Ethanol as a neurochemical surrogate of conventional reinforcers: The dopamine-opioid link

Various lines of evidence support the view that ethanol is a neurochemical surrogate of conventional reinforcers, such as food and sex. In fact, ethanol activates central neuronal systems that utilize dopamine, opioids, and γ-aminobutyric acid (GABA) as neurotransmitters and also are activated by conventional reinforcers. These neurotransmitter systems are likely to mediate specific aspects of ethanol's reinforcing properties. Activation of the mesolimbic dopamine and endogenous opioid systems might be the substrate of the incentive and rewarding (ergotropic) properties of ethanol (arousal, euphoria, motor stimulation) and of the process of acquiring ethanol-related secondary reinforcers (incentive learning) and ethanol self-administration habits. Stimulation of the endogenous GABAergic system might mediate the sedative and drive-reducing (trophotropic) properties of ethanol. The dopamine and opioid systems are largely interconnected. Thus, pharmacological blockade of the endogenous opioid system by mu- or delta-opioid receptor antagonists prevents ethanol's activation of the dopamine system and reduces ethanol consumption. This interaction might contribute to naltrexone's effectiveness in reducing alcohol craving in humans.     

Does a relationship exist between the quality of stress and the motivation to ingest alcohol?

Previous experimental findings on the relationship between emotional stress and motivation to ingest alcohol are contradictory. To obtain information about this relationship we tested the effects on alcohol consumption in rats subjected to two types of chronic unavoidable stressors, intermittent immobilization and social isolation, which differ in their influence on the functional state of the endogenous opioid system. To characterize the nature and magnitude of the stress induced by these stressors, we measured their effects on functional parameters which have a close relationship to the regulatory influence of endogenous opioid peptides (endogenous opioid dependence, pain sensitivity, blood pressure). Our investigations have shown that chronic intermittent immobilization, which induced development of endogenous opioid dependence, presumably due to activation of endogenous opioid systems, did not produce increased alcohol consumption. On the contrary, chronic social isolation, which did not induce development of endogenous opioid dependence, was followed by a significant increase in alcohol consumption. It is concluded that not all types of stress produce increased alcohol consumption, but that the effect on the endogenous opioid system may be a decisive factor in determining whether a stressor produces increased alcohol consumption.     

Reduced alcohol consumption in mice lacking preprodynorphin.

Many studies suggest a role for endogenous opioid peptides and their receptors in regulation of ethanol intake. It is commonly accepted that the kappa-opioid receptors and their endogenous ligands, dynorphins, produce a dysphoric state and therefore may be responsible for avoidance of alcohol. We used mutant mice lacking preprodynorphin in a variety of behavioral tests of alcohol actions. Null mutant female, but not male, mice showed significantly lower preference for alcohol and consumed lower amounts of alcohol in a two-bottle choice test as compared with wild-type littermates. In the same test, knockout mice of both sexes showed a strong reduction of preference for saccharin compared to control mice. In contrast, under conditions of limited (4 h) access (light phase of the light/dark cycle), null mutant mice did not show any differences in consumption of saccharin, but they showed significantly reduced intake of sucrose. To determine the possible cause for reduction of ethanol preference and intake, we studied other ethanol-related behaviors in mice lacking the preprodynorphin gene. There were no differences between null mutant and wild-type mice in ethanol-induced loss of righting reflex, acute ethanol withdrawal, ethanol-induced conditioned place preference, or conditioned taste aversion to ethanol. These results indicate that deletion of preprodynorphin leads to substantial reduction of alcohol intake in female mice, and suggest that this is caused by decreased orosensory reward of alcohol (sweet taste and/or palatability).     

Enkephalinase inhibition: regulation of ethanol intake in genetically predisposed mice

This is the first report of alteration in alcohol intake in mice with a genetic predisposition to alcohol preference and known to have innate brain enkephalin deficiencies. We have been able to significantly attenuate both volitional and forced ethanol intake respectively by acute and chronic treatment with hydrocinnamic acid and D-phenylalanine, known carboxypeptidase (enkephalinase) inhibitors. Since these agents, through their enkephalinase inhibitory activity, raise brain enkephalin levels, we propose that excessive alcohol intake can be regulated by alteration of endogenous brain opioid peptides.     

Genetic analysis of the mu-opioid receptor in alcohol-dependent individuals.

