Christopher J. Franks

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

ABSTRACT 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 A chimeric transgene in which the C-terminal end of the protein has been replaced by the human dystrophin sequence is able to partly suppress the phenotype of the dys-1 mutants, showing that both proteins share some functional similarity.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.

A nematode FMRFamide-like peptide, SDPNFLRFamide (PF1), relaxes the dorsal muscle strip preparation of Ascaris suum

PF1 (SDPNFLRFamide) is a FMRFamide-like peptide extracted from the free-living nematode Panagrellus redivivus. Here we show that this peptide causes a hyperpolarization of somatic muscle cells of the parasitic nematode Ascaris suum and a relaxation of the somatic muscle strip preparation. We have assessed whether or not the relaxation of Ascaris dorsal muscle strip by PF1 is due to (i) inhibition of the release of the excitatory neuromuscular junction transmitter acetylcholine (ACh), (ii) potentiation of the release of the inhibitory neuromuscular junction transmitter gamma-aminobutyric acid (GABA) or (iii) a direct inhibitory action of the peptide on the muscle cells. Under the experimental conditions described here, tonic ACh release does not seem to be involved in determining the resting membrane potential or resting tone of the Ascaris dorsal muscle strip and thus inhibition of tonic ACh release is unlikely to explain the relaxation elicited by the peptide. Furthermore, PF1 (100 nM-1 microM) inhibited the contraction of the muscle strip elicited by bath application of ACh, suggesting either a direct inhibitory action of the peptide on the muscle cells or a potentiation of GABA release. In electrophysiological experiments, the reversal potential for the PF1 hyperpolarization was not the same as that for GABA. Thus, PF1 hyperpolarizes Ascaris muscle by a mechanism that does not involve stimulation of GABA release from inhibitory pre-synaptic terminals.

The effects of the nematode peptide, KHEYLRFamide (AF2), on the somatic musculature of the parasitic nematode Ascaris suum

AF2 is an endogenous RFamide-like peptide from the parasitic nematode Ascaris suum. The potent stimulatory effects of this peptide on the somatic musculature of Ascaris strongly suggest that it may have an important role in the motornervous system. Here we have investigated the possibility that AF2 may elicit a stimulatory action on Ascaris muscle by potentiating the actions of the excitatory cholinergic motonervous system either pre-synaptically, post-synaptically or both. In in vitro pharmacological experiments AF2 produced a dose-dependent increase in the frequency and amplitude of spontaneous contractions of Ascaris muscle strip which lasted for more than 1 h after a 3 min application of AF2 (10 nM-10 microM; N = 7). In addition, AF2 (100 nM) potentiated the contraction elicited by ACh by 43 +/- 9% (P < 0.01; N = 8). In electrophysiological recordings from muscle cells, AF2 (10-100 nM; N = 10) potentiated the amplitude of EJPs (excitatory junction potentials). For 100 nM AF2, the potentiation of the EJP was 218 +/- 48% (N = 7; P < 0.01). This effect reversed after a wash of 10 min. AF2 did not potentiate the depolarization of the muscle cell elicited by bath applied ACh. These latter two observations are consistent with a presynpatic action of AF2. AF2 (10-100 nM) generated spontaneous muscle cell action potentials in previously quiescent cells. This effect took more than 1 h to wash out. These observations are discussed in terms of the paralysis of Ascaris that is elicited by AF2.

Anatomy, physiology and pharmacology of Caenorhabditis elegans pharynx: a model to define gene function in a simple neural system

Invertebrate neuroscience has provided a number of very informative model systems that have been extensively utilized in order to define the neurobiological bases of animal behaviours (Sattelle and Buckingham in Invert Neurosci 6:1–3, 2006). Most eminent among these are a number of molluscs, including Aplysia californica, Lymnaea stagnalis and Helix aspersa, crustacean systems such as the crab stomatogastric ganglion and a wide-range of other arthropods. All of these have been elegantly exploited to shed light on the very important phenomenon of the molecular and cellular basis for synaptic regulation that underpins behavioural plasticity. Key to the successful use of these systems has been the ability to study well-defined, relatively simple neuronal circuits that direct and regulate a quantifiable animal behaviour. Here we describe the pharyngeal system of the nematode C. elegans and its utility as a model for defining the genetic basis of behaviour. The circuitry of the nervous system in this animal is uniquely well-defined. Furthermore, the feeding behaviour of the worm is controlled by the activity of the pharynx and this in turn is regulated in a context-dependent manner by a simple nervous system that integrates external signals, e.g. presence or absence of food, and internal signals, e.g. the nutritional status of the animal to direct an appropriate response. The genetics of C. elegans is being effectively exploited to provide novel insight into genes that function to regulate the neuronal network that controls the pharynx. Here we summarise the progress to date and highlight topics for future research. Two main themes emerge. First, although the anatomy of the pharyngeal system is very well-defined, there is a much poorer understanding of its neurochemistry. Second, it is evident that the neurochemistry is remarkably complex for such a simple circuit/behaviour. This suggests that the pharyngeal activity may be subject to exquisitely precise regulation depending on the animal’s environment and status. This therefore provides a very tractable genetic model to investigate neural mechanisms for signal integration and synaptic plasticity in a well-defined neuronal network that directs a quantifiable behaviour, feeding.

The effect of the nematode peptides SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) on synaptic transmission in the parasitic nematode Ascaris suum.

