Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities.

In mammals, synchronization of the circadian pacemaker in the hypothalamus is achieved through direct input from the eyes conveyed by intrinsically photosensitive retinal ganglion cells (ipRGCs). Circadian photoentrainment can be maintained by rod and cone photoreceptors, but their functional contributions and their retinal circuits that impinge on ipRGCs are not well understood. Using mice that lack functional rods or in which rods are the only functional photoreceptors, we found that rods were solely responsible for photoentrainment at scotopic light intensities. Rods were also capable of driving circadian photoentrainment at photopic intensities at which they were incapable of supporting a visually guided behavior. Using mice in which cone photoreceptors were ablated, we found that rods signal through cones at high light intensities, but not at low light intensities. Thus, rods use two distinct retinal circuits to drive ipRGC function to support circadian photoentrainment across a wide range of light intensities

Cytoskeletal scaffolds in neuronal development

Neuronal polarization is one of the most studied topics in neuroscience. In less than seven days neurites sprout out from the neuron, explore the surrounding environment and mature in axon or dendrites. This process is possible because the neuronal cytoskeleton can rapidly modify its architecture changing neuronal shape and length. Among all, two main proteins are involved: tubulin that supports the neurite elongation and builds a solid frame, while actin supports pathfinding. In this period of important cytoskeletal changes, it is possible to observe the actin waves (AW) that are highly dynamic structures emerging at the neurite base which move up to its tip, causing a transient retraction of the growth cone (GC). Since their discovery in 1988, there have been only few studies about AWs, usually linked to the neurite outgrowth and axon elongation. In the present work, I used long term live cell imaging to investigate alternative roles of such cytoskeletal phenomena. I examined in details AWs and I concluded that they do not promote the neurite outgrowth and that neurites can elongate for hundreds of microns without the AWs. Super resolution nanoscopy indicates that myosin II shapes the GC like AWs structure. The highly concentrated myosin inside the wave can bend the tubulin that support the neurite provoking twists and kinks in the microtubular cytoskeleton. These tubulin twists (TT) cause the GC retraction and are completely abolished with the inhibition of myosin II, that compromises the AW morphology. My results indicate that myosin II has an important role in the AWs dynamics and can bend the tubulin in a way that was not previously observed. Finally, we suggested a role for AWs and TTs in GC exploration and in neurite maturation. Part of these results have already been published in Frontiers in Cellular Neuroscience in the article: Actin waves do not boost neurite outgrowth in the early stages of neuron maturation. We have a second manuscript in preparation entitled \u201cTubulin twists drive Growth Cone retraction and promote tubulin mixed polarity\u201d

New opportunities for photon storage and detection: an exploration of a high-efficiency optical quantum memory and the quantum capabilities of the human eye

The field of quantum information has grown in recent years, due to tremendous technological advancements toward quantum networking and quantum computation. Nevertheless, there is still a great need for creative research that explores possibilities for new capabilities. Particularly, we look towards quantum optics research to develop new ways of manipulating and detecting photons. Here, we discuss our efforts toward developing two separate quantum optics experiments that can provide great insight into the development of quantum devices. We begin by discussing our work to investigate the lower threshold of human vision and the eye's potential as a single-photon detector, using a custom-built single-photon source, and a novel two-alternative forced-choice experimental design. Our preliminary findings show promising data that support previous results found from a similar experiment using a somewhat different approach. We then discuss our second project, where we have developed a robust reconfigurable optical delay line quantum memory that compares favorably with competing methods. Our memory is capable of photon storage with an unprecedentedly high time-bandwidth product, high free-space transmission over the range of 10 $\mu$s, and high fidelity. These attributes, plus the memory's capability for multi-mode storage, make this system a strong candidate for a critical component in the large-scale heterogeneous quantum networks we hope to see developed in the next ten years

