Activity-based anorexia alters leptin signaling in dorsal and ventral hippocampus of adolescent female rats

Background: Anorexia nervosa (AN) is a serious psychiatric disorder, mainly affecting adolescent females, characterized by self-induced starvation and excessive physical activity1. The aetiology of AN remains unclear; however, from a neurobiological standpoint, it has been suggested a dysregulation of appetite modulators that, via adaptive mechanisms, may support the response of the body to starvation. Among them, leptin, an adipokine mainly produced in white fat cells in proportion to the overall amount of adipose tissue, is a strong candidate. Indeed, studies on adolescent females with AN have shown a decrease in serum leptin levels compared to their healthy counterparts and a negative correlation between hypoleptinemia and hyperactivity2 . Objective: It is still unclear how the leptin receptor (LepR) and its JAK2-STAT3 downstream pathway are modulated in brain areas other than the hypothalamus, such as the dorsal (dHip) and ventral (vHip) hippocampus, crucial for memory and emotion regulation, respectively. Thus, we exposed adolescent female rats to the Activity-Based Anorexia (ABA) model3 , to investigate the impact of the anorexic phenotype on the LepR pathway and on cognitive- and reward-related behaviours. Methods: ABA phenotype was induced by exposing rats to a combination of food restriction and free access to an activity wheel. Daily body weight and food intake have been tracked and analysed with a Two-way ANOVA, followed by Bonferroni’s multiple comparisons test. Molecular analyses were conducted at two different time points: post-natal-day 42 (PND42), at the acute phase, and PND49, after body-weight recovery. Leptin plasma levels were determined using an ELISA assay. Proteins from dHip and vHip tissues were extracted and analysed by Western blot in the post-synaptic density, nuclear and cytosolic fractions. Statistical analyses were conducted using a Two-way ANOVA followed by Tukey’s multiple comparisons test. To test hippocampal functionality, Spatial Order Object Recognition Test (SOR) and Palatable-food-induced conditioned-place preference Test (CPP) were performed and analysed with an unpaired Student’s T test and a Mann Whitney test, respectively. Results: Following the beginning of ABA induction, ABA rats lost more weight than only food- restricted (FR) rats and constantly increased running activity over days4 . Leptin plasma levels were significantly reduced in ABA rats in the acute phase (-705,83ng/ml p<0,0001 vs CTRL), while increased after body-weight recovery (+712,87ng/ml p=0,0005 vs CTRL). Concurrently, in the dHip, the combination of food restriction and hyperactivity led to an overall decrease in the intracellular LepR-JAK2-STAT3 pathway of ABA rats. Conversely, in vHip, the whole pathway underwent a shift from hypo- (PND42) to hyper-activation (PND49) in ABA animals5 . In parallel, ABA rats demonstrated spatial cognitive impairment in the SOR test at both timepoints as shown by the discrimination index (PND42:-0,19 p=0,0002 vs CTRL; PND49:-0,208 p=0,0004 vs CTRL). Moreover, ABA rats showed reduced CPP score (-12,3% p=0,0274 vs CTRL) highlighting their inability to associate the conditioned rewarding experience of food. Conclusion: Taken together, these data suggest a possible link between leptin levels, anorexic phenotype, hippocampal-dependent cognitive impairments, and persistent maladaptive alterations that may lay the foundation for comorbid mental disorders and individual susceptibility, frequently observed in AN patients.