Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD). In this study, we show that, in terms of their Ca2+ responses to 0.5？mM methadone, SH-SY5Y cells can be pooled into four different groups. In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, -opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment. 1. Introduction Methadone (D,L-methadone hydrochloride) is frequently used in different therapies including opioid addiction [1], long-lasting analgesics in cancer and neuropathic pain syndromes [1–3]. However, numerous reports indicate a negative impact on human cognition by chronic exposure to opioid drugs. Patients subjected to methadone maintenance programs show impaired cognitive abilities in aspects such as psychomotor performance, information processing, attention, problem solving, memory, decision making, reaction time, and emotional facial expression recognition [4–10]. Changes in the cytosolic free-calcium concentration ([Ca2+]cyt) are involved in control of a large number of cellular and physiological processes including neuronal excitability, synaptic plasticity, and gene transcription [11, 12]. However, the physiological Ca2+ signal can switch to a death signal when the [Ca2+]cyt？？increases dramatically. For example, excitotoxic high glutamate concentrations result in an initial transient increase in [Ca2+]cyt？？that is followed by a delayed, irreversible rise in [Ca2+]cyt？？known
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