Model organisms usually possess a small nervous system but nevertheless execute a large array of complex behaviors, suggesting that some neurons are likely multifunctional and may encode multiple behavioral outputs. Here, we show that the C. elegans interneuron AIY regulates two distinct behavioral outputs: locomotion speed and direction-switch by recruiting two different circuits. The “speed” circuit is excitatory with a wide dynamic range, which is well suited to encode speed, an analog-like output. The “direction-switch” circuit is inhibitory with a narrow dynamic range, which is ideal for encoding direction-switch, a digital-like output. Both circuits employ the neurotransmitter ACh but utilize distinct postsynaptic ACh receptors, whose distinct biophysical properties contribute to the distinct dynamic ranges of the two circuits. This mechanism enables graded C. elegans synapses to encode both analog- and digital-like outputs. Our studies illustrate how an interneuron in a simple organism encodes multiple behavioral outputs at the circuit, synaptic, and molecular levels.

Here, we investigated these questions in C. elegans using a multifaceted approach by integrating calcium imaging, optogenetics, molecular genetics, laser ablation, and electrophysiology at the single neuron resolution. By characterizing a model interneuron AIY that regulates both analog- and digital-like behavioral outputs, we show that at the circuit level, one strategy for a neuron to control multiple behavioral outputs is to recruit multiple downstream circuits with each regulating a specific behavioral output. At the synaptic level, we show that worm synapses display distinct dynamic ranges, which contributes to their ability to encode both analog- and digital-like behavioral outputs. At the molecular level, we demonstrate that the biophysical properties of postsynaptic receptors contribute to the distinct dynamic ranges of the synapses. These results reveal a mechanism by which graded C. elegans synapses encode both analog- and digital-like behavioral outputs. Our studies define the circuit, synaptic, and molecular mechanisms by which an interneuron encodes multiple behavioral outputs in a genetic model organism.

As the nematode C. elegans represents the only organism whose connectome has been reconstructed by electron microscopy, it has emerged as a popular model for dissecting the neural and genetic basis of behavior at the single neuron resolution (). C. elegans has a very small nervous system with merely 302 neurons. Despite this simplicity, worms execute a broad spectrum of behaviors, ranging from motor and sensory behaviors to the more complex mating, social, sleep, and drug-dependent behaviors, as well as associative learning and memory (). This large repertoire of complex behaviors places a high demand on the functionality of the worm nervous system. As such, many worm neurons are thought to be multifunctional (). This notion, however, provokes some intriguing questions. First, what are the circuit and synaptic mechanisms by which single neurons encode multiple behavioral outputs? Additionally, similar to many types of synapses in mammalian sensory organs, most, if not all, C. elegans synapses are believed to be graded; yet some worm behavioral outputs are manifested in an all-or-none digital-like fashion (). This raises the question: how could graded synapses encode digital-like behavioral outputs?

How the nervous system and genes generate behavior is one of the most fundamental questions in neuroscience. Unlike the human brain with billions of neurons, lower organisms such as Caenorhabditis elegans, Drosophila, crustaceans, mollusks, and zebrafish possess a much smaller nervous system and are therefore widely used as models to study the neural and genetic control of behavior. Although equipped with a small nervous system, these model organisms are capable of performing a diverse range of complex behaviors, many of which are analogous to those manifested by humans (). This suggests that neurons in these organisms may not all be specialized and some may regulate multiple behavioral outputs (). Interestingly, this phenomenon is not restricted to simple model organisms, as many neurons in the mammalian brain, especially in the cortex, also have multifunctional properties (). However, how single neurons encode multiple behavioral outputs is not well understood.

Lastly, we asked what molecular mechanisms may underlie the nonlinear transformation at the AIY-AIZ synapse. In light of the nature of worm synapses, presynaptic ACh release from AIY is likely to be graded. Indeed, optogenetic tuning of AIY activity resulted in graded calcium responses in AIY ( Figure 7 C). This suggests that the nonlinear transformation may primarily occur at the postsynaptic site. Because of this and the fact that it has not been technically feasible to optically or electrically monitor presynaptic ACh release, we focused on the postsynaptic neurons AIZ and RIB and recorded their electric responses to varying concentrations of ACh in dissected animals by whole-cell patch-clamping ( Figures 7 H and 7I). In this case, as ACh acted as a ligand to gate ion channels, we fit the data with a Hill equation ( Figure 7 I). Notably, the Hill slope (Hill coefficient) of the AIZ curve was much greater than that of the RIB curve (4.0 versus 0.66), indicating that the ACh-gated Clchannel ACC-2 in AIZ operated over a much narrower dynamic range than did the ACh-gated cation channels ACR-16 and UNC-29 in RIB. Furthermore, the channel in AIZ showed a much higher sensitivity to ACh than did the RIB channel (EC: 11 μM versus 131 μM) ( Figure 7 I). Consequently, the AIZ curve is not only steep with a narrow dynamic range, but also shifted more to the left because of its low ECvalue ( Figure 7 I). Given that ACh suppresses AIZ activity, this low ECvalue suggests that AIZ would be tonically inhibited and locked at an inactive or low activity state over a wide range of ACh inputs from AIY. The narrow dynamic range would then render AIZ to transition from an inactive or low activity state to an active state within a very small window, as if the neuron fired in an all-or-none fashion with a threshold ( Figures 3 H and 7 F). Thus, the features of the AIY-AIZ and AIY-RIB synapses can be explained at least in part by the distinct biophysical properties of ACh-gated channels expressed in AIZ and RIB. These results uncover a molecular mechanism underlying the nonlinear transformation of AIY signals by the AIY-AIZ synapse. Thus, even if presynaptic release might be graded at a synapse in C. elegans, the postsynaptic neuron may adopt a mechanism to transform analog signals into a digital-like output.

