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Co-transmission of neuropeptides and monoamines choreograph the C. elegans escape response

['Jeremy T. Florman', 'Department Of Neurobiology', 'Umass Chan Medical School', 'Worcester', 'Massachusetts', 'United States Of America', 'Mark J. Alkema']

Date: 2022-05

Co-localization and co-transmission of neurotransmitters and neuropeptides is a core property of neural signaling across species. While co-transmission can increase the flexibility of cellular communication, understanding the functional impact on neural dynamics and behavior remains a major challenge. Here we examine the role of neuropeptide/monoamine co-transmission in the orchestration of the C. elegans escape response. The tyraminergic RIM neurons, which coordinate distinct motor programs of the escape response, also co-express the neuropeptide encoding gene flp-18. We find that in response to a mechanical stimulus, flp-18 mutants have defects in locomotory arousal and head bending that facilitate the omega turn. We show that the induction of the escape response leads to the release of FLP-18 neuropeptides. FLP-18 modulates the escape response through the activation of the G-protein coupled receptor NPR-5. FLP-18 increases intracellular calcium levels in neck and body wall muscles to promote body bending. Our results show that FLP-18 and tyramine act in different tissues in both a complementary and antagonistic manner to control distinct motor programs during different phases of the C. elegans flight response. Our study reveals basic principles by which co-transmission of monoamines and neuropeptides orchestrate in arousal and behavior in response to stress.

Co-transmission is a form of neuronal communication where multiple signaling molecules are released from an individual cell. It is commonly found in nervous systems across species that monoamines and neuropeptides are co-released and affect the properties of target cells. In humans, adrenaline and neuropeptide Y are co-released from sympathetic neurons where they increase blood pressure and heart rate during the fight-or-flight response. The complexity of the human nervous system makes it very hard to figure out how these signaling molecules interact to change behavior and physiology. Here, we use the nematode C. elegans to study how co-transmission of the invertebrate analog of adrenaline, tyramine, and a neuropeptide, FLP-18, coordinate the distinct motor programs of the flight response. We find that FLP-18 and tyramine act together to shape different phases of the flight response. Our findings illuminate the cellular mechanisms by which co-transmission of monoamines and neuropeptides orchestrate a flight response.

Funding: This work was supported by NIH grant GM140480 and NS107475 from the National Institutes of Health (MJA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Florman, Alkema. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here we investigate the role of FLP-18 in the orchestration of the C. elegans escape response. We find that FLP-18 plays a central role in coordinating distinct phases of the escape response in C. elegans. FLP-18 is co-released with tyramine from the RIM neurons in response to mechanical stimuli. FLP-18 activates the GPCR NPR-5 in body wall muscle to enhance turning behavior and locomotion speed during the escape response by increasing muscle excitability. Our result show that FLP-18 and tyramine act in different tissues in both a complementary and antagonistic manner to orchestrate distinct motor programs during different phases of the C. elegans flight response.

In response to a mechanical stimulus C. elegans can engage in a flight- or an escape-response, where the worm quickly reverses while suppressing oscillatory head movements. The reversal is followed by a deep ventral bend of the head, and a subsequent slide of the head along the ventral side of the body (omega turn). After the omega turn, the animal moves forward in the opposite direction, away from the noxious stimulus. Tyramine plays a crucial role in the coordination of independent motor programs, which increases the animal’s chances to escape from predatory fungi that use hyphal nooses to entrap nematodes [ 26 – 29 ]. The escape response triggers the release of tyramine from a single pair of neurons called the RIM. Tyramine release activates the inhibitory tyramine-gated chloride channel, LGC-55, in neck muscles, cholinergic head motor neurons and the AVB pre-motor interneuron. LGC-55 activation induces the suppression of oscillatory head movements during long reversals in response to anterior touch [ 26 , 28 , 30 , 31 ]. In addition, tyramine release facilitates ventral bending during the omega turn through the activation of the SER-2 GPCR in GABAergic motor-neurons [ 29 ]. The tyraminergic RIM neurons co-express a NPY like peptide, FLP-18 [ 32 , 33 ]. The RFamide FLP-18 and its associated receptors are related to the NPY/NPYR signaling system [ 34 ]. In vitro experiments have shown that FLP-18 can activate a human NPYR and, conversely, human NPY can activate worm neuropeptide receptors [ 35 ]. In C. elegans, FLP-18 has been shown to play a role in foraging and metabolism [ 36 ], arousal and homeostasis [ 37 – 39 ], chemosensation [ 40 ], reversal behavior [ 41 , 42 ], and swimming [ 33 , 43 ]. FLP-18 acts through several neuropeptide receptors including NPR-1, NPR-4, and NPR-5 [ 32 , 36 , 44 ].

