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. 2016 Mar 15;113(11):3024-9.
doi: 10.1073/pnas.1506012113. Epub 2016 Feb 22.

Convergent evidence from alcohol-dependent humans and rats for a hyperdopaminergic state in protracted abstinence

Natalie Hirth  1 , Marcus W Meinhardt  1 , Hamid R Noori  1 , Humberto Salgado  2 , Oswaldo Torres-Ramirez  2 , Stefanie Uhrig  1 , Laura Broccoli  1 , Valentina Vengeliene  1 , Martin Roßmanith  1 , Stéphanie Perreau-Lenz  1 , Georg Köhr  1 , Wolfgang H Sommer  3 , Rainer Spanagel  1 , Anita C Hansson  4
Affiliations

Convergent evidence from alcohol-dependent humans and rats for a hyperdopaminergic state in protracted abstinence

Natalie Hirth et al. Proc Natl Acad Sci U S A. .

Abstract

A major hypothesis in addiction research is that alcohol induces neuroadaptations in the mesolimbic dopamine (DA) system and that these neuroadaptations represent a key neurochemical event in compulsive drug use and relapse. Whether these neuroadaptations lead to a hypo- or hyperdopaminergic state during abstinence is a long-standing, unresolved debate among addiction researchers. The answer is of critical importance for understanding the neurobiological mechanism of addictive behavior. Here we set out to study systematically the neuroadaptive changes in the DA system during the addiction cycle in alcohol-dependent patients and rats. In postmortem brain samples from human alcoholics we found a strong down-regulation of the D1 receptor- and DA transporter (DAT)-binding sites, but D2-like receptor binding was unaffected. To gain insight into the time course of these neuroadaptations, we compared the human data with that from alcohol-dependent rats at several time points during abstinence. We found a dynamic regulation of D1 and DAT during 3 wk of abstinence. After the third week the rat data mirrored our human data. This time point was characterized by elevated extracellular DA levels, lack of synaptic response to D1 stimulation, and augmented motor activity. Further functional evidence is given by a genetic rat model for hyperdopaminergia that resembles a phenocopy of alcohol-dependent rats during protracted abstinence. In summary, we provide a new dynamic model of abstinence-related changes in the striatal DA system; in this model a hyperdopaminergic state during protracted abstinence is associated with vulnerability for relapse.

