Analysis of the 5-HT2 Receptor Ligands Dimethoxy-4-indophenyl-2-aminopropane and Ketanserin in Ethanol Discriminations

Previous studies have suggested a modulatory role of the 5-HT₂ receptor system in the behavioral effects of ethanol. The present study examined the discriminative stimulus effects of the 5-HT_2A/2C agonist (–)-dimethoxy-4-indophenly-2-aminopropane (DOI) and the 5-HT_2A antagonist ketanserin in rats trained to discriminate either 1.5 g/kg of ethanol from water (intragastrically, n = 7) or 2.0 g/kg of ethanol from water (intragastrically, n = 8). In substitution tests, neither DOI (0.3 to 1.0 mg/kg, ip) nor ketanserin (3.0 to 17.0 mg/kg, ip) produced discriminative stimulus effects similar to either training dose of ethanol, although decreases in rates of responding were significant at the highest doses tested. Likewise, when given in combination with ethanol, neither 5-HT₂ ligand shifted the ethanol-dose response determination in either the 1.5 or 2.0 g/kg ethanol training groups. DOI in combination with ethanol did not alter rates of responding, whereas ketanserin in combination with ethanol significantly decreased response rates. Thus, the 5-HT_2A receptor ligands do not appreciably affect the discriminative stimulus effects of ethanol, in contrast to previous results with 5-HT_1B ligands.     

Diethyldithiocarbamate Methyl Ester Sulfoxide, an Inhibitor of Rat Liver Mitochondrial Low Km Aldehyde Dehydrogenase and Putative Metabolite of Disulfiram

S-methyl N,N -diethylthiolcarbamate sulfoxide (DETC-MeSO) is a potent inhibitor of rat liver mitochondrial low K _m aldehyde dehydrogenase (ALDH₂) both in vivo and in vitro, and has been proposed to be the metabolite responsible for ALDH₂ inhibition by disulfiram. Diethyldithiocarbamate methyl ester (DDTC-Me), a key intermediate in the metabolism of disulfiram, has been shown to be bioactivated by microsomal monooxygenases to diethyldithiocarbamate methyl ester sulfoxide (DDTC-Me sulfoxide). Studies were conducted to determine if DDTC-Me sulfoxide was also an active metabolite of disulfiram and inhibitor of ALDH₂. DDTC-Me sulfoxide inhibited ALDH₂ in vitro with an IC₅₀ of 10 μm, and in vivo with an ID₅₀ of 31 mg/kg (170 μmol/kg). Maximal ALDH₂ inhibition in vivo was observed 8 hr after the administration of 45.2 mg/kg DDTC-Me sulfoxide, with ALDH₂ activity returning to control levels after 48 hr. Although DDTC-Me sulfoxide inhibited ALDH₂ in vivo, DDTC-Me sulfoxide was not detected in plasma from rats treated with either disulfiram (75 mg/kg), DDTC-Me (122.25 mg/kg), or DDTC-Me sulfoxide (45.2 mg/kg). However, DDTC-Me and S -methyl N,N -diethylthiolcarbamate (DETC-Me) were detected in plasma from rats treated with DDTC-Me sulfoxide. In rats treated with DDTC-Me sulfoxide and challenged with ethanol, a small increase of ∼9 μm in blood acetaldehyde and an inconsistent drop in blood pressure was observed. In conclusion, DDTC-Me sulfoxide inhibited ALDH₂ in vitro and in vivo, was less potent than DETC- MeSO, and was not detected after disulfiram administration.     

Possibility of 5-HT3 Receptor Involvement in Alcohol Dependence: A Microdialysis Study of Nucleus Accumbens Dopamine and Serotonin Release in Rats with Chronic Alcohol Consumption