The nematode Caenorhabditis elegans provides an excellent system to study the co-transmission of aminergic and peptidergic neuromodulators due to its compact and completely defined nervous system and wealth of genetic tools [ 20 , 21 ]. The C. elegans genome encodes a large family of NPY related peptides and G-protein coupled receptors (GPCRs) [ 22 , 23 ]. Like other invertebrates, C. elegans lacks NA, however the structurally related tyramine fulfills a similar role to NA in coordinating stress responses and flight behavior [ 24 – 26 ].

Co-localization of classical neurotransmitters and neuropeptides is a common feature of the animal nervous system [ 1 ]. Co-transmission is thought to provide flexibility to the output of hard-wired neural circuits [ 2 ]. In mammals, for instance, the acute fight-or-flight response leads to the activation of the sympathetic nervous system (SNS) and the co-release of different “stress hormones” [ 3 ]. Adrenalin and noradrenaline release in the SNS trigger an increase in heart rate, blood flow, respiration and release of glucose from energy stores, which prepare the animal for vigorous muscle activity and physical exertion [ 4 , 5 ]. Neuropeptide Y (NPY), one of the most abundant neuropeptides in the mammalian nervous system, is a co-transmitter with noradrenaline (NA) in many neurons of the SNS [ 6 – 8 ]. The sympathetic co-transmission of NA and NPY suggest that they may coordinate aspects of the flight response. However, the physiological and behavioral impact of co-transmission can be difficult to dissect. Co-transmitted signaling molecules can activate receptors on a common target (convergence) or different targets (divergence) which induce synergistic or opposing effects, making the functional outcome of co-transmission challenging to anticipate. Studies in mammals have shown that the stress related modulatory functions of NPY and NA co-transmission are complex, with complementary actions in some tissues and antagonistic actions in others. For example, both NA and NPY increase blood pressure through peripheral vasoconstriction, however NPY inhibits presynaptic NA release from sympathetic neurons and opposes the action of NA on cardiac contraction [ 9 – 19 ]. Co-transmission of NPY and catecholamines may induce a longer lasting state of arousal that enhances alertness and the ability to deal with environmental threats. Unraveling the precise effects of co-transmission of neural stress hormones is very challenging in vertebrates given the complexity of the nervous system, the multiple central and peripheral release sites and the diversity of target tissues expressing NA and NPY receptor (NPYR) subtypes.

Results

Mechanical stimulation transiently increases forward velocity Handling of C. elegans, such as the transfer with a platinum wire, bumping the plate on microscope stage or removing the lid of a plate can induce an increase in locomotion rate that persists for several minutes [45,46]. This indicates that mechanical stimulation can lead to longer lasting changes to the internal state of the animal. To quantify locomotion patterns upon mechanical stimulation we analyzed the locomotion rate of animals subjected to a tap to the side of the plate. Mechanical tap can trigger an escape response, in which C. elegans reverses, turns and resumes forward locomotion in the opposite direction [47,48]. We compared behavior of animals before, during and after spontaneous reversals and tap-induced reversals. In animals that initiated a spontaneous reversal, forward locomotion rate remained the same before and after the reversal (Fig 1A). The absolute velocity during spontaneous reversals was similar to the velocity of forward movement. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Increase in locomotion speed following a mechanical stimulus. Backward- and forward-velocity upon a spontaneous or tap-induced reversal. Wild-type animals show a persistent increase in locomotion speed in response to a strong tap stimulus (“forward run”). flp-18 mutants do not show a robust increase locomotion speed in response to a strong tap stimulus. (A-B) Schematic illustrating behavioral sequence (top) and velocity traces (bottom) from wild type animals, flp-18, and tdc-1 mutants, aligned to reversal events. Negative velocity indicates backward locomotion. Dark lines represent mean velocity and shaded region represents standard error. Black lines at the top of the graph indicate the time periods when reversal and forward run speed were quantified. Reversals were spontaneous (A) or induced by a tap stimulus (B). (C) Peak backward velocity during the reversal phase was quantified by identifying the maximum backward locomotion rate for each animal during a 1 second period following the tap. (D) Forward run velocity was quantified as the average speed for each animal during the time period between 7 to 10 seconds following the tap. (E) Quantification of the percentage of animals initiating a reversal within 1 second after a tap was delivered. Graphs represent mean ± SEM, significance was calculated using ANOVA with Šidák’s (C and D) and Dunnett’s (E) multiple comparison test (P> 0.05 = ns, P<0.005 = **, P<0.0001 = ****). Sample sizes: Spontaneous reversals and escape response (A-D), (n = # animals). Spontaneous (A, C, and D): wild type (n = 506), flp-18 (n = 512), tdc-1 (n = 512). Escape (b-d): wild type (n = 275), flp-18 (n = 208), tdc-1 (n = 124). % Reversing (E), (n = # of experiments, 20 worms per experiment): wild type (n = 30), flp-18 (n = 23), tdc-1 (n = 11). https://doi.org/10.1371/journal.pgen.1010091.g001 When an escape response was triggered with a tap stimulus, animals initiated a rapid reversal; double the speed of spontaneous reversals (Fig 1B and 1C). Furthermore, following a tap-induced reversal, wild-type animals exhibited a markedly elevated forward locomotion rate for approximately 3 minutes (S1A Fig);—a behavior we refer to as the ‘forward run’ (Fig 1B and 1D). The sustained increase in forward velocity in response to a tap indicated longer lasting changes in the internal state of the animal.

