Dissection of Affective Catecholamine Circuits Using Traditional and Wireless Optogenetics

Parsing the complexity of the mammalian brain has challenged neuroscientists for thousands of years. In the early 21st century, advances in materials science and neuroscience have enabled unprecedented control of neural circuitry. In particular, cell-type selective manipulations, such as those with optogenetics and chemogenetics, routinely provide answers to previously intractable neurobiological questions in the intact, behaving animal. In this two-part dissertation, I first introduce new minimally invasive, wireless technology to perturb neural activity in the ventral tegmental area dopaminergic system of freely moving animals. I report a series of novel devices for studying and perturbing intact neural systems through optogenetics, microfluidic pharmacology, and electrophysiology. Unlike optogenetic approaches that rely on rigid, glass fiber optics coupled to external light sources, these novel devices utilize flexible substrates to carry microscale, inorganic light emitting diodes (μ-ILEDs), multimodal sensors, and/or microfluidic channels into the brain. Each class of device can be wirelessly controlled, enabling studies in freely behaving mice and achieving previously untenable control of catecholamine neural circuitry. In the second part of this dissertation, I apply existing cell-type selective approaches to dissect the role of the locus coeruleus noradrenergic (LC-NE) system in anxiety-like and aversive behaviors. The LC-NE system is one of the first systems engaged following a stressful event. While LC-NE neurons are known to be activated by many different stressors, the underlying neural circuitry and the role of this activity in generating stress-induced anxiety has not been elucidated until now. I demonstrate that increased tonic activity of LC-NE neurons is both necessary and sufficient for stress-induced anxiety; a behavior which is driven by LC projections to the basolateral amygdala. Furthermore, this activity and behavior is elicited by corticotropin releasing hormone-containing afferent inputs into the LC from the central amygdala. These studies position the LC-NE system as a critical mediator of acute stress-induced anxiety and offer a potential intervention for preventing stress-related affective disorders. Together these two objectives provide a rich technological toolbox for neuroscientists and yield important knowledge of how small catecholamine structures with widespread forebrain innervation can selectively mediate higher order behaviors

Closed-loop approaches for innovative neuroprostheses

The goal of this thesis is to study new ways to interact with the nervous system in case of damage or pathology. In particular, I focused my effort towards the development of innovative, closed-loop stimulation protocols in various scenarios: in vitro, ex vivo, in vivo

Photogenetic Retinal Prosthesis

The last few decades have witnessed an immense effort to develop working retinal implants for patients suffering from retinal degeneration diseases such as retinitis pigmentosa. However, it is becoming apparent that this approach is unable to restore levels of vision that will be sufficient to offer significant improvement in the quality of life of patients. Herein, a new type of retinal prosthesis that is based on genetic expression of microbial light sensitive ion channel, Chanelrhodopsin-2 (ChR2), and a remote light stimulation is examined. First, the dynamics of the ChR2 stimulation is characterized and it is shown that (1) the temporal resolution of ChR2-evoked spiking is limited by a continuous drop in its depolarization efficiency that is due to (a) frequency-independent desensitization process and (b) slow photocurrent shutting, which leads to a frequency-dependent post-spike depolarization and (2) the ChR2 response to light can be accurately reproduced by a four-state model consisting of two interconnected branches of open and close states. Then, a stimulation prototype is developed and its functionality is demonstrated in-vitro. The prototype uses a new micro-emissive matrix which enables generating of two-dimensional stimulation patterns with enhanced resolution compared to the conventional retinal implants. Finally, based on the micro-emitters matrix, a new technique for sub-cellular and network-level neuroscience experimentations is shown. The capacity to excite sub-cellular compartments is demonstrated and an example utility to fast map variability in dendrites conductance is shown. The outcomes of this thesis present an outline and a first proof-of-concept for a future photogenetic retinal prosthesis. In addition, they provide the emerging optogenetic technology with a detailed analysis of its temporal resolution and a tool to expand its spatial resolution, which can have immediate high impact applications in modulating the activity of sub-cellular compartments, mapping neuronal networks and studying synchrony and plasticity effects

Low-frequency local field potentials in primate motor cortex and their application to neural interfaces

