William Schafer et al. (2002) European Worm Meeting "In vivo imaging of mechanoreceptor function."

Christian Frokjar-Jensen, HIROSHI SUZUKI, William Schafer, Rex Kerr

[European Worm Meeting, 2002]

To fully understand behavior at the molecular and cellular level, it is necessary to determine how specific gene products affect the activity of identified neurons, and to correlate the activity of these neurons with behavior. Genetically-encoded optical sensors, such as the FRET-based, ratiometric calcium-sensitive protein cameleon, have many potential advantages for cell-specific non-invasive neural imaging. The use of optical indicators is particularly attractive in C. elegans due to the animal's transparency, the ease with which transgenic animals can be generated, and the difficulty of electrophysiological methods. However, because of their relatively slow kinetics and small signal size, it has been difficult to use genetically-encoded sensors like cameleon in excitable cells. We have recently overcome these hurdles and developed imaging methods that have allowed us to detect and measure in vivo calcium transients in mechanosensory touch receptor neurons in response to sensory stimulation. Using this technique, we have begun to address the molecular basis for these neurons' response to touch stimuli. We have found that calcium influx occurs in both the cell body and sensory dendrite of the touch neuron, suggesting that calcium mediated action potentials may play a role in mechanotransduction. Recordings of calcium transients from mutants defective in the putative mechanotransduction channel MEC-4 showed occcasional calcium transients in response to touch, suggesting that the touch neurons may contain a mec-4-independent sensory modality, perhaps involved in harsh touch detection. Finally, we have observed that repeated high-frequency stimulation of the touch cells results in an accumulation of baseline calcium and a subsequent reduction in the calcium response to successive stimuli. Experiments to determine the mechanistic basis for these experience dependent changes in touch neuron function are in progress..

William Schafer et al. (2002) Mid-west Worm Meeting "Serotonin promotes Go-dependent neuronal migration in Caenorhabditis elegans"

Katie S Kindt, William Schafer, Tobey Tam, Shaleah Whiteman

[Mid-west Worm Meeting, 2002]

The directed migration of neurons during development requires attractive and repulsive cues, which control the direction of migration, as well as permissive cues that potentiate cell motility and responsiveness to guidance molecules. We have found that the neurotransmitter serotonin functions as a permissive signal for embryonic and post-embryonic neuronal migration in the nematode C. elegans. In serotonin-deficient mutants (tph-1), the migrations of the ALM, BDU, SDQR and AVM neurons were often foreshortened or misdirected, indicating a serotonin requirement for normal migration. Moreover, exogenous serotonin could restore motility to AVM neurons in serotonin-deficient mutants as well as induce AVM-like migrations in the normally non-motile neuron PVM, indicating that serotonin was functioning as a permissive cure to enable neuronal motility. The migration defects of serotonin deficient mutants were mimicked by ablations of serotonergic neuroendocrine cells, implicating humoral release of serotonin in these processes. Mutants defective in Gq and Go signaling, or in N-type voltage-gated calcium channels, showed migration phenotypes similar to serotonin-deficient mutants, and these molecules appeared to genetically function downstream of serotonin in the control of neuronal migration. Thus, serotonin is important for promoting directed neuronal migration in the developing C. elegans nervous system. We hypothesize that serotonin may promote cell motility through G-protein-dependent modulation of voltage-gated calcium channels in the migrating cell.

William Schafer et al. (2002) West Coast Worm Meeting "Serotonin promotes Go-dependent neuronal migration in Caenorhabditis elegans"

Katie S Kindt, Shaleah Whiteman, William Schafer, Tobey Tam

[West Coast Worm Meeting, 2002]

The directed migration of neurons during development requires attractive and repulsive cues, which control the direction of migration, as well as permissive cues that potentiate cell motility and responsiveness to guidance molecules. We have found that the neurotransmitter serotonin functions as a permissive signal for embryonic and post-embryonic neuronal migration in the nematode C. elegans. In serotonin-deficient mutants (tph-1), the migrations of the ALM, BDU, SDQR and AVM neurons were often foreshortened or misdirected, indicating a serotonin requirement for normal migration. Moreover, exogenous serotonin could restore motility to AVM neurons in serotonin-deficient mutants as well as induce AVM-like migrations in the normally non-motile neuron PVM, indicating that serotonin was functioning as a permissive cure to enable neuronal motility. The migration defects of serotonin deficient mutants were mimicked by ablations of serotonergic neuroendocrine cells, implicating humoral release of serotonin in these processes. Mutants defective in Gq and Go signaling, or in N-type voltage-gated calcium channels, showed migration phenotypes similar to serotonin-deficient mutants, and these molecules appeared to genetically function downstream of serotonin in the control of neuronal migration. Thus, serotonin is important for promoting directed neuronal migration in the developing C. elegans nervous system. We hypothesize that serotonin may promote cell motility through G-protein-dependent modulation of voltage-gated calcium channels in the migrating cell.

