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Ethanol tolerance and synaptic plasticity
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Ethanol tolerance and synaptic plasticity L. Judson Chandler, R. Adron Harris and Fulton T. Crews Current concepts of the mechanisms underlying many of the pharmacological effects of ethanol on the CNS involve disruption of ion channel function via the interaction of ethanol with specific hydrophobic sites on channel subunit proteins. Of particular clinical importance is the development of tolerance and dependence to ethanol, and it is likely that adaptive changes in synaptic function in response to ethanol’s actions on ion channels play a role in this process. In this article, Judson Chandler, Adron Harris and Fulton Crews discuss potential mechanisms of ethanolinduced changes in synaptic function that might provide a cellular basis for ethanol tolerance and dependence. It is proposed that multiple mechanisms are involved that include both transcriptional and post-translational modifications in NMDA and GABAA receptors. Despite the fact that ethanol is one of the oldest pharmacological agents known and its abuse continues to be a major social, economic and public health problem worldwide, the molecular mechanism(s) by which ethanol produces its effects within the CNS are only now beginning to be understood. Theories concerning the site of action of ethanol and anaesthetics have progressed from membrane lipids (the ‘lipid theory’ of action in which the primary event was thought to be bulk membrane fluidization), to the protein–lipid interface (disruption of lipid–protein interactions) and finally to proteins themselves as primary sites of action. Although the effects of ethanol have traditionally been considered to be relatively nonspecific, recent results show that certain membrane ion channels and signal-transduction systems are particularly important target sites of alcohol. These observations are helping to reveal how ethanol can produce adaptations at the cellular level as well as at the level of gene expression. Ethanol-induced alterations in synaptic plasticity might play an important role in the neurobiological and neurodegenerative processes that can accompany prolonged exposure to ethanol. A characteristic feature of the effects of ethanol on the CNS is the development of tolerance. Tolerance is defined as a reduction in the intensity of the effect of a drug over time and is usually associated with repeated exposure to that drug. Ethanol tolerance has a number of important clinical implications. For example, the initial sensitivity of an individual to the intoxicating effects of ethanol, which could relate to acute tolerance development,
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is a predictor of whether some individuals will become alcoholics in the future. In addition, development of tolerance during chronic ethanol consumption means that larger amounts of ethanol are needed to produce the same pre-tolerant intoxicating effect, leading to increased ethanol consumption and ethanol-related pathologies. Finally, tolerance development can result in the characteristic ethanol hyperexcitability–withdrawal syndrome upon cessation of alcohol consumption. This syndrome, in addition to being a health hazard in itself, can act as a negative reinforcer to promote continued ethanol consumption. Although tolerance to ethanol intoxication probably occurs at multiple levels, alterations in synaptic function (synaptic plasticity) are believed to play a major role. In particular, compensatory changes in excitatory NMDA and inhibitory GABAA receptors probably contribute to the development of ethanol tolerance.
NMDA and GABAA receptors The major excitatory and inhibitory neurotransmitters in the brain are glutamate and GABA, respectively, and ionotropic glutamate [NMDA, (RS)-a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) and kainate] and GABAA receptors are among the most widely distributed and abundant receptor-operated ion channels in the CNS. Both NMDA and GABAA receptors are composed of multiple subunit proteins, which are thought to assemble as hetero-pentameric structures that exhibit distinct properties depending upon the particular subunit composition. Identified subunits include a1–a6, b1–b4, g1–g4, d and r1–2 for GABAA receptors and NR1, NR2A–NR2D and NR3A for NMDA receptors. Additional variants of both GABAA and NMDA receptors are generated by alternative splicing. Various molecular aspects of GABAA and NMDA receptors have been the subject of previous reviews1,2. Convincing evidence has shown that excitatory NMDA and inhibitory GABAA receptors are important sites of action of ethanol. Studies using a variety of tissue preparations (i.e. heterogeneous neuronal preparations and cells transfected with receptor subunits) demonstrate that pharmacologically relevant concentrations of ethanol can potentiate GABAA receptor currents and antagonize NMDA receptor currents3,4. It is possible that the combination of reduced excitatory glutamate-mediated activity and enhanced inhibitory GABA-mediated activity contributes to ethanol intoxication. Brain regional differences in the ethanol-sensitivity of NMDA and GABAA receptors have been noted5,6, leading to the suggestion that the ethanol-sensitivity of native NMDA and GABAA receptors is determined, at least in part, by the subunit composition of the receptor. This is supported by studies using recombinant expression systems showing that the ethanol-sensitivity of these receptors varies with the particular subunits expressed7,8. There is also considerable evidence that specific hydrophobic sites of the receptor polypeptides are crucial for
0165-6147/98/$ – see front matter © 1998 Elsevier Science. All rights reserved. PII: S0165-6147(98)01268-1
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L. J. Chandler, Assistant Professor, Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130, USA, R. A. Harris, Professor, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA, and F. T. Crews, Director, Bowles Center for Alcohol Studies, and Professor, Department of Pharmacology and Psychiatry, The University of North Carolina School of Medicine, CB# 7178, Thurston-Bowles Building, Chapel Hill, NC 27599, USA.
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Box 1. Regulation of NMDA receptors by Modification of NMDA and GABAA receptor function appears to play an important role in ethanol tolerance and dependence, and a number of receptor regulatory processes are potential targets of ethanol. Two important post-translational processes that might be affected by prolonged exposure to ethanol are receptor association with the synaptic cytoskeleton and receptor phosphorylation. NMDA receptors bind to cytoskeletal-associated proteins, and receptor–cytoskeletal linkage is essential for subcellular receptor targeting and synaptic localization. In addition, receptor–cytoskeletal interaction is a dynamic process that appears to be coupled to receptor activity. The C-terminal region of the NMDA receptor NR1 subunit binds a-actinin 2 (Ref. 1) and a recently described protein termed yotiao2, both presumably functioning as linker proteins for cytoskeletal attachment, and neurofilament-light (NF-L), which might link NMDA receptors with the subsynaptic cytoskeleton3 (Fig.). Studies using transfected QT6 cells have shown that NMDA receptor clustering is dependent upon the presence of a NR1 splice variant containing the C1-cassette4. Phosphorylation of the C1-cassette by protein kinase C (PKC) could regulate receptor–cytoskeleton attachment as phosphorylation of serine residues within the C1-cassette disperses NR1 receptor clusters. Ca2+ influx following NMDA receptor activation can disrupt the receptor–cytoskeletal interaction via actin depolymerization leading to downregulation of channel activity5. Furthermore, Ca2+–calmodulin can bind to the C1-cassette in a phosphorylation-dependent and competitive manner with a-actinin 2, also resulting in disruption of NR1–actin association and inhibition of channel activity6. In addition to these modulatory processes involving the C1-cassette, alternative splicing of C1 (i.e. variants lacking C1) could play a role in long-term regulation of receptor clustering, stabilization and trafficking, as well as in other cytoskeletal-related processes. NMDA receptor NR2 subunits bind to the PDZ domains of a family of closely related postsynaptic density proteins (PSDs) [PSD-95/synapse-associated protein (SAP)-90, Chapsyn-110/PSD-93, and SAP102]7. These proteins appear to function in localization of receptors to the postsynaptic density and as scaffolding proteins. PSD-95 proteins can undergo head-to-head disulphide linkage resulting in a multimodular scaffold for clustering receptors and/or ion channels and coupling receptor–enzyme complexes and receptor–downstream signalling molecules8. Phosphorylation is important in direct and indirect modulation of NMDA receptors, and might also play a role in synaptic modifications underlying ethanol tolerance. Tyrosine phosphorylation within the C-terminal region of NR2 subunits enhances NMDA currents9–12 and tyrosine phosphatases
modulation of ionotropic glutamate and GABAA receptor function by ethanol and volatile anaesthetics9,10. Finally, phosphorylation and dephosphorylation play an important role in regulating NMDA and GABAA receptors, and second-messenger phosphorylating systems that can directly or indirectly modulate receptor function can be disrupted by ethanol (see Box 1).
