Benzodiazepines and Anxiety-Like Behaviour in Gastropod Molluscs Assignment

Background

Background: Anxiety disorders are the most prevalent mental health conditions globally. Understanding the underlying mechanisms of anxiety, including its evolutionary origins, is critical for advancing both treatment and prevention strategies.

Environmental threats trigger fear and prompt immediate responses like freezing, escape, or aggression. When the source of threat is absent or unclear, threats can also result in anxiety. Anxiety manifests as anticipatory fear, leading to behavioural changes aimed at minimizing future threats, even in the absence of an immediate threat (Bateson et al., 2011).

The neurobiological and pharmacological substrates of anxiety have been widely studied in both humans and rodents (López-Muñoz et al., 2011). Anxiolytic drugs have been discovered that target either serotonergic neurotransmission or the benzodiazepine site of GABA receptors. The plus maze was developed to study anxiety-like behaviour in rodents and is still a widely used behavioural test. It works by exploiting the animal’s natural aversion to open spaces, which triggers anxiety-like behaviour (see Figure 1).

Emerging studies suggest that anxiety-like behaviours may also occur in invertebrate species. For instance, crayfish exhibit anxiety-like responses in an aquatic version of the plus maze (Fossat et al., 2014). Flatworms exhibit anxiety-like behaviours in an aquatic version of a light-dark box (Zewde et al., 2018). More recently, Talk and Vanatheeva (2024) observed anxiety-like behaviour in Great Ramshorn snails (Planorbarius corneus) in a light-dark box when exposed to predator scent. These findings suggest that anxiety-like behaviour may be a widespread phenomenon across different phyla. However, it is not clear the extent to which the underlying pharmacological mechanisms for anxiety are similar across phyla. To address this, we will perform a study to determine whether anxiety-like behaviour of snails can be elicited by stress and whether it is sensitive to a benzodiazepine drug that is anxiolytic in humans.

Project Objectives and Hypotheses: The purpose of this study is to investigate whether Great Ramshorn snails display heightened anxiety-like behaviour in a plus maze after having been stressed with substrate agitation as opposed to exposed to a non-stressful Fig 1: Aquatic plus maze as reported in a study of crayfish (Fossat et al., 2014). Anxiety-like behaviour was indexed as percent time the animals spent in the enclosed and darkened arms rather than the exposed and lit arms. Avoidance of the lit arms was enhanced by stressing the animals and reduced by administering drugs that are anxiolytic in humans. control procedure. A second purpose of the study is to determine whether a benzodiazepine receptor indirect agonist will reduce anxiety-like behaviour.

It is hypothesised that:

1. Snails exposed to high-speed orbital rotation on a lab mixer will subsequently spend significantly less time in the lit arms of a plus-maze than unstressed snails.

2. Administration of Chlordiazepoxide (CDZ), a benzodiazepine receptor indirect agonist that is anxiolytic in humans, will reduce anxiety-like behaviour in the plus maze.

Methods: Great Ramshorn snails will be randomly assigned to one of eight groups (12 snails per group). Snails in each group will first receive either a stress procedure, involving bursts of substrate agitation using a lab shaker, or a control procedure with no agitation. Then, each group will be placed in an aquatic plus maze scaled to the size of the snails. The plus maze will be filled with artificial pond water as a control, or artificial pond water with addition of one of three increasing concentrations of CDZ, an anxiolytic drug. CDZ is lipophilic and should cross from the surrounding water into and be distributed throughout the bodies of the snails. We will videorecord the snails using infrared and time-lapse. The dependent measure will be percent time the snails spend in the lit arms as opposed to the dark arms.