University of Genova – Italian Institute of Technology：–意大利热那亚大学工学院

University of Genova – Italian Institute of Technology：–意大利热那亚大学工学院 ANNEX A-3 University of Genova – Italian Institute of Technology Doctoral School on “Life and Humanoid Technologies” Academic Year 2010-2011 Doctoral Course on “Neuroscience and Brain Technologies” Research Themes 10 positions available with scholarship INTRODUCTION TO RESEARCH THEMES Candidates are asked to prepare a research project of their choice related to one or more topics of the general project of the Department of Neuroscience and Brain Technologies (Director Prof. Fabio Benfenati) reported below. The soundness and originality of the project will be part of the evaluation process. A. BASIC NEUROSCIENCE RESEARCH: GENOMICS, POSTGENOMICS AND CONNEC- TOMICS A1. Molecular and cellular determinants of synaptic plasticity and information processing in neural networks Neuronal networks are capable of adaptation and learning, although a thorough study of circuit activ-ity has been hindered by the complexity of mammalian networks. Network plasticity can be defined as the shaping of network morphology and function primarily induced by experience. This process is based on complex activity-dependent changes in neurons that modulate the ability of the neural net-work to transfer, elaborate and store information. We propose to clarify the mechanisms underlying synaptic transmission and plasticity in networks of live neurons with the purpose of understanding the changes in the information flow and processing involved in higher brain functions. The investigations on the molecular basis of synaptic plasticity will include the molecular analysis of the neurotransmit-ter release machinery, the functional characterization of key synaptic proteins and of the roadmap of signal transduction and protein phosphorylation processes that mediate the changes in the efficiency of synaptic transmission. These studies will be carried out using leading edge biotechnologies, includ-ing viral-infected neuronal cell cultures, live imaging of neuronal cells coupled to patch-clamp record-ings as well as generation and phenotypic characterization of genetically-altered mice lacking specific neuronal proteins. A2. Development of genetically-encoded probes to specifically stimulate or inhibit neuronal ac-tivity Optogenetics is a novel approach to monitor and manipulate neuronal activity, based on a family of fast light-responsive microbial opsins including proton pumps (Arch and Mack), chloride pumps (eNpHR) and cation channels (ChR-2). Light-driven activation of optogenetic probes allows to selec-tively activate / inhibit neuronal communication and to study the consequences of this manipulation on the activity of the entire network. Sub-cellular targeting of optogenetic probes is a crucial step to understand the computational and integrative properties of single cells and neuronal circuits. Such targeting can be easily obtained by fusing the probes to sequences that would mediate their localisa-tion to specific compartments such as the pre-synaptic terminals or axon domains. We aim to engineer a series of probes addressed to distinct sub-cellular domains, and to test their efficacy in modulating the excitability of the expressing neuron as well as the surrounding network. The project will involve the use of molecular and cell biology techniques as well as electrophysiology, and will be carried out both in primary neuronal cultures and in vivo. A3. Induction of plasticity in neural networks by chronic environmental stimuli Synaptic strength can be finely tuned over a short-to-long time scale by a combination of factors in-cluding previous activity of the network, generation of second messengers, functional changes in pre- and post-synaptic proteins as well as regulation of the expression of genes implicated in growth, sur-vival and synaptic transmission. These changes profoundly affect the processing between input and output information and, ultimately, shape the information flow within the network. We propose to subject random and engineered neuronal networks obtained from wild-type or genetically altered mice to chronic, spatially-defined, patterns of electrical stimulation in the presence or absence of controlled changes in the extracellular environment (ions, neurotransmitters, lipid messengers, hormones, etc). During and after the conditioning sessions, the functional and structural changes induced by experi- ence will be evaluated by live imaging coupled with electrophysiology, in order to define a constella-tion of environmental stimuli with negative/positive influences on neural development and plasticity and capable to modify neural connectivity and information processing through the network. Photic stimulation paradigms will also be employed through the localized application of light stimuli with high spatio-temporal resolution. Systems entail photoreceptive neurons of retinal origin or cerebral neurons rendered light-sensitive by transfection of specific light-sensitive ion channels or ion trans-porters. A4. Study of experience-dependent plasticity in ex-vivo and in-vivo models A4.1 Synaptic basis of experience-dependent plasticity in vivo We will investigate the functioning and plasticity of excitatory and inhibitory microcortical circuits in the intact brain by focusing on the visual cortex of mammals as an anatomically and functionally well-characterized model system. To this aim, we will estimate excitatory and inhibitory synaptic conductances by recording voltages while injecting different currents intracelluarly in vivo. This ap-proach will be complemented by recordings of the spike activity of genetically labelled interneurons and by the study of the effects of monocular deprivation using intracellular blockers of inhibitory neu-rotransmission. This knowledge is crucially lacking and is absolutely required for any attempt to re- brain-machine interfaces. cover cortical function after lesion through the use of A4.2 Cross-modal plasticity in vivo We will study if cortical neurons in area V1 receive subthreshold inputs from other sensory modalities (somatosensory, acoustic) that affect visually driven spiking responsiveness when the different sen-sory stimulations are presented simultaneously. We will investigate this issue in normal animals, in animals subjected to a generalized increase of sensory stimulations (environmental enrichment) or to the opposite condition, a lack of visually evoked activity from birth (dark rearing). We will also inves-tigate the presence of mirror neurons in the prefrontal cortex of rodents. These results will be of utmost importance due to the possibility of studying receptive field modifications in geneti- cally-modified