Behavioral control by depolarized and hyperpolarized states of an integrating neuron

  1. Aylesse Sordillo
  2. Cornelia I Bargmann

  • Curated by eLife

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    Evaluation Summary:

    The interneuron RIM affects many behaviours in C. elegans. Attempts to understand or manipulate its function have sometimes led to conflicting and difficult to interpret results. Sordillo and Bargmann investigate the role of the RIM in locomotion by manipulating it's signaling properties in multiple ways. The strength of this approach is that targeting multiple biological signaling mechanisms, they are able to conduct a nuanced analysis of RIM's signaling functions that goes beyond simplistic ON/OFF distinctions. They reach two primary conclusions: 1. RIM depolarization extends reversals via synaptic (glutamatergic) and secretory (tyraminergic) signaling; 2. RIM hyperpolarization promotes forward locomotion via electrical signaling through gap junctions. As a result, RIM was shown to act for stabilizing both forward and backward movement, which is important for understanding of C. elegans in general. Also, the implication that interneurons can be multifunctional in this way is intriguing and potentially impactful.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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Abstract

Coordinated transitions between mutually exclusive motor states are central to behavioral decisions. During locomotion, the nematode Caenorhabditis elegans spontaneously cycles between forward runs, reversals, and turns with complex but predictable dynamics. Here, we provide insight into these dynamics by demonstrating how RIM interneurons, which are active during reversals, act in two modes to stabilize both forward runs and reversals. By systematically quantifying the roles of RIM outputs during spontaneous behavior, we show that RIM lengthens reversals when depolarized through glutamate and tyramine neurotransmitters and lengthens forward runs when hyperpolarized through its gap junctions. RIM is not merely silent upon hyperpolarization: RIM gap junctions actively reinforce a hyperpolarized state of the reversal circuit. Additionally, the combined outputs of chemical synapses and gap junctions from RIM regulate forward-to-reversal transitions. Our results indicate that multiple classes of RIM synapses create behavioral inertia during spontaneous locomotion.

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  1. Version published to 10.7554/elife.67723 on eLife
  2. eLife

    Author Response:

    Reviewer #3:

    Sordillo and Bargmann report a detailed study of mechanisms by which RIM interneurons control foraging by C. elegans. A comparison of the effects of knocking out the vesicular glutamate transporter in RIMs to the effects of knocking out synthesis of the monoamine transmitter tyramine leads the authors to conclude that glutamate/monoamine cotransmission is required for RIM function. The authors further find that acute perturbation of RIMs by chemogenetics has a surprising effect. Manipulation of RIMs with HisCl - a histamine-gated ion channel that permits rapid and reversible inhibition of target cells - had the opposite effect of RIM ablation or mutations that affect RIM neurotransmission. The effects of HisCl-mediated perturbation require gap junctions, leading the authors to conclude that gap-junction connectivity between RIMs and their targets promotes specific foraging behaviors while neurochemical signaling from RIMs to their targets promotes antagonistic behaviors.

    This study has several strengths. Sophisticated genetic tools are developed to perturb RIMs. Measurements of RIM-dependent foraging behaviors are made using high-resolution video tracking systems, and these rich datasets are clearly presented and rigorously analyzed. The manuscript is clearly written and beautifully illustrated. And the overarching hypothesis that RIM interneurons support distinct behavioral programs when depolarized and hyperpolarized is provocative and significant. The study also has weaknesses, some of which significantly impact the strength of the authors' conclusions. These weaknesses are listed below.

    1. One major conclusion is that RIMs use both glutamate and tyramine as co-transmitters. This conclusion is based on the observed effects of VGLUT knockout in RIMs. It is known, however, that VGLUT facilitates the loading of monoamine neurotransmitters into vesicles, raising the possibility that the observed effects of VGLUT knockdown are via effects on tyraminergic signaling. The authors discuss this point and argue that an observed difference between the effects of tdc-1 mutation and RIM-specific VGLUT mutation indicates separable functions of glutamate and tyramine, i.e. co-transmission. However, most of the data are also consistent with a model in which VGLUT facilitates VMAT function. This point is critical for one of the study's main conclusions and should be resolved. For example, a clear role for a known glutamate receptor in RIM-mediated behavior would support the authors' conclusions.

