andrione, mara et al. (2019) International Worm Meeting "RIS dynamics during sleep and wakefulness in freely-moving worms."

andrione, mara, Zimmer, Manuel, nichols, annika

[International Worm Meeting, 2019]

In both vertebrates and invertebrates, sleep-active neurons depolarize at sleep onset and, with their interplay with wake-promoting centers, guarantee fast and reliable switches between global brain states. However, how sleep homeostatic or circadian pressure influences their activity remains elusive1. While quiescent states in C. elegans are typically accompanied by downregulation of most neuronal activity2,3, the GABAergic and peptidergic interneuron RIS is both sleep-active and sleep-inducing4,5. Several evidences also point to a function of RIS more generally in sleep promotion2,3,6. These aspects represent discrete non-mutual exclusive hypotheses about RIS function, as either a final effector of the brain-state switch or a mediator of sleep pressure. The upregulation of RIS was first observed during lethargus sleep, but recent evidences show similar modulations occurring in different types of sleep, both in adulthood and during the larval stages3,7. Here, we first report about a previously uncharacterized type of spontaneous sleep in adult C. elegans. Then, through Ca2+ imaging of RIS in freely-moving animals, we draw a comparison between RIS dynamics seen during immobility periods in L4 lethargus and in adulthood. By using an arousing stimulation paradigm - O2 concentration shifts in worms with an npr-1 mutant background - we compare basal RIS activity patterns with those following sleep-deprivation. With this approach, we aim to disentangle to what extent sleep pressure and sleep induction are represented in RIS activity. 1: H Bringmann (2018) Genetics 2: ALA Nichols et al (2017) Science 3: S Skora et al (2018) Cell Reports 4: M Turek et al (2013) Current Biology 5: M Turek et al (2016) Elife 6: J Spies & H Bringmann (2018) Scientific Reports 7: Y Wu et al (2018) Current Biology

Steuer Costa, W. et al. (2017) International Worm Meeting "The peptidergic RIS neuron acts as a locomotion stop neuron in the adult."

Fischer, S., Gottschalk, A., Steuer Costa, W., Schueler, C., Miller III, D., Beets, I., Glock, C., Tolstenkov, O., Oranth, A., van der Auwera, P., Liewald, J., Mandal, J.

[International Worm Meeting, 2017]

Animals must be able to halt locomotion, e.g. to wait for certain events or to abruptly change directionality. In vertebrates, descending signals from the brain stem can induce a stop of locomotion, but allow the animal to maintain muscle tone. We suggest that the peptidergic/GABAergic interneuron RIS, well-known for its role in developmentally timed sleep (DTS) [1], attains the role of a stop neuron in adult behavior. In animals expressing Channelrhodopsin-2 (ChR2) solely in the RIS neuron, photo-depolarization led to acute and persistent locomotion and pharyngeal pumping inhibition with no effect on the defecation cycle. We showed that the photo-evoked phenotype required neuropeptides, but not GABA. Interestingly, in contrast to photodepolarization of all GABAergic neurons, evoking general muscle relaxation, RIS::ChR2 photoactivation allowed the animal to maintain its body posture. This was confirmed by Ca2+ imaging as well as recording of body wall muscle currents. However, RIS::ChR2 photoactivation reduced muscle tone in the head region, causing slight anterior body elongation. To identify peptides required for RIS' effects, we analyzed its L4 transcriptome. We characterized several enriched neuropeptides and found that FLP-11, which is also implicated in DTS regulation, is required for the optogenetically induced stop phenotype. Since this optogenetically evoked phenotype may be non-physiological, we assessed RIS' activity during spontaneous locomotion. Combining Ca2+ imaging and animal tracking [2] with custom-written software, we analyzed Ca2+ dynamics specifically in the RIS process, despite deformations caused by locomotion. Ca2+ signals in the RIS neural process preceded the cell body, indicating that they were due to synaptic activity. Temporal analysis of Ca2+ dynamics and locomotion speed suggested RIS as being instructive for locomotion stop before initiation of reverse locomotion. We are currently dissecting the role of individual RIS-expressed neuropeptides in the sequence of events associated with reversals. In sum, RIS, exploiting the molecular mechanisms present in DTS, acquires or maintains a role in adulthood to coordinate movement inhibition and reversal initiation. Hence, RIS spontaneous activity and its signaling physiology suggest a role in inhibiting a central unit of locomotion initiation and/or maintenance, without directly controlling ventral cord motor neurons. Refs: 1 Turek et al. Curr Biol 2013 2 Faumont et al. PLoS ONE 2011

Gendrel, Marie et al. (2011) International Worm Meeting "Development and Function of RIS, a Caenorhabditis elegans GABAergic interneuron."

