Abstract Introduction Drug reinforcement, a form of behavioral plasticity in which behavioral changes happen in response to a reinforcing drug, would finally lead to drug addiction after chronical drug exposure. Drug reinforcement is affected by genetic and environmental factors. Social hierarchy has been reported to regulate drug reinforcement and drug-seeking behaviors, but the underlying molecular mechanism is almost unknown. Methods We take advantage of the tube test to assess the social hierarchy between two co-housed rats. And then, we investigated the drug reinforcement between dominant and subordinate rats via conditioned place preference (CPP). Then we adopted 4-D label-free mass spectrometry to explore the complex phosphoproteome in the nucleus accumbens (NAc) between dominant and subordinate rats. Functional enrichment, protein-protein, motif analysis and kinase prediction interaction analysis were used to investigate the mechanism between substance use disorder and social hierarchy. Specifically, we identified histone deacetylase 4 (HDAC4) which has been previously shown to play critical roles in drug addiction as a key node protein by phosbind-SDS. Finally, we forcibly altered the social hierarchy of rats through behavioral training, follow by which we accessed the HDAC4 phosphorylation levels and drug reinforcement. Results In this study, we found that methamphetamine exhibited stronger reinforcement in the subordinate rats. We identified 660 sites differing between dominant and subordinate rats via 4-D label-free mass spectrometry. Functional enrichment and protein-protein interaction analysis revealed that synaptic remodeling related pathways and substance use disorder related pathway are significantly characterized by social hierarchy. Motif analysis and kinase prediction showed that CaMKIIδ and its downstream proteins maybe the central hub. Phosbind-SDS revealed that higher HDAC4 phosphorylation levels in dominants. After the social hierarchy of rats were forcibly altered by behavioral training, the differences in HDAC4 phosphorylation levels induced by social hierarchy were eliminated, correspondingly the drug reinforcement is also reversed between the two group rats. Discussion In conclusion, our research proves that protein phosphorylation in the NAc may be a vital link between social hierarchy and drug reinforcement. Keywords: social hierarchy, drug reinforcement, phosphoproteomics, drug addiction, HDAC4 Introduction Addiction is a chronic and relapsing disorder characterized by compulsive seeking and taking of a drug, even in the presence of adverse consequences ([36]Koob and Volkow, 2016; [37]Nestler and Landsman, 2001). However, drug use does not inevitably lead to addiction; only 15%–17% of individuals who use drugs progress to this condition ([38]Swendsen and Le Moal, 2011). The transition from drug use to addiction is influenced by a complex interplay of factors, including drug pharmacology, social and environmental contexts, and genetic and epigenetic vulnerabilities ([39]Bardo et al., 2013a; [40]Volkow et al., 2019). Among these, social hierarchy—a ubiquitous phenomenon in both humans and animals that influences physiology and health ([41]Sapolsky, 2005) and governs access to critical resources such as food and mates—has garnered significant attention ([42]Milewski et al., 2022). Evidence suggests that individuals like monkey occupying lower social ranks, or subordinates, are more prone to drug abuse ([43]Gould et al., 2017; [44]Morgan et al., 2002; [45]Nader et al., 2008), although the mechanisms underlying this increased vulnerability remain poorly understood. The nucleus accumbens (NAc), a key hub in the regulation of addiction, motivation, and social hierarchy,is the site where all drugs with addictive potential increase dopamine (DA) levels, either directly or indirectly. This increase activates low-affinity dopamine D1 receptor medium spiny neurons (MSNs) ([46]Castro and Bruchas, 2019; [47]Choi et al., 2024; [48]Volkow et al., 2019). Additionally, the NAc is well-known as an interface between motivational salience and behavioral output. Functional changes in the NAc caused by drug abuse continue to evolve during the abstinence stage, contributing to drug-seeking behavior and relapse due to emotional and motivational alterations ([49]Schall et al., 2021). Neurons from the medial prefrontal cortex (mPFC) that project to the NAc (mPFC-NAc) encode social winning behavior ([50]Choi et al., 2024). Recent studies have also revealed that the metabolic profile of the NAc is associated with social hierarchy ([51]Larrieu et al., 2017). Protein phosphorylation is a critical post-translational modification (PTM) that regulates diverse cellular processes, including signal transduction, transcription, apoptosis, and autophagy, and is implicated in various neuropsychological disorders ([52]Bilbrough et al., 2022). It also plays a pivotal role in molecular networks underlying drug addiction ([53]Lee and Messing, 2008). The phosphorylation of membrane receptors, such as μ-opioid peptide receptors (MOR) and AMPA receptors, modulates their activity and signal transduction efficiency, thereby influencing drug tolerance and addictive behaviors ([54]Cadet et al., 2015; [55]Duarte and Devi, 2020; [56]Kibaly et al., 2016). For instance, the phosphorylation of GluA1-Ser831by CaMKII in the caudate and putamen (CPu) is essential for behavioral sensitization following nicotine exposure in rats ([57]Kim et al., 2022). Protein kinases and phosphatases are central to maintaining phosphorylation homeostasis. Notably, numerous studies have demonstrated that drugs of abuse can activate protein kinases, leading to alterations in gene transcription and protein