OPTIMIZATION OF TIME-RESPONSE AND AMPLIFICATION FEATURES OF EGOTs FOR NEUROPHYSIOLOGICAL APPLICATIONS

In device engineering, basic neuron-to-neuron communication has recently inspired the development of increasingly structured and efficient brain-mimicking setups in which the information flow can be processed with strategies resembling physiological ones. This is possible thanks to the use of organic neuromorphic devices, which can share the same electrolytic medium and adjust reciprocal connection weights according to temporal features of the input signals. In a parallel - although conceptually deeply interconnected - fashion, device engineers are directing their efforts towards novel tools to interface the brain and to decipher its signalling strategies. This led to several technological advances which allow scientists to transduce brain activity and, piece by piece, to create a detailed map of its functions. This effort extends over a wide spectrum of length-scales, zooming out from neuron-to-neuron communication up to global activity of neural populations. Both these scientific endeavours, namely mimicking neural communication and transducing brain activity, can benefit from the technology of Electrolyte-Gated Organic Transistors (EGOTs). Electrolyte-Gated Organic Transistors (EGOTs) are low-power electronic devices that functionally integrate the electrolytic environment through the exploitation of organic mixed ionic-electronic conductors. This enables the conversion of ionic signals into electronic ones, making such architectures ideal building blocks for neuroelectronics. This has driven extensive scientific and technological investigation on EGOTs. Such devices have been successfully demonstrated both as transducers and amplifiers of electrophysiological activity and as neuromorphic units. These promising results arise from the fact that EGOTs are active devices, which widely extend their applicability window over the capabilities of passive electronics (i.e. electrodes) but pose major integration hurdles. Being transistors, EGOTs need two driving voltages to be operated. If, on the one hand, the presence of two voltages becomes an advantage for the modulation of the device response (e.g. for devising EGOT-based neuromorphic circuitry), on the other hand it can become detrimental in brain interfaces, since it may result in a non-null bias directly applied on the brain. If such voltage exceeds the electrochemical stability window of water, undesired faradic reactions may lead to critical tissue and/or device damage. This work addresses EGOTs applications in neuroelectronics from the above-described dual perspective, spanning from neuromorphic device engineering to in vivo brain-device interfaces implementation. The advantages of using three-terminal architectures for neuromorphic devices, achieving reversible fine-tuning of their response plasticity, are highlighted. Jointly, the possibility of obtaining a multilevel memory unit by acting on the gate potential is discussed. Additionally, a novel mode of operation for EGOTs is introduced, enabling full retention of amplification capability while, at the same time, avoiding the application of a bias in the brain. Starting on these premises, a novel set of ultra-conformable active micro-epicortical arrays is presented, which fully integrate in situ fabricated EGOT recording sites onto medical-grade polyimide substrates. Finally, a whole organic circuitry for signal processing is presented, exploiting ad-hoc designed organic passive components coupled with EGOT devices. This unprecedented approach provides the possibility to sort complex signals into their constitutive frequency components in real time, thereby delineating innovative strategies to devise organic-based functional building-blocks for brain-machine interfaces.Nell’ingegneria elettronica, la comunicazione di base tra neuroni ha recentemente ispirato lo sviluppo di configurazioni sempre più articolate ed efficienti che imitano il cervello, in cui il flusso di informazioni può essere elaborato con strategie simili a quelle fisiologiche. Ciò è reso possibile grazie all'uso di dispositivi neuromorfici organici, che possono condividere lo stesso mezzo elettrolitico e regolare i pesi delle connessioni reciproche in base alle caratteristiche temporali dei segnali in ingresso. In modo parallelo, gli ingegneri elettronici stanno dirigendo i loro sforzi verso nuovi strumenti per interfacciare il cervello e decifrare le sue strategie di comunicazione. Si è giunti così a diversi progressi tecnologici che consentono agli scienziati di trasdurre l'attività cerebrale e, pezzo per pezzo, di creare una mappa dettagliata delle sue funzioni. Entrambi questi ambiti scientifici, ovvero imitare la comunicazione neurale e trasdurre l'attività cerebrale, possono trarre vantaggio dalla tecnologia dei transistor organici a base elettrolitica (EGOT). I transistor organici a base elettrolitica (EGOT) sono dispositivi elettronici a bassa potenza che integrano funzionalmente l'ambiente elettrolitico attraverso lo sfruttamento di conduttori organici misti ionici-elettronici, i quali consentono di convertire i segnali ionici in segnali elettronici, rendendo tali dispositivi ideali per la neuroelettronica. Gli EGOT sono stati dimostrati con successo sia come trasduttori e amplificatori dell'attività elettrofisiologica e sia come unità neuromorfiche. Tali risultati derivano dal fatto che gli EGOT sono dispositivi attivi, al contrario dell'elettronica passiva (ad esempio gli elettrodi), ma pongono comunque qualche ostacolo alla loro integrazione in ambiente biologico. In quanto transistor, gli EGOT necessitano l'applicazione di due tensioni tra i suoi terminali. Se, da un lato, la presenza di due tensioni diventa un vantaggio per la modulazione della risposta del dispositivo (ad esempio, per l'ideazione di circuiti neuromorfici basati su EGOT), dall'altro può diventare dannosa quando gli EGOT vengono adoperati come sito di registrazione nelle interfacce cerebrali, poiché una tensione non nulla può essere applicata direttamente al cervello. Se tale tensione supera la finestra di stabilità elettrochimica dell'acqua, reazioni faradiche indesiderate possono manifestarsi, le quali potrebbero danneggiare i tessuti e/o il dispositivo. Questo lavoro affronta le applicazioni degli EGOT nella neuroelettronica dalla duplice prospettiva sopra descritta: ingegnerizzazione neuromorfica ed implementazione come interfacce neurali in applicazioni in vivo. Vengono evidenziati i vantaggi dell'utilizzo di architetture a tre terminali per i dispositivi neuromorfici, ottenendo una regolazione reversibile della loro plasticità di risposta. Si discute inoltre la possibilità di ottenere un'unità di memoria multilivello agendo sul potenziale di gate. Viene introdotta una nuova modalità di funzionamento per gli EGOT, che consente di mantenere la capacità di amplificazione e, allo stesso tempo, di evitare l'applicazione di una tensione all’interfaccia cervello-dispositivo. Partendo da queste premesse, viene presentata una nuova serie di array micro-epicorticali ultra-conformabili, che integrano completamente i siti di registrazione EGOT fabbricati in situ su substrati di poliimmide. Infine, viene proposto un circuito organico per l'elaborazione del segnale, sfruttando componenti passivi organici progettati ad hoc e accoppiati a dispositivi EGOT. Questo approccio senza precedenti offre la possibilità di filtrare e scomporre segnali complessi nelle loro componenti di frequenza costitutive in tempo reale, delineando così strategie innovative per concepire blocchi funzionali a base organica per le interfacce cervello-macchina

