Fig 1.
Aldicarb preconditioning reduces drug-induced inhibition of pharyngeal pumping.
A) Pharyngeal pumping rate was quantified at indicated times for synchronized nematodes exposed to a sub-lethal dose of aldicarb. Nematodes were transferred to non-drug containing plates where the recovery of the pharyngeal function was observed at the indicated times. The shaded box indicates period of treatment. Data are shown as mean ± SEM of 6 worms in 3 independent experiments (N = 6). Statistical significance between exposed and non-exposed nematodes was calculated by two-way ANOVA test followed by Bonferroni correction. **p≤0.01. B) Synchronized L4 worms were incubated on 50 μM aldicarb plates. Non-exposed nematodes were used as controls. After 24 hours, worms were transferred to non-drug plates to allow the recovery of the pharyngeal function before subsequent exposure to 250 μM aldicarb for measurement of pharyngeal pumping C) Preconditioned worms exhibited higher pharyngeal pumping rate than non-preconditioned animals 6 and 24 hours after being transferred to plates containing 250 μM aldicarb. Data are mean ± SEM of 21 worms in at least 11 independent experiments (N = 21). Statistical significance between preconditioned and non-preconditioned nematodes was calculated by two-way ANOVA test followed by Bonferroni correction. **p≤0.01; ***p≤0.001.
Fig 2.
Nematodes preconditioned with paraoxon-ethyl are sensitized to subsequent organophosphate inhibition of pharyngeal pumping.
A) Synchronized L4 worms were incubated on either non-drug or 20 μM paraoxon-ethyl containing plates. After 24 hours, they were transferred to non-drug containing plates to allow the recovery of pharyngeal function. They were transferred to plates containing 100 μM paraoxon-ethyl where the pharyngeal pumping was scored. B) Paraoxon-ethyl-preconditioned nematodes exhibited a greater reduction of the pharyngeal pumping following transfer to 100 μM paraoxon-ethyl plates compared to the non-preconditioned animals. Data are shown as mean ± SEM of 7 worms in at least 4 independent experiments (N = 7). C) Nematodes were preconditioned as indicated in A, however, obidoxime was added to the recovery plate to promote the rescue of the acetylcholinesterase activity after paraoxon-ethyl inhibition. D) Preconditioned nematodes exhibited a similar response to maximal dose of paraoxon-ethyl when they were allowed to recover in the presence or in the absence of obidoxime. Data are shown as mean ± SEM of 6 worms in at least 3 independent experiments (N = 6). Statistical significance between preconditioned and non-preconditioned nematodes was calculated by two-way ANOVA test followed by Bonferroni corrections. ***p≤0.001.
Fig 3.
Aldicarb-preconditioned and non-preconditioned nematodes exhibit a similar pharyngeal function when subsequently exposed to paraoxon-ethyl.
A) Synchronized L4 worms were incubated on either vehicle control or 50 μM aldicarb plates for 24 hours. After recovery on non-drug containing plates, they were exposed to 100 μM paraoxon-ethyl and pumping was measured. B) Aldicarb pre-exposed nematodes exhibit a similar sensitivity to paraoxon-ethyl as non-preconditioned worms. Data are shown as mean ± SEM of 7 worms in at least 4 independent experiments (N = 7).
Fig 4.
Pharyngeal and body wall neuromuscular behaviours exhibit a paraoxon-ethyl intoxication pattern characterized by three phases, an initial inhibition, a spontaneous in drug recovery and a subsequent inhibition.
