Differential alterations in the expression of NMDA receptor subunits following chronic ethanol treatment in primary cultures of rat cortical and hippocampal neurones

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Abstract

In our previous experiments, severe cellular damages and neuronal cell loss were observed following 24 h of alcohol withdrawal in primary cultures of rat cortical neurones pre-treated with ethanol (50–200 mM) repeatedly for 3 days. Increased NMDA induced cytosolic calcium responses and excitotoxicity were also demonstrated in the ethanol pre-treated cultures. Thus, the enhancement in functions of NMDA receptors was supposed to be involved in the adaptive changes leading to the neurotoxic effect of alcohol-withdrawal. In this study, we investigated the effect of the 3-day repeated ethanol (100 mM) treatment on the function and subunit composition of the NMDA receptors. Here, we demonstrate that the maximal inhibitory effect of ethanol was significantly increased after ethanol pre-treatment. Similarly, the inhibitory activity of the NR2B subunit selective antagonists threo-ifenprodil, CP-101,606 and CI-1041 was also enhanced. On the contrary, the efficiency of the channel blocker agent MK-801 and the glycine-site selective antagonist 5,7-dichlorokynurenic acid was the same as in control cultures. According to these observations, a shift in subunit expression in favour for the NR2B subunit was suggested. Indeed, we provided evidence for increased expression of the NR2B and the C1 and C2′ cassette containing splice variant forms of the NR1 subunit proteins in ethanol pre-treated cultures in further experiments using a flow cytometry based immunocytochemical method. These changes may constitute the basis of the increased NMDA receptor functions and subsequently the enhanced sensitivity of ethanol pre-treated cortical neurones to excitotoxic insults resulting in increased neuronal cell loss after ethanol withdrawal. Such alterations may play a role in the neuronal adaptation to ethanol as well as in the development of alcohol dependence, and might cause neuronal cell loss in certain areas of the brain during alcohol withdrawal.

Introduction

Alcohol abuse is one of the major sources of public health, social and medical problems all over the world. However, the cellular mechanisms underlying neuronal adaptation to ethanol are only poorly understood. Long-term use of alcohol can lead to development of alcohol dependence—the need for continued alcohol drinking to avoid the adverse signs of withdrawal (Nestler et al., 1993). During long-term ethanol exposition several homeostatic changes are supposed to occur in neurones, especially in those systems mediating the acute effect of ethanol. These maladaptive cellular alterations consequently lead to an altered state of the cell, when the near-normal function can be attained only in the presence of ethanol. These changes may constitute the basis of cellular-level adaptation to ethanol (Koob and Bloom, 1988) and the development of “alcohol dependence” at the cellular level.

We observed similar kind of cellular-level alcohol dependence in our previous experiments, when primary cultures of cortical neurones from embryonic rat brain were treated with 25–200 mM ethanol repeatedly (daily) for three consecutive days (Nagy et al., 2001, Nagy and Laszlo, 2002). The 24 h of ethanol withdrawal following the chronic ethanol treatment resulted in serious cellular damage and neuronal cell death. On the contrary, neuronal cell loss was not observed in cultures into which ethanol was re-added. These observations suggested that the withdrawal from ethanol was responsible for the enhanced neurotoxicity. Cortical neurones pre-treated with ethanol required the further presence of ethanol to survive.

According to several other observations, alterations in the excitatory (glutamatergic) and/or the inhibitory (GABAergic) neurotransmitter systems may underlie the hyperexcitability observed during ethanol withdrawal and may play a central role in alcohol dependency (Lovinger, 1993, Crews et al., 1996). Moreover, there is an abundance of evidence to suggest that excitatory amino acid neurotransmission is highly involved in the adaptive responses that underlie the pathogenesis of alcohol dependence and alcoholic brain damage (Iorio et al., 1992, Chandler et al., 1993, Freed and Michaelis, 1978). This hypothesis was also supported by our previous observations. In our in vitro model system, sensitivity of primary cultures of cortical neurones for N-methyl-d-aspartate (NMDA) was increased after ethanol pre-treatment (Nagy and László, 2002). Furthermore, besides ethanol, several NMDA receptor antagonists (MK-801, threo-ifenprodil), but not the GABAA receptor agonist muscimol reduced the withdrawal induced neuronal cell loss (Nagy et al., 2001). According to these results, alterations in the function of NMDA receptors were supposed to be involved in the neurotoxic effect of alcohol-withdrawal observed in ethanol pre-treated cultures and were presumed to underlie the development of alcohol dependence in vitro.

NMDA receptors belong to the family of ionotropic glutamate receptors. These receptors are tetra or pentameres containing both NR1 and NR2 subunits (Bigge, 1999). The NR1 subunit has three splice cassettes (N1, C1 and C2 or C2′) in the coding sequence, therefore, it can be transcribed in eight splice variant forms. NR2 subunits are coded by four different genes: NR2A–D. Further subunits designated as NR3A and NR3B were recently shown to contribute to native receptors (Das et al., 1998, Cull-Candy et al., 2001). According to the subunit compositions, numerous heteromeric receptors can be formed, which have distinctive properties with respect to ligand binding and function (Mori and Mishina, 1995).

