Gene expression in the hippocampus of inbred alcohol-preferring and -nonpreferring rats.

The hippocampus is sensitive to the effects of ethanol and appears to have a role in the development of alcohol tolerance. The objective of this study was to test the hypothesis that there are innate differences in gene expression in the hippocampus of inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats that may contribute to differences in sensitivity to ethanol and/or in the development of tolerance. Affymetrix microarrays were used to measure gene expression in the hippocampus of alcohol-naive male iP and iNP rats in two experiments (n=4 and 6 per strain in the two experiments). Combining data from the two experiments, there were 137 probesets representing 129 genes that significantly differed (P < or = 0.01); 62 probesets differed at P < or = 0.001. Among the 36% of the genes that were expressed more in the iP than iNP rat at this level of significance, many were involved in cell growth and adhesion, cellular stress reduction and anti-oxidation, protein trafficking, regulation of gene expression, synaptic function and metabolism. Among the 64% of the genes that had lower expression in the hippocampus of iP than iNP rats were genes involved in metabolic pathways, cellular signaling systems, protein trafficking, cell death and neurotransmission. Overall, the data indicate that there are significant innate differences in gene expression in the hippocampus between iP and iNP rats, some of which might contribute to the differences observed in the development of alcohol tolerance between the selectively bred P and NP lines.

Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats.

The neuropeptide Y (NPY) gene in rat chromosome 4 has been shown to play an important role in alcohol-seeking behavior. NPY knockout mice drink more alcohol than wild-type mice, implicating a link between NPY deficiency and high alcohol intake. This is supported by recent studies showing that intracerebroventricular injections of NPY reduce alcohol intake in both alcohol-preferring (P) and high alcohol-drinking rats. However, it is unknown which anatomical NPY systems are involved in alcohol preference. This study was designed to investigate whether there are innate differences in NPY mRNA in cerebral cortical areas, dentate gyrus (DG) of the hippocampus and medial habenular nucleus (MHb) between P and alcohol-nonpreferring (NP) rats, as these discrete brain regions are rich in NPY mRNA. [(33)P]-labeled 28-mer oligodeoxynucleotide probe was applied for the in situ hybridization study to detect the NPY mRNA, measured using quantitative autoradiography. This study revealed an absence of NPY mRNA in the MHb of P rats. We found that NPY mRNA was significantly lower in the DG of P rats than NP rats. This innate difference of NPY mRNA expression in the DG between P and NP rats is region specific. For example, in most of the cerebral cortical areas examined, an innate difference was not seen. Our study suggests that lower NPY gene expression in the DG and MHb of P rats may be factors contributing to some of the phenotypic differences observed between the P and NP lines of rats.

Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference.

The role of amygdaloid corticotropin-releasing factor (CRF) in alcoholism is not clear. Alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats are selectively bred for high alcohol preference, and have been considered suitable animal models for studying alcoholism. The CRF neurons in the central nucleus of the amygdala (CeA) of P rats and HAD rats were studied in comparison with those of their respective counterparts, namely, alcohol-nonpreferring (NP) rats and low alcohol-drinking (LAD) rats. Specifically, CRF-immunoreactivity (ir) in the CeA and paraventricular hypothalamic nucleus (PVN) was assessed using radioimmunohistochemical (RIH) assay in alcohol-naive P/NP rats, and HAD/LAD rats. Furthermore, CRF mRNA was examined using in situ hybridization in the CeA of P/NP rats. Anxiety levels were also evaluated using an elevated plus maze. Results of the present study showed that CRF-ir was significantly lower in the CeA of P rats than NP rats. Moreover, CRF mRNA in the CeA was also much lower in P rats than NP rats. Such differences were not seen in the PVN. Interestingly, those P rats exhibited higher anxiety than NP rats. In contrary, there were no innate differences of CRF-ir in both the CeA and PVN between HAD and LAD rats whose anxiety levels were similar. This study is consistent with the literature showing CRF knockout (KO) induces alcohol drinking, and central administrations of CRF reduce alcohol intake. Collectively, the present study suggests that reduced CRF gene expression in the CeA of P rats is associated with their alcohol preference and anxiety.

