CAFFEINE

Concentration-dependent behavioural effects of caffeine in the novel in vivo organism, Lumbriculus variegatus 

Shawn Shabu, Aidan Seeley, Nia A. Davies & Melisa J. Wallace  

Background and Purpose: 

Caffeine is the most widely consumed central-nervous-system stimulant which acts through antagonism of adenosine receptors [1]. Previously, invertebrate models such as C. elegans have been used to study the effects of caffeine [2] and here we report that the novel invertebrate model, Lumbriculus variegatus, can be utilised for studying the potential effects of caffeine.

L. variegatus, a species of aquatic worm, 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]. Here we demonstrate concentration-dependent effects of caffeine on L. variegatus stereotypical movements and free, unstimulated, locomotor activity.

Method 

Stereotypical movements were recorded following tactile stimulation of anterior and posterior regions before ethanol exposure, after 10-minute exposure to caffeine (0-10 mM), and 10 minutes and 24 hours after removal of caffeine and subsequent incubation in pondwater. 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 5mM caffeine significantly inhibited body reversal (p=.008, n=8) and helical swimming (p=.008, n=8), with similar results observed in L. variegatus exposed to 10mM caffeine. The effects of 5mM caffeine were readily reversible 10-minutes after removal and incubation in artificial pondwater (p>.05, n=8). 

However, 10mM caffeine resulted in significant inhibition of body reversal (p=.001, n=8) and helical swimming (p=.002, n=8) after 10-minutes removal from caffeine. 10 mM caffeine demonstrated significant long-term effects 24 hours after exposure, with the ability of L. variegatus to perform body reversal (p=.002, n=8) and helical swimming (p=.004, n=8) behaviours significantly reduced from pre-exposure conditions. 

Moreover, unstimulated free locomotor activity was similarly inhibited by 27.51±1.25% at 5mM (p=.002, n=3) and 30.31±5.89% at 10mM ethanol (p=.036, n=3). However, 10 mM demonstrated no prolonged effects on free locomotion after removal of caffeine and incubation in artificial pondwater for 24h (p>.05, n=3). 

Conclusions 

This work demonstrates the concentration-dependent effects of caffeine on L. variegatus behaviours and highlights the wider potential utility of L. variegatus for pharmacological research. 

References:

1. Nehlig, A., Daval, J.L. & Derby, G. (1992) Caffeine and the central nervous system: mechanisms of action, biochemical, metabolic and psychostimulant effects. Brain Res Brain Res Rev; 17(2):139-70.

2. Manalo, R.V.M. & medina, P.M.B. (2020) Caffeine reduces deficits in mechanosensation and locomotion induced by L-DOPA and protects dopaminergic neurons in a transgenic Caenorhabditis elegans model of Parkinson’s disease. Pharm. Biol.; 58(1), 721-737.

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