On the basis of various study results, it is suggested that the ethanol-induced activation of the endogenous opioid system may play an important role in mediating the reinforcing effects of ethanol. The mesolimbic dopamine reward system is activated by both ethanol and opioids, and genetic differences in the sensitivity of the endogenous opioid system to alcohol may be an important factor determining the risk for the development of excessive alcohol consumption. Thus, variants of the mu-opioid receptor (muOR) gene may confer vulnerability to alcohol dependence. Five exon 1 variants of the muOR were investigated in 327 alcohol-dependent and 340 healthy control subjects. The Val6 variant of the +17C/T polymorphism and the Asp40 variant of the +118A/G polymorphism showed a trend to an increased allele frequency in alcohol-dependent subjects. The latter polymorphism was investigated in more detail. The dopamine receptor agonist apomorphine causes an increase in growth hormone (GH) levels in the blood by stimulating the release of growth hormone-releasing hormone. beta-endorphin also activates this regulatory circuit. We found a blunted response in intoxicated alcohol-dependent subjects, but no difference in GH response between the groups of alcohol-dependent subjects with and without the variant Asp allele. However, alcohol-dependent subjects with the Asp allele showed a significantly higher GH response at day 7 after alcohol withdrawal and a tendency to lower novelty seeking. These results suggest to us that there is reduced dopaminergic neuronal activity in alcohol-dependent subjects with the muOR Asp40 allele, along with a compensating increase in dopamine receptor activity. The difference between the two groups of alcohol-dependent subjects can be demonstrated only under certain conditions such as alcohol withdrawal, which necessitates the adaptation of the neurones to a new homeostasis.     

Differences in ethanol drinking between mice selected for high and low swim stress-induced analgesia

Alcoholism is a complex disorder, still not fully understood, in which environmental and inherited risk factors play essential roles. Of particular importance may be chronic exposure to stress thought to increase preference for ethanol in genetically susceptible individuals. Animal and human data suggest that the opioid system may be involved in the development of alcohol dependence. We studied the effects of chronic mild stress (CMS) on the voluntary intake of 8% ethanol in the mouse lines displaying high (HA) or low (LA) swim stress-induced analgesia. These lines differ in the activity of the endogenous opioid system. Normally, 8% ethanol is aversive to rodents. We found that LA mice with the low opioid system activity exposed to CMS manifested greater ethanol intake than under no stress conditions. No such effect of CMS on ethanol consumption was observed in HA mice that display the enhanced opioid system activity. We conclude that CMS imposed on individuals with a genetically determined low opioid activity may favor the development of ethanol abuse.     

Differences in basal cannabinoid CB1 receptor function in selective brain areas and vulnerability to voluntary alcohol consumption in Fawn Hooded and Wistar rats

AIM: To specify the functional activity of cannabinoid CB1 receptor in alcohol-preferring Fawn Hooded and alcohol nonpreferring Wistar rats under na?ve conditions. METHOD: Cannabinoid CB1 (WIN-55,212)-stimulated [35S]-GTPgammas binding autoradiography, and cannabinoid CB1 receptor gene expression were measured in rats of both strains that received only water. RESULTS: Cannabinoid CB1 receptor stimulated [35S]-GTPgammas binding was significantly lower in cingulate cortex (Cg), caudate-putamen (CPu), nucleus accumbens (Acc), ventromedial hypothalamic nucleus (VMN), amygdaloid area (AMG), fields (CA1, CA3) of the hippocampus and dentate gyrus (DG) in Fawn Hooded than in Wistar rats, whereas no differences were found either in substantia nigra pars reticulata (SNr) nor CA2 field of the hippocampus. In addition, cannabinoid CB1 receptor gene expression was lower in Cg, CPu, VMN and CA3 field of the hippocampus in Fawn Hooded than in Wistar rats. CONCLUSIONS: We speculate that lower cannabinoid function appears to be related to greater vulnerability to alcohol consumption. Cannabinoid CB1 receptor may represent a key target in the treatment of alcohol dependence.     

