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NEUROTEC, Karolinska Institutet, Stockholm, Sweden
1Correspondence: M46 Huddinge University Hospital, S-14186 Huddinge, Sweden. E-mail: Markus.Heilig{at}neurotec.ki.se
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: alcoholism • gene chip • expression profiling • cingulate cortex • amygdala
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This points to the importance of identifying novel treatment targets. Genetic factors predispose for alcoholism, but the actual disease phenotype develops over years, requires extensive ethanol intake, and involves long-term allostatic changes in brain function. Numerous neurotransmitter pathways are affected by ethanol, but it is unclear how long-term changes within these encode the profound and most likely irreversible transition from low to high voluntary alcohol intake (2
, 3)
.
To address this issue, we searched for long-term changes in gene expression in brain areas implicated in regulation of ethanol intake after the induction of alcohol preference in rats. Continuous alcohol vapor exposure resulting in blood alcohol concentration (BAC) > 150 mg/dl leads to alcohol preference in experimental animals (4
, 5)
. However, this phenomenon is transient and thus may not fully replicate long-term transcriptional changes within the central nervous system (CNS) underlying alcohol dependence. We examined whether repeated cycles of intoxication and mild withdrawal rather than continuous exposure may better reflect the real-life course of developing alcohol dependence and trigger processes of neuronal plasticity underlying the progression from low to high alcohol preference.
Despite some pitfalls, aspects of alcoholism can be modeled in experimental animals (6
, 7)
. We hypothesized that behavioral and neural adaptations in our model might be relevant for human alcoholism if the behavioral phenotype is antagonized by compounds that are clinically effective in dependent patients. Acamprosate, an allosteric modulator of glutamatergic N-methyl-D-aspartate (NMDA) receptors through actions at the spermidine site (8)
, reduces ethanol consumption in human alcoholics presumably by interfering with craving and relapse (9)
. We therefore examined the ability of acamprosate to inhibit voluntary ethanol consumption after intermittent exposure to ethanol vapor.
Finally, we addressed the possibility that induction of an alcohol-preferring phenotype may involve transcriptional regulation events in key brain areas. Identifying transcripts regulated in this process might point to pathways underlying the phenotypic change and thus to novel targets for pharmacological treatment. DNA microarray technology has made it possible to identify genes whose expression in the brain is regulated by experiential factors such as age (10)
, for example, or enriched environment (11)
. We used a microarray containing probes for > 1200 transcripts expressed in the rat CNS, and searched for genes whose expression in amygdala and cingulate frontal cortex is regulated in parallel with the transition to a high alcohol drinking phenotype.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethanol exposure
Exposure was in glass/steel chambers (1x1x1 m). HPLC pumps (Knauer, Berlin, Germany) delivered ethanol into electrically heated stainless steel coils (60°C) connected to an airflow of 18 l/min. Ethanol concentration was adjusted by changing pump flow and monitored via a spectrometer (Wilks,;1> South Norwalk, CT). Exposure was for 17 h during each 24 h period (on 16.00; off 09.00). Rats were allowed to habituate to the chambers for 1 wk, then exposed to low ethanol concentration for 1 wk, and finally exposed for 7 wk to ethanol vapor levels yielding BACs between 150 and 250 mg/dl. The duration of exposure was based on independently obtained time course data, indicating that whereas shorter periods of exposure increase the proportion of high drinking subjects, 6 wk of exposure are required to induce robust high drinking in most rats. Controls were kept in identical chambers with normal air flow. Rats were weighed weekly, and blood collected from the lateral tail vein. Serum was assayed for ethanol using an NAD/NADPH enzyme/spectrophotometric assay kit (Sigma Aldrich) according to the manufacturer’s instructions.
24 h voluntary ethanol intake
A modification of the procedure described in ref 12
was used. Ethanol was made continuously available in 0.2% saccharin. Ethanol concentration was gradually increased to 6% (v/v). A 0.2% saccharin solution without ethanol was continuously available in a second bottle providing a free choice situation. Ethanol and water intake were measured Monday, Wednesday, and Friday over 26 days.
Operant ethanol self-administration
Training and testing were as described (13)
, except saccharin was not faded out, since it helps maintain response rates and has been shown not to affect the pharmacokinetic properties of ethanol (14)
. One rat was excluded because it never acquired any degree of operant responding for either ethanol or saccharin. With this exception, all rats were included irrespective of ethanol response rates. This increases variability but avoids possible selection artifacts. After training, animals were ranked for ethanol responses and divided into groups balanced for response rates.