Long-lasting alterations of the mesolimbic dopamine system after periadolescent ethanol drinking by alcohol-preferring rats.

BACKGROUND: This study tested the hypothesis that ethanol consumption by alcohol-preferring (P) rats during the periadolescent period causes persistent alterations in the mesolimbic dopamine (DA) system. After ethanol drinking during periadolescence, P rats were examined for alterations in basal locomotor activity, changes in extracellular DA levels and extraction fraction in the nucleus accumbens (NAc) by using no-net-flux (NNF) microdialysis, and changes in the response of the mesolimbic DA system to ethanol. METHODS: Male P rat pups were given 24-hr free-choice access to 15% (v/v) ethanol from postnatal day (PD) 30 through PD 60. On PD 70, rats were assessed for locomotor activity. On PD 70 to 80, rats were implanted with bilateral guide cannulas aimed above the NAc. After at least 5 days, microdialysis probes were inserted bilaterally; on the following day, NNF microdialysis experiments were conducted. On the day after the NNF experiment, conventional microdialysis experiments were conducted to measure extracellular levels of DA in response to intraperitoneal injection of saline or ethanol 2.5 g/kg. RESULTS: Compared with the ethanol-naive group, ethanol drinking by P rats during periadolescence did not alter basal locomotor activity, nor did it alter the basal extracellular concentration of DA. There was, however, a significant increase in the extraction fraction of DA of ethanol-drinking animals relative to the controls (57.4 +/- 2.7% and 45.8 +/- 2.3%, respectively). Additionally, compared with controls, P rats with exposure to ethanol during the periadolescent period showed a prolonged increase in the extracellular levels of DA after a challenge dose of ethanol. CONCLUSIONS: The results of the microdialysis experiments suggest that periadolescent ethanol drinking by P rats increases basal DA neurotransmission (as indicated by higher DA clearance while maintaining the same extracellular DA concentrations) and prolongs the response of DA neurotransmission to ethanol.

Low-dose stimulatory effects of ethanol during adolescence in rat lines selectively bred for high alcohol intake.

BACKGROUND: The low-dose stimulatory effect of ethanol (EtOH) in rats has been hypothesized to reflect its hedonic effects and to be associated with a genetic predisposition toward high alcohol preference. To test the hypothesis that phenotypes associated with high alcohol preference in adulthood are also present in adolescent rats at the time of onset of alcohol drinking, the current study examined the effects of EtOH on locomotor activity (LMA) during adolescence in lines of rats selectively bred for divergent alcohol intakes. METHODS: Subjects were adolescent (31-40 days of age) rats from the alcohol-preferring (P) and -nonpreferring (NP) lines and from the high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) replicate lines. On day 1, all subjects (n = 8-10/line/gender/dose) received intraperitoneal saline injections and were placed in the activity monitor for 30 min. On day 2, subjects received intraperitoneal saline or 0.25, 0.50, 0.75, 1.0, or 1.5 g EtOH/kg. RESULTS: The LMA of male and female P rats was increased with low doses (0.25-0.75 g/kg) and decreased at the highest dose (1.5 g/kg) of EtOH. Similar effects were observed with low doses of EtOH on the LMA of HAD-1 and HAD-2 rats. None of the EtOH doses stimulated LMA in the NP, LAD-1, or LAD-2 rats, although all of the low-alcohol-intake lines of rats showed decreased LMA at the highest dose of EtOH. Only the P rats among the high-alcohol-consuming lines of rats showed decreased LMA at the highest dose of EtOH. CONCLUSION: Selective breeding for high alcohol consumption seems to be associated with increased sensitivity to the low-dose stimulating effects of EtOH and reduced sensitivity to the high-dose motor-impairing effects of ethanol. The expression of these phenotypes emerges during adolescence by the age of onset of alcohol-drinking behavior.