A) Pharyngeal pumping in nematodes exposed to 500 μM paraoxon-ethyl display a complete inhibition of pumping at 3 hours and an in-drug recovery at 6 hours. The complete inhibition of pumping is observed after 24 hours of exposure. Data are shown as mean ± SEM of 34 worms in at least 17 independent experiments (N = 34). B) Nematodes were exposed to 500 μM paraoxon-ethyl and body length recorded 1, 3, 6 and 24 hours of exposure. Percentage of body length was referenced against the corresponding age-matched untreated control. Similar to pumping the three phases of intoxication were observed encompassing: initial shrinkage of nematodes at 1 hour, spontaneous body length recovery after 6 hours and the subsequent shrinkage of length at 24 hours of exposure. Data are shown as mean ± SEM of 6 worms in 4 independent experiments (N = 6). Statistical significance was calculated by two-way ANOVA test followed by Bonferroni corrections. **p≤0.01; ***p≤0.001.
Fig 5.
The paraoxon-ethyl induced changes in pharyngeal pumping are not elicited by components underpinning pharyngeal neuromuscular function.
N2 wild type nematodes continuously exposed to 500 μM paraoxon-ethyl exhibited in drug recovery of the pharyngeal function at 6 hours followed by a subsequent inhibition at 24 hours. Data are shown as mean ± SEM of 16 worms in at least 8 independent experiments (N = 16). The acetylcholinesterase ACE-3 of C. elegans is specifically expressed in the isthmus of the pharynx [41]. ACE-3 deficient nematodes exposed to 500 μM paraoxon-ethyl exhibit a similar in-drug recovery followed by inhibition of the pharyngeal pumping rate compared to wild-type worms. Data are shown as mean ± SEM of 5 worms in at least 3 independent experiments (N = 5). Nematodes lacking EAT-2 did not exhibit the in drug recovery observed in wild type worms. Data are shown as mean ± SEM of 6 worms in at least 3 independent experiments (N = 6). The muscarinic receptor GAR-3 is expressed in the isthmus and is involved in the feeding movement [46]. Mutant nematodes lacking GAR-3 exhibited a similar paraoxon-induced plasticity of the pumping rate compared to the wild type worms. Data are shown as mean ± SEM of 6 worms in at least 3 independent experiments (N = 6). E) avr-15 encodes a glutamate-gated chloride channel subunit responsible for the relaxation of the pharyngeal muscle upon contraction [45]. avr-15 mutant worms exhibit a similar pattern of pharyngeal pumping rate than wild type animals intoxicated on 500 μM paraoxon-ethyl plates. Data are shown as mean ± SEM of 6 worms in at least 3 independent experiments (N = 6). Statistical significance was calculated by two-way ANOVA test followed by Bonferroni corrections. nsp>0.05; *p≤0.05.
Fig 6.
Nematodes deficient in the non-alpha subunits of the L-type body wall muscle receptor, LEV-1 and UNC-29, exhibited a sustained in drug recovery of the pharyngeal pumping.
Paraoxon-ethyl induced plasticity of the pharyngeal function in wild type nematodes exposed to 500 μM and 1 mM. Data are shown as mean ± SEM of 15 worms in 8 independent experiments (N = 15) or 18 worms in 9 independent experiments (N = 18), respectively. Nematodes deficient in the chaperone protein RIC-3 exhibited resistance to the inhibition of the pumping in the presence of 500 μM of paraoxon-ethyl. The exposure to 1 mM concentration inhibited the pumping rate after 3 hours and showed an in drug spontaneous recovery sustained for up to 24 hours. Data are shown as mean ± SEM of 6 worms in 3 independent experiments for each concentration (N = 6). lev-1 encodes a non-alpha subunit of the L-type receptor. LEV-1 lacking nematodes phenocopy the paraoxon-ethyl induced sustained in drug recovery of ric-3 deficient nematodes. Data are shown as mean ± SEM of 6 worms in 3 independent experiments (N = 6) for 500 μM exposure or 8 worms in 4 independent experiments for 1 mM exposure. UNC-29 is the other non-alpha subunit of the L-type receptor. Nematodes deficient in UNC-29 exhibited wild type resistance to paraoxon-ethyl but a sustained in drug recovery of the pharyngeal pharyngeal pumping after 24 hours in 500 μM and 1 mM. Data are shown as mean ± SEM of 18 worms in 9 independent experiments (N = 18) or 11 worms in 6 independent experiments (N = 11), respectively. Statistical significance was calculated by two-way ANOVA test followed by Bonferroni corrections. nsp>0.05; *p≤0.05; **p≤0.01; ***p≤0.001.
