<p>Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.</p> / Dissertation

<p>Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive. </p><p>Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity. </p><p>We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.</p> / Dissertation

<p>The late phase of long-term synaptic potentiation (LTP) at glutamatergic synapses, which is thought to underlie the long lasting memory (at least hours), requires gene transcription in the nucleus. However, it remains elusive how signaling initiated at synapses during induction of LTP is transmitted into the nucleus to commence transcription. Using a combination of two-photon glutamate uncaging and a genetically encoded FRET sensor, I found that induction of synapse-specific LTP at only a few (3-7) dendritic spines leads to pronounced activation of extracellular signal-regulated kinase (ERK) in the nucleus and downstream phosphorylation of transcription factors, cAMP-response element-binding protein (CREB) and E26-like protein-1 (Elk-1). The underlying molecular mechanism of this nuclear ERK activation was investigated: it seems to involve activation of NMDA receptors, metabotrophic glutamate receptors, and the classical Ras pathway. I also found that the spatial pattern of synaptic stimulation matters: spatially dispersed stimulation over multiple dendritic branches activated nuclear ERK much more efficiently than clustered stimulation within a single dendritic branch. In sum, these results suggest that biochemical signals could be transmitted from individual spines to the nucleus following LTP induction and that such synapse-to-nucleus signaling requires integration across multiple dendritic branches.</p> / Dissertation