Ethanol drinking and deprivation alter dopaminergic and serotonergic function in the nucleus accumbens of alcohol-preferring rats.

The alcohol deprivation effect is a temporary increase in the intake of, or preference for, ethanol after a period of deprivation that may result from persistent changes in key limbic regions thought to regulate alcohol drinking, such as the nucleus accumbens. The present study tested the hypothesis that chronic alcohol drinking under continuous 24-h free-choice conditions alters dopamine and serotonin neurotransmission in the nucleus accumbens and that these alterations persist in the absence of alcohol. Using the no-net-flux microdialysis method, the steady-state extracellular concentration (point of no-net-flux) for dopamine was approximately 25% higher in the adult female alcohol-preferring P rats given prior access to 10% ethanol, even after 2 weeks of ethanol abstinence, compared with the P rats gives access only to water. However, the extracellular concentration of serotonin was approximately 35% lower in animals given 8 weeks of continuous access to ethanol compared with water controls and animals deprived of ethanol for 2 weeks. The effect of local perfusion with 100 microM sulpiride (D(2) receptor antagonist) and 35 microM 1-(m-chlorophenyl)-biguanide (5-hydroxytryptamine(3) receptor agonist) on dopamine overflow were reduced approximately 33% in both groups of ethanol-exposed P rats compared with water controls. Free-choice alcohol drinking by P rats alters dopamine and serotonin neurotransmission in the nucleus accumbens, and many of these effects persist for at least 2 weeks in the absence of ethanol, suggesting that these underlying persistent changes may be in part responsible for increased ethanol drinking observed in the alcohol-deprivation effect.

A genome-wide screen for genes influencing conduct disorder.

While behavioral genetic studies have suggested that childhood conduct disorder is under genetic influence, studies aimed at gene identification are lacking. This study represents the first genome-wide linkage analysis directed toward identifying genes contributing to conduct disorder. Genome screens of retrospectively reported childhood conduct disorder and conduct disorder symptomatology were carried out in the genetically informative adult sample collected as part of the Collaborative Study on the Genetics of Alcoholism (COGA). The results suggest that regions on chromosomes 19 and 2 may contain genes conferring risk to conduct disorder. Interestingly, the same region on chromosome 2 has also been linked to alcohol dependence in this sample. Childhood conduct disorder is known to be associated with the susceptibility for future alcohol problems. Taken together, these findings suggest that some of the genes contributing to alcohol dependence in adulthood may also contribute to conduct disorder in childhood.

Effects of neuropeptide Y on sucrose and ethanol intake and on anxiety-like behavior in high alcohol drinking (HAD) and low alcohol drinking (LAD) rats.

BACKGROUND: In a previous study, neuropeptide Y (NPY) administered into the lateral ventricles decreased ethanol intake in alcohol-preferring (P) rats but not in alcohol-nonpreferring (NP) or unselected Wistar rats. The purpose of the present investigation is to extend these findings in selectively-bred high-alcohol-drinking (HAD)1 and low-alcohol-drinking (LAD)1 rats by examining the effects of intracerebroventricularly administered NPY on the elevated plus maze test of anxiety and on ethanol and sucrose intake. METHODS: Female HAD1 and LAD1 rats were surgically implanted with cannula into the lateral ventricle. Following recovery, a test of anxiety was conducted in which the rats (n = 12-13/group) received either artificial cerebrospinal fluid (aCSF) or NPY (10 microg) 10 min prior to a 5-min test on an elevated plus maze. Following anxiety testing, 11 HAD and 11 LAD rats were trained to self-administer ethanol (8% w/v), and 5 HAD and 8 LAD rats were trained to self-administer sucrose (2.5%) during daily 2-hr sessions. A within-subject design was used in which the rats were pretreated once a week with aCSF, 5 microg NPY, or 10 microg NPY prior to the drinking sessions. RESULTS: HAD and LAD rats treated with aCSF did not differ in time spent in open arms of the plus maze. NPY increased time spent on the open arms to similar degrees in both rat lines. HAD rats consumed more ethanol and sucrose than LAD rats. NPY increased sucrose intake in both rat lines. However, the same doses of NPY reduced ethanol intake in HAD but not in LAD rats. CONCLUSION: The plus maze results indicated that selective breeding for high and low alcohol preference in the HAD1 and LAD1 rats, respectively, did not yield differences in anxiety-like behavior and in response to the anxiolytic effects of NPY. The increases in sucrose intake were consistent with the known orexigenic effects of NPY. The decreased ethanol intake following NPY administration in HAD rats was similar to previous observations with P rats and is consistent with the hypothesis that ethanol intake and NPY activity may be inversely related.

