Alcohol consumption accounts for 4.7% of annual global deaths and for 5.1% of the worldwide disease burden (World Health Organization, 2024). Major depression (MD) ranks as the second leading cause of years lived with disability, contributing to 8% of this parameter at the global level (Vos et al., 2015). Collectively, alcohol use disorder (AUD) and MD contribute to half of the global disease burden caused by mental and substance use disorders (Whiteford et al., 2013). There is a notably high comorbidity between AUD and MD (Brière et al., 2014). Alcohol-related issues in individuals with MD are linked to a more severe course of depression, relapse, heightened risk of suicide and death, and increased healthcare use (Sullivan et al., 2005). Conversely, depressive symptoms frequently occur in AUD, with over one-third of AUD patients meeting the diagnostic criteria for MD at some point in their drinking history (Schuckit, 2006). Compared to those with only one disorder, individuals with co-occurring AUD and MD face a higher risk of alcohol-relapse and dependence, dropping out of treatment, attempting suicide, and experiencing poorer outcomes with antidepressant treatment (Brière et al., 2014; Hasin et al., 2002; Nunes and Levin, 2004).

Research suggests that both AUD and MD, when considered independently, are associated with various immune alterations. However, there is limited understanding of the role neuroimmune function plays in the onset and progression of comorbid AUD and MD. For instance, binge drinking patterns are particularly linked to depression (Boden and Fergusson, 2011), though the precise neurobiological mechanisms underlying this alcohol-induced depression remain unclear. Alcohol significantly impacts the immune system, altering the expression of inflammatory mediators both in the periphery and the central nervous system (CNS). Heavy alcohol use renders the gut wall “leaky”, allowing microbial products like lipopolysaccharides (LPS) to enter the bloodstream (Bala et al., 2014; Enomoto et al., 2000; Keshavarzian et al., 2009; Szabo, 2015). This leaked LPS exacerbates alcohol-induced liver inflammation and activates immune cells, triggering the release of pro-inflammatory cytokines like interleukins IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) (Crews and Vetreno, 2016). Cytokines and chemokines produced in the periphery can relay signals to various brain regions, further activating microglia and astrocytes to produce CNS cytokines. The production of these brain cytokines is regulated by NF-κB pathways (Flores-Bastías and Karahanian, 2018). Notably, immune activation in the brain caused by alcohol persists for several months after withdrawal (Qin et al., 2007; 2008). Increased levels of proinflammatory cytokines are observed in the ventral tegmental area, hippocampus, and amygdala in the brains of people with alcohol use disorders (He and Crews, 2008), all of which are critical areas involved in reward, emotion, and behavior. On the other hand, the initial discovery of immune dysfunction in depressive disorders (Maes et al., 1990) gave rise to extensive research that has confirmed that inflammation plays a role in the development and progression of depression. Numerous studies have consistently shown positive associations between MD and inflammatory markers like C-reactive protein (CRP) and IL-6 (Haapakoski et al., 2015; Stewart et al., 2009), IL-1β (Howren et al., 2009), and TNF-α (Dowlati et al., 2010; Y. Liu et al., 2012). Furthermore, cytokine levels in patients with depression have been found to normalize following treatment (Kim et al., 2007).

Several studies have shown reduced serum levels of brain-derived neurotrophic factor (BDNF) in patients with MD compared to healthy individuals (Brunoni et al., 2008; Molendijk et al., 2014; Sen et al., 2008), and evidence also exists to support renormalization of BDNF levels upon successful anti-depression interventions (Brunoni et al., 2008; 2014). BDNF has also been implicated in alcohol dependence and withdrawal (Davis, 2008), and its levels are altered in AUD individuals (Heberlein et al., 2010). After chronic alcohol consumption, BDNF gene expression decreases in the prefrontal cortex (PFC) (Logrip et al., 2009; Melendez et al., 2012). Neuroinflammation and the overexpression of pro-inflammatory cytokines in the CNS can reduce BDNF levels (Flores-Bastías et al., 2020; Guan and Fang, 2006). Thus, decreased BDNF levels are a common factor between MD and AUD, which could be an important cause of the link between the two conditions.