The present study was performed to examine the involvement of serotonin-3 (5-HT3) receptors in the rat nucleus accumbens (ACC) in alcohol dependence. In alcohol-treated rats, perfusion of 40 mM K⁺ and 100 mM ethanol (EtOH) through the microdialysis probe increased the extracellular levels of ACC dopamine (DA), compared with controls. Perfusion of the serotonin (5-HT) uptake inhibitor sertlarine enhanced the extracellular levels of ACC 5-HT in both groups. Increased 5-HT availability in the synaptic clefts on the ACC further activated ACC DA release in the alcohol-treated rats, in comparison with controls. In the final experiments, perfusion of the 5.0 μ M 5-HT₃ receptor agonist 2-methyl-5-HT (2-Me-5-HT) through the microdialysis probe enhanced the extracellular levels of ACC DA. Magnitude of 2-Me-5-HT-induced DA release was significantly higher in alcohol-treated rats than in controls. On the other hand, 40 mM K⁺- and 100 mM EtOH-induced extracellular 5-HT release in alcohol-treated rats were markedly inhibited. These results show that (1) chronic alcohol intake increases the sensitivity of 5-HT₃ receptors, (2) 5-HT₃ receptors regulate DA release in the ACC, (3) the dopaminergic neuronal systems associated with 5-HT₃ ionophore in the ACC were upregulated after chronic alcohol exposure, and (4) chronic alcohol intake desensitizes the serotonergic neuronal systems in rat ACC. These findings suggest that neurochemical functions of 5-HT₃ receptors in regulating DA release in the ACC after alcohol exposure compensate for the dysfunction of serotonergic activity to restore the original properties in processing alcohol tolerance and that the development of alcohol dependence may be mediated by ACC 5-HT₃ receptors.     

Selective Inhibition of Alcohol Intake in Diverse Alcohol-Preferring Rat Strains by the 5-HT2A Antagonists Amperozide and FG 5974

The present studies sought to elucidate the role of 5-HT_2A receptor antagonists in suppressing alcohol intake by comparing the effects of amperozide and FG 5974 on alcohol, food, and water intake in strains of alcohol-preferring rats: P, Alko Alcohol (AA), and Fawn-Hooded (FH). Both amperozide and FG 5974 have 5-HT_2A receptor antagonist properties, but FG 5974 also shows presynaptic 5-HT_1A receptor agonist activity. After establishment of stable baselines for intake measures in a two-bottle continuous access paradigm, rats ( n = 10) were injected with 1 of 5 doses (0, 1.0, 2.5, 5.0, and 10.0 mg/kg, sc) of amperozide or FG 5974 at weekly intervals. Amperozide dose-dependently reduced alcohol intake, total fluid intake, and alcohol preference in all three strains under continuous access conditions, whereas FG 5974 was less effective. Food intake was also suppressed by amperozide at higher doses, whereas it was increased by FG 5974. Amperozide also dose-dependently reduced alcohol intake when it was available for only 1 hr/day, but FG 5974 tended to increase it. After oral administration, amperozide was also more effective than FG 5974 in reducing alcohol intake. Despite these differences in efficacy in suppressing alcohol intake, both compounds produced taste aversion to a novel saccharin solution. These complex findings suggest that biochemical properties other than 5-HT_2A receptor antagonism (e.g., 5-HT_1A receptor agonism) may be involved in the effects of amperozide and FG 5974 on alcohol intake and other consummatory behaviors.     

Preclinical Evaluation of Riluzole: Assessments of Ethanol Self-Administration and Ethanol Withdrawal Symptoms

Background:  Many of the neurobehavioral effects of ethanol are mediated by inhibition of excitatory N -methyl- d -aspartate (NMDA) and enhancement of inhibitory γ-amino-butyric-acid (GABA) receptor systems. There is growing interest in drugs that alter these systems as potential medications for problems associated with alcoholism. The drug riluzole, approved for treatment of amyotrophic lateral sclerosis (ALS), inhibits NMDA and enhances GABA_A receptor system activity. This study was designed to determine the preclinical efficacy of riluzole to modulate ethanol self-administration and withdrawal.
Methods:  Male C57BL/6J mice were trained to lever press on a concurrent fixed-ratio 1 schedule of ethanol (10% v/v) versus water reinforcement during daily 16-hour sessions. Riluzole (1 to 40 mg/kg, IP) was evaluated on ethanol self-administration after acute and chronic (2 week) treatment. To determine if riluzole influences ethanol withdrawal-associated seizures, mice were fed an ethanol-containing or control liquid diet for 18 days. The effects of a single injection of riluzole (30 mg/kg) were examined on handling-induced convulsions after ethanol withdrawal.
Results:  Acute riluzole (30 and 40 mg/kg) reduced ethanol self-administration during the first 4 hours of the session, which corresponds to the known pharmacokinetics of this drug. Ethanol self-administration was also reduced by riluzole after chronic treatment. Riluzole (30 mg/kg) significantly decreased the severity of ethanol-induced convulsions 2 hours after ethanol withdrawal.
Conclusions:  These results demonstrate that riluzole decreases ethanol self-administration and may reduce ethanol withdrawal severity in mice. Thus, riluzole may have utility in the treatment of problems associated with alcoholism.     