Mechanisms of ethanol tolerance Tolerance can include changes at the behavioural level (behavioural tolerance) and at the cellular level (physio492
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reduce NMDA currents13–15. It is thought that tyrosine kinases and phosphatases participate in a dynamic process that regulates channel activity. Phosphorylation of tyrosine residues within the C-terminal region of the NR2 subunit appears to potentiate NMDA currents by reducing tonic inhibition of the receptor by zinc16. Calmodulin kinase II has also been reported to phosphorylate the Cterminal region of NR2 (Ref. 17), but it is not known how this affects channel function. Phosphorylation of the C1-cassette by PKA can enhance NMDA receptor function. In the absence of synaptic activity, it appears that NMDA receptors are phosphorylated by basally active PKA, thus enhancing the activity of quiescent receptors18–20. Ca2+ influx during receptor activation leads to calcineurinmediated dephosphorylation and receptor downregulation, which can be overcome by b-adrenoceptor-mediated stimulation of PKA activity19. As noted above, PKC phosphorylation of the C1cassette can also regulate channel function. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Wyszynski, M. et al. (1997) Nature 385, 439–442 Lin, J. W. et al. (1998) J. Neurosci. 18, 2017–2027 Ehlers, M. D. et al. (1998) J. Neurosci. 18, 720–730 Ehlers, M. D., Tingley, W. G. and Huganir, R. L. (1995) Science 269, 1734–1737 Rosenmund, C. and Westbrook, G. L. (1993) Neuron 10, 805–814 Ehlers, M. D. et al. (1996) Cell 84, 745–755 Sheng, M. (1996) Neuron 17, 575–578 Hsueh, Y-P., Kim, E. and Sheng, M. (1997) Neuron 18, 803–814 Rosenblum, K., Dudai, Y. and Richter-Levin, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10457–10460 Suzuki, T. and Okumura-Noji, K. (1995) Biochem. Biophys. Res. Commun. 216, 582–588 Rostas, J. A. P. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10452–10456 Chen, S. and Leonard, J. P. (1996) J. Neurochem. 67, 194–200 Wang, Y. T. and Salter, M. W. (1994) Nature 369, 233–235 Wang, Y. T., Yu, X. and Salter, M. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1721–1725 Hall, R. A. and Soderling, T. R. (1997) J. Biol. Chem. 272, 4135–4140 Zheng, F. (1998) Nat. Neurosci. 1, 185–191 Omkumar, R. V. et al. (1996) J. Biol. Chem. 271, 31670–31678 Tong, G., Shepherd, D. and Jahr, C. E. (1995) Science 267, 1510–1512 Raman, I. M., Tong, G. and Jahr, C. E. (1996) Neuron 16, 415–421 Tingley, W. G. et al. (1997) J. Biol. Chem. 272, 5157–5166
logical tolerance). Behavioural tolerance is thought to be a learned response to overcome alcohol-induced behaviours and can be observed in chronic alcoholics and non-alcoholic social drinkers. Physiological tolerance is viewed as a compensatory change at the cellular level in response to the depressant effects of ethanol, and can be temporally divided into rapid (minutes to hours) shortterm changes in response to continuous acute ethanol exposure (acute tolerance) and delayed (hours to days) long-term changes in response to chronic ethanol exposure (chronic tolerance).
phosphorylation
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PP2B PTK
GK
(Fyn, Src)
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ok, G. L. (1993) Neuron 10,
CaMKII
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PSD-95 multimers
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1 1
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yotiao NF-L
C2 PDZ domains 1
2
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R. (1997) J. Biol. Chem. 272,
1, 185–191 Biol. Chem. 271, 31670–31678 ahr, C. E. (1995) Science 267,
P
Ca2+
PO4
6) J. Neurochem. 67, 194–200 1994) Nature 369, 233–235 M. W. (1996) Proc. Natl. Acad.
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Ca2+
ure 385, 439–442 i. 18, 2017–2027 rosci. 18, 720–730 and Huganir, R. L. (1995)
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phosphorylation and cytoskeletal association
It is thought that tyrotases participate in a ulates channel activity. ne residues within the R2 subunit appears to by reducing tonic inhinc16. Calmodulin kinase o phosphorylate the C17), but it is not known nction. Phosphorylation A can enhance NMDA sence of synaptic activA receptors are phose PKA, thus enhancing ceptors18–20. Ca2+ influx leads to calcineurinon and receptor downvercome by b-adrenon of PKA activity19. As horylation of the C1annel function.
4, 745–755 5–578 M. (1997) Neuron 18, 803–814 Richter-Levin, G. (1996) Proc. 7–10460 , K. (1995) Biochem. Biophys.
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PDZ-domain interacting proteins
3 S
ahr, C. E. (1996) Neuron 16,
GK
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Acute ethanol tolerance Acute tolerance can be especially important in determining the initial sensitivity of an individual to the intoxicating effects of alcohol. Acute tolerance to ethanol inhibition of NMDA-mediated excitatory postsynaptic potentials (EPSPs) can be observed in vitro at the cellular level. In the hippocampal slice preparation, application of ethanol causes an initial depression of NMDA-mediated EPSPs in the CA1 region, which gradually recover during the period of continuous ethanol exposure11. Similarly, when DBA/2J mice were pretreated with ethanol
Fig. Phosphorylation and cytoskeletal association of NMDA receptors. CaM, calmodulin; CaMKII, calmodulin kinase II; GK, guanylate kinase; NF-L, neurofilament-light; NOS, nitric oxide synthase; PDZ, PSD-95, Dlg-A, Z0-1; PKA, protein kinase A; PP1/2A, protein phosphatase 1 and 2A; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase.