Role of the endocannabinoid system in the development of tolerance to alcohol

The present review evaluates the evidence that the endocannabinoid system plays in the development of tolerance to alcohol. The identification of a G-protein-coupled receptor, namely, the cannabinoid receptor (CB(1) receptor), which was activated by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), the major psychoactive component of marijuana, led to the discovery of endogenous cannabinoid agonists. Until now, four fatty acid derivatives identified to be arachidonylethanolamide (AEA), 2-arachidonylglycerol (2-AG), 2-arachidonylglycerol ether (noladin ether) and virodhamine have been isolated from both nervous and peripheral tissues. Both AEA and 2-AG have been shown to mimic the pharmacological and behavioural effects of Delta(9)-THC. The role of the endocannabinoid system in the development of tolerance to alcohol was not known until recently. Recent studies from our laboratory have implicated for the first time a role for the endocannabinoid system in development of tolerance to alcohol. Chronic alcohol treatment has been shown to down-regulate CB(1) receptors and its signal transduction. The observed downregulation of CB(1) receptor function results from the persistent stimulation of the receptors by AEA and 2-AG, the synthesis of which has been shown to be increased by chronic alcohol treatment. The enhanced formation of endocannabinoids may subsequently influence the release of neurotransmitters. It was found that the DBA/2 mice, known to avoid alcohol intake, have significantly reduced CB(1) receptor function in the brain, consistent with other studies in which the CB(1) receptor antagonist SR 141716A has been shown to block voluntary alcohol intake in rodents. Similarly, activation of the CB(1) receptor system promoted alcohol craving, suggesting a role for the CB(1) receptor gene in excessive alcohol drinking behaviour and development of alcoholism. Ongoing investigations may lead to a better understanding of the mechanisms underlying the development of tolerance to alcohol and to develop therapeutic strategies to treat alcoholism.     

Opioids in the perifornical lateral hypothalamus suppress ethanol drinking

The opioid system is known to enhance motivated behaviors, including ethanol drinking and food ingestion, by acting in various reward-related brain regions, such as the nucleus accumbens, ventral tegmental area and medial hypothalamus. There is indirect evidence, however, suggesting that opioid peptides may act differently in the perifornical lateral hypothalamus (PF/LH), causing a suppression of consummatory behavior. Using brain-cannulated Sprague–Dawley rats trained to voluntarily drink 7% ethanol, the present study tested the hypothesis that opioids in the PF/LH can reduce the consumption of ethanol, with animals receiving PF/LH injections of the δ-opioid receptor agonist D-Ala2-met-enkephalinamide (DALA), the μ-receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), the κ-receptor agonist (±)-trans-U-50,488 methanesulfonate (U-50,488H), or the general opioid antagonist methylated naloxone (m-naloxone). The consumption of ethanol, lab chow, and water was monitored for 4 h after injection. The results showed that the three opioid receptor agonists injected into the PF/LH specifically and significantly reduced ethanol intake, while causing little change in chow or water intake, and the opposite effect, enhanced ethanol intake, was observed with the opioid antagonist. Of the three opioid agonists, the δ-agonist appears to produce the most consistent and long-lasting suppression of consumption. This effect was not observed with injections 2 mm dorsal to this area, focusing attention on the PF/LH as the main site of action. These results suggest that the opioid peptides have a specific role in the PF/LH of reducing ethanol drinking, which is distinct from their more commonly observed appetitive actions in other brain areas. The additional finding, that m-naloxone in the PF/LH stimulates ethanol intake in contrast to its generally suppressive effect in other regions, focuses attention on this hypothalamic area and its distinctive role in contributing to the variable effects sometimes observed with opioid antagonist therapy for alcoholism.     

Chronic ethanol consumption regulates cannabinoid CB1 receptor gene expression in selected regions of rat brain

AIMS: The aim of this study was to examine the effects of chronic ethanol consumption in cannabinoid CB(1) receptor gene expression in Wistar rats. METHODS: Rats were exposed to a bottle containing a solution of ethanol (10% v/v) and saccharin (0.25% w/v) for 52 days. At the end of this period, rats were killed by decapitation and cannabinoid CB(1) receptor gene expression was measured by in situ hybridization histochemistry. RESULTS: Our results indicated that chronic ethanol consumption reduced cannabinoid CB(1) receptor gene expression in caudate-putamen (CPu) (24%), ventromedial nucleus of the hypothalamus (VMN) (43%), CA1 (27%) and CA2 (22%) fields of hippocampus and increased dentate gyrus (DG) (30%). CONCLUSIONS: These results reveal for the first time that prolonged exposure to ethanol produces marked alterations in cannabinoid CB(1) receptor gene expression in selected regions of the rat brain, supporting an interaction between ethanol consumption and the endogenous cannabinoid receptor. Furthermore, these findings suggest that cannabinoid CB(1) receptor may be considered as a new pharmacological target for treating ethanol dependence.     

