S-nitrosylation mediates synaptic plasticity in the retina

Includes bibliographical references.2015 Summer.Over the course of an entire day, our visual system must accommodate intensities of light that can change by a factor of 10¹⁰. In order to do so, the retina adapts to large, daily changes in natural light intensity by shifting its dynamic range of coding. For example, as morning light intensity increases, the retina implements multiple strategies that result in decreases in overall sensitivity in order to avoid saturation. However, adaptation to bright environments poses the inherent risk of losing visual information carried by dim/weak signals in complex natural scenes. Here we studied whether the light-evoked increase in retinal nitric oxide (NO) production is followed by NO-mediated, direct post-translational modification of proteins called S-nitrosylation and if it contributes to the modulation of the dynamic range of vision. In the central nervous system, including the retina, S-nitrosylation has not been considered to be significant under physiological conditions, and instead, has been primarily associated with neurodegenerative diseases. In this study, we provide immunohistochemical and proteomic evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner. Functionally, we report a novel form of activity-dependent synaptic plasticity via S-nitrosylation: a “weighted potentiation” that selectively increases the output of Mb-type bipolar cells in the goldfish retina in response to weak inputs but leaves the input-output ratio for strong stimuli unaffected. Importantly, the NO action resulted in a weighted potentiation of Mb output in response to small (≤-30 mV) depolarizations. Our data strongly suggest that in the retina, light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant post-translational modification affecting a wide range of proteins under physiological conditions. S-nitrosylation may function to extend the dynamic range of vision by counteracting the decreases in retinal sensitivity during light adaptation ultimately preventing the loss of visual information carried by dim scotopic signals. Finally, our results may set the framework for exploring the role of S-nitrosylation in certain neurodegenerative retinal diseases that are associated with toxic levels of NO

Light inputs to dopaminergic amacrine cells of the mammalian retina

Background: The retina responds to light over a wide range of operational conditions, surpassing 10 units in a logarithmic scale. Adaptation of the retina to the particular presenting light conditions relies considerably on modulation of retinal pathways by dopamine, which is released in response to light or circadian rhythms exclusively from dopaminergic amacrine cells. Rods, cones and intrinsically photoresponsive retinal ganglion cells (ipRGCs) have all been shown to input into dopaminergic amacrine cells. However, the pathways that these photoreceptors employ to ultimately trigger dopamine release in response to light remain unclear. Methods: Ultra-high performance liquid chromatography separation and tandem mass spectrometry detection was used to quantify dopamine, and its primary metabolite 3,4-dihidroxyphenylacetic acid (DOPAC). Retinal dopamine release was assessed under various conditions, in a variety of mouse models, using two complementary experimental designs: in vivo anaesthetised mice and ex vivo explanted retinae. Conclusions: This thesis provides novel evidence about dopamine dynamics in a variety of light conditions, transgenic mouse models and presence of pharmacological agents. Surprisingly, I found that rod input is both necessary and sufficient to evoke light-induced release dopamine across a wide range of light intensities, without quantifiable contribution from cones or ipRGCs, suggesting that electrophysiological inputs do not match dopamine release. Further, this data suggests that the main pathway that drives this increase in light-induced dopamine release at light intensities where rods should be saturated is the primary rod pathway (with smaller contributions from the secondary and tertiary pathways) and involves bleaching adaptation of rods

VLSI analogs of neuronal visual processing: a synthesis of form and function

This thesis describes the development and testing of a simple visual system fabricated using complementary metal-oxide-semiconductor (CMOS) very large scale integration (VLSI) technology. This visual system is composed of three subsystems. A silicon retina, fabricated on a single chip, transduces light and performs signal processing in a manner similar to a simple vertebrate retina. A stereocorrespondence chip uses bilateral retinal input to estimate the location of objects in depth. A silicon optic nerve allows communication between chips by a method that preserves the idiom of action potential transmission in the nervous system. Each of these subsystems illuminates various aspects of the relationship between VLSI analogs and their neurobiological counterparts. The overall synthetic visual system demonstrates that analog VLSI can capture a significant portion of the function of neural structures at a systems level, and concomitantly, that incorporating neural architectures leads to new engineering approaches to computation in VLSI. The relationship between neural systems and VLSI is rooted in the shared limitations imposed by computing in similar physical media. The systems discussed in this text support the belief that the physical limitations imposed by the computational medium significantly affect the evolving algorithm. Since circuits are essentially physical structures, I advocate the use of analog VLSI as powerful medium of abstraction, suitable for understanding and expressing the function of real neural systems. The working chip elevates the circuit description to a kind of synthetic formalism. The behaving physical circuit provides a formal test of theories of function that can be expressed in the language of circuits

