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 glass1. 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 release2,3. 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 RIBEYE4, 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

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 mGluR1α 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

Vision in the ultraviolet range in Carassius auratus (Ostariophysi, Cipriniformes, cyprinidae)an electrophysiological study of the cones-horizontal cells systems

Nas últimas décadas uma série de experimentos realizados em vertebrados tem demonstrado a capacidade que alguns destes possuem de discriminar a luz ultravioleta, ao contrário do acreditado anteriormente. O peixe dourado (Carassius auratus), um bom modelo experimental para pesquisas sobre visão, apresentou em experimentos comportamentais altos níveis de discriminação de cor nas regiões espectrais do azul e do violeta, o que só seria possível com a existência de um receptor adicional para o ultravioleta (Neumeyer, 1985; Hawryshyn & Beauchamp, 1985; Neumeyer & Arnold, 1989; Neumeyer, 1992; Fratzer, Dörr & Neumeyer, 1994). Estes resultados foram confirmados pela determinação microespectrofotométrica (Bowmaker, Thorpe & Douglas, 1991) e eletrofisiológica (Palacios et al., 1998) da existência de cones com pigmentos específicos para luz ultravioleta, sem haver entretanto um estudo da codificação dessa entrada em neurônios de segunda ordem até o momento. Além disso, permanece ainda em discussão na literatura o papel das células horizontais retinianas no processamento cromático. O presente projeto teve por objetivo investigar qual a contribuição da entrada do receptor UV para os perfis de resposta eletrofisiológica das células horizontais e quais tipos celulares da retina externa do Carassius auratus são subjacentes à discriminação observada comportamentalmente. Para tanto, registros intracelulares de células horizontais e bipolares foram obtidos sob estimulaçãomonocromática de diferentes intensidades, ) diâmetros e comprimentos de onda, com o intuito de determinar suas respostas espectrais, bem como o campo receptivo e as possíveis interações entre estas e os cones. As células horizontais mono-, bi- e trifásica apenas hiperpolarizam na região do UV, não havendo oponência cromática entre as regiões do UV e do azul em nenhum destes tipos celulares; tampouco encontramos uma célula horizontal tetrafásica. Através de adaptações cromáticas, observamos serem as respostas eletrofisiológicas à luz das células horizontais mono- e bifásicas resultado de interações entre os sistemas de cones vermelho,verde e azul, não tendo sido identificada nenhuma entrada UV significativa nessas células. Por fim, encontramos uma célula bipolar oponente entre as regiões espectrais do UV e do azul, o que constitui uma base neural compatível com a discriminação comportamentalmente observada. Uma vez que canais oponentes são necessários para a discriminação de cor, e essa oponência não foi encontrada nas células horizontais, mais sim nas bipolares, acreditamos ser esse um indício de que as células horizontais não participam diretamente da codificação da informação UV na retina do Carassius auratus. Essa idéia está de acordo com dados recentes da literatura, que atribuem às células horizontais papel relacionado aos mecanismos de constância e contraste simultâneo de cor, deixando para as células bipolares a função de codificar cores para os neurôniossubseqüentes (Kamermans, Kraaij & Spekreijse, 1998)In the last decades a number of experiments in vertebrates has demonstrated the ability of some of these animals to discriminate ultraviolet light. Among them, the goldfish (Carassius auratus), considered a good model in vision research, presented in behavioural experiments high discrimination rates in the violet and blue spectral regions, which could only be accomplished through an additional UV receptor (Neumeyer, 1985; Hawryshyn & Beauchamp, 1985; Neumeyer & Arnold, 1989; Neumeyer, 1992; Fratzer, Dörr & Neumeyer, 1994). These results were confirmed by microspectrophotometrical (Bowmaker, Thorpe & Douglas, 1991) and electrophysiological (Palacios et al., 1998) determinations of the existence of cones with specific photopigments with a maximum in the UV region of the light spectrum. However, to date there are no data concerning the spectral coding of this input in second order neurons. Furthermore, the role of the retinal horizontal cells in chromatic processing remains under debate. The present study aimed at investigating the contribution of the UV input to the electrophysiological response profiles of the cone-driven horizontal cells, as well as determining which neurons in the outer retina of the goldfish could subserve vision in the UV range, as observed behaviourally. For that purpose, intracellular recordings of horizontal and bipolar cells under monochromatic stimuli of different intensities, diameters and wavelengths were obtained in order to determinetheir action spectra, receptive field sizes and characteristics as well as the possible interactions between them and the different cone systems. Mono-, bi- and triphasic horizontal cells always hyperpolarised in the UV spectral range: there was no chromatic opponency between the UV and the blue zones in any of these cell types. We have also failed to find a tetraphasic horizontal cell. We observed through chromatic adaptation experiments that the electrophysiological responses recorded from mono- and biphasic horizontal cells are the result of interactions between the red, green and blue cone systems. No relevant UV input to these cells could be found. Finally, we found a bipolar cell type with spectral opponency between the UV and blue regions, which could be the neural basis of the discrimination observed behaviourally. Colour-opponent channels are thought to be necessary for colour discrimination, and the absence of a horizontal cell type with opponency between the UV and blue spectral regions provides further evidence that this cell type might not play a major role in the chromatic processing in the goldfish retina. Since this opponency pattern was found in a bipolar cell, we think that this cell type, and not the horizontal cells, might underlie the coding of the UV information in this animal. This idea is in good agreement with some recent literature data that ascribe the horizontal cells a role in colour constancy and simultaneous colour contrast phenomena,leaving the function of colour codification to the bipolar cells (Kamermans, Kraaij & Spekreijse, 1998

Computational neuroscience in the study of cognitive processes

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.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.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.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

A neurociência computacional no estudo dos processos cognitivos

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

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.FAPESP[07/54136-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP[96/12645-9]FAPESP[92/3498-1]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq[523303/95-5]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Financiadora de Estudos e Projetos (FINEP)FINEP[66.94.0107.00

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

Abstract 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