First evidence of an anxiety-like behavior and its pharmacological modulation in a molluscan model organism, Lymnaea stagnalis.

Anxiety, a behavioral consequence of stress, has been characterized in humans and some vertebrates but remains largely unexplored in invertebrates. Here, we demonstrate that after being exposed to fish water, which simulates the presence of predators, pond snails (Lymnaea stagnalis) exhibit a series of sustained fear responses. These include increased aerial respiration, changes in righting behavior, and reduced escape responses. Notably, these behaviors persist even after the stressor (fish water) is removed, indicating that they likely represent an anxiety-like state rather than a simple conditioned reflex. Additionally, exposure to fish water enhances long-term memory formation for the operant conditioning of aerial respiration, suggesting that the predator scent potentially induces a state of heightened alertness, which enhances memory consolidation processes. Furthermore, when snails experience fish water alongside an appetitive stimulus (carrot), they form configural learning-a higher form of learning - where the appetitive stimulus now triggers a fear response instead of eliciting feeding. Importantly, the anxiolytic drug alprazolam prevents these anxiety-like responses. Through dose-response experiments, we found that alprazolam at a concentration of 0.1 µM for 15 min effectively counteracts predator-induced anxiety without causing sedation. This treatment also prevents the effects of predator cues on learning and memory. However, consistent with data from vertebrates - alprazolam induces anterograde amnesia, impairing the formation of new memories for up to 3 h after treatment, though it does not cause long-term memory deficits. Overall, this is the first study showing that a molluscan model organism exhibits anxiety-like behaviors similar to those seen in vertebrates, and these behaviors can be mitigated by an anti-anxiety drug. This suggests that fundamental anxiety mechanisms are evolutionarily conserved across species. By using this simple invertebrate model, our research offers new insights into the biological basis of anxiety and sets the stage for future pharmacological studies.