Cellular thalamic correlates of the slow (< 1hz) sleep rhythm

Sleep and wakefulness form an inherent, biological rhythm that defines our daily lives. Despite the fact that sleep is a constant interruption to the waking state, its purpose and the neural processes occurring during this behavioural state are not fully understood. However, it is now well established that sleep is not a period of the brain 'silence'. During the transition form light to deep sleep, the activity of the corticothalamic network becomes globally synchronised into consistent, characteristic rhythmic activities at <1Hz, quite in contrast to the so-called cortical 'desynchronisation' characterising states of brain alertness. The mechanism by which global synchronisation in the corticothalamic network arises is not fully defined and previous investigation has focused principally on the role of the cortex. However a clear understanding of the activities of thalamic, as well as cortical, neurones during sleep will aid our understanding into how, and why, global synchronisation occurs, and perhaps why sleep is so fundamental to life. In this thesis, I demonstrate a number of novel activities in two types of thalamic neurones recorded in vitro. Firstly, in thalamocortical neurones, the principal cell type and thalamic output neurones, I demonstrate the presence of an mGluRIa dependent slow (<1Hz) oscillation with identical properties to that seen in the intact brain during sleep. Thalamocortical neurones in relay nuclei subserving visual, somatosensory, auditory and motor systems displayed the slow (<1Hz) oscillation suggesting it could be the substrate for global thalamic synchronization at <1Hz. In addition, I provide a full characterisation of the cellular mechanism of this <1Hz oscillatory activity and demonstrate that during mGluRIa activation, the window component of the low-voltage activated Ca2+ current is unmasked, due to a reduction in the constitutive K+ leak current, inducing bistability-mediated activities that underlies the generation of the slow (<1Hz) oscillation. In neurones of the nucleus reticularis thalami, overlying the thalamus and providing an inhibitory drive to thalamocortical neurones, I also demonstrate a slow (<1Hz) oscillation, again with identical properties as seen in this cell type in the intact brain during sleep. I demonstrate that this slow (<1Hz) oscillation is dependent on mGluRIa activation and provide evidence suggesting that it is generated by a bistability-mediated mechanism as occurs in thalamocortical neurones. In light of these findings, I suggests that the thalamus, has a significant role in aiding, as well as maintaining, the global synchronisation of the corticothalamic network at <1Hz during the transition to, and during sleep. The ability of thalamic neurones to generate rhythmic activities at <1Hz due to cortical mGluRIa activation, that results simply in a reduction of the K+ leak current, will provide a strong excitatory drive to organise cortical activity at <1 Hz. A further novel observation was the presence of spikelets and burstlets (compounds of spikelets) in thalamocortical neurones. Investigations into the origin of these events indicated that they were electrophysiological manifestations of interneuronal electrotonic coupling. Furthermore, spikelets and burstlets had the ability to entrain the output of the neurones in which they were observed. Therefore, the presence of electrotonic coupling in thalamic neurones may have a hitherto unrealised role in the synchronization of thalamic activity during both sleep and awake states.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

Cellular thalamic correlates of the slow (<1Hz) sleep rhythm

Sleep and wakefulness form an inherent, biological rhythm that defines our daily lives. Despite the fact that sleep is a constant interruption to the waking state, its purpose and the neural processes occurring during this behavioural state are not fully understood. However, it is now well established that sleep is not a period of the brain 'silence'. During the transition form light to deep sleep, the activity of the corticothalamic network becomes globally synchronised into consistent, characteristic rhythmic activities at <1Hz, quite in contrast to the so-called cortical 'desynchronisation' characterising states of brain alertness. The mechanism by which global synchronisation in the corticothalamic network arises is not fully defined and previous investigation has focused principally on the role of the cortex. However a clear understanding of the activities of thalamic, as well as cortical, neurones during sleep will aid our understanding into how, and why, global synchronisation occurs, and perhaps why sleep is so fundamental to life. In this thesis, I demonstrate a number of novel activities in two types of thalamic neurones recorded in vitro. Firstly, in thalamocortical neurones, the principal cell type and thalamic output neurones, I demonstrate the presence of an mGluRIa dependent slow (<1Hz) oscillation with identical properties to that seen in the intact brain during sleep. Thalamocortical neurones in relay nuclei subserving visual, somatosensory, auditory and motor systems displayed the slow (<1Hz) oscillation suggesting it could be the substrate for global thalamic synchronization at <1Hz. In addition, I provide a full characterisation of the cellular mechanism of this <1Hz oscillatory activity and demonstrate that during mGluRIa activation, the window component of the low-voltage activated Ca2+ current is unmasked, due to a reduction in the constitutive K+ leak current, inducing bistability-mediated activities that underlies the generation of the slow (<1Hz) oscillation. In neurones of the nucleus reticularis thalami, overlying the thalamus and providing an inhibitory drive to thalamocortical neurones, I also demonstrate a slow (<1Hz) oscillation, again with identical properties as seen in this cell type in the intact brain during sleep. I demonstrate that this slow (<1Hz) oscillation is dependent on mGluRIa activation and provide evidence suggesting that it is generated by a bistability-mediated mechanism as occurs in thalamocortical neurones. In light of these findings, I suggests that the thalamus, has a significant role in aiding, as well as maintaining, the global synchronisation of the corticothalamic network at <1Hz during the transition to, and during sleep. The ability of thalamic neurones to generate rhythmic activities at <1Hz due to cortical mGluRIa activation, that results simply in a reduction of the K+ leak current, will provide a strong excitatory drive to organise cortical activity at <1 Hz. A further novel observation was the presence of spikelets and burstlets (compounds of spikelets) in thalamocortical neurones. Investigations into the origin of these events indicated that they were electrophysiological manifestations of interneuronal electrotonic coupling. Furthermore, spikelets and burstlets had the ability to entrain the output of the neurones in which they were observed. Therefore, the presence of electrotonic coupling in thalamic neurones may have a hitherto unrealised role in the synchronization of thalamic activity during both sleep and awake states

