My work has focused on the development and application of fluorescent voltage-sensitive proteins based on microbial rhodopsins. These probes led to the discovery of electrical activity in the bacterium Escherichia coli, the first robust optical recordings of action potentials (APs) in mammalian neurons using a genetically encoded voltage reporter, and the development of a genetically targetable all-optical electrophysiology system. I first introduce an engineered fluorescent voltage sensor based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated. I then present another microbial rhodopsin, Archaerhodopsin 3 (Arch), whose endogenous fluorescence exhibited a twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. In rat hippocampal neurons, Arch detected single electrically triggered APs with an optical signal-to-noise ratio > 10. A mutant, Arch(D95N), lacked endogenous proton pumping and had 50% greater sensitivity than the wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual APs. Finally, I introduce evolved archaerhodopsin-based voltage indicators and a spectrally orthogonal channelrhodopsin actuator, which together enabled all-optical electrophysiology. A directed evolution screen yielded two mutants, QuasAr1 and QuasAr2, that showed improved brightness and voltage sensitivity relative to previous archaerhodopsin-based sensors, and microsecond response times. An engineered channelrhodopsin actuator, CheRiff, showed high light sensitivity and rapid kinetics. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured neurons, the Optopatch system probed membrane voltage across temporal and spatial scales, from the sub-cellular and sub-millisecond dynamics of AP propagation, to the simultaneous measurement of firing patterns of many neurons in a circuit. In brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. In human stem cell-derived neurons, Optopatch measurements revealed homeostatic tuning of intrinsic excitability, a subtle form of plasticity that had yet to be observed in human neurons. The suite of tools and techniques presented here enable high-throughput, genetically targeted, and spatially resolved electrophysiology without the use of conventional electrodes. / Engineering and Applied Sciences

<p> Manual dexterity, the ability to manipulate objects with the hands, and the related position sense, or proprioception, are often impaired after stroke. Associative stimulation of motor points (MPAS) in hand muscles is known to modify motor cortex excitability and improve manual dexterity (McDonnell and Ridding, 2006). </p><p> However, it is not known whether the effect of this peripheral stimulation can be increased by central stimulation of sensorimotor cortex, in terms of function, proprioception, or cortical neurophysiology. Here we compare the functional and neurophysiological consequences of MPAS with and without transcranial direct current (tDCS) in healthy adults. MPAS was applied to two right hand muscles important for manual dexterity: APB and FDI. tDCS, a non-invasive brain stimulation technique, was simultaneously applied over left sensorimotor cortex. Both techniques stimulate motor as well as somatosensory pathways. Neurophysiological measures of motor cortex, including SICI (short intra-cortical inhibition), ICF (intracortical facilitation), and input/output (I/O) curve, were assessed with transcranial magnetic stimulation (TMS). Manual dexterity and proprioceptive acuity were also measured. 14 subjects completed 3 sessions of MPAS in combination with sham, anodal (excitatory) and cathodal (inhibitory) tDCS. 13 subjects completed 2 sessions of sham MPAS with sham or anodal tDCS. In combination with MPAS, anodal tDCS significantly increased the plateau of manual dexterity, increased cortical response to TMS, and tended to improve proprioceptive acuity compared to sham tDCS. The neural basis for the observed functional improvements may thus include somatosensory as well as motor cortex. Neither MPAS nor tDCS alone had any measurable effect. These results suggest that adding tDCS as a central intervention to complement peripheral MPAS may be a promising avenue of treatment for patients with impaired manual dexterity. </p>

Luminance and contrast adaptation are neuronal mechanisms that the retina applies for continuous adjustment to light sensitivity though a collection of cellular and synaptic mechanisms distributed across the retinal network, thus accommodating the wide input range of the visual system within the constricted output range of retinal ganglion cells. Luminance mean adaptation has been demonstrated in the output neurons of the invertebrate eye (eccentric cells), and the aim of the study was to investigate whether the homology in visual processing extends to luminance variance (contrast) adaptation as well. The spike trains of individual eccentric cells were recorded from live horseshoe crabs to white noise stimuli of varying contrast delivered to optically-isolated ommatidial receptors. Linear-nonlinear models estimated from the spike output of eccentric cells decreased in gain with increasing contrast of white noise, suggesting an unknown mechanism of contrast adaptation may operate in the retina. Given the simple organization of the horseshoe crab eye determining whether this mechanism exists in the retina is of fundamental importance to vision research.

