Rozprawy doktorskie na temat „Phosphenes”

I fosfeni, o light flash, sono stati percepiti per la prima volta durante il volo verso la luna della navicella Apollo. In genere sono avvertiti al buio, prima di addormentarsi. In questa tesi si studia l'ipotesi di una eccitazione dei fotorecettori della retina da parte di ioni pesanti (carbonio). Campioni di recettori dei bastoncelli vengono irradiati, e viene studiato l'effetto degli ioni carbonio su topo, attraverso la registrazione della risposta elettrofisiologica (segnale retinico e corticale). La tesi si e' sviluppata nell'ambito del progetto ALTEA (Anomaluos Long Term Effect on Astronauts) e tutti gli esperimenti relativi a questo lavoro sono stati svolti a terra. L’obiettivo di ALTEA e’ piu’ generale e riguarda lo studio del rischio funzionale sul sistema nervoso centrale degli astronauti dovuto alla particelle ionizzanti presenti in condizioni di microgravita’ durante le missioni spaziali a lungo termine; ALTEA include sia esperimenti a terra che in orbita, a bordo della Sazione Spaziale Internazionale. La tesi inizia con una introduzione al progetto ALTEA; nel secondo capitolo e’ descritto lo studio chiamato ALTEA-MICE (Mice intermittent Irradiation with Concurrent Electrophysiological monitoring). Nel terzo capitolo sono descritto il sistema costituito dai segmenti esterni dei bastoncelli bovini in sospensione e il meccanismo di attivazione dei fotorecettori. Nel quarto capitolo e quinto capitolo mi sono occupata della interazione radiazione – materia nei tessuti e del caso specifico dell’irradiazione dei campioni dei segmenti esterni contenenti la rodopsina. Nel quinto e ultimo capitolo viene presentato un modello che spiega l’isomerizzazione (attivazione) del retinale della rodopsina come prodotto dell’azione dei radicali liberi provenienti dalla radiolisi dell’acqua.
Phosphenes, or light flashes, have been reported by astronauts since the first Apollo flights to the Moon. They are usually described as occurring in the dark and typically before falling asleep. The light flashes are thought to originate as an effect of high- energy particles interacting with the visual system. The study presented in this thesis investigates the hypothesis of a direct excitation of the rod photoreceptors inside the retina and in particular the interaction between carbon ions and rods. Also the retinal and cortical electrophysiological response of mice submitted to carbon ions irradiation have been studied. This thesis has been developed as a section of the ’Anomalous Long Term Effects on Astronauts’ (ALTEA) program, and all the experiments concerning this thesis took place on ground. The objective of the ALTEA project is more general, and is aimed to assess the functional risks on the central nervous system due to particle flux in microgravity conditions during long term space mission. ALTEA includes a series of experiments both in orbit and on ground. The experiments on board International Space Station will contribute to define the causes of the anomalous phosphene perception of the astronauts by detecting the incident particles through the head of the astronauts and concurrently monitoring the central nervous system functionality. The experiments on ground include the direct irradiation with carbon ions of the outer segments of the rods, and an animal model to investigate the retina/cortex responses of mice irradiated in controlled laboratory experiments. In these experiments on ground I directly contributed in the preparation, the test and the optimization of the experiments and in the following analysis of the data. The whole project has been supported by the Italian Space Agency. The outline of this thesis includes an introduction to the interdisciplinary ALTEA program in the first chapter. In the second chapter ALTEA-MICE (Mice intermittent Irradiation with Concur- rent Electrophysiological monitoring) study is described: the experimental sessions as well as the evidences in the electrophysiological responses of the mouse retina have been described in detail. My role has been chiefly the experimental set up, and I also collaborated on the data analysis. The main results of this investigations have been published. In the third chapter the system of the rod outer segments in suspension and the mechanism of the activation of the photoreceptors are described. The fourth chapter deals with the radiation effects in the biological tissue, and in the fifth chapter there is particular regard to the effects of radiation on samples containing a suspension of rods extracted from bovine retinae. The effects on the rod photoreceptor rhodopsin has been studied: measurements have been performed irradiating with carbon ions the outer segments of the rods in suspension. The amount of the isomeric transformations (bleaching) of the molecules of chromophore into the photoreceptor gives the extent of the effects of radiation. I collaborated to the experiment and carried out the following data analysis. Finally, in the sixth chapter the process of the photo-isomerization of rhodopsin is explained by a model: the process starts with the water radiolysis and the formation of hydroxyl radicals OH in order to achieve the lipid peroxidation, then there is the subsequent emission of visible photons which are responsible for the photoreceptor bleaching. Since rhodopsin is surrounded by phospholipids, present in high concentration in the disc membranes, the effect of chemiluminescence is proposed to be the cause of the photo-transduction cascade and the light flash perception as a consequence. In order to test this hypothesis, a series of experiments was aimed at studying the effects of the hydroxyl radicals produced in an enzymatic way. The main results of this work are illustrated in chapter six, and have been published.

