The chemical complexity of the nematode neurosystem has become apparent only within the last ten years. The nervous system of C. elegans is the most complex organ in the worm. 37 % of the cells in a hermaphrodite belong to the neuronal system (HOBERT, 2007). The most complex neuropil in the animal is a nerve ring encircling the pharynx. Efferent from this nerve ring, a dorsal and a ventral cord extend almost to the tail. Both contain motor neurons; the ventral cord additionally carries sensory neurons and interneurons. The system is completed by several ganglia, mainly in the pharyngeal and tail regions, and by sublateral cords. All nerves are located immediately beneath the hypodermis (THOMAS and LOCKERY, 1999).
Due to its large size, having a length of 15 – 30 cm and a diameter of 3 – 6 mm, the pig roundworm Ascaris suum is a convenient system for neurological studies. The neuronal system of A. suum consists of three components: the peripheral, central, and enteric nervous systems. Since the nervous systems of nematodes are highly similar, the derived information may be applied to other nematodes. The nervous system of nematodes is a combined nervous and endocrine system with commonly shared messenger molecules, mainly neuropeptides of 3 – 100 amino acids.
Nematodes lack endocrine glands and a circulatory system (BROWNLEE et al., 2000).
3.6.1 Neurotransmitters
Neurotransmitters are compounds synthesized and stored in neurons where they mediate nerval signals into cellular responses. Their release is dependent on calcium ions (Ca2+) and has inhibitory or stimulatory effects on the postsynaptic cells. The neurotransmitters used by nematodes are mainly classical transmitters also known in mammals. However, some transmitters play a role in nematodes but are uncommon in mammals (THOMAS and LOCKERY, 1999). Which transmitters are used depends on the respective neuron. Neurotransmitters have been studied in C. elegans and A. suum in greater detail.
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Synaptic transmitters are stored in vesicles in the presynaptic terminal. Influx of calcium into the cell causes vesicle release. For termination of action the released transmitters are recycled or degraded. The proteins involved in forming the vesicles are also recovered. An overview of the proteins potentially involved in vesicle formation is given by HARRIS and colleagues (2001).
3.6.1.1 Acetylcholine
A major excitatory transmitter in neuromuscular junctions of C. elegans is acetylcholine (ACh). ACh is used by a third of the cells belonging to the nervous system in C. elegans (RAND, 2007). In C. elegans GPCRs (LEE et al., 1999; LEE et al., 2000; PARK et al., 2003) and ion channels have been identified as receptors for ACh. The ion channels are assumed to consist of five subunits, which are arranged around an ion-pore (MARTIN et al., 2002). Dependent on their affinities, receptors are classified in L-, B-, and N-subtypes. L-subtype receptors have the highest affinity to levamisole, whereas B-type receptors bind bephenium and N-type receptors are sensitive to nicotine (MARTIN et al., 2005). The termination of action is mediated by acetylcholine esterase, which hydrolyzes ACh. The degradation products can be recycled (RAND, 2007). In flatworms, ACh is known to act as an inhibitory transmitter (RIBEIRO et al., 2005).
3.6.1.2 Glutamate
As in vertebrates, the main transmitter for rapid excitatory synaptic signaling in nematodes is glutamate. The excitatory action of glutamate in vertebrates is mediated by ionotropic and metabotropic glutamate receptors (NAKANISHI et al., 1998). In C. elegans at least ten subunits of excitatory ionotropic glutamate receptors have been identified, indicating that a number of different functional types of ionotropic glutamate receptors might be expressed in the worm. The other group of receptors known in vertebrates, the metabotropic glutamate receptors, are GPCRs.
Recently, three genes for metabotropic glutamate receptors have been identified in C. elegans (DILLON et al., 2006). Another target for glutamate in nematodes are glutamate-gated chloride (GluCl) channels, which are unique to invertebrates.
Contrary to the excitatory ionotropic and metabotropic glutamate receptors GluCl channels mediate an inhibitory effect of glutamate by forming channels for
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chloride ions (BROCKIE and MARICQ, 2006). In Xenopus oocysts expressed homomers of GluCl channel subunits show different binding capacities, depending on the subunits expressed: GluClα 1 homomeric channels are sensitive to the anthelmintic drug ivermectin but not to glutamate, whereas GluClβ homomeric channels react to glutamate but not to ivermectin (CULLY et al., 1994; CULLY et al., 1996). DENT and coworkers (1997) identified the alternatively spliced subunits GluClα 2A and GluClα 2B. Homomeric channels of these subunits are ivermectin and glutamate sensitive. Another subunit in two splicing variants, GluClα 3A and GluClα 3B, has been identified later by the same group. Only simultaneous mutations of the three genes for GluClα 1, GluClα 2, and GluClα 3 leads to highly resistant animals, mutations of only two of these genes in C. elegans confers only modest or no resistance (DENT et al., 2000). In C. oncophora and H. contortus orthologous genes for GluClα 3 and GluClβ have been identified (CHEESEMAN et al., 2001;
NJUE and PRICHARD, 2004). In expression studies of these genes amplified from ivermectin-susceptible and ivermectin-resistant C. oncophora in Xenopus oocysts, a single mutation was identified to confer resistance to ivermectin (NJUE et al., 2004).
In nematodes the channels are assumed to consist of five subunits, but the composition is still unknown (MARTIN et al., 2002). Ivermectin-sensitive channels were shown to be expressed in the pharynx of nematodes (DENT et al., 1997;
LAUGHTON et al., 1997).
