour of the zebrafish larvae also showed regular post-CS repetitions at the entrained time interval that correlated with rhythmic neuronal ensemble activities in the tectum. Thus, rhythmic activities among specific neuronal ensembles may act as an adjustable 'metronome' for time intervals in the order of seconds, and serve as a mechanism for the short-term perceptual memory of rhythmic sensory experience.
Asunto(s)
Memoria/fisiología , Neuronas/fisiología , Periodicidad , Pez Cebra/fisiología , Animales , Calcio/metabolismo , Condicionamiento Psicológico , Larva/fisiología , Estimulación Luminosa , Colículos Superiores/citología , Colículos Superiores/fisiología , Natación/fisiología , Cola (estructura animal)/fisiología , Factores de Tiempo , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrolloRESUMEN
The retinotectal projection has long been studied experimentally and theoretically, as a model for the formation of topographic brain maps. Neighbouring retinal ganglion cells (RGCs) project their axons to neighbouring positions in the optic tectum, thus re-establishing a continuous neural representation of visual space. Mapping along this axis requires chemorepellent signalling from tectal cells, expressing ephrin-A ligands, to retinal growth cones, expressing EphA receptors. High concentrations of ephrin A, increasing from anterior to posterior, prevent temporal axons from invading the posterior tectum. However, the force that drives nasal axons to extend past the anterior tectum and terminate in posterior regions remains to be identified. We tested whether axon-axon interactions, such as competition, are required for posterior tectum innervation. By transplanting blastomeres from a wild-type (WT) zebrafish into a lakritz (lak) mutant, which lacks all RGCs, we created chimaeras with eyes that contained single RGCs. These solitary RGCs often extended axons into the tectum, where they branched to form a terminal arbor. Here we show that the distal tips of these arbors were positioned at retinotopically appropriate positions, ruling out an essential role for competition in innervation of the ephrin-A-rich posterior tectum. However, solitary arbors were larger and more complex than under normal, crowded conditions, owing to a lack of pruning of proximal branches during refinement of the retinotectal projection. We conclude that dense innervation is not required for targeting of retinal axons within the zebrafish tectum but serves to restrict arbor size and shape.
Asunto(s)
Axones/fisiología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/fisiología , Colículos Superiores/citología , Colículos Superiores/fisiología , Pez Cebra/fisiología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Neurológicos , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The brain is spatially organized into subdivisions, nuclei and areas, which often correspond to functional and developmental units. A segmentation of brain regions in the form of a consensus atlas facilitates mechanistic studies and is a prerequisite for sharing information among neuroanatomists. Gene expression patterns objectively delineate boundaries between brain regions and provide information about their developmental and evolutionary histories. To generate a detailed molecular map of the larval zebrafish diencephalon, we took advantage of the Max Planck Zebrafish Brain (mapzebrain) atlas, which aligns hundreds of transcript and transgene expression patterns in a shared coordinate system. Inspection and co-visualization of close to 50 marker genes have allowed us to resolve the tripartite prosomeric scaffold of the diencephalon at unprecedented resolution. This approach clarified the genoarchitectonic partitioning of the alar diencephalon into pretectum (alar part of prosomere P1), thalamus (alar part of prosomere P2, with habenula and pineal complex), and prethalamus (alar part of prosomere P3). We further identified the region of the nucleus of the medial longitudinal fasciculus, as well as the posterior and anterior parts of the posterior tuberculum, as molecularly distinct basal parts of prosomeres 1, 2, and 3, respectively. Some of the markers examined allowed us to locate glutamatergic, GABAergic, dopaminergic, serotoninergic, and various neuropeptidergic domains in the larval zebrafish diencephalon. Our molecular neuroanatomical approach has thus (1) yielded an objective and internally consistent interpretation of the prosomere boundaries within the zebrafish forebrain; has (2) produced a list of markers, which in sparse combinations label the subdivisions of the diencephalon; and is (3) setting the stage for further functional and developmental studies in this vertebrate brain.
Asunto(s)
Diencéfalo , Pez Cebra , Animales , Pez Cebra/metabolismo , Larva , Diencéfalo/metabolismo , Tálamo , ProsencéfaloRESUMEN
Animals constantly need to judge the valence of an object in their environment: is it potential food or a threat? The brain makes fundamental decisions on the appropriate behavioral strategy by integrating external information from sensory organs and internal signals related to physiological needs. For example, a hungry animal may take more risks than a satiated one when deciding to approach or avoid an object. Using a proteomic profiling approach, we identified the Calmodulin-interacting peptide Pcp4a as a key regulator of foraging-related decisions. Food intake reduced abundance of protein and mRNA of pcp4a via dopamine D2-like receptor-mediated repression of adenylate cyclase. Accordingly, deleting the pcp4a gene made zebrafish larvae more risk averse in a binary decision assay. Strikingly, neurons in the tectum became less responsive to prey-like visual stimuli in pcp4a mutants, thus biasing the behavior toward avoidance. This study pinpoints a molecular mechanism modulating behavioral choice according to internal state.