The catecholamine dopamine (DA) is a neurotransmitter that regulates behavior in both vertebrate and invertebrate animals. In humans, altered DA signaling is involved in several mental disorders. DA signaling mechanisms are regulated by environmental factors and various essential components encoded by genes involved in DA synthesis, release, reuptake, and response. The human SLC6A3 gene encodes the dopamine transporter (DAT), a sodium- and chloride-dependent member of the solute carrier family 6 widely distributed throughout the brain in areas of dopaminergic activity. DAT provides reuptake of DA from the synaptic cleft modulating the neurotransmitter signal. There are data supporting the role of DAT in attention deficit disorder, bipolar disorder, clinical depression and addiction disorders.

In C. elegans, the dat-1 gene encodes a plasma membrane DAT-1, which is predicted to regulate dopaminergic neurotransmission via reuptake of dopamine into the presynaptic neuron. The DAT-1 protein is ortholog of the human SLC6A3. Swimming-induced paralysis (SWIP) is a phenotype of C. elegans described in dat-1-deficient mutants (Mcdonald et al., 2007). It has been proposed that the SWIP phenotype appears in DAT-1-deficient nematodes because in water animals have to exert maximal motor activity, which demand efficient clearance of dopamine mediated by the dopamine transporter (Mcdonald et al., 2007).

Given that the synaptic action of DA is inactivated by reuptake of this neurotransmitter into the nerve terminals from which they are released, the transporter is target for psychostimulants and antidepressant. Thus, methylphenidate is a drug that blocks the human DAT, and it is used widely for treatment of several mental affections including attention deficit hyperactivity disorder.

Here we show that methylphenidate is functional in C. elegans. Figure 1A illustrates a dose-response of C. elegans to methylphenidate indicating that this drug induces SWIP in N2 wild type strain. The SWIP phenotype of N2 animals in presence of methylphenidate (9.3 mM) was similar to that of dat-1(ok157) mutant used as control (Figure 2B). These results suggest that in C. elegans methylphenidate, as in mammals, acts inhibiting the dopamine transporter. In this regard, the worm dopamine transporter DAT-1 protein has 42% aminoacid sequence identity with the human SLC6A3 protein.

In C. elegans dwelling and roaming activities are two examples of distinctive behaviors. During the dwelling phase worms have an intermittent backward and forward movement and present a low/high speed of locomotion. However throughout the roaming phase worms have predominantly high speed forward movement with few changes of direction (Fujiwara et al., 2002; Shtonda and Avery, 2006). These two different behaviors are controlled by the presence or absence of food in the environment (Fujiwara et al., 2002). Both states seem to be regulated in a different way, thus while dwelling is induced by internal metabolic perceptions, roaming is mediated by stimuli detected via the amphid neurons (Ben Arous et al., 2009).

Fig. 1 shows that the roaming behavior of nrx-1–deficient mutants was almost annulled when worms were transferred from OP50 bacteria plates to plate without food. This contrasts with the behavior of N2 wild type strain. Interestingly, transgenic expression in the nrx-1 deficient mutant of cDNAs coding human alpha-NRXN-1 or beta-NRXN-1, all driven by the C. elegans nrx-1 promoter, rescued the mutant phenotype of the nematode (Fig. 1). In these experiments nrx-1(tm1961) transgenic animal expressing beta-NRX-1 from the nematode were used as control. To quantify the exploratory capacity of the worms, twenty L4 larval stage animals of each genotype grown on OP50 E. coli in fresh NGM plates were picked and placed in the middle of a 9 cm plate containing 8 ml of agar medium (2% agar, 0.25% v/v Tween 20, 10 mM HEPES pH 7.2) without bacteria. Plates were incubated from 5 to 7 days at 20 ºC. The fingerprints of the worms were recognized by sight as a consequence of their roaming, and they were quantified by scanning the surface of agar dishes at 600-ppp resolution and analyzing the images in “Image J” software using custom scripts. Images were turned into binary format converting them through a predetermined threshold, and finally a particles analysis was performed. Data was normalized with respect N2 wild-type strain.

A previous report had shown that human and rat neuroligin-1 were functional in C. elegans (Calahorro and Ruiz-Rubio, 2012). Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in some neurological diseases.

Social interactions, learning experiences and responses to wide-ranging environmental stimuli occur through nervous cells via synapses. Several observations have suggested that the alteration of neuron connections during nervous system development could represent the root of many cases of autism spectrum disorder. Neuroligins are synaptic cell adhesion proteins that have been shown to be able to induce synaptogenesis. Single missense and frameshift mutations in neuroligin coding genes have been proposed that may lead to autism or mental retardation with complete penetrance (Baudouin and Scheiffele, 2010; Etherton et al., 2011). In C. elegans, nlg-1 is orthologous to mammal neuroligin genes, and it has been shown that nlg-1 mutants are defective in several sensory behaviors and sensitive to oxidative stress (Calahorro et al., 2009; Hunter et al., 2010).

