Kato-Negishi and Kawahara Figure 2 Heterogenic responses to AβP. A. Patterns of [Ca2+]i increase in 20 hippocampal neurons induced by AβP[1–40].Traces of temporal changes in [Ca2+]i in 20 ran- domly chosen cultured hippocampal neurons in the same ﬁeld of view were analyzed. Numbers represent the number of cells used in the analysis.Arrows indicate the time at which 10 µ M AβP[1–40] was added to the bath solution. B. Immunohistochemical identiﬁcation and localization of AβP[1–40] on cultured hippocampal neurons. Laser confocal microscopy images of cultured hippocampal neurons (29 DIV) after MAP2 binding (Texas Red, red) ((a) and (d)) and AβP binding (FITC, green) ((b) and (e)), and their superimposed images ((c) and (f)). Images (d)-(f) are images of the same areas shown in (a)-(c) at a higher magniﬁcation. Discussion We observed the spatiotemporal changes in [Ca2+] in cul- i tured rat hippocampal neurons induced by AβP[25–35] and AβP[1–40] induces changes in electrophysiological activities in 3- to 4-week-cultured rat cerebral cortical neurons (Hartley et al 1999). Although factors affecting the susceptibility of AβP[1–40], both exhibiting β-sheet formation and neurotoxic- neurons to AβP are under investigation, it is widely accepted ity. Our results indicate that long-term (ca 1 month) cultured that the properties of membrane lipids including membrane neurons are more susceptible to AβP than short-term (ca 1 ﬂuidity and the net charge of membrane surfaces are crucial to week) cultured neurons. We have also demonstrated that the peptide binding and the subsequent channel formation (Terzi responses to AβP[25–35] as well as to AβP[1–40] are highly et al 1994). In particular, the changes in membrane properties heterogeneous among neighboring cultured neurons and that such as the level ratio of cholesterol to other phopholipids AβP[1–40] has an afﬁnity to some type of neuron. Our results during the in vitro development of cultured hippocampal are in accordance with the previous results showing that neurons may be important. In particular, cholesterol decreases 214 Neuropsychiatric Disease and Treatment 2008:4(1)