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This paper presents the design and the utilization of a wireless electro-optic platform to perform simultaneous multimodal electrophysiological recordings and optogenetic stimulation in freely moving rodents. The developed system can capture neural action potentials (AP), local field potentials (LFP) and electromyography (EMG) signals with up to 32 channels in parallel while providing four optical stimulation channels. The platform is using commercial off-the-shelf components (COTS) and a low-power digital field-programmable gate array (FPGA), to perform digital signal processing to digitally separate in real time the AP, LFP and EMG while performing signal detection and compression for mitigating wireless bandwidth and power consumption limitations. The different signal modalities collected on the 32 channels are time-multiplexed into a single data stream to decrease power consumption and optimize resource utilization. The data reduction strategy is based on signal processing and real-time data compression. Digital filtering, signal detection, and wavelet data compression are used inside the platform to separate the different electrophysiological signal modalities, namely the local field potentials (1–500 Hz), EMG (30–500 Hz), and the action potentials (300–5,000 Hz) and perform data reduction before transmitting the data. The platform achieves a measured data reduction ratio of 7.77 (for a firing rate of 50 AP/second) and weights 4.7 g with a 100-mAh battery, an on/off switch and a protective plastic enclosure. To validate the performance of the platform, we measured distinct electrophysiology signals and performed optogenetics stimulation in vivo in freely moving rondents. We recorded AP and LFP signals with the platform using a 16-microelectrode array implanted in the primary motor cortex of a Long Evans rat, both in anesthetized and freely moving conditions. EMG responses to optogenetic Channelrhodopsin-2 induced activation of motor cortex via optical fiber wer

The development of electrodes for the electrical modulation of cell activity has become a research topic of increasing interest, due to an ever-growing list of applications. These include deep brain stimulation for neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease; implantable bionic devices such as the Cochlear ear implant and bionic eye; and promoting cell growth for wound healing or tissue engineering. Electrical stimulation is conventionally achieved by delivering current from an external power source through metallic electrodes to the target biological tissue. However, these types of electrodes have drawbacks. For instance, they typically require cumbersome and invasive wiring, while the stimulating spatial resolution is limited by current spreading. One attractive alternative technique is light-based stimulation, which offers wireless and scalable targeting of cells with superior spatial precision. In particular, light-sensitive electrode materials can be used to transduce light into an electrical stimulus to excite nearby cells. Nitrogen-doped ultrananocrystalline diamond (N-UNCD) is a promising material for this technique, possessing a highly attractive combination of properties including high chemical inertness, durability, conductivity, and biocompatibility. In addition, it has also been shown to produce a photoresponse using near-infrared light, which offers greater optical penetration in biological tissue than shorter wavelengths. In this thesis, we optimise and characterise N-UNCD electrodes for light-based cell stimulation, and then demonstrate their use in the modulation of stem cell cultures. To achieve this, we tested the effect of different chemical surface treatments on the stimulating performance of the N-UNCD electrodes, measured by the capacitance and photoresponse. Of these treatment methods, we found that annealing N-UNCD in oxygen ambient produced dramatically improved properties, with the capacitance and photoresponse

The blood–brain barrier (BBB) limits the delivery of majority of cancer drugs and thereby complicates brain tumor treatment. The nasal-brain-lymphatic system is discussed as a pathway for brain drug delivery overcoming the BBB. However, in most cases, this method is not sufficient to achieve a therapeutic effect due to brain drug delivery in a short distance. Therefore, it is necessary to develop technologies to overcome the obstacles facing nose-to-brain delivery of promising pharmaceuticals. In this study, we clearly demonstrate intranasal delivery of liposomes to the mouse brain reaching glioblastoma (GBM). In the experiments with ablation of the meningeal lymphatic network, we report an important role of meningeal pathway for intranasal delivery of liposomes to the brain. Our data revealed that GBM is characterized by a dramatic reduction of intranasal delivery of liposomes to the brain that was significantly improved by near-infrared (1267 nm) photostimulation of the lymphatic vessels in the area of the cribriform plate and the meninges. These results open new perspectives for non-invasive improvement of efficiency of intranasal delivery of cancer drugs to the brain tissues using nanocarriers and near-infrared laser-based therapeutic devices, which are commercially available and widely used in clinical practice., RSF, RFBR, RF Governmental Grant, Peer Reviewed

In deep tissue imaging, pulsed near-infrared lasers commonly provide high peak powers needed for nonlinear absorption, but average power and linear absorption can be limiting factors for tissue damage through heat. We implemented intra-cavity dumping within a mode-locked Ti:Sapphire laser used for two-photon computer generated holography stimulation. This system enables photostimulation in which pulse energy, average power, and repetition rate can each be varied and harnessed as degrees of freedom. We demonstrate how this system provides a new dimension of temporal control in photostimulation experiments to alter the ratio of nonlinear to linear light-tissue interactions, namely by tuning the laser repetition rate between single-shot and ≈ 3 MHz. Repetition rates below 1 MHz, yielding pulse energies over 60 nJ, facilitated holographic projections with more regions of interest than the highest repetition rate of 3 MHz. Even lower repetition rates ( ≈ 10 kHz) diminished thermal load on the sample, as characterized by quantification of heat shock protein expression in zebrafish tissue., ImPhys/Microscopy Instrumentation & Techniques, ChemE/Chemical Engineering