PhD ThesisFor patients with spinal cord injury and paralysis, there are currently very limited options for clinical therapy. Brain-machine interfaces (BMIs) are neuroprosthetic devices that are being developed to record from the motor cortex in such patients, bypass the spinal lesion, and use decoded signals to control an effector, such as a prosthetic limb. The ideal BMI would be durable, reliable, totally predictable, fully-implantable, and have generous battery life. Current, state-of-the-art BMIs are limited in all of these domains; partly because the typical signals used—neuronal action potentials, or ‘spikes’—are very susceptible to micro-movement of recording electrodes. Recording spikes from the same neurons over many months is therefore difficult, and decoder behaviour may be unpredictable from day-today. Spikes also need to be digitized at high frequencies (~104 Hz) and heavily processed. As a result, devices are energy-hungry and difficult to miniaturise. Low-frequency local field potentials (lf-LFPs; < 5 Hz) are an alternative cortical signal. They are more stable and can be captured and processed at much lower frequencies (~101 Hz). Here we investigate rhythmical lf-LFP activity, related to the firing of local cortical neurons, during isometric wrist movements in Rhesus macaques. Multichannel spike-related slow potentials (SRSPs) can be used to accurately decode the firing rates of individual motor cortical neurons, and subjects can control a BMI task using this synthetic signal, as if they were controlling the actual firing rate. Lf-LFP–based firing rate estimates are stable over time – even once actual spike recordings have been lost. Furthermore, the dynamics of lf-LFPs are distinctive enough, that an unsupervised approach can be used to train a decoder to extract movement-related features for use in biofeedback BMIs. Novel electrode designs may help us optimise the recording of these signals, and facilitate progress towards a new generation of robust, implantable BMIs for patients.Research Studentship from the MRC, and Andy Jackson’s laboratory (hence this work) is supported by the Wellcome Trust

Modern Telemetry

Telemetry is based on knowledge of various disciplines like Electronics, Measurement, Control and Communication along with their combination. This fact leads to a need of studying and understanding of these principles before the usage of Telemetry on selected problem solving. Spending time is however many times returned in form of obtained data or knowledge which telemetry system can provide. Usage of telemetry can be found in many areas from military through biomedical to real medical applications. Modern way to create a wireless sensors remotely connected to central system with artificial intelligence provide many new, sometimes unusual ways to get a knowledge about remote objects behaviour. This book is intended to present some new up to date accesses to telemetry problems solving by use of new sensors conceptions, new wireless transfer or communication techniques, data collection or processing techniques as well as several real use case scenarios describing model examples. Most of book chapters deals with many real cases of telemetry issues which can be used as a cookbooks for your own telemetry related problems

Use of functional neuroimaging and optogenetics to explore deep brain stimulation targets for the treatment of Parkinson's disease and epilepsy

Deep brain stimulation (DBS) is a neurosurgical therapy for Parkinson’s disease and epilepsy. In DBS, an electrode is stereotactically implanted in a specific region of the brain and electrical pulses are delivered using a subcutaneous pacemaker-like stimulator. DBS-therapy has proven to effectively suppress tremor or seizures in pharmaco-resistant Parkinson’s disease and epilepsy patients respectively. It is most commonly applied in the subthalamic nucleus for Parkinson’s disease, or in the anterior thalamic nucleus for epilepsy. Despite the rapidly growing use of DBS at these classic brain structures, there are still non-responders to the treatment. This creates a need to explore other brain structures as potential DBS-targets. However, research in patients is restricted mainly because of ethical reasons. Therefore, in order to search for potential new DBS targets, animal research is indispensable. Previous animal studies of DBS-relevant circuitry largely relied on electrophysiological recordings at predefined brain areas with assumed relevance to DBS therapy. Due to their inherent regional biases, such experimental techniques prevent the identification of less recognized brain structures that might be suitable DBS targets. Therefore, functional neuroimaging techniques, such as functional Magnetic Resonance Imaging and Positron Emission Tomography, were used in this thesis because they allow to visualize and to analyze the whole brain during DBS. Additionally, optogenetics, a new technique that uses light instead of electricity, was employed to manipulate brain cells with unprecedented selectivity

Information transmission in normal vision and optogenetically resensitised dystrophic retinas

Phd ThesisThe retina is a sophisticated image processing machine, transforming the visual scene as detected by the photoreceptors into a pattern of action potentials that is sent to the brain by the retinal ganglion cells (RGCs), where it is further processed to help us understand and navigate the world. Understanding this encoding process is important on a number of levels. First, it informs the study of upstream visual processing by elucidating the signals higher visual areas receive as input and how they relate to the outside world. Second, it is important for the development of treatments for retinal blindness, such as retinal prosthetics. In this thesis, I present work using multielectrode array (MEA) recordings of RGC populations from ex-vivo retinal wholemounts to study various aspects of retinal information processing. My results fall into two main themes. In the rst part, in collaboration with Dr Geo rey Portelli and Dr Pierre Kornprobst of INRIA, I use ashed gratings of varying spatial frequency and phase to compare di erent coding strategies that the retina might use. These results show that information is encoded synergistically by pairs of neurons and that, of the codes tested, a Rank Order Code based on the relative order of ring of the rst spikes of a population of neurons following a stimulus provides information about the stimulus faster and more e ciently than other codes. In the later parts, I use optogenetic stimulation of RGCs in congenitally blind retinas to study how visual information is corrupted by the spontaneous hyperactivity that arises as a result of photoreceptor degeneration. I show that by dampening this activity with the gap junction blocker meclofenamic acid, I can improve the signal-to-noise ratio, spatial acuity and contrast sensitivity of prosthetically evoked responses. Taken together, this work provides important insights for the future development of retinal prostheses

