Physiological role of PRRT2 and its involvement in the pathogenesis of paroxysmal disorders

Mutations in the PRoline-Rich Transmembrane protein 2 gene (PRRT2) underlie a heterogeneous group of paroxysmal disorders encompassing infantile epilepsy, paroxysmal kinesigenic dyskinesia, a combination of these phenotypes and migraine. For the majority of the pathogenic PRRT2 variants, the mutant proteins are not expressed or not correctly targeted to the plasma membrane, resulting in a loss-of function mechanism for PRRT2-related diseases. PRRT2 is a neuron-specific, type II transmembrane protein of 340 amino acids with an important functional role in synapse formation and maintenance, as well as in the regulation of fast neurotransmitter release at both glutamatergic and GABAergic terminals. The PRRT2 knock-out (PRRT2-KO) mouse, in which PRRT2 has been constitutively inactivated, displays alterations in brain structure and a sharp paroxysmal phenotype, reminiscent of the most common clinical manifestations of the human PRRT2-linked diseases. To gain further insights on the pathogenic role of PRRT2 deficiency, I used Multi-Electrode Arrays (MEAs) to characterize neuronal activity generated by primary hippocampal cultures obtained from the PRRT2-KO mouse embryos and to assess the epileptic propensity of cortico-hippocampal slices obtained from the same animal model. This experimental approach revealed a state of heightened spontaneous activity, hyper-synchronization in population bursts of action potentials (APs) and enhanced responsiveness to external stimuli in mutant networks. A complex interplay between (i) a synaptic phenotype, with weakened spontaneous transmission and increased short-term facilitation, and (ii) a marked increase in intrinsic excitability of excitatory neurons as assessed by single-cell electrophysiology, upholds this network phenotype. Furthermore, our group has generated cortical neurons from induced pluripotent stem cells (iPSCs) derived from heterozygous and homozygous siblings carrying the most common C.649dupC mutation. Patch-clamp recordings in neurons from homozygous patients showed an increased Na+ current that was fully rescued by expression of exogenous wild-type PRRT2. A strikingly similar electrophysiological phenotype was observed in excitatory primary cortical neurons from the PRRT2-KO mouse, which was accompanied by an increased length of the axon initial segment (AIS). At the network level, mutant cortical neurons grown on MEAs also displayed a state of spontaneous and evoked hyper-excitability and elevated propensity to synchronize their activity in network bursting events

Deep Brain Stimulation to Increase Generalized Arousal in Intact Mice and a Mouse Model of Traumatic Brain Injury

G protein-coupled receptors (GPCRs) are dynamic membrane proteins that bind extracellular molecules to transduce biological signals. Although GPCRs represent the largest class of targets for therapeutic agents, ligand-binding sites have been precisely defined for only a small percentage of the receptors in the human genome. A general cellbased photocrosslinking approach was developed to investigate the binding interfaces necessary for the formation of GPCR signaling complexes. Amber codon suppression was extended to facilitate the incorporation of photoactivatable unnatural amino acids, pbenzoyl- L-phenylalanine and p-azido-L-phenylalanine, into engineered GPCRs expressed in mammalian cells in culture. Proof-of-concept studies were carried out in chemokine receptors C-X-C chemokine receptor 4 (CXCR4) and C-C chemokine receptor 5 (CCR5), which are known HIV-1 co-receptors required for HIV-1 cellular entry in CD4+ cells. A cyclic peptide CXCR4-specific inhibitor, T140, photocrosslinked primarily to a specific site on CXCR4 and the result was reconciled with existing structural biology data. A small molecule drug, maraviroc, photocrosslinked to multiple sites on CCR5 and the results were extended to develop a computer homology model of the CCR5-maraviroc complex. In summary, the application of a novel targeted cellbased photocrosslinking strategy provided detailed information about receptor-ligand complexes in two chemokine receptors. The general approach described here using genetically-encoded photoreactive molecules to study the binding interactions between GPCRs and ligands represents a significant advance in the application of photocrosslinking reagents to address problems in biochemistry and pharmacology

