Kalervo Kiianmaa

Targeted use of naltrexone without prior detoxification in the treatment of alcohol dependence: a factorial double-blind, placebo-controlled trial

Several studies have shown the opioid antagonist naltrexone to be effective when combined with psychosocial therapies for the treatment of patients who are dependent on alcohol with fixed medication and time (12 weeks). In this study, 121 nonabstinent outpatients with alcohol dependence (DSM-IV) were treated with sessions of cognitive coping skills (N = 67) or supportive therapy (N = 54) and either naltrexone 50 mg/day (N = 63) or placebo (N = 58) daily for the first 12 weeks and thereafter for 20 weeks only when craving alcohol (i.e., targeted medication) in a prospective one-center, dual, double-blind, randomized clinical trial. The dropout rate for all subjects was 16.5% during the first 12-week period and approximately twice that level by the end of the study. There were no significant group differences in study completion and therapy participation rates. After the continuous medication (12 weeks), the coping/naltrexone group had the best outcome, and coping/placebo had the worst. This difference remained during the targeted medication period (the following 20 weeks). Naltrexone was not better than placebo in the supportive groups, but it had a significant effect in the coping groups: 27% of the coping/naltrexone patients had no relapses to heavy drinking throughout the 32 weeks, compared with only 3% of the coping/placebo patients. The authors’ data confirm the original finding of the efficacy of naltrexone in conjunction with coping skills therapy. In addition, their data show that detoxification is not required and that targeted medication taken only when craving occurs is effective in maintaining the reduction in heavy drinking.

The alcohol-preferring AA and alcohol-avoiding ANA rats: neurobiology of the regulation of alcohol drinking.

The AA (alko, alcohol) and ANA (alko, non‐alcohol) rat lines were among the earliest rodent lines produced by bidirectional selection for ethanol preference. The purpose of this review is to highlight the strategies for understanding the neurobiological factors underlying differential alcohol‐drinking behavior in these lines. Most early work evaluated functioning of the major neurotransmitter systems implicated in drug reward in the lines. No consistent line differences were found in the dopaminergic system either under baseline conditions or after ethanol challenges. However, increased opioidergic tone in the ventral striatum and a deficiency in endocannabinoid signaling in the prefrontal cortex of AA rats may comprise mechanisms leading to increased ethanol consumption. Because complex behaviors, such as ethanol drinking, are not likely to be controlled by single factors, system‐oriented molecular‐profiling strategies have been used recently. Microarray based expression analysis of AA and ANA brains and novel data‐mining strategies provide a system biological view that allows us to formulate a hypothesis on the mechanism underlying selection for ethanol preference. Two main factors appear active in the selection: a recruitment of signal transduction networks, including mitogen‐activated protein kinases and calcium pathways and involving transcription factors such as Creb, Myc and Max, to mediate ethanol reinforcement and plasticity. The second factor acts on the mitochondrion and most likely provides metabolic flexibility for alternative substrate utilization in the presence of low amounts of ethanol.

Effect of ethanol on extracellular dopamine in the nucleus accumbens of alcohol-preferring AA and alcohol-avoiding ANA rats

The role of central monoamines in the genetically determined influences on voluntary ethanol consumption were examined by studying the extracellular levels of monoamines in the nucleus accumbens of the alcohol-preferring AA (Alko Alcohol) and alcohol-avoiding ANA (Alko Nonalcohol) rats with in vivo microdialysis. Dialysate samples for the assay of monoamines with small bore HPLC were collected from freely moving animals at 15 min intervals after administration of ethanol (0.5, 1, or 2 g/kg, i.p.). Ethanol significantly increased the extracellular levels of dopamine, DOPAC, and HVA, suggesting stimulation of dopamine release by ethanol, while the effect on 5-HIAA did not reach significance. No difference in the extent or time course of stimulation of dopamine release between the AA and ANA rats was found. The results could so far give no indication that the differential ethanol consumption by AA and ANA rats could be explained in terms of differences in ethanol-induced stimulation of dopamine release in the nucleus accumbens.

