2.3 DHPG stimulation of slices to measure Arc protein expression.
For the DHPG stimulation experiments, brain slices were prepared from WT (n = 4) and ArcKR (n = 4) mice as described in the hippocampal slice preparation section. Hippocampal slices from two mice were pooled together to obtain sufficient tissue to perform an experimental repeat. Hippocampi were isolated from the surrounding tissue, area CA3 was removed, and hippocampal slices were maintained at 34°C bubbled with 95% O2 and 5% CO2for 2-3 hours in aCSF (1 mM MgCl2, 2 mM CaCl2) before use. Hippocampal slices for each experimental group were then incubated in aCSF (1 mM MgCl2, 2 mM CaCl2) containing 50 µM picrotoxin to block GABAA receptors (Tocris) and the NMDA receptor antagonist L-689,560 (5 µM; Tocris) for 30 min followed by incubation with vehicle (control), DHPG (100 µM) to induce LTD or DHPG (20 µM) to prime mGluRs. After 10 min incubation, DHPG was washed out and the slices left to rest for another 30 min before the slices from different groups were homogenized in Eppendorf vials with a pellet pestle in ice-cold solution composed of: 1 mM EDTA, 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM Sodium Orthovanadate, 50 mM Sodium Fluoride, Sodium pyrophosphate, 0.27 M Sucrose, 20% NaN3 and protease inhibitor cocktail (Roche) and rotated for 1 h at 4°C. Homogenate was centrifuged at 13,000 g for 15 min, the supernatant collected and protein levels determined (BCA protein assay kit, Thermo Scientific). Western blotting was performed as previously described (Eales et al. , 2014; DaSilva et al. , 2016; Wall et al. , 2018). Membranes were probed with rabbit anti-Arc (Synaptic Systems, 1:1,000) and mouse anti- Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, ab8245, 1:5,000) antibodies followed by goat anti-Rabbit IgG-HRP H+L (Cell Signaling, 1:10,000) and goat anti-Mouse IgG HRP LC (Jackson ImmunoResearch, 1:20,000) secondary antibodies. GAPDH was used as loading controls. Blots were imaged and analysed using the ChemiDocTM MP Imaging System (Bio-Rad) and volume intensity of each band was calculated using the Image Lab 5.2.1 software.