Reconsolidation of appetitive memory and sleep: functional connectomics and plasticity

Introduzione: La dipendenza da cibo \ue8 un disturbo comportamentale caratterizzato da modelli maladattativi di consumo alimentare, in cui alimenti ricchi in zuccheri, sale e/o grassi possono indurre una dipendenza tale da essere paragonata ai disturbi relativi all\u2019abuso di sostanze. Alla base di questo processo vi \ue8 l\u2019associazione tra questi cibi altamente palatabili e la sensazione piacevole e rinforzante indotta dal loro consumo, che pu\uf2 essere codificata in una nuova memoria maladattativa sottostante il disturbo di dipendenza. Infatti, le nuove informazioni che riceviamo quotidianamente dall\u2019esterno vengono processate dal nostro cervello tramite un primo stadio di codifica e un secondo stadio di consolidamento, durante il quale vengono stabilizzate in una nuova memoria e integrate nella rete cerebrale di conoscenze preesistenti. Tuttavia, dopo il suo consolidamento, una memoria pu\uf2 essere destabilizzata e riportata ad uno stato di labilit\ue0 che ne permette la modifica e l\u2019eventuale integrazione con nuove informazioni. Infine, un nuovo processo di stabilizzazione chiamato riconsolidamento \ue8 necessario affinch\ue9 la traccia mnemonica aggiornata sia nuovamente stabilizzata. Da recenti studi, \ue8 noto come il sonno sia rilevante sia per il consolidamento che per il riconsolidamento della memoria. Tuttavia, mentre \ue8 chiaro come il consolidamento che avviene durante il sonno permetta la stabilizzazione a lungo termine delle tracce mnemoniche, non \ue8 ancora stato del tutto chiarito il ruolo del sonno nel processo di riconsolidamento. Scopo: Date queste premesse, e nota l\u2019importanza dell\u2019interazione tra amigdala basolaterale (BLA) e ippocampo nel riconsolidamento delle memorie appetitive, gli obiettivi della presente tesi erano: i) valutare come l\u2019amigdala BLA e ippocampo interagiscono in termini di potenziali locali durante la riattivazione della memoria strumentale effettuata o durante la fase di attivit\ue0 o durante la fase di inattivit\ue0 del ciclo circadiano dei roditori; ii) valutare come il richiamo della memoria effettuato durante la fase attiva o quella inattiva possa influenzare il successivo processo di riconsolidamento, e iii) trovare, in-vivo, un marker di riattivazione della memoria appetitiva. Metodi: Sono stati utilizzati 32 ratti maschi, ceppo Sprague Dawley, a cui sono stati impiantati due elettrodi profondi: uno in BLA e uno in ippocampo, per la registrazione dei potenziali locali. I ratti sono stati sottoposti ad un protocollo comportamentale in gabbia operante di auto-somministrazione di saccarosio, composto da quattro stadi: addestramento (i), in cui i ratti imparavano l\u2019associazione tra la pressione di una leva e l\u2019emissione di un pellet di saccarosio; astinenza (ii), durante la quale i soggetti non venivano esposti al contesto di addestramento; riattivazione o non riattivazione (iii) della memoria strumentale in gabbia operante, svolta o durante la fase di attivit\ue0, o durante la fase di inattivit\ue0; test di ricaduta (iv). I potenziali locali sono stati analizzati per lo stadio (iii) in modo da ottenere la potenza delle oscillazioni theta e gamma per i due elettrodi profondi; tali frequenze sono state scelte in quanto rilevanti per i processi mnemonici. Infatti, il richiamo della memoria \ue8 correlato alla sincronizzazione delle onde theta (4-12 Hz) tra BLA e altre aree cerebrali quali l\u2019area CA1 dell\u2019ippocampo, ed \ue8 inoltre correlato alle basse gamma (30-60 Hz) nell\u2019ippocampo. Infine, l\u2019accoppiamento tra le onde theta e gamma nell\u2019ippocampo \ue8 un noto metodo di comunicazione tra sotto-aree ippocampali nel corso dei processi di memoria. Risultati: I risultati hanno mostrato la presenza di una correlazione inversa tra la potenza delle basse gamma nell\u2019area CA1 ippocampale e il tasso di risposta durante lo stadio di richiamo della memoria nella fase di attivit\ue0, indipendentemente dal fatto che i soggetti stessero o meno premendo la leva. Le basse gamma potrebbero quindi rappresentare un marker di correlazione per il richiamo della memoria appetitiva. Inoltre, la potenza di basse e alte gamma ippocampali aumenta durante le epoche di pressione di leva quando il richiamo della memoria viene effettuato nella fase di inattivit\ue0, suggerendo che le onde gamma potrebbero essere dei marker correlazionali specifici per la componente strumentale del richiamo della memoria effettuato durante