The inhibitory effect of ethanol is highly dependent on the nature of the NR1 and the NR2 subunits. NMDA receptors containing NR2A and/or NR2B subunits were shown to be more sensitive for ethanol than those including NR2C or NR2D subunits (Masood et al., 1994, Mirshahi and Woodward, 1995). Furthermore, receptors composed of NR1-1a/NR2B subunits were inhibited to a greater extent by ethanol than NR1-1a/NR2A receptors when expressed in L(tk-) mouse fibroblasts or human embryonic kidney (HEK) 293 cells (Smothers et al., 2001). Ethanol sensitivity of the recombinant NMDA receptors composed of NR1 subunits lacking or holding the N1 splice cassette with the NR2A subunit were not different in HEK 293 cells (Popp et al., 1998). On the contrary, homomeric assemblies of NR1 splice variants bearing all the three cassettes expressed in Xenopus oocytes were shown to be more sensitive to ethanol than those containing none of them (Koltchine et al., 1993). A calcium-dependent enhancement in the ethanol sensitivity was also observed when NR1-1a (lacking N1 cassette) or NR1-4a (lacking all the three cassettes) subunits were co-expressed with NR2A in Xenopus oocytes. In addition, the ethanol sensitivity of the NR1/NR2A receptors were significantly reduced when a mutant NR1 subunit that lacked the C0, C1 and C2 domains was used (Mirshahi et al., 1998).

There are several papers presenting evidence for altered NMDA receptor subunit composition due to chronic ethanol treatment. Following chronic ethanol administration, expression of the NR1, NR2A and NR2B subunit proteins were found to be increased in the cortex and hippocampus of rats or mice (Trevisan et al., 1994, Kalluri et al., 1998). According to other reports, the expression of the NR1 subunit was unaltered in the rat brain (Morrow et al., 1994, Hardy et al., 1999). Studies in vitro showed increased NR2B subunit mRNA levels, with no change in NR1 and NR2A subunit transcription (Hu et al., 1996). On the contrary, besides NR2B, the expression of the NR2A (Follesa and Ticku, 1996a) as well as the NR1 subunit proteins (Follesa and Ticku, 1996b, Kalluri et al., 1998) were also shown to be increased. New data concerning the expression of the different NR1 splice variants show a marked decrease in the ratio of the N1 cassette holding splice variant mRNA versus those ones that lacking it. The ratio of the 3′ splice variants (C1 and C2) was shown to be unaltered in cerebral cortex of rats chronically treated with ethanol (Hardy et al., 1999).

All these results suggest that ethanol sensitivity of the NMDA receptor molecule is dependent on its subunit composition, and after chronic ethanol treatment several changes may occur in the make-up of the receptor complex. In this study, we aimed at investigating the alterations in the subunit composition of the NMDA receptors as a consequence of chronic and repeated ethanol treatment using our previously described in vitro model of alcohol dependence. To show ethanol pre-treatment induced alterations in the function and subunit composition of NMDA receptors, we studied the effect of several NR2B subunit-selective and non-selective NMDA antagonists on NMDA evoked cytosolic calcium elevation in primary cultures of cortical neurones. Then we investigated the expression of the different NMDA receptor subunits as well as the different NR1 splice cassettes in control and in 100 mM ethanol pre-treated primary cultures of rat cortical and hippocampal neurones using a flow cytometry based immunocytochemical method.

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. NR subunit specific primary antibodies were purchased from Santa Cruz, and antibodies against N1, C1, C2 and C2′ cassettes of NR1 subunits were from Novus Biologicals. NR2B subunit selective NMDA antagonists CP101,606 and CI-1041 were synthesized by G. Richter Ltd. All other chemicals and secondary antibodies were purchased from Sigma–Aldrich.

Cell cultures

Primary cultures

Effect of ethanol pre-treatment on the inhibitory potential of NMDA receptor antagonists

NMDA (40 μM) produced considerable elevation in the cytoplasmic calcium concentration of rat cortical neurones maintained in primary cultures for 13 days. Similarly to our previous observations (Nagy and László, 2002), the basal calcium levels were slightly elevated (154±12 nM versus 126±7 nM in control, P=0.12, n=23) and the NMDA induced calcium responses were significantly increased (284±10 nM versus 200±9 nM in control, P<0.001, n=23) in primary cortical cultures pre-treated with 100 mM ethanol

Discussion

Long-term exposure to ethanol has been shown to result in compensatory changes in the function and/or subunit expression of glutamate-gated ion channels of neurones. These changes have been thought to lead to the development of alcohol dependence and to contribute to the neurochemical imbalance associated with the hyperactive state of the central nervous system during alcohol withdrawal. In the present study, we investigated the possible alterations in the inhibitory activity of different NMDA

Acknowledgements

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

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