Neurotransmitters synthesis

3.3.2.1 Neurotransmitters synthesis

Acetylcholine (ACh) was the first substance proven to be a neurotransmitter (Loewi, 1921). Electrophysiological, biochemical and genetical experiments suggest that ACh is a major excitatory neurotransmitter at neuromuscular junctions in nematodes (Del Castillo et al., 1967;

Delcastillo et al., 1963). C. elegans contains the enzymes of acetylcholine synthesis (Figure 6) and degradation (Johnson and Russell, 1983; Kolson and Russell, 1985; Rand and Russell, 1985), and, moreover, acetylcholine itself is present in extracts (Hosono and Kamiya, 1991;

Hosono et al., 1987). In addition, the worms are paralyzed by cholinesterase inhibitors (organophosphates and carbamates) and by nicotinic agonists (Nguyen et al., 1995). In C.

elegans, ACh is synthesized by the choline acetyltransferase (ChAT), encoded by the gene cha-1. Partially ChAT-deficient mutants grow slowly and display severe changes in a number of neuromuscular behaviours, such as locomotion, pharyngeal pumping, egg laying, and defecation (Alfonso et al., 1994; Rand and Russell, 1984). Null mutations of cha-1 are lethal;

mutant homozygote embryos are able to hatch, but the young larvae are unable to move or feed normally, thus slowly shrink and die within a few days (Rand, 1989). Based on the immunostaining with the anti-ChAT antibody, more than one-third of C. elegans neurons appear to be cholinergic: all classes of putative excitatory motor neurons in the ventral nerve cord (the DA and DB neurons in the first larval stage and the AS, DA, DB, VA, VB, and VC neurons in the adult) and several interneurons with somas in the tail and processes in the tail or body. Sensory neurons are generally not cholinergic (Duerr et al., 2008).

The biogenic amines such as dopamine, serotonin, octopamine and tyramine can all be detected in C. elegans extracts by HPLC analysis and appear to function as neurotransmitters or neuromodulators (Alkema et al., 2005; Horvitz et al., 1982; Sulston et al., 1975). Histamine, epinephrine and norepinephrine were not yet detected in C. elegans (Rand et al., 2000; Rand and Nonet, 1997; Sanyal et al., 2004). In addition, the presence of N-acetyl serotonin has been reported in several parasitic nematodes. Moreover, tissue extracts of Ascaris and C. elegans contain an enzymatic activity (arylalkyl amine N-acetyl transferase) capable of acetylating serotonin, dopamine, and octopamine (Isaac et al., 1991; Muimo and Isaac, 1993). It is not

known whether the N-acetylated derivatives are only catabolic intermediates or if they might represent additional or alternative neurotransmitters (Rand et al., 2000).

Dopamine (DA) was originally detected in C. elegans by its characteristic formaldehyde-induced fluorescence (FIF), revealing eight DA neurons in the hermaphrodite with an additional six DA neurons specific to male worms (Sulston et al., 1975) reviewed in (McDonald et al., 2006). All of these fourteen neurons are considered mechanosensory neurons. Exogenous dopamine inhibits locomotion, egg laying and decreases enteric muscle contractions in wild type worms resulting in slower defecation (Horvitz et al., 1982; Weinshenker et al., 1995). Dopamine is synthesized in a two-step enzymatic process (Figure 6). In the first step, the amino acid tyrosine is converted to 1-dihydroxypheny-l-alanine (l-DOPA), the immediate DA precursor, by the enzyme tyrosine hydroxylase (TH), encoded by the cat-2 gene (Lints and Emmons, 1999). Secondly, l-DOPA is converted to DA by an aromatic amino acid decarboxylase (AAAD; named also DOPA decarboxylase), encoded by the bas-1 gene (Loer and Kenyon, 1993). The DA-related mutants show difficulties in sensing and responding to the presence of food (Sawin et al., 2000). Males have three additional pairs of dopamine-containing sensory neurons in the tail and subsequently mutants lacking dopamine are deficient in male mating behaviours (Liu and Sternberg, 1995; Loer and Kenyon, 1993).

Octopamine (p-hydroxyphenylethanolamine) is present in C. elegans homogenates, and exogenous octopamine inhibits egg laying and stimulates locomotion (Horvitz et al., 1982).

Octopamine is synthesized from tyramine by the tyramine !-hydroxylase, encoded by tbh-1 gene (Figure 6). Based on the immunostaining of TBH-1, octopamine is limited to the RIC interneurons and the gonadal sheath cells (Alkema et al., 2005).