Reduced Sensitivity to Ethanol Reward, But Not Ethanol Aversion, in Mice Lacking 5=HT1B Receptors

Various serotonergic receptor systems are thought to influence the motivational effects of ethanol. This experiment characterized the acquisition of ethanol-induced conditioned taste aversion and ethanol-induced conditioned place preference in mutant knockout mice lacking 5-HT_1B receptors. In the taste conditioning procedure, adult homozygous knockout mice (-/-) and homozygous wild-type mice (+/+) received access to 0.2 M NaCl solution, followed immediately by intraperitoneal injection of 0 to 4 g/kg of ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. In the place conditioning procedure, knockout and wild-type mice received six pairings of a tactile stimulus with ethanol (2 g/kg, ip). A different tactile stimulus was paired with saline. Ethanol produced increases in locomotor activity, with wild-type mice showing higher levels of ethanol-stimulated activity than knockout mice during conditioning trials 5 and 6. Wild-type mice demonstrated conditioned place preference for the ethanol-paired stimulus. In contrast, knockout mice showed no evidence of place conditioning. These results are generally consistent with an important role for serotonergic systems in ethanol reward and specifically indicate that 5-HT_1b receptors are important for ethanol's rewarding effects but not for ethanol's aversive effects.     

Dexmedetomidine, Diazepam, and Propranolol in the Treatment of Ethanol Withdrawal Symptoms in the Rat

In this study, the effects of dexmedetomidine, a selective α₂-adrenoceptor agonist, on ethanol withdrawal symptoms, were compared with those of diazepam and propranolol. The rats were given highly intoxicating doses of ethanol for 4 days. After the intoxication period, rats were divided into four equal groups: a dexmedetomidine-treated group (30 μg/kg, sc), a diazepam-treated group (2 mg/kg, sc), a propranolol-treated group (5 mg/kg, sc), and a control group with no medication. Medication was given in the withdrawal phase—2, 8, 14, and 20 hr after the onset of the withdrawal symptoms. The severity of the ethanol withdrawal symptoms (rigidity, tremor, irritability, and hypoactivity) was observed up to 33 hr after the onset of the ethanol withdrawal symptoms. Both dexmedetomidine and diazepam significantly relieved tremor compared with the control group. Diazepam reduced irritability significantly, compared with the control group. When measured as the sum score of the three most specific withdrawal signs (rigidity, tremor, and irritability), dexmedetornidine and diazepam significantly relieved the ethanol withdrawal reaction. Propranolol attenuated tremor, but was inefticient against other withdrawal symptoms. Dexmedetomidine may thus represent a new effective drug in the treatment of the ethanol withdrawal syndrome.     

Effect of Ethanol and Vitamin B6 Deficiency on Pyridoxal 5-Phosphate Levels and Fetal Growth in Rat

The effects of chronic ethanol consumption and dietary vitamin B₆ levels on tissue pyridoxal 5-phosphate (PLP) contents and rat fetal development were investigated. Pregnant Sprague-Dawley rats were given 35% ethanol-calorie liquid diet with either adequate B₆ (1.7 mg/liter) or deficient B₆ (0.17 mg/liter), ad libitum, from gestation days 7 to 21. Control groups (adequate control and deficient control) were pair-fed with isocaloric sucrose substituted for ethanol. Rats were killed on gestation day 21. Ethanol groups had smaller fetuses than control groups, regardless of their dietary B₆ levels. However, in B₆ deficiency, ethanol affected fetal weight more severely than in B₆ adequate state. Tissue PLP levels were determined by radioen-zymatic method. In B₆ deficiency, ethanol feeding reduced maternal liver PLP by 22%. PLP in other tissues were not affected by ethanol. These results confirmed that chronic alcohol consumption affected fetal growth and also provided evidence that B₆ deficiency exacerbated ethanol effect.     