Hobert, Oliver, Baison, George, Cai, Diana, Gendrel, Marie, Alden, Darym

[International Worm Meeting, 2011]

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain and dysfunction of GABAergic neurons can have profound pathological implications. In C. elegans, 26 neurons express conserved GABAergic terminal differentiation markers, such as the enzyme producing GABA (GAD/UNC-25), the GABA-specific vesicular transporter (VGAT/UNC-47) and the protein targeting VGAT to the synaptic membrane (a LAMP-like protein/UNC-46). 25 of these are motoneurons and only one neuron, named RIS, is an interneuron which most closely resembles the dominant type of GABA neurons in vertebrates. Preliminary evidence indicates that LIM-6 - a LIM homeobox transcription factor - is partly involved in RIS development, and loss of LIM-6 suggests that RIS might be involved in the control of metabolism and life span. To identify cis-regulatory elements necessary for the expression of known RIS terminal differentiation markers, we used mutational analysis of the gfp reporter genes. Initial results of the unc-47 promoter analysis define a 25bp area that is necessary for unc-47 expression in RIS. Further analysis of this region combine with the study of the other promoters should allow us to define cis regulatory motifs and therefore potential binding sites for transcription factors that control RIS differentiation. In parallel, in order to identify these factors we undertook EMS screens to identify trans-acting factors necessary for the expression of RIS terminal differentiation markers. Using the worm sorter, we isolated one temperature-sensitive mutant, in which unc-47 expression disappears at the adult stage. The whole-genome deep sequencing technique combine with the SNP mapping strategy should allow us to quickly clone the mutant. Altogether we should learn whether the RIS terminal differentiation markers are co-regulated through common cis-regulatory elements and trans-acting factors, with the LIM homeobox gene lim-6 being one but not the only component. In the end, we will use transgenic animals in which RIS is genetically eliminated to determine RIS function. Together, these results should give us a detailed picture of GABA neuron development and function.

Gendrel, Marie et al. (2013) International Worm Meeting "Development and Function of RIS, a Caenorhabditis elegans GABAergic interneuron."

Gendrel, Marie, Hobert, Oliver

[International Worm Meeting, 2013]

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain and dysfunction of GABAergic neurons can have profound pathological implications. In C. elegans, 26 neurons express conserved GABAergic terminal differentiation markers, such as the enzyme producing GABA (GAD/unc-25), the GABA-specific vesicular transporter (VGAT/unc-47) and the protein targeting VGAT to the synaptic membrane (a LAMP-like protein/unc-46). 25 of these are motoneurons and only one neuron, named RIS, is an interneuron that most closely resembles the dominant type of GABA neuron in vertebrates. Preliminary GABAergic cell fate marker analysis has shown that the RIS neuron fails to express terminal differentiation markers characteristic of GABA fate in lim-6 and nhr-67 mutant animals. Loss of LIM-6 -a LIM homeobox transcription factor- suggests that RIS might be involved in the control of metabolism. In parallel, we generated a null allele of nhr-67 -a Tailless/TLX ortholog- using the MosDEL technique in order to determine its role in RIS development. We hypothesize that these lim-6 and nhr-67 broadly control RIS GABAergic fate and that they may do so together. In parallel, to identify cis-regulatory elements necessary for the expression of known RIS terminal differentiation markers (unc-47, unc-25,unc-46, lim-6 and nlr-1), we used mutational analysis of gfp reporter genes. Altogether we should learn whether the RIS terminal differentiation markers are co-regulated through common cis-regulatory elements and trans-acting factors, with either the LIM homeobox gene lim-6 or the Tailles/TLX ortholog nhr-67 or the combination of the two. In addition, using the split-caspase system, we have generated transgenic animals in which RIS is genetically ablated to determine RIS function. We will report on a series of behavioral test undertaken with these animals. Together, these results should give us a detailed picture of a GABA interneuron development and function.