New issues in phototransduction

Light and dark cycles are one of the principal driving forces for the metabolism of both eukaryotic and prokaryotic organisms, which are the evolutionary closest one to the first living beings. Therefore, is reasonable to think that the alternation between light and dark has influenced the evolution of all the organisms on Earth. Cyanobacteria as well as yeasts have evolved oscillating rhythms of gene expression, also known as circadian rhythms, in order to synchronize their physiological processes with Earth\u2019s day/night cycles. Light cues can also drive unicellular organisms\u2019 motility and growth. However, the light perception of these organisms is limited, and the vast majority of them can express a small number of molecules that can detect a limited range of the light spectrum. On the other hand, metazoan and in particular vertebrates have evolved very specialized and sensitive neurons, rods and cones, which enable visual perception in almost all the lightning conditions that occur during the 24 hours of the day. It has been hypothesized that vertebrates have evolved firstly cone photoreceptors, to perceive light changes during the day (photopic vision). However, cones\u2019 sensitivity is very limited at night and in low illumination conditions their functions as photons detectors are drastically reduced. Rods instead can perceive very dim light signals at night (scotopic vision), even single photons, ensuring an approximate visual stimulus also in poor lighting conditions. It has been proposed that rods are the latest evolved photoreceptors, which have conferred to vertebrates an extraordinary sensitive visual system. The cellular and molecular mechanisms of light and dark adaptation in vertebrates\u2019 photoreceptors have been widely studied and accurately described since the early seventies of the last century. Nonetheless, the study of these fascinating and complex dynamics has left some open questions, regarding both physiological and pathological aspects of photoreceptors metabolism as well as the influence of these alternating mechanisms on phototransduction, the set of enzymatic reactions that transform light stimuli into electrical signals. During the course of my PhD, I have focused my attention on rod\u2019s physiology, in particular on the mechanisms that govern light induced degeneration of photoreceptors, in a Xenopus laevis model of retinitis pigmentosa. Frogs carrying a specific genetic mutation displayed an altered turnover of their cellular body as well as some deficits of phototransduction signaling cascade. In our work, we attempted to estimate how many light induced photoisomerization were necessary to see these alterations. Moreover, I have partially continued the assessment of the response of rods photoreceptors to very localized light stimuli, elicited by mean of a special type of metal coated, tapered optical fibers. Through this technique, we have studied light adaptation of Xenopus laevis\u2019 rods to very confined light stimuli, in order to understand if variations of this process may occur along the rod cells bodies. Furthermore, I have assessed the possible contribution of the circadian rhythms on the phototransduction machinery, by altering the light and dark adaptation cycles of Xenopus frogs. Finally, I have initiated the study of the possible coupling between mechano and phototransduction in rod photoreceptors. This last part is the most fascinating one and made me embrace the idea that sensory neurons are surely specialized cells for one sensory stimulus such as light, sound or chemicals molecules, but others sensory stimuli like temperature variations and small mechanical forces could modulate significantly the perception of the principal stimulus

Vertebrate vision : about physical determinants of photoreceptor sensitivity and kinetics

Rod and cone photoreceptors transform information about incoming light into neural signals with broadly similar molecular mechanisms. Yet their sensitivity, response kinetics and adaptation properties are quite different as rods mediate dim-light vision and cones function mainly under daylight. This thesis 1. addresses the functional differences between rods and cones as well as mammalian and non-mammalian photoreceptors and 2. provides novel findings regarding the existence and regulation of rod-cone interactions at the photoreceptor level. Rod and cone photoresponses to brief flashes of light were recorded with electroretinogram (ERG) from isolated rodent and amphibian retinas. Various phototransduction models were used to compare their relevant parameters over a range of adapting conditions. The study focused on how the following physical factors shape and limit photoreceptor function: operating temperature, thermal stability of the amphibian long wavelength sensitive (A1-)visual pigment, outer segment dimensions, morphology and electrical connections between adjacent rods and cones. Mammalian rod photoreceptors generate faster photoresponses but light-adapt less efficiently than amphibian rods. In the rodent and anuran rods studied in this thesis, the main differences could be accounted for by the higher operating temperature and smaller outer segment size of the rodent photoreceptors. Additionally, the slender outer segments of the mammalian rods enabled sufficient quantal responses and high quantum catch despite the observed desensitizing effect of warming. Long wavelength -sensitive cone photoreceptors have been hypothesized to be desensitized by thermal excitation of their visual pigment molecules. However, it has been shown experimentally only in amphibian cones that utilize the A2-chromophore. The relative stability of the A1-based cone pigments - used by all terrestrial vertebrates - has remained unclear, as well as its role in limiting cone function. In this study, thermal isomerization rate of the long wavelength sensitive (A1-)visual pigment was estimated to play at most a minor role in regulating cone sensitivity of the frog Rana temporaria. Finally, ERG light responses originating in mouse cone photoreceptors were found to be suppressed in the dark-adapted retina, apparently through direct electrical coupling between rods and cones. The results indicated this coupling is weakened by moderate background light, explaining a long known phenomenon of unknown origin: light-induced growth of cone flash responses in mammalian ERG. This is indicative of a previously unknown mechanism of retinal adaptation