    We have not examined receptor mutants; there are many excitatory and inhibitory glutamate receptors in this circuit, and each is expressed in multiple neurons, so we believe the only rigorous approach will be single-cell receptor knockouts like those presented here, possibly combined with single-cell eat-4 knockouts. With that said, Li et al. (2020) have identified a glutamate receptor subunit, avr-14, that (1) acts within command neurons and motor neurons to affect spontaneous reversals and (2) shows a genetic interaction with RNAi knockdown of eat-4 in RIM. These results support the suggestion that RIM uses glutamate as a transmitter. We now cite that result on page 14.

    1. The surprising observation that HisCl-silencing of RIMs causes the opposite effect as ablation of RIMs or mutation of the monoamine biosynthetic pathway is the basis for the other major conclusion of the study. The authors conclude that this difference reflects a signaling function for hyperpolarized RIMs that is eliminated by ablation. This difference might also reflect differences between chronic and acute perturbations. Methods exist to chronically silence neurons by expressing hyperpolarizing conductances, and the authors' model suggests that these manipulations would cause effects similar to those caused by acute inhibition via HisCl.

    We acknowledge this point. We added new data to Figure 5 to address it, showing that we saw similar behavioral results upon acute or chronic (48 hours) silencing of RIM.

    1. HisCl silencing of RIMs was performed using a tdc-1::HisCl transgene, which supports expression in RIMs and RICs. The authors should be certain that RICs have no role in the effects they see using this transgene. Similarly, perturbation of RIM gap junctions uses a tdc-1-based transgene, which should be paired with a control that allows the authors to rule out any contribution of RICs.

    The key difference between RIC and RIM(+RIC) chemical synapses is the large effect of RIM on reversal frequencies and length. To ask if that distinction applies to gap junctions, we have now expressed the unc-1(dn) transgene in RIC. This transgene did not affect reversal frequencies and length, unlike unc-1(dn) in RIM(+RIC). It did cause a decrease in reversal speed, forward run speed, and forward run length, results matching those of RIC chemical synapses. These results have been added as Figure 6- figure supplement 3.

    1. The authors' final model proposes that the constellation of chemical and electrical synapses endows RIMs with a kind of 'inertia.' In the absence of any data that report how perturbation of RIMs affects dynamics of the foraging circuit (AIB/RIB/AVA/AVB) is it difficult to assess this model.

    We added calcium imaging data demonstrating that silencing RIM reduces AVA activity as the new Figure 5 – figure supplement 3.

    Minor comments:

    The authors use variants of 'RIM glutamate KO' when referring to to the strain carrying RIM-targeted allele of eat-4/vglut. They should be consistent.

    We have addressed this point.

    A critical set of control experiments for the eat-4 conditional allele is presented in Figure 2S1 but not mentioned in the manuscript until data from Figure 3 are being discussed. These controls should be clearly described earlier in the results section - they are beautiful experiments and establish confidence in the method.

    Thank you -- we added that information to the text on page 5, as it is also relevant to some of the questions about variability between assays addressed in Essential Revisions point 3.

    Line 192 refers to 'a synaptobrevin-dependent transmitter.' This is correct, but it might be more clear to simply say 'another neurotransmitter.'

    Changed.

