An inhibitory acetylcholine receptor gates context dependent mechanosensory processing in C. elegans

SUMMARY

An animal’s current behavior influences its response to sensory stimuli, but the molecular and circuit-level mechanisms of this context-dependent decision-making is not well understood. In the nematode C. elegans, inhibitory feedback from turning associated neurons alter downstream mechanosensory processing to gate the animal’s response to stimuli depending on whether the animal is turning or moving forward [1–3]. Until now, the specific neurons and receptors that mediate this inhibitory feedback were not known. We use genetic manipulations, single-cell rescue experiments and high-throughput closed-loop optogenetic perturbations during behavior to reveal the specific neuron and receptor responsible for receiving inhibition and altering sensorimotor processing. An inhibitory acetylcholine gated chloride channel comprised of lgc-47 and acc-1 expressed in neuron RIM receives inhibitory signals from turning neurons and performs the gating that disrupts the worm’s mechanosensory evoked reversal response.

The nematode C. elegans’s response to mechanosensory stimuli is influenced by its current behavior: it is less likely to reverse in response to a stimulus that it receives when in the midst of a turn compared to a stimulus that it receives when moving forward Fig 1A [2]. Because turns are fast, typically lasting approximately two seconds, we hypothesize that synaptic signaling via neurons may mediate this context dependency. Understanding the molecular and circuit mechanism of this sensorimotor processing will provide insights into how a simple nervous system implements context-dependent decision-making.

Figure 1. LGC-47 and ACC-1 in neuron RIM gate turning-dependent reversals.

A) Optogenetic stimulation is delivered to a worm expressing Chrimson in the six gentle touch neurons during forward or turning locomotion. B) Anatomical wiring diagram of touch receptor neurons, turning-associated neurons and reversal-associated interneurons (adapted from Nemanode [11]). C) Probability of reversal in response to optogenetic stimulation delivered during forward movement or turning onset is shown for different genetic manipulations, including loss-of-function of lgc-47 or acc-1, and their corresponding cell-specific rescues. All animals express Chrimson under mec-4 promoter including “WT.” N > 400 stim events for each condition (Fig S1). D) Reversal response for acc-1 loss-of-function mutants and corresponding cell-specific rescue. Here a different mec-4 Chrimson allele is used, denoted by ^QW. N > 320 stimulation events per condition (Fig S1). *** indicates p < 0.001, ‘n.s.’ indicates p > 0.05 via two proportion Z-test.

The gentle touch mechanosensory circuit in C. elegans has been extensively studied [4] and its downstream interneurons are known Fig. 1B. When the worm turns, inhibitory feedback from turning neurons is thought to disrupt downstream processing of mechanosensory signals by inhibiting some of these interneurons [3]. In particular, inhibitory feedback from a collection of turning-associated neurons SMB, SAA and RIV [5], decrease the likelihood of reversing during a turn [3], but the exact location and mechanism with which these turning signals interact with downstream mechanosensory processing is unknown. Previously we performed optogenetic activation studies showing that inhibitory signals from turning neurons arrive at or upstream of reversal interneuron AVA, because only activation of those neurons, and not AVA evoked reversals in a turning-dependent manner [3]. Here we seek to identify the neurons and receptors that receive inhibition from the turning circuit.

We investigated the outgoing synapses of SAA because that neuron type is known to be involved in turning [3, 5, 6] and it makes synaptic contacts onto reversal neurons including AVA, RIM and AIB [7, 8], Fig 1B. We specifically investigated the role of inhibitory acetylcholine receptors expressed at these synapses because SAA releases acetylcholine [9–11] and we expect it to send an inhibitory signal [3, 5]. We focused on lgc-47 and acc-1, two genes for inhibitory acetylcholine receptors, with known expression in neurons RIM, AIB, and AVA among others [10, 11]. We investigated LGC-47 first because it expresses at higher levels than ACC-1, Supplementary Figure S1A [11]. We therefore measured the response to mechanosensory stimuli in lgc-47 loss-of-function mutants.

We expressed the light-gated ion channel Chrimson in the six gentle touch receptor neurons under the control of a mec-4 promoter, and used a high throughput closed-loop optogenetic delivery system to automatically stimulate animals either when moving forward or triggered upon the onset of a turn [2], Fig 1C. lgc-47 loss-of-function mutants exhibited little or no turning-dependent gating of the reversal response: they were similarly likely to reverse in response to stimuli regardless of whether the stimulus was delivered while the animal was turning or moving forward. By contrast, wild-type background animals showed significant gating: they were significantly less likely to reverse in response to stimuli delivered while turning compared to stimuli delivered during forward movement. These measurements suggest that LGC-47 mediates gating of mechanosensory evoked reversals.

To identify where LGC-47 acts, we performed cell-specific rescues in the reversal associated interneurons AIB, AVA, RIM or all three, by expressing WT lgc-47 cDNA in the background of lgc-47 loss-of-function animals, Supplementary Table S1. For each rescue, we measured the animal’s response to optogenetically induced mechanosensory stimuli, Fig 1C. Only animals that expressed LGC-47 in RIM recapitulated the WT gating behavior. We therefore conclude that LGC-47 mediates the gating of mechanosensory evoked reversals by inhibiting reversal neuron RIM in response to acetylcholine release from SAA. In other words, SAA inhibits RIM via LGC-47 during turns to prevent the initiation of a reversal.

We next investigated the candidate inhibitory acetylcholine receptor gene acc-1 that is expressed at lower levels in a similar pattern of neurons [9, 12]. Intriguingly, recent work suggests that ACC-1 regulates the duration of spontaneous reversals in a manner similar to what we proposed for LGC-47: namely ACC-1 is thought to inhibit RIM upon SAA activation, in that case halting an ongoing reversal [13]. We wondered whether ACC-1 also contributes to turning-dependent gating by potentially stopping reversals before they start.

acc-1 loss-of-function mutants showed no turning-dependence in their mechanosensory evoked responses, Fig 1 C,D, just like lgc-47 mutants. We observed the same effect both in our mec-4::Chrimson background strains Fig 1 C and in a nominally similar but separately generated set of strains from the Quan Wen group, Fig 1 D. Turning dependence reappeared when ACC-1 was rescued in the neuron type RIM, just as it did for LGC-47. Therefore ACC-1 performs the same role as LGC-47 in mediating the turning dependent gating. Recent in vitro electrophysiology studies suggest that LGC-47 and ACC-1 may form a heteromeric ion channel [14], and our finding that LGC-47 and ACC-1 perform the same role in vivo supports this hypothesis.

Taken together, we conclude that neuron type SAA gates mechanosensory evoked reversals by inhibiting reversal neurons RIM via an inhibitory acetylcholine receptor comprised of LGC-47 and ACC-1. Inhibition of RIM during turns is ideally situated to prevent reversals because it makes many gap junctions with neurons AVA and AVE, as well as gap junctions with AIB— all neurons implicated in promoting reversals. Inhibition of RIM may serve as a shunt to inhibit activity across the reversal circuitry. Future imaging studies are needed to reveal the neural dynamics of these neurons in response to mechanosensory stimuli delivered during turns.

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