synthesis ([58]Bernstein et al., 2024). These molecular changes contribute to long-term modifications in synaptic function and neural circuitry, ultimately driving the development and persistence of addiction ([59]Lee and Messing, 2008). Recent studies highlight a significant interplay between social hierarchies and protein phosphorylation. For example, subordinate mice exhibit increased phosphorylation of the NMDA receptor subunit GluN2A and extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain ([60]Yoshida et al., 2022). Similarly, subordinate rainbow trout display heightened activity of liver AMP-activated protein kinase (AMPK), evidenced by an elevated phosphorylated AMPK ratio ([61]Gilmour et al., 2017). Moreover, subordinate rats subjected to repeated defeat stress show increased ERK phosphorylation in the ventral tegmental area (VTA), a key brain region implicated in reinforcement and reward processes ([62]Yap et al., 2015). These findings suggest that protein phosphorylation is influenced by social rank and may intersect with behaviors linked to drug addiction ([63]Yap et al., 2015). What’s more, protein phosphorylation profiles have been identified play a role in drug memory extinction and reconsolidation in the NAc ([64]Torregrossa et al., 2019). Research has shown that protein phosphorylation profiles related to cocaine memory extinction and reconsolidation change in the NAc, but there is no evidence indicating changes in protein phosphorylation profiles in the VTA or prefrontal cortex in the context of drug addiction ([65]Choi et al., 2024). However, a comprehensive understanding of the phosphorylation landscape within the context of social hierarchies remains largely uncharted. In this study, we utilized 4D label-free LC-MS to investigate the proteomic and phosphoproteomic profiles in the NAc of dominant and subordinate animals. While no significant differences were observed in overall protein expression between dominant and subordinate rats, the phosphoproteomic profiles revealed substantial variation. Furthermore, methamphetamine (Meth) demonstrated a more pronounced reinforcing effect in subordinate rats compared to dominant rats at the same dosage. Notably, differentially phosphorylated proteins were enriched in pathways associated with synaptic transmission and neural plasticity, both of which are critical in the development of drug addiction. To validate the phosphoproteomic findings, Phosbind SDS-PAGE analysis was performed. Additionally, behavioral interventions aimed at reversing social hierarchy were shown to modulate both drug reinforcement and protein phosphorylation in rats. Materials and methods Animals Male Sprague-Dawley rats, aged 5–6 weeks, were obtained from Charles River (Beijing, China) for this study. The animals were housed under controlled conditions at a temperature of 22°C–25°C with a 12-hour light–dark cycle. All experimental procedures and animal care protocols were approved by the Animal Care and Use Committee of the Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS). Tube test The tube test, adapted from the previous deviously described method ([66]Fan et al., 2023), was used to assess social hierarchy in rats. Starting at 5–6 weeks of age, animals were pair-housed with bodyweight-matched conspecifics for 2 weeks and handled daily for 7 days prior to testing. An 80-cm acrylic tube with a 60 mm internal diameter was used. Rats were trained to traverse the tube alone, completing 10 trials per day over 2 days, with counterbalanced starting positions. For formal testing, two mice were introduced at both ends and released as they meet in the middle of the tube. The rat that retreated or was pushed out was classified as the “subordinate” or “loser”, while the other was the “dominant” or “winner”. Each pair underwent 6 tests per day for 6 days, with social rank determined by the number of wins. Only pairs with consistent results across at least three consecutive days were included in subsequent experiments. Pairs with inconsistent outcomes were excluded. Forced win/loss After establishing stable social ranks among paired rodents, rats were subjected to a forced win/loss protocol. This involved blocking the side of subordinate rats in the tube, leaving them with no choice but to push forward against their dominant counterpart. In cases where pairs remained locked in a standoff for more than 10 min, gentle force was applied behind the subordinate to encourage them to push the dominant rat out of the tube. This forced win/loss regimen consisted of 4 trials daily for a minimum of 2 weeks, continuing until the original subordinate rats could achieve wins without intervention. Rats that failed to alter their social rank after the forced win/loss sessions were excluded from subsequent formal experiments. Conditioned place preference (CPP) The place preference apparatus comprised two distinct conditioning environments. Each conditioning environment measured 30 (L) × 30 (W) × 30 (H) cm. One environment had a floor consisting of smooth acrylic sheet and walls with alternating 2 cm wide black and white stripes. For another, the floor was rough acrylic sheet, and walls was black and white squares. Rats were handled and weighed the day before the start of an experiment. The activity of each rat was recorded and analyzed by AnyMaze software. On day 1, all rats were placed individually in the conditioned place preference apparatus and allowed to freely explore the entire apparatus for 30 min for acclimatization. On day 2 and 3, Animals were allowed to explore both sides of a custom-designed conditioned place preference (CPP) apparatus for 15 min to establish baseline preferences. Any rat that spent <20% or >80% of the entire