Fig 7.
Body wall muscle rescue of the non-alpha subunits LEV-1 and UNC-29 restores wild type sensitivity to prolonged paraoxon-ethyl exposure.
A) The pharyngeal sensitivity and the three phases characteristic of drug-induced plasticity to paraoxon-ethyl in the lev-1 deficient worms were restored by introducing the wild type version of the gene selectively in the body wall muscles under control of the myo-3 promoter. Data are shown as mean ± SEM of 6 worms in 3 independent experiments for N2 and lev-1 (e211) strains (N = 6); 12 worms in 6 independent experiments of 2 independent lines for lev-1 control line (N = 12) and 22 worms in 11 independent experiments of 4 independent lines for lev-1 BW rescue (N = 22). The genotype of control and BW rescue lines corresponds to CB211 lev-1 (e211) IV; Ex[Punc-122::gfp] and CB211 lev-1 (e211) IV; Ex[Punc-122::gfp; Pmyo-3::lev-1], respectively. B) The introduction of the wild type UNC-29 in the body wall muscles of unc-29 mutant worms rescued the consequent inhibition of the pharyngeal function that follows the spontaneous recovery in paraoxon-ethyl exposed worms. Data are shown as mean ± SEM of 10 worms in 5 independent experiments for N2 (N = 10); 6 worms in 3 independent experiments for unc-29 (e193) (N = 6); 16 worms in 8 independent experiments of 3 independent lines for unc-29 control line (N = 16) and 20 worms in 10 independent experiments of 3 independent lines for unc-29 BW rescue (N = 20). The genotype of control and BW rescue lines corresponds to CB193 unc-29 (e193) I; Ex[Punc-122::gfp] and CB193 unc-29 (e193) I; Ex[Punc-122::gfp; Pmyo-3::unc-29], respectively. Statistical significance was calculated by two-way ANOVA test followed by Bonferroni corrections. nsp>0.05; *p≤0.05; **p≤0.01; ***p≤0.001.
Fig 8.
The efficacy of the L-type receptor is a significant determinant of the spontaneous recovery of pharyngeal pumping in nematodes exposed to paraoxon-ethyl.
Paraoxon-induced pharyngeal plasticity in wild type nematodes incubated with 500 μM organophosphate. Data are shown as mean ± SEM of 14 worms in at least 7 independent experiments (N = 14). lev-1 (e211) mutant strain contains a single point mutation in the fourth transmembrane domain. This confers resistance to paraoxon-ethyl induced inhibition of the pharyngeal pumping. Data are shown as mean ± SEM of 8 worms in at least 4 independent experiments (N = 8). The mutation of lev-1 (x427) strain consists of a rearrangement of the genomic sequence that prevents the transcription of the gene into a protein [49]. This is the reference null-mutant of LEV-1. This mutation does not phenocopy the pharyngeal response to paraoxon-ethyl observed in lev-1 (e211) mutant background. Data are mean ± SEM of 8 worms in at least 4 independent experiments (N = 8). The mutation of unc-29 (e193) strain consists of a single point proline to serine in the loop connecting the second and third transmembrane domain of the subunit. This proline is highly conserved in the cys-loop receptor subunits and is implicated with the gating of the receptor [50–52]. This mutation conferred a sustained paraoxon-ethyl induced plasticity. Data are shown as mean ± SEM of 8 worms in at least 4 independent experiments (N = 8). In contrast, the null-mutant unc-29 (e1072) exhibited resistance to the pharyngeal inhibition by paraoxon-ethyl but did not express paraoxon-ethyl induced plasticity. Data are shown as mean ± SEM of 8 worms in at least 4 independent experiments (N = 8). MOLO-1 is an auxiliary protein implicated in the positive modulation of the L-type receptor [24]. molo-1 lacking worms exposed to paraoxon-ethyl exhibited spontaneous recovery of the pharyngeal function that was sustained compared to wild type worms. Data are shown as mean ± SEM of 7 worms in at least 4 independent experiments (N = 7). Statistical significance was calculated by two-way ANOVA test followed by Bonferroni corrections. ***p≤0.001.
