Acute drug responses in C elegans.
We have developed a simple image-based assay for drug responses in C. elegans which is described in our recent paper. The key findings are summarized here.
In our assay, we take worms in liquid buffer and incubate them with drug solutions in 96-well plates. We begin our assay immediately on adding the drug and use an automated microscope and simple image-analysis software to quantify the degree of worm movement in each well.
By repeatedly sampling the drug responses, at 5 minute intervals, we can build up a time-resolved profile of response. We found that responses are often highly dynamic: approximately a third of the compounds we tested showed a phase of paralysis followed by a some sort of recovery (Figure 1). These recovery phases varied in their properties: sometimes recovery increased in response to increasing dose and sometimes it decreased. Several compounds elicited a response containing several successive phases of paralysis and transient recovery. This variety among responses suggested that a range of mechanisms might underlie these recoveries.
Levamisole (Figure 1A), in particular, captured our interest. Levamisole a widely used nematicidal drug that works by stimulating nicotinic acetylcholine receptors (nAChRs), causing muscle contraction and paralysis in adults. Since the neuromuscular system in C. elegans has been so well characterised, and many drugs are available to manipulate acetylcholine (ACh) neurotransmission, we decided to focus our investigation on recovery from cholinergic stimulation.
Aldicarb, formerly in widespread use as a pesticide, is an inhibitor of acetylcholinesterase, an enzyme that degrades ACh after it has been released into the synapse. Aldicarb, therefore, causes a rise in the synaptic concentration of ACh by preventing its degradation. Nicotine is a selective agonist of nAChRs, with a long pedigree in pharmacological research. Like levamisole, both these compounds cause an initial phase of paralysis in L1 worms, followed by a recovery phase (Figure 2A and Figure 2B). In contrast to levamisole, where higher concentrations cause earlier recovery, increasing the dose of nicotine suppressed the rate and extent of recovery, suggesting that the mechanisms of recovery from these two compounds are likely distinct. The aldicarb response was more closely related to the levamisole response: as aldicarb concentration was increased, recovery continued to increase, up to the limit of aldicarb solubility.
To circumvent the limitations of aldicarb solubility, we tried combining increasing concentrations of aldicarb with a fixed dose of levamisole (Figure 2C). Interestingly, increasing aldicarb concentrations reproduced the recovery-promoting effects of higher levamisole doses. This suggests that aldicarb and levamisole might share a similar mechanism of recovery. Finally, we investigated whether aldicarb might be able to promote recovery from paralysis caused by high doses of nicotine (Figure 2D). This was indeed the case: adding increasing doses of aldicarb to 10 mM nicotine causes increasing degrees of recovery.
The finding that aldicarb could stimulate recovery from nicotine-induced paralysis was pivotal, since it implies that aldicarb possesses some pharmacological property that nicotine does not. More precisely, ACh, the neurotransmitter that increases in concentration in response to aldicarb, has such a property. An obvious candidate for this property is activity at muscarinic acetylcholine receptors (mAChRs). In contrast to nAChRs, which are fast-acting, ligand gated ion channels, mAChRs are slower-acting G-protein coupled receptors. Both receptor types are activated by ACh but, as their names suggest, each receptor has differential sensitivity to other ligands, including nicotine, muscarine, among several others .
To test this hypothesis, we tested the ability of two mAChR-specific compounds to manipulate recovery from cholinergic paralysis. Titrating increasing doses of oxotremorine M, an agonist at mAChRs, onto a fixed dose of nicotine caused increasing recovery from paralysis, reproducing the effects of increasing aldicarb concentrations (Figure 3A). Conversely, titrating a mAChR antagonist, atropine, onto a fixed dose of aldicarb suppressed the characteristic recovery phase of the aldicarb response (Figure 3B). Together, these results suggest that mAChRs are able to mediate recovery from cholinergic paralysis in L1 worms.
The C. elegans genome encodes three mAChRs. We acquired loss of function mutants for each of these and tested their ability to mediate recovery from nicotine induced paralysis (Figure 3C) and the recovery phase of the aldicarb response (Figure 3D). Our data confirmed the role of mAChRs in mediating recovery and implicated gar-3 as the receptor predominantly responsible.
Our findings suggest a model where paralysis is mediated by nicotinic acetylcholine receptors and recovery is mediated by muscarinic acetylcholine receptors (Figure 4).
The role of muscarinic receptors in recovery from paralysis suggests a means to screen for new modulators of muscarinic signalling. Such modulators are of interest as potential treatments for the cognitive dysfunction associated with diseases such as Alzheimer’s disease and schizophrenia. We have performed a small scale, proof-of-principle screen of around 250 compounds with previously demonstrated bioactivity in C. elegans to demonstrate a potential application of this research (details coming soon!).