Estudis previs realitzats en altres espècies, han demostrat que la fotoestimulació amb llum vermella augmenta la motilitat espermàtica. A més, altres estudis indiquen que la capacitat fecundant dels espermatozoides pot incrementar després de la irradiació. Tot i que els mecanismes a través dels quals la llum exerceix els seus efectes sobre els espermatozoides es desconeixen en gran mesura, l'evidència creixent atorga als fotosensibilitzadors cel·lulars endògens, especialment els presents en les mitocòndries, un paper fonamental. Per tot això, l'objectiu d'aquesta tesi doctoral va ser avaluar els efectes de diferents protocols de fotoestimulació amb un llum LED vermella a una longitud d'ona de 620-630 nm sobre els paràmetres de qualitat espermàtica, en semen de ruc i cavall; fresc, refrigerat i congelat/descongelat, a més d'avaluar la influència del medi i del color de la palleta utilitzada en els efectes observats, i determinar si els efectes produïts per l'estimulació amb llum vermella en els espermatozoides estan provocats per canvis en la funció mitocondrial induïts per l'acció de la llum sobre els citocroms mitocondrials com el citocrom C. Amb aquest propòsit es van dissenyar cinc estudis: en el primer es van avaluar els efectes de diferents patrons d'estimulació amb llum LED vermella sobre els paràmetres de motilitat espermàtica, la integritat de la membrana, el potencial de membrana mitocondrial i els nivells intracel·lulars de ROS en semen de ruc fresc i refrigerat. En el segon estudi es van analitzar els efectes de diferents patrons d'irradiació amb llum vermella sobre la motilitat espermàtica, les subpoblacions d'espermatozoides mòbils, el potencial de membrana mitocondrial, la taxa de consum d'O2, els nivells intracel·lulars d'ATP, la integritat de la membrana i la integritat de l'ADN espermàtic en semen fresc de cavall. El tercer estudi tenia com objectiu determinar si la irradiació amb llum vermella de l'esperma de cavall congelada-descongelada podria mi, Estudios previos realizados en otras especies, han demostrado que la estimulación con luz roja aumenta la motilidad espermática. Además, otros estudios han sugerido que la capacidad fecundante de los espermatozoides puede incrementarse después de la irradiación. Aunque los mecanismos a través de los cuales la luz ejerce sus efectos sobre los espermatozoides se desconocen en gran medida, la evidencia creciente otorga a los fotosensibilizadores celulares endógenos, especialmente los presentes en las mitocondrias, un papel crucial. Por todo ello, el objetivo de esta Tesis Doctoral fue evaluar los efectos de diferentes protocolos de fotoestimulación con un luz LED roja a una longitud de onda de 620-630 nm sobre los parámetros de calidad espermática, en semen de burro y caballo; fresco, refrigerado y congelado/descongelado, además de evaluar la influencia del medio/diluyente y del color de la pajuela utilizada en los efectos observados, así como estudiar si los efectos producidos por la estimulación con luz roja en los espermatozoides están provocados por cambios en la función mitocondrial inducidos por la acción de la luz sobre los citocromos mitocondriales como el citocromo C. Con este propósito, se diseñaron cinco estudios: en el primero se evaluaron los efectos de diferentes patrones de estimulación con luz LED roja sobre los parámetros de motilidad espermática, la integridad de la membrana plasmática, el potencial de membrana mitocondrial y los niveles intracelulares de ROS en semen de burro fresco y refrigerado. En el segundo estudio, se analizaron los efectos de diferentes patrones de irradiación con luz roja sobre la motilidad espermática, las subpoblaciones de espermatozoides móviles, el potencial de membrana mitocondrial, la tasa de consumo de O2, los niveles intracelulares de ATP, la integridad de la membrana plasmática y la integridad del ADN espermático en semen fresco de caballo. En el tercer estudio se propuso determinar si la irradiación con luz roja del, Previous studies carried out in other species, showed that stimulating sperm with red light increases their motility. In addition, other studies indicate sperm fertilizing ability can be increased after irradiation. While the mechanisms through which light exerts its effects on sperm are largely unknown, mounting evidence gives endogenous cellular photosensitizers, especially those present in mitochondria, a crucial role. Therefore, the objective of this Dissertation was to evaluate the effects of different irradiation protocols using a red LED light at a wavelength of 620-630 nm on freshly ejaculated, liquid-stored and frozen-thawed sperm from stallions and jackasses. The potential influence of the medium/extender and the colour of the straw was also investigated, as well as whether the effects produced by red light resulted from changes in mitochondrial function via modulation of cytochromes, such as cytochrome C. For this purpose, five studies were designed. In the first one, the effects of different red LED light patterns on sperm motility, integrity of plasma membrane, mitochondrial membrane potential, and intracellular ROS levels were investigated in fresh and stored donkey semen. The second study analysed the effects of separate red light irradiation patterns on motility, membrane integrity, motile sperm subpopulations, mitochondrial membrane potential, O2 consumption rate, intracellular ATP levels and the integrity of DNA of fresh horse sperm. The third study sought to determine whether red light irradiation of frozen-thawed horse semen could improve sperm quality and increase their ability to withstand incubation. In the fourth study, whether the colour of the straw and / or the turbidity of the medium affected the response induced by red light stimulation was examined in liquid-stored horse semen. Finally, the fifth work studied whether the effects produced by red light on sperm were related to changes in mitochondrial function induced by mitochondrial cyt, Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina i Sanitat Animals

