Schafer WR et al. (1993) International C. elegans Meeting "Effects of dopamine and a dopamine antagonist on behavior."

Schafer WR, Kenyon CJ

[International C. elegans Meeting, 1993]

Schafer WR et al. (2000) European Worm Meeting "Regulation of calcium transients in the pharyngeal and vulval muscles"

Schafer WR, Kerr RA, Tsien RY

[European Worm Meeting, 2000]

Genetic and behavioral analysis has defined a number of signal transduction molecules that regulate the activity of the muscle cells involved in feeding and egg-laying behavior. To obtain understand more precisely the effects of these molecules on muscle cell physiology, we are using in vivo calcium imaging to visualize muscle calcium transients in live wild-type and mutant animals. We have expressed the FRET-based calcium-sensitive protein cameleon, developed in Roger Tsien's lab, in the pharyngeal and vulval muscles, and demonstrated that the calcium influx accompanying contraction can be reliably detected through ratiometric video-rate fluorescence imaging. Moreover, by analyzing the fluorescence traces of egl-19 calcium channel mutants previously shown by the Avery lab to affect the duration of the pharyngeal action potential, we have found that we can use cameleon to measure and distinguish effects of specific mutations on either the duration or the magnitude of the pharyngeal calcium influx. Using this approach, we have made the surprising discovery that unc-36 loss-of-function mutations, which eliminate the activity of the conserved large accessory (alpha-2) subunit of the calcium channel, cause a significant increase in the magnitude of the pharyngeal calcium transient. This suggests that the function of the alpha-2 subunit in muscle cells is to negatively regulate calcium influx through the channel. We have also expressed cameleon in the vulval muscles, and been similarly successful in detecting changes in fluorescence ratio associated with calcium transients. Preliminary results using this technique indicate that serotonin, a molecule known to function as a modulator of vulval muscle activity, dramatically increases the frequency of sub-threshold calcium transients (i.e. transients too small to induce an egg-laying event). Further analysis of vulval muscle calcium dynamics will be presented.

Schafer WR et al. (1995) International C. elegans Meeting "A calcium channel required for desensitization to dopamine and serotonin."

Schafer WR, Kenyon CJ

[International C. elegans Meeting, 1995]

Prolonged exposure of an excitable cell to neurotransmitter often causes an attenuation of neurotransmitter response, a process known as adaptation or desensitization. We found that long-term dopamine treatment causes worms to adapt to, and become dependent on, exogenous dopamine. We have identified a number of mutations that cause a defect in dopamine adaptation, including an allele of the gene unc-2. unc-2 animals are strongly defective in dopamine desensitization, although their initial response to dopamine is normal. unc-2 mutants also are hypersensitive, and fail to adapt, to serotonin, and display behaviors similar to those induced by serotonin treatment.

Schafer WR et al. (2000) West Coast Worm Meeting "Calcium Imaging in Excitable Cells of C. elegans."

Lev-Ram V, Tsien RY, Schafer WR, Kerr RA

[West Coast Worm Meeting, 2000]

It is desirable in behavioral studies to record the activity of neurons and muscles, but this has traditionally been difficult in C. elegans. We are using a transfectable calcium indicator, cameleon [Miyawaki et al., Nature 388:882], to overcome this barrier. To develop the technique, we focused on the pharyngeal muscle. The pharynx has been previously characterized behaviorally and electrically, primarily by the Avery lab, and is large enough to simplify imaging. In addition, calcium influx in muscles causes contraction, which provides an independent measure of activity. Using the myo-2 promoter, we expressed cameleon in the pharynx and recorded distinctive calcium transients coupled to muscle contraction in intact animals. The duration of these transients varied in mutants of egl-19, a voltage-gated calcium channel, consistent with previous electrical recordings [Lee et al., EMBO 16:1066]. In mutants of unc-36, a channel-associated a2 subunit, we found that transients were substantially increased in magnitude, suggesting an inhibitory role for the wild-type UNC-36 protein. This was surprising as coexpression studies of vertebrate homologues in Xenopus oocytes had found that the a2 had many relatively subtle effects, including increasing expression levels of the pore-forming subunit. We are currently extending our study of muscular calcium transients to investigate the effects of neurotransmitters and mutations on the vulval muscles and their role in egg-laying behavior. In addition, we expressed cameleon using the pan-neuronal promoter unc-119. We provided direct electrical stimulation through an extracellular electrode inserted through the cuticle and positioned near the nerve ring. Transients were observed coupled to the stimulation, indicating that calcium influx through voltage-gated calcium channels can be observed in neurons using this method. This raises the possibility of constructing a functional map of neuronal connectivity by electrically stimulating single neurons and recording the activity of their synaptic partners using cameleon.

