Lifelong ethanol consumption enhances the age-related changes in rat sympathetic neurons.

The effects of aging and chronic ethanol administration on the histochemical and morphometric features of rat superior cervical ganglion were studied in a rat strain selected for voluntary alcohol consumption. Ethanol was administered to the experimental group ad libitum (10% v/v in drinking water) from 3 months to 28 months of age, the average ethanol intake being 6.4-5.4 g/kg per day. The sympathetic neurons of the ethanol consuming rats showed several signs of enhanced degeneration, e.g. decreased neuronal packing density, increased amount of age-pigment and decreased intensity of catecholamine histofluorescence and tyrosine hydroxylase immunoreactivity. The results may indicate a selective vulnerability of peripheral sympathetic neurons rather than a universal accelerated aging due to chronic ethanol exposure.

Interaction of aging and lifelong ethanol ingestion on ethanol-related behaviors and longevity.

The interactions of aging and long-term voluntary ethanol consumption were studied in the alcohol-preferring AA (Alko Alcohol) rats. The mean daily ethanol intake was 6.45 +/- 0.31 g/kg/day (mean +/- SE) at the beginning of the exposure at 3 months of age. The control animals were given only food and water ad libitum. There was no difference in survival or weight gain between the control and ethanol groups. When tested for voluntary ethanol intake at the age of 24 months, the rats in the ethanol group consumed significantly more ethanol than the controls. The two groups did not differ in ethanol-induced motor impairment, sleep-time, or hypothermia, nor in the rate of ethanol elimination. The 24-month-old animals, however, showed higher sensitivity to ethanol than the 3-4-month-old rats in the sleep-time test. It is concluded that the feeding regimen used in this study did not produce any detectable interactions between ethanol and the aging processes in the AA rats.

Effects of chronic alcohol consumption on the expression of different NR1 splice variants in the brain of AA and ANA lines of rats.

Following chronic alcohol treatment alterations in N-methyl-D-aspartate receptor subunit 1 and 2 (NR1 and NR2), mRNA and protein levels have been reported. The NR1 gene undergoes alternative RNA splicing, resulting in eight splice variants, which were shown to differ in their sensitivity to alcohol. Here, we studied mRNA and protein levels of NR1 splice variants in alcohol-preferring (AA) and alcohol-nonpreferring (ANA) rat lines under basal conditions (alcohol-naive), and following chronic alcohol consumption. mRNA levels of three NR1 splice variants (NR1-1, NR1-2, NR1-4), and the protein levels of NR1 (NR1-1/NR1-2), and of NR1 alternative C-terminus (NR1-3/NR1-4) were determined in the hippocampus and nucleus accumbens by competitive RT-PCR and Western blot analysis, respectively. No significant differences in NR1 mRNA, or protein levels were found in the nucleus accumbens between the two rat lines under basal conditions, or following chronic alcohol consumption. In the hippocampus of alcohol-naive rats, the NR1-4 mRNA content was significantly higher in ANA compared to AA rats, however, no significant difference could be detected at the protein level. Following chronic alcohol consumption, the protein level of the NR1 alternative C-terminus (NR1-3/NR1-4) was significantly higher in AA rats compared to the corresponding control. Taken together, these results suggest: (i) brain site-specific alterations in NMDA receptor subunit composition occur following chronic alcohol consumption. (ii) In the hippocampus, NR1 splice variant mRNA levels differ between AA and ANA rats. (iii) The mRNA levels and protein levels of NR1 splice variants are differentially affected by chronic alcohol consumption.

Characteristics of ethanol tolerance in alcohol drinking (AA) and alcohol avoiding (ANA) rats.

The development of tolerance to ethanol was examined in two rat lines selected for high (AA) and low (ANA) ethanol consumption. In the first experiment, the acquisition of tolerance to the motor-impairment, hypothermic and hypnotic effects of ethanol produced by daily treatment with 5 g/kg ethanol for a period of 24 days was examined. Tolerance to these effects of ethanol was observed in the AA rats while marginal or no tolerance was demonstrated in the ANA rats. In the second experiment the development of rapid tolerance to the hypothermic and hypnotic effects of ethanol was examined. The hypothermic and hypnotic responses to IP injection of 3.5 g/kg ethanol were found to be attenuated in the AA but not the ANA rats by a single equivalent ethanol injection given 24 h earlier. These results suggest some relationship between the capacity to develop tolerance and voluntary ethanol intake.