Multiquantal Glutamate Release from Rod Photoreceptors

Neurons communicate via Ca2+-dependent release of neurotransmitters packaged into vesicles (quanta). Some CNS neurons, especially sensory synapses, can release multiple vesicles at a time, increasing information transmission and overcoming the unreliability of a stochastic process. Ribbon-bearing neurons, including retinal photoreceptors, face the challenge of encoding sensory receptor potentials into an ever-changing train of vesicle release events. We studied release of glutamate using voltage clamp to measure anion currents activated during glutamate reuptake into presynaptic terminals (IA(glu)) of salamander and mouse rods, finding that each employ distinct mechanisms for multiquantal release. In amphibian rods, we found that 1/3 of the spontaneous IA(glu) fusion events involve synchronous fusion of multiple vesicles. By varying intracellular buffering to localize Ca2+-dependent events, we found that multiquantal release occurs near Ca2+ sources. In photoreceptors, Ca2+ influx occurs just below synaptic ribbons. Vesicles house SNARE machinery so we hypothesized that vesicles on the ribbon undergo homotypic fusion prior to exocytosis. Destruction of ribbons and disruption of the SNARE-protein syntaxin3B prevented spontaneous multiquantal release, suggesting that salamander rods are capable of multivesicular release due to homotypic fusion of vesicles along ribbons. In mouse rods, spontaneous release at −70 mV involved the stochastic fusion of single vesicles. With depolarization, glutamate release increased linearly with voltage-gated Ca2+ currents. As the membrane approached the resting potential in darkness of −40 mV, rods began to release glutamate in multivesicular bursts of 17±7 vesicles every 2801±598 ms. Release evoked by brief depolarizations and bursts both involved the same pool of ribbon-associated vesicles with fusion regulated by the vesicular Ca2+ sensor synaptotagmin-1. A second, slower component of release controlled by synaptotagmin-7 is also present in rods but not cones. We hypothesized a v role for coordinated bursts of release in transmitting single photon signals. The rate of bursting was responsive to small voltage changes of 1.0-3.5 mV and the voltage waveform that triggered bursts most effectively was similar to single photon responses. We propose that multiquantal bursts contribute to mechanisms that filter out small noisy events to improve reliable detection of single photons by the retina

Chromatic Properties of Bipolar Cells in the Mouse Retina

The retina performs a wide range of computations to process visual signals. Feature extrac-tion, such as the detection of edges, motion, and color originate in specialized retinal circuits. In this study we investigated the circuits underlying chromatic processing in the mouse retina. Although color vision is wide spread among mammals, its research tends to focus on primates. Studying non-primate mammals can be advantageous in understanding the general principles of retinal chromatic processing. Like most mammals, mice feature dichromatic color vision based on short (S) and medium (M) wavelength-sensitive cone types. It is thought that mammals share a common retinal circuit that compares S- and M-cone output (in trichromats S- and M+L-cone) to generate blue/green (blue/yellow) opponent signals, with distinct bipolar cells providing separate chromatic channels. While S cone selective ON-bipolar cells (in mouse “type 9”) have been anatomi-cally identified, little is known about other cone selective channels, such as, for instance, M-cone selective OFF-bipolar cells. Here, we characterized cone connectivity and light responses of selected mouse bipolar cell types using immunohistochemical and electrophysiological methods. Our anatomical data indicate that four of the five mouse OFF-bipolar cell types (types 2, 3a/b and 4) as well as type 7 (as an example for ON-bipolar cells) indiscriminately contact both S- and M-cones. Using a marker that labels dendrites of both type-1 and -2 OFF-bipolar cells we found reduced immunofluorescence at S-cone, suggesting that type 1 avoids S-cones. Recordings of light responses showed that the chromatic tuning of bipolar cells strongly depended on their position along the dorso-ventral axis – due to the dorso-ventral gradient in S-opsin co-expression in mouse M-cones. In dorsal retina, where co-expression is low, most type-2 (and type-7) cells were green-biased, with a fraction of cells (≈ 14 %) displaying strongly blue-biased responses, likely reflecting S-cone input. Type 1 cells were also green biased but did not include blue-biased “outliers”, consistent with type-1 cells avoiding S-cones. We therefore suggest that type 1 represents the greenOFF pathway in mouse. In addition, we confirmed that type-9 bipolar cells display blueON responses. In ventral retina, all bipolar cell types studied here displayed similar blue-biased responses, suggesting that color vision may be only supported in the dorsal mouse retina. In conclusion, our data supports an antagonistically organized blue/green circuit with bipolar cells functioning as chromatically defined channels, which form the common basis for mammalian dichromatic color vision