Schafer WR et al. (1996) West Coast Worm Meeting "ANALYSIS OF THE TEMPORAL PATTERN OF EGG-LAYING BEHAVIOR"

Schafer WR, Kenyon CJ

[West Coast Worm Meeting, 1996]

Thanks to the efforts of many investigators, a significant amount is known about how worms lay eggs. However, relatively little is known about what controls the timing of egg-laying events in individual animals. We therefore investigated the temporal pattern of egg-laying by observing single animals over long time courses and noting the times when they laid eggs. Several simple patterns were imaginable; for example, egg-laying could be periodic, with a roughly constant interval between egg-laying events, or it could be a Poisson process, with a constant probability of an egg being laid at any given time. In fact, we observed a pattern that was consistent with neither of these models. Rather, the animals appeared to alternate between an inactive state, lasting an hour or more, during which eggs were never laid, and an active state, lasting five minutes or so, during which many eggs were laid in rapid succession. (This "clutching" behavior has been observed anecdotally by many investigators, including Croll (1975)). It is not clear what governs the switch between active and inactive states, but preliminary data suggests that the onset of active phases is neither periodic nor a Poisson process. Thus, the problem of how egg-laying rates are regulated can be broken into several problems: what determines the length of the inactive and active phases, what regulates the number of eggs laid within an active period, and what maintains this overall temporal pattern? Obtaining the data to address these questions by manual observation would be impractical; thus, we have set up a computerized system to track single animals under high magnification and record their behavior on videotape. Using this video record, we can obtain detailed information about the timing of individual egg-laying events over time courses of six hours or longer. Ultimately, we intend to devise a system that will detect egg-laying events automatically, increasing our speed of analysis and decreasing tedium. Using this system, we will investigate the pattern of egg-laying behavior in wild-type animals under different environmental conditions, in mutant animals and in animals with specific cell ablations.

Schafer WR et al. (2000) West Coast Worm Meeting "Serotonin-resistant egg-laying mutants and a receptor knockout in progress"

Shyn SI, Schafer WR

[West Coast Worm Meeting, 2000]

Serotonin (5HT) stimulates egg-laying in the nematode1, and we hypothesize that this occurs by modulation of vulval muscle excitability from a quiescent mode to an active egg-laying state2. The effectors used to accomplish this are largely unknown, so we are mapping and characterizing catalogued as well as more recently isolated mutants (5 lines from our own lab) which are 5HT-resistant for egg-laying. One such mutant is egl-24 (n572)3. Further characterization has confirmed its profound 5HT-resistance, but perhaps more intriguingly that, in the absence of any exogenous 5HT, its temporal pattern of egg-laying is not grossly different from that of N2s. Preliminary data suggests its map interval may be further narrowed to between -0.54 and -0.79 on LGIII, an interval spanned by 26 cosmids and a portions of 2 YACs. From a reverse-genetics approach, we are also preparing to knockout 5HT-Ce, the only known vulval 5HT receptor4. Among vertebrate classes, 5HT-Ce shows the most homology to the 5HT1 subtype, which typically inhibits adenylate cyclase5. This prompted us to look at worms mutant in acy-1, an adenylate cyclase expressed in virtually all neurons and muscles, including the vulva6. nu329 and pk450::Tc1, both only partial loss-of-function, demonstrated 5HT-resistance despite a baseline with wildtype parameters. Whether cAMP stimulates or inhibits egg-laying is still unclear, but pharmacological manipulations are planned to clarify the issue. Finally, assuming 5HT-Ce inhibits adenylate cyclase, downstream G-protein candidates might include goa-1 or gpa-7. Yet, functional goa-1 appears to play an inhibitory role in egg-laying7,8 while testing in our lab of gpa-7 worms did not uncover significant 5HT-resistance. Recently, the Plasterk lab surveyed the complete family of genes encoding worm G-proteins9. One of these genes, gpa-14, is expressed in the vulva and appears to be a novel G-protein whose downstream effectors are unknown. gpa-14 (pk347) mutants were subsequently tested and found to be 5HT-resistant, suggesting that gpa-14 might be part of a 5HT signalling pathway. 1Science 216:1012 2Neuron 21:203 3Genetics 104:619 4Tim Niacaris (Avery lab), pers comm 5J Mol Neurosci 8:53 6J Neurosci 18:2871 7Science 267:1652 8Science 267:1648 9Nat Genetics 21:414

