TiPS -May 1992 [Vol. 231
204 sci. 13,420-423 9 Matsuda, L. A., Lolait, S. J., Bmwnstein. M. J., Young, A. C. and Bonner, T. I. (1990) Nature 346, 561-564 10 Johnson, M. R. and Melvin, L. S. (1986) in Cannabinoids as Therapeutic Agents (Mechoulam,R., ed.), pp. 121-144, CRC Press 11 Bell, M. R. et al. (1991) 1. Med. Chem. 34, 1099-1110 12 Mechoulam, R. et al. (1987) NIDA Res. Mono~. Ser. 79,15-30 13 Razd&, R. K. (1987) NIDA Res. Monogr. Ser. 79,3-14 14 Di Chiara, G. and Imuerato, A. (1988) Proc. Nat1 Acad. Sci. USA 85,5274-&278. 15 Chen, J_ et al. (1990) PFychophannacolopv 102.156-162 16 Che& J., &&es, W., Lowinson, J. H. and Gardner, E. L. (1990) Eur. I. Pharmuco!. 190,2S9-262 17 Gardner, E. L. et al. (1988) Psychouhannucoloav %, 142-144 18 &wee, R.ud. (l&l) in TheBiologicalBasis of Drux Tolerance and Dependence (F%att, J: A., &i.). pp. 232-263, Academic Press 19 Kaymakcakm, S. (1973) Bull. NIX. 25, 39-47
20 Beardsley, P. M., Balster, R. L. and Harris, L. S. (1986) 1. Pharmacol. Exp. Ther. 239,311-319 21 McMillan, D. E., Dewey, W. L. and Harris, L. S. (1971) Ann. NY Acad. Sci. 191,699 22 Camey, J. M., Uwaydah, I. M. and Balster. R. L. (1977) Phurmacol. Biochem. Behuv.~7,357~364 23 Harris, L. S., Dewey, W. L. and Razdan, R. K. (1977) in Handbook of Experimental Pharmucology (Born, G. V. R., Eichler, 0.. Farah. A. and Welch. A. D., eds), DD. r. 1’ 371-429, Springer-Verlag 24 Dill, J. A. and Howlett, A. C. (1988) 1. Ph&mucol. Exp. Ther. 244, 1157~1163~ 25 Westlake, T. M. et aI. (1991) Brain Res. X4.14.5-149 26 Jon&, R. T. (1983) in Cannabis and Heulth Huzurds (Fehr, K. 0. and Kalant, H., eds), pp: 617-689, Addiction Research Foundation 27 Jones, R. T., Benowitz, N. and Bachman, J. (1976) Ann. NY Acad. Sci. 282,221-239 28 Jones, R. T. and Benowitz, N. (1976) in Phurmucology of Murih&n &aide, M. C. and Szara, S., eds), pp. 627-642, Raven Press
Neurobiology of alcohol abuse Herman H. Samson and R. Adron Harris Excessive consumption of beverage alcohol (ethanol) is a major health concern worldwide. Understanding the mechanisms by which ethanol affects neural functioning, after both acute and chronic exposure, has become a major goal in the study of alcoholism. With such an understanding, we should be able to institute more effective treatments and preventative measures for alcohol abuse problems. Recent studies have found, contrary to earlier assumptions, that ethanol has selective, dose-dependent effects on various neurotransmitter systems within the CNS. These effects are observed at all levels of analysis, from molecular to behavioral. This review by Herman Samson and Adron Harris covers these recent findinps. with the intent of generating questions that will focus further resedrch efioks. Alcohol is the second most widely used psychoactive substance in the world after caffeine. While most societies approve of moderate alcohol use, chronic and/or excessive drinking is considered a hazard to both health and public safety. In the USA, the cost of alcohol-related problems was estimated at over $136 billion in 1990, and unless changes in drinking patterns occur, or better treatment and prevention approaches H. H. Samson is Director at the Alcohol and Drug Abuse Institute and Professor in the Depurfment of Psychiatry and Behavioral Sciences, University of Washington School of Medicine. Seattle, Washington, and R. Adron Harris is a Research Career Scientist at the VA Medic42 Center and Professor in the Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado, USA. 0 1992.Elsevier
Science Publishers Ltd (UK)
are found, health costs will be over $150 billion in 1995l. In order to both treat and prevent alcoholrelated problems, basic research into the mechanisms by which alcohol produces its psychotropic activity is essential. Ethyl alcohol (ethanol), unlike most psychoactive drugs, has no known specific receptor system within the CNS. However, it is clear that ethanol can affect a variety of neurotransmitter systems and understanding how this occurs is most likely to be the key to explaining the psychoactive actions of ethanol. It is impossible to cover all of the neurotransmitter systems known to be affected by ethanol within the space limitations of this review. While important findings related to many transmitter
29 Jones, R. T., Benowitz, N. L. and Heming, R. I. (1981) 1. Clin. Pharmacol. 21,143S-1525 30 Howlett, A. C., Qualy, J. M. and Khachatrian, L. L. (1986) Mol. PharmacoI. 29,307-313 31 Biduat-Russell, M., Devane, W. A. and Howlett, A. C. (1990) /. Neurochem. 55, 21-26 32 Pacheco. M.. Childers. S. R.. Arnold. R.. Casiano; F: and Ward, 6. J. (lb91j 1. Phnrmacol. Em. Ther. 257,170-183 33 Eisenstat, M. A: et nf. (1990) NIDA Res. Monogr. Ser. 105,427-428 34 Herkenham. M. et al. (1991) . ,, 1. Neurosci. ~~~ 11.563-583. 35 Herkenham, M. et al. (1990) Proc. Nat! Acad. Sci. USA 87,1932-193& 36 Harris, L. S. (1978) in Psvchovharmacology, A Generation of Progress ilipton, M. A., DiMascio, A. and Killam, K. F., eds). UD. __ 1565-1574. Raven Press 37 Plasse, T. F. et ui. (1991) Pharmucof. Biochem. Behuv. 40.695-700 38 Munson, A. E. and Fehr, K. 0. (1983) in Cannabis and Health Hazards (Fehr, K. 0. and Kalant, H., eds), pp. 257-354, Addiction Research Foundation
systems have been delineated (see Ref. 2 for a more complete discussion of other transmitter systems), this review highlights the recent findings related to the excitatory and inhibitory amino acid transmitters and to dopamine and 5-HT. Molecular pharmacology The search for moiecular actions of alcohol has focused mainly on mechanisms of intoxication and development of tolerance and dependence. A key, and unanswered, question is how these actions are related to alcohol selfadministration in laboratory animals and to alcohol abuse and alcoholism in humans. Membrane actions Alcohols (typified by ethanol) are members of the family of intoxicant anesthetics that includes volatile anesthetics, barbiturates and benzodiazepines; drugs that display a similar spectrum of intoxication as well as substantial cross-tolerance and cross-dependence. Because of the chemical structural diversity of these intoxicant anesthetics, it is difficult to imagine a single receptor that could be responsible for actions of ethanol as well as these other related drugs. Based on the pioneering work of Meyer and Overton carried out 100 years ago, the idea that alcohol acts by partitioning into neuronal mem-
TipS - May
1992 (Vol.
