Methamphetamine (MA) is a powerful psychostimulant characterized by significant reinforcing and abuse potential, with its prominent rewarding effects contributing to a high addictive capacity, making it one of the most extensively abused illicit substances globally (Jayanthi et al., 2021). The conditioned place preference (CPP) paradigm, as a key model in understanding addiction, is widely used in drug-associated memory studies, designed to reflect rewarding environmental connections (Jayanthi et al., 2021; Chen et al., 2023; Nestor et al., 2023). The acquisition phase in the CPP model reflects the initial formation of addiction-related memory, which provides the foundation for the subsequent consolidation of CPP memory. However, studies have mainly focused on the maintenance and relapse of drug-associated memory, whereas comparatively less emphasis has been placed on the acquisition phase. The mechanism that associates drugs with the environment during the acquisition phase is still unclear. Therefore, identifying the neural mechanisms underlying the acquisition phase of MA-induced CPP is of enormous significance in elucidating the formation of addiction memory and providing a theoretical basis for interventions to prevent relapse.

Drug addiction is accompanied by significant disruption to motivational and decision-making systems, especially in the dysregulation of reward, stress, and executive function networks (Murakami et al., 2015). The medial prefrontal cortex (mPFC) is associated with these long-term neuroadaptive changes, playing a crucial role in regulating reward learning and drug-associated behaviors (Koob and Volkow, 2016; Ceceli et al., 2022; Nahar et al., 2021b). As a critical subregion of the mPFC, the prelimbic cortex (PL) plays a critical role in regulating emotion, addictive memory encoding, relapse-related responses, and drug-seeking behaviors (Dalley et al., 2004; Green and Bouton, 2021; Zheng et al., 2025). At the circuit level, the function of the PL relies on coordinated interactions between excitatory and inhibitory neurons, with inhibitory neurons playing a key role in receiving, consolidating, and encoding information to shape output signaling (Hattori et al., 2017). In animals conditioned with cocaine, GABAergic interneurons within the PL are preferentially activated during CPP expression, suggesting a critical role in mediating drug-associated memory retrieval (Miller and Marshall, 2004). Among these inhibitory populations, parvalbumin-positive(PV+) interneuron is one of the primary inhibitory neurons in PL, showing distinct inhibitory effects on excitatory neurons (Kawaguchi and Kubota, 1997). In addition, morphine has been shown to suppress inhibitory synaptic transmission from PV+ interneurons to pyramidal neurons in the PL via the activation of μ-opioid receptors (MORs) expressed in PV+ interneurons, thereby altering local cortical excitability (Jiang et al., 2021). However, whether the PL and its PV+ interneurons contribute to encoding the initial formation of MA-induced CPP memory during the acquisition phase of CPP remains largely unexplored.

PNNs are specialized extracellular matrix structures composed of chondroitin sulfate glycosaminoglycans, forming dense nets surrounding fast-spiking inhibitory interneurons, particularly PV+ interneurons. These structures, which surround the somata and dendrites of PV+ interneurons, are crucial in stabilizing neural circuits and restricting synaptic plasticity (Mulligan et al., 1989; Härtig et al., 1992; Carulli et al., 2020, 2021). Acute cocaine administration reduces PNNs intensity in the PL, while repeated exposure enhances PNNs density, correlating with behavioral sensitization (Slaker et al., 2015, 2018). In CPP models, the degradation of PNNs in the amygdala using chondroitinase ABC (ChABC) prior to extinction training facilitates extinction learning and reduces the reinstatement of morphine- or cocaine-induced CPP (Xue et al., 2014b). Our previous work has demonstrated that the degradation of PNNs within the mPFC alleviates extinction resistance and relapse-like behaviors in MA-induced CPP models, suggesting that PNNs are essential in the maintenance and reconsolidation of addiction-related memories (Yao et al., 2024). However, whether PNNs are involved in the acquisition phase of MA-induced CPP memory remains uncertain.

This study was designed to uncover the role of PL PNNs and PV+ interneurons in the acquisition of MA-associated contextual memory and to provide mechanistic insights into early-phase addiction-related plasticity in female mice. Accumulating evidence shows that females display stronger engagement with drug-associated cues (Towers et al., 2023; Daiwile et al., 2022). Females escalate more rapidly and exhibit more persistent drug-seeking motivation in psychostimulant paradigms. This pattern is commonly described as the “telescoping effect,” reflecting a shortened interval between initial use and the emergence of problematic drug use (Towers et al., 2023). In both humans and rodents, craving and relapse-related responses to drug cues are often more pronounced in females. In preclinical experiments, MA-induced CPP is more reliably facilitated by estradiol, indicating that ovarian hormones actively shape reward learning rather than merely altering general activity (Chen et al., 2003). In detail, we combined c-Fos and ΔFosB immunohistochemistry (IHC) and fiber photometry to examine neuronal activity in the PL during the acquisition phase of MA-induced CPP. Wisteria floribunda agglutinin (WFA) and PV staining were used to assess changes in PNNs and PV+ interneurons in the PL. Immunofluorescent (IF) staining revealed the colocalization between PV+ interneuron and PNNs. Additionally, we enzymatically degraded PNNs in the PL using ChABC to investigate their role in CPP acquisition. To further elucidate the functional contributions of PV+ interneurons, we employed PV-Cre mice injected with AAV-DIO-hM4D(Gi) to selectively inhibit PV+ interneuron activity in the PL, combining this with fiber photometry to monitor neural activity during the formation of MA-induced CPP memory. Collectively, this work establishes a causal link between PL PNNs, PV+ interneuron activity, and the encoding of MA-associated contextual memory, providing a cellular framework for understanding early-stage psychostimulant addiction-related plasticity.