LA MATRICE EXTRACELLULARE COME FONTE DI INIBIZIONE DELLA PLASTICITA' SINAPTICA:EFFETTI DELLA SUA DEGRADAZIONE IN CORTECCIA VISIVA, IN VIVO.

La plasticità del sistema nervoso è definibile come la sua capacità di adattarsi in risposta all’esperienza; questa proprietà è legata a cambiamenti strutturali e funzionali a livello delle sinapsi già esistenti, e alla formazione di nuove sinapsi. E’ ormai dimostrato che la matrice extracellulare (MEC) gioca un ruolo centrale nella regolazione della plasticità, sia durante lo sviluppo che nell’adulto: essa è una struttura complessa, multimolecolare, che occupa lo spazio extracellulare di tutti i tessuti, e nel sistema nervoso è costituita principalmente da glicosamminoglicani (GAG), in particolare condroitin solfato (CSPGs) ed eparan solfato (HSPGs). Recentemente si è arrivati a parlare di una sinapsi tetrapartita: secondo questa nuova visione la comunicazione cellulare dipende dall’interazione di quattro elementi: presinaptico, postsinaptico, cellule della glia e molecole della matrice. Manipolando la matrice extracellulare con metodi genetici o farmacologici e accoppiando paradigmi esperienza-dipendenti, si assiste ad alterazioni della trasmissione sinaptica e della morfologia delle spine, le quali sono i più importanti correlati morfo-funzionali della plasticità. In particolare è stato possibile riaprire la plasticità in corteccia visiva adulta in seguito all’utilizzo dell’enzima batterico CondroitinasiABC, che degrada le catene di CSPGs. Partendo da queste premesse abbiamo voluto utilizzare lo stesso enzima per poter studiare gli effetti della digestione della matrice a livello delle spine dendritiche dei neuroni del V strato della corteccia visiva primaria. Il nostro studio si è concentrato sia sulle conseguenze morfologiche che su quelle funzionali. Nel primo caso, la tecnica della microscopia a due fotoni ci ha consentito di valutare, in vivo, le variazioni della motililtà e delle fluttuazioni della testa delle spine. Il modello utilizzato è un topo transgenico in cui alcune cellule dello strato V esprimono la GFP. L’imaging è stato effettuato sia in acuto che a distanza di tre giorni dalla somministrazione dell’enzima. Per ricercare possibili conseguenze funzionali abbiamo messo a punto un protocollo di LTP in corteccia visiva in vivo, non ancora presente in letteratura. Con questo studio abbiamo dunque voluto valutare il ruolo della MEC sui meccanismi di plasticità della corteccia visiva di topo adulto, svelando, seppur in maniera indiretta, possibili relazioni tra i cambiamenti morfologici delle spine e la trasmissione sinaptica a livello dei dendriti apicali dei neuroni piramidali del V strato. Le osservazioni effettuate possono rappresentare un punto di partenza per ulteriori studi che trovino una relazione di causa-effetto tra la composizione della MEC e le variazioni morfo-funzionali delle spine dendritiche

Quasi-simultaneous multiplane calcium imaging of neuronal circuits

Two-photon excitation fluorescence microscopy is widely used to study the activity of neuronal circuits. However, the fast imaging is typically constrained to a single lateral plane for a standard microscope design. Given that cortical neuronal networks in a mouse brain are complex three-dimensional structures organised in six histologically defined layers which extend over many hundreds of micrometres, there is a strong demand for microscope systems that can record neuronal signalling in volumes. Henceforth, we developed a quasi-simultaneous multiplane imaging technique combining acousto-optic deflector and static remote focusing to provide fast imaging of neurons from different axial positions inside the cortical layers without the need for the mechanical interference of either the objective lens or the specimen. The hardware and the software are easily adaptable to existing two-photon microscopes. Here, we demonstrated that our imaging method can record, at high speed and high image contrast, the calcium dynamics of neurons in two different imaging planes separated axially, with the in-focus and the refocused planes 120 μm and 250 μm below the brain surface respectively

A framework towards resilient Mediterranean eco-solutions for small-scale farming systems

The impacts of climate change on crop and livestock sectors are well-documented. Climate change and its related events (e.g., high temperatures, extreme events, disease outbreaks) afect livestock production in various ways (e.g., nutrition, housing, health, welfare), and tend to compromise the physical productivity and the economic performances. Understanding animal responses to climate change may help planning strategies to cope with the adverse climatic conditions and also to reduce polluting emissions. Through an interdisciplinary approach, we develop a conceptual framework to assess and develop new organisational models for Mediterranean small-scale farming systems so as to mitigate the impacts of climate change, to improve farm management and farming tech‑ nologies, and to achieve an efective adaptation to the climate changes. The conceptual framework consists of four phases: (i) community engagement, (ii) strategies development, (iii) data collection and analysis, (iv) business model generation and sustainability assessment. We assess strengths, weaknesses, opportunities, and threats of the ecosolutions by mean of a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis technique.info:eu-repo/semantics/publishedVersio

Open-source, Python-based, hardware and software for controlling behavioural neuroscience experiments

Laboratory behavioural tasks are an essential research tool. As questions asked of behaviour and brain activity become more sophisticated, the ability to specify and run richly structured tasks becomes more important. An increasing focus on reproducibility also necessitates accurate communication of task logic to other researchers. To these ends, we developed pyControl, a system of open-source hardware and software for controlling behavioural experiments comprising a simple yet flexible Python-based syntax for specifying tasks as extended state machines, hardware modules for building behavioural setups, and a graphical user interface designed for efficiently running high-throughput experiments on many setups in parallel, all with extensive online documentation. These tools make it quicker, easier, and cheaper to implement rich behavioural tasks at scale. As important, pyControl facilitates communication and reproducibility of behavioural experiments through a highly readable task definition syntax and self-documenting features. Here, we outline the system’s design and rationale, present validation experiments characterising system performance, and demonstrate example applications in freely moving and head-fixed mouse behaviour

