Debnath, Arunima et al. (2021) International Worm Meeting "Neuromodulation and the relationship between Ca2+ transients and neuronal states"

Bamber, Bruce, Debnath, Arunima

[International Worm Meeting, 2021]

Neuromodulators (monoamines and neuropeptides) shape nervous system function by regulating neuronal depolarization and synaptic strengths. We are studying neuromodulation in the context of nociception, to better understand pain perception and pain treatment strategies. The ASH neuron is a major nociceptor in C. elegans. ASH senses 1-octanol and drives an aversive response, modulated by the monoamines serotonin (5-HT, potentiating) and octopamine (OA, inhibitory). To better understand neuromodulation, we are focusing on 1-octanol stimulated Ca2+ dynamics in ASH, and the quantitative relationship between Ca2+ signals and depolarization amplitudes. We showed that 5-HT potentiates ASH depolarization, but surprisingly, inhibits ASH Ca2+ transient amplitudes. These effects, like the 5-HT stimulation of behavior, depend on the SER-5 receptor in ASH. This paradoxical finding is explained by existence of a Ca2+-dependent inhibitory feedback loop: Ca2+ inhibits ASH depolarization through SLO-1 IKCa channels, and 5-HT inhibits EGL-19 L-type Ca2+ channels in ASH (via SER-5), thus disinhibiting the neuron. We are currently investigating modulation of 1-octanol responses by OA. OA inhibits 1-octanol behavioral responses, and antagonizes 5-HT potentiation, dependent on the OCTR-1 receptor in ASH. OA also inhibits ASH Ca2+ transients, representing another paradox: how can OA and 5-HT have opposite effects on ASH-dependent aversive behaviors, but the same effect on ASH Ca2+ transients? Our results show that 5-HT and OA utilize distinct signaling pathways in ASH (Galphaq for 5-HT and Galphao for OA), and that OA does not inhibit EGL-19. We are currently testing the hypothesis that OA hyperpolarizes ASH through Galphao-dependent activation of IRK K+ channels. These results further emphasize that neuronal Ca2+ transients, as key reporters of neuronal depolarization, are also critical signaling intermediates in and of themselves, with multiple upstream inputs and downstream consequences.

Bao, Zhirong (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Chiral forces establish left-right asymmetry in C. elegans by uncoupling the midline and anteroposterior axis"

Bao, Zhirong

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Left-right (LR) patterning is an intriguing but poorly understood process of bilaterian embryogenesis. We report a novel mechanism to break LR symmetry, whereby the embryo uncouples its midline from the anteroposterior (AP) axis. Specifically, the eight-cell C. elegans embryo establishes a midline that tilts rightward from the AP axis and positions more cells on the left, allowing subsequent differential LR fate inductions. To establish the tilted midline, cells exhibit LR asymmetric protrusions and a handed collective movement. This process, termed chiral morphogenesis, is based on differential regulation of cortical contractility between a pair of sister cells that are bilateral counterparts fate-wise, and is activated by non-canonical Wnt signaling. Chiral morphogenesis is timed by the division furrow of a neighbor of the sister pair, suggesting a nov el developmental clock and a novel signaling mechanism from the contractile ring to adjacent cells.

Jamie White et al. (2001) International C. elegans Meeting "A reverse genetic approach to neuropeptide function in the ASH sensory circuit"

Jamie White, Anne Hart

[International C. elegans Meeting, 2001]

Backward locomotion in response to nose touch, 1-octanol, benzaldehyde, and high osmolarity is mediated by the ASH sensory neurons (CGC 1705, 2314). Glutamate neurotransmission between ASH and the interneurons of the ASH circuit appears to be absolutely required, since the response to all stimuli is abolished by a mutation in EAT-4, a sodium-dependent P i cotransporter proposed to load glutamate into synaptic vesicles (CGC 3349, PMID 11001057). However, a mutation in the GLR-1 glutamate receptor subunit eliminates the response to only nose touch (PMID 7477294). GLR-1 functions post-synaptically, in the interneurons of the ASH circuit, not in detection in ASH itself, indicating that glutamatergic neurotransmission is modulated in a stimulus-specific manner (PMID 7477294). That is, glutamatergic neurotransmission is necessary but not sufficient for the response to volatile odorants and high osmolarity. To understand how the ASH circuit transmits stimulus-specific information across the ASH-interneuron synapse, we are investigating the function of novel neuropeptides in the ASH circuit. There are at least 32 neuropeptide-like preprotein ( nlp ) genes in the genome, and GFP-reporter studies show at least two of these are expressed in ASH (ECWM 2000ab56). Additionally, electron microscopy has shown dense-core vesicles, presumably containing peptidergic transmitters or modulators, at the ASH-interneuron synapse (PMID 8806). These observations raise the possibility that NLP peptides could modulate glutamate neurotransmission in a stimulus-specific manner. To address the function of putative ASH-specific neuropeptides, we generated a frozen deletion library of 2.4x10 6 genomes, and are currently screening for deletions in the putative neuropeptide genes. Results of the deletion screening and preliminary phenotypic analysis will be presented.