5 min to 1 h prior to decapitation, ethanol no longer potentiated muscimol-stimulated 36Cl– uptake in isolated cerebellar vesicles12,13. These changes in ethanol sensitivity suggest that changes in NMDA and GABAA receptor regulation might underlie acute tolerance. The rapidity of these changes precludes effects at the transcriptional and translational level, or both, and strongly implicate second-messenger modulation of receptor function in the adaptive response. NMDA receptor function is enhanced by tyrosine kinases and reduced by tyrosine phosphatases. Recent
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evidence suggests that acute tolerance to ethanol inhibition of NMDA receptor-mediated EPSPs involves modulation of NMDA receptor tyrosine phosphorylation by the non-receptor tyrosine kinase Fyn14. Fyn-deficient (fynz/fynz) mice, which exhibit enhanced sensitivity to the hypnotic effect of ethanol compared with heterozygotes (+/fynz), do not show acute tolerance to NMDA receptormediated EPSPs. Furthermore, ethanol administration produced an upregulation of tyrosine phosphorylation of the NR2B subunit in heterozygotes but not in Fyndeficient mice. However, it should be noted that loss of righting reflex was the only behavioural parameter examined in the Fyn-deficient mice, and enhanced tyrosine phosphorylation of NMDA receptors was demonstrated only in the hippocampus. Furthermore, it is not known whether the increase in ethanol sensitivity in the Fyn-deficient mice was a direct result of a lack of NR2B phosphorylation by Fyn. Clearly, further work is needed to clarify the role of Fyn and NMDA receptor phosphorylation in acute ethanol tolerance. Studies have also shown that ethanol enhancement of GABAA receptor function requires activation of protein kinase C (PKC)15 and that null mutant mice lacking PKC-g are resistant to the actions of ethanol on GABAA receptors and on behavioural measures16. Whether decreases in the phosphorylation state of GABAA receptors occur during development of ethanol tolerance is not known. Furthermore, in the intact brain, ethanol interaction with GABAA receptors and/or their regulatory processes appears complex, and considerable efforts will be required to define conclusively the role of phosphorylation and subunit composition in acute ethanol tolerance.
Chronic ethanol tolerance Hyperexcitability of the CNS is a characteristic component of ethanol withdrawal, and there is good evidence for both a reduction in GABA-mediated inhibitory and an enhancement in glutamate-mediated excitatory neurotransmission following chronic ethanol exposure. Studies using primary neuronal cultures have shown that prolonged exposure to ethanol leads to a supersensitization of NMDA receptor-mediated events, including Ca2+ influx17 and Ca2+-dependent processes [excitotoxicity18 and nitric oxide (NO) formation19]. Similarly, studies with isolated brain preparations have reported that ethanol-mediated enhancement of GABAA receptorcoupled Cl– flux is decreased following chronic ethanol exposure20. Thus, functional changes in NMDA and GABAA receptors could contribute to physiological tolerance. It is possible that the alterations in NMDA and GABAA receptor function are caused by up- and downregulation, respectively, of receptor density. Several studies have reported increased NMDA receptor binding and/or subunit protein levels following chronic ethanol exposure both in vitro and in vivo21–26, whereas others have failed to find any such changes19,27–29. It was recently reported that exposure of cultured cortical neurones to ethanol leads to enhancement of NMDA494
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T stimulated NO formation (but not that stimulated by kainate, AMPA or ionomycin) without a change in receptor density, and it was suggested that the chronic ethanol effect related to post-translational modification of NMDA receptors19. Similarly, in cells stably transfected with GABAA receptor subunits, ethanol was shown to cause changes in GABAA receptor function similar to those observed in vivo, but no change in surface receptor density was observed30. This finding is in agreement with previous observations in rats31 and mice32 chronically exposed to ethanol. Because the transfected cells contain defined GABAA receptor subunits with expression controlled by the dexamethasone-sensitive promoter, it is unlikely that ethanol affected subunit expression or produced subunit substitution. Thus, it is possible that posttranslational modification(s) underlie these functional changes. No change in GABAA receptor density following chronic ethanol exposure has been observed in the majority of studies. However, changes in GABAA receptor subunit expression in the brain have been reported, suggesting that subunit changes could play an important role in vivo33. An important component of ethanol-induced posttranslational modification of receptor function could relate to second-messenger systems. Tyrosine phosphorylation of GABAA receptors has been shown to enhance GABAA receptor-gated currents34 and, as noted above, tyrosine phosphorylation of NMDA receptors has been linked to the development of acute tolerance of NMDA receptors. PKC, PKA and calmodulin kinase II have been reported to phosphorylate and/or alter the function of NMDA or GABAA receptors, or both (for review, see Ref. 3; Box 1), and phosphorylation could alter their ethanol sensitivity. Whether the phosphorylation state of NMDA and GABAA receptors is altered by chronic ethanol exposure is not yet known. Chronic ethanol can increase PKC levels and activity35–39 and induce heterologous desensitization of cAMP signalling with decreased PKA activity40–42. Some of these effects of ethanol could relate to changes in subcellular translocation and localization. Ethanol has been shown to stimulate translocation to the nucleus of the catalytic subunit of PKA where it remains sequestered for as long as ethanol is present43, and to stimulate translocation of PKC-d and PKC-e to new intracellular sites39. Translocation of PKC and PKA isozymes to subcellular anchoring proteins is thought to be important in targeting specific signalling events. Furthermore, PKA and calcineurin (protein phosphatase 2B) are concentrated in postsynaptic densities via a common Akinase anchoring protein (AKAP79), putting them in position to regulate phosphorylation and/or dephosphorylation of key postsynaptic proteins44. Clearly, changes in PKA and/or PKC activity and subcellular targeting could play an important role in ethanolinduced changes in synaptic function, including modulation of NMDA and GABAA receptors. Another potentially important process in NMDA and GABAA receptor adaptation during ethanol exposure is
V receptor–cytoskeletal interaction (see Box 1). NMDA receptors are required for activity-dependent synaptic remodelling during development, and studies in hippocampal cultures have shown that the subcellular distribution of NMDA receptors is modulated by receptor activity. Chronic treatment with an NMDA receptor antagonist leads to increased NMDA receptor clustering at synaptic sites and, conversely, spontaneous activity leads to decreased synaptic NMDA receptor clustering45. Because studies in primary neuronal cell cultures might more closely model developmental processes, an important question to be addressed is whether this activitydependent redistribution of NMDA receptors also occurs in mature neurones. Furthermore, it is not known whether the functional property of the NMDA receptor itself is altered by clustering and redistribution (i.e. synaptic versus non-synaptic). However, receptor redistribution could represent a novel form of activity-dependent synaptic modification (plasticity), and prolonged inhibition of the NMDA receptor during chronic ethanol exposure might also lead to an increase in NMDA receptor clustering at synaptic sites. Clearly, this is an intriguing hypothesis that needs testing. The specifics of receptor–cytoskeleton interaction is less clear for GABAA receptors. These receptors form clusters in postsynaptic membranes that are not associated with prominent postsynaptic densities, and might anchor to the subsynaptic cytoskeleton via gephyrin or a gephyrin-related protein46. Interestingly, studies in transfected cells have shown that disruption of microtubule polymerization also disrupts GABAA receptor clustering and abolishes the action of ethanol47. Also of interest is the observation that insulin can stimulate a rapid translocation and clustering of functional native GABAA receptors to the postsynaptic domain of inhibitory synapses in cultured hippocampal neurones48. However, whether GABAA receptor clustering is responsive to changes in channel activity and the effect of chronic ethanol on this process remain to be determined.
