Concentrations of ethanol in rebreathed air of rats: correlation with the discriminative stimulus effects of ethanol

We modified the method of Pohorecky and Brick (14) for determination of ethanol (ETOH) concentrations in rebreathed air of rats. Rats were injected with different doses (1-2 g/kg) of ETOH and both arterial blood and rebreathed air samples were collected at various time intervals (15-120 min) after administration. We found a very good correlation (r = .96) between ETOH concentrations in arterial blood and in rebreathed air; the blood/breath conversion factor (+/- SEM) was 3241 +/- 55. In the second part of the study, rats were trained to discriminate between IP administered ETOH (1.2 g/kg) and the saline vehicle (12 ml/kg). Training occurred 15 min after administrations. Once trained to reliably differentiate between ETOH/saline training sessions, different doses (0.6, 0.9 and 1.2 g/kg) of ETOH were examined at various time intervals (1, 7.5, 15, 30, 60, 120 and 240 min) after administrations on certain test days. The results indicated a good correlation (r = .65) between the discriminative stimulus effects of ETOH and the concentrations measured in rebreathed air. The behavioral effects as well as the concentrations of ETOH in rebreathed air have a fast onset. The peak occurred 7.5 min after injection, and both the stimulus effects and concentrations of ETOH were time- and dose-dependent.     

Lack of effects of Ca-acetyl homotaurinate on chronic and acute toxicities of ethanol in rats

In a previous study, it was shown that in rats Ca-acetyl homotaurinate suppresses or eliminates the high oral intake of ethanol induced by earlier chronic ethanol administration, and to a lesser degree the spontaneous ethanol intake of ethanol-naive rats. The present study examines the effect of the drug on the chronic and acute toxicity of ethanol as a possible mechanism in its alteration of the reinforcing properties of ethanol intake. In the first experiment, it was demonstrated that chronic administration of the drug (45 mg/kg per day) for 15 days, alone or combined with chronic intragastric administration of high doses of ethanol, did not significantly alter subsequent ethanol intake. In two other experiments, it was shown that the drug did not interfere with acute ethanol toxicity as tested by ethanol-induced hypothermia, motor impairment and taste aversion in ethanol-naive rats. It is concluded that the acute activity of Ca-AOTA on CNS mechanisms presumably involved in the state of tolerance-dependence, other than those concerned in ethanol-induced hypothermia, motor impairment and taste aversion, may explain its action on the reinforcing property of ethanol intake.     

Lever conditioned stimulus-directed autoshaping induced by saccharin-ethanol unconditioned stimulus solution: effects of ethanol concentration and trial spacing.

Two experiments were designed to evaluate whether brief access to a saccharin-ethanol solution would function as an effective unconditioned stimulus (US) in Pavlovian-autoshaping procedures. In these experiments, the insertion of a lever conditioned stimulus (CS) was followed by the brief presentation of a sipper tube containing saccharin-ethanol US solution. Experience with this Pavlovian-autoshaping procedure engendered lever CS-directed autoshaping conditioned responses (CRs) in all rats. In Experiment 1, the concentration of ethanol [0%, 2%, 4%, 6%, or 8% (vol./vol.)] in 0.1% saccharin was systematically increased within subjects across autoshaping sessions to evaluate the relation between a rat's drinking and lever pressing. In Experiment 2, the mean intertrial interval (ITI) duration (60, 90, 120 s) was systematically increased within subjects across autoshaping sessions to evaluate the effect of ITI duration on drinking and lever pressing. A pseudoconditioning control group received lever CS randomly with respect to the saccharin-ethanol US solution. In Experiment 1, lever-press autoshaping CRs developed in all rats, and the tendency of a rat to drink an ethanol concentration was predictive of the performance of lever-press autoshaping CRs. In Experiment 2, longer ITIs induced more lever CS-directed responding, and CS-US paired procedures yielded more lever CS-directed responding than that observed in CS-US random procedures. Saccharin-ethanol is an effective US in Pavlovian-autoshaping procedures, inducing more CS-directed responding than in pseudoconditioning controls receiving CS-US random procedures. More lever CS-directed responding was observed when there was more drinking of the saccharin-ethanol US solution (Experiment 1); when the CS and US were paired, rather than random (Experiment 2); and with longer mean ITI durations (Experiment 2). This pattern of results is consistent with the hypothesis that lever CS-directed responding reflects performance of Pavlovian-autoshaping CRs.     