  3. eLife

    Evaluation Summary:

    The interneuron RIM affects many behaviours in C. elegans. Attempts to understand or manipulate its function have sometimes led to conflicting and difficult to interpret results. Sordillo and Bargmann investigate the role of the RIM in locomotion by manipulating it's signaling properties in multiple ways. The strength of this approach is that targeting multiple biological signaling mechanisms, they are able to conduct a nuanced analysis of RIM's signaling functions that goes beyond simplistic ON/OFF distinctions. They reach two primary conclusions: 1. RIM depolarization extends reversals via synaptic (glutamatergic) and secretory (tyraminergic) signaling; 2. RIM hyperpolarization promotes forward locomotion via electrical signaling through gap junctions. As a result, RIM was shown to act for stabilizing both forward and backward movement, which is important for understanding of C. elegans in general. Also, the implication that interneurons can be multifunctional in this way is intriguing and potentially impactful.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this work authors focused on RIM neurons which are part of the locomotion circuit of C. elegans but whose role was enigmatic. They put particular focus on the fact that RIM has multiple ways to communicate to other neurons, through glutamate, tyramine gap junctions as well as neuropeptides.

    It is praised that they made precise cell-specific manipulation of each of these transmission pathways and clarified the different roles of the transmission by combining with detailed quantification of behavior.

    There are several interesting observations: lack of AIM glutamate and/or tyramine both caused increased reversal initiation rate. This is similar to the effect of RIM ablation previously reported. Lack of glutamate and/or tyramine also shortened reversal duration. However, of all reversals, lack of tyramine caused decreased rate of omega, while increased pure reversals occurred instead. It is a good contrast to the previously reported role of tyramine in suppressing head swing during reversal.

    Most interestingly, hyperpolarization of RIM by the HisCl channel caused reduced rate of reversal initiation and extended forward run duration. This was opposite to the effect of removal of RIM, and suggested that RIM hyperpolarization has some active effect. This was proposed to be caused through gap junctions. Authors tried to inhibit gap junctions by cell-specific expression of unc-1(n494), which by itself caused increased rate of reversal initiation and shortened forward runs. This effect was antagonistic to effect of unc-1(n494).

    A previous paper from the same lab (Gordus et al. 2015) showed that RIM neurons are activated (or inactivated) along with AVA and AIB in response to odor stimulus and this was assumed to be mediated by gap junctions between these neurons and correspond to the reversal behavior. On the other hand, an opposite activation pattern was sometimes observed for RIM (for example Piggott et al. 2011). The current manuscript solves the confusion to some extent.

    It is important to note that the current manuscript focuses on spontaneous activity changes during "local search" and "global search" (especially the former) in unstimulated animals, because the role of RIM is likely different between evoked and spontaneous behaviors. Under these settings, it looks like glutamate and tyramine mainly acts for prolonging reversal behavior, while gap junction looks to have a major role in prolonging forward motion. Overall, RIM seems to have a role for "behavioral inertia" as proposed by the authors.

    This is a very informative work and the idea of positive and negative regulation causing "behavioral inertia" is novel and interesting.

  5. eLife

    Reviewer #2 (Public Review):

    The interneuron RIM affects many behaviours. Attempts to understand or manipulate its function have sometimes led to conflicting and difficult to interpret results. Sordillo and Bargmann investigate the role of the RIM interneuron in locomotion by manipulating it in multiple ways: chemogenetic hyperpolarization, optogenetic depolarization, and genetic disruption of glutamate, tyramine, syanptobrevin-dependent, or electrical synaptic signaling. They reach two primary conclusions: 1. RIM depolarization extends reversals via synaptic (glutamatergic) and secretory (tyraminergic) signaling; 2. RIM hyperpolarization promotes forward locomotion via gap junctions.

    These conclusions of the study are well supported by the experiments. Overall, it is of interest to the subfield because it resolves some conflicting interpretations of the role of RIM in regulating locomotion. Of broader interest, it illustrates an interesting case of a multifunctional neuron involved in stabilizing behavioral states, the complexity of the interplay between electrical and chemical signaling, insight into network/circuit functions of electrical synapses, and the caution needed in interpreting results from various kinds of neural manipulation.