131
Drugs and synapses: puzztes and paradoxes Drugs discussed in this issue of TiPS vary widely in their potencies. For
example, nanomoIar concentrations of some opioids are sufficient to produce appreciable occupancy of receptors with in vitro binding assays, whereas ethanol ~nc~atio~s of IO-l#tl rn?.sare required for actions both in viva and in vitro. What is the physical meaning of these concentrations at a synapse? If we assume that the synapse is a cylinder with a diameter of IOOOnm and a height of 2Onm, then its volume is 1.6 X Xi” Liters. A concentration of 1 IIM cmwerts to 6 X IOx mofecutes per Literrthus at 1 no there wiII be only one drug molecule per 100 synapses! At the seemingly huge concentration of lOmM, there will be only 100 molecules of ethanol in each synapse. Yet some drugs do indeed exert effects on behavior with brain concentrations in the nanomdar range. How can this occur? It is possible that only a small fraction of the synapses need to be affected by the drug for a behavioral action. However, it seems more likely that the drugs accumulate at synapses leading to local concentrations that are larger than the average concentration. This could occur because of a high hpid solubihty in which case the concentration of the drug in the membrane would be considerably gmater than in the water phase. If the receptor is also buried in the membrane, then the lipid concentration is more relevant than the water concentration. Likewise, slow dissociation of drugs from their receptors would lead to accumulation of drugs in the synapse. ft is interest&g to note that potent drugs are often quite lipid soluble and display slow dissociation kinetics, whereas ethanol, the prototype lowpotency drug, has low lipid solubihty and its effects are rapidly reversed in vitro. Perhaps the synaptic concentrations of the drugs described in this issue are not very different; certainly they may not be as different as the water concentrations (or dosages) would imply. Another puzzle is why some drug actions observed in vitro have any effect on synaptic transmission. Articles in this issue tell us that benzodiazepines and ethanol increase the action .of GABA at GABAA receptors‘ that ethanol increases the action of 5-HT at 5-HTs receptors and that cocaine increases synaptic dopamine, noradrenahne and 5-HT. However, these actions of ethanol and benzodiazepines occur only at submaximal concentrations of the neurotransmitters; once the receptors are fully occupied neither of these drugs (or cocaine} would be expected to affect synaptic transmission. What is the synaptic concentration of a neurotransmitter? This is, of course, unknown, but we can make some estimations. The lowest concentration that a synapse can have is one molecule, which would be 100 nM from the calculations above. However, it is hkely to be mush larger_ Vesicular concentrations of acetylchohne and glutamate are estimated at lOS2QOmru. The volume of synaptic vesicIes (60 nm diameter) is about 1% of the synaptic volume calculated above, thus a synaptic neurotransmitter concentration of lrmur is not unreasonable {and quite saturating for receptors). Perhaps drugs affect extrajunctional receptors where the concentration of ne~ans~~er is not saturating? Alternatively, they may affect the synaptic transmission only when the concentrations of
neurotransmitter are rising or fallingand are not saturating. Are actions of these drugs more subtle than simply enhancing neuro~ans~~ion? New techniques allowing rapid application of ne~ro~ansmi~ers (concentration clamp, quench flow] and measurement of synaptic ~~smission in brain slices (slice patch) should solve this puzzle. Values used for calculations are from McGeer,P. I.., Eccles, J. C. and McGeer, E. G. (2978) Molecular Biology of the Martian Brain, Pleuu~ Press.
branes and altering their physical properties received considerable attention during the 1970s and 1980s. The hypothesis that acute alcohol disorganizes or ‘fluidizes’ membranes and that chronic alcohol rigidifies membranes is supported by numerous studies of brain membranes, but the question remains as to whether these changes in physical proper-
ties are sufficient to alter membrane function2*4. In the 1980s and 199&z, emphasis has shifted to a search for neuronal functions that are sensitive to intoxicating concentrations of alcoho15,6 (see Box). Electrophysiological and neurochemical studies in the brain have identified ion channels, particu-
(,
t ilicotinic acatylcholine(-I-) i
\
7r
oulpurtt 11
I?& 1. Acure eq3osure to alcuhuf enhances action of 5-H-f at 5-M-receptorsand a&on af a~~~t~e at nimtinic chuthwgic receptors. Both these rscspiors actlvste cation chmts, thus 8re sxctta~, and s-i wfmid imrem Cwput of a hy~~~t r?suri~~expressing cmiy fhsse recento~. Data fromti$inaer. D. M. anh White, G. (1991) kai Phamad. 40, 263-270 and Furman. S. A.. F@$i, D. I and Miuet, K. W. (1989~8&c&m. &bpfys. Ada 987,~103.
larlv &and-gated channels, that are especially sensitive to alcohol. Neurotransmission at two types of excitatory receptor, nicotinic acetylcholine and 5HTa, is enhanced by acute alcohol exposure (Fig. 1). Most other excitatory receptors are inhibited by alcohol, while ~~ibito~ systems are augmented. For exampie, acute alcohol exposure inhibits the excitatory action of glutamate at both N&IDA and kainate receptors* inhibits voltage-sensitive Caa” channels and enhances GABA action at some GABAA receptors (Fig. 2). Chronic alcohol treatment results in increased numbers of N&IDA receptors and voltage-sensitive Ca” channels, whereas GABAA function is reduced but kainate responses are unaltered. These change5 in receptor and channel function offset the acute action of alcohol and should result in hyperactivity when alcohol is ~~~a~, providing a piausible mechanism for tolerance and dependence’. The question of which, if any, of these neurochemical responses to alcohol treatment is related to the severity of alcohol withdrawal can be addressed by determining genetic correlations (see Crabbe and Belknap, this issue). Lines of mice have been selectively bred to be either prone or resistant to seizures when withdrawn from chronic alcohol exposure. After
TiPS - May 1992 \Vol. 131
20s
ethanol intoxication
ethanol dependence t NMDA (+)
a\
Fg. 2. Acute exposure to alcohol inhibits action of NIUDA and kainafe on g/ufamate racepmrs, inhibits timcfion of voltage-sensitive Ca” channels and enhances action of &WA at GAB&receptmsBeczwsethethreecation (excitatory) channels are inhibited and the anfon (inhibitory) channel is activafed, acute alcohol would markedly decrease the output of a hypothetical neuron expressing these ion channels. In contra.% chronic exposure to alcohol inMeases NMDA receptors and voltage-sensitive ion channels and demsaws GABA. tinction. ofLsetlino the acute action of alcohol and resulfing in kweased ou@ut &en akwhoi is with&awn tiom this hypothericar cell. Data from Refs 3 4 Wafaht, F. F.. Lovinper, D. M., White, G. and Peoples, R. W. (1991) Ann. NYAcad. %825.>7-107;Brenn& C. H.. Guppy, L. J. and kleton, J. M. (1989) Ann. NYAcad. sci. 560,467-469.