Hapiak, Vera et al. (2009) International Worm Meeting "Multiple monoamine receptors modulate nose touch in C. elegans."

Schafer, William, Chatzigeorgiou, Marios, Komuniecki, Richard, Wragg, Rachel, Hapiak, Vera, Harris, Gareth

[International Worm Meeting, 2009]

The ASH sensory neuron in C. elegans is polymodal, responding to both noxious (high osmolarity/volatile repellents) and mechanical (nose touch) stimuli to initiate backward locomotion. Examination of one ASH-mediated locomotory behavior, avoidance to octanol, has revealed that modulation of the ASH neural circuit is complex and involves multiple monoamines and distinct amine receptors (Wragg et al., 2007; Harris et al., 2009). In the present study, we have identified the monoamine receptors involved in another 5-HT stimulated ASH-mediated aversive response, nose touch. Like responses to octanol, multiple 5-HT receptors also have been identified in the food/5-HT sensitization of nose touch. For example, the expression of ser-5 in the ASHs appears to be essential for 5-HT dependent increases in aversive responses to nose touch. In contrast, both TA and OA inhibit nose touch and two monoamine receptors (F14D12.6 and SER-3) appear to be involved in octopaminergic inhibition. These receptors are currently being localized in the ASH locomotory circuit. Using the calcium indicator cameleon, we analyzed ASH Ca2+ responses to nose touch in ser-5 null animals. ASH Ca2+ transients were independent of exogenous 5-HT and more robust in ser-5 null animals than in wild-type or ser-4; mod-1; ser-7 ser-1 quadruple null animals that presumably only express ser-5. Since we predict that SER-5 stimulates neurotransmitter release at the ASH synapse, the increased Ca2+ signaling in the ASH soma was surprising. We are currently exploring whether SER-5 signaling has other effects on the ASH or whether SER-5 also modulates the release of other ligands, perhaps peptides, that inhibit ASH Ca2+ dynamics.

Kass J et al. (1996) East Coast Worm Meeting "Isolation and characterization of suppressors of glr-1."

Kaplan JM, Hart AC, Kim P, Kass J

[East Coast Worm Meeting, 1996]

How do animals distinguish between sensory stimuli? We are studying a neural circuit in which the ASH sensory neurons detect noxious chemical and mechanical stimuli and signal to the worm to move backward. Three stimuli are detected by the ASH neurons (nose touch, high osmolarity and volatile repellants) and each provokes backward locomotion. Genetic and molecular evidence suggests that different signals are produced at the ASH-interneuron synapses in response to the different stimuli. The glr-1 gene encodes a glutamate receptor required specifically for response to nose touch. GLR-1 is expressed in the command interneurons, AVA, AVD, and AVB, that receive synaptic input from ASH, suggesting that neurotransmitter release from ASH in response to nose touch is different than release in response to osmotic stimuli and volatile repellants. For example, in response to nose touch ASH may release glutamate whereas, in response to high osmolarity ASH may release both glutamate and a neuropeptide. To better understand ASH signaling, we screened for suppressors of glr-1 (n2461). glr-1 might be suppressed by mutations which cause ASH to use the other neurotransmission pathway(s); thus suppressors may give us insight into differential release of neurotransmitters. Approximately 6,500 haploid genomes have been screened and two suppressors have been isolated. One suppressor (nu349) has been mapped to chromosome V and also suppresses a deletion allele, glr-1(ky176). We are currently laser ablating ASH in the suppressed animals to determine the site of action of the suppressors. If the ablation of ASH returns the suppressed animals to the mutant phenotype then the suppressor may be acting in ASH. If the animals are still suppressed then another cell may be taking over the mechanosensory function of ASH or the interneurons may have been sensitized so that the same intensity of signal from ASH results in a greater response in the interneurons. We are also interested in determining whether the suppressors are capable of suppressing all of the defects of glr-1 including loss of responsiveness to harsh touch. We will continue to map and eventually clone the suppressors of glr-1.

