Organotypic hippocampal slice cultures
6 to 7 day old APP transgenic (ArcAβ mice) and non-transgenic littermates were decapitated, brains were removed, hippocampi were isolated and cut into 400 μm thick slices. Three slices were placed per cell culture insert (Millipore). Slices were placed in a culture medium (minimum essential medium Eagle with HEPES modification, 25% basal medium with Earle’s modification, 25% heat-inactivated horse serum, 2 mM glutamine, 50 units per ml penicillin, 50 μg/ml streptomycin, 0.6% glucose, pH 7.2). Culture medium was exchanged every second or third day. On days in vitro (DIV) 11, culture medium was replaced by low-serum Nb-N2 medium (Neurobasal medium, 0.5% heat-inactivated horse serum, 2 mM glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, 0.6% glucose, 1×N2 supplement, pH 7.2) to ensure more defined condition during analysis. On DIV12, slices were infected with sindbis virus.
The following viral constructs were used for experiments: pSinRep5-EGFP, pSinRep5-EGFP-441wt tau. Infection of slices causes neuron-specific expression of EGFP or EGFP-coupled tau.
BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and memantine (3,5-Dimethyl-tricyclo[18.104.22.168,7]decan-1-amine hydrochloride) were purchased from Tocris. Cultures were treated in parallel to EGFP/EGFP-tau expression from DIV12-16.
Memantine and BAPTA (each prepared from the same batch as in the present study) are pharmacologically active at the used concentrations.
On DIV 16, cell culture supernatant from each well was collected for cytotoxicity analysis. In addition, slices were lysed (all three slices per well were pooled) in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% deoxycholate and 0.1% SDS, pH 8.0) containing phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Roche) and centrifuged at 5,000 g for 10 min at 4°C. Lysate and supernatant were frozen in liquid nitrogen and stored at -80°C until further use. Cytotoxicity was measured using CytotoxGlo assay (Promega), according to the manufacturer’s recommendations. EGFP and EGFP-tau signals were determined using microplate reader (Synergy HT; BioTek, Germany). Cytotoxicity assay luminescence was divided by EGFP or EGFP-tau signal to normalize toxicity to EGFP or EGFP-tau viral infection efficiencies.