Chronic ethanol induces changes in opiate receptor function and in met-enkephalin release

Ethanol induces supersensitivity of striatal delta-opiate receptor sites labelled by 3H-Etorphine. This effect may be ascribed to the diminished enkephalin release detected in striatal slices after chronic ethanol consumption. On the other hand, Kd values for 3H-Met-enkephalin and 3H-DHM (mu-opiate receptors) specific binding are enhanced. The different sensitivity of the two classes of opiate receptors to ethanol may be due to specific effects on enkephalinergic transmission. It has been hypothesized that the decrease of 3H-Met-enkephalin and 3H-DHM affinity for their receptors takes place because endogenous substances from ethanol metabolism (for example salsolinol) behave as mu opioid agonists. This hypothesis is confirmed by "in vitro" studies demonstrating that salsolinol displaces 3H-Met-enkephalin and 3H-DHM but not 3H-DADLE binding. On the contrary, it seems that delta-receptors become supersensitive because of the decreased endogenous peptide release.     

Moderate Alcohol Intake: Behavioral and Neurochemical Correlates in Rats

A debate is open in the literature concerning the safety of low (dietary) versus high (intoxicating or addicting) alcohol consumption. Epidemiological data do indeed suggest that moderate ethanol intake may have beneficial effects, at least at cardiovascular level. On the other hand there are few data on the effect of low doses ethanol at brain level and few experimental models to investigate it, in spite of a vast literature on the addicting mechanisms. In the last years we have addressed this question by investigating behavioral and neurochemical parameters in rats consuming low ethanol doses, not producing tolerance and dependence, thus mimicking balanced dietary intake of ethanol. Ethanol exposure (3% vol/vol in drinking water for 8 weeks) ameliorates emotional reactivity, evaluated as decrease in ultrasonic calls, and improves learning in animals undergoing a two-way avoidance task. The concomitant measure of neurochemical parameters indicates plastic changes in receptor (dopamine) and post-receptor (protein kinase C) mechanisms. These changes are in some cases qualitatively different from those observed with intoxicating ethanol doses. This model may be useful for the further characterization of the beneficial versus detrimental effect of moderate ethanol consumption at brain level.     

Cholecystokinin and satiation with alcohol

Release of the brain-gut peptide cholecystokinin (CCK) is stimulated by intragastric instillation of ethanol, and peripheral administration of CCK inhibits ethanol consumption. To assess the temporal specificity of the inhibitory effect of CCK on alcohol intake, water-deprived rats were given 5% ethanol at 20, 10 or 0 min after intraperitoneal injections of CCK octapeptide. Delaying access to ethanol for 20 min prevented a significant effect of CCK on intake. CCK's temporally constrained inhibitory action on alcohol consumption is consistent with an ethanol satiation effect. To test the motivational specificity of CCK's effect on fluid intake, rats were allowed a 2-bottle choice of 2% ethanol and water after CCK injections. Ethanol solution intake was suppressed by CCK, and total water intake was unaffected. The putative alcohol satiation action of CCK is appropriately specific to ethanol solution in free-choice tests. Hungry, but not fluid-deprived rats that were either ethanol experienced or naive received a 2-bottle choice of 4% ethanol or water after CCK or saline injections. CCK again specifically inhibited ethanol intake, but this effect required prior ethanol experience. Doses of CCK and naloxone, an opioid receptor blocker, combined to inhibit ethanol intake in an infra-dose-additive manner in water-deprived rats. CCK may act endogenously, in part on opioid receptor-mediated processes, as a preabsorptive satiety signal of ethanol. The full expression of this action appears to depend on prior conditioning of nutritive expectancy of the postingestive effects of alcohol.     

Effect of natural and synthetic polyamines on ethanol intake in UChB drinker rats.

