Acute ethanol effects on local cerebral glucose utilization in select central nervous system regions of adolescent alcohol-preferring (P) and alcohol-nonpreferring (NP) rats.

BACKGROUND: Alcohol abuse among adolescents is a major health and developmental problem. The 2-[(14)C]deoxyglucose (2-DG) technique allows for the in vivo quantification of local cerebral glucose utilization (LCGU) as a measure of functional neuronal activity. METHODS: Local cerebral glucose utilization rates were examined after acute ethanol administration within selected brain regions of adolescent alcohol-preferring (P) and -nonpreferring (NP) rats. Postnatal day 45 male P and NP rats were injected with saline or 1.0 g/kg ethanol, i.p., 10 minutes prior to an intravenous bolus of [(14)C]-2-deoxyglucose (125 microCi/kg). Image densities were determined using quantitative autoradiography and LCGU values calculated. RESULTS: Acute ethanol injection significantly decreased LCGU rates in select brain regions including the olfactory tubercles, the frontal cortex (Fr), and subregions of the posterior hippocampus (pCA1 and pCA3). Acute ethanol had no significant effects on LCGU rates in any region of the adolescent NP rats. Significant basal LCGU rate differences were apparent between the rat lines in a nearly global fashion with adolescent P rats having much higher basal LCGU rates compared with adolescent NP rats. CONCLUSIONS: These findings suggest that the adolescent P and NP rats are less sensitive to the effects of acute ethanol than their adult counterparts. The adolescent P rat is relatively more sensitive to the initial effects of acute ethanol in select brain regions as compared with the adolescent NP rat. Additionally, the innate hyper-excited state of the adolescent P central nervous system is a likely factor in the development of their high alcohol drinking behaviors.

Differential gene expression in the nucleus accumbens with ethanol self-administration in inbred alcohol-preferring rats.

The current study examined the effects of operant ethanol (EtOH) self-administration on gene expression kin the nucleus accumbens (ACB) and amygdala (AMYG) of inbred alcohol-preferring (iP) rats. Rats self-trained on a standard two-lever operant paradigm to administer either water-water, EtOH (15% v/v)-water, or saccharin (SAC; 0.0125% g/v)-water. Animals were killed 24 h after the last operant session, and the ACB and AMYG dissected; RNA was extracted and purified for microarray analysis. For the ACB, there were 513 significant differences at the p<0.01 level in named genes: 55 between SAC and water; 215 between EtOH and water, and 243 between EtOH and SAC. In the case of the AMYG (p<0.01), there were 48 between SAC and water, 23 between EtOH and water, and 63 between EtOH and SAC group. Gene Ontology (GO) analysis indicated that differences in the ACB between the EtOH and SAC groups could be grouped into 15 significant (p<0.05) categories, which included major categories such as synaptic transmission, cell and ion homeostasis, and neurogenesis, whereas differences between the EtOH and water groups had only 4 categories, which also included homeostasis and synaptic transmission. Several genes were in common between the EtOH and both the SAC and water groups in the synaptic transmission (e.g., Cav2, Nrxn3, Gabrb2, Gad1, Homer1) and homeostasis (S100b, Prkca, Ftl1) categories. Overall, the results suggest that changes in gene expression in the ACB of iP rats are associated with the reinforcing effects of EtOH.

Functional gene expression differences between inbred alcohol-preferring and -non-preferring rats in five brain regions.

The objective of this study was to determine if there are innate differences in gene expression in selected CNS regions between inbred alcohol-preferring (iP) and -non-preferring (iNP) rats. Gene expression was determined in the nucleus accumbens (ACB), amygdala (AMYG), frontal cortex (FC), caudate-putamen (CPU), and hippocampus (HIPP) of alcohol-naïve adult male iP and iNP rats, using Affymetrix Rat Genome U34A microarrays (n = 6/strain). Using Linear Modeling for Microarray Analysis with a false discovery rate threshold of 0.1, there were 16 genes with differential expression in the ACB, 54 in the AMYG, 8 in the FC, 24 in the CPU, and 21 in the HIPP. When examining the main effect of strain across regions, 296 genes were differentially expressed. Although the relatively small number of genes found significant within individual regions precluded a powerful analysis for over-represented Gene Ontology categories, the much larger list resulting from the main effect of strain analysis produced 17 over-represented categories (P < .05), including axon guidance, gliogenesis, negative regulation of programmed cell death, regulation of programmed cell death, regulation of synapse structure function, and transmission of nerve impulse. Co-citation analysis and graphing of significant genes revealed a network involved in the neuropeptide Y (NPY) transmitter system. Correlation of all significant genes with those located within previously established rat alcohol QTLs revealed that of the total of 313 significant genes, 71 are located within such QTLs. The many regional and overall gene expression differences between the iP and iNP rat lines may contribute to the divergent alcohol drinking phenotypes of these rats.

