The new compound GET73, N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide, Regulates hippocampal Aminoacidergic transmission possibly via an allosteric modulation of mGlu5 receptor. Behavioural evidence of its "anti-alcohol" and anxiolytic properties.

The present article attempts to provide, on the basis of data emerging from studies carried out in our laboratories, a summary of the chemical and pharmacological properties of the new compound N-[(4-trifluoromethyl)benzyl]4- methoxybutyramide (GET73). Particular emphasis is given to findings obtained in vivo and in vitro suggesting that an allosteric modulation of metabotropic glutamate receptor 5 (mGlu5 receptor) by GET73 may represent the mechanism underlying the effects of the compound produced on rat hippocampal glutamate and GABA transmission. Furthermore, behavioural findings demonstrating how this new compound reduces alcohol intake, displays anxiolytic properties, and influences spatial memory in rats are also summarized. Since mGlu5 receptors play an important role in regulating several central actions of drugs of abuse, and the hippocampus is a crucial brain area involved in addiction, anxiety, and spatial memory, a possible link between mGlu5 receptor allosteric modulation and the profiles of action of GET73 is proposed, although to date no studies have yet explored GET73 binding at the mGlu5 receptor orthosteric and/or allosteric sites. Following a brief overview of glutamatergic neurotransmission, mGlu receptor structures and activation mechanisms, the general properties of mGlu5 receptor and its allosteric modulators are described in the first part of the review.

GABA(B) receptor-mediated increase of neurosteroids by gamma-hydroxybutyric acid.

Among the pharmacological actions of gamma-hydroxybutyric acid (GHB), some may involve GABA(A) receptor-mediated mechanisms. GHB, however, fails to directly interact with sites for agonists and modulators on the GABA(A) receptor complex. We hypothesized that, in vivo, GHB may interfere with GABA(A) receptor function by altering the brain concentrations of the neurosteroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, AP) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone, THDOC), positive allosteric modulators of GABA-gated chloride currents. In male Wistar rats, GHB dose-dependently (75-1000 mg/kg, i.p.) increased AP, THDOC and their precursors pregnenolone and progesterone in brain cortex and hippocampus. The increases of AP (4-5 fold) and THDOC (3-4 fold) elicited by 300 mg/kg GHB peaked between 30 and 90 min and abated by 180 min. The selective GABA(B) receptor antagonist SCH 50911 (50 mg/kg, i.p.) prevented the action of GHB, while the GABA(B) receptor agonist baclofen (5-10 mg/kg) mimicked it. NCS-382 (50 mg/kg, i.p.), the purported selective antagonist of the GHB receptor, failed to antagonize GHB, but at 300 mg/kg increased brain cortical neurosteroids to the same extent as 300 mg/kg GHB; coadministration of GHB and NCS-382, however, failed to yield an additive effect. These results strongly suggest that GHB, via a GABA(B) receptor-mediated mechanism, increases the brain concentrations of neurosteroids, whose properties as amplifiers of the GABA-gated chloride conductances may play a role in the GABA(A) receptor-mediated pharmacological actions of GHB.

The cannabinoid receptor antagonist SR 141716 prevents acquisition of drinking behavior in alcohol-preferring rats.

The cannabinoid CB(1) receptor antagonist, N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide) (SR 141716); 0.3-3 mg/kg, i.p., twice daily for 10 days), prevented the acquisition of alcohol drinking behavior in rats genetically selected for alcohol preference (Sardinian alcohol-preferring (sP) rats), having the free choice between alcohol (10%, v/v) and water. The results suggest that activation of cannabinoid CB(1) receptors is essential for the acquisition of alcohol drinking behavior in animals with a genetically determined alcohol preference.

Role of GABA(B) receptors in the sedative/hypnotic effect of gamma-hydroxybutyric acid.