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.

Brain ethanol in AA, ANA, and Wistar rats monitored with one-minute microdialysis

A microdialysis system for measuring the ethanol concentration curve in the nucleus accumbens of the rat brain was developed and tested in three different rat lines (AA, ANA, and Wistar) after an intraperitoneal (IP) and an intragastric (IG) dose of 1.0 g/kg ethanol. The flow rate of the modified Ringer solution was set at 5 microliters/min; samples were taken every minute after ethanol administration and analyzed with headspace gas chromatography. After IP administration, the brain ethanol levels rose much more rapidly than tail blood ethanol levels in the same animals. The maximum brain ethanol level after IG administration was lower and occurred later, and were similar to the tail blood levels in AA and ANA rats. No clear difference between the lines was found after IP administration but there was some indication that ANA rats may absorb alcohol after IG intubation faster than AA or Wistar rats.

Enhanced extracellular glutamate and dopamine in the ventral pallidum of alcohol-preferring AA and alcohol-avoiding ANA rats after morphine

The purpose of the present study was to investigate the role of ventral pallidal opioidergic mechanisms in the control of ethanol intake by studying the effects of acute administration of morphine on the levels of GABA, glutamate, and dopamine in the ventral pallidum. The study was conducted using the alcohol-preferring Alko Alcohol (AA) and alcohol-avoiding Alko Non-Alcohol (ANA) rat lines that have well-documented differences in their voluntary ethanol intake and brain opioidergic systems. Therefore, examination of neurobiological differences between the lines is supposed to help to identify the neuronal mechanisms underlying ethanol intake, since selection pressure is assumed gradually to lead to enrichment of alleles promoting high or low ethanol intake, respectively. The effects of an acute dose of morphine (1 or 10 mg/kg s.c.) on the extracellular levels of GABA and glutamate in the ventral pallidum were monitored with in vivo microdialysis. The concentrations of GABA and glutamate in the dialyzates were determined with a high performance liquid chromatography system using fluorescent detection, while electrochemical detection was used for dopamine. The levels of glutamate in the rats injected with morphine 1 mg/kg were significantly above the levels found in the controls and in the rats receiving morphine 10 mg/kg. Morphine 10 mg/kg also increased the levels of dopamine. Morphine could not, however, modify the levels of GABA. The rat lines did not differ in any of the effects of morphine. The data suggest that the glutamatergic and dopaminergic systems in the ventral pallidum may mediate some effects of morphine. Since there were no differences between the AA and ANA lines, the basic hypothesis underlying the use of the genetic animal model suggests that the effects of morphine detected probably do not underlie the different intake of ethanol by the lines and contribute to the control of ethanol intake in these animals.

Effects of ethanol on the accumbal output of dopamine, GABA and glutamate in alcohol-tolerant and alcohol-nontolerant rats

Effects of ethanol on the accumbal extracellular concentrations of dopamine, as well as of the amino acid transmitters gamma-amino butyric acid (GABA), glutamate and taurine, were studied in the alcohol-insensitive (alcohol-tolerant, AT) and alcohol-sensitive (alcohol-nontolerant, ANT) rats selected for low and high sensitivity to ethanol-induced motor impairment. Ethanol (2 or 3 g/kg ip) enhanced the output of dopamine and its metabolites in freely moving rats of both lines as measured by in vivo microdialysis. The effect of ethanol on the metabolites of dopamine tended to be stronger in the ANT rats. The smaller dose of ethanol decreased the output of GABA only in the AT rats, whereas the larger dose of ethanol decreased the output of GABA in rats of both lines to a similar degree. Ethanol at the dose of 2 g/kg slightly, but statistically, significantly decreased the output of glutamate in rats of both lines, but the larger dose of ethanol decreased the output of glutamate only in the AT rats. Ethanol at the dose of 2 g/kg induced a small transient increase in the output of taurine within 2 h after its administration in rats of both lines, but the larger dose of ethanol was without significant effect. These results confirm the previous findings that ethanol suppresses the release of GABA more in the AT than ANT rats. Thus, among the neurotransmitter systems we studied, the effects of ethanol might be the most relevant on GABAergic transmission regarding the sensitivity towards ethanol. However, our findings suggest that glutamate is also involved in this respect.

