Direct Observation of Vesicle Transport on the Synaptic Ribbon Provides Evidence That Vesicles Are Mobilized and Prepared Rapidly for Release.

Synaptic ribbons are thought to provide vesicles for continuous release in some retinal nonspiking neurons, yet recent studies indicate that genetic removal of the ribbon has little effect on release kinetics. To investigate vesicle replenishment at synaptic ribbons, we used total internal reflection fluorescence microscopy to image synaptic vesicles and ribbons in retinal bipolar cells of goldfish (Carassius auratus) of both sexes. Analysis of vesicles released by trains of 30 ms depolarizations revealed that most releasable vesicles reside within 300 nm of the ribbon center. A single 30 ms step to 0 mV was sufficient to deplete the membrane-proximal vesicle pool, while triggering rapid stepwise movements of distal vesicles along the ribbon and toward the plasma membrane. Replenishment only becomes rate-limiting for recovery from paired-pulse depression for interstimulus intervals shorter than 250 ms. For longer interstimulus intervals, vesicle movement down the ribbon is fast enough to replenish released vesicles, but newly arrived vesicles are not release-ready. Notably, the rates of vesicle resupply and maturation of newcomers are among the fastest measured optically at any synapse. Lastly, our data show that the delay in vesicle departure increases and vesicle speed decreases with multiple stimuli. Our results support a role for ribbons in the supply of vesicles for release, provide direct measurements of vesicle movement down the ribbon, and suggest that multiple factors contribute to paired-pulse depression.SIGNIFICANCE STATEMENT Synaptic ribbons are macromolecular scaffolds that tether synaptic vesicles close to release sites in nonspiking neurons of the retina and cochlea. Because these neurons release neurotransmitter continuously, synaptic ribbons are assumed to act as platforms for supplying vesicles rapidly in the face of prolonged stimulation. Yet, ribbon synapses suffer from profound paired-pulse depression, which takes seconds to subside. We investigated the mechanistic origin of this phenomenon by directly imaging triggered vesicle movement and release at ribbon sites in retinal bipolar cells, and find that, although ribbon synapses deliver and prime vesicles faster than most conventional synapses, both vesicle absence and vesicle priming contribute to the long recovery from paired-pulse depression.

Expression and distribution of synaptotagmin family members in the zebrafish retina.

Synaptotagmins belong to a large family of proteins. Although various synaptotagmins have been implicated as Ca2+ sensors for vesicle replenishment and release at conventional synapses, their roles at retinal ribbon synapses remain incompletely understood. Zebrafish is a widely used experimental model for retinal research. We therefore investigated the homology between human, rat, mouse, and zebrafish synaptotagmins 1-10 using a bioinformatics approach. We also characterized the expression and distribution of various synaptotagmin (syt) genes in the zebrafish retina using RT-PCR, qPCR, and in situhybridization, focusing on the family members whose products likely underlie Ca2+ -dependent exocytosis in the central nervous system (synaptotagmins 1, 2, 5, and 7). Most zebrafish synaptotagmins are well conserved and can be grouped in the same classes as mammalian synaptotagmins, based on crucial amino acid residues needed for coordinating Ca2+ binding and determining phospholipid binding affinity. The only exception is synaptotagmin 1b, which lacks 34 amino acid residues in the C2B domain and is therefore unlikely to bind Ca2+ there. Additionally, the products of zebrafish syt5a and syt5b genes share identity with mammalian class 1 and 5 synaptotagmins. Zebrafish syt1, syt2, syt5, and syt7 paralogues are found in the zebrafish brain, eye, and retina, excepting syt1b, which is only present in the brain. The complementary expression pattern of the remaining paralogues in the retina suggests that syt1a and syt5a may underlie synchronous release and syt7a and syt7b may mediate asynchronous release or other Ca2+ -dependent processes in different retinal neurons.

No evidence of UV cone input to mono- and biphasic horizontal cells in the goldfish retina.