Modèle et simulateur à grande échelle d'une rétine biologique, avec contrôle de gain

The retina is a complex neural structure. The characteristics of retinal processing are reviewed extensively in Part I of this work: It is a very ordered structure, which proceeds to band-pass spatio-temporal enhancements of the incoming light, along different parallel output pathways with distinct spatio-temporal properties. The spike trains emitted by the retina have a complex statistical structure, such that precise spike timings may play a role in the code conveyed by the retina. Several mechanisms of gain control provide a constant adaptation of the retina to luminosity and contrast. The retina model that we have defined and implemented in Part II can account for a good part of this complexity. It can model spatio-temporal band-pass behavior with adjustable filtering scales, with the inclusion of plausible mechanisms of contrast gain control and spike generation. The gain control mechanism proposed in the model provides a good fit to experimental data, and it can induce interesting effects of local renormalization in the output retinal image. Furthermore, a mathematical analysis confirms that the gain control behaves well under simple sinusoidal stimulation. Finally, the simulator /Virtual Retina/ implements the model on a large-scale, so that it can emulate up to around 100,000 cells with a processing speed of about 1/100 real time. It is ready for use in various applications, while including a number of advanced retinal functionalities which are too often overlooked.La rétine est une structure neuronale complexe, qui non seulement capte la lumière incidente au fond de l'oeil, mais procède également à des transformations importantes du signal lumineux. Dans la Partie I de ce travail, nous résumons en détail les caractéristiques fonctionnelles de la rétine des vertébrés: Il s'agit d'une structure très ordonnée, qui réalise un filtrage passe-bande du stimulus visuel, selon différents canaux parallèles d'information aux propriétés spatio-temporelles distinctes. Les trains de potentiels d'action émis par la rétine ont également une structure statistique complexe, susceptible de véhiculer une information importante. De nombreux mécanismes de contrôle de gain permettent une adaptation constante à la luminosité et au contraste. Le modèle de rétine défini et implémenté dans la Partie II de ce travail prend en compte une part importante de cette complexité. Il reproduit le comportement passe-bande, à l'aide de filtres linéaires spatio-temporels appropriés. Des mécanismes non-linéaires d'adaptation au contraste et de génération de potentiels d'action sont également inclus. Le mécanisme de contrôle du gain au contraste proposé permet une bonne reproduction des données expérimentales, et peut également véhiculer d'importants effets d'égalisation spatiale des contrastes en sortie de rétine. De plus, une analyse mathématique confirme que notre mécanisme a le comportement escompté en réponse à une stimulation sinusoïdale. Enfin, le simulateur /Virtual Retina/ implémente le modèle à grande échelle, permettant la simulation d'environ 100 000 cellules en un temps raisonnable (100 fois le temps réel)

Developing a new generation of neuro-prosthetic interfaces: structure-function correlates of viable retina-CNT biohybrids

PhD ThesisOne of the many challenges in the development of neural prosthetic devices is the choice of electrode material. Electrodes must be biocompatible, and at the same time, they must be able to sustain repetitive current injections in a highly corrosive physiological environment. We investigated the suitability of carbon nanotube (CNT) electrodes for retinal prosthetics by studying prolonged exposure to retinal tissue and repetitive electrical stimulation of retinal ganglion cells (RGCs). Experiments were performed on retinal wholemounts isolated from the Cone rod homeobox (CRX) knockout mouse, a model of Leber congenital amaurosis. Retinas were interfaced at the vitreo-retinal juncture with CNT assemblies and maintained in physiological conditions for up to three days to investigate any anatomical (immunohistochemistry and electron microscopy) and electrophysiological changes (multielectrode array stimulation and recordings; electrodes were made of CNTs or commercial titanium nitride). Anatomical characterisation of the inner retina, including RGCs, astrocytes and Müller cells as well as cellular matrix and inner retinal vasculature, provide strong evidence of a gradual remodelling of the retina to incorporate CNT assemblies, with very little indication of an immune response. Prolonged electrophysiological recordings, performed over the course of three days, demonstrate a gradual increase in signal amplitudes, lowering of stimulation thresholds and an increase in cellular recruitment for RGCs interfaced with CNT electrodes, but not with titanium nitride electrodes. These results provide for the first time electrophysiological, ultrastructural and cellular evidence of the time-dependent formation of strong and viable bio-hybrids between the RGC layer and CNT arrays in intact retinas. We conclude that CNTs are a promising material for inclusion in retinal prosthetic devices