Acute withdrawal
Behavior was observed 6–7 h after the last cycle of exposure. Presence or absence of behavioral signs (Table 1
) was scored by a trained observer blind to treatment. Locomotor activity was examined 7–8 h after ethanol vapor exposure was discontinued. Activity was recorded for 30 min as described (15)
.
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Oligonucleotide microarray analysis
Subjects were decapitated between 10 and 12 AM, and brains were frozen in -40°C isopentane and kept at -70°C. Bilateral cingulate frontal cortex and amygdala samples were obtained from 2 mm-thick coronal slices (amygdala: 2 mm punch diameter, level -1.8 to -3.8 according to ref 16
; frontal cortex: level +1.7 to -0.3) and stored at -70°C. Total RNA was extracted with TRIzol reagent (Life Technologies, Baltimore, MD), followed by RNeasy clean-up (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. All RNA samples showed A260/280 ratios of 1.9–2.1. RNA integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany), and only material without signs of degradation was used.
To eliminate individual variation, random subjects from each experimental condition were pooled (three/pool, 2–3 µg total RNA from each). Two independent pools from each condition were used for each brain region. Target preparation and hybridization were according to the manufacturer’s technical manual (Affymetrix, Santa Clara, CA). Double-stranded cDNA was synthesized using HPLC purified T7-(dT)24 primer starting with 5–8 µg of total RNA. cDNA was purified, ethanol precipitated, and resuspended in RNase-free water. Biotinylated cRNA targets were in vitro transcribed, yielding 40–80 µg labeled cRNA. After purification and fragmentation, targets (5 µg) were hybridized to Rat Neurobiology U34 Arrays for 16–17 h at 45°C. Chips were washed, stained, and scanned.
Data were analyzed with Microarray Suite 4.0.1. Within each brain region, each of the two pools from the ethanol exposed condition was compared with each of the control pools. Only targets detected as consistently changed in three or four of the four possible comparisons and with an average fold change of two or higher were considered.
Corticosterone and clinical chemistry
Trunk blood samples were obtained 1–3 h into the dark phase. Serum corticosterone was analyzed as described previously (15)
. A range of routine clinical chemistry analyses (Table 1)
was carried out by the SWEDAC accredited clinical chemistry laboratory of Huddinge University Hospital.
Statistics
Where appropriate, analysis of variance (ANOVA) was used followed by Tukey’s HSD post hoc test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, main graph). During the withdrawal period within each 24 h cycle, BACs dropped to undetectable levels (Fig. 1
, inset). Six to 8 h after the last exposure cycle was discontinued, we observed clear signs of acute withdrawal and decreased horizontal locomotor activity (one-way ANOVA for exposure effect, F1,21=23.43; P<0.001) with no change in rearings (exposure effect F1,21=1.46; P<0.2; Table 2
). Similar BACs and withdrawal signs were obtained in experiments 2–5.
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Corticosterone and blood chemistry (n=15 vs. 16, exposed vs. control)
The exposure procedure did not seem to be associated with long-term nonspecific adverse consequences. When elevated drinking was found in parallel groups (3 wk after completion of the exposure), basal corticosterone levels 1–3 h into the dark phase did not differ significantly between the groups (247.2±14.2 vs. 208.7±15.9; control vs. exposed; mean±SE ng/ml; P>0.05). At the same time, an extensive range of routine clinical chemistry analyses did not reveal any pathology. The only difference found between the groups was in serum bilirubin levels, which were significantly subnormal in the exposed group (Table 1)
. Finally, although weight gain in exposed subjects was significantly retarded during the first week of the exposure phase (wk 3), it was subsequently indistinguishable from that of controls during weeks 4–9 of exposure and thereafter (data not shown).
Voluntary ethanol intake after intermittent exposure (n=12 vs. 16, exposed vs. controls)
After a 3 wk period during which previously ethanol-exposed and control rats were left undisturbed, ethanol was made available in the two-bottle free choice paradigm. Exposed subjects consumed markedly higher amounts of ethanol (two-way ANOVA for exposure effect and time; exposure effect F1,26=17.12; P<0.0003; Fig. 2
). During the final phase of the procedure, when 6% ethanol was available, intake was 5.4 ± 0.5 and 2.8 ± 0.2 g/kg/day for exposed rats and controls, respectively. No difference in saccharin intake was seen (F1,26=0.91; P=0.33; data not shown).