<p>It has long been appreciated that the process of learning might invoke a physical change in the brain, establishing a lasting trace of experience. Recent evidence has revealed that this change manifests, at least in part, by the formation of new connections between neurons, as well as the modification of preexisting ones. This so-called structural plasticity of neural circuits – their ability to physically change in response to experience – has remained fixed as a primary point of focus in the field of neuroscience. </p><p>A large portion of this effort has been directed towards the study of dendritic spines, small protrusions emanating from neuronal dendrites that constitute the majority of recipient sites of excitatory neuronal connections. The unique, mushroom-like morphology of these tiny structures has earned them considerable attention, with even the earliest observers suggesting that their unique shape affords important functional advantages that would not be possible if synapses were to directly contact dendrites. Importantly, dendritic spines can be formed, eliminated, or structurally modified in response to both neural activity as well as learning, suggesting that their organization reflects the experience of the neural network. As such, elucidating how these structures undergo such rearrangements is of critical importance to understanding both learning and memory. </p><p>As dendritic spines are principally composed of the cytoskeletal protein actin, their formation, elimination, and modification requires biochemical signaling networks that can remodel the actin cytoskeleton. As a result, significant effort has been placed into identifying and characterizing such signaling networks and how they are controlled during synaptic activity and learning. Such efforts have highlighted Rho family GTPases – binary signaling proteins central in controlling the dynamics of the actin cytoskeleton – as attractive targets for understanding how the structural modification of spines might be controlled by synaptic activity. While much has been revealed regarding the importance of the Rho GTPases for these processes, the specific spatial and temporal features of their signals that impart such structural changes remains unclear. </p><p>The central hypotheses of the following research dissertation are as follows: first, that synaptic activity rapidly initiates Rho GTPase signaling within single dendritic spines, serving as the core mechanism of dendritic spine structural plasticity. Next, that each of the Rho GTPases subsequently expresses a spatially distinct pattern of activation, with some signals remaining highly localized, and some becoming diffuse across a region of the nearby dendrite. The diffusive signals modify the plasticity induction threshold of nearby dendritic spines, and the spatially restricted signals serve to keep the expression of plasticity specific to those spines that receive synaptic input. This combination of differentially spatially regulated signals thus equips the neuronal dendrite with the ability to perform local biochemical computations, potentially establishing an organizational preference for the arrangement of dendritic spines along a dendrite. Finally, the consequences of the differential signal patterns also help to explain several seemingly disparate properties of one of the primary upstream activators of these proteins: brain-derived neurotrophic factor (BDNF). </p><p>The first section of this dissertation describes the characterization of the activity patterns of one of the Rho family GTPases, Rac1. Using a novel Förster Resonance Energy Transfer (FRET)- based biosensor in combination with two-photon fluorescence lifetime imaging (2pFLIM) and single-spine stimulation by two-photon glutamate uncaging, the activation profile and kinetics of Rac1 during synaptic stimulation were characterized. These experiments revealed that Rac1 conveys signals to both activated spines as well as nearby, unstimulated spines that are in close proximity to the target spine. Despite the diffusion of this structural signal, however, the structural modification associated with synaptic stimulation remained restricted to the stimulated spine. Thus, Rac1 activation is not sufficient to enlarge spines, but nonetheless likely confers some heretofore-unknown function to nearby synapses. </p><p>The next set of experiments set out to detail the upstream molecular mechanisms controlling Rac1 activation. First, it was found that Rac1 activation during sLTP depends on calcium through NMDA receptors and subsequent activation of CaMKII, suggesting that Rac1 activation in this context agrees with substantial evidence linking NMDAR-CaMKII signaling to LTP in the hippocampus. Next, in light of recent evidence linking structural plasticity to another potential upstream signaling complex, BDNF-TrkB, we explored the possibility that BDNF-TrkB signaling functioned in structural plasticity via Rac1 activation. To this end, we first explored the release kinetics of BDNF and the activation kinetics of TrkB using novel biosensors in conjunction with 2p glutamate uncaging. It was found that release of BDNF from single dendritic spines during sLTP induction activates TrkB on that same spine in an autocrine manner, and that this autocrine system was necessary for both sLTP and Rac1 activation. It was also found that BDNF-TrkB signaling controls the activity of another Rho GTPase, Cdc42, suggesting that this autocrine loop conveys both synapse-specific signals (through Cdc42) and heterosynaptic signals (through Rac1). </p><p>The next set of experiments detail one the potential consequences of heterosynaptic Rac1 signaling. The spread of Rac1 activity out of the stimulated spine was found to be necessary for lowering the plasticity threshold at nearby spines, a process known as synaptic crosstalk. This was also true for the Rho family GTPase, RhoA, which shows a similar diffusive activity pattern. Conversely, the activity of Cdc42, a Rho GTPase protein whose activity is highly restricted to stimulated spines, was required only for input-specific plasticity induction. Thus, the spreading of a subset of Rho GTPase signaling into nearby spines modifies the plasticity induction threshold of these spines, increasing the likelihood that synaptic activity at these sites will induce structural plasticity. Importantly, these data suggest that the autocrine BDNF-TrkB loop described above simultaneously exerts control over both homo- and heterosynaptic structural plasticity. </p><p>The final set of experiments reveals that the spreading of GTPase activity from stimulated spines helps to overcome the high activation thresholds of these proteins to facilitate nearby plasticity. Both Rac1 and RhoA, the activity of which spread into nearby spines, showed high activation thresholds, making weak stimuli incapable of activating them. Thus, signal spreading from a strongly stimulated spine can lower the plasticity threshold at nearby spines in part by supplementing the activation of high-threshold Rho GTPases at these sites. In contrast, the highly compartmentalized Rho GTPase Cdc42 showed a very low activation threshold, and thus did not require signal spreading to achieve high levels of activity to even a weak stimulus. As a result, synaptic crosstalk elicits cooperativity of nearby synaptic events by first priming a local region of the dendrite with several (but not all) of the factors required for structural plasticity, which then allows even weak inputs to achieve plasticity by means of localized Cdc42 activation. </p><p>Taken together, these data reveal a molecular pattern whereby BDNF-dependent structural plasticity can simultaneously maintain input-specificity while also relaying heterosynaptic signals along a local stretch of dendrite via coordination of differential spatial signaling profiles of the Rho GTPase proteins. The combination of this division of spatial signaling patterns and different activation thresholds reveals a unique heterosynaptic coincidence detection mechanism that allows for cooperative expression of structural plasticity when spines are close together, which in turn provides a putative mechanism for how neurons arrange structural modifications during learning.</p> / Dissertation

