Final Report Summary - ASTROSYN (Roles of astrocytes in synaptic transmission and plasticity) A. Project title: Roles of astrocytes in synaptic transmission and plasticityB. Summary of the project goal:In the central nervous system (CNS), astrocytes are the most numerous glial cell type. They contact most other cell types in the CNS, and the processes of a single astrocyte can envelop as many as 140,000 synapses. Thus, these cells are uniquely able to receive signals from many sources, in particular synapses, via two types of their G protein-coupled receptors (the Gq GPCRs and Gs GPCRs). Additionally, using conventional pharmacological approaches, it has been suggested that activation of astrocyte Gq GPCRs can lead astrocytes to send signals to neurons by release of various transmitters (glutamate, adenosine, D-serine) to modulate synaptic transmission and plasticity. This reciprocal signaling between neurons and astrocytes (gliotransmission) is still a novel concept, and its physiological relevance remains debated in the field. Furthermore, the main energy reserve of the brain is glycogen, which is almost exclusively localized in astrocytes of the adult brain. Breakdown of glycogen leads to increased lactate production in astrocytes, which is then used as an energy source by neurons, and is thought to support synaptic transmission and memory formation. Importantly, activation of the enzymes involved in glycogen catabolism is downstream of Gs GPCR activation. However, these pieces of evidence are mainly derived from studies on cultured astroglia, and its relevance remains to be established in vivo. The main goal of this project has been to understand better the role of astrocytic Gq GPCRs and Gs GPCRs in synaptic transmission and metabolism in the CNS in vivo.C. Description of the work performed to achieve the project objectives, main results, and conclusions:Over the 4-year reintegration period, my new group has focused on addressing the main specific objectives of the project. 1- First, using immunohistochemistry, we characterized at the cellular level all our new AAV-based pharmacogenetic tools (to activate Gq and Gs GPCR signaling) in mice. We found that they are selectively expressed in astrocytes and not in other neural cell types. Thus these tools can be used to selectively activate astrocyte signaling.2- Second, using 2-photon calcium imaging and biochemistry, we tested the functionality of our tools in astrocytes of intact tissues (ex vivo or in vivo). We found that activation of both tools induces mobilization of corresponding downstream signaling messengers, indicating that our pharmacogenetic tools are functional in astrocytes. Thus, we validated the use of our pharmacogenetic tools to address the role of astrocytic Gq and Gs GPCR signaling in synaptic transmission and metabolism in vivo.3- Third, combining these tools with in vivo electrophysiology, we found that activating Gq GPCR calcium (Ca2+) signaling in astrocytes of the visual cortex in awake mice does not statistically modify cortical synaptic transmission. These results suggest that activating astrocyte Gq GPCR does not lead to the release of gliotransmitters to modulate synaptic transmission in vivo or evoke long-term potentiation (LTP). Our data do not support previous findings from two recent exciting in vivo studies that have reported that activation of Gq GPCR Ca2+ in cortical or hippocampal astrocytes induces D-serine or glutamate release from astrocytes leading to long-term potentiation (LTP); therefore other factors must be involved to allow the full initiation of LTP. We reasoned that LTP induction found in previous studies may not rely solely on activation of astrocytic Gq GPCRs but rather that concomitant neuronal and astrocytic Gq GPCR activation may be needed for LTP to occur. We thus tested this hypothesis. Our results showed that neither activating neuronal Gq GPCR nor activating both neuronal and astrocytic Gq GPCR trigger cortical LTP in awake mice. In fact, we found that activating neuronal Gq GPCR induces a depression of synaptic transmission (but not a potentiation), which was not modulated by concomitant astrocytic Gq GPCR activation. An article reporting this work has been submitted and we hope it will be accepted for publication in 2017.4- Fourth, using biochemistry and immunohistochemistry, we found that acute in vivo activation of astrocyte leads to the modulation of enzymes involved in glycogen catabolism, an indication that glycogen may be breakdown into lactate downstream of astrocyte Gs GPCR signaling in vivo. To test this hypothesis, we used amperometry in vivo and observed that astrocyte Gs GPCR activation leads to lactate release. This piece of evidence is important because it is the first in vivo demonstration that activation of astrocytic Gs GPCR triggers glycogen catabolism and lactate release into the extracellular space. An article reporting this work is in preparation.5- Fifth, because we found that acute activation of astrocyte Gq and Gs GPCR signaling does not modulate synaptic transmission in vivo, we decided to stop investigating further this question. As a contingency plan we thus started an alternative aim, which addresses the role of chronic activation of astrocytic Gq and Gs GPCRs on synapse number and maturation. So far, our preliminary results have shown that chronic activation of astrocytic GPCR pathways leads to both the modulation of the number of cortical synapses. Such results have been the starting point of a new project in the lab for which one of my master students was awarded a governmental Ph.D fellowship to development this project. D. Potential socio-economic impact and societal implications of the project: Having addressed the aims of this project has profound implications for researchers in the area, but also and importantly, in the clinic. One of our most significant results shows for the first time that acute activation of astrocyte GPCR signaling leads to lactate production in vivo. Because there is evidence that lactate is an important element for LTP and memory formation, our results may lead to potential treatments (targeting astrocytes and their GPCRs) of diseases, including Alzheimer disease and memory disorders. In conclusion, we believe that our data have helped resolve some important aspects of astrocyte function and controversies, and have led to new questions that we are currently addressing.Contact details:Cendra Agulhon, Ph.DGroup Leader,Glia-Glia & Glia-Neuron Interactions GROUPCNRS FR3636 – Paris Descartes UniversityParis – FranceE-mail: cendra.agulhon@parisdescartes.frPhone : +33 1 42 86 40 08