Tanizawa, Yoshinori et al. (2009) International Worm Meeting "Arousal regulation by sensory stimulation and neuromodulators in adult C. elegans."

Schafer, William, Tanizawa, Yoshinori

[International Worm Meeting, 2009]

Changing behavioral strategy according to environmental and internal context is essential for animals to survive. C. elegans can respond to a wide variety of sensory cues, and modify their behavior from their experience. Another parameter influencing behavior is arousal, which is defined as a state of increased motor activity, enhanced sensory responsiveness and emotional changeability in the case of higher animals. In C. elegans, the best characterized example of arousal regulation is lethargus, a sleep-like state which accompanies every moult during development. In this project, we characterize a new aspect of arousal regulation, evoked by sensory stimulation, in adult C. elegans. To characterize arousal regulation by external cues, we applied sensory stimulation to adult animals and observed their behavior. We are currently using two types of stimuli in different modalities: tapping the agar plate using a mechanical tapper, and artificial activation of ASH nociceptive neurons by specifically-expressed channelrhodopsin-2, a light-gated cation channel. Both of these stimuli induced transient upregulation of motor activity lasting for one to two minutes depending on the stimulus strength. They also caused crossmodal sensitization, in which one stimulus enhanced behavioral response to another stimulus presented later. Arousal is likely to be affected and regulated by neuromodulators such as monoamines and neuropeptides. We examined the available mutants for these molecules, and found that signaling through tyramine and a neuropeptide receptor candidate (ZC412.1) inhibits and promotes arousal change caused by sensory stimulation, respectively. We are now in search for the signaling partners for these molecules by candidate gene approach and ligand screening in heterologous expression system (collaboration with Peter Evans group in Babraham institute, Cambridge). These results suggest that arousal is regulated by sensory cues and neuromodulators in adult animals, and prove that C. elegans is a good model to study arousal regulation in its simplest form.

YANG, Xinyi et al. (2021) International Worm Meeting "Explore novel functions of anti-microbial neuropeptides"

YANG, Xinyi, Schafer, William

[International Worm Meeting, 2021]

Infections caused by pathogens have been shown to alter the behaviour of animals, in part due to interactions between antimicrobial response pathways and the nervous system. Recent research has shown that the activation of immune cells can modulate neuronal circuits, and that the nervous system in turn can regulate both innate and adaptive immune responses. However, it remains unclear how small, infection-related neurotransmitters could contribute to the neuro-immune interactions. Here, we selected 10 epidermally expressed antimicrobial peptides (AMPs) in C. elegans, aiming at study the biological functions of these neuropeptides in worm behaviours. All these neuropeptides contain several YGGYG motifs, which is considered to have an antimicrobial function. Among them, expression level of nlp-24, nlp-29, nlp-31 are known to be up-regulated after fungal infection (N. Pujol et al. 2008). We have generated knockout mutants for each of these antimicrobial peptide genes using Crispr, and failed to observe any effect of these mutations on worm locomotion, or the response to gentle or harsh touch. However, several AMP mutants had significant effects on egg-laying behaviour. Specifically, we discovered that nlp-27, nlp-28, and nlp-30 mutants exhibited increased egg-laying rates, an effect primarily due to increased egg-laying within the egg-laying active phase. On the other hand, nlp-29 mutant showed significantly decreased egg-laying and specifically decreased egg-laying rate within the active phase. These results implicate several AMPs in the control of intensity and/or duration of the active phase of egg-laying. Our next step is to further study if the modulated egg-laying phenotypes caused by the knock-out of the certain neuropeptide will be altered after culturing the worm on pathogenic bacteria. We also aim at discovering the corresponding receptors for these AMPs and further discovering the down-stream signalling pathways that are involved in egg-laying regulation.

Branicky, Robyn et al. (2011) International Worm Meeting "Genetic, pharmacological and calcium imaging analysis of mutants affecting HSN activity."

