Abstract
Astrocytes express a plethora of G protein-coupled receptors (GPCRs) that are crucial for shaping synaptic activity. Upon GPCR activation, astrocytes can respond with transient variations in intracellular Ca2+. In addition, Ca2+-dependent and/or Ca2+-independent release of gliotransmitters can occur, allowing them to engage in bidirectional neuron-astrocyte communication. The development of designer receptors exclusively activated by designer drugs (DREADDs) has facilitated many new discoveries on the roles of astrocytes in both physiological and pathological conditions. They are an excellent tool, as they can target endogenous GPCR-mediated intracellular signal transduction pathways specifically in astrocytes. With increasing interest and accumulating research on this topic, several discrepancies on astrocytic Ca2+ signalling and astrocyte-mediated effects on synaptic plasticity have emerged, preventing a clear-cut consensus about the downstream effects of DREADDs in astrocytes. In the present study, we performed a side-by-side evaluation of the effects of bath application of the DREADD agonist, clozapine-N-oxide (10 µM), on Gq- and Gi-DREADD activation in mouse CA1 hippocampal astrocytes. In doing so, we aimed to avoid confounding factors, such as differences in experimental procedures, and to directly compare the actions of both DREADDs on astrocytic intracellular Ca2+ dynamics and synaptic plasticity in acute hippocampal slices. We used an adeno-associated viral vector approach to transduce dorsal hippocampi of male, 8-week-old C57BL6/J mice, to drive expression of either the Gq-DREADD or Gi-DREADD in CA1 astrocytes. A viral vector lacking the DREADD construct was used to generate controls. Here, we show that agonism of Gq-DREADDs, but not Gi-DREADDs, induced consistent increases in spontaneous astrocytic Ca2+ events. Moreover, we demonstrate that both Gq-DREADD as well as Gi-DREADD-mediated activation of CA1 astrocytes induces long-lasting synaptic potentiation in the hippocampal CA1 Schaffer collateral pathway in the absence of a high frequency stimulus. Moreover, we report for the first time that astrocytic Gi-DREADD activation is sufficient to elicit de novo potentiation. Our data demonstrate that activation of either Gq or Gi pathways drives synaptic potentiation through Ca2+-dependent and Ca2+-independent mechanisms, respectively.
Highlights
Astrocytes are one of the most abundant resident cells of the brain, well known for providing metabolic support, ensuring homeostasis, and maintaining the blood–brain barrier
Viral vector transduction Astrocyte-specific expression of the designer receptors exclusively activated by designer drugs (DREADDs) was obtained using an adeno-associated viral (AAV) vector expressing either the Gq- or Gi-DREADD fused to the red fluorescent protein mCherry under the GFAP promotor (Gq-DREADD: vector AAV8-GFAP-hM3DqmCherry, diluted 1:50 with sterile saline with a final concentration of 2.96 × 1012 gc/ml, plasmid #50478 acquired from Addgene; Gi-DREADD: vector AAV8-GFAPhM4Di-mCherry, diluted 1:50 with sterile saline with a final concentration of 2.46 × 1012 gc/ml, plasmid #50479 acquired from Addgene); or the control vector AAV8GFAP-mCherry
In the past few years, chemogenetic approaches have been used to evaluate the crucial role of astrocytes in higher brain functions and this has drastically broadened our understanding of these star-like cells
Summary
Astrocytes are one of the most abundant resident cells of the brain, well known for providing metabolic support, ensuring homeostasis, and maintaining the blood–brain barrier. It is of utmost importance to understand how astrocytes tune neuronal activity and modulate higher brain functions, as this can aid in the discovery of potential novel therapeutic strategies for multiple brain disorders. It is well-established that increases in astrocytic Ca2+ transients can be elicited by activation of Gq-coupled GPCRs [3, 10,11,12,13]. A reduction in cAMP has been reported upon Gi-GPCR activation in astrocytes [15], suggesting that these receptors are coupled to the expected Gαi/o protein. There is ample evidence showing an increase in astrocytic Ca2+ transients upon Gi-GPCR activation, such as activation of G ABABRs and dopamine D1 and D2-type receptors in the hippocampus [18,19,20,21],
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