Schafer WR et al. (1996) West Coast Worm Meeting "GENES AFFECTING THE SEROTONIN RESPONSE OF THE EGG-LAYING MUSCLES"

Sanchez BM, Waggoner LE, Rakow TL, Schafer WR, Ramirez DS

[West Coast Worm Meeting, 1996]

Modulation of a post-synaptic cell's response to neurotransmitter is an important mechanism for regulating the synaptic strength, a process critical for diverse forms of learning and memory. Our laboratory has been attempting to understand the molecular events that regulate the sensitivity of a specific post-synaptic cell type, the vulval muscles, to the neurotransmitter serotonin, released endogenously by the HSN motorneurons. In earlier work, we found that mutations in two genes encoding putative voltage-gated calcium channel subunits, unc-2 and unc-36, caused hypersensitivity and failure to desensitize to serotonin. This suggested that a voltage-sensitive calcium channel containing the UNC-2 and UNC-36 proteins activates a calcium-dependent process leading to serotonin desensitization. Based on expression studies and mosaic analysis, we proposed that the UNC-2 calcium channel might act by regulating the neurotransmitter release from modulatory neurons, perhaps the VCs. To identify more molecules that participate in the regulation of serotonin response, we surveyed known Egl-c Unc mutants for serotonin hypersensitivity. Mutations in at least 6 genes (unc-8, unc-10, unc-20, unc-35, unc-75, and unc-77) conferred both constitutive egg-laying and hypersensitivity to serotonin. We found that one of these genes, unc-20, is required for the proper guidance of the VC axons and positioning of the VC cell bodies. Mutations in several other genes have also been isolated (by other groups) in screens for aldicarb-resistant mutants, suggesting that the function of their gene products is to facilitate release of ACh and possibly other neurotransmitters. In fact we determined that both snt-1 mutants (defective in synaptotagmin) and cha-1 mutants (defective in choline acetyltransferase) were serotonin hypersensitive, and that cha-1 mutants were also strongly Egl-c, indicating that ACh is an inhibitory modulator of egg-laying behavior and serotonin response. Analysis of nicotinic ACh receptor mutants suggested that this modulation is mediated through a ligand-gated ion channel containing the UNC-38 protein as a subunit. We are currently investigating the site of action of this receptor, as well as the site of ACh release. In the future, we hope to use physiological approaches, such as calcium imaging, to study the processes involved in serotonin desensitization. Progress in developing these techniques will (hopefully!) be discussed.

Walker, D.S. et al. (2017) International Worm Meeting "UNC-7 innexin hemichannels function as mechanosensors in gentle touch and nociception in Caenorhabditis elegans."

Schafer, W.R., Walker, D.S.