Many animal species make use of ultraviolet (UV) light in a number of behaviors, such as feeding and mating. The goldfish (Carassius auratus) is among those with a UV photoreceptor and pronounced UV sensitivity. Little is known, however, about the retinal processing of this input. We addressed this issue by recording intracellularly from second-order neurons in the adult goldfish retina. In order to test whether cone-driven horizontal cells (HCs) receive UV cone inputs, we performed chromatic adaptation experiments with mono- and biphasic HCs. We found no functional evidence of a projection from the UV-sensitive cones to these neurons in adult animals. This suggests that goldfish UV receptors may contact preferentially triphasic HCs, which is at odds with the hypothesis that all cones contact all cone-driven HC types. However, we did find evidence of direct M-cone input to monophasic HCs, favoring the idea that cone-HC contacts are more promiscuous than originally proposed. Together, our results suggest that either UV cones have a more restricted set of post-synaptic partners than the other three cone types, or that the UV input to mono- and biphasic HCs is not very pronounced in adult animals.

Retinal alterations in a pre-clinical model of an autism spectrum disorder.

Background: Autism spectrum disorders (ASD) affect around 1.5% of people worldwide. Symptoms start around age 2, when children fail to maintain eye contact and to develop speech and other forms of communication. Disturbances in glutamatergic and GABAergic signaling that lead to synaptic changes and alter the balance between excitation and inhibition in the developing brain are consistently found in ASD. One of the hallmarks of these disorders is hypersensitivity to sensory stimuli; however, little is known about its underlying causes. Since the retina is the part of the CNS that converts light into a neuronal signal, we set out to study how it is affected in adolescent mice prenatally exposed to valproic acid (VPA), a useful tool to study ASD endophenotypes. Methods: Pregnant female mice received VPA (600 mg/kg, ip) or saline at gestational day 11. Their male adolescent pups (P29-35) were behaviorally tested for anxiety and social interaction. Proteins known to be related with ASD were quantified and visualized in their retinas by immunoassays, and retinal function was assessed by full-field scotopic electroretinograms (ERGs). Results: Early adolescent mice prenatally exposed to VPA displayed impaired social interest and increased anxiety-like behaviors consistent with an ASD phenotype. The expression of GABA, GAD, synapsin-1, and FMRP proteins were reduced in their retinas, while mGluR5 was increased. The a-wave amplitudes of VPA-exposed were smaller than those of CTR animals, whereas the b-wave and oscillatory potentials were normal. Conclusions: This study establishes that adolescent male mice of the VPA-induced ASD model have alterations in retinal function and protein expression compatible with those found in several brain areas of other autism models. These results support the view that synaptic disturbances with excitatory/inhibitory imbalance early in life are associated with ASD and point to the retina as a window to understand their subjacent mechanisms.

Distribution and density of mixed-input ON bipolar cells of the goldfish (Carassius auratus) during growth.

Neurons are continuously produced at different rates and locations in the teleost retina. Goldfish rods are homogeneously distributed and maintain a stable density throughout growth, whereas little is known about their postsynaptic partners. We examined the distribution and density of mixed-input ON bipolar cells (ON mBCs) in 57 goldfish of various sizes by immunolabeling their retinas with an antibody against PKCα and counting PKCα-positive neurons in wholemounts. Cell densities were correlated with morphometric data for the same animals, and the spatial resolution of the ON mBC mosaic was calculated in each case. The distribution of ON mBCs is homogeneous throughout growth. For a 10-fold change in body size (i.e., from 20 to 200 mm), the total number of ON mBCs increases 2.8 times, while retinal area expands around 10 times. As a consequence, the density of ON mBCs in large fish falls to ∼1/3 of that of small animals, and intercellular spacing doubles. The eye and the lens become around three times larger from small to large fish. This causes the retinal magnification factor (and thereby the image projected onto retina) to augment by the same amount. Because the retinal magnification factor rises more than the intercellular spacing in the same animals, the spatial resolution of the ON mBC mosaic improves from 0.8 to 1.4 cycles/degree as the body size increases from 20 to 200 mm. As ON mBCs are mostly rod-driven, our results suggest that the scotopic acuity of the goldfish may improve as the animal grows.

Pharmacokinetics, electrophysiological, and morphological effects of the intravitreal injection of mycophenolic acid in rabbits.