Serotonin (5HT) has been identified in C. elegans neurons by FIF (Horvitz et al., 1982) and by anti-5HT immunostaining (Desai et al., 1988; McIntire et al., 1992). Treatment with exogenous serotonin causes dramatic behavioural effects: stimulates egg laying and pharyngeal pumping and inhibits locomotion and defecation (Mendel et al., 1995; Niacaris and Avery, 2003; Rogers et al., 2001; Sawin et al., 2000; Segalat et al., 1995; Waggoner et al., 1998; Weinshenker et al., 1995). There are eight types of neurons in C. elegans with anti-5HT immunoreactivity, totalling 11 neurons that produce serotonin. These include all categories of neurons: sensory, inter- and motor neurons (Loer and Kenyon, 1993). Serotonin is synthesized by the sequential action of tryptophan hydroxylase, encoded by the tph-1 gene, and the enzyme which is also used for dopamine synthesis – AAAD, encoded by bas-1 (Sze et al., 2000)(Figure 6). Mutant analysis and drug studies have shown that, in addition to regulating egg laying, serotonin mediates the response to starvation and is also required for male mating behaviours (Loer and Kenyon, 1993; Sawin et al., 2000; Schafer et al., 1996; Weinshenker et al., 1995).

Figure 6. Neurotransmitter synthesis

The most abundant synapses in the central nervous system of vertebrates are inhibitory synapses that use the neurotransmitter !-aminobutyric acid (GABA). GABA is found also in C.

elegans but unlike the vertebrates’ ortholog, acts primarily at neuromuscular synapses. The evidences for GABA function in C. elegans include pharmacological studies using GABA-related compounds, immunohistochemical staining of GABA in specific cells and analysis of mutants defective in GABA synthesis and function (McIntire et al., 1993a; McIntire et al., 1993b).

Twenty-six neurons contain GABA immunoreactivity; most of these are inhibitory motor neurons, but some are apparently excitatory GABAergic motor neurons. In each of these cells, the GABA immunoreactivity is uniformly distributed throughout the cytoplasm and is not restricted to synaptic regions (McIntire et al., 1993b). Mutants with GABAergic transmission defects are viable, although they have several motor defects. The most obvious phenotype is a tendency to contract dorsal and ventral body wall muscle simultaneously in response to touch (‘shrinker’ phenotype); this appears to result from lack of function of the GABA-containing DD and VD inhibitory motor neurons (McIntire et al., 1993b).

GABA is synthesized from glutamic acid by the enzyme glutamic acid decarboxylase (GAD). In C. elegans, unc-25 encodes a protein that is 45% identical to the mammalian GAD (Eastman et al., 1999; Jin et al., 1999) the respective mutants have no apparent GABA (McIntire et al., 1993a). Axon morphology of GABA neurons is normal in adult unc-25 mutants (Jin et al., 1999). Moreover, synaptic varicosities, assayed by clusters of fluorescently tagged synaptic vesicles, are at normal positions and densities in the ventral cord GABA motor neurons. A reconstruction of the ventral nerve cord from serial electron micrographs demonstrated that the synaptic ultrastructure and connectivity were correct (Jin et al., 1999). In addition, the post-synaptic GABA receptor also has a normal distribution in unc-25 mutants (Gally and Bessereau, 2003). Rescuing unc-25 function in adults by addition of exogenous GABA provided definitive proof that GABA synapses are functional. This provided the AVL and DVB neurons with an external source of GABA, which was pumped into cells via the plasma membrane transporter and normal behavior was restored (McIntire et al., 1993a). Therefore, all these data suggest that GABA release is not required for axon outgrowth, synapse formation or synapse maintenance in C. elegans.

Glutamate (the carboxylate anion of glutamic acid) is the most abundant and rapid excitatory neurotransmitter in the mammalian nervous system. It can be synthesized from glutamine by the action of phosphate-activated glutaminase (Figure 6). Alternatively, glutamate may be formed from "-ketoglutarate and alanine catalyzed by alanine aminotransferase (reviewed in (Fonnum, 1993). In C. elegans, several of the components of glutamatergic transmission were identified and characterized, but these are mostly components involved in the transport or reception of glutamate. The identification of glutamate releasing neurons and glutamate-dependent behaviours was based on the expression patterns of the receptors and also on the analysis of the phenotypes of their mutants (especially glr-1 which encodes an

AMPA-type receptor). These studies suggest that glutamate is released by many sensory neurons and interneurons. Furthermore, some of the behavioural circuits have been associated with glutamatergic neurotransmission. These include:

1) the response to light mechanical stimulation of the head mediated by the ASH sensory neurons through the GLR-1 postsynaptic receptor (Hart et al., 1995; Maricq et al., 1995);

2) the response to strong mechanical stimulation in the anterior body mediated by the ALM and AVM sensory neurons through the AVR-15 postsynaptic receptor (Lee et al., 1999a);

3) the effects of the M3 motor neuron on the pharyngeal muscle, mediated through the AVR-15 postsynaptic receptor (Dent et al., 1997; Li et al., 1997)