Investigation of feeding behaviour in C. elegans reveals distinct pharmacological and antibacterial effects of nicotine.

Caenorhabditis elegans is an informative model to study the neural basis of feeding. A useful paradigm is one in which adult nematodes feed on a bacterial lawn which has been pre-loaded with pharmacological agents and the effect on pharyngeal pumping rate scored. A crucial aspect of this assay is the availability of good quality bacteria to stimulate pumping to maximal levels. A potential confound is the possibility that the pharmacological agent impacts bacterial viability and indirectly influences feeding rate. Here, the actions of nicotine on pharyngeal pumping of C. elegans and on the Escherichia coli bacterial food source were investigated. Nicotine caused an immediate and concentration-dependent inhibition of C. elegans pharyngeal pumping, IC50 4 mM (95% CI = 3.4 mM to 4.8 mM). At concentrations between 5 and 25 mM, nicotine also affected the growth and viability of E. coli lawns. To test whether this food depletion by nicotine caused the reduced pumping, we modified the experimental paradigm. We investigated pharyngeal pumping stimulated by 10 mM 5-HT, a food 'mimic', before testing if nicotine still inhibited this behaviour. The IC50 for nicotine in these assays was 2.9 mM (95% CI = 3.1 mM to 5.1 mM) indicating the depletion of food lawn does not underpin the potency of nicotine at inhibiting feeding. These studies show that the inhibitory effect of nicotine on C. elegans pharyngeal pumping is mediated by a direct effect rather than by its poorly reported bactericidal actions.

The concentration-dependent effects of ethanol on Caenorhabditis elegans behaviour.

The effects of ethanol on the brain are concentration dependent. Low concentrations (mM) intoxicate, while greater than 100 mM anaesthetize. Of most relevance to human alcohol addiction are mechanisms of intoxication. Previously, Caenorhabditis elegans has been employed in genetic screens to define effectors of intoxication. Here, we inform interpretation of these studies by providing evidence that ethanol rapidly equilibriates across C. elegans cuticle. Importantly, the effect of ethanol on muscle activity rapidly reaches steady-state, and the concentration-dependence of the effect is very similar in intact animals and exposed muscle. Thus the cuticle does not present an absorption barrier for ethanol, and furthermore the internal concentration is likely to approach that applied externally. Thus, modelling intoxication in C. elegans requires exposure to external ethanol less than 100 mM. Furthermore, the permeability of the cuticle to ethanol enables analysis of precisely controlled concentration-dependent effects of acute, chronic, and episodic ethanol exposure on behaviour.

Molecular characterization of the metabotropic glutamate receptor family in Caenorhabditis elegans.

mGluRs (metabotropic glutamate receptors) are G-protein-coupled receptors that play an important neuromodulatory role in the brain. Glutamatergic transmission itself plays a fundamental role in the simple nervous system of the model organism Caenorhabditis elegans, but little is known about the contribution made by mGluR signalling. The sequenced genome of C. elegans predicts three distinct genes, mgl-1, mgl-2 and mgl-3 (designated Y4C6A.2). We have used in silico and cDNA analyses to investigate the genes encoding mgls. Our results indicate that mgl genes constitute a gene family made up of three distinct subclasses of receptor. Our transcript analysis highlights potential for complex gene regulation with respect to both expression and splicing. Further, we identify that the predicted proteins encoded by mgls harbour structural motifs that are likely to regulate function. Taken together, this molecular characterization provides a platform to further investigate mGluR function in the model organism C. elegans.

Molecules of the mind: integrating synaptic biochemistry to understand brain function.

The focused meeting entitled 'Molecular Determinants of Synaptic Function: Molecules and Models' brought together several molecules and experimental models that are furthering our understanding of the biochemical basis of integrative brain function. Invited speakers and short communications from more junior scientists highlighted how individual molecules or protein networks underlie defined subcellular functions (e.g. transmitter release, receptor activation and transmitter uptake) can be used to unravel integrative function at cellular, systems and behavioural levels.

Actions of glutamate and ivermectin on the pharyngeal muscle of Ascaridia galli: a comparative study with Caenorhabditis elegans.