We then asked how AIY activity might encode both analog- and digital-like behavioral outputs. Because AIY regulates locomotion speed and direction-switch through RIB and AIZ, respectively, we examined the input-output relations of the AIY-RIB and AIY-AIZ synapses. To do so, we optogenetically tuned the activity of AIY ( Figure 7 C) and then recorded the postsynaptic response in RIB and AIZ by calcium imaging using the CARIBN system in freely behaving animals ( Figures 7 D and 7E). Based on these data ( Figures 7 C–7E), we plotted the input-output relations of the two synapses ( Figures 7 F and 7G). The slope factor of the AIY-AIZ curve was much smaller than that of the AIY-RIB curve (0.04 versus 0.20), indicating that the former possessed a much narrower dynamic range than did the latter ( Figures 7 F and 7G). In addition, as the Xvalue of the AIY-AIZ curve was smaller than that of the AIY-RIB curve (0.25 versus 0.69), the AIY-AIZ curve was shifted more to the left than the AIY-RIB curve ( Figures 7 F and 7G). Consequently, AIZ only displayed activity in response to a small window of AIY inputs, but exhibited relatively little or no activity across a large range of AIY inputs ( Figure 7 F). This is consistent with our preceding calcium imaging data that AIZ showed relatively low activity beyond reversal periods ( Figure 3 H). As the AIY-AIZ synapse is inhibitory, this analysis suggests that AIZ may be tonically inhibited and locked at an inactive or low activity state over a wide range of inputs from AIY. With a narrow dynamic range, AIZ would then transition from an inactive state to an active state in a very small window, as if it fired in an all-or-none manner with a threshold. This nonlinear transformation strategy would enable the AIY-AIZ synapse to convert graded signals from AIY into a digital-like output in AIZ.

As a first step to approach this question, we examined our imaging data collected from freely behaving worms and analyzed the input-output relations between AIY calcium activity (input) and reversal initiation (output) or locomotion speed (output). Both can be fit with a sigmoidal function which is commonly used to describe the input-output relations of neural networks () ( Figures 7 A and 7B ). Notably, the slope factor of the “reversal” curve, a parameter that describes a curve’s steepness, was much smaller than that of the “speed” curve (0.02 versus 0.11). This indicates that reversal probability displayed a much steeper relationship with AIY activity than did locomotion speed ( Figures 7 A and 7B). In other words, the dynamic range in the “reversal” curve is rather narrow ( Figure 7 A), as if reversals occurred abruptly as a function of AIY activity, which is consistent with the nature of reversal initiation as an all-or-none digital-like behavioral output. By contrast, the “speed” curve exhibited a much wider dynamic range with locomotion speed changing rather gradually with AIY activity ( Figure 7 B), a feature that fits well with the nature of locomotion speed as an analog-like behavioral output. In addition, the lower the AIY activity, the higher the reversal probability, but the lower the locomotion speed ( Figures 7 A and 7B). Thus, AIY activity appears to inversely correlate with reversal probability but positively with locomotion speed. This analysis provides further evidence that AIY activity can differentially encode both analog- and digital-like behavioral outputs.

(H and I) The postsynaptic ACh receptors in AIZ and RIB show distinct biophysical properties. (H) Sample traces of AIZ and RIB whole-cell currents evoked by different concentrations of ACh. Voltage: −60 mV. (I) ACh-gated currents in AIZ and RIB were plotted as a function of ACh concentrations. Data were fit with a Hill equation: I/I max = 1/[1 + (EC 50 /[ACh]) n ], where n represents Hill slope (Hill coefficient).

(F and G) The input-output relations of the AIY-AIZ and AIY-RIB synapses. Data in (C–E) were replotted to derive the input-output relations of the two synapses. Specifically, the relative AIZ and RIB calcium activity values (output) shown in (D) and (E) were replotted as a function of the relative AIY calcium activity values (input), which were shown in (C), to generate (F) and (G), respectively. Both data were fit with a sigmoidal equation: Y output = 1/(1 + exp((X 1/2 − X input )/slope))).

(C–E) Calcium responses in AIY, AIZ, and RIB triggered by optogenetic tuning of AIY activity. Freely behaving worms carrying separate transgenes, which expressed NpHR in AIY (i.e., AIY::NpHR) and GCaMP3 in AIY, AIZ, and RIB, were challenged with varying intensities of yellow light to tune the activity of AIY. Both the presynaptic calcium activity in AIY and the postsynaptic calcium responses in AIZ and RIB were recorded with the CARIBN system. The resulting calcium activities in AIY, AIZ, and RIB were normalized and plotted as a function of yellow light intensities. (C) AIY calcium response curve. (D) AIZ calcium response curve. (E) RIB calcium response curve. n ≥ 4.