Studying the propagation of action potentials across neuronal networks and how information is stored and accessed in their dynamic firing patterns has always been the essence of neuroscience. Emerging evidence shows that information in the brain is encoded in the simultaneous or avalanche-like firing of multiple, spatially separated groups of neurons. Thus, understanding the collective behavior of neurons is essential for understanding how the brain processes information and encodes memory. Since its discovery, the advent of optogenetics has brought upon a revolution in neuroscience, where individual neuronal circuits are able to be selectively probed and their connections decoded. This ability has been used by many groups to great effect, with some groups even using optogenetic stimulation to create phantom sensations, which are typically encoded in the functional activity of distinct neuronal populations. However, in-vivo optogenetic excitation relies inherently on the quality and accuracy of the stimulation method, with many problems arising due to biological effects such as animal motion, the scattering nature of brain tissue, and cell health. Typically, groups either use digital micromirror arrays or spatial light modulators, with the former lacking transmission efficiency and the latter having a high technical skill barrier due to its propensity to induce artifacts into intended patterns of light. This dissertation attempts to reduce the barrier towards the use of spatial light modulators in optogenetics by improving targeting accuracy, reducing the effects of unmodulated light and related artifacts, and developing new methods of stimulation which reduce the power density directed at neurons. To accomplish the first step, improving targeting accuracy, I created and demonstrated a real-time capable particle-based motion tracking algorithm to correct for animal motion. To reduce the effects of optical artifacts, I developed and patented a method of using Fresnel

We present a series of cationic membrane-targeted azobenzene molecules, with the aim to understand how variations in molecular architecture influence the relative optical and biological properties. 1,4-Amino-substituted azobenzene was chosen as the switching unit while the number of linked alkyl chains and their cationic end-group were systematically varied. Their photophysics, membrane partitioning, and electrophysiological efficacy were studied. We found that the polar end group is a key-factor determining the interaction with the phospholipid heads in the plasma membrane bilayer and consequently the ability to dimerize. The monosubstituted photoswitch with a pyridinium-terminated alkyl chain was found to be the best candidate for photostimulation. This study provides a structure-property investigation that can guide the chemical engineering of a new generation of molecular photo-actuators.

Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca2+ signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca2+ concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca2+-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.

Working memory (WM) consists of short-term storage and executive components. We studied cortical oscillatory correlates of these two components in a large sample of 156 participants to assess separately the contribution of them to individual differences in WM. The participants were presented with WM tasks of above-average complexity. Some of the tasks required only storage in WM, others required storage and mental manipulations. Our data indicate a close relationship between frontal midline theta, central beta activity and the executive components of WM. The oscillatory counterparts of the executive components were associated with individual differences in verbal WM performance. In contrast, alpha activity was not related to the individual differences. The results demonstrate that executive components of WM, rather than short-term storage capacity, play the decisive role in individual WM capacity limits. © 2020, The Author(s).

Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca2+ signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca2+ concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca2+-dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.

Covert facial mimicry involves subtle facial muscle activation in observers when they perceive the facial emotional expressions of others. It remains uncertain whether prototypical facial features in emotional expressions are being covertly mimicked and also whether covert facial mimicry involves distinct facial muscle activation patterns across muscles per emotion category, or simply distinguishes positive versus negative valence in observed facial emotions. To test whether covert facial mimicry is emotion-specific, we measured facial electromyography (EMG) from five muscle sites (corrugator supercilii, levator labii, frontalis lateralis, depressor anguli oris, zygomaticus major) whilst participants watched videos of people expressing 9 different basic and complex emotions and a neutral expression. This study builds upon previous research by including a greater number of facial muscle measures and emotional expressions. It is the first study to investigate activation patterns across muscles during facial mimicry and to provide evidence for distinct patterns of facial muscle activation when viewing individual emotion categories, suggesting that facial mimicry is emotion-specific, rather than just valence-based. © 2020, The Author(s).

The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole-cell electrophysiological recordings with laser-scanning photostimulation to test whether peripheral nerve injury in the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity.