A successful restoration of vision should allow the blind to look, to see and to understand. The engineering of a microelectronic vision prosthesis has come a long way over the last forty years, but the understanding of how the restored form of vision would be interpreted and functionally applied to everyday living has made little progress until recent times. Prosthetic vision is not what most people think it would be; it is a visual scene composed of relatively large, isolated, spots of light so-called "phosphenes", very much like a magnified pictorial print. This thesis dissertation seeks to obtain a complete survey of the visual description of phosphenes from the human trial reports in the literature, simulate it, obtain a measure of the functional capacity of such visual perception, and explain the measured performance against design aspects of phosphene presentation, human perception, cognition and behaviour. Specifically, "visual acuity" (VA) was assessed on normally sighted subjects (N=15) administered with "simulated prosthetic vision". VA is a functional measure of vision highly correlated to many daily activities. Aggregating the results from the study with the other VA studies in prosthetic vision, it is shown that in general, the density of the phosphene field determines the affordable VA; however, design aspects relating to the phosphene field lattice (0.03 10gMAR with the hexagonal lattice as opposed to a square lattice) and image processing routines (0.15 10gMAR at optimised settings) can be further fine-tuned to improve VA performance. Significant performance improvement also arose from learning (0.13 10gMAR over ten visitations) and visual scanning adaptation (0.20 10gMAR with a circular scanning strategy). Performance improvements are likely related to various preferences and perceptual preferences of the human visual system. A rehabilitation program targeting the appropriate behavioural adaptation coupled with image processing routine optimised for image comprehension should provide a vision prosthesis recipient with the best functional experience to restored vision.

The convergence and integration of information from the different sensory channels represents a fundamental ability of the human brain: in our daily life, multisensory cues impact our sensory system, shaping our perception of sensory events. Classical models of multisensory perception defer integration until sensory-specific information has been extensively processed. Strikingly, several anatomical studies now suggest that this view may be over-simplistic and that the substrate for multisensory integration is not constrained to the classical area of multisensory convergence; rather, it also occurs in the early stages of sensory processing, rising the intriguing hypothesis that most of the brain, including the primary sensory cortices, is essentially multisensory. The present dissertation inquires the causal involvement of classical ‘sensory-specific’ visual areas in multisensory processing. Using a combination of behavioral, neuromodulatory, and neuropsychological evidence, I seek the behavioral and brain signatures of a causal link between visual cortical excitability and multisensory perception. In parallel, I provide a characterization of the impact of distinct crossmodal stimuli on subjective visual experience. By directly measuring visual cortical excitability via Transcranial Magnetic Stimulation (TMS), the first study shows facilitatory effects by spatially-specific bimodal and trimodal stimuli on visual cortical responses, which, in turn, improve visual perception. Moreover, by using Transcranial Direct Current Stimulation (tDCS), I demonstrate the role played by higher order multisensory cortices in mediating such spatially-specific crossmodal influences on visual perception, reporting the presence of regional preferences for auditory or somatosensory influences on visual responses. In the second experiment, I further extend these findings by showing that multisensory influences on vision not only express themselves through an enhancement of visual perception, but they can also provoke phenomenological changes in conscious visual perception, namely a crossmodal illusion, when incongruent auditory cues are provided. Crossmodal illusory effects show a specific time-course, compatible with the occurrence of early visual-auditory interaction in the primary visual cortex. At complement with evidence in the healthy brain, in the third study I investigate how a well-known crossmodal illusion, the Sound-Induced Flash Illusion (SIFI), is processed by brain-damaged patients with visual field defect (with damage to the primary visual cortex, the sensory visual pathways, or both), and unilateral spatial neglect (with damage to the posterior parietal and fronto-temporal regions). Perception of the SIFI is defective in patients with visual field defects, but not in those with unilateral spatial neglect, further supporting the role of low-level visual areas in integrating multisensory cues. Overall, this set of experiments shows a causal link between the crossmodal modulation of visual perception and the activity of the primary visual areas, which represents a key site for multisensory integration.

Electrical stimulation of the retina affected by photoreceptor loss (e.g., cases of retinitis pigmentosa) elicits the perception of luminous spots (so-called phosphenes) in the visual field. This phenomenon, attributed to the relatively high survival rates of neurons comprising the retina's inner layer, serves as the cornerstone of efforts to provide a microelectronic retinal prosthesis -- a device analogous to the cochlear implant. This thesis concerns phosphenes -- their elicitation and modulation, and, in turn, image analysis for use in a prosthesis. This thesis begins with a comparative review of visual modelling of electrical epiretinal stimulation and analogous acoustic modelling of electrical cochlear stimulation. The latter models involve coloured noise played to normal listeners so as to investigate speech processing and electrode design for use in cochlear implants. Subsequently, four experiments (three psychophysical and one numerical), and two statistical analyses, are presented. Intrinsic signal optical imaging in cerebral cortex is canvassed appendically. The first experiment describes a visual tracking task administered to 20 normal observers afforded simulated prosthetic vision. Fixation, saccade, and smooth pursuit, and the effect of practice, were assessed. Further, an image analysis scheme is demonstrated that, compared to existing approaches, assisted fixation and pursuit (but not saccade) accuracy (35.8% and 6.8%, respectively), and required less phosphene array scanning. Subsequently, (numerical) information-theoretic reasoning is provided for the scheme's superiority. This reasoning was then employed to further optimise the scheme (resulting in a filter comprising overlapping Gaussian kernels), and may be readily extended to arbitrary arrangements of many phosphenes. A face recognition study, wherein stimuli comprised either size- or intensity-modulated phosphenes, is then presented. The study involved unpracticed observers (n=85), and showed no 'size' --versus--'intensity' effect. Overall, a 400-phosphene (100-phosphene) image afforded subjects 89.0% (64.0%) correct recognition (two-interval forced-choice paradigm) when five seconds' scanning was allowed. Performance fell (64.5%) when the 400-phosphene image was stabilised on the retina and presented briefly. Scanning was similar in 400- and 100-phosphene tasks. The final chapter presents the statistical effects of sampling and rendering jitter on the phosphene image. These results may generalise to low-resolution imaging systems involving loosely packed pixels.

