Acute restraint stress causes anxiety-related behaviors and neuronal degeneration in the CA1 and PFC, which are blocked by crocin and D-AP5.

Crocin, a water-soluble carotenoid known for its therapeutic potential in anxiety disorders, exhibits a range of beneficial properties such as anti-inflammatory, neuroprotective, and anti-anxiety effects. Despite its documented efficacy, the precise mechanism behind its anxiolytic action remains incompletely understood. Given crocin's known interaction with NMDA receptors within the glutamatergic system-particularly in cognitive processes-this study aimed to investigate its interplay with NMDA receptor modulators in regulating anxiety-related behaviors in male mice under an acute restraint stress (ARS) model. Mice were implanted with guide cannulas for intracerebroventricular (i.c.v.) drug administration and subjected to 4 h of immobilization to induce ARS. Anxiety was assessed using the elevated plus maze (EPM), which revealed that stressed mice displayed increased anxiogenic behaviors, reflected by reduced percentages of open arm time (%OAT) and open arm entries (%OAE). Additionally, ARS led to a rise in dark cell count in hippocampal CA1 and prefrontal cortical regions. Administration of NMDA (0.5 μg/mouse, i.c.v.) intensified anxiety-like behavior, whereas both D-AP5 (an NMDA antagonist; 0.5 μg/mouse, i.c.v.) and crocin (50 mg/kg, intraperitoneal (i.p.)) attenuated it. A sub-effective dose of NMDA (0.25 μg/mouse) counteracted the anxiolytic effects of crocin across multiple doses, while a sub-threshold dose of D-AP5 (0.25 μg/mouse) enhanced crocin-induced anxiety reduction. Synergistic anxiolytic effects between crocin and D-AP5 were observed in both stressed and non-stressed mice. These findings strongly indicate that crocin modulates anxiety-related behavior, at least partially, via interaction with NMDA receptors.