Metafluor

Calcium imaging experiments were performed on acute tissue slices of GG according to Brechbühl et al. (2008 (link), 2013b (link)). Animals were dissected in fresh ACSF solution at 4°C. The tips of the noses were included in blocks of 4–5% agar at a temperature <41°C and directly placed on ice for solidification. Coronal slices from 60 to 80 μm were cut and collected in ACSF at 4°C with a vibroslicer (VT1200 S, Leica). They were selected under a stereomicroscope (M165 FC, Leica) based on their general morphology, loaded in a solution of 5 μM Fura-2 acetoxymethyl ester (Fura-2AM; #0103, TEFLabs) containing 0.1% of Pluronic® F-127 (#P-6867, Invitrogen) for a minimum of 45 min in an incubator (37°C, 5% CO₂). Slices were placed in a RC-26G bath chamber (#64-0235, Warner Instruments) and immobilized with a slice anchor. Observations were made under an inverted fluorescence microscope (Axiovert 135, Zeiss) with a 25× or 63× objective and a Cool SNAP-HQ camera. A bipolar temperature controller (SC-20/CL-100, Warner instruments) was used to control the bath temperature. The MetaFluor® system (MetaFluor, Visitron Systems) was used to monitor Fura-2AM ratio (F340/380 nm) and to acquire images (Brechbühl et al., 2011 (link)).

As described in Brechbühl et al. [3 (link)], pups (post natal days P0–P7) were killed by decapitation, and the GG regions were removed and carefully placed in artificial cerebrospinal fluid (ACSF), consisting of 118 mM NaCl, 25 mM NaHCO₃, 10 mM d-glucose, 2 mM KCl, 2 mM MgCl₂, 1.2 mM NaH₂PO₄, 2 mM CaCl₂ (pH 7.4), and gassed with oxycarbon (95% O₂, 5% CO₂). They were embedded in 5% agar prepared in phosphate-buffered saline (PBS), and acute 80-μm tissue slices were then processed with the microtome. The tissue slices were then loaded with a solution of 0.1% Pluronic (F-127, Invitrogen) and 5 μM Fura-2 AM (TEFLabs) in ACSF for 45 min. The slices were then placed in the bath chamber (RC-26, Warner Instruments). Calcium variations were observed with an inverted microscope (Axiovert 135, Zeiss) using a 63× objective and a CoolSNAP HQ (Photometrics) camera. MetaFluor (Visitron) software was used to monitor calcium variations.