Self-reported subjective perception of intoxication reflects family history of alcoholism when breath alcohol levels are constant.

BACKGROUND: The premise of this study is that the increased familial risk for alcoholism is associated with genetic determinants of the response to alcohol, characterized by sensitivity and adaptation. Following a single administration, sensitivity is the initial response to alcohol, expressed as the change in dependent measures from baseline. Adaptation of dependent measures within a single exposure to alcohol can be expressed as acute tolerance (recovery of dependent measures toward baseline values) or sensitization (movement of dependent measure further away from baseline values). This study tested the hypothesis that family history-positive (FHP) subjects are more sensitive and more adaptive to alcohol compared with family history-negative (FHN) subjects. METHODS: The initial response and development of adaptation to alcohol were assessed by using self-reported subjective perceptions during a breath alcohol concentration (BrAC) clamp of 60 mg%. The Biphasic Alcohol Effects Scale, the Sensation Scale and a visual analog scale of intoxication were acquired at baseline, after the BrAC clamp was established, and after maintenance of the clamp for 105 min. RESULTS: FHP subjects were more sensitive to alcohol compared with FHNs, as evidenced by greater changes in feelings of intoxication when the BrAC clamp was initially achieved. While the clamp was maintained, the FHP subjects adapted to the effects of alcohol and their perceptions of intoxication became indistinguishable from those of the FHN subjects. The FHP subjects had developed acute tolerance to alcohol, whereas the FHN subjects did not. Other self-reported perceptions of alcohol's effects did not distinguish between the groups. CONCLUSIONS: A differential family history of alcoholism was reflected in self-reported subjective perceptions of intoxication when the brain's exposure to a specified concentration of alcohol was held constant (BrAC of 60 mg%). FHP subjects reported greater intoxication after alcohol and subsequently developed acute tolerance to alcohol compared with FHN subjects.

Response of saccadic eye movements to alcohol in African American and non-Hispanic white college students.

BACKGROUND: The primary goal of this study was to evaluate how race and sex interact with the effects of a moderate dose of alcohol on different ocular control subsystems in African American (AA) and non-Hispanic white American (WA) college students. METHODS: Horizontal visually guided (VG) saccades and antisaccades (AS) of 80 young adult, healthy, AA and WA college students were recorded with an infrared system. Subjects ingested 10 aliquots of ethanol at 3 min intervals, with the aggregate dose precalculated to yield a peak breath alcohol concentration (BrAC) of 80 mg%. Data from the measures performed at baseline and the ascending and descending limbs of the BrAC at approximately 65 mg% were compared across race and sex by multivariate analysis of variance. A no-alcohol control session, performed in 20 of the subjects, documented test-retest reliability of the VG and AS measurements. RESULTS: Both AA and WA groups demonstrated slowing of AS and VG saccades after alcohol administration, but there was no significant effect of 65 mg% alcohol on VG accuracy or AS errors. AS latency recovered toward baseline values, whereas the slowing of VG latency/velocity progressed, during alcohol exposure. There were significant differences between AA and WA groups in the time course of VG latency after alcohol but not in most other dependent measures. No significant effects for sex were observed in any of the saccade measures. The faster disappearance of alcohol in WA compared with AA was replicated, and some measures demonstrated a significant, albeit small, negative correlation between the alcohol disappearance rate and impairing effects of alcohol on saccades. CONCLUSIONS: Prolonged latencies and unchanged percentage of errors reflect a differential effect of alcohol on neural function in specific areas (parietal eye field, superior colliculus, and frontal eye areas). Race may interact with the effect of ethanol on saccadic eye movements in a college student population.