van Swinderen B et al. (1995) International C. elegans Meeting "INTER-BEHAVIORAL QTL STUDY OF ANESTHETIC RESPONSE"

van Swinderen B, Crowder CM

[International C. elegans Meeting, 1995]

We are studying the behavior genetics of anesthesia in C. elegans by using Recombinant Inbred(RI) lines. Based on electrophysiologic studies in vertebrates, the response to anesthetics may involve several genes. Genetic polymorphisms created by mosaic Bristol (N2) and Bergerac (BO) genomes provide an alternative to classical mutagenesis for mapping such loci. Two sets of RIs were examined in a preliminary study of select behaviors (these RIs, kindly provided by Tom Johnson" !Affiliation "[1] and Robert Shmookler Reis" !Affiliation "[2], are already genotyped for Tc1 polymorphic markers). Chemotaxis and other C. elegans behaviors have been shown to be significantly more sensitive than mobility to the volatile anesthetic halothane." !Affiliation "[3] An inter-behavioral Quantitative Trait Loci (QTL) analysis of anesthetic response was initiated by generating dose-response curves for 40 RIs for chemotaxis and mobility. The RIs were polymorphic for both behaviors in halothane: EC50 values for chemotaxis derived from dose-response curves for each RI strain ranged from 0.08 to 1.82 vol % halothane, while the N2 strain had an EC50 of 0.49=B10.064. About one-fourth of the RIs were significantly more sensitive than N2, and a few were more resistant to halothane's effect on chemotaxis. The same RIs were assayed for immobility, and the EC50 range was found to be 3.0 to 5.6 vol % halothane; the EC50 for N2 is 3.58=B10.069. Surprisingly, most RIs tested for immobility were found to be more resistant than N2. Further, patterns of sensitivity and resistance to halothane in the various RIs did not necessarily correlate between the immobility and chemotaxis assays, suggesting that different QTLs may be involved in the different behavioral effects of anesthetics. Marker correlations with EC50s suggest inter-behavioral QTL overlaps on chromosomes II and III, but a mobility-specific QTL on chromosome V. Synthetic gene interactions between N2 and BO alleles appear to be an important factor in creating phenotypic variability in the RIs. 1 Behav. Gen.16(#1): 221-235 (1986). 2 Genetics 135: 1003-1010 (1993). 3 WBG 13(#3): 76 (1994).

Besseling, J. et al. (2015) International Worm Meeting "Mechanisms for sleep neuron specification and sleep induction."

Turek, M., Bringmann, H., Besseling, J., Spies, J.

[International Worm Meeting, 2015]

Sleep is induced by sleep-active neurons that are conserved from mammals to C. elegans. These neurons express GABA and neuropeptides, are active at the onset of sleep and actively promote it. However, little is known about how sleep neuron cell fate is determined and how sleep neurons induce this behavior.In C. elegans, sleep-like behavior occurs during lethargus, preceding the molt (Cassada and Russell 1975, Raizen et al. 2008). It depends on the sleep-active sleep-promoting neuron RIS, which requires the AP2 transcription factor APTF-1 to induce sleep (Turek et al. 2013). Thus, APTF-1 provides a unique starting point to understand sleep neuron specification and sleep induction.We combined transcriptome analysis of aptf-1 mutants with analysis of RIS-expressed genes to find out what determines expression of APTF-1 and how APTF-1 causes RIS to become sleep promoting. We found that the lim box homeodomain transcription factor LIM-6, which broadly specifies GABAergic neuron fate (Hobert et al. 1999), determined the expression of APTF-1 in RIS. Transcriptome analysis of aptf-1 mutants showed that APTF-1 is required for the expression of an FMRFamide-like neuropeptide, FLP-11, in RIS. Deletion of FLP-11 caused substantially reduced sleep behavior during lethargus, an effect that was increased by knockout of GABA. Overexpression of FLP-11 caused ectopic sleep behavior, confirming that this peptide is somnogenic. FLP-11 has been shown to activate three receptors, NPR-22, NPR-4, and FRPR-3 (Peyman et al. 2014). All these receptors appeared to contribute to the induction of sleep behavior downstream of FLP-11 release by RIS.Thus, we present a mechanism of how RIS becomes sleep inducing: LIM-6 causes APTF-1 expression, which in turn causes expression of the somnogenic peptide FLP-11. At the onset of sleep, RIS is strongly activated and releases both GABA and FLP-11, which activates multiple receptors to globally induce sleep. Like mammalian sleep-active neurons, RIS uses both peptidergic and GABAergic signaling to induce sleep, which further supports the view of a common evolutionary origin of sleep neurons.