Effects of Chronic Ethanol Exposure on GABA Receptors and GABAB Receptor Modulation of 3H-GABA Release in the Hippocampus

Chronic ethanol treatment (CET), sufficient for decreasing long-term potentiation (LTP) in rats, also enhances ³H-GABA release from hippocampal slices in these same animals. The mechanism for an increase in GABA release may involve changes in presynaptic receptors. Therefore, we characterized presynaptic autoreceptor modulation of ³H-GABA release in hippocampal slices from control and CET rats. The effects of a GABA_B receptor agonist (baclofen) and antagonist [2-hydroxy (OH)-saclofen] were tested for their ability to modulate electrically stimulated ³H-GABA release from superfused hippocampal slices. Baclofen decreased stimulated release in a dose-dependent manner and 2-OH-saclofen increased release consistent with the existence of presynaptic GABA_B autoreceptors in hippocampus. The GABA_A antagonist bicuculline did not significantly modulate basal or stimulated release. When the effects of baclofen and 2-OH-saclofen were measured in animals 48 hr after withdrawal from CET, presynaptic modulation of release by baclofen and 2-OH-saclofen was decreased. In addition, we examined the density of ³H-baclofen and ³H-bicuculline binding in the hippocampal formation using quantitative autoradiographic techniques. We found that the density of ³H-baclofen binding sites was not affected by CET, whereas the density of ³H-bicuculline binding sites was increased by 28% in ethanol-treated rats. These data may explain how CET increases presynaptic regulation of GABA release from hippocampus that may contribute to the decrease in LTP seen in rats after CET.     

Effects of MDL 72222, a serotonin3 antagonist, on operant responding for ethanol by alcohol-preferring P rats

BACKGROUND: Previous studies indicated that, under continuous access conditions, the 5-HT3 antagonist MDL 72222 (MDL) effectively reduced ethanol drinking of alcohol-preferring P rats. However, MDL was without effect when similar doses were tested under scheduled access conditions, unless the ethanol access period was randomly presented. This study examined the effects of MDL on operant responding for ethanol and water by adult male alcohol-preferring P rats. METHODS: During the dark cycle, subjects in the first experiment were trained to respond concurrently for 15% ethanol and water on a fixed-ratio 5 (FR-5) and FR-1 schedule of reinforcement, respectively. Approximately 30 min before the 4-hr operant session, rats were injected subcutaneously (sc) with saline or MDL (1, 3, or 5 mg/kg). A second experiment tested the effects of 1 mg/kg MDL on operant responding for 15% ethanol in 1-hr sessions when operant access was given at a fixed time each day (fixed scheduled access, FSA group) or at variable time periods throughout the dark cycle (variable scheduled access, VSA group). RESULTS: In the first experiment, only the 5 mg/kg dose of MDL decreased responding for ethanol (approximately 20%) during the first 30 min of the 4-hr session. This dose also reduced total 4-hr responding for ethanol and water. In the second experiment, the 1 mg/kg dose of MDL had no effect on operant responding by the FSA group, but significantly reduced ethanol responding by the VSA group. CONCLUSIONS: These results suggest that 5-HT3 receptors may be involved in mediating the reinforcing effects of ethanol, and that temporal-environmental cues associated with the presentation of ethanol may be one factor involved in reducing the effectiveness of 5-HT3 antagonists to attenuate ethanol intake.     

