Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C87
Are astroglial Gq-protein coupled receptors functionally relevant?
S. Lane1, N. Marina2, A. V. Gourine2, A. G. Teschemacher1, S. Kasparov1
1. School of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom. 2. Neuroscience, Physiology & Pharmacology, University College London, London, United Kingdom.
It is widely accepted that astrocytes release gliotransmitters to modulate synaptic transmission and that this release is at least partially Ca2+-dependent. However, recent publications (1,2) have reported that in two mouse models, MrgA1+ and IP3R2 knockout, Gq protein-coupled receptor (GqPCR) mediated Ca2+ signalling in astrocytes does not affect synaptic transmission or short- and long-term plasticity. These observations questioned the importance of signalling via GqPCR-activated pathways in astrocytes. We have used optogenetics to determine if GqPCRs on astrocytes are functionally relevant. An adenoviral vector (Ad.BS.GfaABC1D.Optoα1.skip.Gal4p65) was generated which employs an enhanced(3) shortened glial fibrillary acidic protein promoter to express an opsin-GqPCR chimera - Opto-α1 adrenergic receptor (Optoα1AR) specifically in astrocytes. To verify that this construct activates phospholipase C (PLC), cultured primary rat astrocytes were transduced with Ad.BS.GfaABC1D.Optoα1.skip.Gal4p65. 48 hours later, they were loaded with the Ca2+indicator Rhod-2AM, superfused with HBSS (34oC) and imaged using Leica SP1 confocal microscope. Stimulation with 470nm light, induced rapid increases in [Ca2+]i (fluorescence increase +187.2%±13.91, n=36 cells from 6 cover-slips). The PLC inhibitor U73122 (10µM) blocked light-induced [Ca2+]i elevations in Optoα1AR-transduced astrocytes (-2.51±0.8%,n=10 cells from 6 cover-slips, p<0.001). These data confirm that Optoα1AR signals through PLC. Next, we investigated if activation of Optoα1AR can induce responses in nearby neurones. We focused on the locus coeruleus (LC), the main noradrenergic (NAergic) nucleus innervating the forebrain. We engineered a viral vector (Ad.SuperI.PRSx8.TN-XXL) to drive expression of a Ca2+ indicator, TN-XXL (4) in NAergic LC neurones. Organotypic rat brainstem slices containing LC were prepared using methods described previously (5) and transduced with Ad.SuperI.PRSx8.TN-XXL and Ad.BS.GfaABC1D.Optoα1.skip.Gal4p65. Stimulation of astrocytes with 445 nm light induced rapid increases in the YFP/CFP ratio in TN-XXL-expressing LC neurones (+58%±3.5,n=25 cells in 10 slices, p<0.01), indicating that they were activated by light-stimulated astrocytes. In vivo, optogenetic activation of Optoα1AR expressed in the rostral ventrolateral medulla, an area containing catecholaminergic neurones involved in blood pressure control, triggered robust cardiovascular and sympathetic responses in α-chloralose anaesthetised rats (100mg/kg I.V, supplemented with 20 mg/kg as required). These experiments indicate that signalling mediated by GqPCRs in astrocytes is physiologically relevant and may be important in central autonomic control.
Where applicable, experiments conform with Society ethical requirements