Alterations in alcohol consumption, withdrawal seizures, and monoamine transmission in rats treated with phentermine and 5-hydroxy-L-tryptophan.

We have previously shown that coadministration of the dopamine (DA) agonist phentermine plus the serotonergic agonist fenfluramine suppresses alcohol intake and withdrawal seizures in rats. In the present study, phentermine and the serotonin (5-HT) precursor, 5-hydroxy-L-tryptophan (5-HTP), were administered alone, or in combination, to rats fed on a 6% alcohol-containing diet or an isocaloric control diet. Following a 9-h withdrawal period from the alcohol-containing diet, phentermine enhanced the effects of 5-HTP on both reduction of alcohol withdrawal seizures as well as changes in striatal serotonin. Food intake was monitored for 24 h after drug treatment, and neurochemical measures were examined at various time points. Phentermine alone reduced food intake in all diet conditions, but this anorectic effect was followed by hyperphagia in control rats. Phentermine plus 5-HTP reduced the consumption of the alcohol-containing diet, while its effects on consumption of control diets were mixed. In vivo microdialysis in rat nucleus accumbens revealed that phentermine increased extracellular DA, whereas 5-HTP caused marked elevations in extracellular 5-HT. Coadministration of phentermine and 5-HTP evoked simultaneous elevations in extracellular DA and 5-HT that mirrored the effects of each drug alone. Collectively, these findings show that coadministered phentermine plus 5-HTP is effective in reducing alcohol intake and suppressing alcohol withdrawal seizures. These therapeutic actions may be related to elevations in synaptic DA and 5-HT in critical brain regions.

Effect of MPEP, a selective mGluR5 antagonist, on the antielectroshock activity of conventional antiepileptic drugs.

MPEP, a selective non-competitive antagonist of group I metabotropic glutamate receptor subtype 5 (mGluR5), administered at doses ranging from 0.75 to 1 mg/kg, failed to influence the electroconvulsive threshold in mice. However, when administered at higher doses (1.25 and 1.5 mg/kg), it significantly increased the threshold. Moreover, MPEP (applied at its highest subprotective dose of 1 mg/kg) did not affect the protective action of valproate, carbamazepine, diphenylhydantoin and phenobarbital against maximal electroshock-induced seizures in mice. The presented results indicate that mGluR5 antagonists should not be considered as good candidates for add-on therapy of generalized seizures.

Dose-dependent up-regulation of rat pulmonary, renal, and hepatic cytochrome P-450 (CYP) 1A expression by nicotine feeding.

In a previous study in which a single 2.5 mg/kg (15.4 micromol/kg) s. c. dose of nicotine effected a transient, lung-specific induction of cytochrome P-450 (CYP) 1A1 in the rat, a dose-response study and assessment of the lung specificity of the induction was limited by toxicity of the acute parenteral nicotine exposure. In the present study, we examined the dose-CYP1A1/2 induction response relationship and the tissue specificity of the induction by orally administered nicotine, which lacks the toxicity of the parenterally administered drug. Nicotine, administered in a nutritionally balanced liquid diet, at a level of 20 (low), 60 (medium), or 200 (high) mg/kg of diet, induced CYP1A1 in the lung and kidney in a dose-dependent manner and in the liver at the high nicotine dose only, whereas CYP1A2 was induced in the liver dose-dependently and in the kidney at the high nicotine dose only. The high nicotine dose up-regulated mRNA level in the three tissues examined, but with the lung being the most responsive to the up-regulation. Induction of the CYP1A1-preferential activity ethoxyresorufin O-deethylase by the low, medium, and high nicotine diets was 1.9-, 4.9-, and 21.6-fold, respectively, in the lung, 1.4-, 1.7-, and 15.9-fold, respectively, in the kidney, and 1.7-, 2.9-, and 5.1-fold, respectively, in the liver. Similarly, albeit to lower extents, the dietary alkaloid induced the CYP1A2-preferential activity methoxyresorufin O-demethylase in all three tissues dose-dependently. Plasma nicotine concentration correlated neither with the dietary nor intake dose of the alkaloid nor with tissue levels of CYP1A, especially with the high-dose diet. Plasma nicotine levels at which CYP1A induction was maximal were comparable to those reported in smokers, suggesting that nicotine may induce CYP1A1 in humans.