Bioengineering models of cell signaling

Strategies for rationally manipulating cell behavior in cell-based technologies and molecular therapeutics and understanding effects of environmental agents on physiological systems may be derived from a mechanistic understanding of underlying signaling mechanisms that regulate cell functions. Three crucial attributes of signal transduction necessitate modeling approaches for analyzing these systems: an ever-expanding plethora of signaling molecules and interactions, a highly interconnected biochemical scheme, and concurrent biophysical regulation. Because signal flow is tightly regulated with positive and negative feedbacks and is bidirectional with commands traveling both from outside-in and inside-out, dynamic models that couple biophysical and biochemical elements are required to consider information processing both during transient and steady-state conditions. Unique mathematical frameworks will be needed to obtain an integrated perspective on these complex systems, which operate over wide length and time scales. These may involve a two-level hierarchical approach wherein the overall signaling network is modeled in terms of effective "circuit" or "algorithm" modules, and then each module is correspondingly modeled with more detailed incorporation of its actual underlying biochemical/biophysical molecular interactions

Characterization of response properties in the mouse lateral geniculate nucleus

The lateral geniculate nucleus (LGN) has been increasingly recognized to actively regulate information transmission to primary visual cortex (V1). Although efforts have been devoted to study its morphological and functional features, the full array of response characteristics in mouse LGN as well as their dependency on subjective state have been relatively unexplored. To address the question we recorded from mouse LGN with multisite-electrode-arrays (MEAs). From a dataset with 185 single units, our results revealed several exceptional response features in mouse LGN. We also demonstrated that subtypes, such as ON-/OFF-centre and transient/sustained cells exhibited functionally distinctive features, which might indicate parallel projections. To further compare response features from the full extent of mouse LGN, we developed a three-dimension (3D) LGN volume through histological approach. This volume explicitly captures morphological features of mouse LGN and provides the preciseness to classify location of single neuron into the anterior/middle/posterior LGN. Based on this categorization, we showed that response features were not regionally restricted within mouse LGN. We further examined neural activity with subjects in high or low isoflurane states. The distinct features in LFPs between the two states indicated that adjusting isoflurane concentration could provide a reliable and controllable experimental model to explore the state-dependent neural activity in mouse visual system. Subsequently, our results demonstrated that properties, including response latency, contrast sensitivity and spatial frequency properties were modulated by isoflurane concentration. Our current work suggests that mouse LGN can dynamically regulate information transmission to the cortex using numerous mechanisms, including responding mode, modulation of neuronal responses according to subjects’ states.Open Acces

Distinct roles for innexin gap junctions and hemichannels in mechanosensation.