Keywords: alcoholism; dopamine release; in silico analysis; postmortem brain tissue; translational studies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Expression analysis of the dopaminergic system in postmortem brains of heavy alcoholics suggests a hyperdopaminergic state. (A–C) Bar graphs showing quantitative analysis of D1 receptors (A), D2-like receptors (B), and DAT-binding sites (C) in postmortem striatal brain sections of heavy alcoholics (red bar in A, blue bar in B, green bar in C) and controls (white bars). D1 and DAT are strongly decreased in striatal brain regions, but D2 is not altered. Data are expressed as means (expressed in femtomoles per milligram) ± SEM, n = 8 or 9 per group. (D) Schematic illustration showing a coronal section of the human striatal forebrain region including the NC and VS (including the accumbens).
Fig. S1.
Fig. S1.
D1 receptor-binding sites are strongly decreased in striatal postmortem tissue of human alcoholics. The bar graphs show D1 receptor-binding sites as measured by [3H]-SCH23390 autoradiography of controls (white bar) compared with not-intoxicated (hatched bar) and intoxicated (black bar) alcoholic subjects. No differences have been detected between not-intoxicated and intoxicated alcoholics. Statistics was performed by ANOVA followed by Fisher’s LSD post hoc test (**P < 0.01, ***P < 0.001 vs. controls). Data (in femtomoles per milligram) are expressed as mean ± SEM; n = 9–26 per group.
Fig. 2.
Fig. 2.
Analyses of the dopaminergic system in alcohol-dependent rats reveals a hyperdopaminergic state in long-term abstinence. (A) The time course of DA release in the Acb was modeled by a meta-analysis of 192 rats derived from 16 animal studies by continuous interpolation of the averages of experimental values with respect to the time of measurement during abstinence. This quantitative analysis suggests a robust pattern of dynamic changes in DA concentrations. During the first 6 d of withdrawal, the DA concentrations decline to 30% of the baseline concentrations (hypodopaminergic state) but then increase again to a hyperdopaminergic state. (B–D) Regulation of D1 receptors (red bars), D2-like receptors (blue bars), and DAT-binding sites (green bars) at different days of abstinence in the AcbS (B), AcbC (C), and CPu (D) of alcohol-dependent rats vs. control rats (set as 0% baseline at each time point). Rats were intermittently exposed to ethanol vapor for 7 wk and were killed immediately after the last exposure cycle (on day 0) or after 1, 3, 7, or 21 d of abstinence. D1 and DAT are dynamically increased and decreased at different times during abstinence, but D2-like binding levels remain unaffected. Statistical analysis was performed by two-way ANOVA followed by Fisher’s post hoc test. Data are expressed as percent of controls ± SEM, n = 4–8 per group. For expression levels in controls at each time point, see Table S3. The shaded areas in the figure indicate a hypo- or a hyperdopaminergic state during abstinence.
Fig. S2.
Fig. S2.
Meta-analysis predicts a hyperdopaminergic state during protracted alcohol abstinence. DA and its metabolites DOPAC and HVA were investigated during alcohol abstinence by a meta-analysis. During the first 6 d of abstinence DA, HVA, and DOPAC concentrations decline by up to 30% of the baseline condition (hypodopaminergic state). Afterward, concentrations rise above baseline levels (hyperdopaminergic state). The inset shows the dynamic regulation of DA, DOPAC, and HVA during the first 24 h of abstinence.
Fig. S3.
Fig. S3.
Representative images showing D1 ([3H]SCH23390), D2-like ([3H]raclopride), and DAT ([3H]mazindol) binding (T; total binding) on a coronal striatal rat brain section. Nonspecific (NS) binding was determined on adjacent section by adding flupenthixol (D1), sulpiride (D2-like), and nomifensine (DAT) to radioligand.
Fig. 3.
Fig. 3.
The hyperdopaminergic state in 3-wk alcohol-abstinent rats. DA microdialysis displays increased DA levels and a blunted response to ethanol treatment in alcohol-dependent rats. (A) Basal extracellular DA levels within the AcbS are markedly increased in alcohol-dependent rats (n = 15 per group). (B) AcbS DA levels after the application of consecutive doses of ethanol (1 or 2 g/kg, i.p.). Control animals show an increase in extracellular DA levels after ethanol (2 g/kg, i.p.), whereas alcohol-dependent rats show a blunted response to the treatment (n = 8 per group). (C and D) Hyperlocomotion in 3-wk-abstinent rats was detected by records of locomotor activity in the open field (C) and home cage (D). The bar graph in C represents the total track length measured over a 60-min interval in the open-field setting. The graph in D represents the number of body movements per hour during a 72-h period detected using a home cage e-motion system. For the respective time courses, see Fig. S4. (E) Enhanced ethanol cue-induced reinstatement in abstinent rats compared with controls. The graph shows the mean number of responses after the presentation of stimuli previously paired with ethanol. (F) Representative EPSCs recorded at −80 mV in MSNs were evoked by electrical stimulation in the AcbS before (baseline) and during perfusion with 25 mM ethanol or 25 mM ethanol plus 5 μM SKF81297. Current traces represent the average of 10 sweeps. (G) Time courses of the effects shown in F for normalized EPSCs. (H) Summary of the effects on EPSCs (control, n = 12; alcohol-dependent, n = 7). Data are expressed as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline or vs. ethanol; #P < 0.05 alcohol-dependent vs. control.
Fig. S4.
Fig. S4.
Increased locomotor activity and TH expression in 3-wk-abstinent rats. (A) The graph represents 5-min intervals in the open-field setting. *P < 0.05, **P < 0.01. (B) The bar graph shows total track length measured during a 60-min interval in alcohol-dependent and control rats. *P < 0.05. (C) Home cage activity was monitored during a 72-h period using a home cage e-motion system. The hyperactivity in the home cage follows a circadian pattern, i.e., is evident only during the active phase of the rats. (D) The bar graph represents the number of body movements per hour during a 72-h period in alcohol-dependent and control rats. **P < 0.01. The bar graphs in B and C are also shown in Fig. 3. (E) In situ hybridization for TH mRNA revealed increased transcriptional levels in the SNc but no change in the VTA in alcohol-abstinent as compared with control rats. **P < 0.01, alcohol-dependent vs. control rats. There appears to be an increase in habituated activity but not in exploratory activity. The former could reflect hyperactivity of nigrostriatal rather than mesolimbic DA systems; this possibility also is supported by the increase in TH mRNA in the SNc, whereas no difference was observed in the VTA. However, the latter finding is difficult to interpret, because in the VTA, as opposed to the SN, the lack of distinct segregation of cells into TH+ and TH− fractions could lead to a dilution of the measured TH signal and could reduce the likelihood of detecting an effect. Also, changes in TH mRNA provide no direct evidence for altered TH synthesis/availability in terminals.
Fig. 4.
Fig. 4.
A hyperdopaminergic state in DAT N157K mutant rats is associated with hyperlocomotion and increased alcohol consumption. (A and B) Quantitative analysis of DAT ([3H]-mazindol)-binding sites (A) and D1 ([3H]-SCH23390)-binding sites (B) (expressed in femtomoles per milligram) in the Acb of WT and DAT N157K mutant rats. (C) Basal extracellular DA levels (expressed in femtomoles per microliter) in the Acb of WT and DAT N157K mutant rats. (D) Total track length (in meters) during 60-min testing in the open field in WT and DAT N157K mutant rats. (E) Intake of total ethanol (expressed in grams per kilogram body weight per day) in WT and DAT N157K mutant rats during 6 wk of continuous concurrent access to water and 5%, 10%, and 20% ethanol solutions. Data are expressed as means ± SEM; *P < 0.05, ***P < 0.001 vs. WT.
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