Lack of changes in beta-endorphin plasma levels after repeated treatment with fluoxetine: possible implications for the treatment of alcoholism--a pilot study.

Clinical and animal studies indicate that selective serotonin-reuptake inhibitors (SSRIs) may help to reduce alcohol intake but investigations led to conflicting results. A few studies indicated that serotonin (5-HT) may modulate the brain beta-endorphin level, which plays an important role in the development of alcohol craving. Our study examined the influence of fluoxetine on the endogenous opioid system. We investigated plasma levels of beta-endorphin in rats with either high alcohol preference (Warsaw High-Preferring; WHP) or low alcohol preference (Warsaw Low-Preferring; WLP) after repeated treatment with fluoxetine (5 mg/kg i.p. for 21 days). We examined the rats 24 hours after fluoxetine treatment in order to determine whether chronic fluoxetine produces a long-term change in the beta-endorphin levels. The animals received either a single dose of ethanol (2 g/kg) or an identical single dose of saline one hour before blood collection. While a few studies observed an increase in the level of beta-endorphin after a single fluoxetine injection, we did not observe any increase in beta-endorphin plasma levels after repeated fluoxetine treatment. We also did not observe any changes in beta-endorphin levels of rats treated with fluoxetine and injected with ethanol. A lack of increase of beta-endorphin levels may explain why fluoxetine has a limited value in the prevention of craving for alcohol.

Lack of ifenprodil anxiolytic activity after its multiple treatment in chronically ethanol-treated rats.

AIMS: The purpose of this study was to assess the anxiolytic activity of ifenprodil in Warsaw high-preferring (WHP) and low-preferring (WLP) rats after chronic ethanol treatment. METHODS: WHP and WLP animals, their paired-ethanol-naive groups and control Wistar rats were treated with ifenprodil (1.0 mg/kg, intraperitoneally) for 21 consecutive days. Anxiolytic activity was evaluated by using the two-compartment exploratory test. In addition, the locomotor activity paradigm was also assessed. RESULTS: Ifenprodil did not affect this paradigm in all investigated groups. The ethanol treatment led to lowering of anxiolytic scores in WHP rats. Multiple ifenprodil administration showed an anxiogenic-like activity in both WHP- and WLP-ethanol-treated groups. CONCLUSIONS: Our results suggest that, under some conditions, the role of ifenprodil in the treatment of alcoholism may be insufficient to support its use.

Changes in ethanol preference by rats treated with gamma1 and gamma2 GABA(A) receptor subunit antisense oligodeoxynucleotides.

Micro-injections (10 nmol/day over 5 days) of antisense oligodeoxynucleotides (aODNs) to gamma-aminobutyric acid A (GABA(A)) receptor alpha1 and gamma2 subunits reduce the mRNA for these subunits in rat brain. In this study, the effects of alpha1 and gamma2 subunit aODNs on rat alcohol preference were investigated. Reduction of the alpha1 subunit mRNA decreased, whereas reduction of the gamma2 subunit mRNA increased, ethanol intake in rats.

Animal model of ethanol abuse: rats selectively bred for high and low voluntary alcohol intake.

The selectively bred alcohol-preferring and alcohol-non-preferring lines of rats have been used to study the biology of alcohol abuse and dependence. In our laboratory new lines of Wistar rats have been selectively outbread for 7 years and 19 generations for high and low ethanol intake (WHP--Warsaw High Preferring) and WLP--Warsaw Low Preferring respectively). After the first selection procedure, the highest scoring females and males were used initiate upward selection, while the lowest scoring pairs were used to initiate downward selection. Mated pairs were housed in breeding cages, pups were allowed to nurse for 3 weeks before weaning, then the pups of each litter were culled to the same-sex cage and allowed to mature until they were subjected to the selection procedure. In order to determine the alcohol intake and preference, the rats were individually housed in wire cages containing two graduated drinking tubes mounted at the front. During the entire investigation, the subjects had free access to standard lab chow (Bacutil, Poland). Ethanol solution was prepared from 95% stock ethanol and tap water. The animals were presented with 10% ethanol solution and water (two-bottle choice test). The drinking tubes were rotated daily to prevent position preference. Alcohol intake was calculated as average g/kg/day (absolute ethanol) while alcohol preference (in %) was calculated as the amount of alcohol consumed/total fluid x 100. Our results (17-19 generations) have shown that mean alcohol intake in WHP rats was higher than 5.0 g/kg/24 h ethanol, while WLP rats generally consumed less than 2.0 g/kg/24 h ethanol. Our results also showed that the total fluid intake in WHP rats slightly but not significantly higher as compared with WLP rats. Maximal ethanol consumption (in both lines) occurred during the natural dark phase three bungs (19.00-20.00 hrs, 23.00-02.00 hrs and 04.00-05.00 hrs). Interestingly, the intakes of high concentrations of sucrose and saccharin solutions were significantly higher in WHP than in WLP rats. Furthermore, the WHP rats reduced their alcohol and water intakes in the presence of 10% sucrose solution. Thus, it appears that high consumption of sweets may be a neurobiological factor promoting increased ethanol intake by WHP rats.