Glutamate and N-methyl-d-aspartate binding sites in the guinea pig hippocampus: Ontogeny and effect of acute in vitro ethanol exposure

The objectives of this study were to characterize the ontogeny of the l-glutamate (glutamate) and N-methyl-d-aspartate (NMDA) binding sites in the developing guinea pig hippocampus, and to determine the effect of acute in vitro ethanol exposure on these binding sites. Specific [³H]glutamate binding and NMDA-sensitive [³H]glutamate binding were determined using a guinea pig hippocampal synaptic membrane preparation (HSMP). To characterize the ontogeny of the density (B_max) and affinity (K_d) of the glutamate and NMDA binding sites, saturation analysis was conducted on HSMP of guinea pigs at gestational day (GD) 50 (immature fetus; term, GD 68), GD 62 (mature, near-term fetus), postnatal day (PD) 13 (neonate), and PD > 60 (adult). To examine the effect of ethanol on the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 50, GD 62, PD 13, and PD > 60 was incubated with ethanol (0–100 mM), followed by determination of specific ['H]glutamate binding and NMDA-sensitive [³H]glutamate binding. To determine the effect of 50 mM ethanol on the B_max, and K_d of the glutamate and NMDA binding sites, HSMP of guinea pigs at GD 62 and PD > 60 was incubated with 0 or 50 mM ethanol followed by saturation analysis. The B_max, values of the hippocampal glutamate and NMDA binding sites were greater at GD 62 and PD 13 compared with GD 50 and PD > 60, but there was no change in the K_d of the binding sites throughout development. Ethanol did not alter hippocampal specific [³H]glutamate binding or NMDA-sensitive [³H]glutamate binding at any of the ages studied, and did not alter the hippocampal B_max or K_d of the glutamate or NMDA binding sites at GD 62 and PD > 60.     

Delta receptor antagonism, ethanol taste reactivity, and ethanol consumption in outbred male rats

Naltrexone, a nonspecific opioid antagonist, produces significant changes in ethanol responsivity in rats by rendering the taste of ethanol aversive as well as producing a decrease in voluntary ethanol consumption. The present study investigated the effect of naltrindole, a specific antagonist of delta opioid receptors, on ethanol taste reactivity and ethanol consumption in outbred rats. In the first experiment, rats received acute treatment of naltrexone, naltrindole, or saline followed by the measurement of ethanol consumption in a short-term access period. The second experiment involved the same treatments and investigated ethanol palatability (using the taste-reactivity test) as well as ethanol consumption. Results indicated that treatment with 3 mg/kg naltrexone significantly affected palatability (rendered ethanol more aversive, Experiment 2) and decreased voluntary ethanol consumption (Experiments 1 and 2). The effects of naltrindole were inconsistent. In Experiment 1, 8 mg/kg naltrindole significantly decreased voluntary ethanol consumption but this was not replicated in Experiment 2. The 8 mg/kg dose produced a significant increase in aversive responding (Experiment 2) but did not affect ingestive responding. Lower doses of naltrindole (2 and 4 mg/kg) were ineffective in altering rats' taste-reactivity response to and consumption of ethanol. While these data suggest that delta receptors are involved in rats' taste-reactivity response to ethanol and rats' ethanol consumption, it is likely that multiple opioid receptors mediate both behavioral responses.     