The present study was aimed at identifying the receptor systems involved in the mediation of the sedative/hypnotic effect of gamma-hydroxybutyric acid (GHB) in DBA mice. Administration of the putative antagonist of the GHB binding site, 6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylideneacetic acid (NCS-382; 50-500 mg/kg, i.p.), significantly increased the duration of loss of righting reflex induced by GHB (1000 mg/kg, i.p.). In contrast, the GABA(B) receptor antagonists, (2S)(+)-5,5-dimethyl-2-morpholineacetic acid (SCH 50911; 25-100 mg/kg, i.p.) and (3-aminopropyl)(cyclohexylmethyl)phosphinic acid (CGP 46381; 12.5-150 mg/kg, i.p.), completely prevented the sedative/hypnotic effect of GHB. SCH 50911 (100 and 300 mg/kg, i.p.) was also capable to readily reverse the sedative/hypnotic effect of GHB (1000 mg/kg, i.p.) in mice that had lost the righting reflex. SCH 50911 (100 mg/kg, i.p.) also completely abolished the sedative/hypnotic effect of the GABA(B) receptor agonist, baclofen. These results indicate that the sedative/hypnotic effect of GHB is mediated by the stimulation of GABA(B) receptors and add further support to the hypothesis that the GABA(B) receptor constitutes a central site of action of GHB.

Procedure of bidirectional selective outbreeding for production of two rat lines differing in sensitivity to the sedative/hypnotic effect of gamma-hydroxybutyric acid.

The exogenous administration of gamma-hydroxybutyric acid (GHB), a constituent of the mammalian brain where it likely functions as a neurotransmitter or a neuromodulator, exerts a number of pharmacological effects, including sedation and hypnosis. The present paper describes a procedure for selective breeding of two rat lines which markedly differ in sensitivity to the sedative/hypnotic effect of GHB. Selective breeding originated from Wistar rats showing opposite sensitivity to the sedative/hypnotic effect of 1 g/kg GHB (i.p.). 'Sensitive' Wistar rats, defined as those individuals displaying values of r = sleep time/onset greater than the upper 15th percentile, were mated to generate the GHB-sensitive (GHB-S) line; conversely, 'resistant' Wistar rats (r-values lower than the lower 15th percentile) were mated to generate the GHB-resistant (GHB-R) line. Upper and lower 15th percentiles were also used to establish the selection cut-offs and criteria for rats of subsequent generations. Specifically, r-values of GHB-S rats were required to be r > or =8 on two separate tests with GHB; r-values of GHB-R rats were required to be r < or =2 on two separate tests with GHB. In each of the three generations produced to date, GHB-S rats showed significantly shorter onset, longer sleep times and greater r-scores than GHB-R rats. The selective breeding of GHB-S and GHB-R rats: (a) suggests that sensitivity to GHB is under genetic control, and (b) may constitute a unique model for investigation of the physiological function of GHB.

Selective breeding of two rat lines differing in sensitivity to GHB and baclofen.

Two Wistar-derived rat lines, one sensitive (GHB-S) and the other resistant (GHB-R) to the anesthetic effect of gamma-hydroxybutyric acid (GHB), have been selectively bred. GHB-S and GHB-R rats were also sensitive and resistant, respectively, to the anesthetic effect of baclofen, the prototype GABA(B) receptor agonist, suggesting that they may be useful to elucidate not only the role of endogenous GHB but also that of GABA(B) receptors in sleep and anesthesia.

Alcohol stimulates motor activity in selectively bred Sardinian alcohol-preferring (sP), but not in Sardinian alcohol-nonpreferring (sNP), rats.

The present study was conducted to evaluate the effect of low doses of ethanol on motor activity in selectively bred Sardinian alcohol-preferring (sP) and Sardinian alcohol-nonpreferring (sNP) rats. Ethanol was acutely administered at the doses of 0, 0.25, and 0.5 g/kg (i.p.) immediately before rat exposure to an open-field arena for 15 min. The number of square crossings, used as index of motor activity, was significantly lower in saline-treated sP than in saline-treated sNP rats, suggestive of a genetically determined higher emotional state in sP than in sNP rats. Ethanol administration resulted in a dose-dependent, significant increase in the number of square crossings in sP rats, whereas it was completely ineffective in sNP rats. These results suggest to us that a positive relationship exists between ethanol preference and ethanol-induced motor stimulation in sP/sNP rat lines.