Schafer, William R., Branicky, Robyn

[International Worm Meeting, 2011]

We are using the egg-laying system as a model to investigate how neural circuits generate behavioural outputs. The egg-laying behaviour is regulated by a simple motor circuit, made up of only a few neurons (the HSNs and the VCs) and muscles (the VMs). Despite the anatomic simplicity of the egg-laying system, genetic, pharmacological, and more recently, calcium imaging studies have revealed several layers of complexity including the involvement of multiple neurotransmitters and their receptors as well as neuropeptides. We have used calcium imaging to monitor the activity of the HSNs and VMs in a collection of characterised and uncharacterised egg-laying defective (Egl) mutants with presumed defects in HSN activity. We have identified mutants acting both upstream and downstream of calcium entry into the HSN. We will present our phenotypic and molecular characterisation of these mutants and propose models for how they could be affecting HSN activity and ultimately the activity of the egg-laying circuit.

Hardege, Iris et al. (2021) International Worm Meeting "Expansion of Cholinergic Signalling Reveals Polymodal and Novel Ligand Gated Ion Channels Involved in Switching Behavioural States"

Morud, Julia, Schafer, William, Hardege, Iris

[International Worm Meeting, 2021]

Ligand-gated ion channels (LGICs) play important roles in synaptic communication and the regulation of behaviours. The cys-loop superfamily of LGICs, which contains mammalian nicotinic acetylcholine and GABA receptors, has undergone vast gene expansion in nematodes and includes channels gated by classical and non-classical neurotransmitters. Yet the majority of C. elegans LGICs remain uncharacterised, with no known ligand or biological function. We have undertaken a deorphanisaiton study of a number of uncharacterised C. elegans LGICs, in particular from a subfamily of 12 channels known as the "diverse" group. Using two-electrode voltage clamp recordings from Xenopus oocytes injected with worm LGICs, we identified ligands for 5 of the 12 channels in the diverse subgroup. All are inhibitory anion-selective channels, yet despite sharing close sequence similarity, they are gated by chemically diverse ligands. We identify three receptors for choline, GGR-1, GGR-2 and LGC-40, which despite binding the same ligand differ in their pharmacological and neuronal expression profiles. This may point towards a potential role for choline in the regulation of the nervous system. Interestingly, we found a single channel, LGC-39 to be gated not only by acetylcholine but also by aminergic ligands, in particular octopamine and tyramine. Thus LGC-39 has the capacity to form a polymodal receptor activated by chemically disparate neurotransmitters. The expression pattern of lgc-39 reveals that it is present in a number of neurons which receive both aminergic and cholinergic input, including AVA, the major synaptic target of the octopamine producing RICs. Finally, we find that LGC-41 is not activated by any classical neurotransmitter, but is activated by betaine, a metabolite chemically related to choline. LGC-41, along with putative betaine synthesis genes, is widely expressed in the nervous system, in particular in a number of neurons associated with regulating search behaviours including ASI. Strikingly, we find that lgc-41 and betaine synthesis mutant worms show defects in transitioning from local to global search behaviour in the absence of food. This implicates betaine and its receptor in regulating complex behaviours which rely on the integration of a number of sensory inputs. Taken together our findings highlight the remarkable functional and behavioural diversification amongst the LGICs in C. elegans.

Hardege, Iris et al. (2021) International Worm Meeting "LGC-50 - a new serotonin receptor involved in aversive olfactory learning that displays regulated plasma membrane trafficking"

Schafer, William, Hardege, Iris, Morud, Julia

[International Worm Meeting, 2021]

Across animal phyla, monoamines signal through both metabotropic and ionotropic receptors. In worms as well as in humans, metabotropic monoamine receptors, which modulate neuronal activity through G-protein-mediated second messenger pathways, have received most attention. However, expression studies have indicated that the vast majority of C. elegans neurons postsynaptic to aminergic neurons do not express metabotropic amine receptors. This implies that synaptic monoamine transmission may be mediated by as yet uncharacterized ionotropic receptors. We have in our recent work identified endogenous ligands for five new amine-gated ion channels (LGC), all of which are found localised postsynaptically to aminergic neurons. In particular we have shown the serotonin-gated receptor LGC-50 to be a cation channel that is localised in the interneuron RIA, which is strongly innervated by the serotonergic neuron ADF. Previous work has indicated a role for ADF and RIA in aversive pathogen learning, through which animals learn to avoid odours released by pathogenic bacteria following infection. We have also shown that lgc-50 mutants show a strong defect in pathogen learning, which can be rescued by expression of LGC-50 in RIA. These results suggest that serotonin may act through LGC-50 to modify the strength of specific synapses in the olfactory navigation circuit. Our recent work also indicated that the plasma membrane localisation of LGC-50 is tightly regulated, and that potential disruption of this trafficking influences memory formation. We have now identified a 17-amino acid long motif in the intracellular M3/4 domain of LGC-50 that conveys this regulated membrane localisation. Further, we have evidence that this motif might act as a binding site for the protein NRA-1 and that this protein-protein interaction might be involved in moving LGC-50 to the plasma membrane during memory formation. LGC-50 thus provides an entry point to define the molecular and neural changes underlying learning and memory in the worm.

