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.