The spinal dorsal horn is the first relay structure coding for pain transmission and modulation. Previous anatomical and electrophysiological studies have examined spinal dorsal horn circuit connections and network activity. Further work is required to understand spinal cord sensory information processing that underlies pathological neuropathic pain states. Our previous studies suggest that peripheral nerve injury enhances presynaptic excitatory input onto spinal superficial dorsal horn neurons, which in turn contributes to pathologic nociception. The potential changes in local postsynaptic circuits in the dorsal horn that lead to pathologically heightened behavioral responses to pain remain largely unexplored. We combined whole-cell electrophysiological recordings with laser-scanning photostimulation to test whether peripheral nerve injury in the spinal nerve ligation (SNL) mouse model of neuropathic pain leads to alterations in the functional connectivity of spinal cord circuits including lamina II excitatory interneurons. Here we show that SNL enhances excitation and decreases inhibition to lamina II excitatory interneurons along with their increased glutamate-evoked excitability. The enhanced excitatory postsynaptic input and connectivity evoked by SNL eventually return to normal levels concurrently with the resolution of the neuropathic pain states. The physiological pattern highly correlates with mouse pain behaviors following SNL, supporting a neurophysiological mechanism of central sensitization and neuropathic pain that is functionally localized to the spinal dorsal horn. Together, these data support that SNL induces functional changes in synaptic input and connectivity to lamina II excitatory interneurons that code for pain perception, and thus provide new insights into the mechanism and locus of pain hypersensitivity.

The use of light for triggering skeletal and cardiac muscles allows lower invasiveness higher selectivity and unprecedented possibility to target individual cells or even subcellular compartments in a temporally and spatially precise manner. Because cells are in general transparent, this requires the development of suitable interfaces that bestow light sensitivity to living matter. In the present work, successfully demonstrated is the use of conjugated polymer films as transducer to optically enhance the contraction rate of a human and patient-specific cardiac in vitro cell model. By different experimental approaches, the coupling mechanism to the photothermal effect is assigned. This work extends the range of application of the polymer-mediated cell photostimulation phenomenon to cardiac muscle cells, opening up possible applications in cardiac therapy and for implementation of in vitro studies.

Preclinical prediction of autistic spectrum disorders (ASD) is one of the priorities of current research. Children at risk of ASD develop an atypical visual perception profile early in their lives, which influences their visual responsiveness, distribution of attention, and social orienting. In this study we have compared the oculomotor behavior in an infant at familial risk of ASD with data from two 10-month infants with typical development. The SMI RED500 eye tracker was used for acquisition. Most parameters of visual perception in the at-risk infant were found to differ significantly from these of the controls. The strategy of visual search in the at-risk infant was generally less successful (13% of attempts vs 31% and 56% in the controls) with a tendency to focus predominantly on social stimuli (50% of the total gaze time). The said changes together with longer fixation duration (576.41 ms vs 527.77 and 386.72 ms in the two controls), lower saccadic frequency (1.74 counts/ms vs 1.84 and 2.18 counts/ms), and shorter scan path length (2774.24 px vs 3612.58 and 3985.43 px) may result in difficulties in switching tasks and processing information.

A distorted body representation is a core symptom in eating disorders (EDs), though its mechanism is unclear. Allocentric lock theory, emphasising the role of reference frame processing in body image, suggests that ED patients may be (b)locked to an (allocentric) representation of their own body stored in long-term memory (e.g., my body is fat) that is not updated (modified) by the (real-time egocentric) perception-driven experience of the physical body. Employing a well-validated virtual reality-based procedure, relative to healthy controls, ED patients showed deficits in the ability to refer to and update a long-term stored (allocentric) representation with (egocentric) perceptual-driven inputs.

Spontaneous network activity is believed to sculpt developing neural circuits. Spontaneous giant depolarizing potentials (GDPs) were first identified with single-cell recordings from rat CA3 pyramidal neurons, but here we identify and characterize a large-scale spontaneous network activity we term global network activation (GNA) in the developing mouse hippocampal slices, which is measured macroscopically by fast voltage-sensitive dye imaging. The initiation and propagation of GNA in the mouse is largely GABA-independent and dominated by glutamatergic transmission via AMPA receptors. Despite the fact that signal propagation in the adult hippocampus is strongly unidirectional through the canonical trisynaptic circuit (dentate gyrus [DG] to CA3 to CA1), spontaneous GNA in the developing hippocampus originates in distal CA3 and propagates both forward to CA1 and backward to DG. Photostimulation-evoked GNA also shows prominent backward propagation in the developing hippocampus from CA3 to DG. Mouse GNA is strongly correlated to electrophysiological recordings of highly localized single-cell and local field potential events. Photostimulation mapping of neural circuitry demonstrates that the enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA and reveals the underlying pathways accounting for GNA backward propagation from CA3 to DG. The disappearance of GNA coincides with a transition to the adult-like unidirectional circuit organization at about 2 weeks of age. Taken together, our findings strongly suggest a critical link between GNA activity and maturation of functional circuit connections in the developing hippocampus.