Investigation of semiconductor-metal heteronanostructured assemblies and their photoelectrochemical properties

To reduce carbon dioxide emissions in energy consumption, there is a need to employ renewable energy carriers with no carbon footprint. Heterogeneous catalysts accelerate chemical reactions or facilitate energetically unfavorable ones to produce such renewable energy carriers. Hydrogen, a promissing primary energy carrier, demands energy for its production, which could be sourced from electric or solar energy via electrolyzers or photocatalysts, respectively. This study focused on synthesizing and characterizing novel nanoparticle-based photocatalytic materials, utilizing CdSe and CdS semiconductors in various forms alongside metal cocatalysts. These nanoparticles can form three-dimensional porous gel-like networks, constructed through shock-freezing or chemical destabilization methods. Photoelectrochemical investigations emphasize the importance of homogeneous metal-semiconductor contact and distributed metal domains for efficient charge carrier separation. Furthermore, the effectiveness of semiconductor and semiconductor-metal hybrid gel structures in photocatalytic hydrogen production is demonstrated. Nanoparticles embedded in hydrogels facilitate efficient photocatalysis, ensuring diffusion within the network without requiring colloidal stability. Platinum as a cocatalyst significantly enhances hydrogen production rates. Moreover, the limiting factors in semiconductor-metal hybrid networks appear to be diffusion within the network or the catalytic reaction itself rather than semiconductor-metal electron transfer. Fine-tuning the distribution of metal domains is crucial for optimizing properties tailored to photocatalytic applications

Investigations of primary active transporters expressed in Xenopus laevis oocytes : Wilson Disease Protein, a p-type ATPase and Proteorhodopsin, a light driven proton pump