Absence of tolerance to the aversive stimulus properties of ethanol following oral ethanol self-administration

Depending on the situation, ethanol can serve as a reinforcer in one paradigm and an aversive stimulus in another. The relationships between the two stimuli are not clear, particularly the behavioural adaptation following chronic ethanol exposure. We report on two experiments using an oral-self administration (OSA) paradigm and a conditioned taste aversion (CTA) paradigm. Male Wistar rats were exposed to ethanol using either the OSA or the CTA paradigm, and the consequences were examined in the same groups of rats by performing the other corresponding experiment. Thus, sensitisation or tolerance to the respective stimulus properties of ethanol would be detectable. For OSA experiments, rats were presented, under a free-choice setting, tap water and an ascending series of ethanol concentrations (2–10%) for up to 4 days per concentration. The amounts of ethanol and water consumed in 23-h sessions were measured. For CTA, a two-bottle procedure was employed. Distinctively flavoured solutions (saccharin or saline) were paired with IP injections of either ethanol (1.5 g/kg) or saline (1 ml/kg). Tests for aversion were made after two pairings, when both solutions were presented simultaneously for 10 min. At low concentrations of ethanol, drinking solution consumption was high, decreasing gradually with increasing concentrations; however, daily intake of orally self-administered ethanol remained stable. No significant differences could be established between the two groups tested. Ethanol preference [EtOH/EtOH + H₂O] was attenuated in rats experienced with the CTA procedure before the OSA experiment. Injections of ethanol produced marked CTAs, even in rats that had consumed ethanol in the OSA experiment. The absence of tolerance to the aversive stimulus effects suggests that this stimulus property may not play a significant role in the consumption of ethanol.     

Classically conditioned ethanol stimulus control of a motor behavior

Rats with extensive unilateral 6-hydroxydopamine lesions of substantia nigra rotate (turn in circles) in response to administration of dopamine agonists. Here apomorphine administration, but not single or repeated administrations of ethanol, resulted in rotation in such lesioned animals. When apomorphine and ethanol were administered simultaneously to lesioned animals, rapid contralateral rotation resulted. After five of these conditioning trials in which ethanol and apomorphine were paired, ethanol alone elicited apomorphine-like rotation. Saline administration did not result in rotation in the conditioned animals, indicating that ethanol rather than the injection procedure exerted stimulus control over the behavior. Control animals which were treated five times with apomorphine and ethanol in an unpaired manner showed no conditioned rotation to ethanol.     

Discriminative stimulus effects of 5.6 mg/kg pregnanolone in DBA/2J and C57BL/6J inbred mice.

Neurosteroids represent a class of endogenous compounds that exert rapid, nongenomic effects through neurotransmitter receptor systems such as gamma-aminobutyric acid(A) (GABA(A)). Two neurosteroids, allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) and pregnanolone (3alpha-hydroxy-5beta-pregnan-20-one), possess anxiolytic and sedative properties and show substitution for ethanol, benzodiazepines, and barbiturates in drug discrimination assays. A previous study examining the discriminative stimulus effects of 10 mg/kg pregnanolone in DBA/2J and C57BL/6J mice showed pregnanolone's discriminative stimulus to be mediated primarily through GABA(A) positive modulation. This study examined the discriminative stimulus effects of a lower training dose (5.6 mg/kg) of pregnanolone in DBA/2J and C57BL/5J mice. Twelve male DBA/2J mice and 12 male C57BL/6J mice were trained to discriminate 5.6 mg/kg pregnanolone. GABA(A)-receptor positive modulators, neuroactive steroids, NMDA receptor antagonists, and 5-HT(3) receptor agonists were tested for pregnanolone substitution. In DBA/2J and C57BL/6J mice benzodiazepine, barbiturate, and GABAergic neuroactive steroids all substituted for pregnanolone. In the DBA/2J mice, NMDA receptor antagonists showed generalization to the discriminative stimulus cues of pregnanolone, an effect not seen in the C57BL/6J mice. 5-HT(3) receptor agonists and zolpidem failed to substitute for pregnanolone's discriminative stimulus in either strain. AlloTHDOC and midazolam were more potent in producing pregnanolone-like discriminative stimulus effects in DBA/2J mice. These results provide a comprehensive look at pregnanolone's discriminative stimulus effects in two commonly used strains of mice. The present data suggest GABA(A)-receptor positive modulation as the predominant receptor mechanism mediating the discriminative stimulus effects of pregnanolone. NMDA receptor antagonism was suggested in the DBA/2J mice and may represent a heterogenous cue produced by the lower training dose of pregnanolone.     

