In the current preclinical study, we combined longitudinal behavioral paradigms with longitudinal PDE10A microPET imaging, to directly evaluate in vivo changes of PDE10A activity in rats during different stages of AUD, in combination with behavioral assessments such as anxiety and decision-making. Overall, we found that two weeks of voluntary alcohol consumption induced a reversible increased PDE10A enzymatic availability in the dorsal and ventral striatum. At both week 4 of the alcohol exposure and relapse, the striatal PDE10A decrease was related to alcohol consumption and preference. Also, the rats who showed a greater future alcohol intake corresponded to those rats with an initial impaired decision-making and a lower locomotor activity, possibly due to a more anxious state, during the abstinence and relapse phase.
To induce alcohol consumption in our experimental group of rats, we used the IAE model with a 2-bottle choice paradigm, one of the most prominent and validated animal models used in ethanol research nowadays [32, 34]. The IAE model successfully induced an escalation in alcohol consumption after the first weeks of alcohol administration, following the threshold criteria of high ethanol consumption (> 4.5 g/kg/day) suggested by Carnicella et al. . Moreover, after the two weeks of abstinence, the average alcohol intake returned to the same level as before the period of abstinence, suggesting that IAE paradigm induces adaptations which are maintained after abstinence, in line with previous findings [32, 38]. However, although half of the group did not exploit high alcohol consumption during the first intake phase, observing the progress of the individual ethanol intake, a subgroup of rats clearly developed high alcohol consumption over time. So far, little is known about the predisposing behavioral traits of high alcohol intake in rats, except rats with higher impulsivity traits are known to have a higher alcohol intake . Noteworthy, in this study a low baseline rIGT performance, hence impaired initial decision-making, are related to an higher alcohol consumption over time. When looking at behavioral outcomes in function of the alcohol exposure over time; six weeks of alcohol intake had no effects on decision-making performances, while on the other hand a decreased locomotor activity (hence increased anxious behavior) was found starting from week 3 on, including the abstinence and relapse period.
Regarding alcohol consumption and PDE10A availability over time, our longitudinal [18F]JNJ42259152 PDE10A imaging data showed that two weeks of voluntary alcohol consumption induced an increased PDE10A enzymatic availability in the striatum and NuAc. We also found that this alcohol-induced PDE10A upregulation was reversible upon a longer period of alcohol administration and was mostly present in those rats that consumed a bigger amount of alcohol. A similar regional decrease in PDE10A availability towards normalization was observed after one week of relapse, extending to the lateral globus pallidum.
The observed PDE10A changes might be a result of alterations in dopamine neurotransmission and subsequent PDE10A modulation through feedback on the cAMP/PKA pathway. PDEs can promote alcohol intake through reduction of cyclic nucleotide activity . Considering the limited distribution of PDE10A to the striatal GABAergic MSNs, together with the role of PDE10A in cAMP-PKA-DARPP-32 signaling cascade and metabolism in the MSNs, several studies have demonstrated a relation between abnormal striatal dopaminergic transmission characterizing neurodegenerative and neuropsychiatric basal ganglia diseases and the loss of PDE10A enzyme levels and expression [18, 21–23, 27]. Upregulated striatal signaling, is PDE10A-regulated in processes controlling reward-motivated behaviors and furthermore, incentive salience . In the striatum the increased dopamine release upon alcohol exposure stimulates both the dopamine D1 and D2 receptor pathways in the postsynaptic density of the MSNs resulting in increased (via D1) or decreased (D2) cAMP levels. It is of central question whether our findings are mostly a consequence of increased stimulation of the direct D1 pathway, or via the opposite indirect D2 pathway resulting in the activation of Gαi/o, and cAMP inhibition. Ooms et al.  observed increased PDE10A binding after repeated D-amphetamine treatment, suggesting a potential pharmacological interaction between PDE10A enzymes and drugs modifying dopamine neurotransmission. This was suggested to be a compensation mechanism secondary to alterations of cAMP-levels being caused by increased D1 receptor stimulation over D2 stimulation. Here, we showed that PDE10A availability in the striatum and NuAc increases at the first stage of alcohol exposure (week 2) and then gradually decreases. This might indicate a short-term neuronal adaptation due to alcohol-induced increased dopamine release, which results in increased stimulation of adenylyl cyclase and thus increased cAMP levels. The MSNs react to increased cAMP levels by up-regulating cyclic nucleotide hydrolyzing enzymes. As a consequence, this leads to an increased PDE10A enzymatic availability that subsequently decreases cAMP levels. Due to the long-term chronic alcohol effect, dopamine release decreases leading to less cAMP production and to a loss of PDE10A. Since we did not observe an increased PDE10A availability during the relapse phase after abstinence, this suggests neuroadaptational changes within specific neurocircuits take place in a long-term fashion.