Since neuroinflammation could play an important role in depression triggered by excessive alcohol consumption, the identification of neuroimmune targets to address this condition is highly desirable. In this regard, in recent studies we have shown that the administration of fenofibrate during the withdrawal stage after chronic alcohol consumption in rats reverses ethanol-induced neuroinflammation and brain oxidative stress, along with an 80% decrease in alcohol consumption at relapse (Ibáñez et al., 2023; Villavicencio-Tejo et al., 2021). Fenofibrate is a synthetic agonist that activates peroxisome proliferator-activated receptor alpha (PPAR-α), which negatively regulates NF-κB activity decreasing neuroinflammation (Collino et al., 2006). Another effect of PPAR-α activation is to increase peroxisomal activity of fatty acid degradation, which is why this drug is approved by the FDA and is being used for several years in the clinic for the treatment of dyslipidemia (Cignarella et al., 2006). PPAR-α agonists have been reported in animal models of depression induced by social isolation (B. Jiang et al., 2017), stress (B. Jiang et al., 2015; Ni et al., 2018; Scheggi et al., 2016), and LPS administration (Yang et al., 2017), however the effect of PPAR-α agonists on alcohol-induced depression had not been studied so far. Therefore, given the impact of neuroinflammation in MD, in this work we hypothesized that fenofibrate administration during the withdrawal stage following chronic alcohol consumption in rats will have a positive effect on ethanol-induced depression-associated symptoms.

Given the high rate of comorbidity between AUD and MD, and that both conditions are themselves strongly related to neuroinflammatory processes, in this study we evaluated the ability of fenofibrate -a drug that we had already demonstrated its anti-inflammatory capacity in the brain of rats treated with ethanol (Ibáñez et al., 2023; Villavicencio-Tejo et al., 2021)- to decrease behavioral symptoms associated with ethanol-induced depression, normalizing BDNF expression and dendritic arborization in the hippocampus and PFC.

In this study, we opted for i. p. administration of alcohol to Sprague-Dawley rats as one of the available methods to ensure that all animals are exposed to the same ethanol dose. In various studies of alcohol-induced depression and anxiety in murine models, other methods have been reported with the same objective: exposure to ethanol vapor (Hartmann et al., 2020; Yao et al., 2021) or exclusive provision of a liquid diet containing alcohol (Huang et al., 2010). In all these studies, the animals developed depressive-like behaviors similar to those reported here, and similar to voluntary consumption studies (see a current review by Mashayekhi-Sardoo et al., 2025).

To assess behavioral symptoms, we first evaluated animals treated with ethanol and fenofibrate using the open field test, designed to measure the levels of anxiety (Gould and Dao, 2009), which is one of the hallmarks of MD. Ethanol treatment for 3 weeks markedly decreased the time that animals spent in the center of the open field box compared to their own basal levels prior to ethanol exposure (Figures 1A–C). We had anticipated this observation, since ethanol treatment and subsequent withdrawal is known to increase the levels of anxiety in both rats and mice (Kliethermes, 2005). It can also be observed in Figure 1A that the control animals gradually increased the time spent in the center on the second and third occasions in which they were subjected to the open field test. This same observation was made (although it does not reach a statistically significant difference) in the fenofibrate-treated group. This could reflect the knowledge and habituation to the test box, which leads to less fear and anxiety in exploring it compared to the first time. Interestingly, fenofibrate administration for the last 5 days of the 2-week withdrawal stage increased time spent in the center to levels that were similar to pre-EtOH measure in the same group. These results would indicate that fenofibrate was able to reverse the anxiety-associated depressive symptoms generated by alcohol administration. Furthermore, the average displacement speeds do not show a reduction due to the ethanol treatment, and even increased post-EtOH, which suggests that treatment with alcohol or fenofibrate does not produce musculoskeletal biases that could account for the observed results (Figure 1D).