La Fragile X Mental Retardation Protein (FMRP) est une protéine régulant la traduction locale de nombreux ARNm dans le cerveau. Elle est absente dans le Syndrome de l'X Fragile (SXF), première cause de retard mental héréditaire. J'ai étudié le rôle de FMRP dans la plasticité structurelle des cellules granulaires (CG) produites à l'âge adulte dans le bulbe olfactif (BO) en réponse à un apprentissage chez la souris. L'apprentissage perceptif (AP) induit de profonds changements structurels des nouveaux neurones du BO adulte. En absence de FMRP dans les CG nouvellement générées, l'apprentissage et la plasticité induite par cet apprentissage sont abolis. αCamKII est une cible traductionnelle connue de FMRP impliquée dans la plasticité synaptique et structurelle. En absence de l'ARNm de αCamKII dans les neurites, l'AP et la plasticité structurelle associée ne sont pas permis. De plus, l'AP augmente la traduction locale de αCamKII de façon dépendante de FMRP. De manière inattendue, αCamKII est présente dans 50% des CG. La plasticité structurelle des CG induite par l'apprentissage ne se produit que dans les CG qui expriment αCamKII (αCamKII+). De manière intéressante, l'AP active les deux populations de manière similaire. Ces résultats révèlent un rôle nouveau de la traduction locale dans la plasticité structurelle induite par l'apprentissage. De plus, l'AP produit des effets différents sur les deux populations que nous avons identifiées, qui pourtant participent probablement toutes deux à l'AP. / The Fragile X Mental Retardation Protein (FMRP) is a major regulator of local translation in neurons. It is absent in the Fragile X Syndrom (FXS), which is the main cause of inherited intellectual deficiency. I studied the role of FMRP in structural plasticity of adult-born granule cells (abGC) of the mouse olfactory bulb (OB) in response to learning. Perceptual learning (PL) induces profound structural changes in abGC. In absence of FMRP in adult-born neurons, learning and associated structural plasticity are prevented. αCamKII is a well known translational target of FMRP, which is involved in synaptic and structural plasticity. In absence of αCamKII mRNA in neurites, PL and associated structural plasticity are abolished. Besides, PL increases the dendritic local translation of αCamKII in an FMRP-dependent manner. Unexpectedly, αCamKII is present in 50% of the total GC population of the OB. Learning-associated structural plasticity occurs only in αCamKII expressing GC. Interestingly, PL activates similarly both populations. These results reveal a new role for local translation in learning-induced structural plasticity. Moreover, PL induces different effects in the two subpopulations we identified, which probably both participate to PL.

Le champ des neurosciences connaît depuis quelques décades un développement très important dans la compréhension des corrélats neuronaux de la perception. Le cerveau adulte répond aux variations de l'environnement et à l'expérience par des modifications fonctionnelles et structurales, regroupées sous le terme générique de plasticité, plasticité qui sous-tend l'apprentissage. Cette plasticité affecte la perception sensorielle, olfactive puisque c'est cette modalité qui va nous intéresser, mais également la perception de stimuli dans d'autres modalités sensorielles. Contrairement à des convictions longtemps érigées en dogme mais maintenant dépassées sur la nature fixe du cerveau, il est établi désormais que le cerveau adulte est capable de générer tout au long de la vie de nouveaux neurones qui s'intègrent dans la circuiterie cérébrale complexe, en particulier dans le bulbe olfactif et pourraient jouer un rôle dans l'apprentissage. Des travaux antérieurs de l'équipe ont démontré que l'acquisition de l'apprentissage perceptif dépend de la présence des neurones formés chez l'adulte. Par ailleurs, les systèmes neuromodulateurs comme les systèmes noradrénergique et cholinergique innervent massivement le bulbe olfactif et en particulier les neurInhibiting noradrenergic fibers duroing post learning discrimination testing lblmocked ones cibles de la neurogenèse adulte, les interneurones granulaires. Ils sont depuis longtemps connus pour leur implication dans les processus d'apprentissage en général et olfactif en particulier. Un objectif de la thèse était de déterminer le pattern temporal et spatial de l'innervation des neurones formés chez l'adulte dans le bulbe olfactif et sa modification potentielle par l'apprentissage, par des approches comportementales combinées à des approches neuro-anatomiques. Un autre objectif était d'évaluer le rôle fonctionnel des contacts noradrénergiques mis en place par l'apprentissage en utilisant l'outil optogénétique. Les résultats indiquent que l'innervation des neurones formés chez l'adulte s'installent dès le huitième jour après la naissance des neurones pour le système cholinergique, comme pour le système noradrénergique. L'apprentissage induit une augmentation massive des contacts noradrénergiques sur les neurones formés chez l'adulte qui n'est pas retrouvée pour les fibres cholinergiques, pointant le système noradrénergique comme un acteur majeur de la plasticité induite par l'apprentissage perceptif / The field of neuroscience has experienced explosive growth over the past decade toward understanding the neural correlates of perception. More specifically, the adult brain responds to environmental experience by significant functional and structural modifications, called "neural plasticity" which underlies learning. A main issue in neuroscience is to understand the cellular basis of perceptive plasticity and subsequent behavioral adaptations. Contrary to previously held beliefs about its static nature, the adult brain is in fact capable of generating new neurons that can integrate into its complex circuitry. The birth of new neurons constitutively occurs in two specific regions of the adult mammalian brain (OB and hippocampal dentate gyrus). Adult neurogenesis is a sophisticated biological process whose function has remained a mystery to neuroscience researchers but a role in learning and memory has been proposed. Previous work in our group have shown that perceptive olfactory learning depends on adult neurogenesis. In addition, neuromodulatory systems, including noradrenergic and cholinergic systems massively innervate the olfactory bulb and more specifically the inhibitory interneurons targeted by adult neurogenesis and are long-known for their role in learning and memory. One objective of the present work was to determine the spatial and temporal pattern of the innervation by noradrenergic and cholinergic inputs of developing adult-born neurons and to investigate its modulation by learning. For that purpose, we used behavioral and neuro-anatomical approaches. Another objective was to assess the functional role of centrifugal contacts using an optogenetic approach. Results indicate that the noradrenergic innervation is selectively increased on adult born neurons following perceptual olfactory learning, a phenomenon that was not observed for cholinergic innervation, pointing the noradrenergic system as a key mechanisms involved in perceptual learning. Interestingly, noradrenergic contacts on neurons born during ontogenesis were not affected by learning, suggesting a very specific part played by adult-born neuron in learning associated plasticity. In the same brains, we have analyzed the structural plasticity induced by learning in adult-born and pre-existing neurons. The major finding is that mirroring the increased number of noradrenergic contacts, learning induced an increase in dendritic spines on adult-born, but not on pre-existing neurons