Cette these illustre la synthese de nouveaux phosphetanes chiraux et leur utilisation comme ligands pour la catalyse enantioselective par les complexes du palladium et du rhodium. Apres une etude bibliographique des voies de synthese du cycle phosphetane, la preparation de l'oxyde de 1-menthyl-2, 2, 3, 3-tetramethylphosphetane est decrite. Ce compose est utilise comme produit de depart pour la synthese des differents ligands fonctionnalises en du phosphore. Divers electrophiles sont en effet introduits sur le cycle phosphore par une reaction de metallation-alkylation, conduisant a des phosphetanes monodentes ou potentiellement bidentes. Ces ligands sont ensuite testes dans les reactions d'hydrosilylation des olefines et de substitution nucleophile allylique catalysees par les complexes du palladium. L'hydrogenation de l'acide -acetamidocinnamique par les complexes du rhodium est egalement abordee.

Au cours de ce travail, nous avons developpe la synthese des 1,2-bis(2,4-dialkylphosphetano)benzenes et 1,2-bis(2,4-dialkylphosphetano)ethanes chiraux. L'hydrogenation catalytique enantioselective de dicetones 1,3 nous a permis un acces aise et efficace aux diols 1,3 anti symetriques optiquement purs. Ces diols ont servi a la synthese d'une serie de bisphosphetanes portant sur les cycles phosphores des substituants alkyles d'encombrements varies. Nous avons montre l'efficacite des bisphosphetanes en tant que ligands dans l'hydrogenation catalytique enantioselective. Des exces enantiomeriques proches de 90% sont obtenus dans l'hydrogenation de cetones fonctionnalisees catalysee par le ruthenium. Les complexes de ruthenium utilises jusqu'ici comme precurseurs ne generent pas une espece catalytique unique et bien definie, laissant esperer une optimisation des resultats par un choix plus approprie du systeme catalytique. Dans l'hydrogenation de dehydroaminoesters par des complexes rhodium-bisphosphetane, l'enantioselectivite varie fortement selon les ligands et les substrats. Cependant, on observe des effets tres significatifs de la pression, de la temperature et de la nature du solvant sur les exces enantiomeriques, effets qui permettent d'optimiser ces hydrogenations et d'atteindre des enantioselectivites elevees. De plus, ces effets etonnants ont porte notre attention sur le mecanisme de l'hydrogenation asymetrique catalysee par les phosphines riches en electrons. Des hypotheses mecanistiques ont ete formulees sur la base d'observations experimentales. Enfin, la synthese du (r,r)-1-2-(r,r)-2,4-dimethylazetidinophenyl-2,4-dimethylphosphetane et du (r,r)-1-2-(r,r)-2,4-dimethylazetidinoethyl-2,4-dimethylphosphetane, ligands mixtes p-n analogues des bisphosphetanes, a ete mise au point. Leur etude donnera des indications sur l'influence de la difference d'electronegativite entre phosphore et azote sur l'activite et/ou la selectivite des reactions catalytiques.

Ce manuscrit décrit la synthèse et les caractérisations physico-chimiques de nouveaux systèmes hétérocycliques π-conjugués contenant un atome de phosphore. Le chapitre d'introduction présente les différentes voies de synthèse du cycle phospholes pouvant être incorporé dans des systèmes π-conjugués. Le chapitre I décrit la synthèse et les propriétés physico-chimiques de dérivés du pyrène contenant soit un phosphole, soit un silole. La synthèse de ces nouvelles structures a été réalisée à l'aide de réactions métallo-catalysées. Deux types de régioisomères ont ainsi été synthétisés afin de déterminer l'impact de la nature de l'hétéroatome (P ou Si) sur leurs propriétés optiques et électrochimiques et sur l'organisation des molécules à l'état solide. Le chapitre II décrit la synthèse et les propriétés physico-chimiques de nouveaux complexes d'Ir(III) ortho-métallés contenant un ligand phosphole. La présence de deux centres stéréogènes induit la formation de diastéréoisomères, dont la formation peut être contrôlée par les conditions réactionnelles. De plus, les propriétés électroniques du ligand phosphole permettent de moduler à façon les propriétés redox et optiques du complexe. Le chapitre III se focalise sur l'utilisation de la phosphépine comme sous-unité pour la construction de nouveaux systèmes π-conjugués. Deux systèmes π-conjugués ont été synthétisés : un squelette de PAH contenant la phosphépine et les dithieno-phosphépines. Les propriétés optiques et redox de ces dérivés ont été étudiées et discutées dans le manuscrit
This manuscript describes the synthesis and characterisation of new heterocyclic π-conjugated systems built around the phosphorus atom. Firstly, an introduction to the phosphole chemistry (synthesis methods and integration into π-conjugated systems) is presented. The chapter I describes the synthesis and physico-chemical properties of pyrene functionalised either by a phosphole or silole ring. The synthesis of these new structures was achieved by using a transition-metal catalysis. Two types of regioisomers were synthesised in order to study the effect of the heteroatom (P or Si) on the optical and electrochemical properties together with the impact on the solid-state organisation. The chapter II describes the synthesis and the physical properties of the first phosphole-based Ir(III) ortho-metallated complexes. The presence of two stereogenic centres in the complex induced diastereoselectivity issues that could be solved by carefully choosing the reaction conditions. Furthermore, it was demonstrated that the nature of the ligands can tune the optical and the redox properties. The chapter III describes the use of P-containing seven-membered rings (phosphepines) for the development of new π-conjugated systems. Two original π-systems have been synthesised and studied: (i) phosphepine based PAH and (ii) dithieno-phosphepines. The optical and redox properties have been investigated and discussed in the manuscript