The actions of glutamate and ivermectin were examined in the pharynx of Ascaridia galli and the results compared with those on the pharynx of Caenorhabditis elegans. In both preparations glutamate elicits a depolarization and inhibition of pharyngeal pumping, but the response of the pharynx of A. galli was much less than for C. elegans. This may be either because the pharyngeal membrane potential of the former is closely linked to the equilibrium potential for chloride ions (E(Cl)) while that of C. elegans is independent of E(Cl), or that there is a lower density of glutamate receptors on the pharyngeal muscle of A. galli compared with C. elegans. The maximum depolarization to glutamate of the pharyngeal muscle was 4.5+/-0.8 mV in A. galli while it was >25 mV in C. elegans. Picrotoxin was a weak antagonist of the glutamate response in both species. Flufenamic acid, pentobarbitone and flurazepam had no significant effect on either preparation at concentrations up to 100 microM. Three glutamate receptor agonists, ibotenate, kainate and quisqualate were all more potent than glutamate on the A. galli pharyngeal muscle. In contrast, only ibotenate was more potent than glutamate in C. elegans pharynx, the other two agonists being approximately 20 times less potent. The potency of ivermectin differed markedly between the two species, being approximately three orders of magnitude less potent on the pharynx of A. galli compared with C. elegans. This study demonstrates clear differences between the properties of the pharyngeal muscle of the two species and shows that care must be taken when extrapolating data from free-living to parasitic species of nematode.

Mechanisms of action of emodepside.

The research of the class of cyclic octadepsipeptides started at the beginning of the 1990s. PF1022A, the starting material of emodepside, is a natural secondary metabolite of the fungus Mycelia sterilia, which belongs to the microflora of the leaves of Camellia japonica. PF1022A consists of four N-methyl-L-leucins, two D-Iactic acids and two D-phenyllactic acids, which build up a cyclic octadepsipeptide with an alternating L-D-L-configuration. Emodepside is a semisynthetic derivative of PF1022A, which contains a morpholine attached in para position at each of both D-phenyllactic acids. Emodepside is efficacious against a variety of gastrointestinal nematodes. Emodepside binds to a presynaptic latrophilin receptor in nematodes. The following presynaptic signal transduction occurs via activation of Gqalpha protein and phospholipase-Cbeta, which leads to mobilization of diacylglycerol (DAG). DAG then activates UNC-13 and synaptobrevin, two proteins which play an important role in presynaptic vesicle-functioning. This finally leads to the release of a currently unidentified transmitter. The transmitter (or modulator) exerts its effects at the postsynaptic membrane and induces a flaccid paralysis of the pharynx and the somatic musculature in nematodes.

The effect of the anthelmintic emodepside at the neuromuscular junction of the parasitic nematode Ascaris suum.

Here we report on the action of the novel cyclo-depsipeptide anthelmintic, emodepside, on the body wall muscle of the parasitic nematode, Ascaris suum. Emodepside caused (i) muscle relaxation, (ii) inhibition of muscle contraction elicited by either acetylcholine (ACh), or the neuropeptide, AF2 (KHEYLRFamide) and (iii) a rapid relaxation of muscle tonically contracted by ACh. The inhibitory action of emodepside on the response to ACh was not observed in a denervated muscle strip, indicating that it may exert this action through the nerve cord, and not directly on the muscle. Electrophysiological recordings showed emodepside elicited a Ca(++)-dependent hyperpolarization of muscle cells. Furthermore, the response to emodepside was dependent on extracellular K+, similar to the action of the inhibitory neuropeptides PF1 and PF2 (SDPNFLRFamide and SADPNFLRFamide). Thus emodepside may act at the neuromuscular junction to stimulate release of an inhibitory neurotransmitter or neuromodulator, with a similar action to the PF1/PF2 neuropeptides.

Regulation of the pharynx of Caenorhabditis elegans by 5-HT, octopamine, and FMRFamide-like neuropeptides.