Because of the important glutamatergic mediation of the behavioral effects of ethanol, glutamatergic agents have attracted attention for the treatment of ethanol abuse and dependence in preclinical and clinical studies. In the present study, we investigated the effect of pharmacological doses of the natural polyamines putrescine, spermine, and spermidine and the synthetic polyamine N,N'-bis-(3-aminopropyl)cyclohexane-1,4-diamine (DCD) on alcohol consumption in a free-choice paradigm carried out in genetically high-ethanol-consumer UChB rats. Short 3-day treatment with either polyamine, administered p.o., significantly reduced ethanol intake without modifying water and food intakes. Neither polyamine was able to increase markedly blood acetaldehyde in rats submitted to a standard challenge dose of ethanol, to rule out a disulfiram-like effect. Besides, blood ethanol disappearance after a test dose of ethanol was not affected by the synthetic polyamine DCD. Long-term treatment with DCD dose-dependently reduced ethanol intake in UChB rats without producing any observable effect on overt behavior, food consumption, and total fluid intake. The present results indicate that pharmacological doses of polyamines can reduce ethanol consumption in genetically drinking rats without producing significant side effects, suggesting that modulation of brain N-methyl-d-aspartate receptors by polyamines could represent a suitable strategy to reduce appetite for ethanol. However, caution must be exercised in interpreting the results because polyamines can also affect neuronal excitability by acting at other receptor targets, such as AMPA and kainate receptors, as well as at some voltage-dependent ion channels.     

Intermittent ethanol access schedule in rats as a preclinical model of alcohol abuse

One of the major challenges in preclinical studies of alcohol abuse and dependence remains the development of paradigms that will elicit high ethanol intake and mimic the progressive transition from low or moderate social drinking to excessive alcohol consumption. Exposure of outbred rats to repeated cycles of free-choice ethanol intake and withdrawal with the use of intermittent access to 20% ethanol in a 2-bottle choice procedure (IA2BC) has been shown to induce a gradual escalation of voluntary ethanol intake and preference, eventually reaching ethanol consumption levels of 5–6 g/kg/24 h, and inducing pharmacologically relevant blood ethanol concentrations (BECs). This procedure has recently been gaining popularity due to its simplicity, high validity, and reliable outcomes. Here we review experimental and methodological data related to IA2BC, and discuss the usefulness and advantages of this procedure as a valuable pre-training method for initiating operant ethanol self-administration of high ethanol intake, as well as conditioned place preference (CPP). Despite some limitations, we provide evidence that IA2BC and related operant procedures provide the possibility to operationalize multiple aspects of alcohol abuse and addiction in a rat model, including transition from social-like drinking to excessive alcohol consumption, binge drinking, alcohol seeking, relapse, and neuroadaptations related to excessive alcohol intake. Hence, IA2BC appears to be a useful and relevant procedure for preclinical evaluation of potential therapeutic approaches against alcohol abuse disorders.     

Alcohol addiction (alcoholism) and the opioid system

Alcohol interacts with many neurotransmitter systems, including the endogenous opioid peptide system, which influences the mesolimbic dopamine system and is believed to be central to drug-induced reward. These observations emphasize the need to further characterize the alcohol-opioid system link. Two important clinical studies have shown that treatment with the systemically active opioid antagonist naltrexone reduced the frequency as well as the severity of an alcohol slip (i.e., prevented relapse) among alcohol-dependent subjects who were enrolled in outpatient alcoholism treatment programs. Multiple interactions between opioid peptides and various receptors complicate our understanding of these brain mechanisms. Two possible therapeutic approaches to alcohol addiction include interfering with alcohol-induced reward and reducing sensitivity to alcohol by administering drugs that selectively block or reinforce only one selected opioid system.     

Endorphins and experimental addiction

Animal studies suggest that the endogenous opioid systems in the brain play an important role in the initiation and maintenance of drug dependence. Opioids in the ventral tegmental area (VTA) may be involved in rewarded behaviors and, consequently, in the initiation of drug self-administration that may be associated with addiction proneness. Opioids in the limbic forebrain are particularly implicated in subsequent drug self-administration, which may be associated with craving, maintenance, and relapse. Alcohol intake in monkeys is reduced after treatment with naltrexone in a graded, dose-dependent manner. Naltrexone also is associated with a greater decrease in alcohol consumption after imposed abstinence. These findings support the idea that endorphins play a role in alcohol-drinking behavior, particularly after a period of abstinence during the so-called catch-up phenomenon. Recent studies of recovering alcoholic patients provide evidence that opiate antagonists attenuate the craving for alcohol and decrease and/or postpone relapse into addictive behavior.     