Differential effects of monoaminergic agonists on alcohol intake in rats fed a tryptophan-enhanced diet.

The goal of the present study was to determine if enhancement of tryptophan levels in a nutritionally balanced liquid diet would affect alcohol intake in a two-bottle choice procedure. Furthermore. the monoaminergic agonists amphetamine, phentermine (dopaminergic- and noradrenergic-releasing drugs), and fenfluramine (a serotonin releaser) were administered to determine if these drugs reduced alcohol intake in animals fed the tryptophan-enhanced diet compared to those fed an alcohol-containing diet without added tryptophan. Amphetamine 0.5 and 2 mg/kg and phentermine 4 mg/kg selectively reduced alcohol intake in animals fed the tryptophan-enhanced diet; higher doses also reduced alcohol intake in animals fed the control alcohol diet. Three hours after drug administration, phentermine 2 and 4 mg/kg produced increases in consumption of the nonalcoholic diet in animals fed the control diet without affecting consumption in animals fed the tryptophan-enhanced diet. Finally, animals in the tryptophan-enhanced group gained less weight than those animals fed an identical diet without the added tryptophan. Neurochemical analysis revealed that the tryptophan-fed groups showed increased 5-HIAA concentrations and serotonin turnover in the striatum. hypothalamus, and frontal cortex compared to animals fed the control diet. The tryptophan-alcohol group also showed almost double the tryptophan levels in the hypothalamus compared to the tryptophan-isocaloric group. These results indicate that, whereas increasing tryptophan levels by itself was not sufficient to alter consumption of an alcohol-containing diet, the administration of monoaminergic agonists significantly interacted with tryptophan in a dose-dependent manner to reduce intake of an alcohol-containing diet without reducing intake of an isocaloric diet.

Efficacy of providing nicotine in a liquid diet to rats.

To determine if rats would consume nicotine at psychoactive levels, a nutritionally balanced diet with 0, 20, 60, or 200 mg of nicotine tartrate per kg of diet was provided. Diet consumption and body weight differences were recorded for 14 days after which, following 16 hr of withdrawal, animals were given access to a two-bottle choice of the previously presented diet and a nicotine-free diet. Spontaneous horizontal motor activity was recorded 8, 16, and 24 hr after withdrawal. By Day 14, all animals showed a significant increase in diet consumption and significant weight gain compared to Day 1. Animals consumed an average of 2.1, 6.8, or 19.5 mg/kg/day of nicotine on the low, medium, and high-nicotine diets, respectively. However, animals receiving the high-nicotine diet consumed less diet and gained less weight than the control, low, and medium nicotine groups. During only the first 4 hr of the two-bottle choice (16-20 hr postwithdrawal), the high-nicotine group consumed significantly higher amounts of nicotine base than the other groups, but also consumed more of the control diet during the first 2 hr. In a replicate experiment, animals receiving the medium-nicotine diet showed an increased consumption of the nicotine diet and increased preference for nicotine following a 14-day exposure compared to the control-fed animals and compared to a baseline preference test. Also, this group showed differences in locomotor activity consistent with other studies using an injection regimen or subcutaneuos pumps to induce dependence. Finally, animals in all three groups exhibited high plasma nicotine and cotinine (a major nicotine metabolite) levels. Because animals in all groups tolerated the diet well, gained weight, selected the nicotine diet in a choice test, and showed withdrawal symptoms, we conclude that the liquid diet proved to be a satisfactory method of inducing nicotine dependence in rats.

Dietary cholesterol and the activity of stearoyl CoA desaturase in rats: evidence for an indirect regulatory effect.