La recherche qui traite de la mémoire visuo-perceptive en terme d'acquisition de nouvelles connaissances est peu documentée. De plus, les tests d'apprentissage visuel sont généralement composés de stimuli de faible niveau de complexité et susceptibles d'être encodes verbalement. L'évaluation de la mémoire visuelle à l'aide de la figure complexe de Rey (FCR) permet d'éviter ce problème mais ne renseigne pas sur le taux d'apprentissage comme le fait le test des quinze mots de Rey (QMR) pour la modalité verbale. La présente recherche se propose donc de transposer et d'adapter la procédure d'administration des QMR à celle de la FCR afin de vérifier si elle favorise l'apprentissage de matériel visuel chez des adultes normaux et de comparer ces résultats à ceux d'individus frontolésés à droite. Une préexpérimentation vise d'abord à contrôler la durée de l'exposition de la FCR et de procurer des informations sur le taux d'apprentissage et de rétention en mémoire visuo-perceptive chez des participants normaux. Un échantillon de 51 adultes (F=34, H=17) divisé en six groupes a rappelé la FCR après des temps d'exposition différents et variant de 5 à 30 s par tranche de 5 s. La FCR est présentée à cinq reprises où chaque présentation est suivie d'un rappel libre immédiat à un temps d'exposition donné. Le temps d'exposition retenu est de 30 s et correspond à celui où le taux de réussite à la FCR équivaut à la moyenne des mots rappelés au cinquième essai des QMR pour des adultes âgés entre 20 et 69 ans. Des analyses de variance montrent un effet d'apprentissage significatif au fil des cinq présentations de la FCR et ce pour tous les temps d'exposition et tous les groupes d'âge. Par contre, l'âge corrèle négativement avec le niveau de rappel et la performance au premier essai est indépendante du temps d'exposition de la FCR et de l'âge. L'expérimentation porte sur deux femmes présentant une lésion circonscrite au lobe frontal droit et appariées à des participants contrôles. L'administration des épreuves comprend l'évaluation de la capacité d'apprentissage de matériel non-verbal à l'aide de la FCR présentée selon la technique d'administration des QMR. De plus, l'apprentissage verbal est évalué à l'aide des QMR. L'index de latéralité manuelle est mesuré à l'aide de l'Inventaire d'Edinburgh. Le Baycrest Assessment of Neuropsychological Status est utilisé afin de recueillir des informations sur la santé psychologique, physique et psychiatrique. L'acuité visuelle est évaluée à l'aide du Rosenbaum Pocket Vision Screener. Les gnosies visuelles sont évaluées à l'aide du Boston Naming Test. Le quotient intellectuel est mesuré à l'aide de la forme abrégée à deux sous-tests du Weschler Adult Intelligence Scale-Revised. Des calculs de régression linéaire simple montrent que la présentation itérative de la FCR crée une courbe d'apprentissage à pente positive chez les participants normaux et cliniques. L'analyse des écarts aux résultats moyens montre que la performance aux cinq rappels de la FCR du participant clinique #1 ne diffère pas de celle des participants préexpérimentaux de la condition 30 s, alors que la capacité d'apprentissage du participant clinique #2 est inférieure à plus d'un écart-type, sauf au premier rappel. Les résultats supposent que les participants cliniques sont sensibles à l'interférence proactive et non à l'interférence rétroactive. De plus, la présentation successive de la FCR semble contribuer au maintient à long terme de l'information chez les participants expérimentaux. Finalement, la mémoire de reconnaissance des frontolésés est normale lorsque ce test évalue leur capacité à reconnaître la FCR dans son entier.