Role of initial sensitivity and genetic factors in the development of tolerance to ethanol in AT and ANT rats.

The role of initial sensitivity and genetic factors in the development of tolerance to ethanol were examined in rats selected for low (AT) and high (ANT) sensitivity to the motor impairment effect of ethanol. Following chronic ethanol treatment (5 g/kg PO, daily for 20 days), the AT and ANT rats acquired tolerance to the motor impairment effect of ethanol at a similar rate. The AT rats, however, acquired tolerance to the hypothermic effect of ethanol at a higher rate than the ANT rats. Such ethanol treatment did not produce any metabolic tolerance to ethanol in these animals. Since there is no difference in the initial response to the hypothermic effect of ethanol between the AT and ANT rats, the observed differences in the rate of tolerance development might be related to a direct genetic factor. The similar rate of tolerance development to the motor impairment effect of ethanol between the two lines was attributed to an interaction between an indirect (initial sensitivity) and a genetic factor in tolerance development.

Intoxicating effects of lorazepam and barbital in rat lines selected for differential sensitivity to ethanol.

The motor impairing effects and plasma concentrations of barbital and lorazepam were studied in the alcohol tolerant (AT) and alcohol non-tolerant (ANT) rat lines developed for low and high sensitivity to motor impairment from ethanol. The mixed (M) line, from which the AT and ANT rats were derived, was also included in the study. Like ethanol, barbital and lorazepam impaired the performance of the ANT rats more than that of the AT rats. The motor performance of the M rats was relatively more impaired after barbital than after lorazepam administration at the same dose used in the AT and ANT rats. At the two latter time points (2.5 and 3.5 h) the sensitive ANT rats had significantly higher serum barbital concentrations than the AT rats. The serum barbital concentrations of the AT and ANT rats did not differ, however, at the two first time points (0.5 and 1.5 h) of the tilting plane tests, although the ANT rats were significantly more intoxicated. The concentrations of lorazepam in plasma do not explain the differential motor impairment either, since the sensitive ANT rats had lower plasma concentrations than the insensitive AT rats. The results, thus, suggest that the selection involved in the development of the AT and ANT lines has not been specific for ethanol. The results also support the idea that ethanol, barbiturates and benzodiazepines have some modes of action in common.

Antagonism of the behavioral effects of ethanol by naltrexone in BALB/c, C57BL/6, and DBA/2 mice.

The effects of naltrexone on the increase in locomotor activity induced by a low dose (1.35 g/kg IP) of ethanol and on the duration of loss of righting reflex after a high dose (3.5 g/kg) of ethanol were studied in BALB/c, DBA/2, and C57BL/6 mice. Ethanol increased locomotor activity in DBA and BALB mice, but not in C57BL mice. Naltrexone, at a dose of 0.1 mg/kg, antagonized the ethanol-induced increase in locomotion similarly in DBA and BALB mice. The duration of loss of righting reflex was, however, differentially affected in all three strains by naltrexone. The BALB mice affected in all three strains by naltrexone. The BALB mice were the most sensitive strain (1 mg/kg naltrexone significantly counteracted ethanol hypnosis), the C57BL mice were intermediate (8 mg/kg naltrexone required to antagonize this effect of ethanol), and the DBA mice were least sensitive (no effect evident even at the highest dose of 8 mg/kg) to naltrexone. Thus, naltrexone could antagonize the behavioral effects of a low and high dose of ethanol, but the three strains, which differ in their behavioral response to ethanol, also were differentially sensitive to the effect of naltrexone in reversing ethanol-induced hypnosis and ethanol-induced changes in locomotor activity.

Neonatal desipramine or zimeldine treatment causes long-lasting changes in brain monoaminergic systems and alcohol related behavior in rats.