Is an alpha-conotoxin MII-sensitive mechanism involved in the neurochemical, stimulatory, and rewarding effects of ethanol?

Ethanol and nicotine are the most commonly abused drugs among human beings, and a large body of evidence, from both epidemiologic and preclinical studies, indicates that there is a positive correlation between intake of both drugs. Findings of studies from our research group have demonstrated that nicotinic acetylcholine receptors, especially those located in the ventral tegmental area, are important for the stimulatory, rewarding, and dopamine-enhancing effects of ethanol. Furthermore, results of recent work indicate that the alpha4beta2* and the alpha7* receptor subunits of the nicotinic acetylcholine receptors do not seem to be involved in the neurochemical and behavioral effects of ethanol in rodents. The aim of the current study was to investigate further the role of different nicotinic acetylcholine receptor subunits in the stimulatory, dopamine-enhancing, and rewarding effects of ethanol in rodents by using the peptide alpha-conotoxin MII (5 nmol; an antagonist of the alpha3beta2*, beta3*, and alpha6* subunits of the nicotinic acetylcholine receptor) administered locally into the ventral tegmental area. A significant reduction of ethanol-induced accumbal dopamine overflow, measured by means of in vivo microdialysis, and of locomotor stimulation was observed in mice. Furthermore, alpha-conotoxin MII was demonstrated to reduce voluntary ethanol intake significantly in both rats and mice. These results indicate that alpha-conotoxin MU-sensitive receptors may be important in mediating the stimulatory, dopamine-enhancing, and rewarding effects of ethanol, and that alpha-conotoxin MII-sensitive receptors may constitute targets for development of new adjuvant for treatment of ethanol dependence.     

The endocannabinoid system: physiology and pharmacology

The endogenous cannabinoid system is an ubiquitous lipid signalling system that appeared early in evolution and which has important regulatory functions throughout the body in all vertebrates. The main endocannabinoids (endogenous cannabis-like substances) are small molecules derived from arachidonic acid, anandamide (arachidonoylethanolamide) and 2-arachidonoylglycerol. They bind to a family of G-protein-coupled receptors, of which the cannabinoid CB(1) receptor is densely distributed in areas of the brain related to motor control, cognition, emotional responses, motivated behaviour and homeostasis. Outside the brain, the endocannabinoid system is one of the crucial modulators of the autonomic nervous system, the immune system and microcirculation. Endocannabinoids are released upon demand from lipid precursors in a receptor-dependent manner and serve as retrograde signalling messengers in GABAergic and glutamatergic synapses, as well as modulators of postsynaptic transmission, interacting with other neurotransmitters, including dopamine. Endocannabinoids are transported into cells by a specific uptake system and degraded by two well-characterized enzymes, the fatty acid amide hydrolase and the monoacylglycerol lipase. Recent pharmacological advances have led to the synthesis of cannabinoid receptor agonists and antagonists, anandamide uptake blockers and potent, selective inhibitors of endocannabinoid degradation. These new tools have enabled the study of the physiological roles played by the endocannabinoids and have opened up new strategies in the treatment of pain, obesity, neurological diseases including multiple sclerosis, emotional disturbances such as anxiety and other psychiatric disorders including drug addiction. Recent advances have specifically linked the endogenous cannabinoid system to alcoholism, and cannabinoid receptor antagonism now emerges as a promising therapeutic alternative for alcohol dependence and relapse.     

Alteration in the function of cerebral neurotransmitter receptors during the establishment of alcohol dependence: neurochemical aspects

Chronic alcohol treatment induces a variety of effects on the metabolism of neurotransmitters and the function of their receptors. Recent studies on alcohol dependence and/or functional tolerance to alcohol have clearly demonstrated that neurotransmitter-gated and receptor-coupled ion channels, as well as neurotransmitter receptor coupled with intracellular mediator systems, such as phosphatidylinositides and cyclic nucleotide-generating systems, are invariably suppressed during alcohol dependence. Future studies on the central actions of alcohol and on alcohol dependence should therefore be directed at the molecular changes in synaptic membrane-bound components such as neurotransmitter receptors, as well as on neurotransmitter release and relevant membrane-bound enzymes.