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Operant responding for ethanol after intermittent exposure (n=12 vs. 16, exposed vs. controls)
Animals were trained to respond for 10% ethanol, then subjected to the described sequence of 2 wk adaptation, 7 wk intermittent ethanol exposure, and 3 wk recovery. They were subsequently allowed to respond for ethanol or control solution for 5 days in 30 min daily sessions. An elevated level of responding for ethanol was found in exposed subjects (two-way ANOVA, exposure effect F1,25=4.55; P<0.04; mean±SE 38±5 vs. 23±3 responses/30 min). These subjects were then introduced to the two-bottle free choice paradigm 42 days after termination of ethanol vapor exposure. Over the next 26 days, previously exposed subjects still consumed markedly more ethanol (treatment effect F1,25=15.12; P<0.0006; data not shown).
Reversal of exposure-induced but not basal ethanol intake by acamprosate (nonexposed vehicle: n=8; nonexposed acamprosate: n=7; exposed vehicle: n=8; exposed acamprosate: n=7)
Rats were subjected to the same 2 wk adaptation, 7 wk exposure, 3 wk recovery sequence, but daily acamprosate treatment (200 mg/kg, i.p.) was initiated 2 wk after ethanol exposure was discontinued (Fig. 3
, phase I). After 1 wk of treatment, animals were introduced to the two-bottle free choice paradigm and ethanol consumption was measured while drug treatment was continued for two more weeks (Fig. 3
, phase II). This design was chosen because acamprosate treatment is normally initiated clinically in a drug-free state to prevent relapse and the treatment is given for a limited time. Overall, acamprosate reduced ethanol consumption (three-way ANOVA for exposure, drug treatment, and time; drug treatment effect F1,25=8.17, P<0.008). Post hoc analysis showed this was due to a selective effect in exposed acamprosate-treated rats, which differed from exposed, saline-treated subjects (P=0.02) but was indistinguishable from nonexposed saline-treated (P=0.97) or nonexposed acamprosate-treated rats (P=0.94). The acamprosate effect lasted after cessation of treatment (Fig. 3
; exposed acamprosate vs. exposed saline, P=0.02 and 0.0008 on days 16 and 18, respectively). The effect of acamprosate on alcohol drinking did not appear to be the result of nonspecific performance factors or suppressed general motivation to drink, since saccharin intake was not affected to any extent (F1,26=0.66, P=0.42).
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Regulation of gene expression in frontal cortex and amygdala (n=6 vs. 6, exposed vs. control)
Animals were subjected to the same adaptation-exposure-recovery sequence as above. After recovery, tissue was processed for microarray analysis. Restricted sets of genes whose expression was altered by the exposure procedure were identified (Table 3
A, B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. There are no indications of long-term nonspecific adverse health effects. Thus, basal corticosterone levels are unaffected by ethanol exposure and, if anything, show a tendency to be lower in this group when elevated drinking is found. A battery of clinical chemistry tests does not reveal any pathology. Although suppressed during the first week of ethanol exposure, weight gain in exposed subjects eventually returns to and remains normal. Finally, drinking is presumably not to alleviate withdrawal symptoms, since it is present after a prolonged recovery period. The high alcohol drinking in our model may therefore mimic the relapse in alcoholics that commonly follows prolonged sobriety.
Although nonselected rats do not readily consume enough ethanol to achieve pharmacological effects, procedures have been described to induce high ethanol drinking. These procedures mimic important aspects of alcoholism and have a predictive validity with regard to clinical efficacy of pharmacological treatments for alcoholism (17
18
19)
. However, high consumption in these models is transient, whereas it is persistent in the clinical condition. In contrast, the procedure presented here induces long-lasting high drinking and increased motivation to obtain alcohol, as shown by increased ethanol-reinforced lever pressing (7)
. It therefore may be advantageous for modeling neural processes underlying dependence.
Repeated cycles of intoxication and withdrawal mimic the clinical course of alcoholism and induce a persistent neuronal hyperexcitability in rats (20)
. Underlying molecular events are unknown but may be related to hyperactive glutamatergic signaling during withdrawal (21)
, a known signal for neuronal plasticity (3)
. Exposure-induced but not basal ethanol intake was blocked by acamprosate, a dissociation that agrees with previous reports (19)
. This supports the concept that mechanisms underlying high drinking in rats after ethanol exposure are likely to be relevant for human alcoholism. The finding that repeated cycles of intoxication and withdrawal are sufficient to induce a long-lasting high drinking phenotype may have important implications for how this disorder is managed clinically, since routine clinical care of alcohol-dependent patients involves alternating cycles of relapse and detoxifications. In view of the present data, this may be less than ideal.