The present work wishes to contribute with information on two members of the primary active transporter group, which differ both in structure and function: Wilson Disease Protein which uses the energy released by ATP hydrolysis to transport copper across cell membranes, and Proteorhodopsin, which uses the energy of light to build up a proton gradient across the bacterial cell membrane, both heterologously expressed in Xenopus laevis oocytes. The surface detection experiments using HA-tagged WNDP confirm the proposed topology of WNDP. The HA-tag per se does not interfere with the function of WNDP, as shown for WNDP HA56 by ATP-dependent phosphorylation after expression in Sf9 cells. Sequence modifications within the WNDP HA56 template-construct reveal some interesting features: i) the N-terminal domain, which contains the 6 metal binding sites, is not necessary for plasma membrane targeting; ii) elevated surface expression of WNDP was observed when the carboxy terminus containing the tri-Leu motif is missing, which suggests that this motif might be involved in the retrieval of the protein from the plasma membrane; iii) the mutations TGE>AAA (proposed to lock the protein in the E1 conformation and lead to constitutive plasma membrane localisation) and D1027A (phosphorylation deficient) did not interfere with the surface localisation of the protein; iv) the mutations CPC>SPS (copper transport deficient) and H1069Q (phosphorylation deficient, most common mutation in Wilson Disease) reduced plasma membrane expression to less then 50%. Western blot analysis shows that the overall expression level of all constructs is similar to that of the reference construct WNDP HA56. These findings suggest that motifs involved in copper binding and catalytic activity do not interfere with plasma membrane targeting of WNDP in Xenopus oocytes. However, the H1069Q mutation could interfere with the distribution of WNDP protein within the cells. In the case of Proteorhodopsin, data presented in this work support earlier observations according to which proteorhodopsin can operate as an outwardly and inwardly directed light-driven ion pump. The residues proposed to play the roles of proton donor (E108) and acceptor (D97) are important for proton translocation. In the absence of an anionic residue at position 97 no outward pumping takes place, but inward charge translocation may occurs under appropriate conditions. An M-like state similar to that known from BR detectably accumulates under neutral pH conditions or under conditions where reprotonation of the Schiff base from the cytoplasmic side is slowed down, as in case of the mutants at position 108. Under acidic conditions PR pumps inwardly under the concerted action of pH and transmembrane potential. The experiments performed in parallel with PR and BR wild-types brought not only interesting information about similarities and differences between the two retinylidene ion pumps, but also led to the observation that the life-time of the M state in BR wild-type can be extended in addition to hyperpolarising transmembrane potentials also by extracellular acidic pH, when the proton gradient through the cell membrane is directed opposite to the ion transport (i.e. when the electrochemical gradient opposing the direction of proton transport increases). Direct photocurrent measurements of HA-tagged PR and BR have shown that the inserted tag may interfere with the functionality of the protein. Next to E108 and D97 in PR other residues in the vicinity of the retinal binding pocket contribute to the translocation of protons, as exemplified by the mutant L105Q: additionally to changing the absorption maximum of the protein, this mutant is a less effective proton pump than the wild type. The example of PR suggests that transduction of light energy by – and reaction mechanisms of retinylidene ion pumps have not been entirely deciphered by the extensive studies of bacteriorhodopsin.Die vorliegende Arbeit beinhaltet Experimente zum Studium zweier primär aktiver, in Membran lokalisierter Transportproteinen. Sie wurden heterolog in Oozyten von Xenopus laevis exprimiert. Hierbei handelt es sich um eine Schwermetall-ATPase, das „Wilson Disease Protein“, und eine lichtgetriebene Protonenpumpe, „Proteorhodopsin“. Das „Wilson Disease Protein“ (WNDP) ist eine, aus 1465 Aminosäuren bestehende P1-Typ Cu+-ATPase, die wichtig ist, um während der Proteinsynthese im Trans-Golgi-Netzwerk (TGN) Kupfer Ionen (Cu+) bereitzustellen, und im Falle einer Erhöhung des intrazellulärem Kupfer- Konzentrazion trägt, durch nicht ausreichend geklärten Mechanismen, zur Wiederherstellung des physiologischen Cu-Spiegels bei. Durch Mutationen bedingte Fehlfunktionen von WNDP führen zu der Erbkrankheit Morbus Wilson. Die vorliegende Arbeit versucht, die Xenopus-Oozyten – ein bekanntes System zum Studium von Transportproteinen der Plasmamembran – als ein alternatives Expressionssystem zu evaluiren. Die Experimente zeigen, dass das Wilson Disease