Effects of amphetamine on locomotor activity in adult and juvenile alcohol-preferring and -nonpreferring rats.

The objective of this study was to determine whether functional differences exist in amphetamine-induced locomotor activity between alcohol-naive alcohol-preferring (P) and -nonpreferring (NP) rats during postnatal development and during adulthood. Using a between-subjects design, 20- and 28-day-old P and NP rats (male and female counterbalanced, n=11-16/line) were habituated for 30 min in a photocell activity field. Each rat received subcutaneous injections of saline or 0.3, 0.6 or 1.2 mg/kg d-amphetamine (AMPH) and were then tested for an additional 30 min. Because of age and line differences in basal locomotor activity, total activity counts during the 30-min postdrug period were standardized using Z-score transformations. In the 20- and 28-day-old rats, dose-dependent locomotor activity increases after AMPH injections were obtained at both ages, although activity levels were greater in the 20-day-old pups. The 20-day-old female NP rats showed greater AMPH-induced increases in locomotor activity than P rats, whereas at 28 days of age, male NP rats showed greater activity levels than P rats to AMPH. For the adult P and NP rats (n=8/line/gender), a within-subject design was used. In the adults, the NP line had higher locomotor activity than the P line following AMPH injection, and male rats were activated more by AMPH than female rats. The results suggest that functioning of the DA system in the adult P line is reduced compared to the adult NP line, and this line difference is also observed to some degree at an early postweaning developmental period.

Ethanol-stimulated serotonin release in the ventral hippocampus: an absence of rapid tolerance for the alcohol-preferring P rat and insensitivity in the alcohol-nonpreferring NP rat.

This study examined the acute effects of intraperitoneal administration of ethanol on the extracellular levels of serotonin (5-HT) in the ventral hippocampus (vHIP) of adult, male alcohol-preferring P and -nonpreferring NP rats. Using in vivo microdialysis coupled with HPLC and electrochemical detection, the effects of acute administration of saline or 1.0, 1.75, or 2.5 g/kg ethanol on the extracellular levels of 5-HT in the vHIP were examined. Saline and 1.0 g/kg ethanol did not alter the extracellular levels of 5-HT. However, the 1.75-g/kg dose resulted in a transient increase in 5-HT levels in the vHIP of P rats only. Administration of 2.5 g/kg ethanol increased 5-HT levels to 180% of baseline in P rats (P<.05), but was without effect on NP rats. The 2.5-g/kg dose also significantly increased the extracellular levels of 5-HT in the vHIP of P rats, which had been pretreated with the same dose of ethanol 18-24 h earlier (P<.05). Comparison of the response of ethanol pretreated P rats with animals that had been pretreated with saline 24 h earlier did not reveal any significant differences in ethanol-stimulated increases in 5-HT levels between the groups. These data suggest that ethanol may activate terminals of the median raphe 5-HT system in P rats because the vHIP receives its 5-HT inputs primarily from the median raphe nucleus (MRN). Rapid tolerance does not develop to this activation of the system in the vHIP of P rats. In addition, the data suggest that the 5-HT system in the vHIP of NP rats may be relatively insensitive to the stimulating effect of acute ethanol of 5-HT release.