Doss, Argenia et al. (2015) International Worm Meeting "Neural regulation of innate immune response using optogenetics."

Doss, Argenia, Aballay, Alejandro

[International Worm Meeting, 2015]

Previously, our laboratory showed that OCTR-1, a G protein-coupled catecholamine receptor, suppresses the innate immune response in Caenorhabditis elegans. As a result, animals expressing the loss-of-function octr-1 allele, ok371, exhibited an enhanced resistance to killing by the human opportunistic pathogen Pseudomonas aeruginosa. Despite OCTR-1 being expressed in five neurons, data from our laboratory suggests that OCTR-1 functions in chemosensory neurons ASI and/or ASH to suppress innate immunity. However, the specific role of ASI and ASH neurons is unclear. Unpublished data from our laboratory showed that ASI neurons do not affect the susceptibility of C. elegans to P. aeruginosa. Therefore, we examined the role of ASH neurons during infection. With the use of optogenetics, we were able to assess whether activation of ASH neurons via Channelrhodopsin-2 alters the susceptibility of C. elegans to P. aeruginosa-mediated killing. Preliminary data from our experiments revealed that activation of ASH neurons during exposure to P. aeruginosa alters the susceptibility of C. elegans to P. aeruginosa-mediated killing. This suggests that ASH neurons can regulate the innate immune response in C. elegans and OCTR-1 may suppress innate immunity by acting through ASH neurons. Ongoing experiments are directed towards determining how ablation of ASH neurons and OCTR-1 expression in ASH neurons alters immune response. Currently, our data suggests that ASH neurons, and less likely ASI neurons, possess an immunoregulatory function. Data from these studies will help clarify the cellular process(es) involved in OCTR-1's regulation of innate immune response in C. elegans.

Jagan Srinivasan et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Evolution of sensory polymodality in free-living nematodes"

Paul Sternberg, Jagan Srinivasan

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Despite the diversity of the nematode phylum, most nematode nervous systems have around 300 neurons. Some neurons (polymodal) respond to multiple stimuli. C. elegans possesses classical "polymodal" neurons that respond to multiple types of stimuli, including chemo - and mechanosensory (1-3). To trace the evolutionary history of polymodality, we tested in six diverse species of free-living nematodes, three different avoidance behaviors; osmotic avoidance, response to nose touch, and volatile chemical repellence in C. elegans (N2), C. briggsae (AF16), Caenorhabditis sp. 3 (PS1010), P. pacificus (PS312) C. tripartitum (PS1351) and P. redivivus (PS2298). Nearly all species tested show conserved avoidance behaviors. In C. elegans, response to these stimuli are mediated by the ASH neurons. Ablation of the ASH neuron almost completely abolishes osmotic avoidance response in C. elegans (4). However, ASH ablation does not completely abolish osmotic avoidance in other species, suggesting the existence of ASH-independent mechanisms regulating osmotic avoidance in these species. Ablation of additional nociceptive neurons (4) along with ASH resulted in complete abolishment of osmotic avoidance in these species suggesting that additional neurons regulate osmotic avoidance in these other species. In C. elegans, nose touch is mediated by ASH, OLQ and FLP (1). Ablation of the ASH neurons resulted in loss of nose touch avoidance in most species. However in Caenorhabditis sp. 3, ASH-ablated animals completely lack the ability to respond to nose touch. In Caenorhabditis sp. 3, ablation of the FLP and OLQ cells did not result in any significant differences to nose touch than ASH ablated animals, suggesting that the circuitry mediating nose touch might have converged from three sensory neurons (ASH, FLP, OLQ) onto a single sensory neuron (ASH). Repellence to the volatile odorant 1-octanol was mediated by the ASH neuron in all the species. Hence, our findings suggest that polymodality evolved early during diversification of the rhabditids. However, changes in behavior seen at the cellular level might result from loss or gain of certain components of the sensory signaling or changes in the downstream signaling of the sensory stimulus at both the cellular and synaptic level during evolution. This suggests that polymodality as a trait in nematodes is under negative selection but the underlying cellular network evolves to help survive their respective environments.

Geffeney, Shana et al. (2011) International Worm Meeting "DEG-1 and not OSM-9 is the major mechanoeletrical transduction channel in ASH."

