TOBACCO

Tests to Observe the Behavioural Effects of AcetylCholine and related Chemical messengers On worms (TOBACCO) is a research project within SWIRL which is investigating the cholinergic system within Lumbriculus variegatus. Nicotine acts on nicotinic acetylcholine receptors (nAChRs) and we have previously demonstrated the concentration-dependent effects of the cholinergic agonists acetylcholine and nicotine (Carriere et al., 2022). 

TOBACCO aims to examine the behavioural effects of cholinergic drugs in L. variegatus and investigate the presence or absence of a cholinergic system in this species.

Below is an abstract which highlights some of our current work within TOBACCO

Concentration-dependent cholinergic responses in the novel in vivo research animal Lumbriculus variegatus

Julanta Carriere, Craig Dickson & Aidan Seeley  

Swansea Worm Integrative Research Laboratory

Swansea University Medical School

Swansea University 

Introduction 

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 [1]. Cholinergic signalling is highly conserved in animals and signalling can be excitatory, inhibitory, or modulatory depending on the receptor and cell type [2]. Here we report L. variegatus is responsive to the canonical agonist, acetylcholine, and the agonist, nicotine, and that these effects can be inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist, mecamylamine. 

L. variegatus displays two stereotypical behaviours when stimulated. Stimulation of the posterior region elicits helical swimming, whereas stimulation of the anterior region results in body retraction and reversal [3]. Previously, rapid-image collection has been used to measure unstimulated, free locomotion of L. variegatus [1].

Here we demonstrate the concentration-dependent effects of acetylcholine and nicotine on L. variegatus stereotypical movements and free, unstimulated, locomotor activity, and that administration of nicotinic receptor antagonists can decrease these effects. 

Method 

Stereotypical movements were recorded following tactile stimulation of anterior and posterior regions before acetylcholine or nicotine exposure, after 10-minute exposure to acetylcholine (0-100 mM) or nicotine (0-1 mM), and 10 minutes and 24 hours after removal of drug compounds and subsequent incubation in pondwater. The antagonism of these responses was determined by pre-treatment with mecamylamine (0.01-10 mM) before stereotypical movements were quantified. Unstimulated free locomotion was measured by rapid image collection of L. variegatus under the same conditions. Statistical significance was determined by paired t-tests or a two-way ANOVA. 

Results

Exposure to 25 mM acetylcholine significantly inhibited body reversal (p<0.05, n=8) and helical swimming (p<.01, n=8) but had no effect on free, unstimulated, movement (p>.05, n=8). Exposure to 0.1 mM nicotine significantly inhibited body reversal (p<.05, n=8) and helical swimming (p<.01, n=8). Additionally, free locomotion was inhibited by 91.0±2.4% (p<.0001, n=8). 

Inhibition of body reversal could be prevented by exposure to mecamylamine (0.01-5 mM) with levels indistinguishable from pre-exposure conditions (p>.05, n=6). However, the ability of L. variegatus to perform helical swimming stereotypical movements remained significantly inhibited (p<.05, n=6) in the presence of nicotine after mecamylamine exposure. 

Conclusions 

This work demonstrates the concentration-dependent effects of acetylcholine and nicotine, and inhibition of these effects by the nAChR antagonist mecamylamine on L. variegatus behaviours and highlights the wider potential utility of L. variegatus for pharmacological research. 

References

1. 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 

2. Treinin & Jin (2021) Cholinergic transmission in C. elegans: Functions, diversity, and maturation of ACh-activated ion channels. J Neurochem; 158(6):1274-1291. doi: 10.1111/jnc.15164

3. Ding, J., Drewes, C.D. & Hsu, W.H. (2001) Behavioral effects of ivermectin in a freshwater oligochaete, Lumbriculus variegatus. Environ Toxicol Chem; 20(7): 1584-90. https://doi.org/10.1002/etc.5620200724