animals. This project is in collaboration with the BMI project coordinated by Luciano Fadiga and the RBCS-Sandini. A4.3 Synaptic correlates of behavioural plasticity The overall goal is to understand how specific molecular pathways in the brain modulate synaptic plasticity to produce behavioural changes. The main focus will be on the mechanisms underlying learning of goal-directed behaviours, the flexible use of actions and how they become habitual. This is of particular interest given the proposed dysfunction of habitual control over behaviour under patho-logical conditions, including Parkinson’s disease and drug addiction. B. APPLICATIONS TO THE UNDERSTANDING OF HUMAN CENTRAL NERVOUS SYS- TEM DISEASES The basic properties of information processing at the synapse are precociously altered in an array of human central nervous system diseases and many synaptopathies are believed to be underlie the early stages of the pathogenesis of epilepsy, neurodegenerative diseases (such as Parkinson’s or Alzhei- mer’s diseases), autism, schizophrenia, etc. Main goal of this project is to investigate key neuropatho- logical mechanisms of neurodegenerative diseases, with the view of understanding the pathological processes and identifying potential novel targets for drug discovery. The main diseases that will be studied mostly using genetically altered mice will include: - epilepsy, to understand the mechanisms of epileptogenesis and its relationships with an array of epi-lepsy-associated mutations involving pre- and post-synaptic proteins; - neurodegenerative diseases with special reference to Alzheimer’s disease, Down syndrome, tauopa- thies, synucleinopathies and autosomal dominant leukodystrophy, by focusing on: (i) the mechanisms mediated by ,-amyloid aggregates and neuroinflammation, (ii) the potential role of adult neurogenesis; (iii) dysfunction and aggregation of other neuronal proteins such as the microtubule-associated protein tau, mutated ,-synuclein and the nuclear protein lamin B1; - autism, with special reference to the mutations in synaptic proteins found to be associated with the abnormal neural connectivity that is believed to underlie such disease; - schizophrenia and Parkinson’s disease, attention deficit hyperactivity disorders (ADHD) and depres- sion, with special reference to the associated dysfunctions of monoaminergic systems, including novel systems using trace amines as neurotransmitters. C. APPLICATIONS TO BIO-HYBRID AND BIO-INSPIRED TECHNOLOGIES C1. Set-up of neuroelectronic interfaces for high-efficiency coupling C1.1 Generation of artificial networks Attention will be paid to the optimization of the cell-solid state interaction, in order to achieve long- lasting conditions of cell survival and an optimal transfer of forward and backward signals from neu-rons to the solid-state device. We plan to obtain targeted positioning of neuronal terminals, oriented cell motion and neurite outgrowth, which would allow us to record signals related to pathfinding and stabilization of organized contacts and networks. This will be obtained through the development of new solid-state substrates favoring neuronal growth along specific pathways and the optimization of cell solid-state interfacing by micropatterning of various guidance proteins. We also intend to develop 3D neuronal cultures by growing neurons onto porous membranes or on scaffold-like polymeric sub-strates. In particular, we propose the realization of a chip-based technology for realizing this 3D experi-mental models to reconstruct functional neuronal circuits by adopting a bottom-up approach and to interact with this tissue with a microelectrode-based device C1.2 High-resolution microelectrode devices for recording and stimulating individual neurons We aim at the design, realization and testing of novel neuroelectronic interfaces based on micro/nano-fabrication technologies and CMOS integrated systems. We will also design and implement experi-mental platforms for interfacing the realized devices and for exploiting the enabled features in the analysis of multi-dimensional electrophysiological data. We will also apply post-processing technolo-gies for improving the electrode-neuron interface (e.g. carbon nanotubes coating). Similar strategies will be applied also to electrodes for in vivo recordings in order to improve the electrode biocompati-bility and the signal-to-noise ration in long-term recordings, such as coating the electrode surface with autologous glial or neuronal cells in order to prevent the scar-induced long-term loss of electrode sensitivity (collaboration with Luciano Fadiga and Davide Ricci, RBCS-Sandini). C1.3 Exploration of alternative non-invasive electro-optical signal transduction mechanisms for the optical readout of neuronal activity. Towards the development of a kilo- to mega-pixel recording array with embedded optical sensors (e.g., voltage-sensitive dyes or refractive materials) that respond to changes in membrane potential. Resolution will only be limited by spatio-temporal camera characteristics. The main advantages will be arbitrary sampling of neural activity at any location within the network, high stability of the sensor units and enhanced biocompatibility. C2. Embodied networks and bidirectional neurorobotic interfaces Learning capabilities in complex neuronal systems usually require a two-way interaction with the en-vironment (i.e. a closed loop architecture) and the contribution of specialized and coordinated activity of large neuronal assemblies. We will develop efficient neuroelectronic interfaces allowing a bidirec-tional interaction between “the brain” and artificial devices. The research will investigate the mechan- isms which allow to reliably modify synaptic connections in neuronal preparations by using the tech-nology of multi-electrode arrays and efficient coding and decoding schemes. In vitro neurons from different brain areas, extracted from rat/mouse embryos and plated onto microelectrode arrays will be interconnected to external entities (e.g. a computer, a virtual environment, a robot or a biological ac-tuator such as the octopus arm), thus allowing real-time closed-loop interactions. The software tools for the on-line/off-line analysis of the behavior of the neurorobotic system will be also developed and the built hybrid system will be used for studying the computational properties of biological neuronal networks and for investigating the mechanisms underlying complex behaviors such as learning at network level. For any further information please contact: Dr. Luisa Franco PhD Course in Neuroscience and Brain Technologies Fondazione Istituto Italiano di Tecnologia Via Morego, 30 - 16163 Genova Tel. +39 010 71781432 Fax. +39 010 7170817 Email: luisa.franco@iit.it