Fig. 1: Long-lasting and sex-selective effects of omega-3 supplementation on anxiety and cognition.

a Schematic representation of experimental timeline. b Impact of Prenatal THC on Birth Weights (8 pups/dam, approximately even sex distribution): Prenatal THC exposure (THCct) significantly reduced birth weights at postnatal day 1 (PD1) in both male and female pups (p < 0.05). Perinatal omega-3 supplementation (THCω3) prevented this reduction (p < 0.05), with a significant effect in females, but males, compared to VEHct. By PD21, only male VEHω3 offspring showed significant weight differences compared to THCct males. c–e, Anxiety-like behaviors assessed in adulthood (n = 12-20/treatment/sex) using EPM and LDB tests, while anxiety and motility was assessed using OFT: c Elevated Plus Maze (EPM): No significant anxiety-like behavior was observed in THCct males (p > 0.05). However, omega-3 supplementation resulted in reduced anxiety in both VEHω3 and THCω3 males (p < 0.05). d Light-Dark Box (LDB): THCct males exhibited increased anxiety-like behavior compared to VEHct males (p < 0.05). Omega-3 supplementation in THCω3 males normalized this effect (p < 0.05). e Open Field Test (OFT): No significant differences were found in anxiety (first 5 minutes of center entries) or locomotor activity over 30 minutes among the groups (p > 0.05). f Sensorimotor Processing Assessed by Prepulse Inhibition (PPI): Schematic illustrates the PPI protocol with a 100 ms interstimulus interval at prepulse intensities of 72 dB, 76 dB, and 80 dB. Baseline startle magnitudes: VEHω3 females > VEHct and THCω3; males > females (p < 0.05). No significant group differences were observed in PPI. g–i, Cognitive and Memory Assessments in Adulthood: g Three-Chamber Social Interaction Test: Schematic depicts the assessment of social motivation and recognition. THCct offspring showed impaired social motivation and memory compared to VEHct, VEHω3, and THCω3 groups (p < 0.05). Omega-3 supplementation rescued social motivation deficits in both sexes (p < 0.05) but improved social memory only in males (p < 0.05). h Y-Maze Spontaneous Alternation (SA) Test: Prenatal THC exposure led to deficits in spatial working memory in both sexes (p < 0.05). Omega-3 supplementation prevented these deficits in males but not females (p < 0.05). i Temporal Order Novel Object Recognition (TONOR) Test: THCct offspring exhibited impairments in object recognition memory in both sexes. Omega-3 supplementation ameliorated these deficits in males but not in females (p > 0.05). Estrous cycles were assessed for all female offspring, but no effect on estrous cycle and behavior were observed. Outliers were excluded using the ROUT method. Statistical analyses were conducted using three-way ANOVAs with factors of sex, THC treatment, and omega-3 diet, followed by Tukey’s HSD post hoc tests. Treatment effects are indicated with solid lines; sex effects with dashed lines. Significance levels are denoted as ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Data are presented as individual points with mean±SEM.