GABA(B) receptor inhibition causes locomotor stimulation in mice.

The present study investigated the effect of the administration of the GABA(B) receptor antagonists, SCH 50911 [(2S)(+)-5,5-dimethyl-2-morpholineacetic acid], CGP 46381 [(3-aminopropyl)(cyclohexylmethyl)phosphinic acid] and CGP 52432 (3-[[(3,4-dichlorophenyl)methyl]amino]propyl]diethoxymethyl)phosphinic acid), on spontaneous locomotor activity in mice. All drugs were acutely administered at the doses of 10 and 30 mg/kg (i.p.). The dose of 30 mg/kg of all drugs resulted in a significant stimulation of locomotor activity. The locomotor stimulation elicited by SCH 50911 was completely blocked by haloperidol (0.1 mg/kg, i.p.), suggesting that hyperactivity induced by blockade of the GABA(B) receptor is mediated by enhanced dopamine release. These results suggest the existence of a GABA(B) receptor-mediated tonic inhibition of dopamine neurons.

Different sensitivity to ethanol in alcohol-preferring sP and -nonpreferring sNP rats.

BACKGROUND AND OBJECTIVES: Clinical research has proposed that initial sensitivity to ethanol may be negatively correlated with levels of subsequent ethanol intake; consistently, alcohol-preferring P rats were found to be less sensitive to the ataxic and sedative/hypnotic effects of ethanol than -nonpreferring NP rats. The present study investigated the initial sensitivity to the ataxic and sedative/hypnotic effects of ethanol and to the sedative/hypnotic effects of pentobarbital and diazepam in selectively bred Sardinian alcohol-preferring sP and -nonpreferring sNP rats. METHODS: In experiment 1, time to lose (onset) and regain (sleep time) the righting reflex after the acute intraperitoneal (ip) administration of 3.0 and 3.5 g/kg ethanol were measured in sP and sNP rats. In experiment 2, sP and sNP rats were required to perform a motor coordination task on a Rota-Rod after the acute intragastric administration of 2.0, 2.5, and 3.0 g/kg ethanol. Experiment 3 assessed onset and sleep time in sP and sNP rats after the acute injection of pentobarbital (40 mg/kg; ip) and diazepam (15 and 20 mg/kg; ip). RESULTS: In experiment 1, sP rats took shorter times to lose the righting reflex and regained this reflex over longer periods of time and at lower blood ethanol levels than sNP rats. In experiment 2, ethanol affected motor coordination to a greater extent in sP than sNP rats. In contrast, results from experiment 3 showed that sP and sNP rats were not differentially sensitive to the sedative/hypnotic effects of pentobarbital and diazepam. CONCLUSIONS: The results of experiments 1 and 2 suggest that sP rats possess a genetically determined, greater sensitivity to the motor impairing and sedative/hypnotic effects of ethanol than sNP rats. Although caution should be adopted before hypothesizing any comparison to humans, these results may feature sP rats as an experimental model of those subsets of human alcoholics with initial high sensitivity to ethanol challenges. Finally, the results of experiment 3 suggest a minimal involvement of the benzodiazepine and barbiturate recognition sites in the differential sensitivity to ethanol of sP and sNP rats.

Potential use of medicinal plants in the treatment of alcoholism.

The present paper briefly reviews the most relevant experimental data on the reducing effect of some medicinal herbs on voluntary alcohol intake in animal models of alcoholism. Pueraria lobata, Tabernanthe iboga, Panax ginseng, Salvia miltiorrhiza and Hypericum perforatum proved to be effective in decreasing alcohol consumption. Reduction of alcohol absorption from the gastrointestinal system appears to be a common feature among most of the above plants. These data suggest that medicinal plants may constitute novel and effective pharmacotherapies for alcoholism.