In the sucrose intake test, all three groups of rats decreased sucrose consumption in the post-EtOH (or saline for controls) stage compared to their basal sucrose consumption prior to alcohol treatment (Figure 1E). The decrease in the alcohol-treated groups was expected given the known anhedonia generated by alcohol exposure (Boutros et al., 2014; Olney et al., 2018), However, the control group also decreased its sucrose consumption One explanation could be the isolation to which the animals were subjected after the recording of initial basal sucrose consumption; it has been reported that rats raised in individual cages have lower amounts of liking responses to sucrose compared to those raised with environmental enrichment (Wukitsch et al., 2020). Interestingly, both alcohol-treated groups (but not the controls) markedly reduced sucrose consumption after withdrawal compared to the pre-withdrawal stage, regardless of whether they received fenofibrate or not. In this line, there are reports in both humans and animal models that the levels of depression induced by alcohol are even higher during protracted abstinence than immediately after chronic consumption (Oliva et al., 2018; Walker et al., 2010). With these results we can conclude that anhedonia was triggered mainly after withdrawal following alcohol treatment, and that fenofibrate was not able to reverse it in our experimental model. The explanation for why fenofibrate showed no effect in the sucrose intake test is not clear; one possibility is that administration of fenofibrate during only the last 5 days of the 2-week withdrawal phase was insufficient to generate the neurobiological changes associated with sucrose reward (e.g., normalization of the dopaminergic tone in the brain reward circuit). Experiments to assess whether fenofibrate administration normalizes dopamine levels in the nucleus accumbens of rats chronically treated with alcohol are currently underway. These results should be taken into consideration when considering fenofibrate’s translational potential for the treatment of alcohol-induced depression. We believe that longer treatment with fenofibrate during the abstinence period could show better results against ethanol-induced anhedonia.

The third behavioral test corresponded to the tail suspension test, where we obtained results practically identical to those of the open field test (Figure 1F): ethanol treatment increased the time that animals spent immobile hanging by the tail, and fenofibrate reduced immobility time to similar levels as pre-EtOH. This high concordance would somehow validate the results obtained with one or the other test. Our results are consistent with previous reports where ethanol treatment increases immobility time in the tail suspension test in rats and mice, and the administration of anti-inflammatory and antioxidant molecules or proteins reversed this effect of ethanol (Ebokaiwe et al., 2023; X. Jiang et al., 2023).

Regarding proinflammatory cytokine levels, as we had previously reported (Ibáñez et al., 2023), fenofibrate treatment during withdrawal decreased the expression of the proinflammatory cytokines TNF-α and IL-1β in the hippocampus and PFC, while IL-6 decreases only in the hippocampus (Figure 2). The relatively smaller response in the PFC, when compared to the hippocampus may be because the PFC has shown a noticeably lower immunologic reaction to alcohol administration (Villavicencio-Tejo et al., 2021). Similarly, it was reported that ethanol administration does not significantly elevate the expression of pro-inflammatory cytokines such as chemokine C-C motif ligand 2 (CCL2), IL-6, and TNF-α in the mouse cerebral cortex (Kane et al., 2014). Similar to what we reported in a recent study (Ibáñez et al., 2023), for some genes and in certain brain areas, fenofibrate reduced the expression of some proinflammatory cytokines to levels even lower than in controls. We do not have a precise explanation for this consistent observation, but we can attribute it to the potent anti-inflammatory effect of fenofibrate, since it acts by inhibiting the activity of NF-κB (Collino et al., 2006), the master transcription factor for the activation of the innate immune response.