Fig 9.
The mutant genes in lev-1 (e211) and unc-29 (e193) encode for subunits that assemble L-type receptors with reduced function.
Current amplitude to 300 μM of acetylcholine was quantified in different populations of L-type receptors in Xenopus oocytes expressed from cRNA encoding either wild type, lev-1 (e211) or unc-29 (e193) mutations (dark blue). The substitution of the cRNA for wild type lev-1 or unc-29 subunits for their respective lev-1 (e211) or unc-29 (e193) mutations reduced the current amplitude of the L-type receptor. The co-expression of wild type and mutant genes in a 1:1 ratio causes a reduction of the amplitude to acetylcholine-evoked currents, indicating both subunits compete for inclusion into the mature ion channel. Data are shown as the mean ± SEM. Numbers above bars indicate the number of oocytes recorded for each condition. Statistical significance was calculated by two-tail t-test. ***p≤0.001.
Fig 10.
Mutants that underpin the synaptic organization of the L-type receptors modifies the pharyngeal pumping observed in nematodes exposed to paraoxon-ethyl.
The pharyngeal function of wild type nematodes exposed to paraoxon-ethyl exhibited drug-induced plasticity. Data are shown as mean ± SEM of 12 worms in at least 6 independent experiments (N = 12). OIG-4 is a muscle-secreted protein involved in the location of the L-type receptor at the body wall NMJ [55]. Oig-4 lacking nematodes are deficient in the in-drug recovery of pharyngeal pumping observed in the wild type worms. Data are shown as mean ± SEM of 8 worms in at least 4 independent experiments (N = 8). RSU-1 is a cytosolic muscle protein involved in maintaining the equilibrium between synaptic and extra-synaptic nicotinic receptors [54]. rsu-1 lacking nematodes exposed to paraoxon-ethyl are deficient in the within drug recovery of the pharyngeal pumping. Data are shown as mean ± SEM of 7 worms in at least 4 independent experiments (N = 7). Statistical significance was calculated by two-way ANOVA test followed by Bonferroni corrections. ***p≤0.001.
Fig 11.
Hypothesised mechanism underpinning paraoxon-induced plasticity in nematodes exposed to 500 μM paraoxon-ethyl.
A) The body wall motor neuron releases acetylcholine to the neuromuscular junction that activates L-type and N-type nicotinic receptors causing the muscle contraction. In the current study this determinant is measured by scoring pharyngeal function as body wall muscle contraction leads to an associated inhibition of pharyngeal function. At the level of the body wall muscle acetylcholinesterases catalyse the breakdown of acetylcholine. This controls cholinergic transmission from the motor neuron that allows balanced excitation at the body wall muscle. B) The inhibition of the acetylcholinesterase by paraoxon-ethyl causes an increase of acetylcholine at the neuromuscular junction. This leads to the overstimulation of the cholinergic receptors expressed in the muscle fibres and, therefore the hypercontraction of the body wall muscle and an associated inhibition of pharyngeal pumping. C) Molecular determinants that impact the location and sensitivity of L-type receptors at the body wall neuromuscular junction alter the consequence of organophosphate intoxication. Further investigations are required to identify if these determinants encompassing hypo-functional L-type receptors, MOLO-1, OIG-4, RSU-1 execute their impact thought structural or functional changes of the cholinergic synapse.
Table 1.
Primer sequences and PCR conditions for mutation analysis of alleles in lev-1 and unc-29.