The topic of this thesis is the design and synthesis of biaryl-based self adaptableligands for asymmetric metal catalysis. The results discussed in papers I-III are covered, together with some unpublished results concerning substrate-adaptable catalysts. A general survey of self-adaptable catalysts is presented first. The second chapter of this thesis starts with a survey of inversion barriers in biphenyl-based ligands and catalysts. Thereafter, the determination of barriers to conformational adaptation in dibenzoazepines and dibenzophosphepines is described. Palladium complexes with a diphosphine ligand or a diamine ligand, as well as the free diamine ligand, were studied. Entropies and enthalpies of activation were determined with variable temperature NMR spectroscopy. The mechanism of conformational change in the metal complexes was elucidated. The third chapter describes the synthesis of semiflexible and rigid phosphinite ligands, as well as their application in rhodium-catalysed asymmetric hydrogenation. Modest enantioselectivities (up to 63% ee) were obtained. The semiflexible ligand was found to behave like the most active rigid diastereomer. The fourth chapter describes the behaviour of amine and phosphoramidite ligands in model complexes relevant to the palladium-catalysed asymmetricallylic alkylation of benchmark substrates. Diphosphoramidite and aminephosphoramiditeligands were designed and synthesised. Pd(olefin) complexesof diamine and diphosphoramidite ligands were studied, and their symmetry determined. It was found that both types of ligands are able to adapt their conformation to the substrate.
QC20100630

This thesis deals with the design and synthesis of ligands for asymmetric catalysis: palladium catalyzed allylic alkylations, and rho-dium and iridium catalyzed hydrogenations of olefins. Chirally flexible phosphepine ligands based on biphenyl were synthesized and their properties were studied. The rotation barrier for configurationally flexible phosphepines was determined by NMR spectroscopy. The ratio of the atropisomers was shown to depend on the group bound to phosphorus. Only complexes with two homochiral ligands bound to the metal center were observed upon complexation with Rh(I). It was shown that one diastereomer of the flexible ligand exhibits higher activity but lower selectivity than its diastereomer in the rhodium catalyzed hydrogenation of methyl alfa-acetamidocinnamate. These ligands were also tested in nickel catalyzed silabora-tions. Chiral P,N-ligands with pseudo-C2 and pseudo-CS symmetry based on pyrrolidines-phospholanes or azepines-phosphepines were synthesized and studied in palladium catalyzed allylic alkylations. Semi-flexible azepine-phosphepine based ligands were prepared and their ability to adopt pseudo-C2 or pseudo-CS symmetry depending on the substrate in allylic alkylations was studied. It was shown on model allyl systems with flexible N,N-ligands that the ligand prefers CS-symmetry in compexes with anti-anti as well as syn-syn allyl moieties, but that for the latter type of complexes, according to computations, the configuration of the ligand is R*,R* in the olefin complexes formed after addition of a nucleophile to the allylic group. A preliminary investigation of the possibilities to use a su-pramolecular approach for the preparation of P,N-ligands with pseudo-C2 and pseudo-S symmetry was made. An N,N-ligand with C2 symmetry was prepared and its activity in palladium catalyzed ally-lic alkylation was studied. Pyridine-based P,N-ligands were tested in iridium catalyzed hy-drogenations of unfunctionalized olefins with good activities and se-lectivities. In order to attempt to improve the selectivity, ligands with a chirally flexible phosphepine fragment were prepared and applied in catalysis with promising results.
QC 20100929

In che modo le persone vedono? In molti risponderebbero “con gli occhi!” In realtà gli occhi rappresentano solamente il primo stadio nell'elaborazione dell’informazione visiva. Questa tesi cerca di incrementare le conoscenze sui processi corticali coinvolti nella percezione visiva. Piuttosto che presentare stimoli visivi, abbiamo utilizzato la TMS sulla corteccia occipitale per indurre dei percetti visivi. Questi percetti, chiamati fosfeni e scotomi, hanno caratteristiche di luminanza ben distinte e quindi dovrebbero simulare la normale elaborazione visiva. Nel primo studio abbiamo testato se entrambi i percetti potessero essere generati nella corteccia occipitale utilizzando gli stessi parametri sperimentali, poiché, in precedenza, sono stati testati solamente in condizioni sperimentali differenti. Abbiamo indagato la loro influenza sui tempi di reazione (TR), e l’effetto, sia dell’intensità della luminanza sia dell’intensità della TMS, sulla capacità di evocare il percetto. Ogni percetto è stato indotto sotto ogni condizione sperimentale e manipolato nello stesso modo, ed è risultato che i TR sono influenzati dalla luminanza del percetto. I risultati di questo primo studio hanno condotto all’ipotesi da cui è partito il secondo studio: esaminare gli effetti sia dell’intensità della luminanza fisica e percepita sia del contrasto sulla comparsa dei percetti. A questo scopo, è stata utilizzata un’illusione di luminosità al fine di misurare gli effetti della luminanza percepita sui percetti. I risultati hanno mostrato che l’illusione di luminosità modulava i TR agli stimoli reali e la frequenza di comparsa dei percetti artificiali quando la luminosità indotta dall’illusione era percettivamente differente dalla luminanza fisica. I risultati del secondo studio hanno gettato le basi per il terzo studio: testare se un’illusione di contrasto fra grandezze, ottenuta mostrando degli indizi di profondità, possa influenzare le caratteristiche del percetto. Dal momento che questo tipo d’illusioni viene elaborata sia nella via ventrale, a partire dalla corteccia occipitale, sia nella via dorsale, a livello della corteccia parietale, per il terzo studio sono stati evocati dei fosfeni stimolando anche la corteccia parietale, oltre a quelli indotti dalla stimolazione del lobo occipitale. Come risultato di questo esperimento abbiamo trovato che l’illusione influenzava la grandezza percepita e i TR ai fosfeni evocati da stimolazione occipitale, mentre non aveva alcun effetto sui fosfeni indotti stimolando la corteccia parietale. Nel complesso questi risultati suggeriscono che la corteccia visiva elabora le informazioni in maniera standardizzata, indipendentemente dal fatto che esse provengano dall’occhio o che siano generate direttamente nella corteccia occipitale del cervello.