To study the relationship between neonatal antidepressant administration, active (REM) sleep and adult alcohol-related behavior, rat pups were treated daily with 5 mg/kg desipramine (DMI) or 25 mg/kg zimeldine SC from the 6th to the 19th postnatal days. Movement sensitive mattress ("SCSB") measurements showed that zimeldine treatment suppressed active sleep throughout the whole treatment period, but DMI was more effective during the first 8 days than during the last treatment days. At the age of 70 days, the zimeldine-treated rats expressed a selective increase of some components of activity in the open field test, and the DMI rats had a higher defecation score compared to the controls. Furthermore, the zimeldine-rats responded with a decrease in ambulation in the open field to an alcohol dose which generally stimulates locomotion in rats. At the age of 3 months the DMI and zimeldine rats showed increased voluntary intake of 10% (v/v) alcohol. Measurement of brain monoamines revealed that the neonatal treatment with DMI or zimeldine interfered with the normal development and function of the monoamine neuronal systems: the concentrations of noradrenaline, dopamine and 5-hydroxytryptamine (5-HT), and their metabolites were altered in several brain regions. The results thus suggest that neonatal treatment with DMI or zimeldine suppresses active sleep and has an influence on later alcohol-related behavior, possibly due to a long-lasting defect in brain monoaminergic transmission.

Alcohol intake and ethanol intoxication in the rate: effect of a 6-OHDA-induced lesion of the ascending noradrenaline pathways.

The ascending noradrenaline (NA) pathways were lesioned by injecting 6-hydroxydopamine (6-OHDA) 16 micrograms/4 microliters bilaterally into the posterior mesencephalon in male Long Evans rats. Another group of rats was pretreated with protriptyline (25 mg/kg), a NA uptake blocking agent, 15 min before they received the intracerebral injections of 6-OHDA. The controls received the vehicle only. Spectrofluorimetric determination of the catecholamine concentrations in various parts of the brain revealed a marked degeneration of the ascending NA systems in the group receiving 6-OHDA. Unexpectedly, the DA systems were also affected by the 6-OHDA treatments. Three weeks after the operation the 6-OHDA group showed a transient increase in ethanol intake. In the tilting-plane test, ethanol (2 g/kg. i.p.) impaired the performance of the 6-OHDA-treated rats significantly more than that of the controls. In contrast, the hypothermic effect of ethanol (4 g/kg, i.p.) was significantly smaller in the lesioned rats. Furthermore, the catecholamine levels in various parts of the brain could be significantly correlated with both the extent of ethanol intoxication and the hypothermia. However, the duration of ethanol-induced narcosis (4 g/kg, i.p.) was affected by the present treatments. These results give further support for the view that the central NA neurons are important in the control of ethanol intake, and that they are involved in the expression of the acute effects of ethanol administration.

Effect of prior ethanol experience on dopamine overflow in accumbens of AA and ANA rats.

The purpose of this study was to investigate the effect of repeated ethanol administration on dopamine overflow in the nucleus accumbens of alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Nonalcohol) rats. Dopamine is a possible mediator of the reinforcing effects of ethanol, but it has previously been shown that ethanol-naïve alcohol-preferring AA and alcohol-nonpreferring ANA rats do not differ in their dopaminergic reaction to an intraperitoneal ethanol injection (0.5-2.0 g/kg), as assessed by measuring extracellular dopamine in the nucleus accumbens with in vivo microdialysis. Here a group of AA rats drank 10% (v/v) ethanol voluntarily-continual access for 5-15 days, limited access for 3 weeks-while a yoked group of AA rats and a yoked group of ANA rats received the same amount intragastrically by intubation. The rats were implanted with guide cannulas on the fourth week of limited access. Dopamine overflow was monitored in the microdialysis perfusate after 1 g/kg i.p. ethanol. The AA and the ANA rats that received ethanol non-contingently showed the same dopaminergic response to this as naïve animals have before. The group that had ingested the ethanol voluntarily showed, however, a significantly smaller increase in dopamine after 1 g/kg ethanol i.p. This suggests that the active behavior associated with obtaining the contingent drug may have an important impact on the reactions of the dopamine system to the drug, producing different results than when the same drug is administered by other routes. The hypothesis that dopamine mediates ethanol reinforcement in AA rats is not supported by the results.

