Investigating the role of fast-spiking interneurons in neocortical dynamics

PhD ThesisFast-spiking interneurons are the largest interneuronal population in neocortex. It is well documented that this population is crucial in many functions of the neocortex by subserving all aspects of neural computation, like gain control, and by enabling dynamic phenomena, like the generation of high frequency oscillations. Fast-spiking interneurons, which represent mainly the parvalbumin-expressing, soma-targeting basket cells, are also implicated in pathological dynamics, like the propagation of seizures or the impaired coordination of activity in schizophrenia. In the present thesis, I investigate the role of fast-spiking interneurons in such dynamic phenomena by using computational and experimental techniques. First, I introduce a neural mass model of the neocortical microcircuit featuring divisive inhibition, a gain control mechanism, which is thought to be delivered mainly by the soma-targeting interneurons. Its dynamics were analysed at the onset of chaos and during the phenomena of entrainment and long-range synchronization. It is demonstrated that the mechanism of divisive inhibition reduces the sensitivity of the network to parameter changes and enhances the stability and exibility of oscillations. Next, in vitro electrophysiology was used to investigate the propagation of activity in the network of electrically coupled fast-spiking interneurons. Experimental evidence suggests that these interneurons and their gap junctions are involved in the propagation of seizures. Using multi-electrode array recordings and optogenetics, I investigated the possibility of such propagating activity under the conditions of raised extracellular K+ concentration which applies during seizures. Propagated activity was recorded and the involvement of gap junctions was con rmed by pharmacological manipulations. Finally, the interaction between two oscillations was investigated. Two oscillations with di erent frequencies were induced in cortical slices by directly activating the pyramidal cells using optogenetics. Their interaction suggested the possibility of a coincidence detection mechanism at the circuit level. Pharmacological manipulations were used to explore the role of the inhibitory interneurons during this phenomenon. The results, however, showed that the observed phenomenon was not a result of synaptic activity. Nevertheless, the experiments provided some insights about the excitability of the tissue through scattered light while using optogenetics. This investigation provides new insights into the role of fast-spiking interneurons in the neocortex. In particular, it is suggested that the gain control mechanism is important for the physiological oscillatory dynamics of the network and that the gap junctions between these interneurons can potentially contribute to the inhibitory restraint during a seizure.Wellcome Trust

Bridging from single to collective cell migration: A review of models and links to experiments

Mathematical and computational models can assist in gaining an understanding of cell behavior at many levels of organization. Here, we review models in the literature that focus on eukaryotic cell motility at 3 size scales: intracellular signaling that regulates cell shape and movement, single cell motility, and collective cell behavior from a few cells to tissues. We survey recent literature to summarize distinct computational methods (phase-field, polygonal, Cellular Potts, and spherical cells). We discuss models that bridge between levels of organization, and describe levels of detail, both biochemical and geometric, included in the models. We also highlight links between models and experiments. We find that models that span the 3 levels are still in the minority.Comment: 39 pages, 5 figure

Influence of Connexin Expression/Co-Expression Levels on Electrical Impulse Propagation Investigated in the HL-1 Cell Model

In the myocardium action potentials are transmitted from cell-to-cell through gap junctions. These specialised junctions play a pivotal role in regulating the speed and safety of impulse propagation by controlling the amount of depolarised current that is passed from excited to non-excited regions of the heart. In mammalian hearts gap junction proteins connexin43, connexin40 and connexin45 are co-expressed in distinctive combinations and relative quantities in functionally specialised subsets of cardiac myocyte. The functional consequences of these connexin expression/co-expression patterns in modulating impulse propagation are poorly understood. To study the relative importance of membrane excitability and electrical coupling in relation to propagation velocities, clones of the HL-1 mouse atrial myocyte tumour line were used as an in vitro cell model. Five clones were characterised for expression of myocytic markers, calcium handling proteins and connexins, two of which (#2 and #6) displayed large differences in conduction velocities using microelectrode arrays. To ascertain which factor(s) were the main determinants of speed of conduction, the membrane excitability (voltage-gated channels) and electrical coupling (gap junctions) between the two clones were compared. Sodium, L- and T-type calcium channels were present in both clones but no significant differences were found in the current densities. However, large differences were seen in expression levels of connexin43, connexin40 and connexin45. RNA interference combined with microelectrode arrays was employed to establish the relative importance of each connexin in impulse propagation. The results indicate that electrical coupling by gap junctions is a major determinant of conduction velocities in HL-1 cell lines. Further experiments using RNA interference to suppress the expression of proteins thought to play a role in the action potential parameters should help in defining the part played by either the active or passive electrical properties in action potential propagation

Rapid synaptic and gamma rhythm signature of mouse critical period plasticity

Published versio

Understanding spiking and bursting electrical activity through piece-wise linear systems

In recent years there has been an increased interest in working with piece-wise linear caricatures of nonlinear models. Such models are often preferred over more detailed conductance based models for their small number of parameters and low computational overhead. Moreover, their piece-wise linear (PWL) form, allow the construction of action potential shapes in closed form as well as the calculation of phase response curves (PRC). With the inclusion of PWL adaptive currents they can also support bursting behaviour, though remain amenable to mathematical analysis at both the single neuron and network level. In fact, PWL models caricaturing conductance based models such as that of Morris-Lecar or McKean have also been studied for some time now and are known to be mathematically tractable at the network level. In this work we proceed to analyse PWL neuron models of conductance type. In particular we focus on PWL models of the FitzHugh-Nagumo type and describe in detail the mechanism for a canard explosion. This model is further explored at the network level in the presence of gap junction coupling. The study moves to a different area where excitable cells (pancreatic beta-cells) are used to explain insulin secretion phenomena. Here, Ca2+ signals obtained from pancreatic beta-cells of mice are extracted from image data and analysed using signal processing techniques. Both synchrony and functional connectivity analyses are performed. As regards to PWL bursting models we focus on a variant of the adaptive absolute IF model that can support bursting. We investigate the bursting electrical activity of such models with an emphasis on pancreatic beta-cells