How do people see? Most would answer ‘with the eyes’. In fact, the eyes are just the first step in processing visual information. The current thesis seeks to build on our knowledge of cortical processes involved with visual perception. Instead of using visual information presented to the eye, we used TMS to the occipital cortex of the brain to induce visual percepts. These percepts, phosphenes and scotomas, have distinct luminance characteristics and therefore should mimic normal visual processing. In Study One, we tested whether both percepts could be generated in the occipital cortex using the same experimental parameters, as they have previously been tested only under different conditions. We investigated their influence on RT, and the effects of luminance intensity and TMS intensity on percept appearance. Each percept was induced under all experimental conditions, was manipulated in a similar manner, and RT were influenced by percept luminance. The results from Study One led to the hypothesis for Study Two, examining the effects of physical versus perceived luminance intensity and contrast on percept appearance. To this end, illusory brightness was used to gauge the effects of perceived luminance on percepts. Results showed that illusory brightness modulated RT to real stimuli and the frequency of artificial percepts when illusory brightness was perceptually different from physical luminance. The findings of Study Two ushered in the design for Study Three, testing if a size contrast illusion exhibiting depth cues could influence percept characteristics. As size contrast illusions are processed in the ventral stream beginning in the occipital cortex and the dorsal stream of the parietal cortex, Study Three incorporated parietally induced phosphenes as well as those arising from occipital stimulation. Here, we found the illusion affected perceived size of and RTs to occipital-induced phosphenes but not parietal-induced phosphenes. Taken together, these findings imply that the visual cortex processes information in a standardized manner, irrespective of whether the information is delivered to the eye or to the occipital cortex of the brain.

Questa tesi di dottorato si focalizza sullo studio sperimentale dei correlati neurali relativi alla percezione visiva e spaziale nell’uomo. Nello specifico, l’obiettivo di questo progetto è quello di identificare i marker elettrofisiologici della consapevolezza, sia in condizioni normali, sia quando la consapevolezza viene persa a causa di una lesione cerebrale reale o “virtuale”. In entrambi gli studi condotti è stato adottato un approccio causale multimodale, ovvero la coregistrazione dell’elettroencefalogramma durante la Stimolazione Magnetica Transcranica (EEG-TMS), che è in grado di fornire importanti informazioni rispetto ai correlati neurali della consapevolezza visiva e spaziale. Il primo studio condotto è centrato sula consapevolezza spaziale, e in particolare si focalizza sullo studio dei meccanismi alla base della sindrome del neglect. Il neglect viene definito come un deficit di consapevolezza in cui il paziente ignora gli stimoli che vengono presentati nel lato opposto rispetto a quello della lesione cerebrale. Uno dei modelli più influenti di spiegazione della disfunzione alla base di questa sindrome prende in considerazione il concetto di rivalità interemisferica, secondo cui l’emisfero intatto risulterebbe patologicamente iperattivato a causa della riduzione di inibizione precedentemente esercitata da parte dell’emisfero leso. L’obiettivo del presente studio è quello di testare questi modelli di rivalità interemisferica, investigando l’effetto che l’applicazione della TMS ripetitiva (rTMS) a bassa frequenza esercita sull’emisfero stimolato e su quello controlaterale nel processamento di stimoli visivi. In particolare, questo studio si pone l’obiettivo di indagare il contributo della corteccia parietale destra e sinistra in un funzionamento deficitario tipo neglect indotto attraverso l’applicazione della rTMS a bassa frequenza in un campione di soggetti sani. A quattordici volontari sani è stato richiesto di eseguire un test di bisezione di linee e un compito di detezione di stimoli visivi lateralizzati. Entrambi i compiti sono stati somministrati sia prima che dopo 30 minuti di rTMS a bassa frequenza (1 Hz) applicata sulla corteccia parietale posteriore destra. Il segnale EEG è stato registrato continuativamente per tutta la durata dell’esperimento. L’efficacia della rTMS nell’indurre sintomi tipo neglect è stata confermata dai risultati del compito di bisezione di linee, in cui i partecipanti hanno mostrato una performance simile a quella dei pazienti con neglect in questo test, ovvero con una deviazione verso destra del punto di bisezione dopo la rTMS. Considerando il compito di detezione, i risultati hanno mostrato come l’effetto della rTMS sia un rallentamento dei tempi di reazione sia per gli stimoli presentati a sinistra che per quelli presentati a destra e una riduzione dell’ampiezza della componente P200 registrata sui siti parietali sia sinistri che destri. I potenziali evocati dalla TMS (TEPs) registrati durante i 30 minuti di stimolazione hanno mostrato come la rTMS a bassa frequenza abbia indotto una riduzione dell’eccitabilità corticale sia della corteccia parietale destra (quella direttamente stimolata), sia delle aree omologhe controlaterali. Di conseguenza, i nostri risultati non hanno evidenziato una iperattivazione dell’emisfero sinistro conseguente all’inibizione dell’emisfero destro (come postulato dai modelli di rivalità interemisferica). Al contrario, l’inibizione della corteccia parietale destra ha indotto una propagazione dell’inibizione alle aree omologhe dell’emisfero sinistro. Il secondo studio condotto è focalizzato sulla consapevolezza visiva e, in particolare, si pone l’obiettivo di indagare i correlati neurali della percezione di fosfeni. La TMS a singolo impulso applicata sulla corteccia visiva primaria è in grado di indurre delle