flp-18 mutants have defects in locomotory arousal Neuropeptides are potent neuromodulators, whose action can lead to longer lasting changes in behavior. We focused our attention on FLP-18 neuropeptides, since the flp-18 gene is known to be expressed in the tyraminergic RIM neurons and the AVA pre-motor interneurons [32,33]. The RIM and AVA play a central role in the escape response to mechanical touch [25,26,47,49]. flp-18 mutants and tyramine deficient tdc-1 mutants initiated reversals in response to tap stimuli similar to wild type (Fig 1E). This indicates that FLP-18 peptides and tyramine are not required for mechanosensation or the initiation of the escape response. In response to a tap stimulus, flp-18 mutants reversal velocity was slightly reduced compared to wild type and tdc-1 mutant animals (Fig 1B and 1C). Both flp-18 and tdc-1 mutants exhibited shorter reversal duration compared to wild type (Fig 1B). tdc-1 mutants had a reduced basal velocity, but following a tap, increased velocity during the forward run, similar to the wild type. In contrast, flp-18 mutants had significantly slower forward run velocity compared to the wild type (Fig 1B and 1D). tdc-1; flp-18 double mutants behaved similar to tdc-1 single mutants during the initial phase of the escape response and like flp-18 single mutants failed to maintain forward velocity at later stages of the forward run (S1B Fig).

The escape response induces FLP-18 release from the AVA and RIM flp-18 is expressed in the AVA and RIM neurons [32,33], which are activated during the escape response [25,49,51]. To analyze FLP-18 release, we generated transgenic animals with a fluorescently tagged the FLP-18 pro-peptide with the YFP variant Venus (Pflp-18::FLP-18::Venus). Venus is resistant to quenching in low pH environments and has been used to monitor neuropeptide secretion from dense core vesicles in C. elegans [52–54]. FLP-18::Venus fluorescence was observed in the RIM and AVA escape circuit neurons, as well as the AIY and RIG (Fig 4B). FLP-18::Venus fluorescence was also observed in the coelomocytes, in contrast to a Pflp-18::GFP transcriptional reporter which did not produce detectable coelomocyte fluorescence [32]. This is consistent with the idea that FLP-18::Venus protein is secreted and taken up by coelomocytes, which are endocytic scavenger cells that internalize material from the pseudocoelomic fluid [55]. We measured changes in fluorescent intensity in the AVA and RIM neurons after repeated activation of the escape response (Fig 4A and 4C). Mechanical tap to the plate caused a progressive reduction in FLP-18::Venus fluorescent intensity in the AVA and RIM neurons compared to animals that did not receive the tap stimuli (Fig 4D and 4E), suggesting that activation of the escape response elicits the release of FLP-18 from the AVA and RIM. Tap treatment did not change the fluorescent intensity in coelomocytes (Fig 4F). This could be due to protein break down in the coelomocytes and/or the high basal levels of coelomocyte fluorescence due to tonic FLP-18 release from other neurons. PPT PowerPoint slide

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TIFF original image Download: Fig 4. The escape response stimulates FLP-18 release from the AVA and RIM. Animals expressing a FLP-18::Venus fusion protein were subjected to repeated mechanical plate taps. The decrease in fluorescent intensity of the RIM and AVA cell bodies and the anterior-most coelomocyte were quantified as a readout of FLP-18 release. (A) Schematic illustrating the processing and exocytosis of the FLP-18::Venus fusion protein. (B) Representative image showing an animal expressing FLP-18::Venus, DIC and GFP overlay. Scale bar represents 10 μm. (C) Average velocity of a population of animals as they are subjected to mechanical plate taps. Green arrows indicate the delivery of a tap every 2 min. The spikes in velocity coincide with the initiation of the escape response (D-E) Quantification of fluorescence (arbitrary units–a.u.) in the AVA (D), RIM (E), and coelomocytes (F), at 0-, 1- and 2-hour time points. Graphs represent mean ± SEM, significance was calculated using ANOVA with Dunnett’s multiple comparison test (P<0.005 = **, P<0.0005 = ***, P<0.0001 = ****). Sample size: (C) n = 3 replicates, >30 animals per replicate. (D-F) Control (n = 32), Tap 1 hr. (n = 26), Tap 2 hr. (n = 35). https://doi.org/10.1371/journal.pgen.1010091.g004

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