[International Worm Meeting, 2017]

Mechanosensation is central to a wide range of functions, including tactile and pain perception, hearing and proprioception, but identifying the mechanotransducer molecules responsible has proved challenging. The response to gentle body touch in C. elegans is mediated by the touch receptor neurons (TRNs). We find that, in addition to the well-established role of MEC-4, a second mechanosensor, the innexin UNC-7, is also essential for gentle touch. A mutant unc-7 transgene, encoding a derivative that lacks four cysteine residues necessary for gap junction formation, robustly rescues the unc-7 mechanosensory defect, indicating that UNC-7 acts as hemichannels to sense gentle touch. An isoform known to make gap junctions does not rescue, suggesting that the functions of UNC-7 in gap junctions and mechanosensory hemichannels are genetically separable. UNC-7 is also required for harsh touch in both the TRNs and the PVD nociceptors, but not for thermal responses in PVD, indicating that it functions specifically in mechanosensation. We present evidence that UNC-7 and MEC-4 function independently. Heterologous expression of unc-7 in touch-insensitive chemosensory neurons confers ectopic touch sensitivity, demonstrating that UNC-7 hemichannels are sufficient for mechanosensation. The unc-7 touch defect can be rescued by the mouse Panx1 gene, suggesting that innexin/pannexin proteins may play a broadly conserved role in neuronal mechanotransduction.

Walker, D.S. et al. (2017) International Worm Meeting "Role of a MEC-4-actin interaction in the gentle touch neurons of Caenorhabditis elegans."

Walker, D.S., Schafer, W.R.

[International Worm Meeting, 2017]

The response to gentle body touch requires the touch receptor neurons (TRNs); and components of the presumed mechanotransduction apparatus have been identified via mutant screens. MEC-4 and MEC-10 are members of the DEG/ENaC family of amiloride sensitive sodium channels. Proper, punctate localisation of MEC-4 along the length of the TRNs requires 15 protofilament microtubules (MEC-7 and MEC-12), and extracellular matrix components (MEC-1, MEC-5 and MEC-9). These were thought to act together to anchor the channel and thus allow mechanical gating, but MEC-4 does not localise to the ends of microtubules, so their precise role is unclear. While MEC-4 is essential for gentle touch, MEC-10 has a more limited, spatially confined role, is differently localised, and is additionally required for the response to harsh touch. We are interested in understanding the cellular basis of differential MEC-4 and MEC-10 localisation and how their distinct functions are determined. We describe the identification of a novel interaction between the C-terminal cytoplasmic domain of MEC-4 and actin. We show that the C-terminal domain of MEC-4 determines its localisation; and that actin is required for correct localisation of MEC-4. Expression of a competing peptide indicates that the interaction is required for the gentle touch response. We are investigating whether actin colocalises with MEC-4 and thus could perform an anchoring role in gating and whether the interaction is a target for regulation.

Daniel L Chase et al. (2003) International Worm Meeting "Dopamine-resistant mutants of C. elegans identify components of Go and Gq signaling pathways"

Michael R Koelle, Daniel L Chase, Judy S Pepper

[International Worm Meeting, 2003]

Dopamine, a major neurotransmitter in the mammalian central nervous system, acts through two classes of G protein-coupled receptors known as D1-like and D2-like receptors. These two classes of receptor signal to antagonistically regulate neural activity. Despite intensive research, the signaling mechanisms responsible for the antagonistic effects of dopamine in the brain remain debated. We describe the first genetic screen designed to identify proteins required for dopamine signaling. C. elegans uses endogenous dopamine to inhibit locomotion behavior1, and treatment with excess exogenous dopamine paralyzes worms2. We isolated mutants unresponsive to exogenous dopamine and found that they are also defective for endogenous dopamine signaling. Eleven mutations identified five different genes. Four of these genes encode components of the Go and Gq signaling pathways, including the G protein Go itself and subunits of the RGS complex that inhibits Gq signaling. The Go and Gq signaling pathways act antagonistically in C. elegans to control behavior and their components are conserved in humans. Mutations in these signaling components that decrease Go signaling or increase Gq signaling cause dopamine resistance, while mutations that have opposite effects on these two signaling pathways cause dopamine hypersensitivity. Thus the physiological effects of dopamine are mediated by Go in C. elegans and are antagonized by Gq signaling. Such a dopamine signaling mechanism in mammals could explain many of the opposing effects of D1-like and D2-like receptor activation. Our screen also identified one gene that appears to encode a new Go/Gq signaling component. 1. Sawin, E.R., Ranganathan, R., and Horvitz, H.R. (2000). Neuron 26, 619-631; 2. Schafer, W.R., and Kenyon, C.J. (1995). Nature 375, 73-78.