Effects of Chronic Ethanol Consumption and Aging on 5-HT2A Receptors and 5-HT Reuptake Sites

The serotonergic system in brain is adversely affected by both aging and chronic ethanol consumption. The present study examined the combined effects of aging and chronic ethanol consumption on two components of the serotonergic system. Serotonin (5-HT) reuptake sites and 5-HT_2A receptors were quantitated in brain areas of 5-, 14-, and 24-month-old male Fischer 344 rats that were pair-fed a control or 6.6% (v/v) ethanol-containing liquid diet on a chronic basis. The regions examined include those containing the cell bodies and projections of serotonergic neurons. These experiments demonstrated the sensitivity of the serotonergic system of male Fischer 344 rats to both aging and chronic ethanol consumption. In control rats, aging was associated with a decline in the concentration of 5-HT_2A receptors in the nucleus accumbens and four cortical regions: frontal, parietal, piriform, and cingulate cortex. 5-HT_2A receptors were also reduced in the frontal, parietal, and cingulate cortex of aged ethanol-fed rats. In contrast, 5-HT reuptake sites were increased in older rats in the frontal cortex, nucleus accumbens, amygdala, and CA3 region of the hippocampus. If comparable changes in 5-HT_2A receptors and 5-HT reuptake sites occur in elderly humans, they may contribute to ethanol consumption, and lead to cognitive and other age-related problems. These changes may also alter the effectiveness of serotonergic drugs used in the treatment of alcoholism and mental disorders. The effects of chronic ethanol consumption were more limited. The only significant ethanol effect was an increase of 5-HT_2A receptors in the nucleus accumbens of 5-month-old ethanol-fed rats.     

MK-801 can exacerbate or attenuate behavioral alterations associated with neonatal alcohol exposure in the rat, depending on the timing of administration

BACKGROUND: We have reported that administration of MK-801, an NMDA receptor antagonist, during ethanol withdrawal in the developing rat attenuates ethanol's adverse effects on behavioral development. In the present study, we altered the timing of MK-801 delivery in relation to the last alcohol dose to determine if its protective effects were specific to the ethanol withdrawal phase. METHODS: Five groups of rats were artificially reared and exposed to alcohol in a binge-like manner on postnatal day (PD) 6, producing peak blood alcohol levels of 335 mg/dl that cleared to 0 mg/dl by 33 hours. Four groups received MK-801 at various times after alcohol treatment (0, 9, 21, or 33 hr post-ethanol). The fifth alcohol-treated group received saline. Two artificially reared control groups were included: one was injected with saline and the other injected with 0.5 mg/kg MK-801. Finally, a normally reared suckle control group was also included. Activity level and performance on a spatial discrimination reversal-learning task were evaluated at PD 18 and PD 40, respectively. RESULTS: Administration of MK-801 at the same time as ethanol treatment (0 hr) produced a high rate of mortality. Ethanol exposure on PD6 increased activity level relative to controls. Administration of MK-801 at 0 hr exacerbated this ethanol-induced overactivity, whereas administration of MK-801 at 21 and 33 hr reduced the severity of ethanol-related overactivity. Similarly, ethanol exposure on PD 6 significantly increased the number of errors committed on a spatial discrimination reversal-learning task. MK-801 injections 9 hrs after ethanol exacerbated this effect, whereas MK-801 treatment 33 hrs after ethanol attenuated this effect. Thus, MK-801 administration at the time of ethanol treatment was highly toxic, whereas during the withdrawal period it was protective. CONCLUSION: These data are consistent with the hypothesis that ethanol exposure in the neonatal rat inhibits the NMDA receptor, producing a subsequent rebound in NMDA receptor activation and possible excitotoxicity during withdrawal. Both the acute inhibitory effects of ethanol and the excitatory effects of withdrawal may contribute to fetal alcohol effects.     

Clinical Efficacy of the 5-HT3 Antagonist Ondansetron in Alcohol Abuse and Dependence

Medications that act on the serotonergic system have been found to be of benefit in the treatment of alcohol-dependent individuals. In a randomized, placebo-controlled study, the efficacy of 6 weeks of ondansetron, a 5-HT₃ antagonist (0.25 mg bid or 2.0 mg bid), in the treatment of 71 nonseverely alcohol-dependent males was tested. The results showed reduction of drinking differences were steadily increasing toward the end of the treatment period approached significance at week 7 in the 0.25 mg group ( p = 0.06). Twice as many patients in this group showed >2 standard deviations decrease in drinking compared with the other groups. When patients drinking >10 drinks/drinking day at baseline ( n = 11) were excluded from the analysis, significant group differences were found at both treatment and follow-up, with the lower ondansetron dose producing the greatest reduction from baseline (i.e., 2.8 standard drinks; –35% compared with baseline and –21% compared with placebo; p < 0.02–0.001). Within this group, there was an almost 4-fold greater number of patients showing a clinically meaningful decrease in drinking. Lower baseline drinking and higher level of education were significant and strong predictors of drinking reduction during treatment. Ondansetron was very well tolerated; hence, further long-term studies with 5-HT₃ antagonists alone or in combination with other treatment components may offer promise for treatment of alcoholism.     