Preclinical studies suggested that pharmacological inhibition of PDE10A activity with TP-10 dose-dependently reduce alcohol self-administration in rats with stress history, alcohol dependence, or genetic predisposition to high levels of alcohol intake . This action may result, at least partially, from modulation of PDE10A activity in the dorsolateral striatum (DLS), the region that showed a non-recovered PDE10A loss in our findings. Furthermore, systemic TP-10 administration significantly increased dopamine turnover in the DLS and NuAc, with greater potency in the DLS . Consistently, PDE10A inhibition reduces intake of highly palatable high-fat diets in mice , supporting PDE10A is involved in the motivational regulation of highly reinforcing substances.
Our findings suggest that it may be a therapeutic target of interest at the initial stage of AUD. Further research is required to advance our understanding of these findings. There is no increase in PDE10A signal after relapse and therefore, we hypothesize an irreversible neuroadaptational change has taken place due to the MSNs down-regulating cyclic nucleotide hydrolyzing enzymes, such as PDE10A. This provides further insight into the molecular mechanisms that are at play in alcoholism and that are so crucial to be fully understood in order to tackle alcoholism as a disease.
The negative emotional state that arises during acute abstinence from alcohol exposure includes elevations in anxiety-like behavior . Previous studies showed that PDE10A may mediate the relation between stress history and elevated relapse risk . Papaverine, a PDE10A inhibitor, has been shown to reduce anxiety-like behavior . Stress history differentially increased PDE10A expression in low versus high drinking rats, with greater PDE10A expression in the prefrontal cortex correlating with greater relapse-like alcohol intake, and greater PDE10A expression in the basolateral amygdala correlating with increased alcohol preference ratios in rats . In agreement with these findings, our results showed that those rats with decreased locomotor activity over the first initial alcohol and relapse phase and therefore, increased anxiety levels, displayed reduced PDE10A changes during both the protracted alcohol exposure (4 weeks) and relapse. This indicates elevated PDE10A levels are associated with heightened anxiety-like behavior. On the other hand, anxiety is a complex collection of behaviours and aspects that cannot be entirely captured by a single test. Although it would be interesting to confirm our findings using other testing, such as the elevated plus maze (EPM) or the light/dark boxes (LDB). Factor analyses revealed that similar anxiety- and locomotion-related factors were produced by the three tests OFT, EPM and LDB applied either separately or in combination .
A limitation of our study is the small group size of animals used in this study. Increasing the number of animals would help to improve characterization of the subgroups divided by high and low alcohol consumption. Another potential limitation is the lack of the pre-exposure baseline PDE10A measurements, resulting in only limited information on studying the dynamics of PDE10A changes occurring during exposure to alcohol. Nevertheless, a sham control group scanned under the same conditions was used to investigate the effect of chronic alcohol administration.
Finally, although sham animals were not subjected to behavioral tasks, limiting therefore the interpretability of the current behavioral data, the validation of both OFT and rIGT tests was obtained in a FDG PET sub-study (data not shown).
To our knowledge, this is the first PDE10A microPET study investigating in vivo changes of PDE10A activity in rats during different stages of AUD, in relation to apparent risk factors for AUD such as decision-making and anxiety. Our findings indicate that chronic alcohol consumption induces a reversible increased PDE10A enzymatic availability in the striatum that is related to higher alcohol preference. Secondly, we showed that poor decision-making (i.e. high risk-taking and gambling-prone behavior) may be a predisposing factor for a higher vulnerability towards alcohol abuse and AUD, and decreased locomotor activity (i.e. increased anxiety) may be a consequence of chronic alcohol use. Thus, PDE10A-mediated signaling plays an important role in modulating the reinforcing effects of alcohol, and the data suggest that PDE10A inhibition may have beneficial behavioral effects on alcohol intake.