The deposition of amyloid β peptide (Aβ) in the CNS is a major feature of Alzheimer’s disease. As Aβ peptides are toxic to neuronal cultures, we set out to explore of the underlying cellular processes that lead to toxicity. In the present series of experiments, we examined the action of Aβ on [Ca²⁺]_c homeostasis in cultures consisting of mixed glia and neurons or monocultures of astrocytes prepared from the hippocampi or cortices of neonatal rats which were humanely killed according to Home Office Guidelines (Vergun et al. 1999).

Cells were loaded with the [Ca²⁺] indicator fura-2 AM and images acquired using a cooled CCD camera. Aβ was applied either as the native peptide (amino acids 1-42) or as the 25-35 peptide fragment, while the reverse peptide fragment (35-25) served as a control. After a delay of ~5-10 min, both Aβ 25-35 and 1-42 (1-50 µM) increased [Ca²⁺]_c in cortical and hippocampal astrocytes in monoculture or in co-culture with neurons, but had no effect on [Ca²⁺]_c in neurons in the same experiments. The responses consisted of sporadic increases in [Ca²⁺]_c seen as either low amplitude (100-200 nM) [Ca²⁺]_c fluctuations or larger sustained increases in [Ca²⁺]_c (1-2 µM). The reverse peptide Aβ 35-25 had no effect. The Aβ-induced [Ca²⁺]_c signals persisted for hours after washout of Aβ. The [Ca²⁺]_c response to Aβ was entirely dependent on external calcium (n = 231), and neither U73122 (5 µM; n = 89), an inhibitor of PLC, nor 2-APB (40 µM; n = 107), an inhibitor of IP₃ Ca²⁺ channels, had any effect on the amplitude or shape of the [Ca²⁺]_c signals. Moreover, depletion of ER stores with the SERCA inhibitor thapsigargin did not prevent changes in [Ca²⁺]_c in response to Aβ (n = 301). The response was not affected by inhibitors of either ionotropic or metabotropic glutamate receptors, including CNQX, MK-801 or (S)MCPG (n = 178). However, responses were blocked by Zn²⁺ (1 mM, n = 253), which inhibits Aβ-induced pore formation (Lin et al. 2001) and by clioquinol (1 µM; n = 207), a chelator of heavy metal ions which prevents the formation of Aβ in complex with Cu²⁺ and Fe³⁺ (Cherny et al. 2001).

These data strongly suggest that Aβ causes [Ca²⁺]_c signals in astrocytes, but not in neurons, by insertion into the plasma membrane, where it forms a pore which opens sporadically. Such fluctuations of [Ca²⁺]_c over a long period of time may induce pathological changes in astrocytes and could be a trigger for neurotoxicity.

We thank The Wellcome Trust for support.