Behavioural and molecular evidence of GABAergic modulation of nocifensive behaviours in Bombus terrestris

The possibility that insects experience pain remains controversial. A key step towards answering this question is determining whether insect nocifensive behaviours are centrally modulated, a criterion that distinguishes nociception from pain. In this study, we investigated the role of the GABAergic system in modulating nocifensive responses in the bumble bee Bombus terrestris . We tested two drugs acting on distinct components of the GABAergic pathway: gabapentin, which increases GABAergic tone and reduces excitatory transmission via calcium channel modulation, and saclofen, which antagonizes GABA B receptors and disrupts metabotropic inhibitory signalling. Bees were orally administered gabapentin (24 mg/mL), saclofen (0.16 mg/mL) or a sucrose-only control and exposed to a noxious stimulus (a tunnel heated to 50 °C), while their behavioural responses were quantified. To determine whether the behavioural effects may reflect central mediation, we combined behavioural observations with molecular analysis of brain tissue. Specifically, we used reverse-transcription quantitative polymerase chain reaction (RT-qPCR) to quantify the expression of 10 genes related to GABAergic neurotransmission ( Gad1 , Abat , GABA A R ), synaptic activity ( CaMKII , Syn ) and drug-related pathways ( GABA B R1-3 , vGlut , Stj ). Gabapentin suppressed nocifensive behaviours, supporting an analgesic effect, while saclofen induced partial modulation without increasing sensitivity to the noxious stimulus. At the molecular level, RT-qPCR analyses revealed that gabapentin-treated bees exposed to the noxious stimulus showed increased expression of Syn and GABA A R , suggesting potential activation of central inhibitory circuits. In addition, both drug treatments in bees not exposed to the noxious stimulus showed significantly reduced vGlut expression, a trend that may indicate modulation of excitatory signalling in the absence of a nociceptive stimulus. Together, these findings show that pharmacological manipulation of GABAergic signalling alters nocifensive behaviours and brain gene expression in bumble bees. These integrated behavioural and molecular results are consistent with central neuromodulatory mechanisms that may regulate responses to noxious stimuli, raising the possibility of insect nociception beyond peripheral processing.