On temporal scale-free non-periodic stimulation and its mechanisms as an infinite improbability drive of the brain’s functional connectogram

Rationalized development of electrical stimulation (ES) therapy is of paramount importance. Not only it will foster new techniques and technologies with increased levels of safety, efficacy, and efficiency, but it will also facilitate the translation from basic research to clinical practice. For such endeavor, design of new technologies must dialogue with state-of-the-art neuroscientific knowledge. By its turn, neuroscience is transitioning—a movement started a couple of decades earlier—into adopting a new conceptual framework for brain architecture, in which time and thus temporal patterns plays a central role in the neuronal representation of sampled data from the world. This article discusses how neuroscience has evolved to understand the importance of brain rhythms in the overall functional architecture of the nervous system and, consequently, that neuromodulation research should embrace this new conceptual framework. Based on such support, we revisit the literature on standard (fixed-frequency pulsatile stimuli) and mostly non-standard patterns of ES to put forward our own rationale on how temporally complex stimulation schemes may impact neuromodulation strategies. We then proceed to present a low frequency, on average (thus low energy), scale-free temporally randomized ES pattern for the treatment of experimental epilepsy, devised by our group and termed NPS (Non-periodic Stimulation). The approach has been shown to have robust anticonvulsant effects in different animal models of acute and chronic seizures (displaying dysfunctional hyperexcitable tissue), while also preserving neural function. In our understanding, accumulated mechanistic evidence suggests such a beneficial mechanism of action may be due to the natural-like characteristic of a scale-free temporal pattern that may robustly compete with aberrant epileptiform activity for the recruitment of neural circuits. Delivering temporally patterned or random stimuli within specific phases of the underlying oscillations (i.e., those involved in the communication within and across brain regions) could both potentiate and disrupt the formation of neuronal assemblies with random probability. The usage of infinite improbability drive here is obviously a reference to the “The Hitchhiker’s Guide to the Galaxy” comedy science fiction classic, written by Douglas Adams. The parallel is that dynamically driving brain functional connectogram, through neuromodulation, in a manner that would not favor any specific neuronal assembly and/or circuit, could re-stabilize a system that is transitioning to fall under the control of a single attractor. We conclude by discussing future avenues of investigation and their potentially disruptive impact on neurotechnology, with a particular interest in NPS implications in neural plasticity, motor rehabilitation, and its potential for clinical translation

Characterization Of Somatosensation In The Brainstem And The Development Of A Sensory Neuroprosthesis

Innovations in neuroprosthetics have restored sensorimotor function to paralysis patients and amputees. However, to date there is a lack of solutions available to adequately address the needs of spinal cord injury patients (SCI). In this dissertation we develop a novel sensor-brain interface (SBI) that delivers electric microstimulation to the cuneate nucleus (CN) to restore somatosensory feedback in patients with intact limbs. In Chapter II, we develop a fully passive liquid metal antenna using gallium-indium (GaIn) alloy injected in polydimethylsiloxane (PDM) channels to measure forces within the physiological sensitivity of a human fingertip. In Chapter III, we present the first chronic neural interface with the CN in primates to provide access to long-term unit recordings and stimulation. In Chapter IV, we demonstrate that microstimulation to the CN is detectable in a Three Alternative Force Choice Oddity task in awake behaving primates. In Chapter V, we explore the downstream effects of CN stimulation on primary somatosensory cortex, in the context of spontaneous and evoked spindles under sedation. In summary, these findings constitute a proof-of-concept for the sensory half of a bidirectional sensorimotor prosthesis in the CN

Seeing emotions in others: Improving cognitive and neuronal mechanisms of facial expression processing