la fase di inattivit\ue0. Conclusioni e limitazioni: Per concludere, i risultati hanno mostrato l\u2019importanza delle frequenze basse gamma nel richiamo delle memorie appetitive, tuttavia non hanno mostrato alcuna differenza a livello delle onde theta, n\ue9 a livello della BLA. Di conseguenza, si conclude che il protocollo utilizzato nella presente tesi non ha mostrato una sensibilit\ue0 sufficientemente elevata nell\u2019evidenziare i cambiamenti ipotizzati a livello dei potenziali locali. Lo svolgimento di ulteriori esperimenti che andranno a determinare misure di connettomica quali coerenza e accoppiamento, sia intra- che inter- area, aiuter\ue0 a determinare se e come le due aree comunicano tra di loro.Introduction: Food addiction is a behavioural disorder in which individuals develop maladaptive patterns of food consumption. Particularly, food containing processed sugars, salt, fat etc. can be addictive, and refined food consumption behaviours may meet the criteria for substance use disorders. For these characteristics, food addiction can also be considered a memory disorder. Memories in the brain are processed as follows: new information is encoded and then long-term consolidated through a process allowing its integration into already existing knowledge networks. After a memory has been consolidated, it can be destabilized and brought back to a labile state, requiring a new re-stabilization process called reconsolidation. Memory consolidation is known to require sleep. In fact, sleep allows new memory traces to long-term stabilize. Sleep also seems to influence memory reconsolidation; however, its involvement in this process is not yet clear. Aim: Given these premises, the goals of the project were: to evaluate how basolateral amygdala (BLA) and hippocampus interact in terms of local field potentials (LFPs) when appetitive instrumental memory is retrieved either during active or inactive phase of rats circadian rhythm; to evaluate how retrieving the memory in the activity vs inactivity phase influences following memory reconsolidation; and to find an in vivo electrophysiological marker of appetitive memory retrieval. In fact, it has been shown that BLA and dorsal hippocampus interaction is crucial for appetitive memory reconsolidation. Methods: Thirty-two male Sprague Dawley rats were implanted with in-depth electrodes for LFPs recordings in BLA and dorsal hippocampal CA1 and subject to a behavioural protocol apt to induce appetitive memory retrieval. The behavioural procedure consisted of four stages: training (i), in which animals learned lever pressing \u2013 sucrose reward association; abstinence (ii), during which subjects were not exposed to the training context; memory retrieval or no retrieval (iii): instrumental memory reactivation or no reactivation, performed either during active or inactive phase; and relapse test (iv), during which sucrose-seeking behaviour was analysed. Theta and gamma oscillations powers were analysed during stage (iii). In fact, they are known to be involved in memory processes. Memory retrieval has been shown to correlate with theta (4-12 Hz) synchronization between BLA and other brain areas (such as hippocampal CA1) and with low gamma (30-60 Hz) in hippocampus. Particularly, theta-gamma cross-frequency coupling has been shown to be used as a mean of communication between hippocampal sub-areas during memory processing. Results: Results showed an inverse correlation between hippocampal CA1 low gamma power and reactivation rate of responding (either when rats were lever pressing or not) when reactivation was performed during the active phase. This suggests that low gamma may be a correlational marker of instrumental sucrose memory retrieval, independent of whether rats were lever-pressing or not. Moreover, hippocampal CA1 gamma bands increased when lever pressing during instrumental memory reactivation while in the inactive phase, suggesting that both low and high gamma bands may be correlational markers to actual instrumental responding retrieval during the inactive phase. Conclusions and limitations: In conclusion, results showed that low gamma is relevant in sucrose appetitive memory retrieval. However, no difference was observed in the theta frequency band, nor at the level of BLA. Therefore, the current protocol did not have the sensitivity to detect predicted changes in LFPs. Further experiments would help investigating if and how the two areas interact, by determining connectomics measures such as coherence and coupling within and between areas