Developing neuronal circuits have many interesting properties; understanding the development process can give us insights on functioning of complex adult neural circuits. One of major features of immature neural circuits is the spontaneous activity, which has also been considered a driving force of circuit maturation. Giant Depolarizing Current (GDP) is the major spontaneous activity at single neuron level of developing hippocampus. What is missing, however, is whether hippocampus has large scale network level spontaneous activity that precedes the formation of mature hippocampal circuits, i.e. unidirectional trisynaptic flow. In chapter 1 of this study, we use Voltage Sensitive Dye Imaging to investigate the initiation and propagation of global network activity (GNA) in developing hippocampus. GNA originates in distal CA3 and propagates both forward to CA1 and backward to DG. Single-cell and local field potential recording confirmed GNA is closely related to neuronal activation. Further, enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA. Thus Bi-directional GNA precedes the maturation of the mouse hippocampal circuit, and it is correlated to maturation of functional circuit connections. To further understand the underlying pathway for back-projection from CA3 to DG in the developing hippocampus, in the study of chapter 2, we use laser scanning photostimulation technique to map synaptic circuit development of mossy cells. We observed major regional sources of circuit excitation and inhibition to hilar mossy cells were changed from CA3 to DG during development. The excitatory back projection from CA3 to mossy cell were greatly reduced in adult hippocampus. Thus, hilar mossy cells can play important role in circuit signal back-propagation from CA3 to DG. In chapters 3 and 4, we demonstrate an innovative technology which automatically detects and extracts EPSC and IPSC events in electrophysiological recordi

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Neural Circuits 7 (2013): 160, doi:10.3389/fncir.2013.00160., Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice., This work was supported by a CRP grant from the National Research Foundation of Singapore and by the World Class Institute (WCI )Program of the National Research Foundation of Korea (NRF )funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI2009-003).

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Neural Circuits 7 (2013): 160, doi:10.3389/fncir.2013.00160., Here we characterize several new lines of transgenic mice useful for optogenetic analysis of brain circuit function. These mice express optogenetic probes, such as enhanced halorhodopsin or several different versions of channelrhodopsins, behind various neuron-specific promoters. These mice permit photoinhibition or photostimulation both in vitro and in vivo. Our results also reveal the important influence of fluorescent tags on optogenetic probe expression and function in transgenic mice., This work was supported by a CRP grant from the National Research Foundation of Singapore and by the World Class Institute (WCI )Program of the National Research Foundation of Korea (NRF )funded by the Ministry of Education, Science and Technology of Korea (MEST) (NRF Grant Number: WCI2009-003).

Developing neuronal circuits have many interesting properties; understanding the development process can give us insights on functioning of complex adult neural circuits. One of major features of immature neural circuits is the spontaneous activity, which has also been considered a driving force of circuit maturation. Giant Depolarizing Current (GDP) is the major spontaneous activity at single neuron level of developing hippocampus. What is missing, however, is whether hippocampus has large scale network level spontaneous activity that precedes the formation of mature hippocampal circuits, i.e. unidirectional trisynaptic flow. In chapter 1 of this study, we use Voltage Sensitive Dye Imaging to investigate the initiation and propagation of global network activity (GNA) in developing hippocampus. GNA originates in distal CA3 and propagates both forward to CA1 and backward to DG. Single-cell and local field potential recording confirmed GNA is closely related to neuronal activation. Further, enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA. Thus Bi-directional GNA precedes the maturation of the mouse hippocampal circuit, and it is correlated to maturation of functional circuit connections. To further understand the underlying pathway for back-projection from CA3 to DG in the developing hippocampus, in the study of chapter 2, we use laser scanning photostimulation technique to map synaptic circuit development of mossy cells. We observed major regional sources of circuit excitation and inhibition to hilar mossy cells were changed from CA3 to DG during development. The excitatory back projection from CA3 to mossy cell were greatly reduced in adult hippocampus. Thus, hilar mossy cells can play important role in circuit signal back-propagation from CA3 to DG. In chapters 3 and 4, we demonstrate an innovative technology which automatically detects and extracts EPSC and IPSC events in electrophysiological recordi

Spontaneous network activity is believed to sculpt developing neural circuits. Spontaneous giant depolarizing potentials (GDPs) were first identified with single-cell recordings from rat CA3 pyramidal neurons, but here we identify and characterize a large-scale spontaneous network activity we term global network activation (GNA) in the developing mouse hippocampal slices, which is measured macroscopically by fast voltage-sensitive dye imaging. The initiation and propagation of GNA in the mouse is largely GABA-independent and dominated by glutamatergic transmission via AMPA receptors. Despite the fact that signal propagation in the adult hippocampus is strongly unidirectional through the canonical trisynaptic circuit (dentate gyrus [DG] to CA3 to CA1), spontaneous GNA in the developing hippocampus originates in distal CA3 and propagates both forward to CA1 and backward to DG. Photostimulation-evoked GNA also shows prominent backward propagation in the developing hippocampus from CA3 to DG. Mouse GNA is strongly correlated to electrophysiological recordings of highly localized single-cell and local field potential events. Photostimulation mapping of neural circuitry demonstrates that the enhancement of local circuit connections to excitatory pyramidal neurons occurs over the same time course as GNA and reveals the underlying pathways accounting for GNA backward propagation from CA3 to DG. The disappearance of GNA coincides with a transition to the adult-like unidirectional circuit organization at about 2 weeks of age. Taken together, our findings strongly suggest a critical link between GNA activity and maturation of functional circuit connections in the developing hippocampus.

