Functional connectivity analysis in health and brain disease using in vivo widefield calcium imaging

Stroke is one of the leading causes of death and its prevalence is still raising with aging society in future. Despite its major impact, only two specific therapies are approved in clinical practice, today.Thus, hundreds of possible therapies were identified in experimental research, none of them has proven efficiency on human patients. To address this loss in translation from experimental research to clinical practise, several fronts can be scrutinized. Among several options, the establishment of translational methods to assess functional clinical outcome in preclinical research is inevitable. To approach this one option is to develop modalities of functional imaging of the brain activity. Functional brain imaging not only allows to assess translational parameters for functional regeneration after stroke but also to investigate pathophysiological mechanisms in the brain. Hence, the analysis of functional brain activity in experimental stroke research could both identify new therapeutic targets and validate their effectiveness by creating a translational read-out. Functional brain imaging is a frequently used method which strongly advanced our knowledge in neuroscience and as well in human stroke research. Its aim in general is a better understanding of brain functions, identification of functionally connected brain regions and their dynamic changes under certain conditions. In stroke research, the dynamic changes of functional network and its association with regeneration is of major interest. To investigate functional brain activity, functional magnetic resonance imaging (fMRI) is predominantly used in human research. fMRI faces great technical challenges and essential limitations for use in small rodents such as laboratory mice which are the most frequently used animals to study brain disease. This is why there is interest and need for alternative imaging modalities in experimental research. To benefit of the insights from human research in experimental research, we adapted and evolved the imaging modality of in vivo widefield calcium imaging. This imaging modality is based on transgenic animals who permit to investigate brain activity directly via GCaMP fluorescence. GCaMP is a genetically encoded calcium sensor which is well-known to mirror calcium fluctuations during action potential and with this neuronal activity. Via a customized imaging system, it is possible to acquire cortical neuronal activity and analyse it with comparable methods as used in human brain research. Hence, this method allows the repetitive investigation of brain activity in vivo in a translational manner. In three studies we adapted and enhanced existing protocols to establish a reliable transgenic approach to assess functional brain connectivity. In a first study, we investigated the effect of anaesthesia on brain function and characterized the relationship of different frequency-based imaging parameters, functional connectivity and depth of anaesthesia. Subsequently, we established a stringent protocol for light sedation which is easy to use and results in reproducible imaging parameters. In a second study, we identified functional brain areas by using independent vector analysis (IVA) on resting state imaging data. Therefore, we validated the identified areas with help of an anatomical atlas and stimulus-evoked brain activity. This validation justifies the usage of our unbiasedly selected cortical areas as functional seeds. Finally, we implemented the assessment of functional connectivity values after stroke. In this third study, we investigated repetitively the changes in functional connectivity up to 56 days after an ischemic lesion in the motor cortex induced by a photothrombotic model. We demonstrate both acute and chronic effects of ischemia to cortical functional connectivity. In the acute phase on the first day after stroke we demonstrate transient increase in contralateral functional connectivity. A second transient effect is the increase in contralateral motor cortex size. Third, chronic reduction in interhemispheric functional connectivity is present only in functionally but not anatomically close regions of the brain. And last, changes in both functional connectivity values and the size of contralateral motor cortex size are associated with the deficits assessed by behavioural testing. Hence, the identified parameters are of major relevance for the clinical outcome. The results establish two major facts: preclinical investigation of brain function is possible on a routinely basis and adds additional insight on pathophysiological mechanisms in brain disease which are associated with behavioural outcome. Consequently, the application of this translational imaging modality will not only be of great interest to stroke research but also to several brain diseases where pathophysiological mechanisms still need to be elucidated