More than fifty FMRFamide-like neuropeptides have been identified in nematodes. We addressed the role of a subset of these in the control of nematode feeding by electrophysiological recording of the activity of C. elegans pharynx. AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), AF8 (KSAYMRFamide), and GAKFIRFamide (encoded by the C. elegans genes flp-8, flp-14, flp-6, and flp-5, respectively) increased pharyngeal action potential frequency, in a manner similar to 5-HT. In contrast, SDPNFLRFamide, SADPNFLRFamide, SAEPFGTMRFamide, KPSVRFamide, APEASPFIRFamide, and AQTVRFamide (encoded by the C. elegans genes flp-1; flp-1; flp-3; flp-9; flp-13, and flp-16, respectively) inhibited the pharynx in a manner similar to octopamine. Only three of the neuropeptides had potent effects at low nanomolar concentrations, consistent with a physiological role in pharyngeal regulation. Therefore, we assessed whether these three peptides mediated their actions either directly on the pharynx or indirectly via the neural circuit controlling its activity by comparing actions between wild-type and mutants with deficits in synaptic signaling. Our data support the conclusion that AF1 and SAEPFGTMRFamide regulate the activity of the pharynx indirectly, whereas APEASPFIRFamide exerts its action directly. These results are in agreement with the expression pattern for the genes encoding the neuropeptides (Kim and Li, 1999) as both flp-8 and flp-3 are expressed in extrapharyngeal neurons, whereas flp-13 is expressed in I5, a neuron with synaptic output to the pharyngeal muscle. These results provide the first, direct, functional information on the action of neuropeptides in C. elegans. Furthermore, we provide evidence for a putative inhibitory peptidergic synapse, which is likely to have a role in the control of feeding.

Characterization of glutamate-gated chloride channels in the pharynx of wild-type and mutant Caenorhabditis elegans delineates the role of the subunit GluCl-alpha2 in the function of the native receptor.

Glutamate-gated chloride (GluCl) channels are the site of action of the anthelmintic ivermectin. Previously, the Xenopus laevis oocyte expression system has been used to characterize GluCl channels cloned from Caenorhabditis elegans. However, information on the native, pharmacologically relevant receptors is lacking. Here, we have used a quantitative pharmacological approach and intracellular recording techniques of C. elegans pharynx to characterize them. The glutamate response was a rapidly desensitizing, reversible, chloride-dependent depolarization (EC(50) = 166 microM), only weakly antagonized by picrotoxin. The order of potency of agonists was ibotenate > L-glutamate > kainate = quisqualate. Ivermectin potently and irreversibly depolarized the muscle (EC(50) = 2.7 nM). No further depolarization was seen with coapplication of maximal glutamate during the maximal ivermectin response, indicating that ivermectin depolarizes the muscle by the same ionic mechanism as glutamate (i.e., chloride). The potency of ivermectin on the pharynx was greater than at any of the GluCl subunits expressed in X. laevis oocytes. This effect of ivermectin was abolished in the mutant avr-15, which lacks a functional GluCl-alpha2 subunit. However, a chloride-dependent, nondesensitizing response to glutamate persisted. Therefore, the GluCl-alpha2 subunit confers ivermectin sensitivity and a high-affinity desensitizing glutamate response on the native pharyngeal GluCl receptor.

Characterization of 5-HT receptors in the parasitic nematode, Ascaris suum.

The pharmacological profiles of the 5-hydroxytryptamine (5-HT) receptors on Ascaris suum pharyngeal and somatic body wall muscles were investigated. The mechanisms involved following activation of these receptors were also studied. 5-HT activated and maintained pumping in isolated pharynxes with an EC-50 value of 44+/-1.7 microM. The 5-HT agonists, tryptamine, sumatriptan 8-OH-DPAT and 5-carboxyamidotryptamine all failed to stimulate pumping. The 5-HT2 antagonist, ketanserin, initially excited and then inhibited pumping while the 5-HT3 antagonist, ondansetron, had no effect. 5-HT and 5-HT agonists, 8-OH-DPAT, 5-carboxyamidotryptamine, alpha-methyl-5-HT and tryptamine all inhibited ACh-induced contractions of a somatic body wall muscle strip. Ketanserin partially blocked the inhibitory effect of alpha-methyl-5-HT and ACh-induced contractions while the 5-HT uptake blocker, fluoxetine, potentiated the effect of 5-HT on ACh-induced contractions. Basal levels of cAMP, 1540+/-232 pmol/mg, in pharyngeal muscle and 1721+/-134 pmol/mg, somatic body wall muscle, were both increased by forskolin. 5-HT had no effect on pharyngeal muscle cAMP levels but raised cAMP levels in somatic body wall muscle, e.g. 100 micron 5-HT, raised the level to 2851+/-212 pmol/mg and 1000 microM raised levels to 4578+/-1234 pmol/mg. 5-HT, 1000 microM, increased inositol phosphate levels in pharyngeal muscle. These results provide some evidence for a 5-HT2-like receptor on pharyngeal muscle. In contrast, the situation on somatic body wall muscle is more confusing since the pharmacological profile partly indicates a 5-HT2-like receptor but this receptor is linked to a rise in cAMP levels. Further studies are required to resolve the position but they must be based on the rational design of ligands specifically for nematode 5-HT receptors and not simply using ligands developed for the classification of mammalian 5-HT receptors. Such a design must take into account data from molecular biology studies of nematode 5-HT receptors.