many generations of selection, alleles responsible for alcohol withdrawal seizures (and other signs of withdrawal) have segregated and the neurochemical effects of chronic alcohol should be different in these mice lines, provided these neurochemical changes are related to the development of physical dependence (as measured by withdrawal). Use of these selected lines for testing neurochemical hypotheses revealed three consequences of chronic alcohol exposure that are greater for Seizure-Prone mice than for Seizure-Resistant mice: increased density of nitrendipinebinding sites (i.e. upregulation of voltage-sensitive Ca2+ channels); increased efficacy of DMCM, an inverse agonist at benzodiazepine receptors; and decreased mRNA coding for the arl subunit of GABA* receptors (Fig. 3). By contrast, Seizure-Prone and SeizureResistant mice exhibit two similar neurochemical changes produced by chronic alcohol: an increase in dizocilpine-binding sites (i.e. upregulation of NMDA receptors) and an increase in membrane rigidity as detected by diphenylhexatriene fluorescence polarization (Fig. 3). However, the density of NMDA receptors is higher in Seizure-Prone mice than in
Seizure-Resistant mice before exposure to alcohol. Such genetic correlations provide strong support for the involvement of NMDA receptors and some types of voltage-sensitive Ca” channels and GABA* receptors in the development of physical dependence on alcohol. Identification of these actions of alcohol leads to the question of how either acute or chronic exposure to this relatively simple molecule can alter the function of ion channels. Our current understanding of alcohol’s action is most advanced for the GABA* receptor. Both behavioral and neurochemical studies provide evidence of enhancement of GABA receptor activity by alcoho!. Studies of selected lines of mice and rats indicate that genetic differences in acute (and chronic, Fig. 3) actions of alcohol are related to the GABA* receptor7. However, electrophysiological studies have not consistently detected augmentation of GABA* responses by alcoho12. An explanation of these discrepancies may be found in molecular biological studies of the multiple GABA* receptor subunits that have been cloned and sequenced. Although a variety of different subunit combinations
are able to form receptors that respond to GABA or pentobarbital, benzodiazepine responsiveness requires a member of the y family of subunits. A recent study8 using electrophysiological measurement of oocytes expressing cloned GABA receptor subunits indicates that a particular y subunit, y2~, is required for alcohol enhancement of GABA action. It is ironic that the simplest molecule (ethanol) has the most specific subunit requirement for action at the GABA receptor complex! The yzL subunit differs from y2s by only eight amino acids, yet the latter subunit does not confer alcohol sensitivity. The difference between these subunits is that y2~ contains a serine that can be phosphorylated by protein kinase C, and this appears to be critical for alcohol action. Even in these (relatively) simple the molecular action systems, of alcohol remains elusive, but effects on protein kinases or phosphatases could be responsible for changes in function of several ion channels, including those coupled to GABA* receptors. In addition, molecular mechanisms of neuroadaptation to chronic alcohol exposure are beginning to be unraveled. For the GABAA receptor, levels of mRNA for specific subunits are altered, suggesting that chronic alcohol ingestion may affect gene expression’. Chronic alcohol also alters GABAn receptor function in an oocyte system expression of GABAA where receptor genes is bypassed by injection of mRNAlO. Recent evidence that chronic alcohol alters expression of genes for intracellular regulators such as Fos, heat shock protein and the (Y, subunit of G proteins provides exciting prospects for molecular studies of other neuroadaptive mechanisms4. Mechanisms of alcohol abuse and alcoholism The reinforcing action of alcohol, as for other drugs of abuse, appears to involve activation of the mesolimbic dopamine system. For example, alcohol increases the firing of neurons in the ventral tegmental area and increases extracellular concentrations of dopamine in the nucleus accumbens, but the molecular mechanisms responsible for these actions
TiPS - May 1992 /VoL 231 remain to be defined*. Selected lines of rats that should prove useful for such research include alcohol-Preferring Nonand Preferring, alcohol-Avoiding and Non-Avoiding, and High-AlcoholDrinking and Low-AIcohol-Drinking rats”. Studies of these rat lines suggest that an elevated level of dopamine in the nucleus accumbens encourages alcohol drinking, as does a lower level of 5-HT in the nucleus accumbens and several other brain regions7. In addition, recent data show that the alcohol-Preferring rat and High-Alcohol-Drinking rat have a higher density of GABA-containing terminals in the nucleus accumbens than their opposite pairsl*. These results suggest that the increase in transmission at GABA receptors produced by alcohol (discussed above) could be related to the reinforcing actions of the drug. Animal studies demonstrate that alcohol acutely enhances adenylyl cyclase activity due to an action on G, and chronically reduces activity of brain adenylyl cyclase 13. Extension of these studies to human alcoholics demon-
100
75
209
strated a decreased activity of adenylyl cyclase in platelets and lymphocytes. A similar decrease can be produced in neurons cultured in the presence of alcohol. Most importantly, platelets from individuals with a positive family history of alcoholism have a lower content of platelet adenylyl cyclase even if they are not actively drinking13. Thus, this marker may detect the ‘trait’, rather than the ‘state’, of alcoholism. The molecular basis for this defect in platelets of human alcoholics, and whether it occurs in brain, are not yet known. Because of the role of dopamine systems in drug reinforcement, the possible role of brain D2 receptors in alcoholism is emerging as an important area of study. Blum, Noble and co-workers’4 found that one allele (AZ) of the human brain D2 receptor was associated with alcoholism and that the presence of this allele reduced the density of dopamine receptors in human caudate mlcleus. Other workers have confirmed an increased incidence of this allele in alcoholics with severe medical problems compared with
0
WithdrawalSeizure-Resistant
a
WithdrawalSeizure-Prone
50 25
-25
fig. 3. The effect of genetic differences in susceptibility to seizures upon ethanol withdrawal on neurochemicat responses to chronic ethanol treatment. Withdrawal Seizure-Prone mice showed the same or greater changes in response to chronic ethanol exposure than Withdrawal Seizure-Resistant mice. Data fromRefs 4; 10; Brennan,C. H., Crab& J. C. and Littleton, J. M. (1990) Neurophannacofogy 29, 429-432; Valverius, P., Crabbe, J. C., Hoffman, P. L. and Tabakoff, B. (1990) Eur. J. Pharmeco/. 164, 195-189; Harris, R. A., Crabbe, J. C. and McSwigan,J. D. (1994) Life Sci. 35,2601-2606.
nonalcoholic controls, but it was not increased in alcoholics without medical complications and there was no evidence of linkage or cosegregation of the AZ allele with alcoholismi5. It is possible that the AZ allele has a modifying effect that increases the severity of alcoholism. Because alcoholism is polygenic, clinically heterogeneous, and strongly influenced by environment, it is not surprising that this single allele does not demonstrate a major causative role in most cases of alcoholism. However, this finding represents an important initiative in the search for brain genes related to alcoholism. Behavioral pharmacology Ethanol has a variety of behavioral effects, many of which are dependent upon dose, route and type of administration. For a majority of the animal studies, ethanol has usually been administered by the experimenter, using an i-p. or i.v. injection or intubation. While assuring the delivery of a standard, fixed dose, these administration methods generally fail to mimic the blood ethanol curves typical of orally self-administered alcohol consumption. Therefore, caution is required in interpretation of the results in relation to oral selfadministration of alcohol. Ethanol as a reinforcer Over the past 15 years, many studies have shown that ethanol, like other drugs of abuse, can function as a reinforce+i7. Initial studies demonstrated that several different routes of administration (oral, intra-gastric and i.v.) were effective. However, since the oral route is typically used by humans, recent studies have focused on the oral self-administration reinforcement processes. In the past, demonstration that oral ethanol consumption can be reinforcing in nondeprived animals presented problems, but the development of a variety of initiation procedures has overcome many of these difficulties”. While it is difficult to ascertain in animal studies what the psychological nature of alcohol reinforcement might be, recent studies in animals fed ad lib suggest that the calorific properties of alcohol play only a minimal role in its reinforcing actions.
