Snarf 5 am

The inhibitor AR-C155858, tetrodotoxin citrate (TTX) and sulforhodamine 101 (SR101) were acquired from Tocris, Wiesbaden-Germany, Abcam Biochemicals, Cambridge, UK, and Baseclick, Tutzing, Germany, respectively. Fluo-4-AM, SNARF-5-AM, and pluronic acid were obtained from Invitrogen. The fluorescent dyes, pluronic acid, AR-C155858 and SR101 were dissolved in DMSO.

Primary cultures of Wistar rat pups (p2) cerebral astrocytes (adapted from62 ) transduced with AVV.sGFAP.optoβ₂AR, seeded onto coverslips, were loaded with either Rhod-2AM (30 μM; Invitrogen, UK) or SNARF-5AM (30 μM; Invitrogen, UK) 1 h before visualization. Rhod-2AM was used as a Ca²⁺ indicator to avoid spectral interference with EGFP. The pH indicator SNARF-5 responds to acidification with a shift in its emission towards shorter wavelengths. Light was collected from two channels set to 550–580 nm (channel 1) and 620–700 nm (channel 2). Ratio of channel 1: channel 2 is indicative of a pH change.
Imaging experiments were carried out at 34 °C under continuous superfusion (150 ml h^–1) with pH 7.4 HBS buffer in a tissue chamber mounted on a Leica-SP confocal microscope. Astrocytes were imaged in time-lapse mode (0.5 Hz) using the 543 nm laser line to excite Rhod-2 and SNARF-5. To activate optoβ₂AR- or ChR2-transduced astrocytes, a 470 nm light-emitting diode-illumination system (Rapp Optoelectronic) coupled to the microscope via the epifluorescence port was employed. Changes of [Ca²⁺]_i in individual cells were assessed by changes in relative fluorescence intensity (F/F₀).