Cryptotanshinone and Its Metabolite Ameliorate Rewarding Effect ofNitrous Oxide via Nucleus Accumbens D1 Receptor and TrkB Pathway

Introduction: Nitrous oxide (N2O) abuse is a growing international concern, with limited effective intervention strategies. This study investigates the potential of Cryptotanshinone (CTS) and its metabolite tanshinone IIA (TAN IIA) in alleviating N2O-induced conditioned place preference (CPP) behavior and modulating dopamine D1 receptor (D1R) and TrkB pathway Methods: In vivo, a CPP mouse model was established via N2O exposure, followed by CTS administration via gavage for 4 days. In vitro, PC12 cells were exposed to D1R agonist SKF 81297 and TrkB agonist 7,8-DHF, with subsequent CTS or TAN IIA treatment. Results: In vivo, N2O exposure significantly increased CPP scores, upregulated the expression of proteins associated with the D1R and TrkB pathways (such as p-CREB and p-CaMKII), and increased the number of p-CaMKII and p-CREB-positive cells in the Nucleus Accumbens (NAc). These effects were attenuated by CTS treatment. Additionally, N2O exposure increased the colocalization of c-fos with ChAT and GAD67, while CTS treatment significantly decreased the number of ChAT/c-fos positive neurons. Molecular docking indicated no significant difference in binding affinity between CTS/TAN IIA and D1R/TrkB. In vitro, stimulation with D1R and TrkB agonists elevated pathway protein expression, Ca2+ activity, and the density of c-fos, all of which were notably suppressed by CTS and TAN IIA. Discussion: CTS and TAN IIA effectively reverse behavioral and molecular markers of N2O reward, potentially via downstream regulation of the D1R and TrkB pathways. This action mitigates NAc neuronal hyperactivity, supporting N2O extinction Conclusion: Our findings demonstrate that CTS and TAN IIA promote the extinction of N2Oinduced CPP by functionally inhibiting the D1R and TrkB pathways, highlighting their potential for treating substance use disorders.