Characterization of a putative nitric oxide synthase in the neuromuscular system of the parasitic nematode, Ascaris suum.

In this paper we report on the biochemical presence of nitric oxide synthase (NOS)-like activity in Ascaris suum tissue and examine the pharmacological effect of NO donors on A. suum muscle strip preparation. NOS activity was determined by monitoring the formation of [3H]citrulline from [3H]L-arginine and NO formation via the oxyhaemoglobin assay. Neuromuscular tissue from A. suum which stained positively for NADPH diaphorase, contained NOS activity. Neither NOS activity nor NADPH diaphorase staining was detected in intestinal tissue. The absence of Ca2+, NADPH and other co-factors normally required for mammalian neuronal NOS activity only partially reduced the formation of both citrulline and NO by A. suum neuromuscular homogenate. The results of the biochemical assays indicate the presence of an enzyme capable of producing NO and citrulline, but with a different profile from that of rat neuronal NOS. We also present preliminary evidence for the action of NO (NO donors) in the neuromuscular system of A. suum.

Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans.

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.

Physiological and pharmacological studies on nematodes.

Classical transmitters and neuroactive peptides act as transmitters or modulators within the central and peripheral nervous systems of nematodes, for example Ascaris suum and Caenorhabditis elegans. Acetylcholine (ACh) and gamma-aminobutyric acid (GABA) are respectively the excitatory and inhibitory transmitters onto somatic body wall muscle while 5-hydroxytrypamine (5-HT) is the excitatory transmitter onto pharyngeal muscle. 5-HT also reduces ACh-induced contractions of somatic muscle and this action of 5-HT is mediated through activation of adenylate cyclase while that on pharyngeal muscle is mediated through inositol phosphate activation. Glutamate, dopamine and octopamine also have transmitter roles in nematodes. Neuroactive peptides of the RFamide family can excite somatic muscle, for example, AF-1 (KNEFIRFamide), AF-2 (KHEYLRFamide), AF-3 (AVPGVLRFamide) and AF-4 (GDVPGVLRFamide) or inhibit and relax this muscle, for example, PF-1 (SDPNFLRFamide), PF-2 (SADPNFLRFamide) and PF-4 (KPNlRFamide). In addition PF-3 (AF-8) (KSAYMRFamide) has a biphasic action on pharyngeal muscle, excitation followed by inhibition while AF-1 only inhibits this muscle. The peptide effects can be either pre- or postsynaptic or both and are likely to be mediated through second messenger systems. In addition these peptides modulate the action of classical transmitters, particularly ACh.

The range and biological activity of FMRFamide-related peptides and classical neurotransmitters in nematodes.

Nematodes include both major parasites of humans, livestock and plants in addition to free-living species such as Caenorhabditis elegans. The nematode nervous system (especially in C. elegans) is exceptionally well defined in terms of the number, location and projections of the small number of neurons in the nervous system and their integration into circuits involved in regulatory behaviours vital to their survival. This review will summarize what is known about the biological activity of neurotransmitters in nematodes: the biosynthetic pathways and genes involved, their receptors, inactivation mechanisms and secondary messenger signalling systems. It will cover the 'classical' transmitters, such as acetylcholine (ACh), GABA, glutamate, serotonin, dopamine, octopamine, noradrenaline and nitric oxide. The localization of peptides throughout the nematode nervous system is summarized, in addition to the isolation of nematode neuropeptides by both traditional biochemical techniques and more modern genetic means. The major contribution of the completion of the C. elegans genome-sequencing program is highlighted throughout. Efforts to unravel neurotransmitter action in various physiological actions such as locomotion, feeding and reproduction are detailed as well as the various inactivation mechanisms for the current complement of nematode transmitters.

The inhibitory action of tamoxifen on the contraction of Ascaris suum somatic muscle in response to acetylcholine.