(A and B) The input-output relations between AIY activity (input) and reversal initiation (output) or locomotion speed (output). We analyzed the data from AIY calcium imaging and behavioral traces obtained with the CARIBN system ( Figure 2 A). AIY calcium data points were extracted from the CARIBN traces, normalized, and binned into ten groups (bin width: 0.1). Reversal probability and locomotion speed was tabulated for each group based on the data from the behavioral traces, normalized, and plotted as a function of the relative AIY calcium activity of individual groups to make (A) and (B), respectively. Both data were fit with a sigmoidal equation: Y= 1/(1 + exp((X− X)/slope))). Xrepresents the X value where Y shows 50% of its maximal value. slope describes the steepness of the curve.

While the above study identified a circuit and synaptic mechanism by which AIY regulates locomotion speed and direction-switch, it raised one interesting question: In C. elegans, although muscle cells fire action potentials (), most, if not all, neurons are nonspiking and form graded synapses (). While this feature may well explain AIY modulation of analog-like behavioral outputs such as locomotion speed, then how is direction-switch, a digital-like behavioral output, regulated by AIY?

By contrast, ACh elicited a different type of current in AIZ ( Figures 6 E and 6F). This ACh-gated current was outward when clamped at 0 mV with a negative reversal potential, close to the equilibrium potential of Cl Figures 6 E and 6F). In addition, increasing the Clconcentration in the pipette solution shifted the reversal potential close to 0 mV ( Figure 6 F), indicating that the current was primarily carried out by a Clchannel. Furthermore, mutation in the Clchannel ACC-2 nearly eliminated the ACh-gated Clcurrent in AIZ ( Figures 6 G and 6H). These electrophysiological data further support ACC-2 as a key postsynaptic ACh receptor that mediates the inhibitory response in AIZ.

To provide further evidence, we directly recorded ACh-evoked electric responses in AIZ and RIB neurons in dissected animals by whole-cell patch-clamping. ACh elicited an inward current in RIB ( Figure 6 A). This ACh-gated current exhibited a nearly linear I-V relationship with a positive reversal potential ( Figure 6 B), consistent with the view that it was mediated by a cation channel. As was the case with our calcium imaging data, mutations in unc-29 and acr-16 nearly abolished the ACh-gated current in RIB ( Figures 6 C and 6D), providing further evidence supporting UNC-29 and ACR-16 as key postsynaptic ACh receptors that mediate the excitatory response in RIB.

We then set out to identify the postsynaptic ACh receptor in AIZ. The preceding data suggest that the nature of the AIY-AIZ synapse is inhibitory. Then how might ACh, which is best known as an excitatory neurotransmitter, mediate an inhibitory postsynaptic response? Interestingly, the C. elegans genome encodes four ACh-gated Clchannels: ACC-1, ACC-2, ACC-3, and ACC-4 (). We thus tested mutants lacking these ACC channels. Loss of ACC-2 abrogated the ability of AIY (AIY::NpHR) to trigger reversals ( Figure 5 H), while mutations in the other three ACC channels did not ( Figure S3 E), uncovering a critical role of ACC-2 in mediating this behavioral output. In addition, worms lacking acc-2, but not other acc genes, showed an elevated reversal frequency, consistent with the role of ACC-2 as an inhibitory ACh receptor ( Figure S3 F). We also found that ACC-2 was expressed in AIZ ( Figure S3 G), and expression of wild-type acc-2 gene specifically in AIZ rescued the reversal defect of acc-2 mutant worms ( Figure 5 H). Again, we recorded AIY-evoked postsynaptic response in AIZ by calcium imaging using the CARIBN system. Loss of ACC-2 greatly diminished AIZ’s calcium response triggered by AIY (AIY::NpHR), and transgenic expression of wild-type acc-2 gene in AIZ rescued this defect ( Figures 5 I and 5J). Thus, both behavioral and functional imaging data identify ACC-2 as a key component of the postsynaptic ACh receptor in AIZ.

