Astrocytic modulation of the synaptic and the extrasynaptic space

Neuron-astrocyte interactions are now recognized as essential to brain functioning and information processing. They occur at multiple levels, at different cellular locations, and upon different physiological states. Hence, the general aim of this thesis was to unravel structural and molecular interactions of astrocytes with neurons that contribute to synaptic function and memory processing. To that end: In Chapter 2, I aimed to describe the implementation of a recently developed new technique, NAPA (neuron-astrocyte proximity assay), that measures and characterizes astrocyte leaflets proximity to synapses in living tissue. Hence, I present a stepwise protocol on how to perform and measure sensitized emission Förster resonance energy transfer (SE-FRET) in cell lines and acute brain slices. After in vitro testing in HEK293T cells, we used the approach to visualize and track the static and dynamic proximity of striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons (MSNs) in living tissue. In Chapter 3, I aimed to examine astrocyte leaflet-synapse spatial interaction in the hippocampus during the formation and consolidation of fear memories, and to determine whether this proximity is critical for synaptic transmission and fear memory expression. I manipulated astrocyte leaflets in vivo by depleting Ezrin, a leaflet-structural protein, using CRISPR-saCas9 gene editing. Next, I used multidisciplinary approaches, including FRET analysis, electron microscopy, genetically encoded glutamate sensors, whole-cell patch-clamp recordings and contextual fear conditioning to show that the apposition of astrocyte leaflets to the synaptic cleft influences contextual fear memory expression and gates neuronal activation in a context- and time-dependent manner. In Chapter 4, I aimed to characterize hippocampal high-affinity glutamate transporter (GLT-1) protein complexes by combining interactive proteomics approaches. We showed that GLT-1 form at least four stable distinct protein complexes that differ in size and protein composition. Next, we revealed that GLT-1 protein complexes are dynamically assembled during contextual fear conditioning, and differently regulated in the early stages of AD. In Chapter 5, I aimed to develop an automated high-throughput analysis method to measure reactive astrogliosis in vitro, and to determine the effect of nutritional supplementation (FC) on the induction of astrocyte reactivity by pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). We found that extracellular factors, such as extracellular matrix proteins, and culture media have a great influence on the development of the reactive phenotype. In addition, we showed that treatment with FC has an acute beneficial effect on cytokine-induced reactive astrogliosis. Taken together, with this research I deepened our understanding on the structural and molecular interactions of hippocampal astrocytes with neurons, and how this dynamic interplay contribute to synaptic function and mnemonic processing. Furthermore, this data sheds the light on the role of astrocytes in maladaptive/dysfunctional neuronal networks and strengthens the importance to therapeutically target astrocytes to tackle neurological disorders.