Background Caenorhabditis elegans locomotion is a simple behavior that has been widely used to dissect genetic components of behavior, synaptic transmission, and muscle function. Many of the paradigms that have been created to study C. elegans locomotion rely on qualitative experimenter observation. Here we report the implementation of an automated tracking system developed to quantify the locomotion of multiple individual worms in parallel.Methodology/Principal FindingsOur tracking system generates a consistent measurement of locomotion that allows direct comparison of results across experiments and experimenters and provides a standard method to share data between laboratories. The tracker utilizes a video camera attached to a zoom lens and a software package implemented in MATLAB®. We demonstrate several proof-of-principle applications for the tracker including measuring speed in the absence and presence of food and in the presence of serotonin. We further use the tracker to automatically quantify the time course of paralysis of worms exposed to aldicarb and levamisole and show that tracker performance compares favorably to data generated using a hand-scored metric.Conclusions/SignficanceAlthough this is not the first automated tracking system developed to measure C. elegans locomotion, our tracking software package is freely available and provides a simple interface that includes tools for rapid data collection and analysis. By contrast with other tools, it is not dependent on a specific set of hardware. We propose that the tracker may be used for a broad range of additional worm locomotion applications including genetic and chemical screening.

Abstract. Recent studies using the fungal metabolite brefeldin A (BFA) have provided important insights into the dynamics and the organization of the ER/Golgi membrane system. Here we examined the effect of BFA on the functional integrity of the distal part of the secretory pathway, i.e., transport between transGolgi cisternae and the cell surface. To assay export via the constitutive pathway, we followed the movement of vesicular stomatitis virus (VSV) G glycoprotein that had been accumulated in the trans-Golgi network (TGN) by incubation of infected BHK-21 cells at 20~ Addition of BFA rapidly and reversibly inhibited cell surface transport of G protein. The block to secretion was not due to redistribution of externalized G protein to internal pools. It was also not due to collapse of TGN to the ER, since VSV G protein blocked in treated cells resided in compartments that were distinct from the ER/Golgi system. Similar effects were found with a bulk-flow marker: BFA blocked constitutive secretion of glycosaminoglycan chains that had been synthesized and sulfated in the trans-Golgi cisternae. To examine export via the regulated secretory pathway, we assayed secretion of [35S]SO4 labeled secretogranin II from PC12 cells, a marker that has been used to study secretory granule budding from the TGN (Tooze, S. A., U. Weiss, and W. B. Huttner. 1990. Nature [Lond.]. 347:207-208). BFA potently inhibited secretion of sulfated secretogranin II induced by K + depolarization. Inhibition was at the level of granule formation, since BFA had no effect on regulated secretion from preformed granules. Taken together, the results suggest that BFA blocks export via both the constitutive and the regulated pathways. In contrast, endocytosis and recycling of VSV G protein were not blocked by BFA, consistent with previous studies that endocytosis is unaffected (Misumi, Y., Y.

Serotonin (5-HT) is a neuromodulator that regulates many aspects of animal behavior, including mood, aggression, sex drive, and sleep. In vertebrates, most of the behavioral effects of 5-HT appear to be mediated by G-protein-coupled receptors (GPCRs). Here, we show that SER-1 is the 5-HT GPCR responsible for the stimulatory effects of exogenous 5-HT in two sexually dimorphic behaviors of Caenorhabditis elegans, egg laying and male ventral tail curling. Loss of ser-1 function leads to decreased egg laying in hermaphrodites and defects in the turning step of mating behavior in males. ser-1 is expressed in muscles that are postsynaptic to serotonergic neurons and are known to be required for these behaviors. Analysis of the ser-1 mutant also reveals an inhibitory effect of 5-HT on egg laying that is normally masked by SER-1-dependent stimulation. This inhibition of egg laying requires MOD-1, a 5-HT-gated chloride channel. Loss of mod-1 function in males also produces defects in ventral tail curling and enhances the turning defects in ser-1 mutant males. Sustained elevations in 5-HT levels result in behavioral adaptation to both the stimulatory and inhibitory actions of the neurotransmitter, indicating that both SER-1 and MOD-1 signaling can be modulated. Removal of wild-type animals from high levels of exogenous 5-HT produces a SER-1-dependent withdrawal response in which egg laying is significantly decreased. These studies provide insight into the role of 5-HT in behavior and the regulation of 5-HT 2 receptor function.

Abstract. To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-intact PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 #M [Ca2+]fr~ to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SglI) labeled in the TGN, was inhibited by depletion of ATP, or by the inclusion of 100 #M GTP3'S, 50 #M AIF3.5 or 5 #g/ml BFA. When added after immature granules had formed, GTP3,S stimulated rather than inhibited exocytosis. Thus, exocytosis of immature granules in this system resembles the characteristics of fully matured granules. Transport of SglI via the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SglI is sorted to some extent upon exit from the TGN. Addition of A23187 to release Ca 2÷ from the TGN had no significant effect on sorting of SglI into immature granules. In contrast, depletion of lumenal calcium inhibited the endoproteolytic cleavage of POMC and proinsulin. These results establish the importance of intra-cisternal Ca 2÷ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of SglI into immature granules. Disruption of organelle pH gradients with an ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.

C. elegans will orient and travel in a straight uninterrupted path directly towards the negative pole of a DC electric field. We have sought to understand the strategy worms use to navigate to the negative pole in a uniform electric field that is fixed in both direction and magnitude. We examined this behavior by quantifying three aspects of electrotaxis behavior in response to different applied field strengths: the mean approach trajectory angles of the animals’ tracks, turning behavior (pirouettes) and average population speeds. We determined that C. elegans align directly to the negative pole of an electric field at sub-preferred field strength and alter approach trajectories at higher field strengths to maintain taxis within a preferred range we have calculated to be ~ 5V/cm. We sought to identify the sensory neurons responsible for the animals’ tracking to a preferred field strength. eat-4 mutant animals defective in glutamatergic signaling of the amphid sensory neurons are severely electrotaxis defective and ceh-36 mutant animals, which are defective in the terminal differentiation of two types of sensory neurons, AWC and ASE, are partially defective in electrotaxis. To further elucidate the role of the AWC neurons, we examined the role of each of the pair of AWC neurons (AWCOFF and AWCON), which are functionally asymmetric and express different genes. nsy-5/inx-19 mutant animals, which express both neurons as AWCOFF, are severely impaired in electrotaxis behavior while nsy-1 mutants, which express both neurons as AWCON, are able to differentiate field strengths required for navigation to a specific field strength within an electric field. We also tested a strain with targeted genetic ablation of AWC neurons and found that these animals showed only slight disruption of directionality and turning behavior. These results suggest a role for AWC neurons in which complete loss of function is less disruptive than loss of functional asymmetry in electrotaxis behavior within a uniform fixed field.