(CA, USA). These peptides were dissolved in distilled water at 0.2 mM and stored at −80 °C. DHEA-S, DHEA, and preg- nenolone were obtained from Sigma Ltd. (St. Louis, USA), and dissolved in dimethylsulfoxide (DMSO) at 10 mM. Cell culture The hippocampus was dissected out from fetal rats (18-day embryonic stage) and neurons were cultured by the method of Banker and Cowan with slight modiﬁcations (Banker and Cowan 1977; Muramoto et al 1993). Brieﬂy, after the removal of meninges, tissues were dissociated by mechanical trituration following digestion with 0.15 units/ml papain in phosphate-buffered saline (PBS) containing 0.02% L-cystein, 0.02% BSA, and 0.5% glucose. Dissociated neurons were placed on polyethylenimine-coated coverslips and cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) supple- mented with 5% horse serum, 5% newborn calf serum, and 1 mM sodium pyruvate. The culture was maintained in a humidiﬁed atmosphere of 93% air and 7% CO2 at 37 ° C. After 3 days in vitro, the medium was replaced with serum-free DMEM supplemented with B27 (Gibco BRL, USA). The medium was changed every 3 days. Intracellular free-calcium concentration measurements The culture medium was replaced with a basal salt solution (BSS; in mM: 130 NaCl, 5.5 glucose, 5.4 KCl, 1.8 CaCl , 2 20 sodium HEPES, pH 7.4) containing 1.5 mM fura-2 AM (acetoxymethylester cell-permeant form, Molecular Probes, Oregon, USA). The cultured neurons were incubated with fura-2 AM at 37 °C for 60 min, washed and replaced with BSS containing 1 µ M TTX to inhibit the endogenous changes in [Ca2+]i related to spontaneous synaptic ﬁrings (Robinson et al 1993), and then observed under an inverted ﬂuorescence microscope (Nikon, Japan) equipped with a high-resolution video camera (C-2400, Hamamatsu Photonics Co., Japan). Fluorescence images correspond to [Ca2+]i were recorded at a rate of 30 images/sec on VCR tapes and analyzed using an ad- hoc hardware and software system controlled by a computer (FC-300, Mitsubishi Kasei, Japan). Each image represents the ﬂuorescence intensity from a rectangular optical ﬁeld (360 × 420 mm2), which enabled the monitoring of approximately 50 neurons simultaneously. The system enabled the recording of the ratio of the intensity emitted at 510 nm from pixels of images obtained by alternating the excitation wavelength between 340 and 360 nm using a rotating ﬁlter wheel. Each ratio was converted into [Ca2+]i using a calibration curve (ratio versus [Ca2+] ). i Neuropsychiatric Disease and Treatment 2008:4(1) Neurosteroids block AβP neurotoxicity As a rule, prior to AβP addition to the bath solution of the cells, we recorded the resting levels of neuronal [Ca2+]i during a 5-min period. During the experiments, we added aliquots of the peptide stock solution directly to the bath solution (approximately 0.4 ml) of the cells. All recordings were obtained at 37 °C. Immunohistochemistry AβP[1–40] (2.5 µ M) was applied onto hippocampal neurons cultured for 29 days in vitro (DIV). After 48 h, the cultured neurons were ﬁxed with 4% paraformaldehyde in Ca2+- and Mg2+-free PBS. After ﬁxation, the cultured neurons were incubated in PBS containing 0.5% saponin (Sigma Aldrich, St. Louis, USA) and 5% normal goat serum (Vector, UK) at 37 °C for 30 min to render the cell membranes perme- able and to block nonspeciﬁc binding sites. The cultured neurons were incubated with primary antibodies diluted with 10% Block Ace (Dai-nihon Seiyaku, Japan) in PBS at 4 °C overnight. For double-immunoﬂuorescence staining, a combination of a mouse monoclonal antibody and a rabbit polyclonal antibody was added to the cell preparation. An anti-microtubule associated protein 2 (MAP2) antibody (1:500, monoclonal, obtained from Boehringer Mannheim, Darmstadt, Germany) and an anti-AβP antibody (1:100, polyclonal, anti-N terminus of human AβP[1-40], obtained from IBL, Tokyo, Japan) were used as primary antibod- ies. Thereafter, the cells were incubated with anti-mouse Ig, biotinylated species-speciﬁc whole antibody (1:500, Amersham Pharmacia Biotech Ltd., Buckinghampshire, England), and then with an FITC-conjugated goat anti-rabbit IgG antibody (1:500, Cappel) and Texas red-conjugated streptavidin (1:500, Bio-Rad Laboratories, California, USA). Stained cells were observed under a confocal laser scanning microscope (LSM 510; Zeiss). Pretreatment with neurosteroids Prior to the 20–30 min exposure to AβP, the solutions of DHEA-S, DHEA, and pregnenolone were diluted with BSS and were added to the bath solution of the neurons. There- after, we carried out the same protocol designed to test the effects of AβP. As a control, 0.5% DMSO was applied to the cultured neurons, followed by 20 µ M AβP[25–35]. Statistical analyses Our system enabled the accurate measurement of the ampli- tude of the peak [Ca2+]i increase (∆ [Ca2+]i) in the response. To construct histograms from these data, we grouped the cell responses in bins (bin width, 20 nM) and then plotted 211