TiPS - May 1992 [Vol. 131
210 Many of alcohol’s reinforcement properties can be modified by orior treatment with CNS neurohansmitter receptor agonists and antagonists, particularly those affecting the mesolimbic dopamine system”. A critical behavioral component that occurs during the development of oral ethanol consumption is the conditioning of environmental cues that signal the availability of ethanol to its pharmacological effects. These conditioned cues can then elicit ethanolseeking behaviors, even in the absence of ethanol*820. Similarly, in human alcoholics, the intensity of cravings for alcohol increases in the presence of cues related to alcohol availabili@‘. The study of the development of these conditioned behavioral functions is of major importance in understanding their relation to excessive drinking patterns in humans”. Ethanol as a discriminative stimulus While the physiological effects of ethanol can function to reinforce behavior, these effects can also serve as discriminative stimuli to direct behavior selection related to other reinforcers. In his classic study, Barry demonstrated that ethanol could function as a discriminative stimulus under a variety of conditions”. However, as discussed above, there are a variety of CNS cues that result from the pharmacological actions of ethanol. Any or all of these altered CNS effects could underlie the cue that the animal uses. One valuable use of the drugdiscrimination procedure is to test other drugs to determine whether they produce similar or different cues=. If other drugs produce cues that are the same as alcohol (i.e. the animal responds as if it has received alco’nol), then a mechanism that is common to both the drug and alcohol can be hypothesized to underlie generation of the cue. A variety of analgesics and tranquilizers have been found to partially substitute for the ethanol cue, while drugs such as stimulants and neuroleptics do not substitute”. This suggests that the major discrimination cues resulting from ethanol are those that relate to its sedative and anesthetic actions. These actions may
be the result of an interaction of ethanol with the GABA* receptor benzodiazepine modulatory site. However, recent studies using neurotransmitter antagonists have that both the demonstrated NMDA and 5-HTs receptor systems are involved in ethanol discrimination24,25. This suggests that a complex, multiple set of cues is responsible for the ethanol cue. Understanding the relative contributions of these different CNS mechanisms could provide a clearer picture of how ethanol’s pharmacological cues interact environmental cues to with excessive drinking produce behavior. Evidence for tolerance Repeated administration of alcohol results in decreased sensitivity, or tolerance, to many of its effects. It has been suggested that the development of tolerance to the ‘negative’ effects of ethanol (i.e. motor incoordination, sedation, nausea, etc.) is necessary for the generation of excessive drinking behaviorsz6, but clear validation of this hypothesis remains to be provided. The development of tolerance to repeated administration has been well documented for most of the physiological effects of ethanol. Tolerance to the motor, anesthetic, hypothermic, sedative and anxiolytic effects has been shown when ethanol was administered to the animal by the experimenter. Tolerance to the disruptive effects of such experimenter-administered ethanol on the performance of behavior controlled bv food reinforcement has also been found27. Using oral ethanol selfadministration induced by the presentation of food pellets on a 90-second fixed time schedule in food-restricted rats (a scheduleinduced polydipsia paradigm), tolerance to the motor discoordination effects was also demonstrated2’. However, when examining the effects of tolerance to ethanol, it is important to select carefully the procedures used to evaluate tolerance development. Failure to use doses and routes that might be related to oral consumption can make interpretation difficult. For example, Cunningham and colleagues recently demonstrated that the acute hypothermic re-
sponse to an i.p. injection of lg kg-l ethanol failed to occur when the animal orally selfadministered the same dose over a five-minute period”. Cunningham suggests that the tolerance to the acute response seen in injection studies may be specific to the injection procedure30 or to a handling component that accompanies it31. Thus, doses of ethanol that produce tolerance when administered by the experimenter may not produce tolerance when orally self-administered by the experimental animal. It should be noted that no evidence of tolerance to the reinforcing properties of alcohol has been demonstrated. Any observed increase in alcohol intake over repeated drinking opportunities could be a result of tolerance to the reinforcing properties or tolerance to the motor and sedative effects, either of which could result in increased intakes. From studies using the schedule-induced polydipsia paradigm, it would appear that the tolerance is developed to the motor-disrupting effects, with no change to the reinforcing properties17. A variety of environmental stimuli can be conditioned to predict the administration of ethanol. These learned cues can result in physiological responses that are counter to the effects of the ethanol and these counter-responses are a possible mechanism of tolerance32. This conditioned or learned tolerance has been demonstrated as a factor for several different measures of ethanol tolerance, including ataxia, hypothermia and ethanol’s anticonvulsive properties. The development of learned tolerance to the motor discoordination effects of orally consumed alcohol has been shown in humans33, and there is evidence that counter or compensatory responses can also be measured in humans32. However, the exact role of learned tolerance to the development of excessive drinking has yet to be elucidated. The phenomenon of rapid, or acute, tolerance has been observed for several behavioral measures. For example, after an ethanol dose, ethanol levels are lower when ataxia is first observed on the rising phase of the blood ethanol curve than when the ataxia disappears on the fall-
TiPS - May 1992 [Vol. 131 ing limb of the blood ethanol curve. This acute tolerance can be influenced by genetic selection, such that animals selected for higher ethanol preference demonstrate a greater acute tolerance than animals selected for ethanol aversion=. This suggests there may be a direct relationship between acute ethanol tolerance and ethanol preference that is under genetic regulation. Evidence for addiction In humans, alcoholism has as a major diagnostic feature the loss of control when drinking has begun [as classified by the Diagnostic and Statistical Manual of Mental Disorders (Third Edn Revised) (1987) American Psychiatric Association]. Furthermore, the craving for alcohol during periods of abstinence has often been considered a prime factor underlying excessive alcohol use. Unfortunately, these two behavioral features are not easy to examine using experimental animal models, partly because of the difficulty in defining craving and loss of control for humans or other animals. Therefore, most animal models have concentrated on the development of physical dependence to demonstrate that excessive alcohol consumption has resulted in dependence. However, for most models that produce physical dependence (as demonstrated by withdrawal phenomena), little evidence has been found that the animal will seek out ethanol or drink excessively once dependence is achieved. For example, in the schedule-induced model, which results in dependence, removal of the schedule results in withdrawal and little or no ethanol intake, even when ethanol is freely available35. Animals going through withdrawal will not perform an obtain response to operant ethanol17, even after prolonged experience in obtaining ethanol in this manner. Thus, there does not appear to be a good correlation between the development of physical dependence and either craving or loss of control in experimental animal models. This result has been noted in monkeys that
self-administered ethanol i.v., and in human alcoholics in an experimental laboratory situation36.
211 Therefore, the relation between various components of human alcohol addiction remains to be elucidated. q
q
q
Our view of the action of alcohol has shifted from regarding it as a nonselective compound producing diverse actions by perturbing membrane lipids to a drug that selectively alters the function of key cellular proteins. Critical questions include the molecular mechanisms by which alcohol influences these proteins, what determines the alcohol sensitivity or resistance of a protein, and which molecular actions are responsible for specific behavioral consequences of alcohol ingestion and abuse. The developing area of combining classical and molecular genetics (see Crabbe and Belknap, this issue) is leading to rapid progress in understanding some of alcohol’s action in laboratory animals and will provide candidate genes for linkage analysis in alcoholic families. The development of more appropriate behavioral procedures, coupled with determination of CNS receptor systems that underlie the behavioral changes observed, is also beginning to provide insight into the mechanisms of excessive drinking behavior. This broad multidisciplinary approach to the study of alcohol abuse and alcoholism has made important contributions in the last decade, contributions that have led to many important changes in the conceptualization and treatment of alcoholism. It can only be hoped that with continued effort, the ever-growing trend of alcoholrelated costs and problems can be reversed in the future.
References 1 Seventh Specinl Report to the US Con2 3 4 5 6
gress on Akohol and Health (1990) US Public Health Service Deitrich, R. A.: Dunwidde, T. V., Harris, R. A. and Erwin, V. G. (1989) Pharmacol. Rev. 41,491-537 Lipnick, R. L. (1986) Trends Dharmacol. Sci. 7.161-164 Buck; K. J, and Harris, R. A. (1991) Alcohol. Clin. Exv. Res. 15,460-470 Forman, S. A. and Miller, K. W. (1989) Trends Pharmacol. Sci. 10,447-452 Gonzales, R. A. and Hoffman, P. L.
