Chemically induced oxidative stress affects ASH neuronal function and behavior in C. elegans

Oxidative stress (OS) impact on a single neuron’s function in vivo remains obscure. Using C. elegans as a model organism, we report the effect of paraquat (PQ)-induced OS on wild type worms

on the function of the ASH polymodal neuron. By calcium (Ca2+) imaging, we quantified ASH activation upon stimulus delivery. PQ-treated worms displayed higher maximum depolarization (peak of

the Ca2+ transients) compared to untreated animals. PQ had a similar effect on the ASH neuron response time (rising slope of the Ca2+ transients), except in very young worms. OS effect on

ASH was partially abolished in vitamin C-treated worms. We performed octanol and osmotic avoidance tests, to investigate the OS effect on ASH-dependent behaviors. PQ-treated worms have

enhanced avoidance behavior compared to untreated ones, suggesting that elevated ASH Ca2+ transients result in enhanced ASH-mediated behavior. The above findings suggest a possible hormetic

effect of PQ, as a factor inducing mild oxidative stress. We also quantified locomotion parameters (velocity, bending amplitude), which are not mediated by ASH activation. Bending amplitude

did not differ significantly between treated and untreated worms; velocity in older adults decreased. The differential effect of OS on behavioral patterns may mirror a selective impact on

the organism’s neurons.

Oxidative stress (OS) is one of the most significant types of stress an organism experiences throughout its life. It is the result of exposure to numerous environmental factors, including

chemical compounds1. The effect of OS on the nervous system is of special interest, since it has been associated with neurodegenerative diseases2,3, such as Alzheimer’s4 and Parkinson’s5.

Moreover, there is evidence that exposure to certain herbicides, namely paraquat (PQ), known to induce OS6, is strongly associated with higher frequency of neurodegenerative diseases in

humans7.

However, the effect of OS on sensory neurons physiology remains obscure. Altered function of sensory neurons could lead to misperception of the organism’s environment, resulting in a

modified behavior, not uncommon in neurodegenerative diseases. A strong correlation between hyperexcitability of sensory neurons and characteristic symptoms (e.g. photophobia in migraine)

has been shown8, as well as a strong connection between OS and behavioral changes9,10. Monitoring in vivo neuronal function under OS could deepen our understanding on the impact of OS on an

organism’s nervous system as well as on the engendered behaviors. Nevertheless, there is a lack of studies exploring in vivo the effects of OS on single neuron functionality.

In parallel, C. elegans has been broadly used in numerous studies which explore the phenomenon of hormesis, caused, among other harmful conditions, by mild OS11. Beneficial effects of low

doses of oxidative factors have been reported to result in increased lifespan and increased resistance to other types of stress12,13. However, the possibility of OS-driven hormetic effects

on neuronal Ca2+ transients has not been investigated yet.

Here, we investigate the effects of chemically induced OS in the nematode Caenorhabditis elegans, using state-of-the-art microfluidic technology and in vivo calcium (Ca2+) imaging. C.

elegans has been widely used to investigate both OS and neurodegenerative diseases14,15 and is therefore an ideal model organism to examine the interplay between OS and neuronal functional

physiology16,17. We exposed wild type worms of different ages to PQ and to vitamin C (VitC), a well-established antioxidant18,19. We chose PQ concentrations that are known to cause changes

in the worms’ physiology due to oxidative damage16,20, since PQ is known to interfere with electron transfer and catalyze the production of reactive oxygen species (ROS). Using a

microfluidic chip21, we delivered a hyperosmotic stimulus (glycerol) to the worm’s nose and recorded Ca2+ transients from the ASH sensory neuron. We chose ASH, the well-studied worms’ main

nociceptor21,22,23,24,25,26,27,28, because it can be activated by a wide range of repellents25,29,30, thus being especially significant for C. elegans survival and environmental perception.

With the exception of very young worms, stimulus-evoked calcium transients from the ASH neuron are elevated in OS-treated worms, when compared to non-treated animals. This effect is

partially abolished when the worms are simultaneously treated with PQ and VitC. Octanol avoidance test and glycerol drop assay confirmed that behaviors directly mediated by ASH are enhanced

by OS, and this effect is also partially abolished in the presence of VitC. In younger worms, VitC does not reverse the enhancement of the avoidance to glycerol, whereas it reverses the

effect on the response to octanol. In contrast, we found that OS-exposed worms did not display significant differences in their locomotive parameters (velocity and bending amplitude)

compared to untreated ones, with the exception of the average velocity in older worms.

We conclude that exposure to chemically induced OS significantly affects the function of ASH neuron, as well as avoidance behaviors directly mediated by ASH. The observation that behaviors

independent of ASH are not correspondingly affected, could strongly suggest that neurons are not uniformly affected by chemically induced OS. This could further imply a differential

susceptibility to OS among neurons, neuronal circuits and subsequently, behavioral patterns. Our results can pave the way for further experiments on the interplay between OS and neuronal

function.

Last, we suggest that calcium imaging and microfluidic platforms can be a powerful tool for revealing OS-dependent changes in neuronal function in C. elegans, and potentially in other model

organisms as well.

We used the microfluidic platform to quantify the effect of rearing on 0.1 mM PQ to the function of the ASH neuron (Fig. 1). We monitored the activity of ASH using the TNXL indicator in

response to a 30 sec stimulus pulse (1 M glycerol). We examined four groups of age-synchronous worm populations: treated with 0.1 mM PQ, treated with 1 mM VitC, treated with 0.1 mM PQ + 1 

mMVitC and untreated (control) ones (Fig. 2A). We imaged a total of 256 worms, aged L4 + 1 day (marked here as “Day 1” worms), L4 + 4 days (referred to as “Day 4” worms) and L4 + 5 days

(mentioned here as “Day 5” worms).

(A) The 4-flow microfluidic chip is used for immobilizing single worms and delivering a chemical stimulus (glycerol) to their nose21 to induce neuronal activation. Before and after the

delivery of the stimulus, a control buffer solution is being delivered to the worm’s nose. Scale bar: 200 μm. (B) Fluorescence image of a trapped worm. The ASH neuron (highlighted by the

dotted circle) is labelled with the FRET indicator TNXL. Top Image: YFP channel, bottom image: CFP channel, scale bar: 5 μm. (C) A typical Ca2+ transient response of the ASH neuron. The

stimulus is delivered at 10 sec and removed at 40 sec. The maximum FRET ratio change upon delivery of the stimulus is considered as the “peak” response. The ASH neuron has a biphasic

(ON/OFF) response21.

(A) Ratiometric calcium transients of the ASH neuron upon delivery of hyperosmotic stimulus (1 M glycerol) at 3 different ages (L4 animals are considered as Day 0 worms). Worms studied:

untreated (control worms), VitC worms (grown at the presence of 1 mM vitamin C), PQ worms (grown at the presence of 0.1 mM PQ) and VitC/PQ worms (grown at the simultaneous presence of 1 mM

vitamin C and 0.1 mM PQ). The dashed line represents the presence of the stimulus. (B) The maximum peak and rising slope of the on-response (calculated from A). The number in each column

represents the number of worms tested. Error bars indicate standard error of mean. ***p