Concluding remarks Inhibition of NMDA and potentiation of GABAA receptors by acute ethanol results in reduced synaptic activity that is likely to contribute to the intoxicating effects of ethanol. It is probable that alteration in synaptic function by ethanol exposure contributes to the development of physiological tolerance that involves changes in excitatory glutamate-mediated and inhibitory GABAmediated neurotransmission. Acute tolerance might involve post-translational modifications such as receptor phosphorylation and/or dephosphorylation and receptor clustering and/or unclustering. Although up- and downregulation of NMDA and GABAA receptor density, respectively, could be a prominent mechanism underlying changes in receptor function during chronic ethanol exposure, alterations in receptor density cannot account exclusively for the functional changes.
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We suggest that multiple mechanisms could contribute to the alterations in NMDA and GABAA receptor function during ethanol exposure. While acute physiological tolerance might involve only post-translational processes, chronic physiological tolerance could involve both post-translational modifications and changes in gene expression. In addition to changes in receptor density, potential mechanisms include subunit substitution and alternative splicing, alterations in second-messenger phosphorylating and/or dephosphorylating systems and altered receptor clustering and subcellular redistribution. Although it has yet to be determined whether many of the post-translational events observed in vitro actually occur in vivo, these types of observations are beginning to reveal the dynamic nature and potential underlying mechanisms of adaptations of the CNS to the depressant effects of ethanol on synaptic function. Selected references 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Smith, G. B. and Olsen, R. W. (1995) Trends Pharmacol. Sci. 16, 162–167 Sucher, N. J. et al. (1996) Trends Pharmacol. Sci. 17, 348–355 Crews, F. T. et al. (1996) Int. Rev. Neurobiol. 39, 283–367 Diamond, I. and Gordon, A. S. (1997) Physiol. Rev. 77, 1–20 Simson, P. E. et al. (1993) Brain Res. 607, 9–16 Criswell, H. E. et al. (1993) J. Pharmacol. Exp. Ther. 267, 522–537 Massood, K. et al. (1994) Mol. Pharmacol. 54, 324–329 Harris, R. A. et al. (1997) Alcohol. Clin. Exp. Res. 21, 444–451 Minami, K. et al. (1998) J. Biol. Chem. 273, 8248–8255 Wick, M. J. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6504–6509 Grover, C. A. et al. (1994) Brain Res. 642, 70–76 Allan, A. M. and Harris, R. A. (1987) Pharmacol. Biochem. Behav. 27, 665–670 Morrow, A. L. et al. (1988) J. Pharmacol. Exp. Ther. 246, 158–164 Miyakawa, T. et al. (1997) Science 278, 698–701 Weiner, J. L. et al. (1997) J. Neurochem. 68, 1949–1959 Harris, R. A. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3658–3662 Iorio, K. R. et al. (1992) Mol. Pharmacol. 41, 1142–1148 Chandler, L. J. et al. (1993) J. Neurochem. 60, 1578–1581 Chandler, L. J. et al. (1997) Mol. Pharmacol. 51, 733–740 Buck, K. J. and Harris, R. A. (1991) Alcohol. Clin. Exp. Res. 15, 460–470 Grant, K. A. et al. (1990) Eur. J. Pharmacol. 176, 289–296 Gulya, K. et al. (1991) Brain Res. 547, 129–134 Snell, L. D. et al. (1993) Brain Res. 602, 91–98 Trevisan, L. et al. (1994) J. Neurochem. 62, 1635–1638 Hu, X-J. and Ticku, M. K. (1995) Brain Res. 30, 347–356 Chen, X. et al. (1997) J. Neurochem. 69, 1559–1569 Tremwel, M. F., Anderson, K. J. and Hunter, B. E. (1994) Alcohol. Clin. Exp. Res. 18, 1004–1008 Carter, L. A. et al. (1995) J. Neurochem. 64, 213–219 Rudolph, J. G. et al. (1997) Alcohol. Clin. Exp. Res. 21, 1508–1519 Harris, R. A. et al. (1998) J. Pharmacol. Exp. Ther. 284, 180–188 Rastogi, S. K. et al. (1986) Neuropharmacology 25, 1179–1184 Buck, K. J. and Harris, R. A. (1990) J. Pharmacol. Exp. Ther. 253, 713–719 Morrow, A. L. (1995) Int. Rev. Neurobiology 38, 1–41 Valenzuela, C. F. et al. (1995) Mol. Brain Res. 31, 165–172 Messing, R. O., Henteleff, M. and Park, J. J. (1991) J. Biol. Chem. 266, 23428–23432 Roivainen, R. T., McMahon, T. and Messing, R. O. (1993) Brain Res. 624, 85–93 DePetrillo, P. B. and Liou, C. S. (1993) Alcohol. Clin. Exp. Res. 17, 351–354 Coe, I. R. et al. (1996) J. Biol. Chem. 271, 29468–29472 Gordon, A. S. et al. (1997) Mol. Pharmacol. 52, 554–559 Gordon, A. S., Collier, K. and Diamond, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2105–2108 Rabin, R. A. et al. (1992) J. Pharmacol. Exp. Ther. 262, 257–262 Coe, I. R. et al. (1996) J. Pharmacol. Exp. Ther. 276, 365–369 Dohrman, D. P. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10217–10221 Coghlan, V. M. et al. (1995) Science 267, 108–111 Rao, A. and Craig, A. M. (1997) Neuron 19, 801–812 Whatley, V. J. and Harris, R. A. (1996) Int. Rev. Neurobiol. 39, 113–143 Whatley, V. J. et al. (1996) J. Neurochem. 66, 1318–1321 Wan, Q. et al. (1997) Nature 388, 686–690
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Ethanol tolerance and synaptic plasticity L. Judson Chandler, R. Adron Harris and Fulton T. Crews Current concepts of the mechanisms underlying many of the pharmacological effects of ethanol on the CNS involve disruption of ion channel function via the interaction of ethanol with specific hydrophobic sites on channel subunit proteins. Of particular clinical importance is the development of tolerance and dependence to ethanol, and it is likely that adaptive changes in synaptic function in response to ethanol’s actions on ion channels play a role in this process. In this article, Judson Chandler, Adron Harris and Fulton Crews discuss potential mechanisms of ethanolinduced changes in synaptic function that might provide a cellular basis for ethanol tolerance and dependence. It is proposed that multiple mechanisms are involved that include both transcriptional and post-translational modifications in NMDA and GABAA receptors. Despite the fact that ethanol is one of the oldest pharmacological agents known and its abuse continues to be a major social, economic and public health problem worldwide, the molecular mechanism(s) by which ethanol produces its effects within the CNS are only now beginning to be understood. Theories concerning the site of action of ethanol and anaesthetics have progressed from membrane lipids (the ‘lipid theory’ of action in which the primary event was thought to be bulk membrane fluidization), to the protein–lipid interface (disruption of lipid–protein interactions) and finally to proteins themselves as primary sites of action. Although the effects of ethanol have traditionally been considered to be relatively nonspecific, recent results show that certain membrane ion channels and signal-transduction systems are particularly important target sites of alcohol. These observations are helping to reveal how ethanol can produce adaptations at the cellular level as well as at the level of gene expression. Ethanol-induced alterations in synaptic plasticity might play an important role in the neurobiological and neurodegenerative processes that can accompany prolonged exposure to ethanol. A characteristic feature of the effects of ethanol on the CNS is the development of tolerance. Tolerance is defined as a reduction in the intensity of the effect of a drug over time and is usually associated with repeated exposure to that drug. Ethanol tolerance has a number of important clinical implications. For example, the initial sensitivity of an individual to the intoxicating effects of ethanol, which could relate to acute tolerance development,