We next sought to identify the postsynaptic ACh receptors expressed in AIZ and RIB. We first searched for candidate receptors in RIB. Our observation that the AIY-RIB synapse is excitatory suggests that the postsynaptic ACh receptor in RIB is probably a cation channel such as nAChR. Two major groups of nAChRs were identified in C. elegans: the levamisole-type nAChRs represented by UNC-29 and UNC-38 and the nicotine-type nAChRs such as ACR-16 (). We decided to examine UNC-29 and ACR-16 because they both are expressed in RIB (). Loss of UNC-29 modestly but significantly compromised the ability of AIY (AIY::ChR2) to promote locomotion speed, consistent with a role for UNC-29 in regulating locomotion speed ( Figures 5 D and 5E). This also suggests the presence of additional nAChRs in RIB, which prompted us to test a potential contribution from ACR-16. However, unc-29; acr-16 double mutant worms are severely paralyzed (), preventing us from directly assaying locomotion behavior in the double mutant. We therefore resorted to the RNAi approach by introducing acr-16 RNAi as a transgene specifically in RIB in the unc-29 mutant background and vice versa. acr-16(RNAi);unc-29 worms showed nearly no increase in locomotion speed in response to ChR2 stimulation of AIY ( Figures 5 D and 5E). Further, although acr-16 single mutant worms did not exhibit a notable defect in this behavioral response, acr-16;unc-29(RNAi) worms displayed a strong phenotype ( Figures 5 D and 5E). Thus, UNC-29 and ACR-16 appear to function redundantly. To obtain additional evidence, we recorded AIY-evoked postsynaptic response in RIB by calcium imaging using the CARIBN system. While ChR2 stimulation of AIY elicited a robust postsynaptic response in RIB in wild-type worms, such a response was greatly diminished in worms deficient in both acr-16 and unc-29 ( Figures 5 F and 5G). Thus, both behavioral and functional imaging data suggest ACR-16 and UNC-29 as key components of the postsynaptic ACh receptors in RIB.

To provide further evidence, we recorded AIY-evoked postsynaptic responses in AIZ and RIB by calcium imaging using the CARIBN system. RNAi of cha-1 in AIY greatly diminished the postsynaptic calcium response in RIB triggered by AIY (AIY::ChR2) ( Figures 5 F and 5G). A similar result was obtained with the postsynaptic calcium response in AIZ evoked by AIY (AIY::NpHR) ( Figures 5 I and 5J). Thus, both behavioral and functional imaging data support ACh as a key neurotransmitter in both the AIY-AIZ and AIY-RIB circuits.

Having identified a circuit mechanism by which AIY encodes two distinct behavioral outputs, we then set out to characterize the underlying synaptic mechanisms. We first explored the presynaptic side, asking what neurotransmitter in AIY is critical for controlling direction-switch and locomotion speed. AIY is a cholinergic neuron (). We thus examined whether and how blockade of ACh release from AIY would affect these two behavioral outputs. cha-1 encodes the sole C. elegans choline acetyltransferase essential for ACh synthesis (). As cha-1 is expressed in dozens of neurons and cha-1 mutant worms are severely paralyzed, we were unable to directly assay locomotion behavior in mutant worms. We thus took an RNAi approach by introducing a transgene expressing cha-1 RNAi specifically in AIY ( Figures S3 C and S3D). RNAi of cha-1 in AIY blunted the ability of AIY inhibition (by NpHR) to trigger reversals ( Figure 5 A) and also suppressed the effect of AIY stimulation (by ChR2) on promoting locomotion speed ( Figures 5 B and 5C). Furthermore, RNAi of cha-1 in AIY increased reversal frequency and also reduced locomotion speed ( Figures S3 A and S3B), an effect similar to that observed in AIY-ablated worms ( Figures 1 B and 1C). Thus, cha-1 appears to be required in AIY to control direction-switch and locomotion speed, supporting ACh as a key neurotransmitter in AIY for regulating these two behavioral outputs.

(E) acc-2, but not other acc genes, is required for AIY to trigger reversals. All genotypes carried a transgene expressing NpHR in AIY under the ttx-3 s promoter. The assay was conducted as described in Figure 1 D. Yellow light was used to excite NpHR to trigger reversals. No light illumination was used as a control. Error bars: SEM n ≥ 5.p < 0.0001 (ANOVA with Tukey’s HSD test).

(C and D) RNAi of cha-1 knocks down the protein level of CHA-1::mCherry fusion in AIY. To evaluate the effect of cha-1 RNAi in AIY, we crossed a transgene expressing CHA1::mCherry protein fusion in AIY and then quantified its fluorescence level. cha-1 RNAi inhibited the protein expression level of CHA-1::mCherry fusion by ∼90%. Error bars: SEM n ≥ 32. ∗∗ p < 0.0001 (t test).

(A and B ) RNAi of cha-1 in AIY increases reversal frequency (A), and reduces locomotion speed (B). cha-1 RNAi were expressed as a transgene specifically in AIY under the ttx-3 s promoter. Locomotion behavior was assayed using an automated worm tracking system. Error bars: SEM n ≥ 5. ∗ p < 0.02; ∗∗ p < 0.0001 (t test).

Taken together, our data suggest a model in which AIY regulates direction-switch and locomotion speed by forming two separate downstream circuits: the AIY-AIZ circuit is inhibitory and regulates direction-switch, while the AIY-RIB circuit is excitatory and modulates locomotion speed ( Figure 5 K).

(I and J) Calcium imaging shows that cha-1 and acc-2 mediate the synaptic transmission between AIY and AIZ. All worms carried two transgenes with one expressing NpHR in AIY and the other expressing GCaMP in AIZ. (I) AIZ GCaMP calcium traces. (J) Bar graph. n ≥ 8. ∗∗ p < 0.0006 (ANOVA with Tukey’s HSD test).