(1991) Trends Pharmacol. Sci. 12, l-3 Allan, A. M. and Harris, R A. (1991) in The Genetic Basis for Alcohol and Drag Actions (Crabbe, J. C. and Harris, R. A., eds), pp. 105-152, Plenum Press Wafford, K. A. et al. (1991) Neuron 7, 27-33 9 Montpied, P. et al. (1991) Mol. Phnrmacol. 39, 157-163 10 Buck, K. J. and Harris, R. A. (1991) Mol. Neurophnrmacol. 1,59-&l 11 Phillips, T. J. and Crabbe, J. C. (1991) in The Genetic Basis for Alcohol and Drug Actions (Crabbe, 1. C. and Harris, R. A. eds), pi. 2%lti,plenum Press 12 f-twang, B. H., Lumeng, L., Wu, J-Y. and Li, T-K. (1990) Alcohol. Clin. Exp. Res. 14,503-507 13 Hoffman, P. L. and Tabakoff, 8. (1990) FASEB !. 4,2612-2622 14 Noble, E. P., Blum, K., Ritchie, T., Montgomery, A. and Sheridan, P. i (1991) Arch. Gen. Psychiafry 48,648-654 15 Paaian, A. et al. (1991) Arch. Gen. Psychiatry 48, 655-663 16 Meisch, R. A. (1984) in Research Advances in Alcohol and Drag Problems (Vol. 8) (Smart, R. G. et al., eds), pp. 23-45, Plenum Press 17 Samson, H. H. (1987) in Advances in .?rhr;vioral Pharmacology: Neurobehazior;iJ Pharmacology (Vol. 6) (Thompson, T., Dews, P. 8. and Barrett, I. E.. eds), UD. 221-248. Erlbaum 18 %arnr,on,..fi: H., Pfeffer, A. 0. and Tolliver, G. A. (1988) Alcohol. Clin. Exp. Res. 12.591-598 19 Samson, H. H., Tolliver, G. k and Schwirrz-Stevens, K. (1990) AJcohoJ 7, 187-191 20 Samson, H. H. and Grant, K. A. (1990) Drug Alcohol Depend. 25,141-144 21 Kaplan, R. F. et al. (1985) J. Stud. AJcohoJ 46,267-272 22 Barrv. H. (1974) Fed. Proc. 33.1814-1824 23 Bra&, J. ‘V., &enz, R. D.. and Ator, N. A. (1990) Drw Dev. Res. 20,231-249 24 Grant,.K. AI, KnLely, J. S., Tabakoff, B., Barrett, J. E. and Balster, R. L. (1991) Behav. Phnrmacol. 2.87-95 25 Grant, K. A. and Barrett, J. E. (1991) PsuchovhannacoJoav 104,451456 26 T;bak&f, B. and tioffman, P. L. (1988) in Theories of Alcoholism (Chaudron, C. D. and Wiinsos, D. A., eds), pp. 2%72, ARC Foundation Press __ 27 Holloway, F. A., King, D. A., Michaelis, R. C., Harland, R. D. and Bird, D. C. (1989) Psychopharmacology 99,479-485 28 Samson, H. H. and Falk, I. L. (1974) Pharmacol. Biochem. Behav. i, 791-801 29 Cunningham, C. L. and Bischof, L. L. (1986) Alcohol. CJin. Exp. Res. lo,1008 30 Linakis, J. G. and Cunningham, C. L. (1979) Psychopharmacology 64,614 31 Perk, J. and Cunningham, C. L. (1987’) Alcohol Drug Res. 7,187-193 32 Le. A. D. 11990) Ann. Med. 22.265-268 33 Vogel-Sprhtt, h. and Sdao-Jarvie, K. (1989) Psychopharmacology 98,289-296 34 Waller, M. B., McBride, W. J., Lumeng, L. and Li, T-K. (1983) Pharmacol. Biothem. Behav. 19, 683-686 35 Falk, J. L. and Tang, M. (1988) Alcohol. Clin. Exp. Res. 12,591-598 36 Mello, N. K. (1983) in The Biology of Alcoholism: The Pathogenesis of Alcoholism (Vol. 7) (Kissin, B. and Begleiter, H., eds), pp. 133-198, Plenum Press DMCM: methyl-6,7-dimethoxy-4-ethyl-bcarboline-3-carboxylate
204 sci. 13,420-423 9 Matsuda, L. A., Lolait, S. J., Bmwnstein. M. J., Young, A. C. and Bonner, T. I. (1990) Nature 346, 561-564 10 Johnson, M. R. and Melvin, L. S. (1986) in Cannabinoids as Therapeutic Agents (Mechoulam,R., ed.), pp. 121-144, CRC Press 11 Bell, M. R. et al. (1991) 1. Med. Chem. 34, 1099-1110 12 Mechoulam, R. et al. (1987) NIDA Res. Mono~. Ser. 79,15-30 13 Razd&, R. K. (1987) NIDA Res. Monogr. Ser. 79,3-14 14 Di Chiara, G. and Imuerato, A. (1988) Proc. Nat1 Acad. Sci. USA 85,5274-&278. 15 Chen, J_ et al. (1990) PFychophannacolopv 102.156-162 16 Che& J., &&es, W., Lowinson, J. H. and Gardner, E. L. (1990) Eur. I. Pharmuco!. 190,2S9-262 17 Gardner, E. L. et al. (1988) Psychouhannucoloav %, 142-144 18 &wee, R.ud. (l&l) in TheBiologicalBasis of Drux Tolerance and Dependence (F%att, J: A., &i.). pp. 232-263, Academic Press 19 Kaymakcakm, S. (1973) Bull. NIX. 25, 39-47
20 Beardsley, P. M., Balster, R. L. and Harris, L. S. (1986) 1. Pharmacol. Exp. Ther. 239,311-319 21 McMillan, D. E., Dewey, W. L. and Harris, L. S. (1971) Ann. NY Acad. Sci. 191,699 22 Camey, J. M., Uwaydah, I. M. and Balster. R. L. (1977) Phurmacol. Biochem. Behuv.~7,357~364 23 Harris, L. S., Dewey, W. L. and Razdan, R. K. (1977) in Handbook of Experimental Pharmucology (Born, G. V. R., Eichler, 0.. Farah. A. and Welch. A. D., eds), DD. r. 1’ 371-429, Springer-Verlag 24 Dill, J. A. and Howlett, A. C. (1988) 1. Ph&mucol. Exp. Ther. 244, 1157~1163~ 25 Westlake, T. M. et aI. (1991) Brain Res. X4.14.5-149 26 Jon&, R. T. (1983) in Cannabis and Heulth Huzurds (Fehr, K. 0. and Kalant, H., eds), pp: 617-689, Addiction Research Foundation 27 Jones, R. T., Benowitz, N. and Bachman, J. (1976) Ann. NY Acad. Sci. 282,221-239 28 Jones, R. T. and Benowitz, N. (1976) in Phurmucology of Murih&n &aide, M. C. and Szara, S., eds), pp. 627-642, Raven Press
Neurobiology of alcohol abuse Herman H. Samson and R. Adron Harris Excessive consumption of beverage alcohol (ethanol) is a major health concern worldwide. Understanding the mechanisms by which ethanol affects neural functioning, after both acute and chronic exposure, has become a major goal in the study of alcoholism. With such an understanding, we should be able to institute more effective treatments and preventative measures for alcohol abuse problems. Recent studies have found, contrary to earlier assumptions, that ethanol has selective, dose-dependent effects on various neurotransmitter systems within the CNS. These effects are observed at all levels of analysis, from molecular to behavioral. This review by Herman Samson and Adron Harris covers these recent findinps. with the intent of generating questions that will focus further resedrch efioks. Alcohol is the second most widely used psychoactive substance in the world after caffeine. While most societies approve of moderate alcohol use, chronic and/or excessive drinking is considered a hazard to both health and public safety. In the USA, the cost of alcohol-related problems was estimated at over $136 billion in 1990, and unless changes in drinking patterns occur, or better treatment and prevention approaches H. H. Samson is Director at the Alcohol and Drug Abuse Institute and Professor in the Depurfment of Psychiatry and Behavioral Sciences, University of Washington School of Medicine. Seattle, Washington, and R. Adron Harris is a Research Career Scientist at the VA Medic42 Center and Professor in the Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado, USA. 0 1992.Elsevier
Science Publishers Ltd (UK)
are found, health costs will be over $150 billion in 1995l. In order to both treat and prevent alcoholrelated problems, basic research into the mechanisms by which alcohol produces its psychotropic activity is essential. Ethyl alcohol (ethanol), unlike most psychoactive drugs, has no known specific receptor system within the CNS. However, it is clear that ethanol can affect a variety of neurotransmitter systems and understanding how this occurs is most likely to be the key to explaining the psychoactive actions of ethanol. It is impossible to cover all of the neurotransmitter systems known to be affected by ethanol within the space limitations of this review. While important findings related to many transmitter
29 Jones, R. T., Benowitz, N. L. and Heming, R. I. (1981) 1. Clin. Pharmacol. 21,143S-1525 30 Howlett, A. C., Qualy, J. M. and Khachatrian, L. L. (1986) Mol. PharmacoI. 29,307-313 31 Biduat-Russell, M., Devane, W. A. and Howlett, A. C. (1990) /. Neurochem. 55, 21-26 32 Pacheco. M.. Childers. S. R.. Arnold. R.. Casiano; F: and Ward, 6. J. (lb91j 1. Phnrmacol. Em. Ther. 257,170-183 33 Eisenstat, M. A: et nf. (1990) NIDA Res. Monogr. Ser. 105,427-428 34 Herkenham. M. et al. (1991) . ,, 1. Neurosci. ~~~ 11.563-583. 35 Herkenham, M. et al. (1990) Proc. Nat! Acad. Sci. USA 87,1932-193& 36 Harris, L. S. (1978) in Psvchovharmacology, A Generation of Progress ilipton, M. A., DiMascio, A. and Killam, K. F., eds). UD. __ 1565-1574. Raven Press 37 Plasse, T. F. et ui. (1991) Pharmucof. Biochem. Behuv. 40.695-700 38 Munson, A. E. and Fehr, K. 0. (1983) in Cannabis and Health Hazards (Fehr, K. 0. and Kalant, H., eds), pp. 257-354, Addiction Research Foundation
systems have been delineated (see Ref. 2 for a more complete discussion of other transmitter systems), this review highlights the recent findings related to the excitatory and inhibitory amino acid transmitters and to dopamine and 5-HT. Molecular pharmacology The search for moiecular actions of alcohol has focused mainly on mechanisms of intoxication and development of tolerance and dependence. A key, and unanswered, question is how these actions are related to alcohol selfadministration in laboratory animals and to alcohol abuse and alcoholism in humans. Membrane actions Alcohols (typified by ethanol) are members of the family of intoxicant anesthetics that includes volatile anesthetics, barbiturates and benzodiazepines; drugs that display a similar spectrum of intoxication as well as substantial cross-tolerance and cross-dependence. Because of the chemical structural diversity of these intoxicant anesthetics, it is difficult to imagine a single receptor that could be responsible for actions of ethanol as well as these other related drugs. Based on the pioneering work of Meyer and Overton carried out 100 years ago, the idea that alcohol acts by partitioning into neuronal mem-
TipS - May
1992 (Vol.
