Episodic memory, the ability to recall personal experiences, relies heavily on the hippocampus, specifically the dentate gyrus (DG). Pattern separation (PS), the process of distinguishing between similar memories, is crucial for this function and is regulated by adult neurogenesis in the DG. Obesity is linked to episodic memory impairments, potentially due to alterations in PS. Orexin-A and its downstream endocannabinoid 2-arachidonoylglycerol (2-AG) signaling are known to be dysregulated in obese mice. This study hypothesizes that the altered orexin-A/2-AG/CB1 receptor signaling cascade in obese mice contributes to the dysfunction of adult hippocampal neurogenesis (AHN), leading to impaired plasticity, PS, and episodic memory. The researchers aim to elucidate the molecular mechanisms underlying these impairments and test whether interventions targeting orexin-A signaling can restore hippocampal function in obese mice. The importance of this study lies in its potential to reveal novel therapeutic targets for memory deficits associated with obesity, a growing global health concern. Understanding the neurobiological mechanisms underlying these memory impairments is crucial for developing strategies to improve cognitive function and promote healthier eating behaviors in obese individuals.

Prior research has established a correlation between obesity and impaired episodic memory in both humans and animal models. Studies have demonstrated deficits in memory formation and retrieval in obese individuals, potentially influencing food choices and intake. Adult neurogenesis in the DG plays a significant role in PS, and its disruption has been implicated in various neurological disorders. The endocannabinoid system, particularly 2-AG and its CB1 receptor, is involved in regulating neurogenesis and synaptic plasticity, impacting memory function. Furthermore, orexin-A, a hypothalamic neuropeptide involved in sleep-wake cycles, appetite, and energy homeostasis, interacts with the endocannabinoid system. Previous work has shown alterations in orexin-A signaling in obese mice, suggesting a potential link to hippocampal dysfunction. This study builds upon these findings by investigating the causal relationship between orexin-A/2-AG/CB1 signaling and the observed AHN and memory impairments in obesity.

The study utilized both in vivo and in vitro experimental approaches. The researchers employed several behavioral tasks to assess pattern separation in obese mice. The novel object recognition (NOR) task, a modified version using objects with varying degrees of similarity, was used to evaluate short-term and long-term memory. A modified water maze task, sensitive to alterations in DG neurogenesis, was also used to assess spatial learning and memory. To characterize anxiety-like behavior, which can impact results, the open-field and light-dark box tests were conducted. Immunohistochemical techniques were employed to quantify adult neurogenesis in the DG by measuring the density of Ki67 (proliferation marker), DCX (neuroblast marker), and NeuroD (neurodifferentiation marker)-immunoreactive cells. Morphometric analysis of DCX-positive neurons was performed to assess dendritic development and complexity. The functional integration of newborn neurons was evaluated using PSD95/DCX colocalization analysis. In vitro electrophysiological recordings from hippocampal slices were performed to measure long-term potentiation (LTP) at medial perforant path (MPP)-DG synapses, a crucial process in PS. Both in vitro and in vivo LTP experiments were conducted. Finally, ELISA assays were employed to measure orexin-A levels, and LC-MS was used to quantify 2-AG levels in the hippocampus. The study used leptin receptor knockout (ob/ob) mice and wild-type mice fed a high-fat diet (HFD) as obese models, with age-matched lean mice serving as controls. Pharmacological interventions, including orexin-1 receptor (Ox1-R) antagonist (SB334867), CB1 receptor antagonist (AM251), and DAGLa inhibitor (O-7460), were utilized to manipulate the orexin-A/2-AG/CB1 signaling pathway. In vitro experiments were conducted using human iPSC-derived neural stem cells to further elucidate the role of OxA, SB334867, and O-7460 on neurogenesis. Correlative light and electron microscopy (CLEM) was employed to examine the ultrastructural localization of Ox1R, CB1, and DAGLa within the DG. Statistical analysis, including ANOVA, Kruskal-Wallis, and post-hoc tests, was performed to determine statistical significance.

The study found that obese mice (both ob/ob and HFD) exhibited impaired pattern separation in both the NOR and modified water maze tasks, indicating deficits in episodic memory. Adult neurogenesis was altered in obese mice, showing reduced proliferation but increased neurodifferentiation. The density of DCX-positive cells and the complexity of their dendritic processes were significantly increased in obese mice compared to lean controls. In vivo LTP was impaired at MPP-DG synapses in obese mice, while in vitro LTP did not show a significant difference. Orexin-A levels were significantly higher in the hippocampus of obese mice, and this increase was mimicked by exogenous orexin-A administration in lean mice and reversed by leptin treatment in ob/ob mice. Exogenous orexin-A administration in lean mice increased neurodifferentiation, an effect blocked by Ox1-R antagonism. In vitro studies using human iPSC-derived neural stem cells confirmed that orexin-A promotes neurodifferentiation via Ox1-R/DAGLa/2-AG/CB1 signaling. In hippocampal slices from lean mice, orexin-A inhibited LTP, an effect that was prevented by Ox1-R or CB1 antagonism and DAGLa inhibition. In vivo, Ox1-R antagonism restored LTP in obese mice. CLEM analysis revealed the presence of Ox1-R, CB1, and DAGLa in close proximity at MPP-DG synapses. The study also found a significant reduction in NR2B expression in ob/ob mice, which was prevented by leptin treatment. Finally, Ox1-R antagonism partially rescued the pattern separation deficits in obese mice in the NOR task, primarily at the short-term memory phase.

The findings provide compelling evidence for a causal link between dysregulated orexin-A/2-AG/CB1 signaling and impaired hippocampal function in obesity. The increased orexin-A levels in obese mice stimulate 2-AG production, leading to CB1 receptor overactivation and subsequent inhibition of glutamate release at MPP-DG synapses, thus impairing LTP and PS. The observed increase in neurodifferentiation despite reduced proliferation suggests a compensatory mechanism, yet it is insufficient to overcome the functional deficits. The partial rescue of pattern separation deficits by Ox1-R antagonism highlights the therapeutic potential of targeting orexin-A signaling to alleviate cognitive impairments associated with obesity. The difference in the effect of AM251 between HFD and ob/ob mice may be related to differences in NR2B expression levels, suggesting a complex interplay between different signaling pathways in obesity.

This study demonstrates a novel mechanism linking orexin-A signaling, endocannabinoid dysregulation, and impaired hippocampal function in obesity. The findings highlight the importance of orexin-A/2-AG/CB1 signaling in regulating AHN, synaptic plasticity, and episodic memory. The observed rescue of cognitive deficits by Ox1-R antagonism suggests potential therapeutic strategies for obesity-associated memory impairments. Future research should investigate the long-term effects of Ox1-R antagonism and explore the potential interaction between orexin-A signaling and other metabolic factors in influencing hippocampal function.

The study primarily used animal models, which may not fully translate to human conditions. Further research is needed to validate these findings in humans. The focus on a specific signaling pathway may not fully encapsulate the complex interplay of factors involved in obesity-related cognitive dysfunction. The study mainly focused on short-term effects, and further investigation into long-term effects is warranted. Additionally, the small sample size used in some experimental procedures could limit the generalizability of the findings.