(F and G) Calcium imaging shows that cha-1, unc-29 and acr-16 mediate the synaptic transmission between AIY and RIB. All worms carried two transgenes with one expressing ChR2 in AIY and the other expressing RCaMP in RIB. (F) RIB RCaMP calcium traces. (G) Bar graph. n ≥ 11. ∗∗ p < 0.0001 (ANOVA with Tukey’s HSD test).

(D and E) unc-29 and acr-16 act redundantly to promote locomotion speed in RIB. acr-16 RNAi was introduced as a transgene specifically in RIB. n ≥ 12. ∗ p < 0.02; ∗∗ p < 0.0001 (ANOVA with Tukey’s HSD test).

(B and C) RNAi of cha-1 in AIY blunted the ability of AIY to promote locomotion speed. ChR2 and cha-1 RNAi were expressed as separate transgenes specifically in AIY and crossed together. no TG: nontransgenic siblings. n ≥ 11. ∗∗ p < 0.0006 (ANOVA with Tukey’s HSD test).

(A) RNAi of cha-1 blunted the ability of AIY to promote reversal initiation. NpHR and cha-1 RNAi were expressed as separate transgenes specifically in AIY and crossed together. n = 5. ∗∗ p < 0.0001 (ANOVA with Tukey’s HSD test).

The above results point to a model that AIY regulates locomotion speed by forming a circuit with the downstream interneuron RIB. The fact that both AIY and RIB promote locomotion speed suggest that the AIY-RIB circuit is likely excitatory and that AIY may promote locomotion speed by stimulating RIB. To test this model, we recorded the activity of RIB in response to optogenetic stimulation or inhibition of AIY in freely behaving worms using the CARIBN system. ChR2 stimulation of AIY promoted RIB activity ( Figures 4 L and 4M), while NpHR inhibition of AIY suppressed it ( Figures 4 N and 4O). Thus, AIY promotes locomotion speed by forming an excitatory circuit with RIB ( Figure 4 P).

To provide further evidence, we imaged the activity of RIB in freely behaving worms using the CARIBN system. RIB activity paralleled locomotion speed ( Figure 4 I). Cross-correlation analysis revealed a strong correlation between RIB activity and forward locomotion speed (but not backward speed) ( Figures 4 J and 4K). Thus, data from calcium imaging, laser ablation and optogenetics all support a role for RIB in promoting locomotion speed.

The observation that ablation of RIB reduced locomotion speed suggests that RIB acts to promote locomotion speed. To further test this, we optogenetically interrogated the role of RIB in locomotion speed by expressing ChR2 and NpHR as a transgene specifically in this neuron. ChR2 stimulation of RIB increased locomotion speed ( Figures 4 D and 4E), while NpHR inhibition of RIB reduced it ( Figures 4 F and 4G), providing additional evidence that RIB promotes locomotion speed. By contrast, neither NpHR inhibition nor ChR2 stimulation of RIB triggered reversals ( Figure 4 H), suggesting a specific role for RIB in modulating locomotion speed. Thus, both laser ablation and optogenetic data suggest that RIB promotes locomotion speed.

We next sought to identify the neurons that act downstream of AIY to regulate locomotion speed. Laser ablation of RIB, but not AIZ or RIA, abolished the ability of AIY (ChR2) to promote locomotion speed ( Figures 4 A and 4B ), indicating that RIB is required for AIY to regulate locomotion speed. As was the case with AIY, ablation of RIB also reduced locomotion speed, and double ablation of AIY and RIB did not exhibit an additive effect ( Figure 4 C), consistent with the notion that AIY and RIB act in the same pathway to modulate locomotion speed. Notably, laser ablation of RIB does not have a major effect on reversal frequency (). These results suggest that RIB regulates locomotion speed by acting downstream of AIY.

(N and O) Inhibition of AIY suppresses the activity of RIB. Worms carrying two transgenes (one expressing NpHR in AIY and the other expressing GCaMP3.0 in RIB) were imaged with the CARIBN system. Control: worms carrying the RIB::GCaMP3.0 transgene only. (N) RIB GCaMP3.0 calcium traces. (O) Bar graph. n ≥ 10. ∗∗ p < 0.0001 (t test)

(L and M) Stimulation of AIY promotes the activity of RIB. Worms carrying two transgenes (one expressing ChR2 in AIY and the other expressing RCaMP in RIB) were imaged with the CARIBN system. Control: worms carrying the RIB::RCaMP transgene only. (L) RIB RCaMP calcium traces. (M) Bar graph. n ≥ 17. ∗∗ p < 0.0001 (t test).

(H) Optogenetic manipulation of RIB activity does not trigger reversals. Neither stimulation of RIB by ChR2 nor inhibition of RIB by NpHR triggered reversals. The value for RIB::ChR2 is zero under this condition. n = 5. ∗∗ p < 0.02 (t test).

(A and B) Laser ablation shows that RIB is required for AIY to promote locomotion speed. Worms expressing ChR2 as a transgene in AIY were assayed for locomotion speed in response to blue light. (A) Speed traces. no TG: nontransgenic siblings. (B) Bar graph. n ≥ 16. ∗∗ p < 0.0001 (ANOVA with Tukey’s HSD test).