No description available.

La théorie standard de la consolidation postule que l’information est initialement encodée dans le réseau hippocampo-cortical, créant une trace mnésique au sein de l’hippocampe (HIP). Au cours du temps, la trace est transférée au cortex préfrontal médian (CPFm), et notamment au cortex cingulaire antérieur (CCA). À la suite de lésion des noyaux reuniens et rhomboide (ReRh), réciproquement connectés à l’HIP et au CPFm, le souvenir spatial se forme normalement mais ne persiste pas dans le temps. Ainsi, nous avons évalué l’impact de la lésion ReRh sur la plasticité structurale sous-tendant la persistance du souvenir spatial. Des rats lésés ReRh ont été entraînés en piscine de Morris et testés pour un rappel récent (5j) ou ancien (25j). La plasticité structurale a été évaluée par coloration de Golgi dans l’HIP et le CPFm. La lésion ReRh n'avait aucun effet sur l’apprentissage et le souvenir récent, mais a altéré celui du souvenir ancien. Dans le CA1 des rats Sham, le nombre d'épines dendritiques a été augmenté aux deux délais (5 et 25j) post-acquisition comparé au niveau basal. Après la lésion, cette augmentation n’a pas persisté entre 5 et 25j. Dans le CCA des rats Sham, le nombre d'épines dendritiques a été augmenté uniquement à 25j comparé au niveau de base, une modification non observée chez les rats lésés. Ainsi, à la lésion des noyaux ReRh perturbe la plasticité structurale sous-tendant le souvenir spatial ancien indiquant un rôle crucial de ces noyaux dans l’établissement d’un souvenir persistant. / The standard model of systemic consolidation posits that information is initially encoded in the hippocampo-neocortical network, the memory trace being first created in the sole hippocampus (HIP). Over time, the trace is progressively transferred to modules of the medial prefrontal cortex (mPFC), particularly to the anterior cingulate cortex (ACC). Following lesions of the thalamic reuniens and rhomboid nuclei (ReRh), which are reciprocally connected with both the Hipp and mPFC, a spatial memory forms normally but does not persist (Loureiro et al 2012). Therefore, we assessed the impact of ReRh lesions on structural plasticity underlying spatial memory persistence. Male Long-Evans rats subjected to NMDA lesions of the ReRh nuclei were trained in the Morris Water Maze and tested for retrieval of recent (5 days) or remote (25 days) memory. Structural plasticity was assessed on Golgi-stained material in the HIP and CPFm. ReRh lesions had no effect on learning and recent memory, but altered remote memory. In the HIP (CA1) of sham-operated rats, the spine number was increased at both 5 and 25 days post-acquisition vs baseline. After ReRh lesion, the increase did not persist from 5 to 25 days. In the mPFC (ACC) of sham-operated rats, the spine number was increased only at 25 days vs baseline, a modification not observed in ReRh lesioned rats. Thus, following lesion of ReRh nuclei, structural plasticity underlying remote spatial memory formation does not operate correctly in the mPFC and Hip, pointing to a crucial role of ReRh in memory persistence.