Lack of generalizability of sensitivity and specificity with treatment effects

In biomarker development, two types of summary measures are often used to describe marker accuracy. Positive and negative predictive values describe how well a marker predicts clinical states of interest, while sensitivity and specificity describe how well a marker discriminates between the two states. Insofar as predictive values depend heavily on the prevalence of the clinical states and sensitivity and specificity may not, sensitivity and specificity are preferred in early biomarker development.In many applications, an ideal property of a biomarker is fulfillment of the first Prentice criterion. Under this condition, predictive values do not depend on a covariate (such as treatment) because the biomarker captures all relevant information about the clinical state of interest. A similar condition can be defined for sensitivity and specificity which states that these measures do not depend on a covariate (e.g. treatment). This condition, which we refer to as the equal discriminatory accuracy (EDA) condition, is desirable because it allows sensitivity and specificity from one treatment setting (or covariate value) to be applied to a different setting. We demonstrate, however, that the Prentice condition and EDA are incompatible. Further, under a simple proportional hazards model for a time-to-event outcome, EDA will not be satisfied. We present numerical examples as well as examples of a potential marker in late-stage prostate cancer and another for cervical cancer screening. These results demonstrate that evaluating sensitivity and specificity within treatment (or other covariate) groups is necessary even when simple proportional hazards models or the Prentice criterion holds.     

Effects of ethanol on flash-evoked potentials of rats: lack of antagonism by naltrexone.

The present study examined the effects of ethanol and naltrexone hydrochloride (a nonselective opiate receptor antagonist) on flash-evoked potentials recorded from both the visual cortex (VC) and the superior colliculus (SC) of chronically implanted hooded rats. There were four treatment conditions administered on separate days: Either saline or naltrexone (10 mg/kg; volume of 1.0 ml/kg) was given 10 min before either saline or ethanol (2.0 g/kg; 20% ethanol solution in a volume of 1.26 ml/100 g). Evoked potentials were recorded 15 min after the intraperitoneal injections were completed. Animals were tested at 23.1 degrees C room temperature. In the VC, ethanol significantly decreased the amplitude of components N1, P3, and N3, whereas it increased the amplitude of P2. Components P1 and N2 were unaffected by ethanol treatment. The SC components P3 and N4 were reduced in amplitude by ethanol, but component P1 was not altered. Latencies of all components in both structures were increased by ethanol. Naltrexone alone did not significantly affect the potentials, nor did naltrexone pretreatment significantly alter the effects of ethanol on the potentials. Naltrexone produced a modest hypothermia of about 0.25 degrees C, whereas ethanol resulted in hypothermia of about 1.0 degrees C. Ethanol, either alone or in combination with naltrexone, significantly reduced body movement during the evoked-potential recording sessions. The results indicate that endogenous opioid systems do not play a major role in the acute effects of ethanol on flash-evoked potentials recorded from primary areas of the visual system.     

Cholecystokinin-induced satiation with ethanol: effects of lighting cycle and limited access procedures

Cholecystokinin (CCK) is a brain-gut neuropeptide and hormone previously shown to inhibit alcohol intake in water- or food-deprived rats. The effects of CCK and the phase of lighting cycle on alcohol intake in rats were investigated in a comparison of limited access and water-restriction procedures. The limited access procedure (LAP) is a recently developed technique for inducing free-choice alcohol consumption in nondeprived animals. Two groups of 12 male rats each were maintained in either normal or reversed 12:12 L:D lighting cycles and simultaneously given 40 minutes' access to 6% w/v ethanol and water in nonhome cages. After adaptation to this procedure, CCK octapeptide (0.5-16 micrograms/kg) was injected IP prior to access to fluids. During LAP, CCK reduced alcohol intake and increased water intake more potently in the dark phase. These effects of CCK were more reliable when the design was replicated, which suggests the importance of acquired expectancies for the development of CCK's actions. CCK more effectively reduced alcohol intake in LAP, than in a 23.3-h water-deprivation procedure for inducing alcohol intake in a 2-bottle choice test with water. However, CCK was less so effective in LAP, than in the water-deprivation procedure when alcohol was presented alone in a 1-bottle test. The alcohol satiation effect of CCK is independent of prior deprivation and not an artifact of thirst reduction, debilitation, or conditioned aversion, because CCK strongly increased water intake in the limited access procedure, and ethanol preference remained robust after experience with CCK. CCK may operate endogenously as a specific factor in satiation with ethanol.     