The above results suggest a model in which AIY controls direction-switch by forming a circuit with the downstream interneuron AIZ. The fact that AIY suppresses direction-switch but AIZ promotes it suggests that AIY may regulate this behavioral output by inhibiting AIZ. In this case, the nature of the circuit formed by AIY and AIZ would be inhibitory. If so, inhibition of AIY should suppress AIY’s inhibitory output to AIZ, which would result in stimulation of AIZ, leading to reversal initiation. To test this model, we recorded the activity of AIZ in response to optogenetic inhibition of AIY in freely behaving worms using the CARIBN system. NpHR inhibition of AIY activated AIZ ( Figures 3 K and 3L), indicating that AIY inhibited AIZ and disinhibition of AIY promoted reversals. This also supports the prediction that the synaptic connection formed between AIY and AIZ is inhibitory. Interestingly, ChR2 stimulation of AIY did not elicit a notable effect on AIZ activity ( Figures 3 M and 3N), revealing a nonlinearity of the AIY-AIZ synapse (also see below). Together, our results support the model that AIY regulates direction-switch by forming an inhibitory circuit with the downstream interneuron AIZ ( Figure 3 O).

To obtain further evidence supporting a role for AIZ in direction-switch, we imaged the activity of AIZ in freely behaving worms using the CARIBN system. We found that AIZ increased its activity when worms initiated reversals ( Figure 3 H). Interestingly, though AIZ activity tightly correlated with reversal initiation ( Figure 3 I), no strong correlation was found between AIZ activity and locomotion speed ( Figure 3 J), providing further support for a specific role for AIZ in regulating direction-switch. Unlike AIY, AIZ did not display much activity beyond reversals ( Figure 3 H), suggesting that AIZ probably received inhibitory inputs and thereby remained at a rather inactive or low activity state during most time periods (also see below). This imaging data, together with those from laser ablation and optogenetics, strongly suggests that AIZ regulates direction-switch.

If AIZ acts downstream of AIY, this neuron should also regulate direction-switch. The observation that laser ablation of AIZ reduced reversal frequency is consistent with this view ( Figure 3 C). This data also suggests that AIZ promotes direction-switch. To provide further evidence, we optogenetically interrogated the role of AIZ in direction-switch by expressing ChR2 and NpHR as a transgene specifically in AIZ. Stimulation of AIZ by ChR2 triggered reversals ( Figure 3 D), while inhibition of AIZ by NpHR did not and instead suppressed spontaneous reversal frequency ( Figure 3 E), suggesting that AIZ promotes direction-switch. Notably, inhibition of AIZ by NpHR did not affect locomotion speed ( Figures 3 F and 3G), revealing a specific role for AIZ in regulating direction-switch. Thus, both laser ablation and optogenetic studies support that AIZ promotes direction-switch.

Then how does AIY regulate locomotion speed and direction-switch? To approach this question, we set out to identify the neurons that act downstream of AIY to regulate these two behavioral outputs. Based on the wiring diagram, AIZ, RIA, and RIB are the primary downstream interneurons to which AIY sends synaptic outputs ( Figure 3 A). We thus ablated AIZ, RIA, and RIB and assayed whether and how this operation may affect the ability of AIY to regulate locomotion speed and direction-switch. We first examined direction-switch. Laser ablation of AIZ, but not RIA or RIB, abolished the ability of AIY (NpHR) to trigger reversals ( Figure 3 B). Thus, AIZ is required for AIY to regulate direction-switch, suggesting that AIZ acts downstream of AIY. Additional evidence came from spontaneous locomotion behavior: while ablation of AIY led to an increase in reversal frequency, simultaneous ablation of AIZ and AIY fully suppressed this hyperreversal phenotype ( Figure 3 C), further suggesting that AIZ acts downstream of AIY.

(M and N) Stimulation of AIY does not affect the activity of AIZ. Worms carrying two transgenes (one expressing ChR2 in AIY and the other expressing RCaMP in AIZ) were imaged with the CARIBN system. Control: worms carrying the AIZ::RCaMP transgene only. (M) AIZ RCaMP calcium traces. (N) Bar graph. n = 6.

(K and L) Inhibition of AIY activates AIZ. Worms carrying two transgenes (one expressing NpHR in AIY and the other expressing GCaMP3.0 in AIZ) were imaged with the CARIBN system. Control: worms carrying the AIZ::GCaMP3.0 transgene only. (K) AIZ GCaMP calcium traces. (l) Bar graph. n ≥ 6. ∗∗ p < 0.0001 (t test).

(F and G) Optogenetic inhibition of AIZ does not affect locomotion speed. Because optogenetic stimulation of AIZ triggered reversal initiation, we were unable to test its effect on locomotion speed. (F) Speed traces. (G) Bar graph. n ≥ 18.

(D and E) Optogenetic inhibition of AIZ activity promotes reversal initiation (D), while optogenetic stimulation of AIZ suppresses it (E). ChR2 and NpHR was expressed as a transgene specifically in AIZ. Control: nontransgenic siblings. n = 5. ∗∗ p < 0.0001 (t test)

(B) Laser ablation shows that AIZ is required for AIY to promote reversal initiation. Worms expressing NpHR as a transgene in AIY were tested for reversal initiation triggered by yellow light. n = 5. ∗∗ p < 0.0001 (ANOVA with Tukey’s HSD test).