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is a predictor of whether some individuals will become alcoholics in the future. In addition, development of tolerance during chronic ethanol consumption means that larger amounts of ethanol are needed to produce the same pre-tolerant intoxicating effect, leading to increased ethanol consumption and ethanol-related pathologies. Finally, tolerance development can result in the characteristic ethanol hyperexcitability–withdrawal syndrome upon cessation of alcohol consumption. This syndrome, in addition to being a health hazard in itself, can act as a negative reinforcer to promote continued ethanol consumption. Although tolerance to ethanol intoxication probably occurs at multiple levels, alterations in synaptic function (synaptic plasticity) are believed to play a major role. In particular, compensatory changes in excitatory NMDA and inhibitory GABAA receptors probably contribute to the development of ethanol tolerance.
NMDA and GABAA receptors The major excitatory and inhibitory neurotransmitters in the brain are glutamate and GABA, respectively, and ionotropic glutamate [NMDA, (RS)-a-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) and kainate] and GABAA receptors are among the most widely distributed and abundant receptor-operated ion channels in the CNS. Both NMDA and GABAA receptors are composed of multiple subunit proteins, which are thought to assemble as hetero-pentameric structures that exhibit distinct properties depending upon the particular subunit composition. Identified subunits include a1–a6, b1–b4, g1–g4, d and r1–2 for GABAA receptors and NR1, NR2A–NR2D and NR3A for NMDA receptors. Additional variants of both GABAA and NMDA receptors are generated by alternative splicing. Various molecular aspects of GABAA and NMDA receptors have been the subject of previous reviews1,2. Convincing evidence has shown that excitatory NMDA and inhibitory GABAA receptors are important sites of action of ethanol. Studies using a variety of tissue preparations (i.e. heterogeneous neuronal preparations and cells transfected with receptor subunits) demonstrate that pharmacologically relevant concentrations of ethanol can potentiate GABAA receptor currents and antagonize NMDA receptor currents3,4. It is possible that the combination of reduced excitatory glutamate-mediated activity and enhanced inhibitory GABA-mediated activity contributes to ethanol intoxication. Brain regional differences in the ethanol-sensitivity of NMDA and GABAA receptors have been noted5,6, leading to the suggestion that the ethanol-sensitivity of native NMDA and GABAA receptors is determined, at least in part, by the subunit composition of the receptor. This is supported by studies using recombinant expression systems showing that the ethanol-sensitivity of these receptors varies with the particular subunits expressed7,8. There is also considerable evidence that specific hydrophobic sites of the receptor polypeptides are crucial for
0165-6147/98/$ – see front matter © 1998 Elsevier Science. All rights reserved. PII: S0165-6147(98)01268-1
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L. J. Chandler, Assistant Professor, Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130, USA, R. A. Harris, Professor, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA, and F. T. Crews, Director, Bowles Center for Alcohol Studies, and Professor, Department of Pharmacology and Psychiatry, The University of North Carolina School of Medicine, CB# 7178, Thurston-Bowles Building, Chapel Hill, NC 27599, USA.
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Box 1. Regulation of NMDA receptors by Modification of NMDA and GABAA receptor function appears to play an important role in ethanol tolerance and dependence, and a number of receptor regulatory processes are potential targets of ethanol. Two important post-translational processes that might be affected by prolonged exposure to ethanol are receptor association with the synaptic cytoskeleton and receptor phosphorylation. NMDA receptors bind to cytoskeletal-associated proteins, and receptor–cytoskeletal linkage is essential for subcellular receptor targeting and synaptic localization. In addition, receptor–cytoskeletal interaction is a dynamic process that appears to be coupled to receptor activity. The C-terminal region of the NMDA receptor NR1 subunit binds a-actinin 2 (Ref. 1) and a recently described protein termed yotiao2, both presumably functioning as linker proteins for cytoskeletal attachment, and neurofilament-light (NF-L), which might link NMDA receptors with the subsynaptic cytoskeleton3 (Fig.). Studies using transfected QT6 cells have shown that NMDA receptor clustering is dependent upon the presence of a NR1 splice variant containing the C1-cassette4. Phosphorylation of the C1-cassette by protein kinase C (PKC) could regulate receptor–cytoskeleton attachment as phosphorylation of serine residues within the C1-cassette disperses NR1 receptor clusters. Ca2+ influx following NMDA receptor activation can disrupt the receptor–cytoskeletal interaction via actin depolymerization leading to downregulation of channel activity5. Furthermore, Ca2+–calmodulin can bind to the C1-cassette in a phosphorylation-dependent and competitive manner with a-actinin 2, also resulting in disruption of NR1–actin association and inhibition of channel activity6. In addition to these modulatory processes involving the C1-cassette, alternative splicing of C1 (i.e. variants lacking C1) could play a role in long-term regulation of receptor clustering, stabilization and trafficking, as well as in other cytoskeletal-related processes. NMDA receptor NR2 subunits bind to the PDZ domains of a family of closely related postsynaptic density proteins (PSDs) [PSD-95/synapse-associated protein (SAP)-90, Chapsyn-110/PSD-93, and SAP102]7. These proteins appear to function in localization of receptors to the postsynaptic density and as scaffolding proteins. PSD-95 proteins can undergo head-to-head disulphide linkage resulting in a multimodular scaffold for clustering receptors and/or ion channels and coupling receptor–enzyme complexes and receptor–downstream signalling molecules8. Phosphorylation is important in direct and indirect modulation of NMDA receptors, and might also play a role in synaptic modifications underlying ethanol tolerance. Tyrosine phosphorylation within the C-terminal region of NR2 subunits enhances NMDA currents9–12 and tyrosine phosphatases
modulation of ionotropic glutamate and GABAA receptor function by ethanol and volatile anaesthetics9,10. Finally, phosphorylation and dephosphorylation play an important role in regulating NMDA and GABAA receptors, and second-messenger phosphorylating systems that can directly or indirectly modulate receptor function can be disrupted by ethanol (see Box 1).