131
Drugs and synapses: puzztes and paradoxes Drugs discussed in this issue of TiPS vary widely in their potencies. For
example, nanomoIar concentrations of some opioids are sufficient to produce appreciable occupancy of receptors with in vitro binding assays, whereas ethanol ~nc~atio~s of IO-l#tl rn?.sare required for actions both in viva and in vitro. What is the physical meaning of these concentrations at a synapse? If we assume that the synapse is a cylinder with a diameter of IOOOnm and a height of 2Onm, then its volume is 1.6 X Xi” Liters. A concentration of 1 IIM cmwerts to 6 X IOx mofecutes per Literrthus at 1 no there wiII be only one drug molecule per 100 synapses! At the seemingly huge concentration of lOmM, there will be only 100 molecules of ethanol in each synapse. Yet some drugs do indeed exert effects on behavior with brain concentrations in the nanomdar range. How can this occur? It is possible that only a small fraction of the synapses need to be affected by the drug for a behavioral action. However, it seems more likely that the drugs accumulate at synapses leading to local concentrations that are larger than the average concentration. This could occur because of a high hpid solubihty in which case the concentration of the drug in the membrane would be considerably gmater than in the water phase. If the receptor is also buried in the membrane, then the lipid concentration is more relevant than the water concentration. Likewise, slow dissociation of drugs from their receptors would lead to accumulation of drugs in the synapse. ft is interest&g to note that potent drugs are often quite lipid soluble and display slow dissociation kinetics, whereas ethanol, the prototype lowpotency drug, has low lipid solubihty and its effects are rapidly reversed in vitro. Perhaps the synaptic concentrations of the drugs described in this issue are not very different; certainly they may not be as different as the water concentrations (or dosages) would imply. Another puzzle is why some drug actions observed in vitro have any effect on synaptic transmission. Articles in this issue tell us that benzodiazepines and ethanol increase the action .of GABA at GABAA receptors‘ that ethanol increases the action of 5-HT at 5-HTs receptors and that cocaine increases synaptic dopamine, noradrenahne and 5-HT. However, these actions of ethanol and benzodiazepines occur only at submaximal concentrations of the neurotransmitters; once the receptors are fully occupied neither of these drugs (or cocaine} would be expected to affect synaptic transmission. What is the synaptic concentration of a neurotransmitter? This is, of course, unknown, but we can make some estimations. The lowest concentration that a synapse can have is one molecule, which would be 100 nM from the calculations above. However, it is hkely to be mush larger_ Vesicular concentrations of acetylchohne and glutamate are estimated at lOS2QOmru. The volume of synaptic vesicIes (60 nm diameter) is about 1% of the synaptic volume calculated above, thus a synaptic neurotransmitter concentration of lrmur is not unreasonable {and quite saturating for receptors). Perhaps drugs affect extrajunctional receptors where the concentration of ne~ans~~er is not saturating? Alternatively, they may affect the synaptic transmission only when the concentrations of
neurotransmitter are rising or fallingand are not saturating. Are actions of these drugs more subtle than simply enhancing neuro~ans~~ion? New techniques allowing rapid application of ne~ro~ansmi~ers (concentration clamp, quench flow] and measurement of synaptic ~~smission in brain slices (slice patch) should solve this puzzle. Values used for calculations are from McGeer,P. I.., Eccles, J. C. and McGeer, E. G. (2978) Molecular Biology of the Martian Brain, Pleuu~ Press.
branes and altering their physical properties received considerable attention during the 1970s and 1980s. The hypothesis that acute alcohol disorganizes or ‘fluidizes’ membranes and that chronic alcohol rigidifies membranes is supported by numerous studies of brain membranes, but the question remains as to whether these changes in physical proper-
ties are sufficient to alter membrane function2*4. In the 1980s and 199&z, emphasis has shifted to a search for neuronal functions that are sensitive to intoxicating concentrations of alcoho15,6 (see Box). Electrophysiological and neurochemical studies in the brain have identified ion channels, particu-
(,
t ilicotinic acatylcholine(-I-) i
\
7r
oulpurtt 11
I?& 1. Acure eq3osure to alcuhuf enhances action of 5-H-f at 5-M-receptorsand a&on af a~~~t~e at nimtinic chuthwgic receptors. Both these rscspiors actlvste cation chmts, thus 8re sxctta~, and s-i wfmid imrem Cwput of a hy~~~t r?suri~~expressing cmiy fhsse recento~. Data fromti$inaer. D. M. anh White, G. (1991) kai Phamad. 40, 263-270 and Furman. S. A.. F@$i, D. I and Miuet, K. W. (1989~8&c&m. &bpfys. Ada 987,~103.
larlv &and-gated channels, that are especially sensitive to alcohol. Neurotransmission at two types of excitatory receptor, nicotinic acetylcholine and 5HTa, is enhanced by acute alcohol exposure (Fig. 1). Most other excitatory receptors are inhibited by alcohol, while ~~ibito~ systems are augmented. For exampie, acute alcohol exposure inhibits the excitatory action of glutamate at both N&IDA and kainate receptors* inhibits voltage-sensitive Caa” channels and enhances GABA action at some GABAA receptors (Fig. 2). Chronic alcohol treatment results in increased numbers of N&IDA receptors and voltage-sensitive Ca” channels, whereas GABAA function is reduced but kainate responses are unaltered. These change5 in receptor and channel function offset the acute action of alcohol and should result in hyperactivity when alcohol is ~~~a~, providing a piausible mechanism for tolerance and dependence’. The question of which, if any, of these neurochemical responses to alcohol treatment is related to the severity of alcohol withdrawal can be addressed by determining genetic correlations (see Crabbe and Belknap, this issue). Lines of mice have been selectively bred to be either prone or resistant to seizures when withdrawn from chronic alcohol exposure. After
TiPS - May 1992 \Vol. 131
20s
ethanol intoxication
ethanol dependence t NMDA (+)
a\
Fg. 2. Acute exposure to alcohol inhibits action of NIUDA and kainafe on g/ufamate racepmrs, inhibits timcfion of voltage-sensitive Ca” channels and enhances action of &WA at GAB&receptmsBeczwsethethreecation (excitatory) channels are inhibited and the anfon (inhibitory) channel is activafed, acute alcohol would markedly decrease the output of a hypothetical neuron expressing these ion channels. In contra.% chronic exposure to alcohol inMeases NMDA receptors and voltage-sensitive ion channels and demsaws GABA. tinction. ofLsetlino the acute action of alcohol and resulfing in kweased ou@ut &en akwhoi is with&awn tiom this hypothericar cell. Data from Refs 3 4 Wafaht, F. F.. Lovinper, D. M., White, G. and Peoples, R. W. (1991) Ann. NYAcad. %825.>7-107;Brenn& C. H.. Guppy, L. J. and kleton, J. M. (1989) Ann. NYAcad. sci. 560,467-469.