PURPOSE: To determine the half-life of mycophenolic acid (MPA) in the vitreous of New Zealand albino rabbits after intravitreal injection and the retinal toxicity of different doses of MPA. METHODS: Ten micrograms of MPA (Roche Bioscience, Palo Alto, CA) was injected in the vitreous of 16 rabbits, animals were sacrificed at different time-points, and vitreous samples underwent high-performance liquid chromatography. For functional and morphological studies, 5 doses of MPA (0.05, 0.5, 2, 10, and 100 µg) were injected in the vitreous of 20 rabbits. As control, contralateral eyes were injected with aqueous vehicle. Electroretinograms (ERGs) were recorded before injection and at days 7, 15, and 30. Animals were sacrificed on day 30 and retinas were analyzed under light microscopy. RESULTS: MPA half-life in the vitreous was 5.0±0.3 days. ERG revealed photoreceptor functional impairment in eyes injected with 0.5 µg and higher on day 30, while eyes injected with 100 µg presented the same changes already from day 15. No morphological change was found. CONCLUSIONS: MPA vitreous half-life is 5.0 days. Intravitreal injection of 0.5 µg MPA and higher causes dose- and time-related photoreceptor sensitivity decrease in rabbits. The MPA dose of 0.05 µg may be safe for intravitreal use in rabbits.

A neurociência computacional no estudo dos processos cognitivos / Neuroscience computationnelle dans l'étude des processus cognitifs / La neurociencia computacional en el estudio de los procesos cognitivos / Computational neuroscience in the study of cognitive processes

Resumo Nas últimas décadas o estudo de processos cognitivos vem sendo influenciado por duas tendências: a legitimação de diversas formas e níveis de estudo e a tentativa de integração multidisciplinar. A primeira teve grande importância na segunda metade do século XX, quando linhas de pesquisa na psicologia cognitiva e nas neurociências fortaleceram-se. Nesse sentido, destacam-se os três níveis de Marr (computacional, algorítmico e implementacional) como forma de estruturar o estudo dos processos cognitivos. A segunda tendência é mais recente e busca, apoiada na primeira, aprofundar o entendimento dos processos cognitivos em suas diversas escalas e integrar diversos paradigmas de estudos, buscando consiliência teórica. O intento deste artigo é apresentar a neurociência computacional e suas possíveis contribuições para a psicologia cognitiva, articulando, por meio dos três níveis de Marr, uma base teórica que explicite o papel de cada uma das disciplinas e as suas possíveis interações.

Résumé Au long des dernières décennies, l'étude des processus cognitifs se voit influencé par deux tendances : la légitimation de plusieurs formes et niveaux d'études et l'essai d'intégration multidisciplinaire. La première a eu une grande importance pendant la deuxième moitié du XXe siècle, quand des lignes de recherche en psychologie cognitive et en neurosciences ont gagné force. Dans ce sens, on peut souligner les trois niveaux de Marr (computationnel, algorithmique et implémentationnel) comme moyens de structurer l'étude des procédés cognitifs. La deuxième tendance est plus récente et cherche, avec l'aide de la première, à approfondir la connaissance des procédés cognitifs et ses différentes échelles et à intégrer plusieurs modèles d'études, en cherchant des convergences théoriques. Le but de cet article est donc de présenter la neuroscience computationnelle et ses possibles contributions pour la psychologie cognitive en articulant, par les trois niveaux de Marr, une base théorique qui puisse expliciter le rôle de chacune des disciplines et de ses possibles interactions.

Resumen En las últimas décadas, el estudio de procesos cognitivos se ha visto influenciado por dos tendencias: la legitimación de diversas formas y niveles de estudio, y el intento de integración multidisciplinar. La primera tuvo gran importancia en la segunda mitad del siglo XX, cuando varias líneas de investigación en la psicología cognitiva y en las neurociencias se fortalecieron. En ese sentido, destacan los tres niveles de Marr (computacional, algorítmico e implementacional) como una manera de estructurar el estudio de los procesos cognitivos. La segunda tendencia es más reciente y busca, apoyada en la primera, profundizar la comprensión de los procesos cognitivos en sus diversas escalas e integrar diversos paradigmas de estudios, buscando consiliencia teórica. En este artículo, se intenta presentar la neurociencia computacional y sus posibles contribuciones para la psicología cognitiva, articulando, a través de los tres niveles de Marr, una base teórica que ponga de manifiesto el papel de cada una de las disciplinas y sus posibles interacciones.

Abstract In recent decades the study of cognitive processes has been influenced by two tendencies: legitimation of several forms and levels of study and the attempt of multidisciplinary integration. The first had great importance in the second half of the 20th century, when research lines in cognitive psychology and neuroscience were strengthened. In this sense, Marr's three levels of analysis (computational, algorithmic, and implementation) are one way to structure the study of cognitive processes. The second tendency is more recent and, supported by the first one, seeks to deepen the understanding of cognitive processes in their different scales and to integrate several paradigms of studies in order to reach theoretical consilience. This article aims to introduce computational neuroscience and its possible contributions to cognitive psychology, articulating, through Marr's three levels, a theoretical basis that explains the role of each of the disciplines and their possible interactions.