Chatzigeorgiou, Marios et al. (2011) International Worm Meeting "Lateral facilitation between primary mechanosensory neurons controls nose touch perception in C. elegans."

Rabinowitch, Ithai, Schafer, William, Chatzigeorgiou, Marios

[International Worm Meeting, 2011]

The nematode C. elegans senses head and nose touch using multiple classes of mechanoreceptor neurons that are electrically-coupled through a network of gap junctions. Using modelling, in vivo neuroimaging, and genetic tools capable of modifying neural circuits we have found that one sensory neuron class, the multidendritic FLP nociceptors, respond to harsh touch throughout their receptive field but respond to gentle touch only at the tip of the nose. Whereas the FLP harsh touch response depends solely on cell-autonomous mechanosensory channels, gentle nose touch responses require the activities of additional mechanoreceptor classes, OLQ and CEP, which are electrically-coupled to FLP through a gap junction network. Conversely, FLP activity is required to indirectly facilitate nose touch and harsh head touch responses in the OLQs, demonstrating that information flow across the network is bidirectional. Thus, nose touch perception involves a hub-and-spoke network that allows individual sensory neurons to integrate information from multiple interconnected mechanoreceptors. A model of a hub-and-spoke network predicts that inactive neurons will inhibit other cells in the network through shunting; artificial rewiring experiments using ectopic connexin expression support this hypothesis. Thus, the nose touch circuit uses electrical connectivity to detect and generate responses to coincident mechanoreceptor activity.

Ezcurra, Marina et al. (2009) International Worm Meeting "Modulation of chemosensation in ASH through extrasynaptic dopaminergic and neuropeptide signaling."

Schafer, William, Swoboda, Peter, Ezcurra, Marina

[International Worm Meeting, 2009]

C. elegans avoids a variety of repellents, of which many are detected by the neuron ASH. Avoidance responses are influenced by modulators, and previous studies have shown that food affects responses to nose touch and dilute octanol by acting on ASH through 5-HT signaling. Since ASH is polymodal, we asked if other ASH modalities are modulated by food. Using behavioral assays and calcium imaging, we investigated behavioral avoidance and ASH neuronal responses to CuCl2. We found that food affects CuCl2 avoidance; responses are higher when food is present than when food is absent. Surprisingly, we could not find evidence supporting that 5-HT is required, suggesting that other molecules are involved. Dopamine has been implicated in behavioral plasticity, so we tested the role of dopamine in modulation of CuCl2 avoidance. We found that ASH responses, measured by behavioural assays and calcium imaging, were not modulated by food in the dopamine-deficient mutant cat-2(e1112); cat-2 mutants on or off food resembled N2 animals off food. Moreover, exogenous dopamine mimicked the effect of food on N2 animals, suggesting that dopamine is involved in enhancing ASH responses on food. Behavioral candidate dopamine (and other monoamine) receptor mutants revealed that dop-3(vs106) and dop-4(tm1393), like cat-2(e1112) have defects in food modulation of repellent avoidance, suggesting that dopamine affects avoidance behaviors through these receptors. We are using rescue and imaging experiments to investigate if dopamine acts on ASH through DOP-3 and DOP-4 directly or via other neurons. Neuropeptides have modulatory effects on the nervous system. Using behavioral and imaging experiments we have shown that in the absence of food, mutants for the neuropeptide receptors NPR-1 and NPR-2 adapt slower to repeated stimulation with CuCl2 compared to N2. However, in the presence of food this difference is abolished, indicating that NPR-1 and NPR-2 are required to promote faster adaptation in ASH in the absence of food, in contrast to dopamine which promotes slower adaptation in the presence of food. Both npr-1 and npr-2 mutant phenotypes can be rescued by sra-6 promoter control, indicating that neuropeptide signaling acts directly on ASH to modulate adaptation. We are investigating how dopaminergic and neuropeptide modulatory pathways interact through double mutant analysis and cell-specific knockdown of candidate signal transduction molecules.