The somatic muscle of Ascaris suum is principally under the excitatory control of neuromuscular junction transmitter, acetylcholine (ACh). However, it has recently been shown that neuropeptides also play an important role in the motor-nervous system and one of these, AF3 (AVPGVLRFamide), also contracts muscle. The events which trigger contraction to ACh and AF3 would appear to be different, with ACh activating a nicotinic acetylcholine receptor whilst the response to AF3 is most likely to involve a G-protein coupled receptor negatively coupled to adenylate cyclase. In order to further elucidate differences in the cellular signalling pathways through which ACh and AF3 elicit muscle contraction, we investigated the actions of protein kinase C inhibitors, tamoxifen and chelerythrine, on the dorsal somatic muscle strip of A. suum. Contractions in response to 1 microM AF3 were potentiated by 17% in the presence of 10 microM tamoxifen (P < 0.05; n = 8); however, contractions in response to 10 microM ACh were markedly inhibited (tamoxifen IC50 44 +/- 18 microM; n = 6). Tamoxifen also blocked muscle cell depolarizations to 5 microM ACh (IC50 4 +/- 1 microM; n = 6) and 1 microM levamisole (IC50 14 +/- 6 microM; n = 4). This was unlikely to be a non-specific effect on the membrane as hyperpolarizations to 10 microM GABA were unaffected (93% of control with 10 microM tamoxifen; n = 6; P > 0.05). However, another inhibitor of mammalian protein kinase C, chelerythrine, did not affect the response either to ACh or AF3 (n = 6).

The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms.

The pharmacological diversity of the different isoforms of the nicotinic acetylcholine receptor arises from the diversity of the subunits that assemble to form the native receptors. The aim of this study was to investigate the actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle, mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression system. Oocytes were injected with cRNAs for alpha, beta, gamma, delta subunits (the native foetal muscle subunit combination), or with cRNAs for alpha, beta, epsilon, delta subunits (the native adult muscle subunit combination), or with cRNAs for alpha4beta2 subunits (a putative native neuronal subunit combination). Acetylcholine had a similar potency at all three subunit combinations (EC50 11.6, 17.4 and 19.1 microM, respectively). At all three receptor types, d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine in a reversible manner. Furthermore, the inhibition of the acetylcholine currents for the foetal and adult nicotinic acetylcholine receptor by pancuronium and d-tubocurarine was independent of the holding voltage over the range -100 to -40 mV. In oocytes expressing the foetal muscle nicotinic acetylcholine receptors the inhibition of the current in response to 100 microM acetylcholine by 10 nM d-tubocurarine was 29 +/- 5% (mean +/- S.E.M.; n = 7), and the inhibition by 10 nM pancuronium was 39 +/- 6% (mean +/- S.E.M.; n = 8; P > 0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both more effective at blocking the response to 100 microM acetylcholine compared to the foetal muscle nicotinic acetylcholine receptor, with values of 55 +/- 5% (P < 0.01; n = 12) and 60 +/- 4% (P < 0.001; n = 10), respectively. Thus the developmental switch from the gamma to the epsilon subunit alters the antagonism of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine. Vecuronium was more potent than pancuronium. One nM vecuronium reduced the response to 100 microM acetylcholine by 71 +- 6% (n = 10) for foetal and 63 +/- 5% (n = 4) for adult nicotinic acetylcholine receptors. In the alpha4beta2 neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a more effective antagonist of the response to 100 microM acetylcholine (69 +/- 6%, n = 6) than 10 nM d-tubocurarine (30 +/- 5%; n = 6; P < 0.05 compared to pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine receptor, where pancuronium and d-tubocurarine were equieffective. The expression of the beta2 subunit with muscle alpha, epsilon and delta subunits formed a functional receptor which was blocked by pancuronium and d-tubocurarine in a similar manner to the alphabeta1epsilondelta subunit consistent with the hypothesis that the beta subunit is not a major determinant in the action of this drug at the adult muscle nicotinic acetylcholine receptor.

Immunocytochemical localization of a putative inhibitory amino acid receptor subunit in the parasitic nematodes Haemonchus contortus and Ascaris suum.

A rabbit antiserum was raised against a synthetic peptide corresponding to a region near the N-terminus of the Haemonchus contortus inhibitory amino acid receptor subunit, HG1. The antiserum recognized a recombinant form of the N-terminal domain of the subunit on Western blots and reacted with the ventral nerve cord of H. contortus in immunofluorescence experiments. Immunofluorescence was also detected in specific head neurons of H. contortus: these were tentatively identified as ring motor- and inter-neurons, plus a possible sensory neuron equivalent to the AQR cell of Caenorhabditis elegans. In the roundworm Ascaris suum, immunoreactivity was limited to the muscle arms, the post-synaptic component of the neuromuscular junction. The possible ligand of receptors containing the HG1 subunit is discussed in the light of this expression pattern.

The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascaridia galli.