While the data from both laser ablation and optogenetic interrogation strongly support a role for AIY in regulating locomotion speed and direction-switch, both approaches relied on the manipulation of AIY function and thus may not necessarily reveal exactly how AIY acts under native conditions. If AIY truly modulates locomotion speed and direction-switch, then the activity of AIY should correlate with these two behavioral outputs during locomotion. To address this question, we simultaneously recorded the activity of AIY and locomotion behavior in freely behaving worms using our calcium ratiometric imaging of behaving nematodes (CARIBN) system (). Notably, the calcium transients in AIY paralleled the speed of forward locomotion of the animal ( Figure 2 A; Movie S1 ). In addition, when the animal switched to backward movement (reversal), we observed a sharp decrease in AIY calcium level ( Figures 2 A and S2 Movie S1 ). This suggests that the activity of AIY may correlate with both locomotion speed and direction-switch. Indeed, cross-correlation analysis revealed that AIY activity nicely correlated with forward locomotion speed (but not backward speed) ( Figures 2 B and 2C). In addition, cross-correlogram detected a strong correlation between the activity of AIY and the initiation of reversals ( Figure 2 D). Thus, the activity pattern of AIY appears to correlate with both locomotion speed and direction-switch, further supporting that AIY regulates these two motor outputs.

The segments in calcium and velocity traces surrounding reversal events were analyzed. The dotted line in red marks the time point at which animals initiate reversals. The average calcium level in AIY begins to decline at ∼1 s before the initiation of reversals. Shades along the traces represent error bars (SEM). n = 36.

(D) Cross-correlogram showing that reversal initiation correlates with AIY activity decrease. Because reversal initiation occurs in an all-or-none fashion, cross-correlogram was used to analyze correlation using a method similar to that reported previously (). The occurrence of a reversal event and the decrease/increase in calcium level at a given time point were analyzed for correlation. A negative value indicates that a reversal event coincided with a decrease in calcium level and vice versa.

(B and C) Cross-correlation analysis shows that AIY calcium activity correlates with forward but not backward locomotion speed. The shades along the curves represent error bars (SEM). n = 8.

If AIY regulates both speed and direction-switch as suggested by laser ablation, acute interrogation of AIY activity should affect both behavioral outputs. To test this, we took an optogenetic approach by expressing ChR2 and NpHR specifically in AIY as a transgene (). Transient inhibition of AIY activity by NpHR triggered reversals and suppressed locomotion speed ( Figure 1 D and Figures S1 A and S1B available online), while activation of AIY by ChR2 suppressed the initiation of spontaneous reversals and stimulated locomotion speed ( Figures 1 D–1F). Prolonged optogenetic inhibition and stimulation of AIY also promoted and suppressed the initiation of spontaneous reversals, respectively ( Figure S1 C). The optogenetic data are more robust than those from laser ablation, probably because optogenetics tests the acute effects while laser ablation reports the chronic outcome, in which case animals may develop compensatory and adaptive mechanisms. Nevertheless, these two types of data together strongly suggest that AIY regulates both locomotion speed and direction-switch.

(C) Prolonged inhibition and stimulation of AIY by NpHR and ChR2 promotes and suppresses reversal initiation during spontaneous locomotion, respectively. This assay was performed differently from that in Figure 1 D, but similarly to that in Figure 1 B. Specifically, instead of using 5 s light pulses, we applied prolonged light stimulus (2 min of yellow light at 2 mW/mm2 or blue light at 0.5 mW/mm2) to animals expressing NpHR or ChR2 in AIY, respectively. The number of reversal events was then quantified over 2 min of scoring window. NpHR inhibition of AIY increased the reversal frequency to ∼250% of control. This effect is more potent than that obtained with AIY laser ablation, which increased reversal frequency by ∼50% (see Figure 1 B). Conversely, ChR2 stimulation of AIY nearly eliminated all spontaneous reversal events. Control: nontransgenic siblings. Error bars: SEM n ≥ 10.p < 0.0001 (t test).

(A and B) AIY::NpHR reduced forward locomotion speed under a low intensity of yellow light (0.3 mW/mm2). Note: when AIY was further inhibited by brighter yellow light (2 mW/mm2), a reversal was triggered (see Figure 1 D for details).

As a first step, we sought to identify a neuron that regulates these two behavioral outputs. This would offer us an opportunity to dissect the mechanisms underlying encoding of two distinct behavioral outputs by one neuron. Interneurons are known to play a pivotal role in controlling locomotion, particularly spontaneous locomotion (). Based on the wiring diagram, they are organized in a hierarchy-like pattern and send outputs to motor neurons that drive locomotion (). We focused our initial attention on the 1layer interneurons (i.e., AIA, AIB, AIZ, and AIY), as they remain at the very top of the interneuron circuitry () ( Figure 1 A). Laser ablation of these four interneurons all affected reversal frequency ( Figure 1 B), consistent with previous reports (). Among them, AIY is particularly interesting, as loss of AIY led to a significant defect in both locomotion speed and reversal frequency ( Figures 1 B and 1C). Specifically, ablation of AIY reduced the speed of locomotion while increasing the frequency of reversals ( Figures 1 B and 1C). This suggests that while AIY promotes locomotion speed, it also suppresses the initiation of reversals. Thus, AIY likely regulates both locomotion speed and direction-switch.