Protein in Xenopus-Oozyten exprimiert werden kann. Es befindet sich in der Plasmamembran der Oozyten und kann durch Chemilumineszenz und Elektronenmikroskopie detektiert werden. Die Experimente zu Oberflächenexpression, bei denen mit HA-Epitopen markiertes WNDP verwendet wurde, bestätigen die für WNDP vorgeschlagene Topologie. Sequenzänderungen, die in das Konstrukt WNDP HA56 eingefügt wurden, offenbaren einige interessante Eigenschaften des Proteins: i) Die N-terminale Domäne, die 6 Metall-Bindestellen enthält, ist offensichtlich für die Zielsteurung in die Plasmamembran targeting nicht notwendig. ii) Bei fehlendem Carboxy-terminus – wodurch das triple-Leucin-Motiv deletiert wird – kommt es zu einer verstärkten Oberflächenexpression von WNDP, woraus sich Hinweise auf eine Beteiligung dieses Motives bei der Relokalisation des Proteins aus der Plasmamembran zum TGN ergeben. Ausserderm, die Resultate weisen darauf hin, dass die Motive, die für die Kupferbindung und die katalytische Aktivität wichtig sind in Xenopus-Oozyten, keinen Einfluss auf Zielsteuerung zur Plasmamembran von WNDP haben. Allerdings könnte die Mutation H1069Q mit der Verteilung von WNDP innerhalb der Zellen interferieren. Proteorhodopsin (PR), das erste “Bakterio-Rhodopsin“ im eigentlichen Sinne, ist eine lichtgetriebene, auswärtsgerichtete Protonenpumpe, die in Meeresbakterien identifiziert werden konnte. Das Protein besteht aus 249 Aminosäuren und zeigt eine erhebliche Sequenzähnlichkeit mit Bakteriorhodopsin (BR). Die entscheidenden Elemente für die Translokation der Protonen sind konserviert. Die Expression in Xenopus-laevis-Oozyten erlaubt nicht nur die Untersuchung des pHEinflusses auf perfekt orientierte PR Moleküle, sondern auch die des Membranpotentials (beide zusammen bilden das elektrochemische Potential für Protonen, ΔμH+). Wildtyp und PR-Mutanten wurden in diesem System heterolog exprimiert und untersucht, um mehr über die Faktoren, die die Eigenschaften des Protontransfers kontrollieren, zu erfahren. Mittels des Two-Electrode Voltage-Clamp Methode (Zwei-Elektroden Spannungsklemme), wurde das Aktionsspektrum vom PR Wildtyp aufgenommen und mit früheren Messungen an künstlichen Lipid Membranen (BLM) verglichen. Außerdem wurden die Spannungsabhängigkeit der stationären Photoströme von PR Wild-Typ (I-V Kennlinien) bei verschiedene intra- und extrazellulären pHWerten bestimmt. Mutationen der Proton-Akzeptor und –Donor-Gruppen D97 und E108 wurden ebenfalls auf ihre Auswirkungen untersucht. Stationäre Belichtung bei neutralem pH löst bei den Mutanten D97N und D97T nur transiente und stationäre Einwärtsströme, eine Tatsache, die die wichtige Rolle des Protonenakzeptors D97 für das Auswärtspumpen bestätigt. Die Mutante E108G zeigt im Vergleich zum Wild-Typ viel kleinere auswärts gerichtete transiente und stationäre Ströme. Die stationäre Pumpfunktion wird in Anwesenheit von Azid deutlich stimuliert, in Analogie zur BR Mutanten D96G. Die Lebenszeit des M-ähnlichen Intermediates von PR Wildtyp und E108G wurde mittels eines speziellen Belichtungsprotokolls studiert, und dabei BR Photoströme unter gleiche Bedingungen als Kontrolle verwendet. Die Ergebnisse zeigen, dass während der stationären Belichtung bei pH 7.4 blaue Laserblitze deutliche auswärtsgerichtete transiente Ströme bei PR hervorrufen, im Gegensatz zu BR, was darauf hinweist, dass unter gleichen experimentellen Bedingungen immer noch weitere PRMoleküle angeregt werden können. Die Richtung der transienten Ladungstranslokation ist bei negativen Membranpotentialen invertiert (einwärtsgerichtet). Bei extrazellulär saurem pH konnten einwärtsgerichtete transiente Ströme sowohl bei blauen als auch bei grünen Laserblitzen beobachtet werden. Ein M-ähnlicher Zustand wie im Falle von BR kann spektroskopischen Messungen zufolge bei PR nur bei neutralem pH-Wert akkumulieren, oder unter Bedingungen unter denen die Reprotonierung der Schiff’schen Base von der zytoplasmatischen Seite her verlangsamt ist, wie im Fall der Mutationen des Protonendonors E108 (unterstüzt von den Experimenten mit PR E108G in der An- oder Abwesenheit von Azid, und unterschiedliche pH Werte). Diese Ergebnisse bestätigen die früheren Aussagen von Friedrich et al. (2002), dass die Richtung des Protonentransports durch den Protonenkonzentrationsgradienten und das Membranpotential bestimmt wird. Die Experimente, die parallel an PR- und BR-Wildtypen ausgeführt wurden, haben nicht nur interessante Informationen über Ähnlichkeiten und Unterschiede zwischen den beiden Retinyliden- Ionenpumpen gebracht. Außerdem führten sie zu der Beobachtung, dass die Lebenszeit des M-Zustand in BR – außer durch hyperpolarisierende Membranpotentiale (Geibel et al, 2001) – auch durch einen der Richtung der Ionenpumpe entgegengesetzten Protonengradienten verlängert werden kann. Direkte Photostrom-Messungen von HA-Epitop markiertem PR und BR haben gezeigt, dass das HA-Epitop abhängig von der Insertionsstelle die Funktion des Proteins beeinträchtigen kann

AN INVESTIGATION OF THE PHOTOTRANSDUCTION CASCADE AND TEMPORAL CHARACTERISTICS OF THE RETINA OF THE CUTTLEFISH, SEPIA OFFICINALIS

Cephalopods have extremely well developed visual systems which are of particular interest due to the well known morphological similarity of the cephalopod eye to the vertebrate eye. This similarity ends at the level of the photoreceptors where vertebrates and invertebrates have been found to use different intracellular second messengers. Although the effect of extracellular ion manipulation on the light response has been examined and some very useful biochemical studies carried out, the pathway has not been investigated by the use of pharmacological intervention; a method which has proved to be useful in other preparations. This study examines various properties of the photoreceptors of the cuttlefish, Sepia officinalis, with particular interest in the second messenger signalling pathway. Both extracellular and whole cell patch clamp recording has been utilised. The second messenger signalling pathway, which mediates phototransduction in the retina of S. officinalis, was investigated by recording the electroretinogram and examining how this changed with the application of various extracellularly applied, membrane permeable pharmacological agents. Invertebrate phototransduction utilises the phosphoinositide (PI) signalling pathway therefore specific activators and inhibitors targeted at precise sites of this pathway were applied to the extracellular bathing solution. These studies indicated that cleavage of phosphatidylinositol-4,5-bisphosphate is essential for the production of a light response and that the inositol trisphophate (IP3) branch of this pathway is of greatest importance in this preparation, as opposed to the diacylglycerol branch. How this second messenger cascade transfers the incoming information into a temporally coded signal was studied by measuring maximum critical flicker fusion frequency. The effect of cell size on this property was investigated and also how cell sensitivity was affected and whether these properties appeared to fit the animal's environmental conditions or whether they were restricted by cellular properties. The animals were found to have relatively "slow" eyes. However the younger age group studied, with shorter photoreceptors, was found to be both faster and more sensitive. This was an unexpected finding considering temporal resolving power is often sacrificed for sensitivity. It is suggested that the observed differences between age groups was attributable to the effects of increased cell size on the cell membrane time constant and that deterioration of signalling molecules with aging may also be a contributing factor. An investigation of the cell signalling pathway at the level of individual cells was also carried out using the whole cell patch clamp technique. Using this technique, two voltage activated currents were found; an inward sodium current characterised by its voltage and tetrodotoxin sensitivity, and an outward potassium current characterised by its tetraethylammonium sensitivity. As well as finding further evidence for the involvement of the IP3 branch of the PI pathway there is also evidence of a role for cyclic guanosine monophosphate. A suitable mode of measuring light-induced fluctuations in the intracellular calcium levels was also investigated with a view to observing the impact of the pharmacological agents on intracellular calcium concentration. This investigation has enhanced the understanding of the S. officinalis visual system by greatly adding to the present knowledge of the second messenger signalling cascade and by giving an insight into how this transfers into the animal's temporal resolving power. Some preliminary information regarding the membrane currents activated by light has also been presented. This has all been possible by the development of a versatile retinal slice preparation that has been proven to be accessible to extracellular recording and whole cell patch clamp recording combined with pharmacological manipulation.The Marine Biological Associatio