Mechanosensation is central to a wide range of functions, including tactile and pain perception, hearing, proprioception, and control of blood pressure, but identifying the molecules underlying mechanotransduction has proved challenging. In Caenorhabditis elegans, the avoidance response to gentle body touch is mediated by six touch receptor neurons (TRNs), and is dependent on MEC-4, a DEG/ENaC channel. We show that hemichannels containing the innexin protein UNC-7 are also essential for gentle touch in the TRNs, as well as harsh touch in both the TRNs and the PVD nociceptors. UNC-7 and MEC-4 do not colocalize, suggesting that their roles in mechanosensory transduction are independent. Heterologous expression of unc-7 in touch-insensitive chemosensory neurons confers ectopic touch sensitivity, indicating a specific role for UNC-7 hemichannels in mechanosensation. The unc-7 touch defect can be rescued by the homologous mouse gene Panx1 gene, thus, innexin/pannexin proteins may play broadly conserved roles in neuronal mechanotransduction

Recombinant AAV-mediated Gene Therapy Approaches to Treat Progressive Familial Intrahepatic Cholestasis Type 3

Among contemporary gene transfer vehicles, non-pathogenic recombinant adeno-associated viral vectors (rAAV) show exceptional promise for liver-targeted therapeutic approaches. The broad focus of studies described in this thesis was the development of rAAV-mediated gene therapy to treat Progressive Familial Intrahepatic Cholestasis type 3. This autosomal recessive condition, caused by mutations of ABCB4, results in deficient hepatocanalicular phosphatidylcholine translocation and leads to progressive cholestatic liver disease with approximately 50% of patients requiring liver transplantation before reaching adulthood. Using an Abcb4-knockout mouse model, in vivo liver transduction with rAAV2/8 vectors encoding hABCB4 led to increased biliary phosphatidylcholine in disease-free heterozygous, but not in homozygous adults with established liver disease, despite varying vector genome size and routes of administration. Maximal transduction was achieved prior to onset of liver disease, optimally in neonates. However, loss of transgene expression occurs following neonatal vector delivery, due to rAAV episomal degradation during rapid liver growth. A novel, hybrid rAAV-piggyBac transposon vector strategy was devised to sustain hABCB4 expression in neonatally-treated homozygotes. Successful correction of liver disease was demonstrated in Abcb4-/- mice up to 9 months post-inoculation, with preliminary results indicating reduction in disease-related hepatocarcinogenic risk. These results demonstrate that rAAV-mediated gene therapy has the potential to offer patients with this heritable cholestatic liver disease an effective alternative treatment to liver transplantation, but also illustrate the importance of addressing challenges, such as the impact of liver pathology on vector performance, which is vital before this potential can be realised for this and related conditions

Dynamic mechanostimulation of live cells during real-time microscopy

Our body’s functioning depends on the ability of cells to sense and react to their local mechanical environment; this process is known as mechanotransduction. Despite the importance of understanding how cells interact with mechanical stimuli, the specific mechanisms governing such processes have yet to be elucidated. Using microscopy to detect the early responses of living cells to mechanical loads and forces would be a critical step towards further understanding cellular mechanotransduction. Dynamic and high-frequency cyclical loads are relevant to human physiology and disease. Yet, modern microscopy systems are not capable of delivering the appropriate mechanical stimuli to live cell cultures. To address this deficiency, we developed a suite of mechanostimulation platforms that provide precise and relevant loads and forces to cell cultures during simultaneous microscopic analysis. We developed a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Using this system, we found that vibration of osteoblastic cells does not elicit acute elevation of cytosolic free calcium, but did desensitize responses to later stimulation with extracellular ATP. We next developed and validated a technique for the practical fabrication of microfluidic channels. In contrast to the effect of vibration, osteoblastic cells were found to respond to changes in fluid shear stress with transient elevation in the concentration of cytosolic free calcium. Lastly, we developed a system to apply disturbed fluid flow to live cells during real-time imaging. This system was used to demonstrate changes in the concentration of cytosolic free calcium in human endothelial cells exposed to laminar and disturbed flow. Our findings indicate that different forms of mechanical stimuli activate distinct signaling pathways in cells. Moreover, these new technologies will facilitate investigations of the signaling pathways activated by dynamic mechanical stimulation of a variety of cell types, in particular those of the skeletal and vascular systems

Structural and functional alterations of cortical neurons in Alzheimer’s disease transgenic mice assessed by two-photon in vivo imaging