Effects of CB1 cannabinoid receptor blockade on ethanol preference after chronic ethanol administration

BACKGROUND: Chronic ethanol administration results in neurobiological alterations similar to those observed after chronic cannabinoid exposure. The purpose of this study was to investigate alcohol drinking and the withdrawal responses after pulmonary chronic alcoholization with intraperitoneal or oral administration of a cannabinoid CB1 receptor antagonist. METHODS: The cannabinoid receptor antagonist SR141716A, 1, 3 or 10 mg/kg/day intraperitoneally or orally, was administered to Wistar rats either during a 30-day chronic ethanol exposure or at the cessation of this procedure. Motility was recorded during 18 hr after the cessation of chronic alcoholization just before the beginning of the free-choice paradigm (water versus alcohol 10% v/v). RESULTS: A significant increase in ethanol preference was observed during the free-choice paradigm after chronic alcoholization with concurrent SR141716A administration (3 or 10 mg/kg/day). A significant decrease in withdrawal motility after administration of SR141716A was observed with only the highest dose (10 mg/kg/day). The administration of SR141716A, 3 or 10 mg/kg/day, after chronic pulmonary alcoholization significantly decreased the preference for alcohol. Finally, a significant decrease in ethanol preference was seen during the free-choice paradigm of nonalcoholized rats treated with SR141716A, 3 or 10 mg/kg/day, during 30 days before the free-choice paradigm. CONCLUSIONS: The concurrent administration of the CB1 antagonist together with the chronic alcoholization increases the preference for ethanol. Also, the administration of the CB1 antagonist after the chronic alcoholization or at the time of withdrawal drastically diminishes the ethanol preference.     

Potential Role of 5HT1C and/or 5HT2 Receptors in the Mianserin-Induced Prevention of Anxiogenic Behaviors Occurring During Ethanol Withdrawal

A single dose of mianserin (a 5HT_1C/5HT₂ antagonist), administered 1 hr, 48 hr, or 7 days before testing, was evaluated for its efficacy in alleviating or preventing the occurrence of anxiogenic behaviors observed during ethanol withdrawal. Other behavioral experiments using selected drug interactions were conducted to examine whether the effect of mianserin was related to a long-term modification of 5-hydroxy-tryptamine (5HT) receptor function. Rats were fed a liquid diet containing 4.5% ethanol for 4 days. They were tested on the elevated plus-maze (EPM) 12 hr (acute withdrawal) and 5 days (protracted withdrawal) after the last ethanol dose. Ethanol withdrawal induced a pattern of “anxiogenic” behavior that consisted of reduced activity (total entries) and a reduced proportion of open arm activity. Mianserin, injected as a single dose given either 1 hr (0.16-5 mg/kg, ip) before testing or given (20 mg/kg, ip) on the morning of the 3rd day of ethanol administration, i.e., 48 hr and 7 days before testing, dose-dependently prevented or reversed the ethanol withdrawal induced reduction in open-arm activity. In contrast, the 5HT₁C/5HT₂ receptor agonist (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane HCI (DOI) did not affect behaviors in the EPM in ethanolnaive rats, nor in those undergoing ethanol withdrawal. However, although there was a marked tolerance to DOI-induced body shakes (a measure of 5HT₂ function) during withdrawal, DOI reversed the action of mianserin in the EPM. The 5HT₁ receptor agonist, 5HT₂ receptor antagonist 1-naphthyl-piperazine (1-NP) reduced open-arm activity in ethanol-naive rats and this action was enhanced during withdrawal. 1-NP reversed the effect of mianserin pretreatment and during ethanol withdrawal the dose-response curve of 1-NP was shifted to the left. The behavioral data indicated a reduced efficacy of 5HT₂ receptors during ethanol withdrawal while anxiogenic behaviors are present, whereas stimulation of 5HT_1C receptors appears anxiogenic. These data support the hypothesis that mianserin may alleviate withdrawal anxiety by direct blockade or down-regulation of 5HT_1C receptors.     