We report here that transgenic expression of rat Nlgn1 and human NLGN1 proteins are functional in C. elegans. The wild type behavior pattern was rescued in nlg-1 deficient mutants by expressing cDNAs from rat Nlgn1 or human NLGN1 genes under C. elegans nlg-1 promoter (Figure 1A). Induced changes Asp396Stop in human NLGN1 or Arg453Cys in worm NLG1 proteins, failed to rescue the wild type phenotype (Figure 1B). These results indicate that mammalian and C. elegans neuroligins seem to be functionally comparable. The results anticipate that the nematode could be useful as an in vivo model for studying specific synapse mechanisms involved in autism. This system will allow the analysis of how mutations in neuroligin genes change phenotypes in different C. elegans genetic backgrounds and the study of their interactions with different environmental factors. It is probable also that this approach could be extended to other genes encoding synaptic proteins implicated in autism spectrum disorder, such as neurexins and shanks.

Table 1. Strains used in this study

Name	Genotype	Reference/Source
N2	wild type reference	CGCa
VC228	nlg-1 (ok259) X	CGCa
CRR1b	nlg-1 (ok259) X	This study
CRR100	nlg-1 (ok259) X; crrEx4 [pPD95.77; pDD04 NeoR (pmyo-2::GFP)]	This study
CRR104c	nlg-1 (ok259) X; crrEx4 [pPD95.77 (Pnlg-1::nlg-1); pDD04NeoR (pmyo-2::GFP)]	This study
CRR105	nlg-1 (ok259) X; crrEx5 [pPD95.77 (Pnlg-1::nlg-1-R437C); pDD04NeoR (pmyo-2::GFP)]	This study
CRR106d	nlg-1 (ok259) X; crrEx6 [pPD95.77 (Pnlg-1::NLGN1); Pnrx-1::GFP]	This study
CRR107	nlg-1 (ok259) X; crrEx7 [pPD95.77 (Pnlg-1::NLGN1-R453C); pDD04NeoR (pmyo-2::GFP)]	This study
CRR108	nlg-1 (ok259) X; crrEx8 [pPD95.77 (Pnlg-1::NLGN1-D396X); pDD04NeoR (pmyo-2::GFP)]	This study
CRR109e	nlg-1 (ok259) X; crrEx9 [pPD95.77 (Pnlg-1::Nlgn1::EGFP); pBCN24NeoR]	This study

^a Caenorhabditis Genetics Center.

^b Obtained by outcrossing VC228 strain with N2 six times.

^cThe cDNA nlg-1 coding region was obtained from clone yk1657a10, Yuji Kohara, National Institute of Genetics, Mishima, Japan.

^dThe cDNA human NLGN1 coding region was obtained from clone KIAA1070 (hj05602), Kazusa DNA Research Institute, Japan.

^e Rat Nlgn-1::EGFP was a gift from Dr. Thomas Dresbach, Univ. Göttingen, Germany.

C. elegans dwells in humus where it feeds on microorganisms. Movement in the worm is the result of environmental and internal signals creating stimulatory and inhibitory output from sensory neurons. Mutants deficient in neurexin (nrx-1) and/or neuroligin (nlg-1) genes exhibit impairment in excitatory and inhibitory neuronal synapses. It has been shown that nlg-1 deficient mutants are defective in detecting osmotic strength (Calahorro et al., 2009) and other sensory behavior (Hunter et al., 2010). Here we illustrate a simple experiment that allows quantifying the roaming behaviour phenotype in neuroligin and neurexin defective mutants of C. elegans. The results of a representative experiment are shown in Figure 1.

The experiment was carried out as follow: 1) Worms were grown synchronously at 20°C on NGM agar plates to L4 stage. Animals were washed three times with CTX buffer (5 mM K₂HPO₄, KH₂PO₄ buffer pH 6.6; 1 mM CaCl₂; 1 mM MgSO₄). 2) One central circle, 1 cm of diameter, was drawn on the back of two types of assay plates: NGM plates without OP50 bacteria, and NGM plates seeding with 100 µl of OP50 outlining a ring (0.5 cm of thickness) in the perimeter, maintaining free of bacteria the centre of the plate. The plates were incubated for 24 hours at 37°C before used. 3) Around 20-30 washed worms were pipetted in the middle of the agar, coinciding with the centre of the circle drawn on the back of the plate. The excess of liquid was gently removed with a small sterile filter paper. 4) The animals, out of the ring, were counted every 15 minutes.

C. elegans, as most animals, changes its behavior depending on the environment. We are characterizing mutants deficient in neurexin (nrx-1) and neuroligin (nlg-1) genes. These proteins are cell adhesion molecules present in excitatory and inhibitory synapses and they are required for correct neuron network function (Sudhof 2008). Previously we have shown that nlg-1 deficient mutants are defective in detecting osmotic strength (Calahorro et al. in press). Here we show a simple qualitative method to identify impaired roaming motion.

Description of the method:
1) Pick twenty L4 larval stage animals of each genotype from a fresh NGM plate containing OP50 E. coli and put them in the middle of a 9 cm plate containing 8 ml of agar medium (2% agar, 0,25% Tween20, 10 mM HEPES pH7,2) without bacteria.
2) Incubate the plate from 5 to 7 days at 20°C.
3) The fingerprinting of the worms is recognized by sight as a consequence of their roaming. A representative example is shown in Figure 1.