Kato-Negishi and Kawahara the number of cells/bin as a function of peak [Ca2+] . We i obtained both the mean amplitude of the early peak [Ca2+]i for each neuron and the mean percentage of cells responding to AβP. Data are expressed as means ± standard error of mean (SEM) of 25 to 50 neurons in one optical ﬁeld. All statistical evaluations were carried out using a two-tailed Student’s t- test using Stat View (SAS Institute, NC, USA). A probability level of 0.05 was considered to be signiﬁcant. Results Effects of culture period on AβP-induced [Ca2+] changes i We applied AβP[25–35] (20 µ M) solution to rat hippo- campal neurons at various culture periods and observed the time course of [Ca2+]i. Little or no changes were observed in [Ca2+]i after AβP exposure in short-term cultured hip- pocampal neurons. On the other hands, a large proportion of long-term cultured neurons exhibited a marked increase in [Ca2+]i. Figure 1A shows traces of the temporal changes in [Ca2+]i induced by 20 µ M AβP[25–35] at 8 DIV and 35 DIV in 10 randomly chosen neurons in one optical ﬁeld. The extent of increase in [Ca2+]i induced by AβP was differ- ent among cultured neurons even in the same optical ﬁeld. Therefore, we obtained the amplitude of the peak [Ca2+]i increase (∆ [Ca2+]i) in each neuron and the percentage of neurons responding to AβP in an optical ﬁeld for statistical analysis. Figure 1B shows the histograms constructed from measurements of ∆ [Ca2+]i in each neuron at 8 DIV and 35 DIV. In the panels, the vertical axes represent the percent- age of neurons responding to AβP, and the horizontal axes represent the amplitude of ∆ [Ca2+]i (bin = 20 nM). From these histograms, we obtained the mean amplitude of the increase in [Ca2+]i over basal [Ca2+]i (∼100 nM) in hippocampal neu- rons and the proportion of cultured neurons (in percentage) that responded with a characteristic [Ca2+]i increase. At 8 DIV, the percentage of cells responding to AβP was 8 ± 7 % (mean ± SEM, n = 5), and the mean [Ca2+]i increase was 19 ± 3 nM (mean ± SEM, n = 250). On the other hand, at 35 DIV, the same AβP[25–35] concentration increased [Ca2+]i to 311 ± 37 nM (mean ± SEM, n = 250) in 79 ± 3% (mean ± SEM, n = 5) of neurons in the same optical ﬁeld. Heterogeneity of cell-to-cell responses to AβP Further detailed analysis of the elevation of [Ca2+]i in many neurons in the same optical ﬁeld revealed that the type of response of one neuron to AβP was not identical to that of 212 another neuron and that not all neurons exhibited [Ca2+]i increase after the AβP addition. As shown in Figure 1A (b), the cell-to-cell responses to AβP were highly heterogeneous among neighboring neurons. The variability in neuronal responses is similar to our previous work using genetically identical GT1–7 cells, which exhibit heterogeneous responses after their exposure to AβP[25–35] as well as AβP[1–40] (Kawahara et al 2000; Kawahara and Kuroda 2001). The responses of 20 randomly chosen neurons in the same ﬁeld of view before and after the application of AβP[1–40] were ana- lyzed and their traces are shown in Fig. 2A. The traces were aligned to the time of AβP[1–40] application. The magnitude of the peak [Ca2+]i increase, latency (the time of onset of the ﬁrst detectable increase in [Ca2+]i from the addition of AβP), and the pattern of [Ca2+]i changes differed among neurons. Some neurons showed an acute and a transient increase in [Ca2+]i within 5 min after the AβP[1–40] exposure (eg, cell nos. 6, 9, 10, 12, and 20 in Figure 2A), whereas [Ca2+] in other i neurons remained high at least 5 min after the exposure (cell nos. 2, 7, and 16). The delayed elevation of [Ca2+]i after the AβP exposure was observed in cell nos. 13, 15, 17, and 18. We observed no detectable elevation of [Ca2+] in neighboring i neurons (eg, cell nos. 4 and 14). Furthermore, we applied AβP[1–40] at a sublethal level (2.5 µ M) to cultured hippocampal neurons and observed AβP binding to membrane surfaces. Cultured neurons were ﬁxed and double-immunostained using antibodies to AβP and MAP2, a neuronal marker. Fluorescence images were observed under a laser confocal microscope. Figure 2B shows that the afﬁnities of AβP to neuronal membrane surfaces are also highly heterogeneous. AβP[1–40] was found to deposit on somata and dendrites in some neurons (labeled by asterisks in Figures 2B (a) and (d)); however, we found no detectable AβP[1–40] deposition on surfaces of neighbor- ing neurons. Note that the morphological characteristics of these neurons were similar. Figures 2B (d)-(f) show higher- magniﬁcation images of the same area shown in Figures 2B (a)-(c). The AβP[1–40] binding to neurons (asterisks) is not homogeneous. Moreover, AβP localizes to restricted areas of the somata and dendrites (Figure 2B (e)), whereas MAP2 is homogeneously distributed (Figure 2B (d)). Protection against AβP-induced [Ca2+]i increase by neurosteroids We pretreated hippocampal neurons at 30–36 DIV with solutions of neurosteroids, namely, DHEA, DHEA-S, or pregnenolone to. After 20–30 min, cultured neurons were exposed to AβP[25–35] (20 µ M) and the changes in [Ca2+]i Neuropsychiatric Disease and Treatment 2008:4(1)