A key concept of contemporary addiction research is that the brain is remodeled over the course of developing dependence, and that transcriptional regulation triggered by drug use plays a major role in this remodeling (22)
. Using DNA microarray technology, we were able to identify transcripts regulated in parallel with induction of ethanol preference. Cingulate frontal cortex and the amygdala were studied as they are part of a cortico-limbic network activated during ethanol self-administration in rats (23)
and humans (24)
; conversely, manipulations within these regions modulate self-administration of ethanol in experimental animals (see refs 13
, 25
).
We found long-term changes in expression of 19 transcripts in the cortex (Table 3A)
whereas expression of 13 genes (Table 3B)
was altered within the amygdala. Two genes, fractalkine and neuronal pentraxine receptor, were induced in both regions.
Genes associated with neurotransmission
We found frontocortical induction of the glial glutamate/aspartate transporter (GLAST or EAAT1) and of glutamatergic AMPA receptor subunit 2 (GluR2), which is expressed mainly in neurons and confers decreased Ca2+ conductivity on the receptor complex (26)
. AMPA receptor subtype 2/3 immunoreactivity is also increased in hippocampal tissue from human alcoholics (27)
. Both of the changes found in the glutamatergic system may represent counter-regulatory processes induced by increased availability of glutamate during cycles of ethanol withdrawal (21)
. These findings seem to emphasize the ability of the GeneChip strategy to identify relevant changes in gene expression, since the glutamatergic modulator acamprosate selectively inhibited exposure-induced drinking in our model. Acamprosate acts as a partial agonist at NMDA receptors, activating them at low physiological levels of glutamate and inhibiting them during high stimulation (8)
. Chronic ethanol exposure favors inhibitory effects of acamprosate (28)
, and may account for its selectivity to inhibit ethanol intake in previously exposed subjects.
Furthermore, we found increased CB1 receptor mRNA in prefrontal cortex. Chronic ethanol exposure increases formation of endocannabinoids (29
, 30)
and results in decreased CB1 binding and affinity (31)
. The CB1 receptor inhibits Ca2+ currents and cAMP production, and exerts its effects through activation of MAP kinase (32
, 33)
and transcription factor NF-
B (34)
, two genes induced in our study (see below). The CB1 antagonist SR141716A reduces responding for ethanol in genetically selected ethanol preferring (sP) rats (35)
, as well as ethanol vapor-exposed Wistar rats (36)
, and reduces breakpoints for ethanol (37)
. Together, these findings suggest that the endocannabinoid system is important in the development of ethanol dependence and that the CB1 receptor might be an interesting potential novel target for the treatment of alcoholism.
ß2-Adrenoreceptor expression was up-regulated in frontal cortex. This receptor subtype increases intracellular cAMP levels through Gs-alpha activation, but also stimulates the mitogen-activated protein kinase (MAPK) cascade. Chronic ethanol exposure uncouples the ß2 receptor from its G-protein in the frontal cortex of both rats and humans (38
, 39)
; the up-regulation found in our study could therefore represent a compensatory mechanism. We also found an up-regulation of frontocortical monoamine oxidase (MAO) A, which preferentially degrades norepinephrine and serotonin. Consistent with our finding, brain MAO A induction after long-term ethanol exposure has been observed by others (40)
. Thus, adaptive changes within monoaminergic signaling accompany the switch to an alcohol-preferring phenotype and could contribute to it.
Signal transduction
Several members of the MAPK superfamily were up-regulated in frontal cortex after intermittent ethanol exposure. These form an interconnecting network regulating the transmission of signals from cell surface to the nucleus. Activation of several neurotransmitter receptor classes—i.e., NMDA, ß2-adrenergic, and CB1—can induce MAPK, extracellular signal-related kinase (ERK), and stress-activated protein kinase (SAPK) MAPK cascades as well as cyclic AMP-responsive element binding protein phosphorylation (32
, 33
, 41
, 42)
. It has been shown that ß2-adrenergic receptor activation through the ERK/MAPK pathway can lead to increased Na+,K+-ATPase expression (43)
, and such an overexpression was indeed found in the present study. However, protein and/or functional data may need to accrue before our findings can be interpreted further, since a poor correlation between mRNA and protein levels has been reported in the MAPK pathway (44)
.
