Briefly, a 25 mm glass coverslip (thickness, 0.08 mm) was glued over a 22 mm hole in the bottom of a 35 mm tissue-culture dish using silicone sealant. Dissociated neuronal cultures from rat hippocampi at P1 and P2 were prepared. Neurons were plated onto prepared 35 mm tissue culture dishes at a density of 1 × 106 cells per dish. The age of cultured neurons

was counted from the day of plating (1 DIV). Neurons at 7–10 DIV were transfected using a standard calcium phosphate precipitation method and allowed to grow to maturity (>3 weeks) to be imaged. Neurons 5 FU were transfected with equivalent concentrations of plasmids encoding one of six different GFP-tagged htau constructs (WT, P301L, AP, AP/P301L, E14, or E14/P301L htau). Neurons transfected with htau constructs were cotransfected with DsRed to visualize dendritic spines. Some neurons were transfected with GFP alone to visualize dendritic spines. The culture dishes fit tightly in a homemade holding chamber on a fixed platform BI 2536 solubility dmso above an inverted epifluorescent microscope sitting on an X–Y translation stage (Burleigh Instruments, Fishers, NY). Following the protocol described in Strasser et al. (2004), hippocampal and glial cultures were prepared from E16 and P1–2 mice, respectively. Glial cultures prepared from P1–2 mouse cortices were plated on tissue culture dishes in glial plating media (minimal essential medium [MEM] with Earle’s salts, 10%

fetal bovine serum [FBS] or newborn calf serum, 2 mM glutamine, 10 mM sodium pyruvate, 10 mM HEPES, 0.6% glucose, 100 U/ml penicillin and 100 μg/ml streptomyocin). For primary hippocampal neuron cultures, approximately 1.5 × 104 cells were plated on sets of 5 × 12 mm coverslips that had been previously coated with Poly-D-Lysine (100 μg/ml) + laminin (4 μg/ml) in neuronal plating media (MEM with Earle’s salts, 10 mM HEPES, 10 mM sodium pyruvate, GPX6 0.5 mM glutamine, 12.5 μM glutamate, 10% FBS, and 0.6% glucose). Each set of 5 coverslips was maintained in a 35 mm dish and each dish corresponded to 1 mouse. Approximately 4 hr after plating, the media was replaced with either neuronal growth medium

(Neurobasal media with B27 supplement, 0.5 mM glutamine) that had been conditioned on glia for 24–48 hr immediately prior to use. Mice were genotyped by PCR analysis of tail snip lysates using transgene-specific primers. Miniature EPSCs were recorded from cultured dissociated rat and mouse hippocampal neurons at 22–30 DIV with a glass pipette (resistance of ∼5 MΩ) at holding potentials of −55 mV and filtered at 1 kHz as previously described (Liao et al., 2005). Input and series resistances were checked before and after the recording of mEPSCs, which lasted 5–20 min. There were no significant difference in the series resistances and input resistance among various groups of experiments. One recording sweep lasting 200 ms was sampled for every 1 s.