Les séquelles d'un traumatisme crânio-cérébral (TCC) sont susceptibles d'engendrer des difficultés affectives, cognitives et comportementales chez la victime et laisser cette dernière dans un état de dépendance. Une personne proche est souvent appelée à prendre en charge la victime et à assumer un rôle d'aidant pour lequel elle n'est pas suffisamment préparée, ce qui peut conduire au développement d'une détresse psychologique chez l'aidant. Les recherches tendent à démontrer que la santé psychologique du proche aidant constitue un facteur essentiel dans l'adaptation de la victime de TCC. Dans cette perspective, la présente étude s'intéresse à la relation pouvant exister entre la détresse psychologique de l'aidant et le fonctionnement cognitif exécutif de la victime de TCC. À cet égard, deux questions de recherche ont été posées : 1) Existe-t-il un effet corrélationnel positif entre la détresse psychologique chez l'aidant et la détresse psychologique chez la victime de TCC? 2) Est-il possible d'observer un effet corrélationnel négatif entre la détresse psychologique observable chez l'aidant et le fonctionnement exécutif de la personne cérébrolésée? Cette étude à trois temps de mesure est exploratoire. Elle a été réalisée auprès d'un échantillon de 13 dyades d'aidants et de victimes d'un TCC. Les participants ont été recrutés par l'entremise de l'Association Renaissance pour les personnes traumatisées crâniennes (ARPTC) au Saguenay-Lac-Saint-Jean. Chaque dyade de participants a été évaluée à 3 reprises, à environ 6 semaines d'intervalle. Le profil descriptif sociodémographique de la situation générale des participants ayant subi un TCC et des aidants a été obtenu. Les variables du fonctionnement cognitif exécutif du participant ayant subi un TCC ont été mesurées à l'aide de quatre instruments reconnus, soit le Controlled Oral Word Association Test (COWA), l'effet Stroop, le Trail Making Test (TMT) et la Tour de Londres (TOL). L'évaluation de la détresse psychologique du participant ayant subi un TCC et de l'aidant a été déterminée à l'aide du Symptom Checklist-90-Revised (SCL-90-R). L'évaluation des facteurs pouvant influencer la manifestation de la détresse psychologique de l'aidant tel que le soutien social et la satisfaction de l'aidant envers certains aspects de son rôle ont été pris en compte à l'aide de cinq questionnaires. L'échantillon a été divisé en deux groupes, selon la présence ou l'absence de détresse psychologique observable chez l'aidant au premier temps de mesure, d'après l'index global de sévérité (GSI) du SCL-90-R. Suite aux différentes analyses statistiques, il a d'abord été observé un effet corrélationnel positif) entre la détresse psychologique de l'aidant et la détresse psychologique de l'individu ayant subi un TCC et dans un second temps, un effet corrélationnel négatif entre la détresse psychologique de l'aidant et certains aspects spécifiques du fonctionnement exécutif du participant cérébrolésé. L'influence positive d'autres variables a également été observée sur la détresse psychologique de l'aidant. Ces variables sont : la satisfaction de l'aidant à l'égard de son support social obtenu, le type de dyades, plus spécifiquement les dyades de type homme (aidant) ? femme (aidé) et le fait que la dyade demeure sous le même toit. En effet, ces conditions sont présentes lors de l'absence de détresse psychologique chez l'aidant. Enfin, l'analyse des résultats suggère que la détresse psychologique de l'aidant et de la victime de TCC pourrait agir tel un double effet sur le fonctionnement cognitif exécutif de la victime de TCC : la détresse psychologique de l'aidant, aurait un effet sur la détresse psychologique de l'individu cérébrolésé et vice-versa. Ensemble, la détresse psychologique de l'aidant et de la victime de TCC aurait un effet potentiel sur certains aspects du fonctionnement exécutif de la victime de traumatisme crânien.

Thesis (PH.D.)--Syracuse University, 2003. / "Publication number AAT 3081640."