The effects of nicotine on locomotor activity and dopamine overflow in the alcohol-preferring AA and alcohol-avoiding ANA rats.

The aim of the study was to investigate the importance of the interaction between central dopaminergic and cholinergic mechanisms for ethanol reinforcement. This was done by comparing the effects of nicotine on locomotor activity and release of dopamine in the nucleus accumbens of the alcohol-preferring Alko Alcohol (AA) and alcohol-avoiding alko non-alcohol (ANA) rats. Nicotine was administered acutely (0.25, 0.50 or 0.75 mg/kg, s.c.) or repeatedly once daily (0.5 mg/kg, s.c.) for 8 days. An acute dose of nicotine increased locomotor activity and the extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) measured with in vivo microdialysis suggesting stimulation of dopamine release by nicotine. No difference in the stimulation of locomotor activity or in the increase in the extracellular concentrations of dopamine or its metabolites by nicotine was found between the rat lines. The concentrations of nicotine in the plasma were also identical. The rats treated repeatedly with nicotine showed a progressive increase in locomotion. On the challenge day, 1 week after termination of nicotine or saline injections, rats previously treated with nicotine were activated more by nicotine than saline-treated rats. This behavioral sensitization was not accompanied by an increase in the amplitude of the neurochemical response to nicotine, but the duration of the increase in the levels of DOPAC was longer in the nicotine than saline-treated animals. The increases in locomotor activity and metabolite levels were, however, similar in both rat lines. These data suggest that differences in the interaction of central dopaminergic and cholinergic mechanisms probably do not contribute to the difference in ethanol self-administration between the AA and ANA rat lines.

5-Hydroxytryptamine neurons and the sleep-wakefulness cycle. Effects of methergoline and zimelidine.

Methergoline, a 5-hydroxytryptamine (5-HT) receptor blocking agent, produced a significant decrease in the number of slow wave sleep 2 (SWS 2) and paradoxical sleep (PS) episodes and an increase in the length of wakefulness (W) episodes. Zimelidine, a specific 5-HT uptake blocking agent, produced a significant reduction of the number of PS episodes and an increase in the number of spisodes spent in W and slow wave sleep 1 (SWS 1), the total time spent in SWS 1 being increased. The findings demonstrate that increases and decreases of 5-HT receptor activity will produce differential effects on SWS 2 events and on W mechanisms.

Drinking habits and prevalence of heavy drinking among primary health care outpatients and general population.

AIMS: To identify the target group for brief alcohol intervention in primary health care and to compare the prevalence of heavy drinking in two different primary health care populations and the general population in the same geographical area. DESIGN: Drinking data were collected from outpatients of primary health care by a questionnaire containing the CAGE test and quantity-frequency alcohol consumption questions and from a sample of the general population by a telephone survey, including the CAGE. The index of heavy drinking was for men three, and for women two, affirmative answers in CAGE which though not specifically a consumption questionnaire is a good marker of heavy drinking. SETTING: Two different primary health care populations (primary health care clinic and occupational health care clinic) and the general population in a Finnish health care area. PARTICIPANTS: Consecutive 1861 primary health care clinic and 2942 occupational health care clinic outpatients and 544 randomly selected adults in the general population, contacted by telephone. FINDINGS: The primary health care clinic patients drank significantly more per occasion than the patients of the occupational health care clinic (75 vs. 66 g. in men; 33 vs. 27 g. in women) and fewer times per week (0.8 vs. 0.9 in men; 0.5 vs. 0.6 in women). The patients in the primary health care clinic also reported drinking more per week (76 vs. 67 g. in men; 23 vs. 19 g. in women); among women the difference was significant. Among men the prevalences of heavy drinking in the primary health care clinic, occupational health care clinic and general population were 20%, 17% and 16%, respectively (p > or = 0.05). Among women the corresponding figures were 9%, 6% and 13% (p < 0.05). CONCLUSIONS: The high prevalence of heavy drinking found in the study confirms the importance of brief intervention by general practitioners. The study also indicates that prevalence and drinking habits depend on the type of clinic and heavy drinkers in general may not be over-represented in primary health care. This study raises the question, especially among women, of how to reach and to provide health advice to those heavy drinkers who do not attend primary health care facilities.