Alcohol intake and brain [3H]muscimol binding sites in alcohol-preferring and non-preferring rats.

The present study has employed in vitro autoradiography to determine the distribution and density of [3H]muscimol binding sites in the brains of alcohol high-preferring line of rats (WHP) and alcohol low-preferring line of rats (WLP). While the density of [3H]muscimol binding was found to be similar in the frontal cortex, caudate-putamen, nucleus accumbens, lateral and medial septum, the density of [3H]muscimol binding was lower in cingulate cortex of alcohol low-preferring rats as compared to alcohol high preferring rats. Moreover, the density of muscimol binding sites within this area was positively correlated with the intensity of ethanol consumption.

Involvement of nicotinic acetylcholine receptors in the regulation of alcohol drinking in Wistar rats.

The aim of the present study was to determine if nicotinic acetylcholine receptors (nAChRs) might be involved in the regulation of alcohol intake by Wistar rats. A non-selective nAChR agonist, nicotine, and a non-competitive nAChR antagonist, mecamylamine, were tested in alcohol-preferring Wistar rats maintained on a limited access (4 h/24 h) to ethanol (10%, v/v). In addition, the effects of nicotine and mecamylamine on intake of standard laboratory chow were studied in a separate control experiment. Nicotine (0.1-0.6 mg/kg, s.c.) decreased ethanol consumption, but had no effect on food intake. In contrast, mecamylamine (1-3 mg/kg, s.c.) did not alter ethanol drinking even at the dose (3 mg/kg) which significantly decreased food intake. These results suggest that activation of nAChRs may selectively reduce ethanol consumption in outbred Wistar rats.

Ethanol-reinforced behaviour in the rat: effects of uncompetitive NMDA receptor antagonist, memantine.

Ethanol has been reported to alter NMDA receptor-mediated biochemical and electrophysiological responses in vitro. The aim of the present study was to evaluate the effects of an uncompetitive NMDA receptor antagonist memantine, in animal models of alcoholism. Male Wistar rats were trained to drink 8% ethanol in a free-choice, limited access procedure. A separate group of animals was trained to lever press for 8% ethanol in an operant procedure where ethanol was introduced in the presence of sucrose. The selectivity of memantine's actions was assessed by studying its effects on food or water consumption in separate control experiments. Memantine (4.5-24 mg/kg) significantly, but not dose dependently, affected ethanol drinking in the limited access procedure. However, only 6 mg/kg memantine selectively decreased ethanol drinking. Memantine did not alter ethanol intake in rats trained to lever press for ethanol in the operant procedure. Only 9 mg/kg memantine reduced operant responding in the extinction procedure in the rats trained to lever press for ethanol. The same dose of memantine significantly reduced the operant behaviour of rats trained to respond for water. These results indicate that: (i) single doses of memantine only moderately and not dose dependently reduce alcohol drinking in the limited access procedure; (ii) memantine produces non-selective effects on operant behaviour in rats trained to lever press for ethanol in an oral self-administration procedure.

Serotonin3 receptor antagonism of alcohol intake: effects of drinking conditions.