Gottschalk, A. et al. (2019) International Worm Meeting "Optogenetic interrogation of a neuronal circuit orchestrating locomotion 'stop' using fast volumetric scanning and dual Ca2+ imaging."

Gottschalk, A., Aghigh, Arash, Steuer Costa, Wagner, Bach, Maximilian, Van der Auwera, Petrus

[International Worm Meeting, 2019]

Identifying specific neurons within a neuronal circuit and linking their activity to a certain behavior remains one of the most challenging tasks in the field of neurobiology. Using an optimized system for fast automated x-,y-tracking of a fluorescent marker in freely moving C. elegans, we found that the GABAergic interneuron RIS, which is known to promote sleep during larval development, is reutilized in adulthood for orchestrating locomotion 'stop'. We could show that RIS exhibits compartmentalized calcium dynamics by performing axonal calcium imaging. By further expanding our setup using a Piezo scanner for simultaneous volumetric scanning we are able to analyze the neuronal network of RIS by recording calcium activity from two distinct neurons within the network at the same time. This is achieved by expressing the genetically encoded calcium indicators (GECIs) GCaMP6 and jRCaMP1b cell specifically in RIS and the neurons upstream (i.e. CEPs) and downstream (i.e. RIB & RIM) of RIS according to its connectome. The observed, possibly linked activities are verified by optogenetic stimulation or inhibition of RIS or the CEPs using microbial rhodopsins, and assessing the correlation of calcium activity in the downstream neuron. The scanner in our system requires less than 200 ms per volume, moving in a triangular scanning pattern, and enabling to account for fast calcium dynamics. The data analysis is based on open source and easy-to-use image analysis software. Image registration is done with ImageJ software using simple stack-slice manipulations and the Scale Invariant Feature Transform (SIFT). Subsequently all the data from the calcium and behavioral imaging is evaluated using customized MATLAB and Mathematica scripts. Measuring spontaneous or evoked calcium activity in one or two distinct neurons at high spatiotemporal resolution in soma, ventral nerve cord and nerve ring processes and extracting behavioral parameters like bending angles, body length and speed will enable deeper insights into the architecture and functional interplay of cells of the described neuronal network.

Busack, I. et al. (2019) International Worm Meeting "A neural circuit controls sleep-active neuron depolarization to switch sleep states."

Besseling, J., Turek, M., Bringmann, H., Maluck, E., Busack, I., Masurat, F.

[International Worm Meeting, 2019]

Sleep-active neurons depolarize at sleep onset to inhibit wakefulness circuits. Wake-active neurons in turn inhibit sleep-active neurons, thus forming a highly conserved flip-flop sleep switch. However, presynaptic neural circuits that directly depolarize sleep-active neurons to switch sleep states have not been identified. Here we used optogenetics and functional imaging in C. elegansto identify and understand the circuit that depolarizes the single sleep-active RIS neuron during developmental sleep. We found the key RIS activators. Depolarization of RIS in turn promotes the activation of these neurons, thus forming a positive feedback loop. In addition to that we found sleep-active neuron depolarization to be mediated by wake-active sleep-promoting interneurons, which can be suppressed by wake-promoting arousal interneurons. This tripartite flip-flop switch circuit thus acts as an amplifier that translates reduced arousal into sleep bouts. A similar circuit mechanism may also underlie sleep control in other animals including mammals.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.