The cytomatrix protein bassoon contributes to fast transmission at conventional and ribbon synapses.

The presynaptic active zone contains a complex web of proteins involved in synaptic transmission. In this issue of Neuron, two articles show evidence that one of these proteins, Bassoon, coordinates multiple functions in a conventional and ribbon-type synapse.

Retinal parallel pathways: seeing with our inner fish.

The general organization of the vertebrate retina is highly conserved, in spite of structural variations that occur in different animal classes. The retinas of cyprinid fish, for example, differ in many aspects from those of primates. However, these differences are in the same order of magnitude as those found among mammalian species. Therefore, it is important to consider whether these changes are minor variations on the same theme or whether they lead to fundamentally different functions. In this light, we compare the retinal organization of teleost fish and mammals as regards parallel processing and discuss their many similarities.

Imaging exocytosis in retinal bipolar cells with TIRF microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43) by co-puffing the dye and a ringer solution containing a high K(+) concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

Interaction between rod and cone inputs in mixed-input bipolar cells in goldfish retina.

One class of goldfish bipolar cells, the mixed-input bipolar cell, contacts both rods and cones. Although the morphology of the different mixed-input bipolar cell subtypes has been described, insight into the interaction between rods and cones at the bipolar cell level is scarce. The aim of this study was to characterize this interaction in the different physiological types of mixed-input bipolar cells. We found mixed-input bipolar cells that depolarized, hyperpolarized, or showed a combination of the two types of response after center stimulation. The relative contributions of rod and cone inputs varied strongly in these cell populations. Depolarizing mixed-input bipolar cells are rod-dominated, having the highest sensitivity and the smallest dynamic range. Hyperpolarizing mixed-input bipolar cells, on the other hand, have a more balanced rod-cone input ratio. This extends their dynamic range and decreases their sensitivity. Finally, opponent mixed-input bipolar cells seem to be mostly cone-dominated, although some rod input is present. The antagonistic photoreceptor inputs form a push-pull system that makes these mixed-input bipolar cells very sensitive to changes in light intensity. Our finding that spectral tuning changes with light intensity conflicts with the idea that the separate non-opponent and opponent channels are related to coding of brightness and color, respectively. The organization of mixed-input bipolar cells into various classes with different dynamic ranges and absolute sensitivities might be a strategy to transmit information about all visual aspects most efficiently, given the sustained nature of bipolar cell responses and their limited voltage range.

Localization of metabotropic glutamate receptors in the outer plexiform layer of the goldfish retina.

We studied the localization of metabotropic glutamate receptors (mGluRs) in the goldfish outer plexiform layer by light-and electron-microscopical immunohistochemistry. The mGluR1alpha antibody labeled putative ON-type bipolar cell dendrites and horizontal cell processes in both rod spherules and cone triads. Immunolabeling for mGluR2/3 was absent in the rod synaptic complex but was found at horizontal cell dendrites directly opposing the cone synaptic ribbon. The mGluR5 antibody labeled Müller cell processes wrapping rod terminals and horizontal cell somata. The mGluR7 antibody labeled mainly horizontal cell dendrites invaginating rods and cones and some putative bipolar cell dendrites in the cone synaptic complex. The finding of abundant expression of various mGluRs in bipolar and horizontal cell dendrites suggests multiple sites of glutamatergic modulation in the outer retina.

Human retinal circuitry and physiology

Every second, in an average daytime light environment, hundreds of millions of photons enter the human eye and arrive at the photoreceptor layer of the retina. All our information about the visible world is contained in this rain of photons. The retina is a complex tissue, literally an extension of the brain, which transforms the rain of photons into bioelectric signals containing all the information available to the brain to interpret and respond to the external visual world. A considerable amount of processing takes place within the retinal tissue itself. Understanding what kind of processing takes place at each retinal stage is crucial for understanding normal vision, vision in the presence of diseases affecting the retina, and, ultimately, for the development of therapies to treat such diseases. This manuscript reviews the relation between structure and function of the different retinal pathways and addresses their possible roles for visual perception.