Rôle des astrocytes dans la décharge rythmique neuronale du noyau sensoriel principal du trijumeau

La communication entre les neurones est fondée sur leur capacité à changer leur patron de décharge pour l’encodage de différents messages. Pour plusieurs fonctions vitales, comme la respiration et la mastication, les neurones doivent pouvoir générer des patrons d’activité répétitifs, et les groupes de neurones responsables de ces décharges rythmiques sont des générateurs de patron central (GPC). En dépit de recherches soutenues, les mécanismes précis qui sous-tendent la rythmogénèse dans les GPCs ne sont pas bien définis. Le plus souvent, la potentielle contribution des astrocytes demeure grandement inexplorée, même si ces cellules sont aujourd’hui connues pour leur implication dans la modulation synaptique neuronale. Pour nos travaux, le noyau sensoriel principal du trijumeau (NVsnpr) a été pris comme modèle à cause de son rôle central dans les mouvements rythmiques de la mastication. Dans ce noyau, des travaux antérieurs ont montré que la décharge en bouffées rythmiques est déclenchée dans les neurones lorsque la concentration de calcium extracellulaire ([Ca2+]e) est artificiellement baissée. Nous fondant sur cette observation, notre première hypothèse a postulé que la baisse de la [Ca2+]e pouvait survenir de façon physiologique en lien avec des stimulations sensorielles pertinentes. Deuxièmement, parce que les astrocytes ont été impliqués dans le tamponnage et l’homéostasie d’ions extracellulaires comme le K+, nous avons postulé que ces cellules pouvaient jouer un rôle équivalent dans le contrôle de la [Ca2+]e. Nos résultats montrent que les astrocytes peuvent réguler la [Ca2+]e et ainsi contrôler la capacité des neurones à changer leur patron de décharge. Premièrement, en stimulant les afférences sensorielles au NVsnpr, nous avons montré que des baisses physiologiques de la [Ca2+]e sont observées en parallèle à l’apparition de bouffées rythmiques neuronales. Deuxièmement, nous avons démontré que les astrocytes répondent aux mêmes stimuli qui induisent l’activité rythmique neuronale, et que leur blocage avec un chélateur de Ca2+ empêche les neurones de générer un patron de décharge en bouffées rythmiques. Cette habilité est rétablie en rajoutant la S100β, une protéine astrocytaire liant le Ca2+, dans le milieu extracellulaire, alors que l’anticorps anti-S100β empêche l’activité rythmique. Ces résultats indiquent que les astrocytes régulent une propriété neuronale fondamentale : la capacité à changer de patron de décharge. Ainsi, les GPCs dépendraient des fonctions intégrées des astrocytes et des neurones. Ces découvertes pourraient avoir des implications transposables à plusieurs autres circuits neuronaux dont la fonction dépend de l’induction d’activité rythmique.Communication between neurons rests on their capacity to change their firing pattern to encode different messages. For several vital functions, such as respiration and mastication, neurons need to generate a repetitive firing pattern, and the groups of neurons responsible for these rhythmic discharges are called central pattern generator (CPG). Despite intense research in this field, the exact mechanisms underlying rhythmogenesis in CPGs are not completely defined. In most instances, the potential contribution of astrocytes is largely unexplored, even though these cells are now well known to be involved in neuronal synaptic modulation. In our work, the trigeminal main sensory nucleus (NVsnpr) was used as a model owing to its central role in the rhythmic movement of mastication. Previous work have shown that rhythmic bursting discharge is triggered in NVsnpr neurons when extracellular calcium concentration ([Ca2+]e) is artificially decreased. Based on this observation, our first hypothesis postulated that the reduction of [Ca2+]e could also happen physiologically in relation to relevant sensory stimulation. Secondly, because astrocytes have been involved in the buffering and the homeostasis of extracellular ions like potassium, we have postulated that these cells could also play a role in the control of [Ca2+]e. The results presented in this thesis show that astrocytes can regulate [Ca2+]e and thus control the ability of neurons to change their firing pattern. First, we showed that stimulation of sensory afferent fibers to the NVsnpr induced neuronal rhythmic bursting and in parallel reduction of [Ca2+]e . Secondly, we have demonstrated that astrocytes respond to the same sensory stimuli that induce neuronal rhythmic activity, and their blockade with a Ca2+ chelator prevents generation of neuronal rhythmic bursting. This ability is restored by adding S100β, an astrocytic Ca2+-binding protein, to the extracellular space, while the application of an anti- S100β antibody prevents generation of rhythmic activity. These results indicate that astrocytes regulate a fundamental neuronal property: that is the capacity to change their firing pattern. Thus, CPG functions result from integrated neuronal and glial activities. These findings may have broad implications for many other neural networks whose functions depend on the generation of rhythmic activity