5-Hydroxyindoleacetic acid and 5-hydroxytryptophol levels in rat brain: effects of ethanol, pyrazole, cyanamide and disulfiram treatment.

The two serotonin metabolites 5-hydroxyindoleacetic acid (5HIAA) and 5-hydroxytryptophol (5HTOL) were measured in two regions of rat brain (pons medulla and diencephalon) using a gas chromatographic-mass spectrometric (GC-MS) method. Acute ethanol intoxication effected an elevation of 5-hydroxytryptophol levels, while 1 week of treatment with ethanol appeared to have no effect on either metabolite when measured 24 h after the last dose. Disulfiram and cyanamide treatment produced an approximately 2-fold increase in 5-hydroxytryptophol and a slight reduction in 5-hydroxyindole-acetic acid. Pyrazole treatment produced an increase in both metabolites. This effect was, however, counteracted by the simultaneous administration of ethanol.

Alcohol intake, ethanol-induced narcosis and intoxication in rats following neonatal 6-hydroxydopamine or 5, 7-dihydroxytryptamine treatment.

Newborn rats were treated with 5,7-dihydroxytryptamine (5,7-HT; 2 x 100 mg/kg s.c., 24 h interval) after pretreatment with desipramine (20 mg/kg s.c.) for depletion of brain 5-hydroxytryptamine (5-HT) or with 6-hydroxydopamine (6-OHDA; 3 x 100 mg/kg s.c., 24 h interval) for selective reduction of brain noradrenaline (NA). The 5,7-HT treatment resulted in a 53% reduction in endogenous 5-HT in the cerebral cortex and a 60% increase in the pons-medulla when determined in adult rats. The 5-HT content in the midbrain was not affected. Endogenous NA in the 6-OHDA treated animals was selectively reduced by 100% in the cerebral cortex, 35% in the midbrain and increased by 117% in the pons-medulla. No difference was found between the voluntary ethanol selection of these groups and that of the controls when measured at the age of 3 months. In a tilting-plane test, ethanol (2 g/kg i.p.) impaired the performance of the 6-OHDA treated rats significantly more than that of the controls. Moreover ethanol (4 g/kg e.p.) produced significantly longer narcosis in these rats. In contrast, the 5,7-HT treated rats were not affected significantly more than the controls in these tests. These results suggest that catecholamine neuronal systems interact with the expression of alcohol intoxication.

Effects of repeated cocaine treatment on striatal dopamine release in alcohol-preferring AA and alcohol-avoiding ANA rats.

Modulation of striatal dopamine (DA) release by acute or repeated cocaine treatment was studied in the nucleus accumbens and caudate-putamen of alcohol-preferring (AA, Alko Alcohol) and alcohol-avoiding (ANA, Alko Non-Alcohol) rats. Cocaine (5-10 mg/kg i.p.) was administered daily for 4 days and the concentrations of extracellular DA measured by in vivo microdialysis on days 1 and 4 in the freely moving rats. The first administration of cocaine increased DA concentration similarly in rats of both lines in both the nucleus accumbens and caudate-putamen. On the 4th day, the effect of cocaine was significantly larger in the nucleus accumbens of AA than in that of ANA rats, whereas no such enhanced effect of cocaine was found in the caudate-putamen of either line. The results suggest that mesolimbic DA release in response to cocaine is sensitized more readily in AA than in ANA rats, which would not only render the former more susceptible to alcohol, but to other drugs of abuse, and might explain our previous findings that AA rats are more susceptible to psychomotor sensitization than ANA rats.

Decreased levels of muscarinic receptors in bladders from the alcohol preferring rat line.