ZEBRAFISH AS AN INNOVATIVE MODEL TO SCREEN THE BEHAVIOURAL EFFECTS OF NOVEL DRUGS

Zebrafish (Danio rerio) is an emerging animal model alternative to rodents for studying human diseases. Its typical shoaling behaviour (tight aggregation of individuals) consisting of forming a tight group in which fish swim together, may represent an excellent model to study social behaviour. Zebrafish appear to be a good model to study learning and memory, too. The neuropeptides oxytocin (OT) and arginine vasopressin (AVP) are two of the most-studied brain signaling molecules encoding information relevant to social behaviour. Isotocin (ISO) and vasotocin (AVT) are the equivalent neurohypophiseal hormones in fish, regulating reproductive and social behaviour. On this basis, we studied the effect of both OT and AVP in comparison with ISO and AVT, on shoaling, fear response to predator and learning and memory. Social behaviour was studied using mutant zebrafish Nacre. Since these peptides are known to affect anxiety in humans and rodents, the same compounds were also tested on fear response to predator, using Astronotus Ocellatus as stimulus fish. OT (2-40 ng/kg), ISO (0.1-10 ng/kg), AVP (0.5-40 ng/kg) and AVT (0.001-20 ng/kg) were given i.m. 10 min before each test. AVT/AVP were more potent to elicit anxiolytic than social effect while ISO and OT were equally potent. To investigate the mechanism of action, different antagonists were given 10 min before each peptide: the OT receptor antagonist Desgly (0.00001-1 ng/kg), the V1a receptor subtype AVP antagonist SR 49059 (0.00001-20 ng/kg) and the V1b receptor subtype antagonist SSR 149415 (0.00001-1 ng/kg). In both tests, treatment with all the peptides increased social preference and decreased fear response in a dose-dependent manner interpolated by symmetrical parabolas. Pre-treatment with SR 49059, SSR 149415 and Desgly dose-dependently blocked the pro-social and anxiolytic effect induced by each peptide. The less selective antagonist appeared to be SSR 149415. All the neuropeptides did not induce any change in swimming activity. Neuronal nicotinic acetylcholine receptors (nAChRs) play a modulatory role in cognition and zebrafish provide a preclinical model to study these cognitive processes. On the other hand, nicotinic receptor has been characterized in this teleost fish. Using a T-maze task, we investigated the effect of cholinergic drugs on spatial memory in zebrafish. Nicotine (0.0002-0.2 mg/kg), given i.p. 20 min before the test, improved the mean running time difference, showing an inverted U dose-response function. Selective and non selective nAChR antagonists, injected i.p. 10 min before nicotine, were used to study the receptor subunits, involved in spatial memory. Nicotine-induced cognitive enhancement was reduced by the selective nAChR subtype antagonists, MLA (0.01 mg/kg) for \u3b17 subunit, MII (0.1 mg/kg) for \u3b16\u3b22 subunit, Dh\u3b2E (0.01 mg/kg) for the \u3b14\u3b22 subunit, the non selective antagonist mecamylamine (0.1 mg/kg) and the muscarinic antagonist scopolamine (0.025 mg/kg), with Dh\u3b2E being more active than MLA or MII. No change in swimming activity was observed for all the nicotinic drugs. Another important cognitive process is the selective attention. It can be assessed in rodents with the novel object recognition (NOR) test. In the standard version of this test, the selection of objects to be used is critical. To overcome the limitation of NOR, we created a modified version of NOR, the virtual object recognition test (VORT) in mice where 3D objects were replaced with stationary geometrical 2D shapes and presented on two Ipods 3.5-inch widescreen displays. A comparable discrimination index as NOR was shown in VORT. 2D shapes that could be highly discriminated and some which could not, were identified. Mice were able to distinguish among different movements (horizontal, vertical or oblique). In fact, the shapes previously found not distinguishable when stationary were better discriminated when moving. Secondly, we focused our attention on zebrafish, which have a good capability to learn and a better visual acuity. Based on this abilities, we investigated in VORT if zebrafish, like mice, were able to discriminate different geometrical 2D shapes (circle, square or triangle), when presented on Ipod-screens, placed at the sides of a water tank. To evaluate the possibility that moving 2D shapes increased the attention of zebrafish, specific movements were applied to the same geometrical shapes. We found that zebrafish, like mice, were able to discriminate different geometrical 2D shapes both stationary and with different movements. In particular, the discrimination index of shapes, previously not discriminate, increased when they were moving. Finally, we investigated if memory performance could be improved by treatment with nicotine both in mice (0.1 mg/kg) and in zebrafish (0.02 mg/kg) or worsened by scopolamine (0.25 mg/kg for mice and 0.025 mg/kg for zebrafish) or by mecamylamine (1 mg/kg). Nicotine improved discrimination index for stationary shapes previously not discriminated while anticholinergic drugs impaired episodic memory in both species. Taken together, these findings showed the pro-social and anxiolytic properties of OT/AVP system mediated by different receptors and confirmed the important role of cholinergic system in the processes of acquisition and memory consolidation in zebrafish similar to mammals. Moreover, we showed, for the first time, both mice and zebrafish could discriminate not only geometrical shapes but also different movements in VORT, allowing a direct comparison between animal model and human to study attention. Zebrafish opens a new avenue of research to rapidly screen new compounds for the treatment of abnormal social behaviours (including autism or schizophrenia) and neurodegenerative diseases