many generations of selection, alleles responsible for alcohol withdrawal seizures (and other signs of withdrawal) have segregated and the neurochemical effects of chronic alcohol should be different in these mice lines, provided these neurochemical changes are related to the development of physical dependence (as measured by withdrawal). Use of these selected lines for testing neurochemical hypotheses revealed three consequences of chronic alcohol exposure that are greater for Seizure-Prone mice than for Seizure-Resistant mice: increased density of nitrendipinebinding sites (i.e. upregulation of voltage-sensitive Ca2+ channels); increased efficacy of DMCM, an inverse agonist at benzodiazepine receptors; and decreased mRNA coding for the arl subunit of GABA* receptors (Fig. 3). By contrast, Seizure-Prone and SeizureResistant mice exhibit two similar neurochemical changes produced by chronic alcohol: an increase in dizocilpine-binding sites (i.e. upregulation of NMDA receptors) and an increase in membrane rigidity as detected by diphenylhexatriene fluorescence polarization (Fig. 3). However, the density of NMDA receptors is higher in Seizure-Prone mice than in
Seizure-Resistant mice before exposure to alcohol. Such genetic correlations provide strong support for the involvement of NMDA receptors and some types of voltage-sensitive Ca” channels and GABA* receptors in the development of physical dependence on alcohol. Identification of these actions of alcohol leads to the question of how either acute or chronic exposure to this relatively simple molecule can alter the function of ion channels. Our current understanding of alcohol’s action is most advanced for the GABA* receptor. Both behavioral and neurochemical studies provide evidence of enhancement of GABA receptor activity by alcoho!. Studies of selected lines of mice and rats indicate that genetic differences in acute (and chronic, Fig. 3) actions of alcohol are related to the GABA* receptor7. However, electrophysiological studies have not consistently detected augmentation of GABA* responses by alcoho12. An explanation of these discrepancies may be found in molecular biological studies of the multiple GABA* receptor subunits that have been cloned and sequenced. Although a variety of different subunit combinations
are able to form receptors that respond to GABA or pentobarbital, benzodiazepine responsiveness requires a member of the y family of subunits. A recent study8 using electrophysiological measurement of oocytes expressing cloned GABA receptor subunits indicates that a particular y subunit, y2~, is required for alcohol enhancement of GABA action. It is ironic that the simplest molecule (ethanol) has the most specific subunit requirement for action at the GABA receptor complex! The yzL subunit differs from y2s by only eight amino acids, yet the latter subunit does not confer alcohol sensitivity. The difference between these subunits is that y2~ contains a serine that can be phosphorylated by protein kinase C, and this appears to be critical for alcohol action. Even in these (relatively) simple the molecular action systems, of alcohol remains elusive, but effects on protein kinases or phosphatases could be responsible for changes in function of several ion channels, including those coupled to GABA* receptors. In addition, molecular mechanisms of neuroadaptation to chronic alcohol exposure are beginning to be unraveled. For the GABAA receptor, levels of mRNA for specific subunits are altered, suggesting that chronic alcohol ingestion may affect gene expression’. Chronic alcohol also alters GABAn receptor function in an oocyte system expression of GABAA where receptor genes is bypassed by injection of mRNAlO. Recent evidence that chronic alcohol alters expression of genes for intracellular regulators such as Fos, heat shock protein and the (Y, subunit of G proteins provides exciting prospects for molecular studies of other neuroadaptive mechanisms4. Mechanisms of alcohol abuse and alcoholism The reinforcing action of alcohol, as for other drugs of abuse, appears to involve activation of the mesolimbic dopamine system. For example, alcohol increases the firing of neurons in the ventral tegmental area and increases extracellular concentrations of dopamine in the nucleus accumbens, but the molecular mechanisms responsible for these actions
TiPS - May 1992 /VoL 231 remain to be defined*. Selected lines of rats that should prove useful for such research include alcohol-Preferring Nonand Preferring, alcohol-Avoiding and Non-Avoiding, and High-AlcoholDrinking and Low-AIcohol-Drinking rats”. Studies of these rat lines suggest that an elevated level of dopamine in the nucleus accumbens encourages alcohol drinking, as does a lower level of 5-HT in the nucleus accumbens and several other brain regions7. In addition, recent data show that the alcohol-Preferring rat and High-Alcohol-Drinking rat have a higher density of GABA-containing terminals in the nucleus accumbens than their opposite pairsl*. These results suggest that the increase in transmission at GABA receptors produced by alcohol (discussed above) could be related to the reinforcing actions of the drug. Animal studies demonstrate that alcohol acutely enhances adenylyl cyclase activity due to an action on G, and chronically reduces activity of brain adenylyl cyclase 13. Extension of these studies to human alcoholics demon-
100
75
209
strated a decreased activity of adenylyl cyclase in platelets and lymphocytes. A similar decrease can be produced in neurons cultured in the presence of alcohol. Most importantly, platelets from individuals with a positive family history of alcoholism have a lower content of platelet adenylyl cyclase even if they are not actively drinking13. Thus, this marker may detect the ‘trait’, rather than the ‘state’, of alcoholism. The molecular basis for this defect in platelets of human alcoholics, and whether it occurs in brain, are not yet known. Because of the role of dopamine systems in drug reinforcement, the possible role of brain D2 receptors in alcoholism is emerging as an important area of study. Blum, Noble and co-workers’4 found that one allele (AZ) of the human brain D2 receptor was associated with alcoholism and that the presence of this allele reduced the density of dopamine receptors in human caudate mlcleus. Other workers have confirmed an increased incidence of this allele in alcoholics with severe medical problems compared with
0
WithdrawalSeizure-Resistant
a
WithdrawalSeizure-Prone
50 25
-25
fig. 3. The effect of genetic differences in susceptibility to seizures upon ethanol withdrawal on neurochemicat responses to chronic ethanol treatment. Withdrawal Seizure-Prone mice showed the same or greater changes in response to chronic ethanol exposure than Withdrawal Seizure-Resistant mice. Data fromRefs 4; 10; Brennan,C. H., Crab& J. C. and Littleton, J. M. (1990) Neurophannacofogy 29, 429-432; Valverius, P., Crabbe, J. C., Hoffman, P. L. and Tabakoff, B. (1990) Eur. J. Pharmeco/. 164, 195-189; Harris, R. A., Crabbe, J. C. and McSwigan,J. D. (1994) Life Sci. 35,2601-2606.
nonalcoholic controls, but it was not increased in alcoholics without medical complications and there was no evidence of linkage or cosegregation of the AZ allele with alcoholismi5. It is possible that the AZ allele has a modifying effect that increases the severity of alcoholism. Because alcoholism is polygenic, clinically heterogeneous, and strongly influenced by environment, it is not surprising that this single allele does not demonstrate a major causative role in most cases of alcoholism. However, this finding represents an important initiative in the search for brain genes related to alcoholism. Behavioral pharmacology Ethanol has a variety of behavioral effects, many of which are dependent upon dose, route and type of administration. For a majority of the animal studies, ethanol has usually been administered by the experimenter, using an i-p. or i.v. injection or intubation. While assuring the delivery of a standard, fixed dose, these administration methods generally fail to mimic the blood ethanol curves typical of orally self-administered alcohol consumption. Therefore, caution is required in interpretation of the results in relation to oral selfadministration of alcohol. Ethanol as a reinforcer Over the past 15 years, many studies have shown that ethanol, like other drugs of abuse, can function as a reinforce+i7. Initial studies demonstrated that several different routes of administration (oral, intra-gastric and i.v.) were effective. However, since the oral route is typically used by humans, recent studies have focused on the oral self-administration reinforcement processes. In the past, demonstration that oral ethanol consumption can be reinforcing in nondeprived animals presented problems, but the development of a variety of initiation procedures has overcome many of these difficulties”. While it is difficult to ascertain in animal studies what the psychological nature of alcohol reinforcement might be, recent studies in animals fed ad lib suggest that the calorific properties of alcohol play only a minimal role in its reinforcing actions.