The action of two peptides isolated from the nematode Panagrellus redivivus, PF1 (SDPNFLRFamide) and PF2 (SADPNFLRFamide) have been studied on synaptic transmission in the motornervous system of the parasitic nematode Ascaris suum. Intracellular recordings were made from Ascaris somatic muscle cells and excitatory junction potentials (EJPs) elicited by stimulation of the ventral nerve cord. The EJPs were cholinergic as they were blocked by the Ascaris nicotinic receptor antagonist, benzoquinonium. PF1 caused a slow hyperpolarization, similar to the action of this peptide first reported by Bowman, Geary & Thompson (1990) and further characterized by Franks et al. (1994). The hyperpolarization was accompanied by a marked decrease in the amplitude of the EJPs with an EC50 of 311 +/- 30 nM (n = 5). This inhibition is unlikely to be due to a post-synaptic site of action of the peptide as the muscle cell input conductance was not significantly altered by PF1 and furthermore the response to bath-applied acetylcholine was not inhibited by PF1 at concentrations up to 10 microM (n = 6). PF2 also inhibited the EJPs in a similar manner to PF1. These studies indicate that both of the peptides isolated from the free-living nematode Panagrellus redivivus have biological activity in the parasitic nematode Ascaris suum. PF1 and PF2 have inhibitory actions in contrast to the predominantly excitatory actions of the Ascaris endogenous peptides AF1 (KNEFIRFamide) and AF2 (KHEYLRFamide). The potent actions of the Panagrellus neuropeptides PF1 and PF2 in Ascaris suggest that peptides with a similar or identical sequence may also occur in Ascaris and have an inhibitory role in the motornervous system.

Physiological and pharmacological studies on annelid and nematode body wall muscle

1. This review covers the pharmacology and physiology of the body wall muscle systems of nematodes and annelids. 2. Both acetylcholine and gamma-aminobutyric acid (GABA) play important roles in the control of body wall muscle in both phyla. In annelids and nematodes, acetylcholine is the excitatory neuromuscular transmitter while GABA is the inhibitory neuromuscular transmitter. In addition, 5-hydroxytryptamine (5-HT) has a modulatory role at annelid body wall muscle but little if any effect on nematode body wall muscle. 3. The acetylcholine receptor of the body wall muscle can be classified as nicotinic-like in both phyla though the annelid receptor has not been analysed in detail. In nematodes, vertebrate ganglionic nicotinic agonists were the most effective of those so far examined while mecamylamine and benzoquinonium were the most effective antagonists. Both neuronal bungarotoxin and neosurugatoxin were potent antagonists of acetylcholine excitation at the nematode receptor. 4. The GABA receptor of the body wall muscle exhibits similarities with the vertebrate GABA-A receptor in both phyla. Picrotoxin is a very weak or inactive antagonist at leech and nematode GABA receptors, while bicuculline methiodide blocks leech GABA receptors but is inactive on nematode GABA receptors. Picrotoxin does block GABA responses of earthworm body wall muscle. All these GABA responses are chloride mediated. 5. Neuroactive peptides of the RFamide family occur in both phyla and FMRFamide has been identified in leeches. RFamides probably have an important role in heart regulation in leeches and in modulation of their body wall muscles. RFamides also modulate nematode body wall muscle activity with KNEFIRFamide raising muscle tone while SDPNFLRFamide relaxes the muscle. It is likely that this family and other neuroactive peptides play an important role in the physiology of body wall muscle throughout both phyla.

Metabotropic glutamate receptors: modulators of context-dependent feeding behaviour in C. elegans

Background: C. elegans encodes three metabotropic glutamate receptors: mgl-1, mgl-2, and mgl-3. Results: mgl-1 and mgl-3, but not mgl-2, modulate activity in the neural circuit underlying feeding behavior. Conclusion: mgl-1 is the major contributor to the inhibitory tone of the feeding circuit and context-dependent feeding behavior. Significance: C. elegans provides a model for systems-level understanding of metabotropic glutamate receptors. Glutamatergic neurotransmission is evolutionarily conserved across animal phyla. A major class of glutamate receptors consists of the metabotropic glutamate receptors (mGluRs). In C. elegans, three mGluR genes, mgl-1, mgl-2, and mgl-3, are organized into three subgroups, similar to their mammalian counterparts. Cellular reporters identified expression of the mgls in the nervous system of C. elegans and overlapping expression in the pharyngeal microcircuit that controls pharyngeal muscle activity and feeding behavior. The overlapping expression of mgls within this circuit allowed the investigation of receptor signaling per se and in the context of receptor interactions within a neural network that regulates feeding. We utilized the pharmacological manipulation of neuronally regulated pumping of the pharyngeal muscle in the wild-type and mutants to investigate MGL function. This defined a net mgl-1-dependent inhibition of pharyngeal pumping that is modulated by mgl-3 excitation. Optogenetic activation of the pharyngeal glutamatergic inputs combined with electrophysiological recordings from the isolated pharyngeal preparations provided further evidence for a presynaptic mgl-1-dependent regulation of pharyngeal activity. Analysis of mgl-1, mgl-2, and mgl-3 mutant feeding behavior in the intact organism after acute food removal identified a significant role for mgl-1 in the regulation of an adaptive feeding response. Our data describe the molecular and cellular organization of mgl-1, mgl-2, and mgl-3. Pharmacological analysis identified that, in these paradigms, mgl-1 and mgl-3, but not mgl-2, can modulate the pharyngeal microcircuit. Behavioral analysis identified mgl-1 as a significant determinant of the glutamate-dependent modulation of feeding, further highlighting the significance of mGluRs in complex C. elegans behavior.

Electrophysiological and Pharmacological Studies on Excitable Tissues in Nematodes

Nematodes include both major parasites of humans, livestock and plants and 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 chapter will summarize what is known about the biological activity of neurotransmitters in nematodes: the biosynthetic pathways and genes involved, their receptors, inactivation mechanisms and second 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 programme 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 the nematode transmitters.