The reinforcing properties of salsolinol in the ventral tegmental area: evidence for regional heterogeneity and the involvement of serotonin and dopamine

Background:  Salsolinol (SAL), the condensation product of acetaldehyde and dopamine, may be a factor contributing to alcohol abuse. Previous research indicated that both ethanol and acetaldehyde are self-administered into the posterior ventral tegmental area (VTA). The current study examined SAL self-infusions into the VTA, and determined the involvement of dopamine neurons and 5-HT₃ receptors in this process.
Methods:  The intracranial self-administration technique was used to determine the self-infusion of SAL into the VTA of adult, male Wistar rats. The rats were placed in 2-lever (active and inactive) experimental chambers, and allowed to respond for the self-infusion of 0, 0.03, 0.1, 0.3, 1.0 or 3.0 μM SAL into the posterior or anterior VTA. In a second experiment, rats self-administered 0.3 μM SAL for the initial 4 sessions, co-administered SAL with ICS-205,930 (a 5-HT₃ receptor antagonist) or quinpirole (a D_2,3 receptor agonist) for sessions 5 and 6, and then only 0.3 μM SAL for session 7.
Results:  Wistar rats, given 0.03 to 0.3 μM SAL, received more infusions per session than did the group given artificial cerebrospinal fluid (aCSF) alone (e.g., 41 infusions for 0.1 μM SAL versus 9 infusions for the aCSF group), and responded more on the active than inactive lever. These effects were observed in the posterior but not in anterior VTA. Co-infusion of 100 μM ICS-205,930, or quinpirole significantly reduced self-infusions and active lever responding.
Conclusions:  SAL produces reinforcing effects in the posterior VTA of Wistar rats, and these effects are mediated by activation of DA neurons and local 5-HT₃ receptors.     

Additive Reduction of Alcohol Drinking by 5-HT1A Antagonist WAY 100635 and Serotonin Uptake Blocker Fluoxetine in Alcohol-Preferring P Rats

We found previously that alcohol-preferring (P) rats have fewer serotonin (5-HT) neurons and fibers in key brain regions than alcoholnonpreferring (NP) rats. Because 5-HT uptake blockers increase synaptic 5-HT content and 5-HT_1A receptor antagonists increase 5-HT release by disinhibiting 5-HT autoinnervation, in the present study, our intent was to determine whether increased synaptic 5-HT content and/or 5-HT release in P rats would effectively reduce alcohol consumption. In experiment 1, the 5-HT antagonist WAY 100635 (WAY) was tested on adult female P rats maintained on 24-hr free-choice access to ethanol (10% v/v) and water. Twice daily doses of WAY (0.05, 0.1, 0.5, and 1.0 mg/kg, subcutaneously) were administered to each rat in a counterbalanced order. Baseline ethanol intake, derived from the mean ethanol intakes of the three previous non-drug days, was approximately 8 g/kg/day. Results indicated that 0.05,0.1, and 0.5 mg/kg doses of WAY reduced 24-hr ethanol drinking by 25-30% ( p < 0.01) without affecting 24-hr water intake or body weight In the second experiment, the effects of WAY (0.5 mg/kg), fluoxetine (1.0 mg/kg), or a combination of both were tested in another group of female P rats. WAY and fluoxetine, each alone, reduced ethanol drinking by around 20% and, when combined, decreased ethanol intake by 50%, whereas the body weight and the total fluid intake were not significantly affected. Taken together, these results indicate that both fluoxetine and WAY preferentially reduce ethanol drinking in the P line of rats and, when administered together, reduce ethanol intake in an additive manner. It is proposed that coadministration of these two compounds with distinct mechanisms of action may be a new strategy for reducing alcohol intake.     

