SERATONIN

Serotonergic responses in the novel in vivo research animal Lumbriculus variegatus

Max Shepherd, Henriette Leser, Romessa Mahmood & Aidan Seeley

Swansea Worm Integrative Research Laboratory

Swansea University Medical School

Swansea University 

Introduction 

Caenorhabditis elegans uses serotonin as a neurotransmitter to regulate locomotion [1] and the study of other invertebrate models may provide model systems with the potential to make important contributions to the study of serotonin signalling. Lumbriculus variegatus, more commonly known as the Californian Blackworm, is a species of aquatic worm which has potential as a novel in vivo pharmacology research animal [2]. Here we report L. variegatus is responsive to exogenous serotonin and the selective-serotonin reuptake inhibitor, fluoxetine. Moreover, we demonstrate the presence of endogenous serotonin within this novel organism.  Behaviour quantification of L. variegatus can be achieved through rapid image collection to measure unstimulated locomotion of L. variegatus and stimulated locomotion by stimulation of the posterior region, promoting helical swimming, or stimulation of the anterior region, resulting in body retraction and reversal [2].

Here we demonstrate the concentration-dependent effects of serotonin and fluoxetine on L. variegatus stereotypical movements and free, unstimulated, locomotor activity, and the presence of endogenous serotonin within L. variegatus by SDS-PAGE and Western Blot analysis. 

Method 

Stereotypical movements were recorded following tactile stimulation of anterior and posterior regions before serotonin or fluoxetine exposure, after 10-minute exposure to serotonin (0-2 mM) or fluoxetine (0-500 μM), and 10 minutes and 24 hours after removal of drug compounds and subsequent incubation in pondwater. Unstimulated free locomotion was measured by rapid image collection of L. variegatus under the same conditions. Expression of endogenous serotonin was determined by snap freezing L. variegatus at -80oC, lysis in RIPA buffer, subjecting samples to SDS-PAGE and Western Blot analysis. Statistical significance was determined by paired t-tests or a two-way ANOVA. 

Results

Exposure to ≥0.25 mM serotonin significantly inhibited both body reversal and helical swimming (p<.05, n=8). 24 hours after serotonin exposure a significant difference was observed for both stereotypical movements in L. variegatus exposed to ≥0.1 mM  (p<.05, n=8). Conversely, the free, unstimulated movement was unaffected by serotonin exposure and L. variegatus movement was indistinguishable from pre-exposure conditions  (p>.05, n=8). 

Moreover, exposure to fluoxetine resulted in inhibition of both movements at ≥250 μM (p<.05, n=8) with effects persisting 24 hours (p<.05, n=8). Free locomotion was similarly acutely affected by ≥250 μM fluoxetine (p<.05, n=8) with inhibitory affects persisting 24 hours of exposure to fluoxetine and then subsequent incubation in drug-free pondwater. Endogenous expression of serotonin in L. variegatus was confirmed by Western Blot analysis. 

Conclusions 

This work demonstrates the concentration-dependent effects of serotonin and fluoxetine on behavioural responses and confirms endogenous serotonin in L. variegatus which highlights the wider potential utility of L. variegatus for pharmacological research. 

References

1. Gurel et al. (2012) Receptors and Other Signaling Proteins Required for Serotonin Control of Locomotion in Caenorhabditis elegans. Genetics; 192(4): 1359–1371. doi: 10.1534/genetics.112.142125

2. Seeley, A. et al. (2021) Lumbriculus variegatus: A novel organism for in vivo pharmacology education. Pharmacol. res. Perspect; 9:e00853. https://doi.org/10.1002/prp2.853