Increased sensitivity to NMDA is involved in alcohol-withdrawal induced cytotoxicity observed in primary cultures of cortical neurones chronically pre-treated with ethanol

doi:10.1016/S0197-0186(01)00131-0

Neurochemistry International

Volume 40, Issue 7, June 2002, Pages 585-591

https://doi.org/10.1016/S0197-0186(01)00131-0 Get rights and content

Abstract

Severe cellular damage and neuronal cell loss were previously observed in cultures of primary cortical neurones after chronic ethanol pre-treatment followed by ethanol-withdrawal. In this study, we investigated the circumstances and the possible cellular changes leading to alcohol-withdrawal induced neuronal cell death. When cultures were pre-treated with ethanol (25–200 mM) once for 24 or 72 h, the amount of the subsequent 24 h alcohol-withdrawal induced cell death—estimated by measuring the release of lactate dehydrogenase (LDH)—was elevated only in cultures pre-treated with 200 mM ethanol for 72 h. On the contrary, as little as 50 mM ethanol produced significant (P<0.01) increase in the withdrawal induced LDH-release in cultures pre-treated repeatedly with ethanol once daily for three consecutive days. When ethanol was re-added to the cultures during the withdrawal period, the LDH-release was dose-dependently reduced to the level of control. In ethanol pre-treated cultures N-methyl-d-aspartate (NMDA) (0.01–1 mM) induced excitotoxicity as well as NMDA evoked elevation of cytosolic calcium ion concentration was increased. In contrast, the depolarising agent veratridine (0.01–1 mM) produced similar extent of neuronal injury and elevation in cytosolic calcium ion concentration in control as in ethanol pre-treated cultures. According to these observations, repeated ethanol treatment appears to cause more robust adaptive changes in cultured neurones leading to more pronounced withdrawal induced cellular damage than chronic but single treatment does. In addition, the glutamatergic neurotransmission, especially the NMDA receptor system seems to be highly involved in the adaptive changes and in the cytotoxic effect of alcohol-withdrawal.

Introduction

Despite the fact that alcohol abuse is one of the major public health, social and medical problems all over the world, cellular mechanisms underlying neuronal adaptation to ethanol are only poorly understood. Several changes occur in a cell during alcohol exposure: disturbances in membrane structure and transport systems, altered signalisation and metabolic processes. During long-term ethanol exposure, these alterations trigger other cellular processes—adaptive cellular changes—to maintain normal state and function of the cell. These processes may constitute the basis of cellular level adaptation to alcohol (Koob and Bloom, 1988).

The hypothesis that ethanol acts as a drug with non-specific membrane effects has been challenged by data demonstrating high sensitivity of various ion channels to acute effect of ethanol at relevant concentrations (<100 mM). Ethanol interacts with voltage-gated Ca²⁺-channels as well as with ligand-gated channels, especially the N-methyl-d-aspartate (NMDA) and the γ-aminobutyric acid type A (GABA_A) receptors (Lovinger, 1993, Hoffman and Tabakoff, 1994, Crews et al., 1996, Smothers et al., 2001). During chronic ethanol treatment, alterations in the function of these channels and receptors are supposed to contribute to the development of ethanol tolerance, dependence and withdrawal symptoms. These changes may lead to the hyper-excitable state of the CNS producing seizures, proconvulsive states and neurotoxicity when alcohol is withdrawn (Beadles-Bohling et al., 2000, de Gortari et al., 2000, Dodd et al., 2000).

Previously, 24 h of alcohol-withdrawal was shown to cause serious cellular damage and neuronal cell loss in primary cultures of rat cortical neurones after a chronic and repeated (three successive days) ethanol (50–400 mM) pre-treatment. The neuronal injury was reduced by re-addition of ethanol, suggesting that the withdrawal from ethanol may be responsible for the subsequent neuronal cell death. Besides of ethanol, NMDA receptor antagonists like MK-801 or threo-ifenprodil, but not the GABA_A receptor agonist muscimol was shown to reduce the alcohol-withdrawal induced neuronal cell death (Nagy et al., 2001).

The objective of the present study was to investigate the role of the glutamatergic neurotransmitter system—especially that of the NMDA receptors—in the alcohol-withdrawal induced neuronal cell death. In addition, it was analysed what experimental conditions are required for the development of alcohol-withdrawal induced cell loss. For this reason, primary cultures of rat cortical neurones were exposed to ethanol. Alcohol was added to the cells either on day 1 for 1 or 3 days or once daily for 3 days, and NMDA, veratridine or 24 h of alcohol-withdrawal induced cell death was determined. NMDA or veratridine evoked elevation of cytosolic calcium ion concentration was also examined after 3 days of periodic ethanol treatment.

Section snippets

Materials

D-MEM (52100), phenol red free D-MEM (11880) and foetal bovine serum were obtained from GIBCO BRL. Fluo-4/AM was from Molecular Probes. All other chemicals were purchased from Sigma–Aldrich.

Cell cultures

Primary cultures of cortical neurones were initiated from neocortices of 17-day-old rat embryos. Pieces of cortical tissue were washed with Ca²⁺/Mg²⁺ free HEPES buffered salt solution (137 mM NaCl, 5 mM KCl, 36 mM d-glucose, 20 mM HEPES, pH=7.4) and incubated in the presence of 0.25% trypsin for 4 min. After

Cytotoxic effect of ethanol treatment and withdrawal

In preliminary experiments, 24 h of 25–400 mM ethanol treatment did not produce significant changes in LDH-release of primary cultures of cortical neurones (data not shown). Furthermore, after 3 days of ethanol exposure the total LDH activity measured after disrupting the cells did not differ significantly in control (8.88±0.04 U per well) and in 100 mM ethanol pre-treated (9.08±0.20 U per well) cultures, respectively. These results suggest that the viability and/or density of the cultures were not

Discussion

To investigate the possible adaptive cellular changes leading to the development of alcohol dependence, primary cultures of cortical neurones were chronically treated with ethanol (25–200 mM). In preliminary experiments, the viability of cortical neurones was not affected by acute ethanol treatment up to the highest ethanol concentration (400 mM) used (Nagy et al., 2001). Similarly, after 1 day ethanol pre-treatment the subsequent 24 h of alcohol-withdrawal did not produce significant cell loss.

Acknowledgements

We are grateful to Dr. István Tarnawa for his invaluable help in discussions. We also thank Mrs. Krisztina Holti Bertalan and Mrs. Piroska Unghy Fejes for their excellent technical assistance.

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Increased sensitivity to NMDA is involved in alcohol-withdrawal induced cytotoxicity observed in primary cultures of cortical neurones chronically pre-treated with ethanol

Abstract

Introduction

Section snippets

Materials

Cell cultures

Cytotoxic effect of ethanol treatment and withdrawal

Discussion

Acknowledgements

Neurochem. Int.

Neurosci. Lett.

Int. Rev. Neurobiol.

Neurochem. Int.

Neurochem. Int.

Eur. J. Pharmacol.

Brain Res.

Eur. J. Pharmacol.

J. Neurosci. Meth.

Drug and Alcohol Dependence

Drug and Alcohol Dependence

Neurochem. Int.