The Bmax for [3H]QNB binding in the bladders of alcohol preferring (AA) rats was only approximately 60% of that in the alcohol non-preferring (ANA) rats. No significant change in Bmax for [3H]QNB binding in bladder was observed between alcohol insensitive (AT) and alcohol sensitive (ANT) rats. No significant change in Kd for [3H]QNB binding in bladder was observed between the four different rat lines studied. Therefore, alcohol preference but not sensitivity is associated with a decrease in muscarinic receptor density in the rat bladder. Because all of the rats used in this study were ethanol-naive, the decrease in muscarinic receptor density in the bladders of alcohol preferring rats is associated with genetic factors inherent to this rat line. Further studies are needed to determine if these observations are tissue specific or specific to the m2 subtype, which predominates in the rat bladder.

Opioid propeptide mRNA content and receptor density in the brains of AA and ANA rats.

Recent evidence has indicated an association between the rewarding effects of ethanol intake and endogenous opioid activity. The present studies examine the presence of differences in opioid peptide mRNA content and mu and kappa opioid receptor densities, between ethanol naive AA and ANA rats bred selectively for their high and low alcohol consumption, respectively. In situ hybridization was used to compare the content of proopiomelanocortin, proenkephalin and prodynorphin mRNA in distinct brain regions known to be involved in the reinforcing properties of addictive drugs, between rats from each line. Results indicated that AA rats had a significantly greater content of proopiomelanocortin mRNA in the arcuate nucleus of the hypothalamus, of proenkephalin mRNA in the prefrontal cortex and of prodynorphin mRNA in the mediodorsal nucleus of the thalamus (p < or = .05). Receptor autoradiography was performed using 3H-labeled ligands specific for mu and kappa opioid receptors. AA rats were found to have a greater density of mu opioid receptors in the shell region of the nucleus accumbens and prefrontal cortex, but a lower density of kappa opioid receptors in the ventromedial hypothalamus, compared to ANA rats. The present data demonstrate the presence of inherited differences in the activity of distinct components of the endogenous opioid system in some brain regions associated with the processes of reward and reinforcement; and as such, may play a role in determining differences in ethanol drinking between AA and ANA rats.

Decreased intoxicating effect of ethanol in rats after 6-hydroxydopamine-induced degeneration of ascending dopamine pathways.

Selective lesion of ascending dopamine pathways was made by injecting the neurotoxin 6-OHDA (8 microgram/4 microliter) bilaterally close to the nigro-striatal dopamine pathway of 18 male Long Evans rats. Similar injections of the vehicle were given to 10 control rats. Two months after the operation intoxication was measured in a tilting-plane test after an injection of ethanol (2 g/kg, IP). Ethanol impaired the performance of the 6-OHDA-treated rats significantly less than that of the controls. This finding suggests a role for the central dopamine neurons in the intoxicating effect of ethanol.

Effects of ethanol, barbital, and lorazepam on brain monoamines in rat lines selectively outbred for differential sensitivity to ethanol.

The acute effects of ethanol, barbital, and lorazepam on the synthesis and metabolism of brain monoamines were studied in the AT (Alcohol Tolerant) and ANT (Alcohol Nontolerant) lines of rats, which have been selected for differential motor impairment after ethanol administration. The ethanol-sensitive ANT rats are also more sensitive than the ethanol-insensitive AT rats to the motor impairment caused by barbital and lorazepam. Ethanol increased, whereas barbital and lorazepam decreased, the synthesis of catecholamines in several regions of the brain. Ethanol did not affect the formation of DOPAC, whereas barbital and lorazepam reduced it. Similarly, the accumulation of 5-HTP was increased after administration of ethanol, but was decreased after administration of barbital or lorazepam. Ethanol, barbital and lorazepam decreased the formation of 5-HIAA. The rat lines did not differ in any of these responses. Some differences could, however, be demonstrated between the AT and ANT rats in the effects of the three drugs on the levels of the brain monoamines. Although the importance of these differences in the differential sensitivity to these drugs between the two lines is difficult to determine, the role of central monoaminergic mechanisms cannot be excluded. These findings also suggest that the motor impairment induced by ethanol, barbiturates, and benzodiazepines is probably not primarily based on the monoaminergic systems.