AF3 (AVPGVLRFamide) is an endogenous RFamide-like peptide isolated from the parasitic nematode Ascaris suum. It has a potent and long lasting excitatory effect in A. suum and Ascaridia galli. This is mediated by a mechanism independent of the nicotinic-like acetylcholine (ACh) receptor, which mediates excitatory transmission at the neuromuscular junction of both nematodes. In addition, AF3 has been found to sensitise A. suum muscle to the contractile effect of ACh. In this study, the involvement of the second messenger cAMP in mediating the action of AF3 on the somatic musculature of A. suum and A. galli has been investigated. Two approaches have been used; the effects of drugs which raise intracellular cAMP levels on the contractile responses to AF3 have been examined and biochemical assays have been used to measure the effects of AF3 on cAMP levels. AF3 contractions were inhibited in A. suum by 10 microM forskolin (by 22% of control; P < 0.05; n = 9) and by 500 microM isobutylmethylxanthine (IBMX, by 27% of control; P < 0.001; n = 6). AF3 decreased cAMP concentrations in A. suum somatic muscle (basal, 1721 +/- 134 pmol mg-1 protein; with 1 microM AF3, 1148 +/- 133 pmol mg-1 protein; P < 0.05, n = 5). AF3 (1 microM) also reduced the 10 microM forskolin induced potentiation of cAMP concentrations in A. suum (forskolin 3242 +/- 471 pmol mg-1 protein; forskolin and AF3, 1524 +/- 143 pmol mg-1 protein; P < 0.001, n = 6) and A. galli (forskolin 291 +/- 32 pmol mg-1 protein, forskolin +AF3, 185 +/- 12 pmol mg-1 protein; P < 0.005, n = 5). These data suggest that in both nematodes the contractile effect of AF3 is, at least in part, regulated by cAMP.

Mutations in the Caenorhabditis elegans dystrophin-like gene dys-1 lead to hyperactivity and suggest a link with cholinergic transmission.

Mutations in the human dystrophin gene cause Duchenne muscular dystrophy, a common neuromuscular disease leading to a progressive necrosis of muscle cells. The etiology of this necrosis has not been clearly established, and the cellular function of the dystrophin protein is still unknown. We report here the identification of a dystrophin-like gene (named dys-1) in the nematode Caenorhabditis elegans. Loss-of-function mutations of the dys-1 gene make animals hyperactive and slightly hypercontracted. Surprisingly, the dys-1 mutants have apparently normal muscle cells. Based on reporter gene analysis and heterologous promoter expression, the site of action of the dys-1 gene seems to be in muscles. A chimeric transgene in which the C-terminal end of the protein has been replaced by the human dystrophin sequence is able to partly suppress the phenotype of the dys-1 mutants, showing that both proteins share some functional similarity. Finally, the dys-1 mutants are hypersensitive to acetylcholine and to the acetylcholinesterase inhibitor aldicarb, suggesting that dys-1 mutations affect cholinergic transmission. This study provides the first functional link between the dystrophin family of proteins and cholinergic transmission.

Actions of the anthelmintic ivermectin on the pharyngeal muscle of the parasitic nematode, Ascaris suum.

The anthelmintic invermectin has a number of effects on nematodes which result in changes in behaviour, particularly locomotion, including paralysis and an inhibition of feeding. This paper describes the application of an in vitro pharmacological approach to further delineate the action of ivermectin on feeding behaviour. Contraction of Ascaris suum pharyngeal muscle was monitored using a modified pressure transducer system which detects changes in intrapharyngeal pressure and therefore contraction of the radial muscle of the pharynx. The pharynx did not contract spontaneously. However, serotonin (5-HT, 100 microM) stimulated rhythmic contractions and relaxations (pumping) at a frequency of 0.5 Hz. gamma-Aminobutyric acid (GABA) and glutamic acid inhibited the pumping elicited by 5-HT. The duration of inhibition was concentration dependent (1-1000 microM) with a threshold of 1 microM and 10 microM respectively (n = 8). Ivermectin also inhibited pharyngeal pumping (1-1000 nM). At lower concentrations, ivermectin (1-10 pM) potentiated the GABA and glutamate inhibition, so that inhibition occurred at concentrations which were below threshold in the absence of ivermectin. These data provide evidence that the pharynx is a site for the action of ivermectin. Thus interruption of pharyngeal processes such as, feeding, regulation of hydrostatic pressure and secretion may provide a new site of anthelmintic action.