As previously reported in other studies (Logrip et al., 2009; Melendez et al., 2012), in our model ethanol treatment also reduced BDNF expression in PFC (Figure 3). In this work, treatment with fenofibrate during the withdrawal stage fully normalized the expression of this neurotrophin. These results are in line with several studies that reported, in models of depression other than that induced by alcohol, that PPAR-α agonists were able to increase the expression of BDNF (B. Jiang et al., 2015; 2017; Ni et al., 2018; Yang et al., 2017), and that this effect would be directly related to the decrease in neuroinflammation (Yang et al., 2017). Unlike our experimental model, in many of these studies the treatment with the different PPAR-α agonists was performed prior to or during the induction of depressive symptoms so this would limit, in our opinion, the translational options. In contrast, in the studies reported here, fenofibrate treatment was initiated during withdrawal once depressive symptoms had already been induced by alcohol exposure. On the other hand, alcohol treatment slightly decreased BDNF expression levels in the hippocampus, although the difference did not reach statistical significance (Figure 3). Unlike PFC, where decreased BDNF expression by chronic alcohol exposure has been reported (reviewed by (Logrip et al., 2015), the effect on BDNF levels in the hippocampus is still poorly understood. There are even reports indicating that BDNF levels are not reduced by ethanol exposure in this brain area (Darcq et al., 2015; Zhang et al., 2000). However, in the present work fenofibrate treatment still was able to increase BDNF expression in the hippocampus (Figure 3).

The effects of fenofibrate on dendritic arborization (Figure 4) are closely related to its effects in increasing BDNF levels decreased by ethanol exposure (Figure 3). One of the main transcriptional factors involved in dendritic morphology and synaptic plasticity is cAMP response element-binding protein (CREB) (Bibel and Barde, 2000). Since the BDNF signaling pathway culminates in the activation of CREB, this would explain why fenofibrate was able to normalize arborization levels especially in the PFC rather than in the hippocampus. This is especially important, since a strong inverse relationship between severity of depression symptoms and the number of synapses exists (Holmes et al., 2019).

One question that may arise is whether 2 weeks of abstinence could be a sufficiently long period of time to reverse by itself all the effects induced by the previous exposure to ethanol. This did not turn out to be the case, since animals exposed to ethanol and not treated with fenofibrate, after abstinence, maintained the deleterious effects in the behavioral tests, expression of proinflammatory cytokines, and reduction in the expression of BDNF and level of dendritic arborization in PFC. These observations agree with our recent study where 2 weeks of abstinence do not reverse per se the severity of relapse, expression of proinflammatory cytokines and levels of oxidative stress in the brain (Ibáñez et al., 2023).

Additionally, we should note that some of the behavioral and molecular changes produced by fenofibrate do not exactly match, as there is a full rescue of the behavioral changes but in some cases the molecular changes are partial or do not occur (e.g., in PFC the normalization of dendritic arborization is not statistically significant, or in hippocampus there are no changes at all (Figure 4). We believe that this may reflect that the rescue of the observed behavioral changes may be the result of a combination of several factors, such as the notable decrease in the expression of proinflammatory cytokines, the normalization of BDNF expression in PFC, etc., so it is feasible that a particular variable analyzed does not show an exact correlation with the observed behavioral variations.

Finally, we acknowledge that we did not include a control group treated only with fenofibrate, which constitutes a limitation of the present study. However, there is evidence that fenofibrate alone does not produce effects on depression-related behaviors; for example, Scheggi et al. (2016) showed that fenofibrate did not modify appetitive motivation for sucrose in rats not exposed to a chronic stress protocol (controls). Fenofibrate is widely used as a lipid-lowering agent in humans, and its adverse effect profile is well documented. Depressive symptoms have not been reported as a frequent or consistent adverse effect in clinical studies or in meta-analyses of fibrate treatments. In databases such as the FDA, EMA, or VigiBase, spontaneous reports of depression as an adverse event associated with fenofibrate use are very rare and anecdotal, and have not been considered statistically significant.

Overall, in this work we provide evidence that would allow glimpse fenofibrate as an adjunct pharmacological therapy for the treatment of depression induced by alcohol abuse. In this sense, fenofibrate has been clinically used worldwide for decades since it was approved in 1994 by FDA for the treatment of dyslipidemia.