Linear and nonlinear approaches to unravel dynamics and connectivity in neuronal cultures

[eng] In the present thesis, we propose to explore neuronal circuits at the mesoscale, an approach in which one monitors small populations of few thousand neurons and concentrates in the emergence of collective behavior. In our case, we carried out such an exploration both experimentally and numerically, and by adopting an analysis perspective centered on time series analysis and dynamical systems. Experimentally, we used neuronal cultures and prepared more than 200 of them, which were monitored using fluorescence calcium imaging. By adjusting the experimental conditions, we could set two basic arrangements of neurons, namely homogeneous and aggregated. In the experiments, we carried out two major explorations, namely development and disintegration. In the former we investigated changes in network behavior as it matured; in the latter we applied a drug that reduced neuronal interconnectivity. All the subsequent analyses and modeling along the thesis are based on these experimental data. Numerically, the thesis comprised two aspects. The first one was oriented towards a simulation of neuronal connectivity and dynamics. The second one was oriented towards the development of linear and nonlinear analysis tools to unravel dynamic and connectivity aspects of the measured experimental networks. For the first aspect, we developed a sophisticated software package to simulate single neuronal dynamics using a quadratic integrate–and–fire model with adaptation and depression. This model was plug into a synthetic graph in which the nodes of the network are neurons, and the edges connections. The graph was created using spatial embedding and realistic biology. We carried out hundreds of simulations in which we tuned the density of neurons, their spatial arrangement and the characteristics of the fluorescence signal. As a key result, we observed that homogeneous networks required a substantial number of neurons to fire and exhibit collective dynamics, and that the presence of aggregation significantly reduced the number of required neurons. For the second aspect, data analysis, we analyzed experiments and simulations to tackle three major aspects: network dynamics reconstruction using linear descriptions, dynamics reconstruction using nonlinear descriptors, and the assessment of neuronal connectivity from solely activity data. For the linear study, we analyzed all experiments using the power spectrum density (PSD), and observed that it was sufficiently good to describe the development of the network or its disintegration. PSD also allowed us to distinguish between healthy and unhealthy networks, and revealed dynamical heterogeneities across the network. For the nonlinear study, we used techniques in the context of recurrence plots. We first characterized the embedding dimension m and the time delay δ for each experiment, built the respective recurrence plots, and extracted key information of the dynamics of the system through different descriptors. Experimental results were contrasted with numerical simulations. After analyzing about 400 time series, we concluded that the degree of dynamical complexity in neuronal cultures changes both during development and disintegration. We also observed that the healthier the culture, the higher its dynamic complexity. Finally, for the reconstruction study, we first used numerical simulations to determine the best measure of ‘statistical interdependence’ among any two neurons, and took Generalized Transfer Entropy. We then analyzed the experimental data. We concluded that young cultures have a weak connectivity that increases along maturation. Aggregation increases average connectivity, and more interesting, also the assortativity, i.e. the tendency of highly connected nodes to connect with other highly connected node. In turn, this assortativity may delineates important aspects of the dynamics of the network. Overall, the results show that spatial arrangement and neuronal dynamics are able to shape a very rich repertoire of dynamical states of varying complexity.[cat] L’habilitat dels teixits neuronals de processar i transmetre informació de forma eficient depèn de les propietats dinàmiques intrínseques de les neurones i de la connectivitat entre elles. La present tesi proposa explorar diferents tècniques experimentals i de simulació per analitzar la dinàmica i connectivitat de xarxes neuronals corticals de rata embrionària. Experimentalment, la gravació de l’activitat espontània d’una població de neurones en cultiu, mitjançant una càmera ràpida i tècniques de fluorescència, possibilita el seguiment de forma controlada de l’activitat individual de cada neurona, així com la modificació de la seva connectivitat. En conjunt, aquestes eines permeten estudiar el comportament col.lectiu emergent de la població neuronal. Amb l’objectiu de simular els patrons observats en el laboratori, hem implementat un model mètric aleatori de creixement neuronal per simular la xarxa física de connexions entre neurones, i un model quadràtic d’integració i dispar amb adaptació i depressió per modelar l’ampli espectre de dinàmiques neuronals amb un cost computacional reduït. Hem caracteritzat la dinàmica global i individual de les neurones i l’hem correlacionat amb la seva estructura subjacent mitjançant tècniques lineals i no–lineals de series temporals. L’anàlisi espectral ens ha possibilitat la descripció del desenvolupament i els canvis en connectivitat en els cultius, així com la diferenciació entre cultius sans dels patològics. La reconstrucció de la dinàmica subjacent mitjançant mètodes d’incrustació i l’ús de gràfics de recurrència ens ha permès detectar diferents transicions dinàmiques amb el corresponent guany o pèrdua de la complexitat i riquesa dinàmica del cultiu durant els diferents estudis experimentals. Finalment, a fi de reconstruir la connectivitat interna hem testejat, mitjançant simulacions, diferents quantificadors per mesurar la dependència estadística entre neurona i neurona, seleccionant finalment el mètode de transferència d’entropia gereralitzada. Seguidament, hem procedit a caracteritzar les xarxes amb diferents paràmetres. Malgrat presentar certs tres de xarxes tipus ‘petit món’, els nostres cultius mostren una distribució de grau ‘exponencial’ o ‘esbiaixada’ per, respectivament, cultius joves i madurs. Addicionalment, hem observat que les xarxes homogènies presenten la propietat de disassortativitat, mentre que xarxes amb un creixent nivell d’agregació espaial presenten assortativitat. Aquesta propietat impacta fortament en la transmissió, resistència i sincronització de la xarxa