The effects of 5-HT3 receptor antagonists on ethanol intake were examined in the selectively bred alcohol-preferring P line of rats under continuous and limited access to 10% (v/v) ethanol with food and water ad lib. Single daily injections of either MDL 72222 (MDL) or ICS 205-930 (ICS) (0.01-3.0 mg/kg, SC) given 60 min before a 4-h scheduled access period for 4 consecutive days failed at all doses to alter the intake of a 10% (v/v) ethanol solution by P rats. However, multiple daily injections of either MDL (1-3 mg/kg, SC) or ICS (3.0 and 5.0 mg/kg, SC), given three times daily at 4-h intervals, significantly reduced ethanol intake under 24-h free-choice conditions on the first treatment day. Additionally, a single administration of 1.0 mg/kg MDL reduced 24-h free-choice ethanol intake by approximately 50% of control values and had no effect on 24-h saccharin intake. The effects of MDL were further examined in a 2-h schedule access paradigm in which rats received the access period at the same time every day (Fixed) or randomly during the dark cycle (Variable). Although 1.0 mg/kg MDL had little effect on ethanol drinking in the Fixed group, ethanol intake was reduced by 55% of control levels in the Variable group. Overall, the data indicate that drinking conditions influence the effectiveness of 5-HT3 antagonists to reduce ethanol consumption. Furthermore, the results suggest that conditions, associated with limited access ethanol drinking, markedly reduce the actions of 5-HT3 antagonists on ethanol intake.

Effects of 5-HT3 receptor agonists on voluntary ethanol intake in rats maintained on a limited access procedure.

Recent evidence from a variety of laboratory studies suggests that the central effects of ethanol (EtOH) are mediated by serotonin 5-HT3 receptors. Notably, EtOH is able to potentiate 5-HT action on 5-HT3 ionophore, and 5-HT3 antagonists are known to reduce certain effects of EtOH. In the present study, we evaluated the effects of two agonists of 5-HT3 receptors, 2-methyl-5-HT (2-Me-5-HT) and m-chlorophenylbiguanide (m-CPBG) that were microinjected i.c.v. and into the nucleus accumbens (NAC) on EtOH intake in Wistar rats with high EtOH preference. 2-Me-5-HT given i.c.v. (1 and 10 microg per rat) and into the NAC (bilaterally 1 and 10 microg per site) significantly reduced EtOH intake in the limited access paradigm (2h session). On the other hand m-CPBG was inactive after intra-NAC administration. It is concluded that central 5-HT3 receptors are involved in the regulation of EtOH consumption.

Evidence that the amygdala is involved in the inhibitory effects of 5-HT3 receptor antagonists on alcohol drinking in rats.

Two 5-HT3 receptor antagonists, tropisetron (1 and 10 ng) and ondansetron (10 and 100 ng) were tested for effects on ethanol drinking in Wistar male rats after bilateral microinjection into the amygdala. The animals had limited access (2 h/day) to the 10% (v/v) ethanol solution, food and water were available ad lib during the scheduled access period. Both drugs caused a decrease in ethanol drinking. Tropisetron (1 and 10 ng) decreased ethanol intake during the first hour of access. The lower dose (10 ng) of ondansetron was more effective than the higher (100 ng) dose. The finding implicates amygdaloid 5-HT3 receptors in the mechanism of ethanol intake in Wistar rats.

The abilities of 5-HT3 receptor antagonist ICS 205-930 to inhibit alcohol preference and withdrawal seizures in rats.

Recent evidence suggests that central 5-HT3 are intimately involved in the ethanol (ETOH) dependence mechanism. In the present study we observed the effects of the 5-HT3 receptor antagonist ICS 205-930 on audiogenic seizure response (ASR) in ETOH-withdrawn rats and on ETOH intake and preference. Low doses of ICS 205-930 (0.001 mg/kg), but not higher doses (0.1 mg/kg), markedly reduced both ASR and ETOH intake in a high preference group of animals. The possible mechanism of different effects of low and high drug doses is discussed.

Densities of dopamine D2 receptors are reduced in CNS regions of alcohol-preferring P rats.

The densities of dopamine D2 recognition sites labelled with [3H]sulpiride were determined in the caudate-putamen, nucleus accumbens (medial and lateral portions), olfactory tubercle, substantia nigra (pars reticulata and pars compacta), and ventral tegmental area (VTA) of alcohol-naive, selectively bred P (N = 7) and NP (N = 7) rats using quantitative autoradiography. The binding of [3H]sulpiride was 20-25% lower (P < 0.05) in the caudate-putamen, medial and lateral nucleus accumbens, and VTA of the P compared with the NP rats. No significant differences were observed between the P and NP rats in the olfactory tubercle or substantia nigra. [3H]Sulpiride binding, using standard membrane preparations, established with Scatchard analysis that the difference in the densities of D2 recognition sites in the caudate-putamen between the P and NP rats was due to lower Bmax values for the P line. The results indicate that the number of dopamine D2 receptor sites is lower in several central nervous system regions of the P rats compared to NP rats.