Effects of lorazepam treatment for multiple ethanol withdrawals in mice

Background: Although many alcohol‐dependent patients present with a history of prior detoxifications, the efficacy and safety of pharmacotherapy in the context of multiple ethanol withdrawal experiences have not been extensively studied. The purpose of this study was to evaluate the ability of lorazepam treatment for multiple withdrawals to prevent or blunt the development/expression of sensitized central nervous system hyperexcitability during a subsequent untreated withdrawal episode. A mouse model of withdrawal sensitization involving repeated ethanol withdrawals was used. Methods: Adult male C3H/He mice were exposed to different patterns of chronic ethanol vapor in inhalation chambers. One group received four cycles of 16 hr of ethanol exposure separated by 8‐hr withdrawal periods, another group was tested after a single 16‐hr exposure period, and a final group served as ethanol‐naïve controls. These groups were further divided into lorazepam dosage (0.25–1.0 mg/kg) conditions. Lorazepam was administered 1 hr into each of the first three withdrawal cycles (or equivalent times); no drug injections were given during the final (fourth) withdrawal cycle. The ability of lorazepam treatment to alter development and expression of sensitized handling‐induced convulsions (HIC), as well as changes in pentylenetetrazol seizure threshold dosage during an untreated withdrawal episode, was examined. Separate animals were used to assess the effects of lorazepam treatment on blood ethanol clearance and plasma levels of the benzodiazepine during the test withdrawal cycle. Results: Lorazepam dose‐dependently reduced HIC activity during successive withdrawal cycles, and this resulted in attenuated expression of the sensitized HIC response during the acute phase of a subsequent untreated withdrawal episode. However, HIC activity was exacerbated at later time points during this final test withdrawal in mice that had received lorazepam treatment for earlier withdrawals. A similar pattern of results was obtained for changes in pentylenetetrazol seizure threshold dosage. These results do not seem to be due to pharmacokinetic factors, because peak blood ethanol levels, rate of ethanol elimination, and plasma levels of lorazepam did not significantly differ among groups during the final test withdrawal cycle. Conclusions: Blocking central nervous system hyperexcitability during repeated ethanol withdrawals with lorazepam effectively blunts the development and expression of sensitized seizure activity during the acute phase of a subsequent unmedicated withdrawal episode. At later time points, withdrawal‐related seizure activity was exacerbated, and this is possibly reflective of an interaction between protracted ethanol withdrawal and withdrawal from the benzodiazepine. The clinical implications of these findings suggest that repeated use of benzodiazepines for treatment of multiple ethanol withdrawals may have some initial beneficial effects, but such treatment may also place patients at increased risk of seizures at later time points.     

Ethanol Acutely Impairs the Renal Conservation of 5-Methyltetrahydrofolate in the Isolated Perfused Rat Kidney

The ethanol-induced increase in urinary folate excretion has been shown to decrease plasma folate levels and may contribute to the development of folate deficiency associated with alcoholism. The mechanism for this effect remains to be elucidated. The present studies were designed to examine the direct effect of ethanol on the renal handling of 5-methyltetrahydrofolate (5-CH₃-H₄PteGlu), the physiological folate. Rats were given four consecutive hourly doses of ethanol (1 g/kg) or isocaloric doses of glucose solution orally. This treatment generated an average plasma ethanol level of 305 mg/dl. Kidneys from male Sprague-Dawley rats were removed 5 hr after initial treatment and perfused in vitro to eliminate any extrarenal effects that could confound interpretation of results. Ethanol was not added to the perfusate. These treatments had no effect on 5-CH₃-H₄PteGlu conservation by the isolated perfused rat kidney in comparison to experiments in which the animal received no treatment. Ethanol was then added directly to the perfusate to generate average concentrations of 293 mg/dl. The in vitro addition of ethanol significantly decreased the percentage reabsorption and increased the fractional excretion of 5-CH₃H₄PteGlu in comparison to controls (kidneys perfused with or without an isocaloric dose of glucose). This effect did not become significant until the renal tissue was exposed to these levels of ethanol for 1 hr. These results indicate that ethanol directly impairs the renal conservation of 5-CH₃-H₄PteGlu.