Mechanisms of ethanol tolerance Tolerance can include changes at the behavioural level (behavioural tolerance) and at the cellular level (physio492
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reduce NMDA currents13–15. It is thought that tyrosine kinases and phosphatases participate in a dynamic process that regulates channel activity. Phosphorylation of tyrosine residues within the C-terminal region of the NR2 subunit appears to potentiate NMDA currents by reducing tonic inhibition of the receptor by zinc16. Calmodulin kinase II has also been reported to phosphorylate the Cterminal region of NR2 (Ref. 17), but it is not known how this affects channel function. Phosphorylation of the C1-cassette by PKA can enhance NMDA receptor function. In the absence of synaptic activity, it appears that NMDA receptors are phosphorylated by basally active PKA, thus enhancing the activity of quiescent receptors18–20. Ca2+ influx during receptor activation leads to calcineurinmediated dephosphorylation and receptor downregulation, which can be overcome by b-adrenoceptor-mediated stimulation of PKA activity19. As noted above, PKC phosphorylation of the C1cassette can also regulate channel function. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Wyszynski, M. et al. (1997) Nature 385, 439–442 Lin, J. W. et al. (1998) J. Neurosci. 18, 2017–2027 Ehlers, M. D. et al. (1998) J. Neurosci. 18, 720–730 Ehlers, M. D., Tingley, W. G. and Huganir, R. L. (1995) Science 269, 1734–1737 Rosenmund, C. and Westbrook, G. L. (1993) Neuron 10, 805–814 Ehlers, M. D. et al. (1996) Cell 84, 745–755 Sheng, M. (1996) Neuron 17, 575–578 Hsueh, Y-P., Kim, E. and Sheng, M. (1997) Neuron 18, 803–814 Rosenblum, K., Dudai, Y. and Richter-Levin, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10457–10460 Suzuki, T. and Okumura-Noji, K. (1995) Biochem. Biophys. Res. Commun. 216, 582–588 Rostas, J. A. P. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10452–10456 Chen, S. and Leonard, J. P. (1996) J. Neurochem. 67, 194–200 Wang, Y. T. and Salter, M. W. (1994) Nature 369, 233–235 Wang, Y. T., Yu, X. and Salter, M. W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1721–1725 Hall, R. A. and Soderling, T. R. (1997) J. Biol. Chem. 272, 4135–4140 Zheng, F. (1998) Nat. Neurosci. 1, 185–191 Omkumar, R. V. et al. (1996) J. Biol. Chem. 271, 31670–31678 Tong, G., Shepherd, D. and Jahr, C. E. (1995) Science 267, 1510–1512 Raman, I. M., Tong, G. and Jahr, C. E. (1996) Neuron 16, 415–421 Tingley, W. G. et al. (1997) J. Biol. Chem. 272, 5157–5166
logical tolerance). Behavioural tolerance is thought to be a learned response to overcome alcohol-induced behaviours and can be observed in chronic alcoholics and non-alcoholic social drinkers. Physiological tolerance is viewed as a compensatory change at the cellular level in response to the depressant effects of ethanol, and can be temporally divided into rapid (minutes to hours) shortterm changes in response to continuous acute ethanol exposure (acute tolerance) and delayed (hours to days) long-term changes in response to chronic ethanol exposure (chronic tolerance).
phosphorylation
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DA receptors by
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Y and S/T phosphatases PKC Actin depolymerization CaM NOS
PTP (PTP1D)
actin CaM
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α-actinin 2
PP2B PTK
GK
(Fyn, Src)
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ok, G. L. (1993) Neuron 10,
CaMKII
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2
PO4
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1 1
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yotiao NF-L
C2 PDZ domains 1
2
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1 2
R. (1997) J. Biol. Chem. 272,
1, 185–191 Biol. Chem. 271, 31670–31678 ahr, C. E. (1995) Science 267,
P
Ca2+
PO4
6) J. Neurochem. 67, 194–200 1994) Nature 369, 233–235 M. W. (1996) Proc. Natl. Acad.
W
Ca2+
ure 385, 439–442 i. 18, 2017–2027 rosci. 18, 720–730 and Huganir, R. L. (1995)
c. Natl. Acad. Sci. U. S. A. 93,
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phosphorylation and cytoskeletal association
It is thought that tyrotases participate in a ulates channel activity. ne residues within the R2 subunit appears to by reducing tonic inhinc16. Calmodulin kinase o phosphorylate the C17), but it is not known nction. Phosphorylation A can enhance NMDA sence of synaptic activA receptors are phose PKA, thus enhancing ceptors18–20. Ca2+ influx leads to calcineurinon and receptor downvercome by b-adrenon of PKA activity19. As horylation of the C1annel function.
4, 745–755 5–578 M. (1997) Neuron 18, 803–814 Richter-Levin, G. (1996) Proc. 7–10460 , K. (1995) Biochem. Biophys.
I
PDZ-domain interacting proteins
3 S
ahr, C. E. (1996) Neuron 16,
GK
l. Chem. 272, 5157–5166
Acute ethanol tolerance Acute tolerance can be especially important in determining the initial sensitivity of an individual to the intoxicating effects of alcohol. Acute tolerance to ethanol inhibition of NMDA-mediated excitatory postsynaptic potentials (EPSPs) can be observed in vitro at the cellular level. In the hippocampal slice preparation, application of ethanol causes an initial depression of NMDA-mediated EPSPs in the CA1 region, which gradually recover during the period of continuous ethanol exposure11. Similarly, when DBA/2J mice were pretreated with ethanol
Fig. Phosphorylation and cytoskeletal association of NMDA receptors. CaM, calmodulin; CaMKII, calmodulin kinase II; GK, guanylate kinase; NF-L, neurofilament-light; NOS, nitric oxide synthase; PDZ, PSD-95, Dlg-A, Z0-1; PKA, protein kinase A; PP1/2A, protein phosphatase 1 and 2A; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase.