Dopamine and serotonin content in select brain regions of weanling and adult alcohol drinking rat lines.

The objective of the present study was to examine innate differences in the tissue content of dopamine (DA), serotonin (5-HT) and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in five brain regions of weanling and adult alcohol-preferring (P), alcohol-nonpreferring (NP), high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) selected rat lines. Adult male and weanling (postnatal day 25) male rats were killed by decapitation and brains were rapidly dissected for the following regions: olfactory tubercles (OTU), nucleus accumbens (ACB), septum (SEP), anterior cerebral cortex (ACTX) and amygdala (AMYG). Tissue extracts were assayed by HPLC with electrochemical detection. Due to significantly higher content levels in the adults, adult and weanling animals were analyzed separately. Significant differences were found in the ACB and OTU between the adult lines in both DA and 5-HT content, with P and HAD rats having lower levels than NP and LAD rats, respectively. Significant differences in DA content between the weanling lines were also found in the OTU, with P and HAD rats having lower DA levels than NP and LAD rats, respectively. These results confirm previous findings of an association between innate low DA content in select limbic regions and high alcohol drinking behavior.

Effects of acute administration of ethanol on cerebral glucose utilization in adult alcohol-preferring and alcohol-nonpreferring rats.

Local cerebral glucose utilization (LCGU) rates, as determined by the [(14)C]-2-deoxyglucose (2-DG) technique, were examined after acute ethanol administration within selected brain regions of alcohol-preferring (P) and alcohol-nonpreferring (NP) rats. Adult male P and NP rats were injected with saline, 0.25 g/kg, or 1.0 g/kg ethanol, intraperitoneally (ip), 10 min before an intravenous bolus of [(14)C]2-DG (125 microCi/kg). Timed arterial blood samples were collected over 45 min and assayed for plasma glucose, ethanol, and [(14)C]2-DG levels. Image densities were determined using quantitative autoradiography and LCGU values calculated. Data were collected from several key limbic, basal ganglionic, cortical, and subcortical structures. Low-dose ethanol (0.25 g/kg) significantly decreased LCGU rates in several brain regions including the medial prefrontal cortex, olfactory tubercles, and the CA1 subregion of the hippocampus of P rats. Low-dose ethanol had no significant effects on LCGU rates in the NP rats. Moderate-dose ethanol (1.0 g/kg) also significantly lowered LCGU rates in many brain regions of P rats, including key limbic structures, such as the medial prefrontal cortex, olfactory tubercles, ventral tegmental area, basolateral nucleus of the amygdala, lateral septum, and ventral pallidum. Moderate-dose ethanol also significantly lowered LCGU rates in the medial prefrontal cortex as well as in the habenula of NP rats. All other regions were unaffected in the NP rats. These findings support the suggestion that certain central nervous system regions of P rats may be more sensitive than those of NP rats to the effects of low to intermediate doses of ethanol.

Adolescent alcohol drinking and its long-range consequences. Studies with animal models.

This chapter reviews findings, mainly obtained from the selectively bred alcohol-preferring (P) line of rats, on (a) the development of alcohol drinking during the peri-adolescent period, (b) neurobiological factors that may contribute to adolescent drinking, (c) interventions to prevent alcohol drinking during adolescence, and (d) some long-lasting consequences of adolescent alcohol drinking. The findings indicate that P rats readily initiate alcohol drinking during the early post-weaning, adolescent and peri-adolescent periods of development. The early age-of-onset of alcohol drinking in the P compared to the NP line is associated with (a) higher densities of serotonin-1A (5-HT1A) receptors in cerebral cortical and hippocampal regions; (b) lower densities of dopamine (DA) D2 receptors in the ventral tegmental area (VTA); (c) higher functional activity in several limbic, cortical and hippocampal regions; and (d) sensitivity to the low-dose stimulating effect of ethanol. Conditioned taste aversion (CTA) training during adolescence produces long-term effects on preventing high alcohol drinking behavior of P rats. Alcohol drinking during peri-adolescence by P rats produces long-lasting effects that increase the acquisition of ethanol self-administration in adulthood, and, in addition, increase craving-like behavior and the potential for alcohol relapse. With suitable animal models, a better understanding of the mechanisms underlying adolescent alcohol drinking and its long-range consequences can be attained.

Regional CNS densities of serotonin 1A and dopamine D2 receptors in periadolescent alcohol-preferring P and alcohol-nonpreferring NP rat pups.