Geffeney, Shana, Lee, Tim, Montoya, Misty, Cueva, Juan, Glauser, Dominique, Goodman, Miriam, Doll, Joseph, Garakani, Arman, Pruitt, Beth, Karania, Snetu

[International Worm Meeting, 2011]

Mechanoreceptor neurons, including nociceptors, detect, encode and transmit mechanical stimuli as electrical signals. Understanding how mechanoreceptors detect force is critical for understanding how these neurons function, however the molecules responsible for mechanotransduction remain obscure except in two C. elegans neurons, PLM and CEP. Our goal was to identify the channels required to detect force in a C. elegans nociceptor ASH. In this study, we combined in vivo whole-cell patch-clamp recording and genetic dissection to deconstruct mechanoreceptor currents (MRCs) in ASH neurons. We found that forces 100-fold larger than those needed to activate a gentle touch receptor neuron, PLM, are required to activate MRCs in ASH. These results demonstrate that, like other nociceptors, ASH has a high threshold for cell activation. MRCs in ASH activate within milliseconds of stimulus application, like MRCs in touch receptor and CEP neurons. These results suggest that the channels carrying these currents are directly activated by force. MRCs in ASH are both Na+-dependent and inhibited by amiloride, properties of DEG/ENaC channels. Indeed in this study, we identify DEG-1, a DEG/ENaC channel, as the major mechano-electrical transduction (MeT) channel in ASH. These findings demonstrate that MRCs in both nociceptors and touch receptors rely on DEG/ENaC channels. But, DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 reduced MRC by 80% and revealed a second minor current, which is not formed by another DEG/ENaC channel. Thus, ASH nociceptors rely on two genetically-distinct, MeT channels. Surprisingly the minor current is also independent of two TRPV channel genes, osm-9 and ocr-2, that are co-expressed in ASH. Though null mutations in osm-9 and ocr-2 inhibit ASH-dependent behavioral responses to noxious stimuli, loss of OSM-9 and OCR-2 channels has no effect on mechanoreceptor currents or potentials in ASH. We propose that TRPV channels are not essential for detecting force, but contribute to encoding and transmitting information. Because mammalian and insect nociceptors as well as other C. elegans nociceptor neurons also co-express DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.

Chokshi, Trushal V. et al. (2011) International Worm Meeting "Effects of electrical stimulation on the ASH chemosensory neuron in Caenorhabditis elegans."

Chokshi, Trushal V., Chronis, Nikos

[International Worm Meeting, 2011]

Electrical stimulation has been widely used to modulate and study the functionality of the nervous system in-vivo and in-vitro. Here, we characterized the effect of electrical stimulation on ASH neuron in C. elegans and employed it to probe the neuron's age dependent properties. We utilized an automated microfluidic-based platform to acquire calcium imaging data from the ASH neuron in response to an electric current passing through the worm's body. We characterized the ASH neuronal activity in response to electric currents of varying strength and electrical polarity. For positive polarities (the worm's head faces the positive electrode), ASH depolarization was proportional to the magnitude of electric current while for negative polarities (the worm's head faces the negative electrode), it was inversely related. Interestingly, the ASH neuron hyperpolarized for higher negative polarity electric currents. Further, the neuronal activity in response to electrical stimulation showed significant differences across worms of different ages, indicating that the effect of electrical stimulation is age-dependent.

Wood, Jordan et al. (2015) International Worm Meeting "Developmental specification of a polymodal nociceptor in C. elegans."

Ferkey, Denise, Wood, Jordan

[International Worm Meeting, 2015]

All animals rely on their ability to sense and respond to the environment to survive. Nociception (the perception of noxious/painful stimuli) serves an important protective function, eliciting withdrawal and avoidance behaviors in response to potentially damaging stimuli. The primary nociceptors in C. elegans are the two ASH head sensory neurons, which detect multiple forms of aversive stimuli. However, while the sensation of pain is particularly valuable in helping an organism to avoid potentially harmful stimuli, there are still large gaps in our understanding of the mechanisms that govern nociceptor development across species. Based upon previously published experiments from other groups, we hypothesized that the paired-like homeodomain transcription factor UNC-42 functions as a terminal selector of ASH identity. However, our data indicate that although UNC-42 regulates a significant portion of the functionally mature ASH state, it does not function as the sole driver of the terminal ASH identity. In addition to UNC-42, a diverse array of transcription factors are expressed in the ASH neurons, which in combination likely comprise the genetic program underlying ASH specification and thus drive expression of the sensory signaling apparatus that endows the adult ASHs with polymodal nociceptive sensitivity. Using molecular, genetic and behavioral approaches, we have identified and are currently characterizing the function of several transcription factors in ASH specification.