Firing dynamics of thalamic neurones during genetically determined experimental absence seizures

Absence seizures (ASs) are the predominant form of seizure featuring in the idiopathic generalised epilepsies, and are the only seizure type of childhood absence epilepsy. They are characterised by behavioural arrest, impairment of consciousness and an electrographic signature of spike-and-wave discharges (SWDs) and are associated with psychosocial and cognitive impairment of development. The seizures are known to arise in the thalamocortical network, but the firing dynamics of thalamic neurones during seizure is not known. In vivo and in vitro studies have yielded contradictory results, suggesting predominant silence and regular burst firing respectively, but no studies have previously recorded from intact, single thalamic neurones in a freely moving model of absence epilepsy. In this thesis it has been shown that, in Genetic Absence Epilepsy Rats from Strasbourg, thalamocortical (TC) neurones are mostly either silent or fire single spikes irregularly but synchronously during AS. T-type calcium channel-mediated bursts in neurones of the reticular thalamic nucleus (nRT) were frequently observed during full seizure expression. These cells expressed varied firing patterns ranging from regular burst firing to predominant silence, with similarly varying degrees of synchrony. It is also suggested that the nRT burst firing observed may be required for seizure generation. T-type calcium channel-mediated burst firing of TC neurones is neither necessary for, nor commonly observed in, the full generation or propagation of absence seizures These results suggest that TC neurones are predominantly silent during AS. This is compatible with the idea of a cortical seizure initiator and driver, as suggested by the cortical initiation site and cortical abnormalities observed in multiple experimental AS models. The observations herein also confirm that the temporal relationship between thalamic firing and SWDs previously observed in anaesthetised animals is maintained in the freely moving condition, but suggest that there is a greater incidence of asynchronous thalamic activity during AS (particularly of nRT neurones) than previously suggested. The firing dynamics of thalamic neurones observed are a crucial step towards understanding TC network activity during AS, and provide a significant insight into the role of the thalamus in alterations of sensation, movement, and consciousness associated with these seizures