Transcriptome analysis of frontal cortex in alcohol-preferring and nonpreferring rats

Although it is widely accepted that alcohol abuse and alcoholism have a significant genetic component of risk, the identities of the genes themselves remain obscure. To illuminate such potential genetic contributions, DNA macroarrays were used to probe for differences in normative cortical gene expression between rat strains genetically selected for alcohol self‐administration preference, AA (Alko, alcohol) and P (Indiana, preferring), or avoidance, ANA (Alko, nonalcohol) and NP (Indiana, nonpreferring). Among 1,176 genes studied, six demonstrated confirmable, differential expression following comparison of ethanol‐naive AA and ANA rats. Specifically, the mRNA level for metabotropic glutamate receptor 3 (mGluR3) was down‐regulated in the AA vs. ANA lines. In contrast, calcium channel subunit α2δ1 (cacna2d1), vesicle‐associated membrane protein 2 (VAMP2), syntaxin 1 (both syntaxin 1a and 1b; STX1a and STX1b), and syntaxin binding protein (MUNC‐18) mRNAs were found to be increased in frontal cortex following comparison of AA with ANA animals. Bioinformatic analysis of these molecular targets showed that mGluR3 and cacna2d1 fall within chromosomal locations reported to be alcohol‐related by the Collaborative Study on the Genetics of Alcoholism (COGA) as well as quantitative trait loci (QTL) studies. To determine further whether these differences were strain specific, the above‐mentioned genes were compared in ethanol‐preferring (P) and ‐nonpreferring (NP) selected lines. VAMP2 was the only gene that displayed statistically different mRNA levels in a comparison of P and NP rats. In conclusion, the altered cortical gene expression illuminated here would have the effect of altering neurotransmitter release in AA rats (compared with ANA rats). Such alterations, however, might not be a universal characteristic of all animal models of alcohol abuse and will also require further investigation in post‐mortem human samples.

Alcohol intake and ethanol intoxication in the rat: 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.

Dose-dependent decrease in glial fibrillary acidic protein-immunoreactivity in rat cerebellum after lifelong ethanol consumption

The effects of aging and lifelong ethanol consumption on astrocytic morphology and glial fibrillary acidic protein-immunoreactivity (GFAP-IR) in the cerebellar vermis obtained from ethanol-preferring Alko, Alcohol (AA) rats were analyzed by using computer-assisted image analysis. The ethanol-consuming animals (both male and female) were given ethanol (10%-12%, vol./vol.) as the only available fluid for 21 months (3-24 months), whereas the young (3 months) and the old (24 months) controls received water. In the male rats, but not in the female rats, an age-related decrease in GFAP-IR was found in folia II, VII, and X of the molecular layer, and in turn, an age-related increase was found in folium X of the granular layer, indicating opposite changes in GFAP-IR for male rats due to aging in adjacent brain regions. In the female rats, 21 months of daily average ethanol consumption of 6.6 g/kg resulted in decreased GFAP-IR in folium VII of the molecular layer, and the decrease in cerebellar GFAP-IR correlated with the average daily ethanol intake (r=-.886, P=.019) when folia II, IV, VII, and X were analyzed together. No effect of ethanol on GFAP-IR was detected in the granular layer or in the central white matter of the female rats. There was no change in GFAP-IR in any of the three cerebellar layers of the male rats with average daily ethanol consumption of 3.2 g/kg. These results indicate that the Bergmann glial fibers are the GFAP-expressing structures of the cerebellum most sensitive to moderate-to-heavy chronic ethanol exposure and that this effect is dose dependent.