Autoshaping of ethanol drinking in rats: effects of ethanol concentration and trial spacing.

In two studies, we evaluated the effects of ethanol concentration and trial spacing on Pavlovian autoshaping of ethanol drinking in rats. In these studies, the brief insertion of an ethanol sipper conditioned stimulus (CS) was followed by the response-independent presentation of food unconditioned stimulus (US), inducing sipper CS-directed drinking conditioned responses (CRs) in all rats. In Experiment 1, the ethanol concentration in the sipper CS [0%-16% volume/volume (vol./vol.), in increments of 1%] was systematically increased within subjects across autoshaping sessions. Groups of rats received sipper CS-food US pairings (Paired/Ethanol), a CS-US random procedure (Random/Ethanol), or water sipper CS paired with food US (Paired/Water). In Experiment 2, saccharin-fading procedures were used to initiate, in the Ethanol group, drinking of 6% (vol./vol.) ethanol in 0.1% saccharin or, in the Water group, drinking of tap water in 0.1% saccharin. After elimination of saccharin, and across days, the duration of access to the sipper CS during each autoshaping trial was increased (5, 10, 12.5, 15, 17.5, and 20 s), and subsequently, across days, the duration of the mean intertrial interval (ITI) was increased (60, 90, 120, and 150 s). In Experiment 1, Paired/Ethanol and Random/Ethanol groups showed higher intake of ethanol, in terms of grams per kilogram of body weight, at higher ethanol concentrations, with more ethanol intake recorded in the Paired/Ethanol group. In Experiment 2, the Ethanol group drank more than was consumed by the Water group, and, for both groups, fluid intake increased with longer ITIs. Results support the suggestion that autoshaping contributes to sipper CS-directed ethanol drinking.     

Is ethanol a neurotoxin?: The effects of ethanol on neuronal structure and function

Although the psychiatric manifestations of heavy ethanol consumption have been well known for decades, studies on the structural changes at the level of the nerve cell which result from chronic ethanol administration are relatively recent. It is now well established that ethanol increases the fluidity of the neuronal membrane by changing the ratio of unsaturated to saturated fats in favour of the latter; in addition, the concentration of cholesterol is increased. These changes in lipid composition appear to be associated with the development of behavioural tolerance to the drug. The resultant change in membrane structure affects transport processes across the cell surface involving calcium and other electrolytes and the active transport of neurotransmitters such as the biogenic amines and GABA; there is evidence that neurotransmitter receptor function is also impaired as a consequence of the alteration in the membrane micro-environment brought about by chronic ethanol exposure. Such effects suggest that alterations in cellular function, and ultimately behaviour, are primarily the result of the changes in nerve membrane structure and function. The possible consequences of this with regard to the development of a therapy to counteract the neurotoxicity of ethanol are discussed.     

Endosulfan: Lack of cytogenetic effects in male rats

Summary Cytogenetic effects of endosulfan (Thiodan^R) a member of the chlorinated hydrocarbon insedticide was tested in male albino rats. The insecticide was administered orally to rats at 0, 11.00, 22.00, 36.60 and 55.00 mg/Kg daily for 5 days. The highest doses were associated with clinical signs of insecticide poisoning and death. Cytogenetic analysis of bone marrow cells and spermatogenial cells did not reveal any significant effect of the insecticides on chromosomes. The ratio of mitotic index and frequency of chromatid break in the two cell types had no correlation with the doses tested and was not very different from those of the control group.     