Processing of local features in the zebrafish optic tectum

The optic tectum is the main visual processing area in zebrafish and is involved in a variety of visually-driven behaviours. A key question is how information about the visual environment is processed and integrated in order to generate guided behaviour. The aim of this study was to explore the response properties of tectal neurons, i.e., their preference for certain features of the visual input. To do this, I developed a custom set-up for calcium imaging and simultaneous visual stimulation in older zebrafish larvae, up to the age of 21 dpf. First, this set-up was employed to measure the spatial receptive fields of tectal neurons with small moving spots. Notably, the results suggested that receptive field development is not completed by 9 dpf as previously believed; instead, receptive field refinement continues beyond this age. The results also confirmed that receptive fields in the optic tectum were relatively large in older larvae. Based on this, I formulated the hypothesis that tectal neurons might process multiple local features simultaneously. To test how the optic tectum encodes local features, I used small, moving oriented bars and combinations of bars, i.e., angles. Tectal responses to these stimuli suggested that, not only does the optic tectum encode local features, but is also tuned to horizontal-oriented local stimuli. Finally, I used a set of moving stimuli, consisting of simple features (i.e., lines and angles) and a composite feature (i.e. square) to test how information about multiple local features was integrated by tectal neurons. The results indicated that local features are spatially integrated in a sublinear fashion. The outcomes of the work presented in this thesis add to our understanding of how visual information provided by the retina is processed within the optic tectum

SOX2 Is Essential for the Maturation and Maintenance of Retinal Müller Glia

Muller glia (MG) are the principal glial cell of the vertebrate retina. The last cell to divide from a multipotent retinal progenitor cell, they maintain many stem cell characteristics including the expression of the HMG-box transcription factor Sox2. In this thesis, we explore the role of Sox2, a marker of pluripotency throughout the CNS, in this population of presumptive neural stem cells, the MG. Through glial specific ablation of Sox2 we demonstrate that SOX2 plays an essential role in the maturation and maintenance of MG in the murine retina. Loss of SOX2 at P5 results in aberrant development and extension of MG side processes that ensheathe the neuronal cell bodies and neurites in the retina. Additionally, MG cell bodies are disorganized and their end feet fail to properly form the limiting membranes of the retina. As a result, neuronal processes in the synaptic plexiform layers are disorganized, accompanied by a marked reduction in inner retinal function. These data indicate a role for Sox2 in guiding the structural development of MG, as well as providing new insights into the role of MG in the maturation of the neural retina. Additionally we address the complex regulation of SOX2 in the retina by examining SOX2 expression in the mildly hypomorphic Sox2Cond line in two different strain backgrounds: on the inbred C57BL6/J background and on a mixed, outbred CD1 background. On a CD1 background, mice heterozygous for the Sox2Cond allele display only a mild reduction in SOX2 expression and display no phenotypic abnormalities. However, SOX2 expression is significantly reduced on the C57BL6/J background compared to wild type levels, accompanied by a marked reduction in retinal function that degenerates over the animal's lifetime. Further, Muller glial specific ablation in the C57BL6/J background results in almost complete loss of retinal function and a slow loss of MG cells over time. Together these results demonstrate the essential role of SOX2 in the maturation of MG and the neural retina, as well as pointing to a role for SOX2 in the maintenance of retinal and MG structure and function in the compromised retina.Doctor of Philosoph