5 min to 1 h prior to decapitation, ethanol no longer potentiated muscimol-stimulated 36Cl– uptake in isolated cerebellar vesicles12,13. These changes in ethanol sensitivity suggest that changes in NMDA and GABAA receptor regulation might underlie acute tolerance. The rapidity of these changes precludes effects at the transcriptional and translational level, or both, and strongly implicate second-messenger modulation of receptor function in the adaptive response. NMDA receptor function is enhanced by tyrosine kinases and reduced by tyrosine phosphatases. Recent
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evidence suggests that acute tolerance to ethanol inhibition of NMDA receptor-mediated EPSPs involves modulation of NMDA receptor tyrosine phosphorylation by the non-receptor tyrosine kinase Fyn14. Fyn-deficient (fynz/fynz) mice, which exhibit enhanced sensitivity to the hypnotic effect of ethanol compared with heterozygotes (+/fynz), do not show acute tolerance to NMDA receptormediated EPSPs. Furthermore, ethanol administration produced an upregulation of tyrosine phosphorylation of the NR2B subunit in heterozygotes but not in Fyndeficient mice. However, it should be noted that loss of righting reflex was the only behavioural parameter examined in the Fyn-deficient mice, and enhanced tyrosine phosphorylation of NMDA receptors was demonstrated only in the hippocampus. Furthermore, it is not known whether the increase in ethanol sensitivity in the Fyn-deficient mice was a direct result of a lack of NR2B phosphorylation by Fyn. Clearly, further work is needed to clarify the role of Fyn and NMDA receptor phosphorylation in acute ethanol tolerance. Studies have also shown that ethanol enhancement of GABAA receptor function requires activation of protein kinase C (PKC)15 and that null mutant mice lacking PKC-g are resistant to the actions of ethanol on GABAA receptors and on behavioural measures16. Whether decreases in the phosphorylation state of GABAA receptors occur during development of ethanol tolerance is not known. Furthermore, in the intact brain, ethanol interaction with GABAA receptors and/or their regulatory processes appears complex, and considerable efforts will be required to define conclusively the role of phosphorylation and subunit composition in acute ethanol tolerance.
Chronic ethanol tolerance Hyperexcitability of the CNS is a characteristic component of ethanol withdrawal, and there is good evidence for both a reduction in GABA-mediated inhibitory and an enhancement in glutamate-mediated excitatory neurotransmission following chronic ethanol exposure. Studies using primary neuronal cultures have shown that prolonged exposure to ethanol leads to a supersensitization of NMDA receptor-mediated events, including Ca2+ influx17 and Ca2+-dependent processes [excitotoxicity18 and nitric oxide (NO) formation19]. Similarly, studies with isolated brain preparations have reported that ethanol-mediated enhancement of GABAA receptorcoupled Cl– flux is decreased following chronic ethanol exposure20. Thus, functional changes in NMDA and GABAA receptors could contribute to physiological tolerance. It is possible that the alterations in NMDA and GABAA receptor function are caused by up- and downregulation, respectively, of receptor density. Several studies have reported increased NMDA receptor binding and/or subunit protein levels following chronic ethanol exposure both in vitro and in vivo21–26, whereas others have failed to find any such changes19,27–29. It was recently reported that exposure of cultured cortical neurones to ethanol leads to enhancement of NMDA494
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T stimulated NO formation (but not that stimulated by kainate, AMPA or ionomycin) without a change in receptor density, and it was suggested that the chronic ethanol effect related to post-translational modification of NMDA receptors19. Similarly, in cells stably transfected with GABAA receptor subunits, ethanol was shown to cause changes in GABAA receptor function similar to those observed in vivo, but no change in surface receptor density was observed30. This finding is in agreement with previous observations in rats31 and mice32 chronically exposed to ethanol. Because the transfected cells contain defined GABAA receptor subunits with expression controlled by the dexamethasone-sensitive promoter, it is unlikely that ethanol affected subunit expression or produced subunit substitution. Thus, it is possible that posttranslational modification(s) underlie these functional changes. No change in GABAA receptor density following chronic ethanol exposure has been observed in the majority of studies. However, changes in GABAA receptor subunit expression in the brain have been reported, suggesting that subunit changes could play an important role in vivo33. An important component of ethanol-induced posttranslational modification of receptor function could relate to second-messenger systems. Tyrosine phosphorylation of GABAA receptors has been shown to enhance GABAA receptor-gated currents34 and, as noted above, tyrosine phosphorylation of NMDA receptors has been linked to the development of acute tolerance of NMDA receptors. PKC, PKA and calmodulin kinase II have been reported to phosphorylate and/or alter the function of NMDA or GABAA receptors, or both (for review, see Ref. 3; Box 1), and phosphorylation could alter their ethanol sensitivity. Whether the phosphorylation state of NMDA and GABAA receptors is altered by chronic ethanol exposure is not yet known. Chronic ethanol can increase PKC levels and activity35–39 and induce heterologous desensitization of cAMP signalling with decreased PKA activity40–42. Some of these effects of ethanol could relate to changes in subcellular translocation and localization. Ethanol has been shown to stimulate translocation to the nucleus of the catalytic subunit of PKA where it remains sequestered for as long as ethanol is present43, and to stimulate translocation of PKC-d and PKC-e to new intracellular sites39. Translocation of PKC and PKA isozymes to subcellular anchoring proteins is thought to be important in targeting specific signalling events. Furthermore, PKA and calcineurin (protein phosphatase 2B) are concentrated in postsynaptic densities via a common Akinase anchoring protein (AKAP79), putting them in position to regulate phosphorylation and/or dephosphorylation of key postsynaptic proteins44. Clearly, changes in PKA and/or PKC activity and subcellular targeting could play an important role in ethanolinduced changes in synaptic function, including modulation of NMDA and GABAA receptors. Another potentially important process in NMDA and GABAA receptor adaptation during ethanol exposure is
V receptor–cytoskeletal interaction (see Box 1). NMDA receptors are required for activity-dependent synaptic remodelling during development, and studies in hippocampal cultures have shown that the subcellular distribution of NMDA receptors is modulated by receptor activity. Chronic treatment with an NMDA receptor antagonist leads to increased NMDA receptor clustering at synaptic sites and, conversely, spontaneous activity leads to decreased synaptic NMDA receptor clustering45. Because studies in primary neuronal cell cultures might more closely model developmental processes, an important question to be addressed is whether this activitydependent redistribution of NMDA receptors also occurs in mature neurones. Furthermore, it is not known whether the functional property of the NMDA receptor itself is altered by clustering and redistribution (i.e. synaptic versus non-synaptic). However, receptor redistribution could represent a novel form of activity-dependent synaptic modification (plasticity), and prolonged inhibition of the NMDA receptor during chronic ethanol exposure might also lead to an increase in NMDA receptor clustering at synaptic sites. Clearly, this is an intriguing hypothesis that needs testing. The specifics of receptor–cytoskeleton interaction is less clear for GABAA receptors. These receptors form clusters in postsynaptic membranes that are not associated with prominent postsynaptic densities, and might anchor to the subsynaptic cytoskeleton via gephyrin or a gephyrin-related protein46. Interestingly, studies in transfected cells have shown that disruption of microtubule polymerization also disrupts GABAA receptor clustering and abolishes the action of ethanol47. Also of interest is the observation that insulin can stimulate a rapid translocation and clustering of functional native GABAA receptors to the postsynaptic domain of inhibitory synapses in cultured hippocampal neurones48. However, whether GABAA receptor clustering is responsive to changes in channel activity and the effect of chronic ethanol on this process remain to be determined.