3.6.1.3 GABA
γ-aminobutyric acid (GABA) is an inhibitory transmitter in mammals. The receptors for GABA are GABAA, a chloride channel, and GABAB, a GPCR. Genes for both are also found in the genomes of nematodes, and GABAA receptors have also been shown to be targets for GABA in nematodes (SCHOFIELD et al., 1987). The activation of GABAA receptors leads, depending on the intracellular chloride concentration, to the influx or efflux of chloride ions and therefore to membrane hyperpolarization or depolarization. In both cases body muscle contraction is inhibited (reviewed in JORGENSEN, 2005). GABAB GPCRs mediate an inhibition of membrane excitability by opening potassium (K+) channels and inhibiting Ca2+ channels (KAUPMANN et al., 1997). Recently an excitatory effect of GABA was discovered in C. elegans. The mechanism involves a cation-selective channel, and the influx of sodium ions causes
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contraction of the enteric muscles (BEG and JORGENSEN, 2003). GABA is cleared from the synaptic cleft by a plasma membrane transporter (SCHUSKE et al., 2004).
3.6.1.4 Dopamine
In mammals the known dopamine receptors are GPCRs. They are classified as D1- and D2-like receptors. D1-like receptors couple positively to adenylate cyclase and therefore increase the level of cyclic adenosine monophosphate (cAMP), whereas D2-like receptors inhibit cAMP formation. Nevertheless, additional second messengers and effector pathways are also recognized (NEVE et al., 2004). In C. elegans four GPCRs for dopamine are currently known, two D1-like and two D2-like receptors (MCDONALD et al., 2006).
3.6.1.5 Serotonin
Serotonin is a modulating transmitter in many physiological mechanisms in C. elegans. According to CARRET-PIERRAT (2006a), C. elegans has 3 – 8 GPCRs for serotonin, which have a predominantly neuronal expression. Another receptor is a serotonin-gated chloride channel (RANGANATHAN et al., 2000).
3.6.1.6 Octopamine and Tyramine
C. elegans was further shown to have physiological processes modulated by biogenic amines other than dopamine and serotonin: octopamine and its biosynthetic precursor tyramine. In some nematode species octopamine is metabolized to noradrenaline, synephrine and epinephrine (FRANDSEN and BONE, 1988). The receptors for octopamine were predicted by database searches, but none have been definitively identified (KOMUNIECKI et al., 2004). Two GPCRs are known for tyramine (REX et al., 2004; REX et al., 2005).
3.6.1.7 Neuropeptides
Neuropeptides are peptides acting as neuromodulators or neurotransmitters. They are the major class of transmitter compounds in nematodes. 75 % of the nerve cells in A. suum (BROWNLEE et al., 1996), and > 50 % in C. elegans (KIM and Li, 2004)
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were shown to express neuropeptides. Like other neurotransmitters they are highly specific in their action but have a much higher potency than many other transmitters. Their synthesis involves proproteins or precursors. The cleavage and posttranslational modification of the precursor molecules occurs in the endoplasmatic reticulum. The neuropeptides are then bound to a carrier protein and transported in vesicles through the Golgi complex to the nerve terminal. The release is, like the release of classical neurotransmitters, Ca2+-dependent. They are stored in vesicles different than those containing classical transmitters; a differential release is therefore likely possible and would be necessary for the modulating activities of neuropeptides on neurotransmitters. In the simple invertebrate polyp Hydra no transmitters other than neuropeptides have been identified, therefore, neuropeptides are thought to be the original transmitter molecules (BROWNLEE et al., 2000). In C. elegans the neuropeptides can be subdivided into three main classes: insulin-like peptides, neuropeptide-like proteins, and FMRFamide-like peptides. Some neuropeptide-like proteins are antimicrobial and are expressed in the hypodermis rather than in neurons. Their expression is induced upon bacterial or fungal infection (HUSSON et al., 2007).
FMRFamide-like peptides (FaRPs or FLPs) are the most complex group of neuropeptides known from metazoans. In free-living and parasitic nematodes these peptides are proposed to play a fundamental role (MCVEIGH et al., 2005). The name is derived from their similarity to a molluscan neuropeptide called FMRFamide, containing the sequence Phe-Met-Arg-Phe-NH2. FLPs contain the C-terminal tetrapeptide motif X-Xo-Arg-Phe-NH2, where X is any amino acid except cysteine and Xo is any hydrophobic amino acid except cysteine (MCVEIGH et al., 2006). In the snail Lymnaea stagnalis two groups of FMRFamide-like peptides are known, the N-terminally extended peptides and the tetrapeptides. They are derived from the same gene by alternative splicing. Only one tetrapeptide has been identified to date in nematodes: FIRFamide in A. suum. All other currently known FMRFamide-like peptides in nematodes are N-terminally extended peptides (BROWNLEE et al., 2000). FLPs differing in a single amino acid can have different receptors, leading to different actions (BOWMAN et al., 2002). Neuropeptides containing an RFamide motif are also known from mammals. Several FLPs in C. elegans are known to act via GPCRs (reviewed in MCVEIGH et al., 2006). Another mechanism of action
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appears to involve an FMRFamide-gated chloride channel (PURCELL et al., 2002).
Termination of action occurs by enzymatic degradation. The examination of FLPs and their receptors is still ongoing. BROWNLEE and coauthors (2000) emphasize that in addition to the currently known FLPs in C. elegans and A. suum other FLPs might exist, which could be unique to parasitic nematodes. These FLPs need to be studied in the various parasitic species.