Intrusive memories are a hallmark symptom of posttraumatic stress disorder (PTSD). They reflect excessive and uncontrolled retrieval of the traumatic memory. Acute elevations of cortisol are known to impair the retrieval of already stored memory information. Thus, continuous cortisol administration might help in reducing intrusive memories in PTSD. Strong perceptual priming for neutral stimuli associated with a "traumatic" context has been shown to be one important learning mechanism that leads to intrusive memories. However, the memory modulating effects of cortisol have only been shown for explicit declarative memory processes. Thus, in our double blind, placebo controlled study we aimed to investigate whether cortisol influences perceptual priming of neutral stimuli that appeared in a "traumatic" context. Two groups of healthy volunteers (N = 160) watched either neutral or "traumatic" picture stories on a computer screen. Neutral objects were presented in between the pictures. Memory for these neutral objects was tested after 24 hours with a perceptual priming task and an explicit memory task. Prior to memory testing half of the participants in each group received 25 mg of cortisol, the other half received placebo. In the placebo group participants in the "traumatic" stories condition showed more perceptual priming for the neutral objects than participants in the neutral stories condition, indicating a strong perceptual priming effect for neutral stimuli presented in a "traumatic" context. In the cortisol group this effect was not present: Participants in the neutral stories and participants in the "traumatic" stories condition in the cortisol group showed comparable priming effects for the neutral objects. Our findings show that cortisol inhibits perceptual priming for neutral stimuli that appeared in a "traumatic" context. These findings indicate that cortisol influences PTSD-relevant memory processes and thus further support the idea that administration of cor

Although epileptic photosensitivity is well known, its genetics and syndromic associations are incompletely understood. Seizures triggered by photic stimulation are usually a manifestation of the idiopathic generalized epilepsies, especially juvenile myoclonic epilepsy (JME), or of the occipital epilepsies. Idiopathic photosensitive occipital epilepsy (IPOE) is a focal epilepsy with colourful elementary visual auras, often with conscious tonic head and eye version; myoclonus is not a feature. All seizures are induced by photic stimuli. We describe four families with phenotypic overlap between JME and IPOE. Families were identified if two or more affected individuals had visual auras and electro-clinical features of an idiopathic epilepsy. Family members underwent detailed electro-clinical assessment. In addition, 40 unrelated JME probands were investigated systematically for unrecognized features of IPOE (visual aura and conscious head version). There were 12 affected individuals in four families; 11 were female. Clinical onset was at 8-21 years of age. Of 10 patients with visual auras, six had conscious head version and five also experienced myoclonic jerks; eight had non-photic induced tonic-clonic seizures (TCS). Of the remaining individuals, one had myoclonic jerks and occipital spikes; the other had TCS without visual aura or myoclonic jerks. Of 10 patients with EEG studies, eight had generalized spike and wave (GSW) and six had occipital spikes. All had photosensitivity with GSW and four had additional occipital spikes. Of the 40 JME probands, six had visual aura and/or conscious head version; five of these were photosensitive. There is overlap between the clusters of clinical features used to diagnose IPOE and JME. Half of the affected individuals in our families with visual aura had myoclonic jerks; the former is characteristic of IPOE and the latter of JME. Importantly, visual aura is not regarded as part of JME, nor myoclonus part of IPOE, but our data emp

Purpose. This study investigates the role of insulin in retinal function by examining the effects of insulin on the a- and b-waves of the electroretinogram (ERG) recorded from an in vitro bovine retina eye-cup preparation. Methods. Bovine eyes were enucleated immediately after exsanguination, hemisected to form an eye cup, and transported to the laboratory in oxygenated medium. Eye cups were then transferred to a perfusion system that provided constant superfusion of oxygenated perfusate solution to the retina, enabling it to remain in a functional state in vitro. The ERG was recorded, as the retinal response to photic stimulation, from electrodes mounted within the perfusion system. The effects of insulin on the ERG were investigated by adding insulin to the perfusate solution. Results. Application of insulin to the in vitro retina preparation decreased the amplitudes of the a- and b-waves of the ERG in a dose dependent manner with a maximal effect at doses of 0.1 U/ml and above. These effects were reversible. Conclusion. The reduction of ERG amplitudes may result from the hyperpolarising effect of insulin reported in other tissues. The findings suggest that insulin may have a regulatory role in retinal activity; however extrapolation of these results to the intact organism is dependent on the presence of insulin in retina.

It has generally been assumed that high-level cognitive and emotional processes are based on amodal conceptual information. In contrast, however, "embodied simulation" theory states that the perception of an emotional signal can trigger a simulation of the related state in the motor, somatosensory, and affective systems. To study the effect of social context on the mimicry effect predicted by the "embodied simulation" theory, we recorded the electromyographic (EMG) activity of participants when looking at emotional facial expressions. We observed an increase in embodied responses when the participants were exposed to a context involving social valence before seeing the emotional facial expressions. An examination of the dynamic EMG activity induced by two socially relevant emotional expressions (namely joy and anger) revealed enhanced EMG responses of the facial muscles associated with the related social prime (either positive or negative). These results are discussed within the general framework of embodiment theory.