Concluding remarks Inhibition of NMDA and potentiation of GABAA receptors by acute ethanol results in reduced synaptic activity that is likely to contribute to the intoxicating effects of ethanol. It is probable that alteration in synaptic function by ethanol exposure contributes to the development of physiological tolerance that involves changes in excitatory glutamate-mediated and inhibitory GABAmediated neurotransmission. Acute tolerance might involve post-translational modifications such as receptor phosphorylation and/or dephosphorylation and receptor clustering and/or unclustering. Although up- and downregulation of NMDA and GABAA receptor density, respectively, could be a prominent mechanism underlying changes in receptor function during chronic ethanol exposure, alterations in receptor density cannot account exclusively for the functional changes.
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We suggest that multiple mechanisms could contribute to the alterations in NMDA and GABAA receptor function during ethanol exposure. While acute physiological tolerance might involve only post-translational processes, chronic physiological tolerance could involve both post-translational modifications and changes in gene expression. In addition to changes in receptor density, potential mechanisms include subunit substitution and alternative splicing, alterations in second-messenger phosphorylating and/or dephosphorylating systems and altered receptor clustering and subcellular redistribution. Although it has yet to be determined whether many of the post-translational events observed in vitro actually occur in vivo, these types of observations are beginning to reveal the dynamic nature and potential underlying mechanisms of adaptations of the CNS to the depressant effects of ethanol on synaptic function. Selected references 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
Smith, G. B. and Olsen, R. W. (1995) Trends Pharmacol. Sci. 16, 162–167 Sucher, N. J. et al. (1996) Trends Pharmacol. Sci. 17, 348–355 Crews, F. T. et al. (1996) Int. Rev. Neurobiol. 39, 283–367 Diamond, I. and Gordon, A. S. (1997) Physiol. Rev. 77, 1–20 Simson, P. E. et al. (1993) Brain Res. 607, 9–16 Criswell, H. E. et al. (1993) J. Pharmacol. Exp. Ther. 267, 522–537 Massood, K. et al. (1994) Mol. Pharmacol. 54, 324–329 Harris, R. A. et al. (1997) Alcohol. Clin. Exp. Res. 21, 444–451 Minami, K. et al. (1998) J. Biol. Chem. 273, 8248–8255 Wick, M. J. et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6504–6509 Grover, C. A. et al. (1994) Brain Res. 642, 70–76 Allan, A. M. and Harris, R. A. (1987) Pharmacol. Biochem. Behav. 27, 665–670 Morrow, A. L. et al. (1988) J. Pharmacol. Exp. Ther. 246, 158–164 Miyakawa, T. et al. (1997) Science 278, 698–701 Weiner, J. L. et al. (1997) J. Neurochem. 68, 1949–1959 Harris, R. A. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3658–3662 Iorio, K. R. et al. (1992) Mol. Pharmacol. 41, 1142–1148 Chandler, L. J. et al. (1993) J. Neurochem. 60, 1578–1581 Chandler, L. J. et al. (1997) Mol. Pharmacol. 51, 733–740 Buck, K. J. and Harris, R. A. (1991) Alcohol. Clin. Exp. Res. 15, 460–470 Grant, K. A. et al. (1990) Eur. J. Pharmacol. 176, 289–296 Gulya, K. et al. (1991) Brain Res. 547, 129–134 Snell, L. D. et al. (1993) Brain Res. 602, 91–98 Trevisan, L. et al. (1994) J. Neurochem. 62, 1635–1638 Hu, X-J. and Ticku, M. K. (1995) Brain Res. 30, 347–356 Chen, X. et al. (1997) J. Neurochem. 69, 1559–1569 Tremwel, M. F., Anderson, K. J. and Hunter, B. E. (1994) Alcohol. Clin. Exp. Res. 18, 1004–1008 Carter, L. A. et al. (1995) J. Neurochem. 64, 213–219 Rudolph, J. G. et al. (1997) Alcohol. Clin. Exp. Res. 21, 1508–1519 Harris, R. A. et al. (1998) J. Pharmacol. Exp. Ther. 284, 180–188 Rastogi, S. K. et al. (1986) Neuropharmacology 25, 1179–1184 Buck, K. J. and Harris, R. A. (1990) J. Pharmacol. Exp. Ther. 253, 713–719 Morrow, A. L. (1995) Int. Rev. Neurobiology 38, 1–41 Valenzuela, C. F. et al. (1995) Mol. Brain Res. 31, 165–172 Messing, R. O., Henteleff, M. and Park, J. J. (1991) J. Biol. Chem. 266, 23428–23432 Roivainen, R. T., McMahon, T. and Messing, R. O. (1993) Brain Res. 624, 85–93 DePetrillo, P. B. and Liou, C. S. (1993) Alcohol. Clin. Exp. Res. 17, 351–354 Coe, I. R. et al. (1996) J. Biol. Chem. 271, 29468–29472 Gordon, A. S. et al. (1997) Mol. Pharmacol. 52, 554–559 Gordon, A. S., Collier, K. and Diamond, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2105–2108 Rabin, R. A. et al. (1992) J. Pharmacol. Exp. Ther. 262, 257–262 Coe, I. R. et al. (1996) J. Pharmacol. Exp. Ther. 276, 365–369 Dohrman, D. P. et al. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10217–10221 Coghlan, V. M. et al. (1995) Science 267, 108–111 Rao, A. and Craig, A. M. (1997) Neuron 19, 801–812 Whatley, V. J. and Harris, R. A. (1996) Int. Rev. Neurobiol. 39, 113–143 Whatley, V. J. et al. (1996) J. Neurochem. 66, 1318–1321 Wan, Q. et al. (1997) Nature 388, 686–690
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