TiPS - May 1992 [Vol. 131
210 Many of alcohol’s reinforcement properties can be modified by orior treatment with CNS neurohansmitter receptor agonists and antagonists, particularly those affecting the mesolimbic dopamine system”. A critical behavioral component that occurs during the development of oral ethanol consumption is the conditioning of environmental cues that signal the availability of ethanol to its pharmacological effects. These conditioned cues can then elicit ethanolseeking behaviors, even in the absence of ethanol*820. Similarly, in human alcoholics, the intensity of cravings for alcohol increases in the presence of cues related to alcohol availabili@‘. The study of the development of these conditioned behavioral functions is of major importance in understanding their relation to excessive drinking patterns in humans”. Ethanol as a discriminative stimulus While the physiological effects of ethanol can function to reinforce behavior, these effects can also serve as discriminative stimuli to direct behavior selection related to other reinforcers. In his classic study, Barry demonstrated that ethanol could function as a discriminative stimulus under a variety of conditions”. However, as discussed above, there are a variety of CNS cues that result from the pharmacological actions of ethanol. Any or all of these altered CNS effects could underlie the cue that the animal uses. One valuable use of the drugdiscrimination procedure is to test other drugs to determine whether they produce similar or different cues=. If other drugs produce cues that are the same as alcohol (i.e. the animal responds as if it has received alco’nol), then a mechanism that is common to both the drug and alcohol can be hypothesized to underlie generation of the cue. A variety of analgesics and tranquilizers have been found to partially substitute for the ethanol cue, while drugs such as stimulants and neuroleptics do not substitute”. This suggests that the major discrimination cues resulting from ethanol are those that relate to its sedative and anesthetic actions. These actions may
be the result of an interaction of ethanol with the GABA* receptor benzodiazepine modulatory site. However, recent studies using neurotransmitter antagonists have that both the demonstrated NMDA and 5-HTs receptor systems are involved in ethanol discrimination24,25. This suggests that a complex, multiple set of cues is responsible for the ethanol cue. Understanding the relative contributions of these different CNS mechanisms could provide a clearer picture of how ethanol’s pharmacological cues interact environmental cues to with excessive drinking produce behavior. Evidence for tolerance Repeated administration of alcohol results in decreased sensitivity, or tolerance, to many of its effects. It has been suggested that the development of tolerance to the ‘negative’ effects of ethanol (i.e. motor incoordination, sedation, nausea, etc.) is necessary for the generation of excessive drinking behaviorsz6, but clear validation of this hypothesis remains to be provided. The development of tolerance to repeated administration has been well documented for most of the physiological effects of ethanol. Tolerance to the motor, anesthetic, hypothermic, sedative and anxiolytic effects has been shown when ethanol was administered to the animal by the experimenter. Tolerance to the disruptive effects of such experimenter-administered ethanol on the performance of behavior controlled bv food reinforcement has also been found27. Using oral ethanol selfadministration induced by the presentation of food pellets on a 90-second fixed time schedule in food-restricted rats (a scheduleinduced polydipsia paradigm), tolerance to the motor discoordination effects was also demonstrated2’. However, when examining the effects of tolerance to ethanol, it is important to select carefully the procedures used to evaluate tolerance development. Failure to use doses and routes that might be related to oral consumption can make interpretation difficult. For example, Cunningham and colleagues recently demonstrated that the acute hypothermic re-
sponse to an i.p. injection of lg kg-l ethanol failed to occur when the animal orally selfadministered the same dose over a five-minute period”. Cunningham suggests that the tolerance to the acute response seen in injection studies may be specific to the injection procedure30 or to a handling component that accompanies it31. Thus, doses of ethanol that produce tolerance when administered by the experimenter may not produce tolerance when orally self-administered by the experimental animal. It should be noted that no evidence of tolerance to the reinforcing properties of alcohol has been demonstrated. Any observed increase in alcohol intake over repeated drinking opportunities could be a result of tolerance to the reinforcing properties or tolerance to the motor and sedative effects, either of which could result in increased intakes. From studies using the schedule-induced polydipsia paradigm, it would appear that the tolerance is developed to the motor-disrupting effects, with no change to the reinforcing properties17. A variety of environmental stimuli can be conditioned to predict the administration of ethanol. These learned cues can result in physiological responses that are counter to the effects of the ethanol and these counter-responses are a possible mechanism of tolerance32. This conditioned or learned tolerance has been demonstrated as a factor for several different measures of ethanol tolerance, including ataxia, hypothermia and ethanol’s anticonvulsive properties. The development of learned tolerance to the motor discoordination effects of orally consumed alcohol has been shown in humans33, and there is evidence that counter or compensatory responses can also be measured in humans32. However, the exact role of learned tolerance to the development of excessive drinking has yet to be elucidated. The phenomenon of rapid, or acute, tolerance has been observed for several behavioral measures. For example, after an ethanol dose, ethanol levels are lower when ataxia is first observed on the rising phase of the blood ethanol curve than when the ataxia disappears on the fall-
TiPS - May 1992 [Vol. 131 ing limb of the blood ethanol curve. This acute tolerance can be influenced by genetic selection, such that animals selected for higher ethanol preference demonstrate a greater acute tolerance than animals selected for ethanol aversion=. This suggests there may be a direct relationship between acute ethanol tolerance and ethanol preference that is under genetic regulation. Evidence for addiction In humans, alcoholism has as a major diagnostic feature the loss of control when drinking has begun [as classified by the Diagnostic and Statistical Manual of Mental Disorders (Third Edn Revised) (1987) American Psychiatric Association]. Furthermore, the craving for alcohol during periods of abstinence has often been considered a prime factor underlying excessive alcohol use. Unfortunately, these two behavioral features are not easy to examine using experimental animal models, partly because of the difficulty in defining craving and loss of control for humans or other animals. Therefore, most animal models have concentrated on the development of physical dependence to demonstrate that excessive alcohol consumption has resulted in dependence. However, for most models that produce physical dependence (as demonstrated by withdrawal phenomena), little evidence has been found that the animal will seek out ethanol or drink excessively once dependence is achieved. For example, in the schedule-induced model, which results in dependence, removal of the schedule results in withdrawal and little or no ethanol intake, even when ethanol is freely available35. Animals going through withdrawal will not perform an obtain response to operant ethanol17, even after prolonged experience in obtaining ethanol in this manner. Thus, there does not appear to be a good correlation between the development of physical dependence and either craving or loss of control in experimental animal models. This result has been noted in monkeys that
self-administered ethanol i.v., and in human alcoholics in an experimental laboratory situation36.
211 Therefore, the relation between various components of human alcohol addiction remains to be elucidated. q
q
q
Our view of the action of alcohol has shifted from regarding it as a nonselective compound producing diverse actions by perturbing membrane lipids to a drug that selectively alters the function of key cellular proteins. Critical questions include the molecular mechanisms by which alcohol influences these proteins, what determines the alcohol sensitivity or resistance of a protein, and which molecular actions are responsible for specific behavioral consequences of alcohol ingestion and abuse. The developing area of combining classical and molecular genetics (see Crabbe and Belknap, this issue) is leading to rapid progress in understanding some of alcohol’s action in laboratory animals and will provide candidate genes for linkage analysis in alcoholic families. The development of more appropriate behavioral procedures, coupled with determination of CNS receptor systems that underlie the behavioral changes observed, is also beginning to provide insight into the mechanisms of excessive drinking behavior. This broad multidisciplinary approach to the study of alcohol abuse and alcoholism has made important contributions in the last decade, contributions that have led to many important changes in the conceptualization and treatment of alcoholism. It can only be hoped that with continued effort, the ever-growing trend of alcoholrelated costs and problems can be reversed in the future.
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