Although epileptic photosensitivity is well known, its genetics and syndromic associations are incompletely understood. Seizures triggered by photic stimulation are usually a manifestation of the idiopathic generalized epilepsies, especially juvenile myoclonic epilepsy (JME), or of the occipital epilepsies. Idiopathic photosensitive occipital epilepsy (IPOE) is a focal epilepsy with colourful elementary visual auras, often with conscious tonic head and eye version; myoclonus is not a feature. All seizures are induced by photic stimuli. We describe four families with phenotypic overlap between JME and IPOE. Families were identified if two or more affected individuals had visual auras and electro-clinical features of an idiopathic epilepsy. Family members underwent detailed electro-clinical assessment. In addition, 40 unrelated JME probands were investigated systematically for unrecognized features of IPOE (visual aura and conscious head version). There were 12 affected individuals in four families; 11 were female. Clinical onset was at 8-21 years of age. Of 10 patients with visual auras, six had conscious head version and five also experienced myoclonic jerks; eight had non-photic induced tonic-clonic seizures (TCS). Of the remaining individuals, one had myoclonic jerks and occipital spikes; the other had TCS without visual aura or myoclonic jerks. Of 10 patients with EEG studies, eight had generalized spike and wave (GSW) and six had occipital spikes. All had photosensitivity with GSW and four had additional occipital spikes. Of the 40 JME probands, six had visual aura and/or conscious head version; five of these were photosensitive. There is overlap between the clusters of clinical features used to diagnose IPOE and JME. Half of the affected individuals in our families with visual aura had myoclonic jerks; the former is characteristic of IPOE and the latter of JME. Importantly, visual aura is not regarded as part of JME, nor myoclonus part of IPOE, but our data emp

Purpose. This study investigates the role of insulin in retinal function by examining the effects of insulin on the a- and b-waves of the electroretinogram (ERG) recorded from an in vitro bovine retina eye-cup preparation. Methods. Bovine eyes were enucleated immediately after exsanguination, hemisected to form an eye cup, and transported to the laboratory in oxygenated medium. Eye cups were then transferred to a perfusion system that provided constant superfusion of oxygenated perfusate solution to the retina, enabling it to remain in a functional state in vitro. The ERG was recorded, as the retinal response to photic stimulation, from electrodes mounted within the perfusion system. The effects of insulin on the ERG were investigated by adding insulin to the perfusate solution. Results. Application of insulin to the in vitro retina preparation decreased the amplitudes of the a- and b-waves of the ERG in a dose dependent manner with a maximal effect at doses of 0.1 U/ml and above. These effects were reversible. Conclusion. The reduction of ERG amplitudes may result from the hyperpolarising effect of insulin reported in other tissues. The findings suggest that insulin may have a regulatory role in retinal activity; however extrapolation of these results to the intact organism is dependent on the presence of insulin in retina.

Performance of reaction time (RT) tasks was investigated in young children and adults to test the hypothesis that age-related differences in processing speed supersede a "global" mechanism and are a function of specific differences in task demands and processing requirements. The sample consisted of 54 4-year-olds, 53 5-year-olds, 59 6-year-olds, and 35 adults from Russia. Using the regression approach pioneered by Brinley and the transformation method proposed by Madden and colleagues and Ridderinkhoff and van der Molen, age-related differences in processing speed differed among RT tasks with varying demands. In particular, RTs differed between children and adults on tasks that required response suppression, discrimination of color or spatial orientation, reversal of contingencies of previously learned stimulus-response rules, and greater stimulus-response complexity. Relative costs of these RT task differences were larger than predicted by the global difference hypothesis except for response suppression. Among young children, age-related differences larger than predicted by the global difference hypothesis were evident when tasks required color or spatial orientation discrimination and stimulus-response rule complexity, but not for response suppression or reversal of stimulus-response contingencies. Process-specific, age-related differences in processing speed that support heterochronicity of brain development during childhood were revealed. © 2008 Elsevier Inc. All rights reserved.

People tend to perceive identical top halves (i.e. above the nose) of two face stimuli as being different when they are aligned with distinct bottom halves. This composite face illusion is generally considered as the most compelling evidence that facial features are integrated into a holistic representation. Here, we recorded eye-movements during the composite face illusion in a delayed matching task of top halves of faces. Behavioural results showed a strong composite face effect, participants making more mistakes and taking longer time to match two identical top halves of faces when they were aligned (vs. misaligned) with different bottom halves. Importantly, fixation sites and eye-movements were virtually identical when the top and bottom parts were aligned (composite illusion) or misaligned (no illusion), indicating that holistic face processing can be independent of gaze behaviour. These findings reinforce the view that holistic representations of individual faces can be extracted early on from information at a relatively coarse scale, independently of overt attention., FLWIN, info:eu-repo/semantics/published

Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2., Hydrogen peroxide (H2O2) is a metastable reactive oxygen species regulating many biological pathways. The role of H2O2 in biological processes depends on its local concentration, in the nM range H2O2 can act as a signalling molecule, while exposure to higher levels of H2O2 can have cytotoxic effects. The aim of this work is to create photosensitive microparticles that would produce physiological amounts of hydrogen peroxide to regulate signalling pathways in cultured cells. The proposed microparticles are substrate-free organic photovoltaic elements that produce H2O2 by photofaradaic reduction of molecular oxygen with the concurrent oxidation of an electron donor present in the electrolyte. The detailed fabrication methods to achieve them were described. The particles have lateral dimensions in the range of micrometres, while a thickness of less than 100 nm. Photofaradaic reactions are driven by tissue-penetrating deep red light (660 nm) to enable potential in vivo applications of the technology. The thesis will discuss optimization of the photofaradaic microparticles production and characterization of the H2O2 evolution.

Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2., Hydrogen peroxide (H2O2) is a metastable reactive oxygen species regulating many biological pathways. The role of H2O2 in biological processes depends on its local concentration, in the nM range H2O2 can act as a signalling molecule, while exposure to higher levels of H2O2 can have cytotoxic effects. The aim of this work is to create photosensitive microparticles that would produce physiological amounts of hydrogen peroxide to regulate signalling pathways in cultured cells. The proposed microparticles are substrate-free organic photovoltaic elements that produce H2O2 by photofaradaic reduction of molecular oxygen with the concurrent oxidation of an electron donor present in the electrolyte. The detailed fabrication methods to achieve them were described. The particles have lateral dimensions in the range of micrometres, while a thickness of less than 100 nm. Photofaradaic reactions are driven by tissue-penetrating deep red light (660 nm) to enable potential in vivo applications of the technology. The thesis will discuss optimization of the photofaradaic microparticles production and characterization of the H2O2 evolution.

Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2., Hydrogen peroxide (H2O2) is a metastable reactive oxygen species regulating many biological pathways. The role of H2O2 in biological processes depends on its local concentration, in the nM range H2O2 can act as a signalling molecule, while exposure to higher levels of H2O2 can have cytotoxic effects. The aim of this work is to create photosensitive microparticles that would produce physiological amounts of hydrogen peroxide to regulate signalling pathways in cultured cells. The proposed microparticles are substrate-free organic photovoltaic elements that produce H2O2 by photofaradaic reduction of molecular oxygen with the concurrent oxidation of an electron donor present in the electrolyte. The detailed fabrication methods to achieve them were described. The particles have lateral dimensions in the range of micrometres, while a thickness of less than 100 nm. Photofaradaic reactions are driven by tissue-penetrating deep red light (660 nm) to enable potential in vivo applications of the technology. The thesis will discuss optimization of the photofaradaic microparticles production and characterization of the H2O2 evolution.

Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2., Hydrogen peroxide (H2O2) is a metastable reactive oxygen species regulating many biological pathways. The role of H2O2 in biological processes depends on its local concentration, in the nM range H2O2 can act as a signalling molecule, while exposure to higher levels of H2O2 can have cytotoxic effects. The aim of this work is to create photosensitive microparticles that would produce physiological amounts of hydrogen peroxide to regulate signalling pathways in cultured cells. The proposed microparticles are substrate-free organic photovoltaic elements that produce H2O2 by photofaradaic reduction of molecular oxygen with the concurrent oxidation of an electron donor present in the electrolyte. The detailed fabrication methods to achieve them were described. The particles have lateral dimensions in the range of micrometres, while a thickness of less than 100 nm. Photofaradaic reactions are driven by tissue-penetrating deep red light (660 nm) to enable potential in vivo applications of the technology. The thesis will discuss optimization of the photofaradaic microparticles production and characterization of the H2O2 evolution.

Peroxid vodíku (H2O2) je metastabilní reaktivní forma kyslíku, která reguluje mnoho biologických drah. Úloha H2O2 v biologických procesech závisí na jeho lokální koncentraci, v malém množství (nM) může působit jako signální molekula, zatímco vyšší koncentrace mohou mít cytotoxické účinky. Cílem této práce bylo vytvoření fotosenzitivních mikročástic, které by produkovaly fyziologické množství peroxidu vodíku pro regulaci signálních drah v kultivovaných buňkách. Navrhované mikročástice jsou organické fotovoltaické struktury bez substrátu, které produkují H2O2 fotofaradickou redukcí molekulárního kyslíku. Produkce peroxidu probíhá za současné oxidace donoru elektronů přítomného v elektrolytu. Částice mají rozměry v řádu mikrometrů, zatímco jejich tloušťka nepřekračuje 100 nm. Fotofaradické reakce jsou poháněny červeným světlem (660 nm), které může pronikat tkáněmi, což umožňuje potenciální aplikace mikročástic in vivo. Práce se bude zabývat optimalizací výroby mikročástic a charakterizací produkce H2O2., Hydrogen peroxide (H2O2) is a metastable reactive oxygen species regulating many biological pathways. The role of H2O2 in biological processes depends on its local concentration, in the nM range H2O2 can act as a signalling molecule, while exposure to higher levels of H2O2 can have cytotoxic effects. The aim of this work is to create photosensitive microparticles that would produce physiological amounts of hydrogen peroxide to regulate signalling pathways in cultured cells. The proposed microparticles are substrate-free organic photovoltaic elements that produce H2O2 by photofaradaic reduction of molecular oxygen with the concurrent oxidation of an electron donor present in the electrolyte. The detailed fabrication methods to achieve them were described. The particles have lateral dimensions in the range of micrometres, while a thickness of less than 100 nm. Photofaradaic reactions are driven by tissue-penetrating deep red light (660 nm) to enable potential in vivo applications of the technology. The thesis will discuss optimization of the photofaradaic microparticles production and characterization of the H2O2 evolution.