sensazioni visive, chiamate appunto fosfeni, che appaiono come brevi lampi di luce senza che effettivamente l’occhio venga stimolato da una luce. Recenti studi hanno dimostrato come la TMS possa produrre delle sensazioni visive non soltanto quando applicata sulle aree visive primarie, ma anche quando viene stimolata la corteccia parietale. Poiché le basi neurali coinvolte nella percezione dei fosfeni parietali sono tuttora sconosciute, rimane da capire se i fosfeni parietali siano generati direttamente da meccanismi locali o se invece la loro generazione dipenda dall’attivazione indiretta di altre aree visive. Al fine di caratterizzare i correlati elettrofisiologici della percezione dei fosfeni occipitali e parietali, abbiamo analizzato i TEPs in un campione di soggetti sani, confrontando i trials in cui i partecipanti riportavano di percepire un fosfene, con i trials in cui all’impulso della TMS non seguiva alcuna sensazione visiva. Quando viene stimolata la corteccia occipitale sinistra, la percezione dei fosfeni inizia a modulare i TEPs ad una latenza tardiva, mentre i fosfeni indotti dalla stimolazione della corteccia parietale sinistra iniziano a modulare i TEPs a delle latenze più precoci. Questa differenza tra fosfeni occipitali e parietali nell’andamento temporale dell’attivazione corticale potrebbe identificare un diverso meccanismo implicato nella loro generazione. L’effetto di latenza precoce osservato quando la TMS viene applicata sulla corteccia parietale potrebbe suggerire che i fosfeni parietali siano il risultato diretto dell’attivazione dell’area stimolata, e non la conseguenza di un’attivazione di tipo feedback della corteccia visiva primaria. Inoltre, abbiamo indagato i correlati elettrofisiologici della percezione di fosfeni parietali in una paziente emianopsica (SL) con una distruzione completa della corteccia visiva primaria sinistra. La percezione di fosfeni indotti dalla stimolazione della corteccia parietale ipsilaterale alla lesione ha mostrato lo stesso pattern di risultati rispetto alla stimolazione parietale del campione di soggetti sani, con una modulazione dei TEPs che emerge ad uno stadio di latenza precoce. Questi risultati supportano quindi la visione della corteccia parietale come un generatore indipendente di esperienze visive consce indotte dalla stimolazione magnetica.
This PhD thesis focuses on attempting to experimentally investigate the neural correlates of awareness related to visual and spatial perception in humans. Specifically, this project aimed at looking into the electrophysiological markers of awareness in normal conditions and when awareness is lost due to a real or a “virtual” lesion. In both the studies conducted we adopted a causal multimodal approach, namely Transcranial Magnetic Stimulation and EEG (TMS-EEG) co-registration, which can provide insights into the neural correlates of visual and spatial awareness. The first study focuses on spatial awareness and specifically on the investigation of the mechanisms underlying neglect syndrome. Neglect is defined as a disorder of consciousness in which patients fail to report, respond to, or orient to stimuli presented on the opposite side of the brain lesion. One of the most influential models to explain the dysfunction underlying this syndrome takes into account the concept of inter-hemispheric rivalry, which postulates a pathological hyperactivation of the unaffected hemisphere due to the reduced inhibitory influences from the lesioned hemisphere. The aim of the present study is to test these models analyzing the effect that low frequency repetitive TMS (rTMS) exerts on the stimulated and contralateral hemispheres in the processing of visual stimuli. Specifically we aim at assessing the contribution of left and right parietal cortices in an impaired neglect-like functioning induced by means of low frequency rTMS in healthy participants. Fourteen healthy volunteers performed a Line Bisection task and a simple detection task of unilateral checkerboards stimuli. Both tasks were performed either before and after 30 minutes of low frequency rTMS (1 Hz) over the right posterior parietal cortex. The EEG signal was continuously recorded throughout the experiment. The efficacy of rTMS in inducing neglect-like phenomena was confirmed by the results of the Line Bisection task where participants showed a rightward deviation after rTMS, a performance comparable to that of neglect patients. Detection task results showed that the effect of rTMS was a lengthening of reaction times for both left and right visual stimuli and a reduction of the amplitude of P200 component registered both on left and right parietal sites. TMS-evoked potentials recorded during 30 minutes of stimulation, showed that low frequency rTMS induced a reduction of cortical excitability both of the stimulated right parietal cortex and of the left contralateral homologous area. Therefore, our results did not show a hyperactivation of the left hemisphere due to the inhibition of the right hemisphere (as theorized by “rivalry models”). Conversely, the inhibition of the right parietal cortex induced a spreading of the inhibition to the homologous area of the left hemisphere. The second study focuses on visual awareness and specifically aimed at investigating the neural correlates of phosphene perception. Single-pulse TMS of the visual cortex is known to induce visual sensations, i.e. phosphenes, which appear as brief flashes of light without light actually entering the eyes. Recent studies have shown that TMS can produce visual sensations not only when it is applied over early visual areas but also when parietal cortex is stimulated. As the pivotal neural basis involved in the perception of parietal phosphenes still remain unknown, the main question is whether parietal phosphenes are generated directly by local mechanisms or emerge through indirect activation of other visual areas. To characterize the electrophysiological correlates of occipital and parietal phosphene