  6. eLife

    Reviewer #3 (Public Review):

    Sordillo and Bargmann report a detailed study of mechanisms by which RIM interneurons control foraging by C. elegans. A comparison of the effects of knocking out the vesicular glutamate transporter in RIMs to the effects of knocking out synthesis of the monoamine transmitter tyramine leads the authors to conclude that glutamate/monoamine cotransmission is required for RIM function. The authors further find that acute perturbation of RIMs by chemogenetics has a surprising effect. Manipulation of RIMs with HisCl - a histamine-gated ion channel that permits rapid and reversible inhibition of target cells - had the opposite effect of RIM ablation or mutations that affect RIM neurotransmission. The effects of HisCl-mediated perturbation require gap junctions, leading the authors to conclude that gap-junction connectivity between RIMs and their targets promotes specific foraging behaviors while neurochemical signaling from RIMs to their targets promotes antagonistic behaviors.

    This study has several strengths. Sophisticated genetic tools are developed to perturb RIMs. Measurements of RIM-dependent foraging behaviors are made using high-resolution video tracking systems, and these rich datasets are clearly presented and rigorously analyzed. The manuscript is clearly written and beautifully illustrated. And the overarching hypothesis that RIM interneurons support distinct behavioral programs when depolarized and hyperpolarized is provocative and significant. The study also has weaknesses, some of which significantly impact the strength of the authors' conclusions. These weaknesses are listed below.

    1. One major conclusion is that RIMs use both glutamate and tyramine as co-transmitters. This conclusion is based on the observed effects of VGLUT knockout in RIMs. It is known, however, that VGLUT facilitates the loading of monoamine neurotransmitters into vesicles, raising the possibility that the observed effects of VGLUT knockdown are via effects on tyraminergic signaling. The authors discuss this point and argue that an observed difference between the effects of tdc-1 mutation and RIM-specific VGLUT mutation indicates separable functions of glutamate and tyramine, i.e. co-transmission. However, most of the data are also consistent with a model in which VGLUT facilitates VMAT function. This point is critical for one of the study's main conclusions and should be resolved. For example, a clear role for a known glutamate receptor in RIM-mediated behavior would support the authors' conclusion.s

    2. The surprising observation that HisCl-silencing of RIMs causes the opposite effect as ablation of RIMs or mutation of the monoamine biosynthetic pathway is the basis for the other major conclusion of the study. The authors conclude that this difference reflects a signaling function for hyperpolarized RIMs that is eliminated by ablation. This difference might also reflect differences between chronic and acute perturbations. Methods exist to chronically silence neurons by expressing hyperpolarizing conductances, and the authors' model suggests that these manipulations would cause effects similar to those caused by acute inhibition via HisCl.

    3. HisCl silencing of RIMs was performed using a tdc-1::HisCl transgene, which supports expression in RIMs and RICs. The authors should be certain that RICs have no role in the effects they see using this transgene. Similarly, perturbation of RIM gap junctions uses a tdc-1-based transgene, which should be paired with a control that allows the authors to rule out any contribution of RICs.

    4. The authors' final model proposes that the constellation of chemical and electrical synapses endows RIMs with a kind of 'inertia.' In the absence of any data that report how perturbation of RIMs affects dynamics of the foraging circuit (AIB/RIB/AVA/AVB) is it difficult to assess this model.

    Minor comments:

    The authors use variants of 'RIM glutamate KO' when referring to to the strain carrying RIM-targeted allele of eat-4/vglut. They should be consistent.

    A critical set of control experiments for the eat-4 conditional allele is presented in Figure 2S1 but not mentioned in the manuscript until data from Figure 3 are being discussed. These controls should be clearly described earlier in the results section - they are beautiful experiments and establish confidence in the method.

    Line 192 refers to 'a synaptobrevin-dependent transmitter.' This is correct, but it might be more clear to simply say 'another neurotransmitter.'

  7. Version published to 10.1101/2021.02.19.431690v1 on bioRxiv