Transcription factors
MAP kinase cascades and other second messenger pathways finally converge in the nucleus on promoter elements regulating gene activity, such as the inducible transcription factor NF-B, which showed a long-term up-regulation in the cortex of ethanol exposed rats. NF-B regulates a broad range of genes and plays a pivotal role in cell death and survival pathways. In the brain, it seems to be involved in regulation of neurotrophin expression (45)
, for example, and plays a major role in the development of brain tolerance to ischemia and epileptic activity (46)
. It has been shown that protein kinase C, p38 MAP kinase, and NF-B form a cascade regulating nitric oxide (NO) synthase expression (47)
, and thereby modulate NMDA receptor activation-induced nitric oxide release (41)
. Increased NO synthase content has previously been found in the frontal cortex and nucleus accumbens of alcoholic postmortem brains (48)
. Inhibition of NO synthase also results in decreased ethanol intake in experimental animals (49)
and thus may be a mechanism through which acamprosate mediates its action.
Retinoblastoma protein Rb is a cell cycle regulator controlled by the phosphatidylinositol 3-kinase (PI3) pathway. It also seems to mediate neuronal death and differentiation (50)
. Notably, substance abuse (i.e., morphine) inhibits and antidepressant treatment induces neurogenesis in the hippocampus (51
, 52)
. The present findings prompt the question of whether cell survival in the prefrontal cortex might be similarly affected by intermittent ethanol exposure and whether up-regulation of Rb might reflect this kind of process.
Within the amygdala, we found up-regulated expression of insulin-like growth factor (IGF) 2, which, within adult CNS tissue, is expressed predominantly in the parenchymal microvasculature (53)
. In parallel, expression of IGF binding protein (IGF-BP) 2 was up-regulated, presumably as an adaptive response limiting the availability of IGF 2 at its receptors. IGF receptors are transmembrane tyrosine kinases activating the PI3 second messenger pathway. We also found an induction of the regulatory subunit of PI3-kinase within the amygdala. Two other genes altered in the amygdala may enhance PI3 signal transduction: increased phosphodiesterase I expression by reducing intracellular cAMP, which is a positive regulator of IGF-BPs (54)
; and decreased expression of Ca2+-ATPase (cytoplasmic Ca2+ pump), which in turn may lead to increased protein kinase C activity. The role of IGF/PI3 signaling in long-term amygdala responses to alcohol is unknown.
Other
Among genes regulated in our model, several may be involved in synaptic plasticity and neurodegenerative processes, and may merit mention.
The cytoplasmic protein ß-arrestin 2 is necessary for inactivation of a range of G-protein coupled receptors including ß2-adrenergic, µ-opioid, and dopamine D1A receptors (55)
. It forms a receptor/arrestin complex for uncoupling and sequestration/recycling of the receptor via endocytosis into clathrin-coated vesicles (56)
. It is thought to be involved in receptor desensitization and endocytosis of ß2-adrenergic and delta-opioid receptors (57)
and is shown to desensitize the µ-opioid receptor and determine morphine tolerance (58)
. ß-Arrestin activity seems to be regulated by phosphoinositides, in particular, IP6 (59
, 60)
.
Finally, transferrin receptor expression was induced in the cortex. Iron is an essential cofactor in neurotransmitter synthesis and in myelination (61)
. The association of disturbances in iron metabolism, iron overload, and alcohol abuse has long been recognized (62)
. The transferrin receptor promotor is activated by PI3, and this second messenger pathway is strongly involved in growth factor protection against ethanol-induced neurotoxicity (63)
.
In summary, we report that repeated exposure to ethanol is sufficient to produce long-lasting ethanol preference, accompanied by long-term transcriptional changes in key brain areas. This finding may have important implications for understanding the development of ethanol dependence and for its clinical management. It also points to a rational strategy wherein candidate genes can be identified in order to establish causal links between these phenomena and to target validated candidates for development of novel pharmacological treatment of alcoholism.
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ACKNOWLEDGMENTS |
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Received for publication August 10, 2001.
Revision received October 9, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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control; • exposed; mean±SE; n=12 vs. 16, P=0.0003; exposed vs. control) after the exposure procedure shown in Fig. 1
nonexposed, acamprosate-treated; 
exposed, acamprosate-treated.