Comparative study of nifedipine and isradipine in animal models of ethanol dependence.

The effects of calcium channel inhibitors on ethanol intake and dependence in Wistar rats were studied. Nifedipine and isradipine were suspended in 1% Tween 40 saline solution and administered ip 60 min prior to testing. Audiogenic seizure response evaluation was performed after 5 days of ethanol treatment. Both nifedipine and isradipine significantly reduced the intensity score of convulsions and, in doses of 2.5 and 5.0 mg/kg, respectively, abolished them. Both drugs significantly reduced ethanol preference and intake in the high preference group of animals. Our results support the observations that certain dihydropyridine derivatives are capable of attenuating both ethanol preference and physical dependence.

Effects of D1 and D2 dopamine receptor agents on ethanol consumption in the high-alcohol-drinking (HAD) line of rats.

Dopamine receptor agonists and antagonists were tested for effects on alcohol drinking in female HAD rats (n = 10) given limited access (4 h/day) to a 10% (v/v) ethanol solution. Food and water were available ad libitum. Subcutaneous drug injections were given 30-60 min before the ethanol access periods. The D2 agonist quinpirole (0.04-2.0 mg/kg) caused a dose-dependent decrease in alcohol drinking throughout the 4-h period. Spiperone, a D2 antagonist, had no effect during the initial part of the session, but by the fourth hour, the 10 micrograms/kg dose tended to increase alcohol intake and the 30 micrograms/kg dose reduced intake. The D1 antagonist SCH-23390 (3-30 micrograms/kg) dose-dependently decreased ethanol drinking during the first hour of access. The D1 agonist SKF-38393 (2-6 mg/kg) also decreased alcohol intake, but it was less effective than SCH-23390. The findings implicate both D1 and D2 receptors in the reinforcing effects of alcohol drinking by the HAD line of rats.

Effects of 5-HT-1A receptor agonists on ethanol preference in the rat.

Pharmacological manipulation of brain serotonin (5-HT) neurons has recently become recognized as an important approach in the treatment of ethanol (ET-OH) dependence. In the present study, we observed the effects of three agonists of 5-HT-1A receptor subtype, 8-OHDPAT, buspirone, and NDO-008, on ET-OH preference in Wistar male rats. Animals received ET-OH intragastrically during the first week of the experiment, and then during 2 consecutive weeks, the only source of fluid (23 h/d) was 5% and 8% wt/vol ET-OH solution, respectively. Then the animals were presented with a free-choice between water and 8% ET-OH solution for a 1-week period. Based on the baseline recordings, two groups of rats were formed: a high preference group (ET-OH intake greater than 50% of total daily fluid intake) and a low preference group of rats (ET-OH intake less than 20%). During week 5 of the experiment, animals were treated with 5-HT receptor agonists (subcutaneous injections twice daily for 4 days). The treatment caused a significant reduction of ET-OH intake in the high preference group, but caused no change in the remaining groups. The effect of highly selective 5-HT-1A receptor agonist, 8-OHDPAT, on ET-OH consumption in the high preference group was antagonized by cyanopindolol, a nonselective antagonist of 5-HT-1 receptor subtype. Our results support the hypothesis that activation of 5-HT-1A receptors reduces ET-OH preference.

5-Hydroxytryptamine1A receptor agonists in animal models of depression and anxiety.