Development of short-lasting alcohol deprivation effect in sardinian alcohol-preferring rats.

Alcohol deprivation effect (ADE), defined as a temporary increase in voluntary alcohol intake following a period of alcohol abstinence, was evaluated in selectively bred Sardinian alcohol-preferring (sP) rats. Alcohol was initially offered in free choice with water for 35 consecutive days (predeprivation phase). Subsequently, one group of rats was deprived of alcohol for 1, 3, 7, 15, 30, 90 or 180 consecutive days, while the second group had continuous access to alcohol (deprivation phase). Once alcohol was re-presented, alcohol intake in alcohol-deprived rats was recorded 1 and 24 h after alcohol re-presentation and compared to that monitored in alcohol-nondeprived rats over the same time periods (postdeprivation phase). Alcohol deprivation for 3 to 30 days resulted in a significant increase in voluntary alcohol intake only in the first hour of re-access. These results demonstrate the development of ADE in sP rats. However, the rapid return of alcohol intake to control levels is discussed as evidence in favor of a set-point mechanism capable of regulating alcohol-drinking behavior in sP rats.

Characterization of the discriminative stimulus effects of gamma-hydroxybutyric acid as a means for unraveling the neurochemical basis of gamma-hydroxybutyric acid actions and its similarities to those of ethanol.

The present paper reviews the drug discrimination studies, both from the literature and from this laboratory, conducted to investigate the pharmacological profile of the discriminative stimulus effects of gamma-hydroxybutyric acid. Collectively, the results of these studies suggest that: (1) the discriminative stimulus effects of gamma-hydroxybutyric acid are composed of different cues, each one being the effect of gamma-hydroxybutyric acid on a specific receptor system; (2) the proportion of each component cue varies as the training dose of gamma-hydroxybutyric acid is increased; (3) the gamma-aminobutyric acid B-mediated cue is a major ingredient of the mixed stimulus of gamma-hydroxybutyric acid, but it is more prominent at high training doses than at low training doses of gamma-hydroxybutyric acid; and (4) positive modulation of the gamma-aminobutyric acid A receptor is a relevant part of the discriminative stimulus effects of low gamma-hydroxybutyric acid doses. Finally, data indicating symmetrical generalization between the discriminative stimulus effects of a specific range of doses of gamma-hydroxybutyric acid and those of ethanol are discussed in regard to their further support of the hypothesis that gamma-hydroxybutyric acid may exert its antialcohol effects through a substitution mechanism.

Mechanism of the antialcohol effect of gamma-hydroxybutyric acid.

Treatment with gamma-hydroxybutyric acid has been reported to effectively decrease alcohol craving and consumption as well as alcohol withdrawal symptoms in alcoholics. We describe the results of animal studies demonstrating the ability of gamma-hydroxybutyric acid to reduce (1) the severity of ethanol withdrawal signs in rats rendered physically dependent on ethanol and (2) voluntary ethanol intake in selectively bred Sardinian alcohol-preferring rats. Furthermore, we review experimental data suggesting that gamma-hydroxybutyric acid and ethanol have several pharmacological effects in common. Relevant similarities are: (1) stimulation of firing rate of dopaminergic neurons and dopamine release in specific rat brain areas; (2) development of cross-tolerance to the motor-impairing effects after repeated administration in rats; 3) abuse potential, as indicated by self-administration of pharmacologically relevant doses of gamma-hydroxybutyric acid in rats and mice; (4) induction of anxiolytic effects in rats; and (5) induction of similar discriminative stimulus effects, as evidenced by symmetrical generalization in a drug discrimination study in rats. These lines of evidence are discussed in relation to gamma-hydroxybutyric acid exerting its antialcohol effects by a substitution mechanism.

Dissociation of ethanol and saccharin preference in sP and sNP rats.