perception we investigated TMS-evoked potentials in a sample of healthy participants by comparing trials in which a phosphene was perceived with trials in which no visual percept was reported. When the left occipital cortex was stimulated, phosphene perception started to affect TMS-evoked potentials at a late latency, whereas phosphenes elicited by left parietal cortex stimulation modulated TMS-evoked potentials at an earlier latency. This difference in the time-course of cortical activation between occipital and parietal phosphenes could underlie a different mechanism in their generation. The early latency of the phosphene effect observed when TMS was applied over the parietal cortex might suggest that parietal phosphenes should be considered as the direct result of the activation of the stimulated area, rather than the consequence of a feedback activation of the early visual cortex. Furthermore, we investigated electrophysiological correlates of parietal phosphene perception in a hemianopic patient (SL) who suffered from a complete destruction of the left primary visual cortex. Ipsilesional parietal phosphene perception in patient SL showed a similar pattern of results to that of parietal phosphene perception in healthy participants, starting to affect TMS-evoked potentials at an early stage of latency. This evidence might thus support the idea of parietal cortex as an independent generator of magnetically induced conscious visual experiences.

Recentemente è stato dimostrato che la Stimolazione Magnetica Transcranica (TMS), applicata anche ad aree diverse da V1, può generare fosfeni. In particolare, sono stati riportati fosfeni in seguito alla stimolazione del Solco Intraparietale (IPS). Tuttavia, non è stato ancora chiarito se i fosfeni parietali siano generati direttamente dalla corteccia parietale indipendentemente dal contributo delle aree occipitali. In primo luogo, è stato svolto un esperimento per confrontare i più comuni metodi di misurazione della soglia di detezione dei fosfeni, dato che la stessa rappresenta il cuore del mio progetto di dottorato. Nello specifico, sono stati misurati il metodo degli stimoli costanti (MOCS), il “Modify Binary Search” (MOBS), ed il “Rapid Estimation of Phosphene Threshold” (REPT) in 17 partecipanti. I dati ottenuti suggeriscono che il MOCS sia il metodo più stabile da utilizzare nei successivi esperimenti: nonostante la sua lunghezza è risultato essere consistente nelle somministrazioni consecutive riportando soglie invariate indipendentemente dal numero di somministrazioni. Il MOBS è risultato invece altamente variabile, mentre il REPT è meno affidabile del MOCS, rimanendo comunque affidabile indipendente dal numero di somministrazioni. Il secondo esperimento si proponeva di descrivere sistematicamente le caratteristiche dei fosfeni parietali in modo da trovare eventuali differenze con i fosfeni classicamente evocati in occipitale in termini di soglia di detezione, eccentricità, grandezza, vividezza e luminosità. I siti di stimolazione sono stati identificati funzionalmente attorno a P3 e O1 e il metodo degli stimoli costanti è stato utilizzato per determinare la soglia dei fosfeni. Ai soggetti è stato anche richiesto di disegnare i fosfeni alla fine della procedura di soglia per determinare eccentricità e grandezza e di stimare vividezza e luminosità delle percezioni evocate. I risultati hanno mostrato una soglia più alta per i fosfeni in parietale (72.4%) rispetto a quelli evocati da stimolazione occipitale (63.1%). Inoltre, le due curve psicofisiche dei due tipi di fosfeni hanno mostrano una diversa forma riportando valori che correlano solo al 50% delle rispettive curve. Luminosità ed eccentricità sono poi risultate essere modulate dal sito di stimolazione, ottenendo rispettivamente, in seguito a stimolazione parietale, fosfeni meno luminosi (e più difficili da percepire) e stabili nella posizione indipendentemente dall’intensità di stimolazione, contrariamente a quanto avviene con i fosfeni in occipitale. Questo dato è probabilmente dovuto ad una peggiore organizzazione retinotopica dello spazio nella regione del solco intraparietale. Dato che l’attivazione di V1 è ritenuta necessaria nella percezione di fosfeni, la possibilità che V1 sia implicata nei fosfeni parietali non può essere totalmente rifiutata con i dati del secondo esperimento. Pertanto, è stata testata una paziente affetta da emianopsia omonima, la cui area visiva primaria danneggiata non mostrava alcuna attività rimanente, per indagare il ruolo del solco intraparietale nell’esperienza visiva cosciente. La paziente ha riportato fosfeni del tutto simili a quelli evocati nell’emisfero sano in seguito a stimolazione parietale dell’emisfero ipsilaterale la lesione occipitale. Inoltre la soglia riportata non è risultata differente da quella ottenuta nel lobo parietale sui soggetti sani. Infine, lo scopo dell’ultimo esperimento era di identificare il network corticale attivato nell’induzione di fosfeni da lobo parietale. In particolare la percezione dei fosfeni parietali è stata esplorata usando la combinazione simultanea di due diverse tecniche: TMS e fast optical imaging (event-related optical signal – EROS), capace di offrire una buona risoluzione spaziale unita ad un’ottima capacità di risoluzione temporale. I dati preliminari ottenuti da cinque partecipanti hanno rivelato pattern di attivazione diversi in base alla presenza o assenza di percezione del fosfene. Tuttavia, poiché molte delle attivazioni evidenziate sono di segno negativo, questi dati risultano di non facile interpretazione e trarre conclusioni certe risulta impossibile prima di un allargamento del campione. Concludendo, questi risultati, valutati insieme a precedenti evidenze sperimentali, sostengono l’ipotesi di un lobo parietale capace di generare esperienze visive coscienti, indipendentemente dalle aree visive primarie, e andando dunque contro le teorie che teorizzano che V1 sia necessaria per la consapevolezza visiva (Tong, 2003).