The objective of the present study was to use quantitative autoradiography to determine binding densities of serotonin(1A) (5-HT(1A)) and dopamine (DA) D(2) receptors in alcohol-naive periadolescent P and NP rat pups. P (n=8) and NP (n=7) rat pups, 25 days of age, from different litters were used. Coronal brain sections were incubated with 2 nM [3H]8-OH-DPAT or 20 nM [3H]sulpiride for 5-HT(1A) or D(2) binding, respectively. Approximately 15-40% higher densities of [3H]8-OH-DPAT binding were observed in the anterior cortical regions of the periadolescent P rat compared with NP rat pups. Similar differences were also observed in posterior cortical regions with P rats having 25-40% higher [3H]8-OH-DPAT binding than NP rats. [3H]8-OH-DPAT binding was approximately 10-20% higher in posterior hippocampal regions of the P rat pups compared with the NP line. [3H]sulpiride binding was significantly different only in the ventral tegmental area (VTA), where binding was approximately 20% lower in the periadolescent P rats compared with the NP rat pups. Overall, these results are very similar to findings observed in adult alcohol-naive P and NP rats, and suggest that the innate differences in the neural systems implicated in high alcohol drinking behaviors may already be established in the periadolescent animal.

Gene expression changes in the nucleus accumbens of alcohol-preferring rats following chronic ethanol consumption.

The objective of this study was to determine the effects of binge-like alcohol drinking on gene expression changes in the nucleus accumbens (ACB) of alcohol-preferring (P) rats. Adult male P rats were given ethanol under multiple scheduled access (MSA; three 1-h dark cycle sessions/day) conditions for 8 weeks. For comparison purposes, a second ethanol drinking group was given continuous/daily alcohol access (CA; 24h/day). A third group was ethanol-naïve (W group). Average ethanol intakes for the CA and MSA groups were approximately 9.5 and 6.5 g/kg/day, respectively. Fifteen hours after the last drinking episode, rats were euthanized, the brains extracted, and the ACB dissected. RNA was extracted and purified for microarray analysis. The only significant differences were between the CA and W groups (p<0.01; Storey false discovery rate=0.15); there were 374 differences in named genes between these 2 groups. There were 20 significant Gene Ontology (GO) categories, which included negative regulation of protein kinase activity, anti-apoptosis, and regulation of G-protein coupled receptor signaling. Ingenuity analysis indicated a network of transcription factors, involving oncogenes (Fos, Jun, Junb had higher expression in the ACB of the CA group), suggesting increased neuronal activity. There were 43 genes located within rat QTLs for alcohol consumption and preference; 4 of these genes (Tgfa, Hspa5, Mtus1 and Creb3l2) are involved in anti-apoptosis and increased transcription, suggesting that they may be contributing to cellular protection and maintaining high alcohol intakes. Overall, these findings suggest that chronic CA drinking results in genomic changes that can be observed during the early acute phase of ethanol withdrawal. Conversely, chronic MSA drinking, with its associated protracted withdrawal periods, results in genomic changes that may be masked by tight regulation of these genes following repeated experiences of ethanol withdrawal.

Drd2 expression in the high alcohol-preferring and low alcohol-preferring mice.

The high alcohol-preferring (HAP) and low alcohol-preferring (LAP) mice were selectively bred for differences in alcohol preference and consumption. Recently, a large-effect QTL was identified on chromosome 9. The peak for this QTL is near the Drd2 (dopamine receptor 2) locus. The present study examined Drd2 mRNA expression differences between the HAP1 and LAP1 mice in brain regions important in the dopaminergic-reward pathway, including the nucleus accumbens, hippocampus, amygdala, and septum. Results show that alcohol-naïve HAP1 mice exhibited lower levels of Drd2 mRNA expression in the nucleus accumbens and the hippocampus compared to LAP1 mice. No differences were found in the amygdala or septum. To determine if a sequence difference might underlie the expression difference, the Drd2 cDNA was sequenced in each line and one single nucleotide polymorphism (SNP) was identified in the 3' UTR. Both HAP and LAP 3' UTR were cloned in the luc-pGL3-promoter-luc vector. The polymorphism in the Drd2 3' UTR was assessed to determine its functional significance in modulating expression. In vitro expression analysis using neuroblastoma SK-N-SH cells resulted in a significant decrease in expression of the HAP 3' UTR luc construct compared with the LAP 3' UTR construct. This decreased expression is consistent with lower levels of Drd2 expression in the nucleus accumbens and the hippocampus as evidenced by qRT-PCR. These results suggest that the SNP may play a role in the differential expression of Drd2 between the HAP and LAP mice and that the polymorphism in Drd2 may contribute to alcohol preference.

Alcohol effects on central nervous system gene expression in genetic animal models.

