Neurosteroids block AβP neurotoxicity Figure 1 Patterns of [Ca2+]i increase in hippocampal neurons cultured at 8 DIV and 35 DIV induced by AβP[25–35]. A. Temporal changes in [Ca2+]i in 10 randomly chosen cultured hippocampal neurons at 8 DIV (a) and 35 DIV (b) in the same optical ﬁeld were analyzed,and their traces are shown. Arrows indicate the time when 20 µ MAβP[25–35] was added to the bath solution. B. Frequency histograms of extent of [Ca2+]i increase at 8 DIV (a) and 35 DIV (b).The vertical axes represent the percentage of neurons responding to AβP, and the horizontal axes represent the amplitude of the peak [Ca2+]i increase (bin = 20 nM). were observed using the same protocol. DHEA, DHEA-S, or pregnenolone at 25 µ M signiﬁcantly inhibited the mean increase in [Ca2+]i induced by AβP[25–35] (Figure 3). The mean increases in [Ca2+] in cultured neurons pretreated with i DHEA, DHEA-S, or pregnenolone were 50 ± 9.2%, 0.7 ± 0.5%, 13 ± 4% that of the control (neurons pretreated with 0.5 % DMSO), respectively (mean ± SEM, n = 200) (Figure 3A). The percentages of neurons responding to AβP among those pretreated with DHEA, DHEA-S, and pregnenolone also decreased (Figure 3B). The inhibition by DHEA-S was more marked than those by DHEA or pregnenolone. Figure 4A shows typical Neuropsychiatric Disease and Treatment 2008:4(1) responses to AβP[25–35] (20 µ M) with (b) or without (a) DHEA-S pretreatment (10 µ M) in the same view ﬁeld. Figure 4B shows the histograms constructed from measurements of the peak [Ca2+]i increase in each neuron. Figures 4C and 4D show the dose-dependent effects of DHEA-S on the mean increase in [Ca2+]i and the percentage of responding neurons induced by AβP[25–35], respectively. DHEA-S at 1 and 10 µ M inhibited the mean [Ca2+]i changes to 58 ± 15% and 35 ± 7% that of control (pretreated with 0.5% DMSO) neurons, respectively (mean ± SEM, n = 250). In the case of using DHEA-S at more than 25 µ M, most neurons exhibited no signiﬁcant increase in [Ca2+]i. 213