<p> Cadmium is a naturally-occurring trace metal frequently found in soil that has been linked with increased prevalence of various cancers via formation of reactive oxygen species. Selenium, a widely-prevalent metalloid, antagonizes the detrimental action of cadmium and has been demonstrated to exert a rescue effect. The present work focuses on the short- and long-term effects of exposure to ecologically-relevant cadmium concentrations on zebrafish development and behavior, and compares this to co-treatment with selenium. This study has demonstrated a decrease in overall brain size, specifically telencephalic area, in response to cadmium exposure, and has documented a sparing effect of selenium treatment. A similar effect is seen in larval body size and eye diameter. This study has also reported an effect on spinal morphology and hatching delay. Longitudinally, cadmium treatment affects survival to six months, and has an impact on adult brain metrics. The results of behavioral assay indicate an effect of larval cadmium exposure on adult learning behavior, as well as a rescue effect of selenium.</p><p>

<p> Developmental processes, genes and environmental factors interact to produce changes in cognition and behavior over the lifespan of an individual. However, the underlying molecular genetic mechanisms that mediate these changes remain to be fully elucidated. DNA methylation is an epigenetic mechanism that defines cell identity and helps regulate gene expression. DNA methylation is dynamic over development and has been shown to mediate experience-dependent changes, including those resulting from learning and memory and early life adversity. Although methylation mainly occurs at genomic cytosines in the CG dinucleotide context, methylation at non-CG sites was recently found in brain tissue. Non-CG methylation is specifically enriched in neurons and accumulates during the early childhood stages of brain development. The biological impact of non-CG methylation in regulating gene expression and regulating cellular function, if any, remains unclear. A major challenge for addressing this question is the complexity and scale of the DNA methylation landscape, which includes nearly one billion cytosines throughout the genome that are potential sites of modification in every cell. Targeted studies of specific candidate genes and genomic loci do not elucidate the overall configuration of the cellular epigenome. Techniques for comprehensively mapping the genome-wide distribution of DNA methylation are powerful, but they require sophisticated new computational methods of analysis that can reliably distinguish and statistically validate epigenomic differences related to developmental and environmental factors. </p><p> In this thesis we develop new approaches to comprehensively analyze DNA methylation throughout the genome and with single base resolution in order to better characterize the role of CG methylation and elucidate the potential role of CH methylation in mammalian brain cells. First, we consider the impact of enriched early life (peri-pubertal) experience on DNA methylation and gene expression in the dentate gyrus of the hippocampus. In addition to its role in experience-dependent gene regulation, DNA methylation also plays a key role in innate developmental processes, including female X chromosome inactivation. We provide the first allele-specific DNA methylomes from the active and inactive X chromosomes in female brain, and use comprehensive genomic analyses to gain insight into the functional relationship between allele-specific DNA methylation and transcription. These two studies provide new evidence of the dynamic changes in DNA methylation in whole brain tissue caused by environmental and innate developmental factors. However, they do not address the heterogeneity of brain cell types, a hallmark of mammalian brain organization. To address the role of DNA methylation in brain cell diversity, we develop computational methods to analyze data from a new assay that measures single cell methylomes. Using these data, we show that brain cell methylomes can be clustered and used to assess neuronal heterogeneity in the frontal cortex of mouse and human. Upon clustering cells, we are able to gain insight into the role of methylation in the establishment and maintenance of cellular identity in neuronal types. </p><p> Overall, this thesis adds to the increasing evidence that DNA methylation is a cell type-specific, dynamic epigenomic modification of brain cells that is impacted by, and may in turn help to regulate, neuronal development and adaptation. In addition, this thesis provides new computational methods for analyzing large-scale, whole-genome DNA methylation data sets and demonstrates their use in uncovering new insights into the mammalian brain epigenome.</p><p>