The effects of different doses of buspirone, 3-dipropyl-amino-5-hydrochromar (NDO 008) and 8-hydroxydipropyl-aminotetralin (8-OH-DPAT) (administered intraperitoneally) were studied in tests of anxiolytic and antidepressant action in rats. These tests included the elavated plus maze test, the forced swim test, stress-induced suppression of open-field behavior, and the differential-reinforcement-of-low-rates-of-behaviour-72 sec (DRL 72 s) test. Buspirone (0.125 mg/kg) and NDO 008 (1.0 to 2.0 mg/kg) produced anxiolytic activity in the elevated plus maze, whereas 8-OH-DPAT did not in the doses employed. All three compounds increased activity in the forced swim test, although buspirone did so at a lower dose than NDO 008 and 8-OH-DPAT. In the stress-induced suppression test of open field activity all three compounds induced an antidepressant-like effect at different doses dependent on whether footshock (stressor) was presented 24 hr before or just prior to the open-field test. All three compounds even caused some reduction of activity in the non-shocked rats. 8-OH-DPAT (1.0 mg/kg) produced a significant and reliable increase in the Reinforcement/Response rate quotient in the DRL 72s test. These diverse results may provide an indication of potential clinical efficacy of the 5-HT1A agonists in the treatment of anxiety and depression.

The involvement of NMDA receptors in acute and chronic effects of ethanol.

Recent evidence indicates involvement of excitatory amino acid receptors sensitive to N-methyl-d-aspartate (NMDA) in the action of ethanol (EtOH). Pronounced inhibition of NMDA receptor function is seen in vitro with concentrations of EtOH corresponding to those present during alcohol intoxication in humans. The present study was devoted to investigate the role of NMDA receptors in the action of EtOH in rats. Acute experiments showed antagonism by EtOH of convulsions induced by intracerebroventricular injection of NMDA. A similar effect was seen with a high dose of diazepam. Convulsions induced by an agonist of another excitatory amino acid receptor subtype, kainate, were also inhibited by EtOH. An uncompetitive antagonist of NMDA receptors, 5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801), potentiated EtOH-induced loss of righting, but attenuated the hypothermic action of EtOH. Moreover, MK-801 inhibited audiogenic convulsions in EtOH withdrawn rats. At the same time the effect of a proconvulsive dose of NMDA was not enhanced. Tolerance to the myorelaxant action of both EtOH and MK-801 upon repetitive administration was seen. Also some degree of cross-tolerance was observed. Moreover, MK-801 failed to modify EtOH preference in rats. The present results support involvement of NMDA receptors in expression of some acute and subchronic actions of EtOH and in expression of EtOH withdrawal.

MIF-1 potentiates the action of tricyclic antidepressants in an animal model of depression.

In the present paper, the effect of simultaneous treatment of rats with low doses of MIF-1 and tricyclic antidepressants on rat behavior in the forced swim test was studied. It was found that MIF-1 stimulated in a dose-dependent manner "active" behavior of animals in this paradigm. The effect of MIF-1 appeared to be independent of changes in rats' locomotion in the open field test. The combined treatment of rats with MIF-1 (0.01 mg/kg IP) and amitriptyline (5 mg/kg IP) or desipramine (1.25 mg/kg) IP) significantly stimulated active behavior in the forced swim test above the level obtained with each of the drugs given separately. The present data suggest the potential clinical efficacy of a combined therapy of depressive patients with MIF-1 and small doses of tricyclic antidepressants.

Activity of diltiazem and nifedipine in some animal models of depression.

The effect of two calcium channel inhibitors, diltiazem and nifedipine in animal models of depression: a) behavioral despair test and b) behavioral deficit produced by uncontrollable footshock was investigated. Additionally, the influence of both drugs on mouse killing (muricide) behavior induced by chronic isolation was studied. Both drugs given in single doses increased the active behavior of rats in behavioral despair test. Nifedipine but not diltiazem was partially effective in the test when administered chronically (14 days). Both drugs also attenuated stress-induced behavioral depression in the open field and forced swim test. Diltiazem was markedly more active in the former whereas nifedipine in the latter test. Neither compound influenced killing behavior in muricidal rats. Our data support the notion that calcium channel inhibitors may possess antidepressant activity, although there appear to exist certain differences in their scope of action depending on the model applied.

The effect of microinjections of clonidine into the locus coeruleus on cortical EEG in rats.

Microinjections of clonidine into the locus coeruleus (LC) area synchronized rat cortical EEG. This effect of clonidine was attenuated by local pretreatment of rats with idazoxan, an alpha-2 adrenoceptor antagonist. The data indicate functional role of alpha-2 adrenoceptors within LC area in regulation of ceruleocortical activity, and point at the LC as one of brain targets for the sedative action of clonidine.