title: Comparative analysis of optogenetic actuators in cultured astrocytes.
abstract: Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, D-serine and L-lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca(2+) ([Ca(2+)]i) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca(2+) permeability (Ca(2+) translocating channelrhodopsin, CatCh). We show here that [Ca(2+)]i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca(2+) from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca(2+)]i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (Gs-coupled) resulted in astrocytic [Ca(2+)]i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca(2+)]i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes.
authors.affiliation: School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK.
authors.forename: Melina
authors.initials: M
authors.lastname: Figueiredo
authors.affiliation: School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK.
authors.forename: Samantha
authors.initials: S
authors.lastname: Lane
authors.affiliation: Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama, Birmingham, AL 35294, USA; The Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Randy F
authors.initials: RF
authors.lastname: Stout
authors.affiliation: School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK.
authors.forename: Beihui
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama, Birmingham, AL 35294, USA; Department of Biotechnology, University of Rijeka, 51000 Rijeka, Croatia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Parpura
authors.affiliation: School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK. Electronic address: anja.teschemacher@bristol.ac.uk.
authors.forename: Anja G
authors.initials: AG
authors.lastname: Teschemacher
authors.affiliation: School of Physiology and Pharmacology, School of Medical Sciences, University of Bristol, BS8 1TD, UK. Electronic address: sergey.kasparov@bristol.ac.uk.
authors.forename: Sergey
authors.initials: S
authors.lastname: Kasparov
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D000075402:Channelrhodopsins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D062308:Optogenetics; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015220:Calcium Channels; D000075402:Channelrhodopsins; D008164:Luminescent Proteins; D018340:Receptors, Adrenergic, alpha-1; D018343:Receptors, Adrenergic, beta-2; C054562:yellow fluorescent protein, Bacteria; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D002118:Calcium
keywords: Astrocyte; Calcium; CatCh; ChR2; OptoARs
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