The effect of cholesterol on stearoyl CoA desaturase (SCD) was investigated. Previous work had shown that the addition of cholesterol to the diet of rats produced higher liver SCD activity compared to non-cholesterol-fed controls. We have confirmed this result and investigated the mechanism responsible for this cholesterol-induced higher SCD activity. Rats were fed either a 10% corn oil (CO) or a 10% corn oil/1% cholesterol (CO/CH) diet for 1, 3, or 7 days. SCD mRNA abundance was 3.3, 1.9, and 2.4 times greater in livers from CO/CH-fed animals after 1, 3, and 7 days, respectively. Northern hybridization of RNA from kidney, intestinal mucosa, heart, adipose, and liver demonstrated that cholesterol feeding specifically altered liver SCD mRNA. Liver esterified cholesterol content increased 27-fold with cholesterol feeding. This esterified cholesterol increase was accompanied by a proportionately greater increase in oleic acid compared to other fatty acids. These studies indicate that cholesterol does influence the expression of SCD specifically in the liver and suggest that the product, oleic acid, is preferentially esterified to cholesterol in the liver. Preliminary liver nuclear run-on assays from rats fed CO or CO/CH diets for 1 and 3 days indicate that transcription regulation is not a factor.

Alcohol utilization and dependence with special reference to protein level in a liquid diet for rats.

Experiments were carried out with a nutritionally balanced diet to test the response of rats to levels of ethanol between 0% and 6%, and to different levels and sources of protein and amino acid supplements in relation to alcohol utilization and withdrawal seizures. The high-calorie/high-carbohydrate liquid diet was well tolerated when the alcohol level was less than 30% of total calories, or 4.5% of diet. When alcohol was provided at 6% of diet, or 33% of total calories, growth and withdrawal seizure rates were negatively affected in comparison with the lower ethanol levels, even though ethanol consumption (in g/kg/day) was not different. The 6% alcohol diet was then altered through the addition of more protein calories, from 13% to 20%. This supplementation improved growth rate of the animals and reduced the rate of withdrawal seizures. The improvement from the additional protein was observed with both casein and soy protein, and was not attributable to any one or even several amino acids that might serve as transmitter precursors. A mixture of all essential amino acids representing the difference in amino acids between 13% and 20% casein protein calories was an effective as the equivalent amount of intact protein. The nonessential amino acids equivalent to 7% casein protein calories, when added to the 13% protein calories diet, increased the rate of withdrawal seizures, presumably by exacerbating the protein deficiency in the 13% protein diet. It was concluded that a 1000-1200 kcal/kg diet with 20% kcal from protein and 50% kcal from carbohydrate provides an optimal nutrient balance for efficient utilization of a 6% ethanol liquid diet for rats.

Amphetamine and fenfluramine suppress ethanol intake in ethanol-dependent rats.

Alcohol intake or preference for alcohol has been attributed to concomitant dopamine and serotonin dysfunction in rats. Amphetamine and fenfluramine, administered alone, have been shown to reduce food and fluid intake as well as alcohol consumption while acute coadministration of these agents has been shown to suppress audiogenic seizure in rats withdrawn from alcohol. The present study was designed to assess the effectiveness of chronic amphetamine and fenfluramine coadministration on reducing alcohol intake. Chronic coadministration of amphetamine (2 mg/kg) and fenfluramine (8 mg/kg) reduced alcohol consumption during choice trials in both alcohol-dependent and alcohol-nondependent rats while not affecting water intake. The findings indicate that coadministration of amphetamine and fenfluramine, a treatment effective in reducing alcohol withdrawal seizures, also selectively attenuates alcohol consumption.

Diet composition, alcohol utilization, and dependence.

Experiments were carried out in which a nutritionally balanced liquid diet previously used in this laboratory was modified as to total calorie content and high or low carbohydrate and fat concentration. Ethanol was added at 4.5% and 6.2% of diet weight and provided either 27% or 34-37% of total calories depending upon the changes in nutrient content. Measurements included 8-day food/calorie and ethanol consumption, plasma ethanol level, liver alcohol dehydrogenase (ADH) activity, and rate of audiogenic-induced withdrawal seizures. The original liquid diet with 4.5% ethanol was consumed in significantly lesser amounts than the alcohol-free diet, and essentially no body weight gain occurred, regardless if the major nonalcohol, nonprotein calorie source was fat or carbohydrate. When the calorie content of the diet was boosted through the addition of extra carbohydrate or fat (at the expense of water), appreciable weight gain was noted; in the case of the higher calorie diet boosted with more carbohydrate (maltodextrin) calories, growth was similar to that observed on the alcohol-free control diet. On this latter diet ethanol calories appeared to be utilized close to their theoretical value of 7 kcal/g. Blood alcohol levels were significantly higher on the lower calorie diets and were lowest on the high-calorie, high-carbohydrate, 4.5% ethanol diet. This diet also allowed for the lowest rate of withdrawal seizures despite an ethanol intake that was as high as on the lower calorie diets. Essentially, no differences were noted among ADH activities for the dietary treatments studied and thus, did not explain the differences observed among blood ethanol levels. When the alcohol concentration in the high-carbohydrate, high-calorie diet was raised to 6.2% from 4.5% to provide 34% of total calories, the rats responded by decreasing their food (and alcohol) intake to the same level as did the animals receiving a much lower calorie diet, but with 37% of caloric alcohol content. This suggests that at a diet alcohol concentration of 34-37%, one or more nutrient metabolites become limiting in the utilization of ethanol, resulting in food intake adjustments that maintain similar amounts of alcohol consumption.