The lost maps: two-photon investigations of the fine scale organization of auditory cortex

The spatial arrangement of neuronal responses in primary auditory cortex (A1) has so far been investigated by using microelectrode recording techniques or imaging of the intrinsic signal, which led to controversial results, at present still discussed. On the other hand, two-photon calcium imaging allows us to investigate the cortical functions at an unprecedented level of spatial detail, and has recently offered new insight into the fine-scale organization of frequency responses in A1. In this thesis, I used two-photon calcium imaging to compare, for the first time, the fine-scale cortical representation of sound frequency to that of two other sound features, crucial for survival and communication in all mammals: differences in intensity between the two ears (interaural level differences; ILDs), and frequency modulation (FM). I found that most neurons in layers II-III of the mouse A1 were tuned to ILDs favouring the contralateral ear, but midline and ipsilateral tuning were present too. Binaural preferences were heterogeneously distributed in space, both on the fine scale (within &amp;Tilde; 200 μm) and on the global one (up to &amp;Tilde; 1 mm). Moreover, A1 neurons were mostly tuned to slow FM sweeps within the range of those used in species-specific calls. Cells activated by similar rates tended to be spatially proximal, indicating a level of local organization similar to the one I found for frequency tuning, and higher than that of ILD responses. Finally, I set the groundwork for two-photon studies of the A1 of the ferret, by presenting the first evidence of the microscopic organization of the tonotopic map in this species. My results shed light on some long-held questions about the response properties of A1, and confirm two-photon imaging as a powerful tool for investigating the processing of sensory signals in the cortex of both small and large mammals.</p

The lost maps: two-photon investigations of the fine scale organization of auditory cortex

The spatial arrangement of neuronal responses in primary auditory cortex (A1) has so far been investigated by using microelectrode recording techniques or imaging of the intrinsic signal, which led to controversial results, at present still discussed. On the other hand, two-photon calcium imaging allows us to investigate the cortical functions at an unprecedented level of spatial detail, and has recently offered new insight into the fine-scale organization of frequency responses in A1. In this thesis, I used two-photon calcium imaging to compare, for the first time, the fine-scale cortical representation of sound frequency to that of two other sound features, crucial for survival and communication in all mammals: differences in intensity between the two ears (interaural level differences; ILDs), and frequency modulation (FM). I found that most neurons in layers II-III of the mouse A1 were tuned to ILDs favouring the contralateral ear, but midline and ipsilateral tuning were present too. Binaural preferences were heterogeneously distributed in space, both on the fine scale (within &Tilde; 200 &mu;m) and on the global one (up to &Tilde; 1 mm). Moreover, A1 neurons were mostly tuned to slow FM sweeps within the range of those used in species-specific calls. Cells activated by similar rates tended to be spatially proximal, indicating a level of local organization similar to the one I found for frequency tuning, and higher than that of ILD responses. Finally, I set the groundwork for two-photon studies of the A1 of the ferret, by presenting the first evidence of the microscopic organization of the tonotopic map in this species. My results shed light on some long-held questions about the response properties of A1, and confirm two-photon imaging as a powerful tool for investigating the processing of sensory signals in the cortex of both small and large mammals.</p

Imaging somatosensory cortex in rodents

The rodent somatosensory cortex has been investigated using a range of electrophysiological techniques, from intracellular recordings to electroencephalography. Nonetheless, their accessible location on the dorsal surface of the brain has more recently made the somatosensory areas popular models for the imaging-based investigation of cortical function. In this chapter, we will outline the general principles of two-photon microscopy applied to the functional study of the rodent somatosensory cortex. This technique allows recording the activity of hundreds of individual neurons simultaneously, with single-cell precision and while knowing their relative positions in the brain. We will place particular emphasis on long-term calcium imaging procedures on awake behaving mice and will introduce advantages and limitations of this technique. Our specific aim is to provide the reader with useful information regarding equipment and experimental procedures, from the choice of the calcium indicator to the post hoc analysis of imaging time series

Fluorescence Transients Acquired via Multiplane Imaging with AOD and Remote Focusing

Fluorescence transients from marked neurons at 120 um and 250 um shown in Fig. 8

Fluorescence Transients Acquired via Multiplane Imaging with AOD and Remote Focusing

Fluorescence transients from marked neurons at 120 um and 250 um shown in Fig. 8

Enhanced bioactivity of internally functionalized cationic dendrimers with PEG cores

Hybrid dendritic-linear block copolymers based on a 4-arm poly(ethylene glycol) (PEG) core were synthesized using an accelerated AB2/CD 2 dendritic growth approach through orthogonal amine/epoxy and thiol-yne chemistries. The biological activity of these 4-arm and the corresponding 2-arm hybrid dendrimers revealed an enhanced, dendritic effect with an exponential increase in cell internalization concomitant with increasing amine end groups and low cytotoxicity. Furthermore, the ability of these hybrid dendrimers to induce endosomal escape combined with their facile and efficient synthesis makes them attractive platforms for gene transfection. The 4-arm-based dendrimer showed significantly improved DNA binding and gene transfection capabilities in comparison with the 2-arm derivative. These results combined with the MD simulation indicate a significant effect of both the topology of the PEG core and the multivalency of these hybrid macromolecules on their DNA binding and delivery capablities