Kato-Negishi and Kawahara homeostasis is crucial to AβP neurodegeneration (Mattson et al 1992; Fraser et al 1997). We previously showed that AβP[1–40] is directly incorporated into membranes excised from GT1–7 cells, which are derived from the tumorigenesis of murine hypothalamic neurons, and forms unregulated cat- ion-selective (including Ca2+) ion channels (Kawahara et al 1997). The characteristics of channels formed by AβP, ie, ‘amyloid channels’, on membranes of GT1–7 cells are similar to those of channels formed on artiﬁcial planar lipid mem- branes (Arispe et al 1993a, 1993b, 1996). Other neurotoxic AβP fragments such as AβP[25–35] and AβP[1–42], and the C-terminal fragment peptide of APP termed CT105 were also reported to form ion channels on artiﬁcial lipid bilayers or membranes of cultured neurons (Furukawa et al 1994; Mirzabecov et al 1994; Fraser et al 1996; Rhee et al 1998; Hirakura et al 1999). Furthermore, we have demonstrated that AβP[1–40], AβP[25–35] and AβP[1–42] cause the increase in intracellular Ca2+ level ([Ca2+]i) in GT1–7 cells (Kawahara et al 2000; Kawahara and Kuroda 2001; Kawahara 2004). Pretreatment with 2-amino-5-phosphonovalerate (D-APV; an antagonist of the N-methyl-D-aspartate (NMDA)-type gluta- mate receptor), tetrodotoxin (TTX; a Na+ channel blocker), or nifedipine (an L-type Ca2+ channel blocker) has no effect on the AβP-induced [Ca2+]i increase (Kawahara 2004). On the basis of these lines of evidences, we hypothesized that the ability of AβP to form unregulated ‘amyloid channels’ across neuronal membranes and the resulting disruption of calcium homeostasis explain the molecular mechanism of AβP neurotoxicity (Pollard et al 1995; Kawahara 2004). This mechanism of AβP neurotoxicity is similar to that of the tox- icity of toxins, venoms, or the complement system (Tomita et al 1992; Bechinger 1997; Kobayashi et al 2004). Here, we applied AβP[1–40] and/or AβP[25–35] to cultured hippocampal neurons and observed the spatiotem- poral changes in [Ca2+]i induced by AβP using a multisite Ca2+-imaging system with fura-2 as a ﬂuorescent probe. This system enables the simultaneous detection of temporal [Ca2+]i changes in cultured neurons facilitating statistical analysis. In this study, we investigated in detail the features of AβP- induced [Ca2+]i changes and observed heterogeneous cell- to-cell responses to AβP in cultured hippocampal neurons. The localization of AβP to the plasma membrane regions of the neurons was also observed by laser confocal micros- copy using immunohistochemistry after AβP exposure. Furthermore, we found that AβP caused an acute increase in [Ca2+]i in many long-term cultured neurons. However, changes in intracellular Ca2+ level after AβP exposure were rarely observed among the short-term cultured hippocampal 210 neurons. Therefore, the difference in susceptibility to AβP among neurons cultured for different periods was also examined. Thus, the search for substances that protect against AβP neurotoxicity is crucial. Our system for observing Ca2+ inﬂux induced by AβP has contributed to the search for such substances (Kawahara and Kuroda 2001). It requires a relatively short time for carrying out assays. The elevation of [Ca2+]i is considered to be the primary event of AβP neuro- toxicity. We previously demonstrated that several lipophilic substances such as phloretin, cholesterol, and 17β-estradiol signiﬁcantly inhibit the AβP-induced elevation of [Ca2+]i in GT1–7 cells (Kawahara and Kuroda 2001). Phloretin, a plant-derived ﬂavonoid, decreases membrane potential and inhibits the electrostatic interaction between AβP and membrane lipids (Hertel et al 1997). Cholesterol decreases membrane ﬂuidity and inhibits channel formation by peptides (Tomita et al 1992). Cholesterol also blocks the increase in [Ca2+]i induced by AβP in cultured neurons (Hartmann et al 1994). 17β-estradiol, a female hormone, is neuroprotec- tive and affects membrane ﬂuidity (Schwartz et al 1996). All these compounds inhibit AβP neurotoxicity (Zhou and Richardson 1996; Hertel et al 1997; Olivieri et al 2002). Therefore, substances that modulate membrane properties such as membrane potential and ﬂuidity may inhibit AβP neurotoxicity. In line with the search for neuroprotective agents, we focused on neurosteroids including dehydro- epiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), and pregnenolone. These neurosteroids are steroid hormones synthesized de novo in the central nervous system from cholesterol or peripheral steroid precursors. Several lines of evidence suggest that neurosteroids modulate various functions of the brain and exhibit neuroprotective activities (Tsutsui et al 2000). For example, DHEA-S could protect neurons from NMDA-induced neurotoxicity (Chara- lampopoulos et al 2006). In addition, there is an age-related decrease in the levels of neurosteroids (Moffat et al 2000), and the levels of neurosteroids in plasma or in the brain are decreased in Alzheimer’s patients (Hillen et al 2000; Marx et al 2006). Thus, neurosteroids have been recognized as anti-aging hormones, and are widely used as supplements for improving the impaired cognitive functions of the elderly (Huppert et al 2000). Experimental procedures Chemicals and reagents AβP[1–40] was obtained from Bachem F.A.G. (Bubendorf, Switzerland). AβP[25–35] was obtained from AnaSpec Inc. Neuropsychiatric Disease and Treatment 2008:4(1)