BACKGROUND: It has been proposed that ethanol intake and consumption of sweet tasting solutions are positively correlated in rodents. Experiment 1 of the present study investigated whether selectively bred ethanol-preferring (sP) and -nonpreferring (sNP) rats differed, consistently with the above hypothesis, as to saccharin intake and preference. Experiment 2 evaluated whether saccharin addition to the ethanol solution, likely resulting in a highly palatable fluid, would result in an increase in voluntary ethanol intake in sP rats. METHODS: The saccharin solution was offered, in free choice with water, at a fixed concentration of 1 g/liter for 6 consecutive days in Experiment 1A or at ascending concentrations (0.002 to 16.4 g/liter, doubling the concentration every day) in Experiment 1B. In Experiment 2, 1 g/liter saccharin was added to the standard 10% ethanol solution and offered to sP rats in free choice with water for 7 consecutive days. RESULTS: In both Experiments 1A and 1B, sP and sNP rats showed avidity for the saccharin solution with marginal line difference in saccharin intake and preference. In Experiment 2, daily ethanol intake remained stable at baseline levels (6-7 g/kg), irrespective of the saccharin addition to the ethanol solution. CONCLUSIONS: The results of Experiments 1A and 1B suggest that saccharin drinking behavior in sNP rats deviates from the hypothesis that saccharin and ethanol intakes may co-vary; thus, at least in sNP rats, saccharin and ethanol intakes do not appear to be influenced by the same genetic factors. The results of Experiment 2 provide further support to the existence of a central set-point mechanism that regulates daily ethanol intake in sP rats, likely based on the pharmacological effects of ethanol.

Ability of baclofen in reducing alcohol intake and withdrawal severity: I--Preclinical evidence.

BACKGROUND: The similarities between the pharmacological effects of the gamma-aminobutyric acid receptor agonist, baclofen, and the alcohol-substituting agent, gamma-hydroxybutyric acid, led us to investigate whether baclofen was capable of reducing (a) ethanol withdrawal syndrome in ethanol-dependent rats and (b) voluntary ethanol intake in ethanol-preferring rats. METHODS: In experiment 1, Wistar rats were rendered physically dependent on ethanol by the repeated administration of intoxicating doses of ethanol for 6 consecutive days. Baclofen was acutely administered intraperitoneally at doses of 10, 20, and 40 mg/kg. In experiment 2, baclofen (0, 2.5, 5, and 10 mg/kg, intraperitoneally) was administered once a day for 14 consecutive days to ethanol-preferring sP rats that had continuous access to ethanol (10%, v/v) and water under the two-bottle free choice regimen. RESULTS: In experiment 1, baclofen dose-dependently decreased the intensity of ethanol withdrawal signs; furthermore, 20 mg/kg of baclofen protected from audiogenic seizures in ethanol-withdrawn rats. In experiment 2, baclofen selectively and dose-dependently reduced voluntary ethanol intake; a compensatory increase in water intake left total fluid intake virtually unchanged. CONCLUSIONS: These results are in close agreement with those of a preliminary clinical study and suggest that baclofen may constitute a novel therapeutic agent for alcoholism.

Properties of benzodiazepine binding sites in peripheral blood lymphocytes.

Benzodiazepine (BDZ) binding sites were studied by using 3H-diazepam and 3H-Ro 5-4864 in intact lymphocytes from peripheral blood (PBL), in comparison to kidney and cerebellum. Experiments with 3H-diazepam performed at equilibrium and measuring kinetics revealed that BDZ binding sites are indeed present in rat PBL. The binding is saturable (Bmax 557 fmoles/10(6) cells), with high affinity (KD = 9.3 nM) and reversible. Specific binding sites are also observed by saturation experiments with 3H-Ro 5-4864 (Bmax 175 fmoles/10(6) cells, KD 2.2 nM). In addition, analysis of saturation isotherms obtained with 3H-diazepam indicates that BDZ binding sites are also present in human PBL. Scatchard plot of binding isotherms revealed an apparent single population of sites in all cases. The pharmacological characterization of BDZ binding sites in PBL, as compared with those of kidney and cerebellum, showed that these sites belong to the so-called "peripheral type."