The role of ligand-gated chloride channels in behavioural alterations at elevated CO₂ in a cephalopod

Projected future carbon dioxide (CO₂) levels in the ocean can alter marine animal behaviours. Disrupted functioning of γ-aminobutyric acid type A (GABA_A) receptors (ligand-gated chloride channels) is suggested to underlie CO₂-induced behavioural changes in fish. However, the mechanisms underlying behavioural changes in marine invertebrates are poorly understood. We pharmacologically tested the role of GABA-, glutamate-, acetylcholine- and dopamine-gated chloride channels in CO₂-induced behavioural changes in a cephalopod, the two-toned pygmy squid (Idiosepius pygmaeus). We exposed squid to ambient (∼450 µatm) or elevated (∼1,000 µatm) CO₂ for seven days. Squid were treated with sham, the GABA_A receptor antagonist gabazine, or the non-specific GABA_A receptor antagonist picrotoxin, before measurement of conspecific-directed behaviours and activity levels upon mirror exposure. Elevated CO₂ increased conspecific-directed attraction and aggression, as well as activity levels. For some CO₂-affected behaviours, both gabazine and picrotoxin had a different effect at elevated compared to ambient CO₂, providing robust support for the GABA hypothesis within cephalopods. In another behavioural trait, picrotoxin but not gabazine had a different effect in elevated compared to ambient CO₂, providing the first pharmacological evidence, in fish and marine invertebrates, for altered functioning of ligand-gated chloride channels, other than the GABA_A R, underlying CO₂-induced behavioural changes. For some other behaviours, both gabazine and picrotoxin had a similar effect in elevated and ambient CO₂, suggesting altered function of ligand-gated chloride channels was not responsible for these CO₂-induced changes. Multiple mechanisms may be involved, which could explain the variability in the CO₂ and drug treatment effects across behaviours.