(E and F) Optogenetic stimulation of AIY promotes locomotion speed. Worms expressing ChR2 as a transgene specifically in AIY were tested under blue light (30 s pulse). Control: nontransgenic siblings. (E) Forward speed traces. The shades along the traces represent error bars. For clarity, the very few reversal events were removed. (F) Bar graph. n ≥ 22. ∗∗ p < 0.0001 (t test).

(D) Optogenetic inhibition or stimulation of AIY promotes or suppress reversal initiation, respectively. Worms expressing NpHR or ChR2 as a transgene specifically in AIY were tested for reversal initiation triggered by yellow or blue light (5 s pulse). Control: nontransgenic siblings (a similar control result was obtained by assaying the transgenic animals reared on retinal-free plates). The low level of reversal events in the control resulted from spontaneous reversals. n = 5. ∗∗ p < 0.0001 (t test).

We focused on locomotion behavior, one of the most prominent behaviors in C. elegans (). Locomotion forms the foundation of most, if not all worm behaviors (e.g., motor, sensory, social, mating, sleep, etc.), as these behaviors are, to varying degrees, all manifested at the locomotion level. During locomotion, worms spend most of their time moving forward and occasionally change locomotion direction by switching to backward movement (reversal) (). We recorded locomotion behavior using an automated worm tracking system () and quantified two simple parameters: speed and reversal initiation. These two parameters represent two distinct motor outputs, with the former reporting the overall activity of locomotion and the latter describing the direction-switch of locomotion. They also bear another distinction in that speed is intrinsically an analog-like parameter, whereas reversal occurs in an all-or-none fashion and thus represents a digital-like output.

Discussion

de Bono and Maricq, 2005 de Bono M.

Maricq A.V. Neuronal substrates of complex behaviors in C. elegans. Jankowska, 2001 Jankowska E. Spinal interneuronal systems: identification, multifunctional character and reconfigurations in mammals. Schiller, 1996 Schiller P.H. On the specificity of neurons and visual areas. Due to the immense complexity of the human brain, simple model organisms with a small nervous system are widely used to study how the brain and genes produce behavior (). The seeming mismatch between the small size of their nervous systems and the large pool of complex behaviors which they execute fuels the suggestion that some of their neurons are probably multifunctional and may regulate multiple behavioral outputs. However, how this is achieved at the circuit and synaptic levels remains largely enigmatic. This is also an important question for mammals, as many neurons in the mammalian brain possess multifunctional properties and can encode multiple outputs ().

Sporns and Kötter, 2004 Sporns O.

Kötter R. Motifs in brain networks. In the current report, using a multifaceted approach we have attempted to investigate this question in C. elegans by characterizing a model neuron AIY that regulates at least two distinct motor outputs: locomotion speed and direction-switch. Our results show that AIY recruits two distinct downstream circuits: one (AIY-AIZ) is inhibitory, which regulates direction-switch, while the other one (AIY-RIB) is excitatory, which modulates locomotion speed ( Figure 5 K). Apparently, at the circuit level, one strategy for a neuron to regulate multiple behavioral outputs is to recruit multiple downstream circuits with each regulating a specific behavioral output. The circuit motifs identified here are highly represented in the nervous system of C. elegans and other organisms including mammals (), raising the possibility that this might be a common strategy adopted by neurons in worms and perhaps other organisms to regulate multiple behavioral outputs.

Chalfie et al., 1985 Chalfie M.

Sulston J.E.

White J.G.

Southgate E.

Thomson J.N.

Brenner S. The neural circuit for touch sensitivity in Caenorhabditis elegans. Piggott et al., 2011 Piggott B.J.

Liu J.

Feng Z.

Wescott S.A.

Xu X.Z.S. The neural circuits and synaptic mechanisms underlying motor initiation in C. elegans. Nevertheless, it should be noted that neural control of behavior is rather complex. For example, the two circuits identified here must involve additional neurons. Indeed, we found that the neurons acting downstream of AIZ in the circuit are backward command interneurons and RIM known to control reversal initiation, while RIB acts through AVB, a forward command interneuron that promotes forward locomotion speed (Z.L. and X.Z.S.X., unpublished data) (). Additionally, as these two circuits have connections with other neurons, they probably do not function in isolation and may engage in crosstalk with other circuits and may be modulated by inputs from other interneurons and sensory neurons. Further, the two circuits may also crosstalk with each other. For example, the fact that AIY inhibition triggers reversals in RIB-ablated worms suggests that AIZ may signal to other neurons in the speed circuit. Lastly, other circuit mechanisms must be employed by worms to encode multiple behavioral outputs.