Modélisation des réponses calciques de réseaux d'astrocytes : Relations entre topologie et dynamiques

Over the last 20 years, astrocytes, a hitherto under-investigated type of brain cells, have gradually rose to prominence owing to multiple experimental discoveries. In contrast with neurons, these cells do not propagate electrical signals but communicate instead through changes in their intracellular calcium concentration. Recent discoveries indicate that, far from being isolated cells, astrocytes respond to neuronal activity and, although this is still controversial, seem to modulate synaptic transmission through the release of `gliotransmitter' molecules (in reference to neurotransmitters). Like neurons, astrocyte are organized in networks and communicate their calcium activity by intercellular diffusion of second messengers, forming intercellular calcium waves. Two networks, one of neurons and the other of astrocytes, thus coexist in the brain; while neuronal networks have been the subject of intense experimental and theoretical investigations, astrocyte networks have been much less investigated. Notably, it was only discovered recently that astrocyte network topology could be more complex than what the hitherto dominant view held (astrocytes organized in a syncytium deprived of any topological specificities). The work presented in this thesis is mainly related to the effect that different network topologies could have on astrocyte calcium signaling. The mechanisms that drive calcium signaling in astrocytes are, at both subcellular and intercellular levels, still not completely understood. Even in the best documented case of astrocyte somatic response to neuronal stimulation, the precise characteristic required from the stimulation to elicit an astrocytic response are still unknown. Similarly, the mechanisms governing intercellular calcium wave propagation in astrocyte networks are not fully known; notably, the effects of the recently documented network heterogeneity on calcium wave propagation have not been investigated. Finally, at the subcellular level, astrocytes display an extremely ramified and complex morphology that also hosts calcium activity. The work presented in this thesis make use of modeling and simulation in order to determine the possible effects of astrocyte network organization on their calcium signaling. We propose that astrocyte network topology: (1) controls single-cell responses to neuronal stimulation; (2) drives the propagation of intercellular calcium waves by favoring it when networks are weakly coupled; (3) can determine the appearance of stochastic resonance phenomena; (4) can be modulated by neuronal activity.Pendant les 20 dernières années, les astrocytes, un type de cellules cérébrales ayant été jusque là relativement ignoré des neuroscientifiques, ont peu à peu gagné en notoriété grâce à de multiples découvertes. Contrairement aux neurones, ces cellules ne transmettent pas de signaux électriques mais communiquent par des changements intracellulaires de leurs concentrations en calcium. Des découvertes récentes semblent indiquer que, loin d'agir en autarcie, les astrocytes répondent à l'activité neuronale et sembleraient, bien que cela soit plus débattu, moduler la transmission synaptique par le relargage de molécules spécifiques appelées `gliotransmetteurs' (en référence aux neurotransmetteurs). Comme les neurones, les astrocytes forment des réseaux et communiquent leur activité calcique par diffusion d'un astrocyte à l'autre, formant ainsi de véritables vagues de calcium intercellulaires. Deux réseaux, de neuronnes et d'astrocytes, cohabitent ainsi dans le cerveau ; mais, alors que les réseaux de neuronnes ont fait l'objet de recherches expérimentales et théoriques, les réseaux d'astrocytes restent encore mal connus. Ainsi, il n'a été découvert que très récement que la topologie de ces réseaux pourrait s'averer plus complexe que la vision qui dominait jusqu'alors : celle d'un syncitium astrocytaire dépourvu de spécificités topologiques. Les travaux présentés dans cette thèse portent principalement sur l'effet que ces différentes topologies pourraient avoir sur la signalisation calcique astrocytaire. En effet, autant au niveau subcellulaire qu'inter-cellulaire, les mécanismes gouvernant l'activité calcique des astrocytes restent mals connus. Même dans le cas le plus documenté de la réponse somatique des astrocytes à une stimulation neuronale, les caractéristiques précises que la stimulation doit avoir pour évoquer une réponse des astrocytes sont inconnues. Il en est de même pour la transmission de vagues de calcium dans des réseaux d'astrocytes : on ignore encore les possibles effets de la complexité récemment documentée des réseaux d'astrocytes sur la propagation de ces vagues. Enfin, au niveau subcelulaire, les astrocytes possèdent une morphologie ramifiée extrèmement complexe qui possède elle-même une activité calcique. Les travaux présentés dans cette thèse utilisent des outils de modélisation et de simulation afin de déterminer les répercussions que l'organisation en réseaux des astrocytes pourrait avoir sur leurs dynamiques calciques. En résumé, nous proposons que la topologie des réseaux d'astrocytes a (1) des répercussion au niveau cellulaire, modulant la réponse des astrocytes à des stimulations neuronales ; (2) contrôle la propagation de vagues de calcium inter-astrocytaire en la favorisant lorsque les réseau sont peu couplés ; (3) joue un rôle important dans l’apparition de phénomènes de résonance stochastique

Calcium Regulation of Cell-Cell Communication and Extracellular Signaling

As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist