Individuals with substance use disorders (SUDs) display maladaptive associative learning, in which contextual cues linked to drug use become strongly connected to the drug's effects through repeated pairings (Cocker et al., 2020; Perry et al., 2014). These drug-context associations persist even after prolonged abstinence, triggering relapse upon exposure to drug-associated contexts through Pavlovian conditioning mechanisms (Cami and Farre, 2003; Nestler, 2001). Consequently, the mere exposure to environments previously linked to drug consumption can elicit intense craving and lead to compulsive drug-seeking behavior (Tzschentke, 2007).

The conditioned place preference (CPP) is a well-established model utilized to study the role of context associations in reward-related behaviors, including both natural rewards and drugs of abuse (McKendrick and Graziane, 2020). Unlike operant conditioning paradigms, which evaluate drug-seeking through increased instrumental responses, CPP provides information about the rewarding effects of contextual cues associated with drug stimuli (Aguilar et al., 2009; Bardo and Bevins, 2000). For example, it was demonstrated that, while increased cocaine self-administration led to enhanced instrumental responding, this elevated drug intake did not necessarily translate to a proportional increase in time spent in the drug-paired chamber during CPP tests (Deroche et al., 1999). These results suggest that CPP focuses more on the presence or absence of drug–context associations, rather than the magnitude of drug exposure, providing a valuable model for studying the emotional components of drug-associated memories (Cardinal et al., 2002).

The development of associations between drugs and contextual cues are critically mediated by the basolateral amygdala (BLA), which is well known to plays a key role in the acquisition of associative memories (Hiroi and White, 1991; Namburi et al., 2015). However, its functional contributions vary depending on the specific subpopulations of BLA neurons that project to other brain regions (Janak and Tye, 2015). BLA projections to the nucleus accumbens (NAc) have been implicated in cue-driven, reward-related behaviors, with distinct functions attributed to projections targeting to the NAc core or shell subregions. (Ambroggi et al., 2008). Neuronal activation in the NAc core induced by reward-predictive cues is significantly attenuated following BLA inactivation (Jones et al., 2010), and chemogenetic studies have demonstrated that the BLA-to-NAc core pathway contributes to reinforcing cue-induced behavioral responses when manipulated during the consolidation of drug-cue associations (Puaud et al., 2021). Conversely, BLA projections to the NAc shell do not typically show neuronal activation in response to reward-predictive cues (Jones et al., 2010). Rather, this pathway appears to play a regulatory role in reward-related behavior, as evidenced by optogenetic studies showing that activation of the BLA-to-NAc shell pathway impairs cue-induced drug-seeking and reduces drug consumption (Millan et al., 2017).

The BLA projection to the prelimbic cortex (PrL), a subregion of the medial prefrontal cortex (mPFC), has been extensively investigated in aversive learning paradigms, but emerging evidence suggests that it may also play a significant role in reward-related behaviors. For instance, pharmacological asymmetric disconnection or optogenetic inhibition of the BLA-to-PrL pathway reduced cue-induced reinstatement in operant paradigms (Mashhoon et al., 2010; Stefanik and Kalivas, 2013). However, these studies have exclusively employed inhibitory manipulations without assessing the potential upregulation and downregulation of neuronal activity that could affect context-reward associations. More importantly, no studies have directly tested whether bidirectional modulation of BLA to PrL activity regulates the expression of pre-established drug-context memories in CPP. Given that CPP and operant conditioning are not interchangeable measures, as they may be differentially influenced by specific manipulations, even when examining the effects of the same drug (Green and Bardo, 2020), it is essential to investigate how modulating neuronal activity within this pathway uniquely affects CPP expression.

While, the reciprocal PrL-to-BLA pathway also warrants investigation, our study focuses on the BLA-to-PrL output pathway. Although recent research has shown that chemogenetic inhibition of PrL–BLA projections reduces social defeat stress-induced CPP enhancement (Saito et al., 2025), the functional role of the BLA-to-PrL output pathway in drug-context memory expression remains unclear. Supporting the importance of this pathway, electrophysiological evidence shows that the BLA-to-PrL projections are essential for governing behavioral responses to concurrent appetitive and aversive stimuli, with PrL-projecting BLA neurons demonstrating superior predictive capacity for behavioral outcomes during cue competition (Burgos-Robles et al., 2017). However, whether modulating activity of BLA-to-PrL pathway in opposite directions (i.e., increasing or decreasing neuronal activity) regulates contextual drug memory expression remains unknown. A clearer understanding of the functional interplay between the BLA and PrL is therefore essential for fully characterizing the neural mechanisms underlying drug-context associations.

To understand the functional specificity of BLA output pathways, we also examined the BLA's projections to the nucleus accumbens (NAc) subregions. While these pathways are known to be involved in cue-driven reward behaviors, their comparative roles in CPP expression remain underexplored. Therefore, our study directly compared BLA projections to the PrL, NAc core, and NAc shell to delineate their specific contributions to CPP expression within the same experimental protocol. To address this question, we employed a chemogenetic approach using designer receptors exclusively activated by designer drugs (DREADDs) (Roth, 2016) to selectively activate or inhibit these BLA projection pathways during the expression of amphetamine(AMPH)-induced CPP.