Effect of chronic maternal ethanol administration on glutamate and N-methyl-d-aspartate binding sites in the hippocampus of the near-term fetal guinea pig

The objective of this study was to determine the effect of chronic maternal administration of ethanol on hippocampal l-glutamate (glutamate) and N-methyl-d-aspartate (NMDA) binding sites in the near-term fetal guinea pig. Starting on gestational day (GD) 2, pregnant guinea pigs received one of the following oral treatments up to and including GD 62 (term, about GD 68): 4 g ethanol · kg maternal body weight⁻¹ · day⁻¹; isocaloric sucrose and pair-feeding; or water. Maternal blood ethanol concentration was determined at 1 h after the daily ethanol dose on GD 59. Fetuses were studied at GD 63 (mature, near-term fetus). Fetal body weight and brain weight were determined. The density (B_max) and affinity (K_d) of the glutamate and NMDA binding sites in the fetal hippocampus were measured using a radioligand membrane binding assay; saturation analysis was conducted on hippocampal synaptic membrane preparation (HSMP). Maternal blood ethanol concentration on GD 59 was 269 ± 111 (SD) mg/dl (59 ± 24 mM). There was no maternal or embryonic fetal lethality in any of the three treatment groups, and ethanol treatment did not affect maternal body weight gain compared with sucrose or water treatment. Fetal brain weight, but not body weight, was decreased in the ethanol treatment group compared with the sucrose and water treatment groups. The B_max values of the glutamate and NMDA binding sites were decreased in the ethanol treatment group compared with the sucrose and water treatment groups; there was no difference in the K_d values of the glutamate and NMDA binding sites among the three treatment groups. The data demonstrate that, in the guinea pig, chronic maternal administration of a binge-type ethanol regimen throughout gestation restricts brain growth and decreases the density of glutamate and NMDA binding sites in the hippocampus, as manifested in the mature fetus.     

Lead/ethanol interactions I: Rate-depressant effects

Adult male rats were exposed to a diet containing 500 ppm added lead as lead acetate (group lead-diet) or a control diet containing no added chemicals (group control-diet) for 61 days prior to commencing fixed-ratio 32 (FR 32) lever press training for water reinforcement. After steady state responding was achieved, all animals received serial administrations of acute doses of ethanol prior to the daily training session. Specifically, lead-diet and control-diet rats received i.p. injections of .25, .5, .75, 1.0, and 1.25 g/kg ethanol, in ascending order, alternating daily with injections of saline. The results revealed a dose-dependent rate-depressant effect, with higher doses of ethanol producing more behavioral suppression than lower doses for both groups. In addition, at the dose of 1.0 g/kg it was observed that the suppressive effects of ethanol on schedule-controlled responding were reduced among lead-treated animals relative to controls. These data are discussed in terms of lead-induced attenuation of the pharmacologic effects of ethanol.     

Combined effects of ethanol and garlic on hepatic ethanol metabolism in mice.

The combined effects of ethanol and components in fresh garlic on ethanol metabolism were investigated in the livers of mice. Male, 11-wk-old C3H/HeNCrj mice were intragastrically administered 2 g ethanol/kg body weight after being administered fresh garlic juice for 8 d (garlic group), and changes in the concentrations of ethanol, acetaldehyde and acetate in the serum, and changes in the activity of hepatic enzymes related to ethanol metabolism in mice were examined. The increases in the concentrations of acetaldehyde and acetate in the serum after ethanol administration tended to be diminished following garlic administration. The microsomal ethanol-oxidizing system (MEOS) in the livers of the garlic groups was significantly lower than that of the control microsomes at 2 h after ethanol administration. It therefore seems that the decrease of MEOS in hepatic microsomes caused a smaller increase in the acetaldehyde concentration in the serum of the garlic groups because cytosolic alcohol dehydrogenase showed no significant difference between the control and garlic groups. After ethanol administration, the content of cytochrome P-450 in the hepatic microsomes of the control groups increased, while that of the garlic groups did not change although cytochrome P-450 (CYP) 2E1 and 1A2 in the hepatic microsomes of the garlic groups increased. These results indicate that the induction of isozymes of cytochrome P-450 other than CYP 2E1 and 1A2 was inhibited following garlic administration. Cytosolic high Km and total aldehyde dehydrogenase (AIDH) in the liver of the garlic groups tended to be lower than those activities of the control groups at 1 and 2 h after ethanol administration. It therefore seems that the decreases of AIDH in the hepatic cytosols diminished the increase of acetate in the serum of the garlic groups after ethanol administration. These results suggest that the ethanol metabolism in the mouse liver is controlled by components in fresh garlic juice.