The study of the molecular organisation of specialised synaptic structures has been considerably advanced by the development of methods for the isolation of synaptic subfractions enriched in synaptic plasma membranes (SPM), synaptic junctional complexes (SJC's) and postsynaptic densities (PSD's). The present thesis deals with one important aspect of this organisation, namely the synthesis of SPM proteins and its modulation by selected chemical agents. SPM, mitochondrial and myelin-enriched fractions of comparable purity to published data have been isolated from adult rat brain. An in vivo35method for monitoring incorporation of L-( S)methionine into the protein of these fractions has been developed. This, involved injection of 35 flooding concentrations of L-( S)methionine into the lateral ventricle via a pre-implanted cannula. Since the precursor specific activity is known and constant for at least 45 minutes the incorporation rates can be reliably measured over this period. The effects of two anaesthetics, halothane and pentobarbitone, on brain protein synthesis were investigated. Halothane (3%) produced a gross inhibition of protein synthesis in total forebrain and SPM, myelin and mitochondrial fractions. Pentobarbitone produced no such inhibition at doses of 60 or 75 mg/Kg body weight. L-DOPA was also found to inhibit incorporation rates by close to 30% in total forebrain and mitochondrial protein in hyperthermic animals (body temperature 39-40&deg;C). Other subcellular fractions were less affected by the drug. Thyroid hormone deficiency during brain development impairs brain structure and function including synapse formation. The present study showed that hypothyroidism decreased protein yields in SPM, myelin and mitochondrial fractions and impaired synthesis of SPM and myelin protein. The rates of incorporation into total forebrain and mitochondrial protein were not significantly affected. SDS polyacrylamide gel, electrophoresis and fluorographic studies tentatively showed that the relative rates of synthesis of specific polypeptides were altered in thyroid deficient animals.

The melanophores in the catfish I. melas are arranged in 3 layers, one epidermal and two dermal. The former are the smallest, and also differ from those in the dermis in their shape and in their reaction-times in response to background change. Hypophysectomy impairs the ability of the animal to adapt to a black background but has apparently no effect on white adaptation. It severely affects the dispersion of pigment in the epidermal melanophores in comparison to the lower dermal ones. Equilibration on different greys and in darkness of hypophysectomised specimens indicates that the pituitary gland plays no role in the initial phases of dispersion or in adaptation to darkness. The removal of the pituitary also appears to accelerate the initial phases of black-to-white adaptation. The nerve fibres controlling the aggregation of pigment in I. melas follow in principle the same path as in Phoxinus. The equilibration to a white background following anterior spinal section requires 6-8 weeks. All melanophore layers of white-adapted chromatically spinal fish fully disperse their pigment in about 24 hr on transfer to a black background. On reversal the white-equilibrated condition is obtained in 7-10 days, epidermal melanophores being slowest to achieve full dispersion and being the first to aggregate it. Hypophysectomised spinal fish remain at an intermediate tint on all backgrounds and after enucleation. Observations suggest that the pituitary contributes to the dispersion of pigment, following transection of the anterior spinal cord. White background adaptation is impaired after interrupting the hypothalamo-hypophyseal tract. It is inferred that in I. melas, as in elasmobranchs and amphibians, the central control on the pars intermedia is of an inhibitory nature. Pinealectomy does not affect the ability of the animals to adapt to illuminated white or black backgrounds. The equilibrium Mi's of white/black adapted and of enucleated pinealectomised animals in darkness are strikingly similar to those of unoperated specimens. Noradrenaline and adrenaline cause pigment aggregation in isolated skin. Adrenergic blocking and anti-adrenaline substances cause a slight dispersion in dermal melanophores and moderate dispersion in epidermal melanophores. Acetylcholine has only a slight dispersion effect. The results of the present investigation can be explained on the assumption of one hormone and mononeuronic control of melanophores in Ictalurus. It is suggested that the epidermal melanophores differ from the dermals in having different threshold levels of response to MSH and to noradrenaline. Finally, it is concluded that melas in its chromatic physiology occupies a position between the teleost species in which the melanophores are predominantly neurally controlled (e.g. Phoxinus) and those in which their co-ordination is mainly hormonal (e.g. Anguilla).