The Role Of The Nmda Receptor In Shaping Cortical Activity During Development

Currently, it is estimated that neuropsychiatric disorders will affect 20-25% of humans in their lifetime. These disorders are a major cause of mortality, suffering, and economic cost to society. Within this broad class, neurodevelopmental disorders (NDDs), including intellectual disability, autism spectrum disorder, and schizophrenia, are estimated to affect 2-5% percent of the world population. Devastatingly, we lack fundamental treatments for NDDs, which have proved some of the most imposing disorders to understand scientifically. The challenge is twofold: first, NDDs affect the most complex aspects of human cognition; second, pathogenesis begins early in neural circuit development, but we lack predictive biomarkers before overt behavioral deficits are apparent. Although we have identified many genes associated with these disorders, how underlying genetic disruptions lead to pathological neural network development and function remains unclear. The overarching framework of this dissertation is that all NPDs are disorders of distributed neural networks, and pathophysiology must be understood at this level to effectively intervene clinically. The cerebral cortex is necessary for complex human capacities, and cortical dysfunction is hypothesized to be central to the pathophysiology of NDDs. NMDA glutamate receptors (NMDARs) are important for the development of local circuit features in the cortex, for normal neurocognitive function, and are strongly implicated in NDDs. However, the role of NMDARs in the development of the large-scale cortical network dynamics that underly higher cognition has not been well examined. Understanding the role of NMDARs at this network level is critical because large-scale “functional connectivity” patterns are thought to be hallmarks of normal cortical function, are hypothesized to be disrupted in NDDs, and may be detectable in humans using non-invasive neuroimaging or electrophysiology. In the studies presented in this dissertation, I (in collaboration and with the support of my colleagues) tested the role of the NMDAR in shaping large-scale cortical network organization using in vivo widefield imaging of whole cortex spontaneous activity in developing mice. I found that NMDAR function in the lineage that includes cortical excitatory neurons and glia, specifically, was critical for the elaboration of normal cortical activity patterns and dynamic network organization. In the first set of experiments, NMDARs were deleted in glutamatergic excitatory neurons (Emx1-cre+/WT/Grin1f/f ; referred to as EX-NMDAR KO mice) or GABAergic inhibitory neurons (Nkx2.1+/WT/Grin1f/f; referred to as IN-NMDAR KO mice). The developing cortex normally exhibits a diverse range of spatio-temporal patterns, reflecting the emergence of functionally associated sub-networks. In EX-NMDAR KO mice, normal patterns of spontaneous activity were severely disrupted and reduced to a nearly one-dimensional dynamic space dominated by large, cortex-wide events. Interestingly, in IN-NMDAR KO mice, the structure and complexity of spontaneous activity was largely normal. In the next set of experiments, I tested the role of extrinsic thalamic neurotransmission on cortical activity during development. Deleting the vesicular glutamate transporter from thalamic neurons while leaving cortical NMDARs intact (Sert-Cre+/−,vglut1−/−,vglut2fl/fl; referred to as TH-VG KO mice) led to a shift in cortical activity patterns towards large domains of activity, reminiscent of patterns observed in EX-NMDAR KO mice. This manipulation also reduced the dimensionality of cortical activity, though not as severally as in EX-NMDAR KO mice. In a final set of experiments, I tested cortical activity in three established mouse models of mono-genetic causes of NDDs in humans: the FMR1-KO mouse based on Fragile X Syndrome, the CNTNAP2-KO mouse, and the TS2-neo mouse based on Timothy Syndrome. In all three of these mouse models, I found that large-scale cortical activity patterns were largely normal, but there was a statistically significant shift towards reduced cortex-wide synchrony and increased dimensionality of spontaneous activity, which may be consistent with the disconnectivity hypothesis of autism. In a final set of experiments, we tested our hypothesis, based on past literature and our results in EX-NMDAR KO and TH-VG KO mice, that the disruptions in cortical activity was predominantly due to the developmental loss of activity-dependent wiring of circuits. To test the developmental versus acute role of NMDAR function in shaping cortical activity, I blocked NMDAR pharmacologically in wild-type mice. I found that acute NMDAR blockade shifted cortical activity to a restricted dynamic space similar to that observed in EX-NMDAR KO mice and more extreme than that observed in TH-VG KO mice. These results strongly reinforce the critical role of NMDAR in shaping cortical activity during development, and suggest that a substantial component of that may be through NMDAR’s role in synaptic transmission and moment to moment cortex-wide circuit function. Overall, these results provide critical insight into the role of NMDARs and the glutamatergic system in cortical network functional organization during development. Specifically, they highlight the essential role of NMDARs in excitatory neurons on the functional connectivity and dynamic repertoire of the cortical network during development. These results make novel contribution to our understanding of how NMDARs may contribute to the pathophysiology of NDDs. Specifically, they contribute powerful new insight into to a critical mechanistic question about the cell-specific role of NMDARs in the pathophysiology of schizophrenia and the mechanisms of NMDAR antagonists, which have transformed psychiatry recently due to their rapid-acting anti-depressant and anti-suicidal properties. Furthermore, they identify a patterns of large-scale network dysfunction that might be detectable in humans using noninvasive functional imaging or electrophysiology

Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer's disease

Dysfunctions of network activity and functional connectivity (FC) represent early events in Alzheimer's disease (AD), but the underlying mechanisms remain unclear. Astrocytes regulate local neuronal activity in the healthy brain, but their involvement in early network hyperactivity in AD is unknown. We show increased FC in the human cingulate cortex several years before amyloid deposition. We find the same early cingulate FC disruption and neuronal hyperactivity in AppNL-F mice. Crucially, these network disruptions are accompanied by decreased astrocyte calcium signaling. Recovery of astrocytic calcium activity normalizes neuronal hyperactivity and FC, as well as seizure susceptibility and day/night behavioral disruptions. In conclusion, we show that astrocytes mediate initial features of AD and drive clinically relevant phenotypes

Analyzing spontaneous oscillatory activity and network organization in cortical slices using the genetically encoded voltage indicator ArcLight

Παρά τον έπαινο τους ως δυνητικό απόγειο στο ερευνητικό πεδίο των νευροεπιστημών, ο αντίκτυπος των γενετικά κωδικοποιημένων δεικτών τάσης (ΓΚΔΤ) στην κατανόηση της φυσιολογίας των νευρικών κυκλωμάτων παραμένει δυσνόητος. Επιπρόσθετα, η συστηματική ανάλυση συνόλων δεδομένων ΓΚΔΤ αποτελεί σημαντική πρόκληση, τόσο λόγω του μεγάλου τους όγκου όσο και της έλλειψης μιας επικρατούσας άποψης όσον αφορά σε κάποια συγκεκριμένη μέθοδο ανάλυσης. Σε αυτή την εργασία, εκφράζοντας επιτυχώς τον ΓΚΔΤ ArcLight παν-νευρωνικά, μπορέσαμε να καταγράψουμε με οπτικό τρόπο αυθόρμητα εγκεφαλικά κύματα σε παρασκευάσματα (τομές) του κινητικού φλοιού μυών, μετά τη χορήγηση βικουκουλίνης. Στη συνέχεια, επιδιώξαμε να αναλύσουμε το προκύπτων σύνολο δεδομένων υπό την προοπτική της θεωρίας δικτύων, αξιοποιώντας την ικανότητα της να αποκαλύπτει πολύπλοκες σχέσεις κι αναδυόμενες ιδιότητες εντός των διασυνδεδεμένων στοιχείων, με στόχο να μελετήσουμε αν και και κατά πόσον η αυθόρμητη εμφάνιση εγκεφαλικών κυμάτων μπορεί να προβλεφθεί μέσω της υποκείμενης δικτυακής δραστηριότητας. Η ανάλυσή που ακολούθησε υποδηλώνει πως εναλλακτικές επιστημονικές διαισθήσεις, αφορώσες στη λειτουργική ερμηνεία των δεδομένων, θα πρέπει να αναζητηθούν, τουλάχιστον μέχρι να επιλυθούν οριστικά οι εγγενείς τεχνικοί περιορισμοί των ΓΚΔΤ.Genetically encoded voltage indicators (GEVIs) have been praised as a potential pinnacle in the field of neuroscience, yet their significant impact on our understanding of circuit physiology remains elusive. In addition, analyzing GEVI datasets poses a significant challenge as these datasets are large and there is no consensus on a specific method of analysis. Here, by expressing the GEVI ArcLight pan-neuronally we were able to optically resolve bicuculline induced spontaneous oscillations in brain slices of the mouse motor cortex. We next sought to analyze the emerged dataset under a network theory perspective, exploiting its ability to uncover complex relationships and reveal emergent properties within the interconnected elements, with our aim being to study whether the spontaneous occurrence of an oscillation can be predicted based on the imaged network activity. Our analysis suggests that alternative interdisciplinary intuitions about the functional interpretation of the data might be more appropriate, at least until the inherent technical limitations surrounding GEVIs are finally resolved

Early postnatal development of neocortex-wide activity patterns in GABAergic and pyramidal neurons