Simultaneous changes in striatal dopamine, serotonin, and metabolites after withdrawal seizures in rats from dependence on alcohol.

Ethanol dependence was achieved in male, Long-Evans rats after 8 days on a balanced liquid diet that supplied 4.5% ethanol. After 1-h access to a solution of 10% ethanol (95%)/5% sucrose, the rats were deprived of food, water, and ethanol for 9 h. Following 30-s key jingling, about 80% of the animals exposed to ethanol experienced tonic-clonic seizures. Neurochemical analyses of striatal tissues revealed a significant (p < 0.05) increase in dopamine (DA) and a significant decrease in serotonin (5-HT) in the ethanol-exposed rats that had seizures compared to control rats. Homovanillic acid concentrations of the ethanol-treated rats with seizures were significantly higher than the levels found in ethanol-treated animals that had experienced no seizures. Daily average ethanol intake of the rats that had seizures vs. those that did not was almost the same at 16 g/kg/day. The findings indicate that rats experiencing ethanol withdrawal-induced seizures manifest opposite alterations in dopaminergic and serotoninergic activity compared to controls. The present results do not reveal if the striatal changes are caused by ethanol rather than by the seizures.

Novel synergistic treatment of ethanol withdrawal seizures in rats with dopamine and serotonin agonists.

A recent observation in this laboratory of a simultaneous increase in striatal dopamine and a decrease in serotonin in ethanol-dependent rats during ethanol withdrawal prompted studies with combined dopaminergic + serotoninergic agonists to stop withdrawal seizures. Amphetamine (2 mg/kg) + fenfluramine (8 mg/kg) given jointly, but not separately, prevented ethanol withdrawal seizures as effectively as benzodiazepines (chlordiazepoxide), the current drugs of choice. The combination of amphetamine and fenfluramine, unlike chlordiazepoxide, significantly reduced intake of ethanol during and immediately following ethanol withdrawal.

Dietary fat ratios and liver plasma membrane lipid composition.

Male Sprague-Dawley weanling rats were fed isocaloric diets consisting of 10% (by wt) fat. The six groups differed in the ratio of corn oil and butter fat present in the diets such that: 10C, 10% corn oil (C); 8C2B, 8% C/2% butter fat (B); 6C4B, 6% C/4% B; 4C6B, 4% C/6% B; 2C8B, 2% C/8% B; and 10B, 10% B. Liver plasma membranes were analyzed for fatty acid composition and cholesterol/phospholipid molar ratio. The 18:2n-6 content was constant in the 10C and 8C2B diets and then decreased linearly through the 2C8B diet. The 20:4n-6 and 18:1n-9 contents were constant except in the 10B diet, in which a significant decrease and increase, respectively, were observed. The cholesterol/phospholipid molar ratio increased between the 10C and 6C4B diets and subsequently (4C6B and 10B diets) remained constant. This data indicates that changes in n-6 fatty acid content in the liver plasma membrane are directly related to dietary intake only for 18:2n-6. Arachidonic acid content in the membrane is maintained at a constant level until the linoleic acid content of the diet is reduced to 0.5% of calories. It also indicates that the cholesterol content of the membrane becomes saturated and does not increase with increasing concentrations of saturated fat in the diet.