Alzheimer’s disease (AD), the most common form of dementia, has been proposed to result from the degeneration of synapses, putatively caused by assemblies of the amyloid-β peptide (Aβ). The spatiotemporal dynamics of this synaptopathy, its potential reversibility as well as its consequences on the function of single neurons and neuronal circuits, however, are not fully understood to date. In order to address these questions, I assessed structural and functional alterations of neurons in the neocortex in a transgenic mouse model of Alzheimer’s disease, namely APP/PS1 (APPswe, PS1L166P) mice, using in vivo two-photon imaging. Chronic imaging of dendrites and axons over the course of four weeks revealed not only a reduction in dendritic spine density close to amyloid plaques (proteinaceous extracellular deposits typical of AD), but I also identified synaptic instability as a main aspect contributing to AD pathology. Importantly, while synapse loss was confined to the immediate plaque vicinity (up to 15µm from the histological plaque border), synaptic instability was evident in a much larger region surrounding plaques (50 µm) and affected both, pre- and postsynaptic compartments. As the prevailing hypothesis in AD holds that Aβ conveys these detrimental effects on synapses one therapeutic approach is based on the pharmacological inhibition of Aβ generation. I thus assessed the impact of a novel selective γ-secretase inhibitor (GSI), a compound that prevents the last cleavage step necessary for the release of Aβ from the longer transmembrane amyloid precursor protein (APP). Notably, the GSI used here primarily interferes with the processing of APP and still allows for processing of other γ-secretase substrates, and hence should largely reduce side effects seen with earlier generations of GSIs before. Daily treatment with the GSI reduced the deposition of Aβ as evidenced by the initial reduction in the number of new plaques and a sustained decrease in the growth of these newly deposited plaques. Importantly, it also ameliorated the plaque-associated synaptic instability, without displaying overt adverse effects on dendritic spines in WT mice. These data represent the first in vivo evidence that selective pharmacological inhibition of the γ-secretase mediated APP cleavage can have beneficial effects on synaptic pathology in AD. Given the widespread impact of Aβ assemblies on neuronal structures, I then asked to which extent these structural alterations affect the function of neurons. To address this question, I recorded neuronal response properties in the primary visual cortex of behaving APP/PS1 mice, employing in vivo two-photon calcium imaging using the genetically encoded calcium indicator GCaMP6m. In order to probe the impact of AD related pathology on specific aspects of information processing, which rely on multiple neuronal circuits, I characterized visually driven and motor-related activity, as well as signals based on mismatches between actual and expected visual input. My data reveal a massive reduction in responsiveness under almost all conditions tested, which is line with the profound impact on neuronal structure. Stimulus selectivity, like orientation or direction tuning, were not altered in APP/PS1 mice, indicating that the main effect is caused by a change in response gain. Along with the massive decrease in feedforward signals, I observed an increase in spontaneous, hence uncorrelated neuronal activity in AD transgenic mice. Both features jointly affected the coding accuracy of the network, and I propose that this combination may represent a common characteristic leading to impaired information processing in AD. Surprisingly, I found that responses elicited after a discordance of actual and expected visual flow during running, i.e. a visuomotor mismatch, were selectively spared in APP/PS1 mice, suggesting a particular resilience of this very signal. Together, both studies demonstrate that global widespread structural changes of neurons in the AD brain are accompanied by a severe impact on information processing, most prominently seen in a strong reduction of feedforward signals. My data, thus, provide a correlate of impaired cognition in AD at the level of single neurons and neural circuits

Transient dynamic mechanical properties of resilin-based elastomeric hydrogels

The outstanding high-frequency properties of emerging resilin-like polypeptides (RLPs) have motivated their development for vocal fold tissue regeneration and other applications. Recombinant RLP hydrogels show efficient gelation, tunable mechanical properties, and display excellent extensibility, but little has been reported about their transient mechanical properties. In this manuscript, we describe the transient mechanical behavior of new RLP hydrogels investigated via both sinusoidal oscillatory shear deformation and uniaxial tensile testing. Oscillatory stress relaxation and creep experiments confirm that RLP-based hydrogels display significantly reduced stress relaxation and improved strain recovery compared to PEG-based control hydrogels. Uniaxial tensile testing confirms the negligible hysteresis, reversible elasticity and superior resilience (up to 98%) of hydrated RLP hydrogels, with Young's modulus values that compare favorably with those previously reported for resilin and that mimic the tensile properties of the vocal fold ligament at low strain (<15%). These studies expand our understanding of the properties of these RLP materials under a variety of conditions, and confirm the unique applicability, for mechanically demanding tissue engineering applications, of a range of RLP hydrogels