Before the onset of sensory experience, developing circuits generate synchronised activity that will not only influence its wiring, but ultimately contribute to behaviour. These complex functions rely on widely distributed cortical that simultaneously operate at multiple spatiotemporal scales. The timing of GABAergic maturation appears to align with the developmental trajectories of cortical regions, playing a crucial role in the functional development of individual brain areas. While local connectivity in cortical microcircuits has been extensively studied, the dynamics of brain-wide functional maturation, especially for GABAergic populations, remain underexplored. In this project, a dual-colour widefield calcium imaging approach was developed to examine the neocortex-wide dynamics of cortical GABAergic and excitatory neurons simultaneously across early postnatal development. This study provides the first broad description of neocortex-wide GABAergic developmental trajectories and their cross-talk with excitatory dynamics during the second and third postnatal weeks. The observed spontaneous activity revealed discrete activity domains, reflecting the modular organisation of the cortex. Both excitatory and GABAergic population exhibited an increase in the size and frequency of activity motifs, as well as changes in motif variability. However, as they matured, the distribution of these spatiotemporal properties displayed divergent trajectories across populations and regions. These findings suggest fundamental differences in the spatial organisation of both populations, indicating potential distinct roles in cortical network function development. Moreover, while excitatory and GABAergic dynamics exhibited high correlations, brief deviations from perfect timing were observed. This correlation patterns changed significantly during development and across regions, with the two populations gradually becoming more correlated as they matured. Manipulating inhibition in vivo disrupted these fluctuations, impacting both local activity and the wider functional network.These findings provide valuable insights into the developmental trajectories of spontaneous activity patterns in excitatory and GABAergic cell populations during early postnatal development. The interplay between both neuronal populations plays a critical role in shaping activity patterns, and understanding the underlying mechanisms of their development can provide valuable insights into neurodevelopmental disorders

Reduced GABAergic Neuron Excitability, Altered Synaptic Connectivity, and Seizures in a KCNT1 Gain-of-Function Mouse Model of Childhood Epilepsy.

Gain-of-function (GOF) variants in K+ channels cause severe childhood epilepsies, but there are no mechanisms to explain how increased K+ currents lead to network hyperexcitability. Here, we introduce a human Na+-activated K+ (KNa) channel variant (KCNT1-Y796H) into mice and, using a multiplatform approach, find motor cortex hyperexcitability and early-onset seizures, phenotypes strikingly similar to those of human patients. Although the variant increases KNa currents in cortical excitatory and inhibitory neurons, there is an increase in the KNa current across subthreshold voltages only in inhibitory neurons, particularly in those with non-fast-spiking properties, resulting in inhibitory-neuron-specific impairments in excitability and action potential (AP) generation. We further observe evidence of synaptic rewiring, including increases in homotypic synaptic connectivity, accompanied by network hyperexcitability and hypersynchronicity. These findings support inhibitory-neuron-specific mechanisms in mediating the epileptogenic effects of KCNT1 channel GOF, offering cell-type-specific currents and effects as promising targets for therapeutic intervention

Chronic T cell proliferation in brains after stroke could interfere with the efficacy of immunotherapies

Neuroinflammation is an emerging focus of translational stroke research. Preclinical studies have demonstrated a critical role for brain-invading lymphocytes in post-stroke pathophysiology. Reducing cerebral lymphocyte invasion by anti-CD49d antibodies consistently improves outcome in the acute phase after experimental stroke models. However, clinical trials testing this approach failed to show efficacy in stroke patients for the chronic outcome 3 mo after stroke. Here, we identify a potential mechanistic reason for this phenomenon by detecting chronic T cell accumulation—evading the systemic therapy—in the post-ischemic brain. We observed a persistent accumulation of T cells in mice and human autopsy samples for more than 1 mo after stroke. Cerebral T cell accumulation in the post-ischemic brain was driven by increased local T cell proliferation rather than by T cell invasion. This observation urges re-evaluation of current immunotherapeutic approaches, which target circulating lymphocytes for promoting recovery after stroke

Influence of Focal Activity on Macroscale Brain Dynamics in Health and Disease

Macroscopic recordings of brain activity (e.g. fMRI, EEG) are a sensitive biomarker of the neural networks supporting neurocognitive function. However, it remains largely unclear what mechanisms mediate changes in macroscale networks after focal brain injuries like stroke, seizure, and TBI. Recently, optical neuroimaging in animal models has emerged as a powerful tool to begin addressing these questions. Using widefield imaging of cortical calcium dynamics in mice, this dissertation investigates the mechanisms by which focal disruptions in activity alter brain-wide functional dynamics. In two chapters, I demonstrate 1) that focal sensory stimulation elicits state-dependent, global slow waves propagating from primary somatosensory cortex (S1). Using a focal ischemic stroke model, I show that bilateral activation of somatosensory cortices is required for initiating global SWs, while spontaneous SWs are generated independent of S1. 2) That regional disruption of cortical excitability induces widespread changes across cortical networks, using chemogenetic manipulation of parvalbumin interneurons to model focal epileptiform activity in S1. We further show that local imbalances in excitability propagate differentially through intra- and interhemispheric connections, and can induce plasticity in large-scale networks. These studies begin to define the mechanisms of macro-scale network disruption after focal injuries, adding to our understanding of how local cortical circuits modulate global brain networks