It has recently been shown that TMS applied to visually responsive areas other than V1 can generate light sensations, called “phosphenes”, in absence of visual stimulation in the environment. In particular, contralateral phosphenes have been obtained by stimulating the intraparietal sulcus. However, a question that still remains open is whether parietal phosphenes are generated by the parietal cortex, independently from the contribution of occipital areas. Since the phosphene threshold represented the heart of my project, a preliminary experiment comparing the most common thresholding methods was carried out in order to assess which methods was the most reliable. We thus tested the “method of constant stimuli” (MOCS), the “Modify Binary Search” (MOBS) and the “Rapid Estimation of Phosphene Threshold” (REPT) in seventeen participants. Each of the three methods was repeated three times per participant in the same week. The data suggested using MOCS in the next experiments because, despite its length, it resulted to be consistent across days, with thresholds not changing depending on the number of administrations. On the other hand, MOBS appeared highly variable, while REPT tended to be less reliable than MOCS but still remaining unaffected by the number of administrations. The aim of the second experiment was to provide a characterization of parietal phosphenes to find possible differences in terms of phosphene threshold, eccentricity, size, vividness and brightness with those evoked by stimulation of the occipital cortex. Single-pulse magnetic stimulations were administered with a figure-of-eight coil, assisted by a neuronavigational system. Individual stimulation sites were functionally identified around P3 and O1 (10-20 system). To determine phosphene threshold the “method of constant stimuli” was used: randomly intermixed intensities were employed (ranging from 45% to 90%) and twenty stimulations were given for each output intensity. Subjects were also requested to draw the phosphenes as to obtain eccentricity and size and to rate vividness and brightness of evoked perceptions. Fourteen subjects participated in the study. Results showed that the threshold was reliably lower for occipital lobe (63.1% of maximum stimulator output, MSO) than for parietal lobe (72.4% MSO) TMS stimulation and that the two psychophysical curves had a significantly different shape, with correlating values only at the 50% threshold. In addition, brightness and eccentricity were modulated by the site of stimulation, respectively obtaining less bright phosphenes (and thus more difficult to perceive) following parietal stimulation and no changes in the location of parietal phosphenes by changing the intensity of stimulation, contrary to what happened for the occipital phosphenes. This was probably due to the coarser retinotopic organization of space within the parietal cortex. Given that the activation of V1 is generally thought to be involved in conscious perception of phosphenes, the possibility that V1 was implicated in parietal phosphenes could not be totally dismissed with the second experiment. Therefore, a hemianopic patient, who showed no residual activity in her left V1, was tested to further investigate the role of intraparietal sulcus in visual conscious experience. She crucially reported parietal phosphenes (broadly similar to those evoked in the intact hemisphere and in sighted individuals) when TMS was applied over the damaged hemisphere (P3), in absence of V1. Additionally, her threshold values were not very different from those of healthy participants. Finally, we sought to identify the cortical neural network activated while inducing phosphenes with parietal TMS. In particular, parietal phosphene perception was explored using a simultaneous combination of TMS and a fast optical imaging tool (event-related optical signal – EROS), which offer a good spatial resolution in conjunction with the ability to map out the time course of feed-forward and feedback processes. The phosphene threshold value was employed to compare phosphene awareness to no phosphene awareness under identical stimulation parameters and to investigate which regions critically determined the parietal phosphene perception. Preliminary data from five participants seemed to reveal different activation patterns depending on the presence/absence of phosphenes but, since a lot of negative activations were highlighted, these data were not easy to be interpreted, as often happens with fMRI data. Therefore, the current sample needs to be enlarged and more data to be collected before any serious inferences can be drawn. In sum, these findings, along with previous evidence, corroborated the hypothesis that the parietal lobe is able to generate conscious visual experience, independently from early visual areas, and that parietal phosphenes may have a different neural basis from those elicited in occipital lobe. Such a result, therefore, goes against theories suggesting that primary visual area is necessary for awareness (Tong, 2003).