In the conceptual framework of affective neuroscience, this thesis intends to advance the understanding of the plasticity mechanisms of other’s emotional facial expression representations. Chapter 1 outlines a description of the neurophysiological bases of Hebbian plasticity, reviews influential studies that adopted paired associative stimulation procedures, and introduces new lines of research where the impact of cortico-cortical paired associative stimulation protocols on higher order cognitive functions is investigated. The experiments in Chapter 2 aimed to test the modulatory influence of a perceptual-motor training, based on the execution of emotional expressions, on the subsequent emotion intensity judgements of others’ high (i.e., full visible) and low-intensity (i.e., masked) emotional expressions. As a result of the training-induced learning, participants showed a significant congruence effect, as indicated by relatively higher expression intensity ratings for the same emotion as the one that was previously trained. Interestingly, although judged as overall less emotionally intense, surgical facemasks did not prevent the emotion-specific effects of the training to occur, suggesting that covering the lower part of other’s face do not interact with the training-induced congruence effect. In Chapter 3 it was implemented a transcranial magnetic stimulation study targeting neural pathways involving re-entrant input from higher order brain regions into lower levels of the visual processing hierarchy. We focused on cortical visual networks within the temporo-occipital stream underpinning the processing of emotional faces and susceptible to plastic adaptations. Importantly, we tested the plasticity-induced effects in a state dependent manner, by administering ccPAS while presenting different facial expressions yet afferent to a specific emotion. Results indicated that the discrimination accuracy of emotion-specific expressions is enhanced following the ccPAS treatment, suggesting that a multi-coil TMS intervention might represent a suitable tool to drive brain remodeling at a neural network level, and consequently influence a specific behavior

Cerebellar Codings for Control of Compensatory Eye Movements

This thesis focuses on the control of the cerebellum on motor behaviour, and more specifically on the role of the cerebellar Purkinje cells in exerting this control. As the cerebellum is an online control system, we look at both motor performance and learning, trying to identify components involved at the molecular, cellular and network level. To study the cerebellum we used the vestibulocerebellum, with visual and vestibular stimulation as input and eye movements as recorded output. The advantage of the vestibulocerebellum over other parts is that the input given is highly controllable, while the output can be reliably measured, and performance and learning can be easily studied. In addition, we conducted electrophysiological recordings from the vestibulocerebellum, in particular of Purkinje cells in the flocculus. Combining the spiking behaviour of Purkinje cells with visual input and eye movement output allowed us to study how the cerebellum functions and using genetically modified animals we could determine the role of different elements in this system. To provide some insights in the techniques used and the theory behind them, we will discuss the following topics in this introduction: compensatory eye movements, the anatomy of pathways to, within and out of the flocculus, the cellular physiology of Purkinje cells in relation to performance and the plasticity mechanisms related to motor learning

Contextual signals in visual cortex:How sounds, state, and task setting shape how we see

What we see is not always what we get. Even though the light that hits the retina might convey the same images, how visual information is processed and what we eventually do with it depend on many contextual factors. In this thesis, we show in a series of experiments how the sensory processing of the same visual input in the visual cortex of mice is affected by our internal state, movements, other senses and any task we are performing. We found that recurrent activity originating within higher visual areas modulates activity in the primary visual cortex (V1) and selectivity amplifies weak compared to strong sensory-evoked responses. Second, visual stimuli evoked similar early activity in V1, but later activity strongly depended on whether mice were trained to report the visual stimuli, and on the specific task. Specifically, adding a second modality to the task demands extended the temporal window during which V1 was causally involved in visual perception. Third, we report that not only visual stimuli but also sounds led to strong responses in V1, composed of distinct auditory-related and motor-related activity. Finally, we studied the role of Posterior Parietal Cortex in an audiovisual change detection task. Despite extensive single-neuron and population-level encoding of task-relevant visual and auditory stimuli, as well as upcoming behavioral responses, optogenetic inactivation did not affect task performance. Whereas these contextual factors have previously been studied in isolation, we obtain a more integrated understanding of how factors beyond visual information determine what we actually see