This thesis examines the rapid, non-genomic effects of estrogens on neural plasticity and learning. Estrogens are classically known to affect gene transcription events, however they have more recently been found to also rapidly activate second messenger systems within 1hr of administration. Therefore, we first examined the rapid effects of 17β-estradiol, and an estrogen receptor (ER) α and ERβ agonist on three different learning paradigms: object placement, object recognition, and social recognition. We found that both systemic injections and intrahippocampal delivery of 17β-estradiol and the ERα agonist improved performance on all 3 learning paradigms within 40min of hormone administration. However, the ERβ agonist administered systemically or intrahippocampally, improved performance only on the object placement learning paradigm, while having no effect on object recognition, and impairing social recognition at high doses. To elucidate how estrogens might rapidly affect learning, we examined how estrogens rapidly affect the neural plasticity of CA1 hippocampal neurons. We found that 17β-estradiol and the ERα agonist increased dendritic spine density in CA1 hippocampal neurons within 40min of administration, suggesting that estrogens rapidly increase the density of synapses within this brain region. Conversely, the ERβ agonist did not affect spine density, or decreased spine density. In addition, by using whole-cell patch clamp recordings of CA1 pyramidal neurons, we were able to determine that 17β-estradiol and the ERα agonist rapidly reduced AMPA receptor (but not NMDA receptor) mediated membrane depolarizations after 15min of hormone application. Similar to above, the ERβ agonist had no effect on AMPA or NMDA receptor mediated membrane depolarizations. These data suggest that estrogens rapidly promote the development of immature synapses (which contain low levels of synaptic AMPA receptors) within the CA1 hippocampus. Immature spines provide synaptic sites at which new memories can be stored and are thought of as “learning spines” (Kasai et al, 2003). Therefore, estrogens (through ERα) may rapidly induce the formation of hippocampal immature spines to promote learning. / Funded by NSERC

Les changements des connections neuronales interviendraient dans la formation de la mémoire. J’ai développé de nouvelles approches basées sur l’imagerie photonique pour étudier (i) les interactions entre les microglies et les épines dendritiques, et (ii) le renouvellement des épines dans l’hippocampe in vivo. Ces deux phénomènes contribueraient au remodelage des circuits synaptiques intervenant dans la mémoire. (i) Les microglies sont impliquées dans de nouvelles fonctions en condition saine. J’ai examiné l’effet de la plasticité synaptique sur la dynamique morphologique des microglies, et sur leur interaction avec les épines. En combinant l’électrophysiologie et l’imagerie bi-photonique dans des tranches aigües de souris transgéniques, je démontre que la microglie intensifie son interaction physique avec les épines. Ainsi pour continuer l’étude de ces interactions et leur impact fonctionnel plus précisément, j’ai optimisé l’imagerie STED dans des tranches aigües. (ii) La plasticité structurale des épines est cruciale pour la mémoire, mais les connaissances à ce sujet dans l’hippocampe in vivo restent limitées. J’ai donc établi une technique d’imagerie chronique STED in vivo pour visualiser les épines dans l’hippocampe. Cette approche a révélé une densité double de celle reportée précédemment à l’aide de la microscopie bi-photonique. De plus j’ai observé un renouvellement des épines de 40% en 5 jours, représentant un taux important de remodelage synaptique dans l’hippocampe. Les approches d’imagerie super-résolutive permettent l’étude des interactions microglie-épine, et du renouvellement des épines hippocampiques avec une résolution inédite chez la souris vivante. / Activity-dependent changes in neuronal connectivity are thought to underlie learning and memory. I developed and applied novel high-resolution imaging-based approaches to study (i) microglia-spine interactions and (ii) the turnover of dendritic spines in the mouse hippocampus, which are both thought to contribute to the remodeling of synaptic circuits underlying memory formation. (i) Microglia have been implicated in a variety of novel tasks beyond their classic immune defensive roles. I examined the effect of synaptic plasticity on microglial morphological dynamics and interactions with spines, using a combination of electrophysiology and two-photon microscopy in acute brain slices. I demonstrated that microglia intensify their physical interactions with spines after the induction of hippocampal synaptic plasticity. To study these interactions and their functional impact in greater detail, I optimized and applied time-lapse STED imaging in acute brain slices. (ii) Spine structural plasticity is thought to underpin memory formation. Yet, we know very little about it in the hippocampus in vivo, which is the archetypical memory center of the mammalian brain. I established chronic in vivo STED imaging of hippocampal spines in the living mouse using a modified cranial window technique. The super-resolution approach revealed a spine density that was two times higher than reported in the two-photon literature, and a spine turnover of 40% over 5 days, indicating a high level of structural remodeling of hippocampal synaptic circuits. The developed super-resolution imaging approaches enable the examination of microglia-synapse interactions and dendritic spines with unprecedented resolution in the living brain (tissue).