Validation of a CUMS paradigm to induce depressive-like behaviour in pond snails and its pharmacological modulation: A pivotal study

Major depressive disorder (MDD) is a leading cause of disability globally, yet its biological mechanisms remain incompletely understood. To bridge this gap, preclinical models are essential for exploring the neurobiology of depression and testing new treatments. While rodent models dominate this field, invertebrate systems like Lymnaea stagnalis (the great pond snail) are gaining attention due to their practical and ethical advantages. Lymnaea offers low maintenance costs, suitability for large-scale behavioral screening, and a simple, well-mapped nervous system. Its open circulatory system enables straightforward drug delivery, and as an invertebrate, it is not subject to the most restrictive animal welfare guidelines—making it compatible with the 3Rs (Replacement, Reduction, and Refinement) in ethical research. This study explored whether Lymnaea could model core symptoms of depression using the Chronic Unpredictable Mild Stress (CUMS) paradigm, a well-established method in rodent research. The goal was to determine if the snail exhibits depressive-like symptoms and whether these could be mitigated pharmacologically. A reliable behavioral protocol was developed where snails were exposed to a rotation of mild stressors (e.g., KCl immersion, thermal shock, predator cues, overcrowding, and vortex agitation). Control snails remained unstressed. To ensure consistency, two experimental groups experienced the same stressors in different orders. Rasping elicited by sucrose was measured daily as a proxy for anhedonia. Moreover, we measured exploratory activity (motivation) and memory ability (via operant conditioning of aerial respiration). Both stressed groups showed a significant decline in feeding and exploration by day 7 of stress exposure, indicating anhedonia and motivational deficits. Additionally, stressed snails did not form memory associations, suggesting cognitive impairment. These results mimic affective and cognitive symptoms typical of a depressive-like phenotype in mammals. To assess pharmacological responsiveness, a daily one-hour treatment of 1 µM fluoxetine (an antidepressant drug belonging to the class of selective serotonin reuptake inhibitors) was administered throughout the stress period. Fluoxetine successfully prevented the stress-induced anhedonia, indicating it alleviated affective symptoms in our model. However, it did not reverse memory deficits, as treated snails still failed to form memories. This mirrors findings in vertebrate systems, where emotional and cognitive symptoms of depression often respond differently to pharmacological treatment. This study provides the first evidence that an invertebrate model can display a chronic stress-induced depressive-like phenotype that responds to antidepressant treatment. The model demonstrates both face validity (behavioral resemblance to human symptoms) and predictive validity (response to known antidepressants). Consistently with vertebrate research, the dissociation between fluoxetine’s effect on ‘mood’ versus memory suggests distinct underlying neural mechanisms. In summary, Lymnaea stagnalis offers a unique platform to investigate the evolutionary basis of depressive behaviors and explore symptom-specific responses to pharmacological interventions.