This article summarizes the proceedings of a symposium presented at the 2004 annual meeting of the Research Society on Alcoholism in Vancouver, British Columbia, Canada. The organizers and chairs were William J. McBride and Michael F. Miles. The presentations were (1) Molecular Triangulation on Gene Expression Patterns in Behavioral Responses to Acute Ethanol, by Robnet T. Kerns; (2) Gene Expression in Limbic Regions After Ethanol Self-Infusion Into the Posterior Ventral Tegmental Area, by Zachary A. Rodd; (3) Microarray Analysis of CNS Limbic Regions of Inbred Alcohol-Preferring and -Nonpreferring rats and Effects of Alcohol Drinking, by Wendy N. Strother and Howard J. Edenberg; and (4) Microarray Analysis of Mouse Lines Selected for Chronic Ethanol Withdrawal Severity: The Convergence of Basal, Ethanol Regulated, and Proximity to Ethanol Quantitative Trait Loci to Identify Candidate Genes, by Joel G. Hashimoto and Kristine M. Wiren.

Neuroimaging of rodent and primate models of alcoholism: initial reports from the integrative neuroscience initiative on alcoholism.

Neuroimaging of animal models of alcoholism offers a unique path for translational research to the human condition. Animal models permit manipulation of variables that are uncontrollable in clinical, human investigation. This symposium, which took place at the annual meeting of the Research Society on Alcoholism in Vancouver, British Columbia, Canada, on June 29th, 2004, presented initial findings based on neuroimaging studies from the two centers of the Integrative Neuroscience Initiative on Alcoholism funded by the National Institute on Alcohol Abuse and Alcoholism. Effects of alcohol exposure were assessed with in vitro glucose metabolic imaging of rat brain, in vitro receptor imaging of monkey brain, in vivo magnetic resonance imaging of monkey brain, and in vivo magnetic resonance spectroscopic quantification of alcohol metabolism kinetics in rat brain.

alpha-Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats.

Total gene expression analysis (TOGA) was used to identify genes that are differentially expressed in brain regions between the alcohol-naive, inbred alcohol-preferring (iP), and -nonpreferring (iNP) rats. alpha-Synuclein, expressed at >2-fold higher levels in the hippocampus of the iP than the iNP rat, was prioritized for further study. In situ hybridization was used to determine specific brain regions and cells expressing alpha-synuclein in the iP and iNP rats. Similar to alpha-synuclein mRNA levels, protein levels in the hippocampus were higher in iP rats than iNP rats. Higher protein levels were also observed in the caudate putamen of iP rats compared with iNP rats. Sequence analysis identified two single nucleotide polymorphisms in the 3' UTR of the cDNA. The polymorphism was used to map the gene, by using recombination-based methods, to chromosome 4, within a quantitative trait locus for alcohol consumption that was identified in the iP and iNP rats. A nucleotide exchange in the iNP 3' UTR reduced expression of the luciferase reporter gene in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the alpha-synuclein gene may contribute to alcohol preference in the iP rats.

Innate differences in protein expression in the nucleus accumbens and hippocampus of inbred alcohol-preferring and -nonpreferring rats.

Two-dimensional gel electrophoresis (2-DE) was used to separate protein samples solubilized from the nucleus accumbens and hippocampus of alcohol-naïve, adult, male inbred alcohol-preferring (iP) and alcohol-nonpreferring (iNP) rats. Several protein spots were excised from the gel, destained, digested with trypsin, and analyzed by mass spectrometry. In the hippocampus, 1629 protein spots were matched to the reference pattern, and in the nucleus accumbens, 1390 protein spots were matched. Approximately 70 proteins were identified in both regions. In the hippocampus, only 8 of the 1629 matched protein spots differed in abundance between the iP and iNP rats. In the nucleus accumbens, 32 of the 1390 matched protein spots differed in abundance between the iP and iNP rats. In the hippocampus, the abundances of all 8 proteins were higher in the iNP than iP rat. In the nucleus accumbens, the abundances of 31 of 32 proteins were higher in the iNP than iP rat. In the hippocampus, only 2 of the 8 proteins that differed could be identified, whereas in the nucleus accumbens 21 of the 32 proteins that differed were identified. Higher abundances of cellular retinoic acid-binding protein 1 and a calmodulin-dependent protein kinase (both of which are involved in cellular signaling pathways) were found in both regions of the iNP than iP rat. In the nucleus accumbens, additional differences in the abundances of proteins involved in (i) metabolism (e.g., calpain, parkin, glucokinase, apolipoprotein E, sorbitol dehydrogenase), (ii) cyto-skeletal and intracellular protein transport (e.g., beta-actin), (iii) molecular chaperoning (e.g., grp 78, hsc70, hsc 60, grp75, prohibitin), (iv) cellular signaling pathways (e.g., protein kinase C-binding protein), (v) synaptic function (e.g., complexin I, gamma-enolase, syndapin IIbb), (vi) reduction of oxidative stress (thioredoxin peroxidase), and (vii) growth and differentiation (hippocampal cholinergic neurostimulating peptide) were found. The results of this study indicate that selective breeding for disparate alcohol drinking behaviors produced innate alterations in the expression of several proteins that could influence neuronal function within the nucleus accumbens and hippocampus.