We hypothesized that tonic signaling from the O₂-sensing neurons URX, AQR, and PQR could reprogram gene expression according to ambient O₂ levels. To investigate this possibility, we analyzed the transcriptomes of npr-1(null) mutants (referred to as npr-1) and gcy-35; npr-1 young adult animals grown at 21% and 7% O₂ using RNA sequencing (see ‘Materials and methods’). URX, AQR, and PQR neurons do not respond to O₂ changes in gcy-35; npr-1 mutants, and exhibit activity levels found in npr-1 animals kept at 7% O₂ (Persson et al., 2009; Zimmer et al., 2009; Busch et al., 2012). To prevent npr-1 animals from aggregating, which induces gene expression changes that would confound our analysis (Andersen et al., 2014), we grew animals at low density. We carried out three sets of comparisons. To identify genes whose expression is O₂-modulated we compared the transcriptomes of npr-1 animals grown at 21% and 7% O₂ (Supplementary file 1). To identify genes regulated by GCY-35-dependent signaling, we compared the transcriptomes of npr-1 and gcy-35; npr-1 animals grown at 21% O₂ (Supplementary file 2). And to identify and exclude genes whose expression is altered by growth at different O₂ levels independently of gcy-35, we compared the transcriptomes of gcy-35; npr-1 animals grown at 21% and 7% O₂ (Supplementary file 3). We then intersected the three comparisons, using as a cut-off q value of 0.05 (see ‘Materials and methods’). If our hypothesis was correct, many of the genes identified as differentially expressed in Supplementary file 1 (npr-1 21% O₂ vs npr-1 7% O₂) should also be identified in Supplementary file 2 (npr-1 21% O₂ vs gcy-35; npr-1 21% O₂) but not in Supplementary file 3 (gcy-35; npr-1 21% O₂ vs gcy-35; npr-1 7% O₂). Consistent with this, we found that 152/210 genes differentially expressed between npr-1 7% O₂ versus npr-1 21% O₂ were also found in the gcy-35; npr-1 21% O₂ versus npr-1 21% O₂ comparison. Expression of 72/152 of these genes was not significantly altered by O₂ experience in gcy-35; npr-1 animals (Supplementary file 4). For almost all of these genes, 71/72, disrupting gcy-35 had a similar effect on gene expression as growing npr-1 animals at 7% O₂ (Supplementary file 4). Genes whose expression was regulated by O₂ experience in a GCY-35-dependent way encoded proteins involved in metabolism (e.g., elo-6, a polyunsaturated fatty acid elongase; folt-2, a folate transporter; mai-2, a mitochondrial intrinsic ATPase inhibitor protein), oxidation–reduction reactions (e.g., the cytochrome p450s, cyp-35C1 and cyp-34A2), and proteolysis (e.g., the cathepsins cpr-4 and cpr-6), suggesting broad changes not limited to the nervous system (Supplementary file 4).

Efficient escape from aversive environments requires coordinated movement that avoids conflicting behaviors. When exposed to 21% O₂ in the presence of food, npr-1 animals not only increased their speed of movement (Busch et al., 2012) (Figure 1A), but also increased the persistence of their forward travel by inhibiting spontaneous short reversals (Figure 1B,C). Mutations in gcy-35, which abolish the O₂ responsiveness of URX, AQR, and PQR neurons, disrupted O₂ modulation of both speed (Busch et al., 2012) and spontaneous reversal frequency (Figure 1B). Expressing gcy-35 selectively in URX neurons restored rapid movement and sustained inhibition of reversals at 21% O₂. URX neurons thus provide a defined entry point to the circuit controlling the behavioral state switch evoked by 21% O₂ in npr-1 animals.

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Among the three O₂-sensing neurons, URX neurons uniquely make gap junctions and reciprocal synaptic connections with the RMG interneurons (White et al., 1986; wormwiring.org; Figure 1—figure supplement 1). The RMG interneurons respond to a 7–21% rise in O₂ with a sustained increase in Ca²⁺, and ablating RMG disrupts behavioral responses evoked by this O₂ switch (Busch et al., 2012). To show that O₂-evoked Ca²⁺ responses in RMG reflect input from URX, we ablated URX and imaged RMG using the YC2.60 Ca²⁺ sensor (Horikawa et al., 2010). When URX was ablated, O₂ stimuli failed to evoke Ca²⁺ responses in RMG (Figure 1D), and RMG Ca²⁺ resembled that found in tax-4 mutants, which lack the cGMP-gated channel required for URX, AQR, and PQR neurons to transduce O₂ stimuli (Figure 1D). These data suggest that O₂-evoked Ca²⁺ responses in RMG are driven by URX input.

Is depolarizing RMG sufficient to switch C. elegans behavioral state? To address this we used channelrhodopsin-2 (ChR2; Nagel et al., 2005) to selectively stimulate RMG in npr-1 animals kept at 7% O₂. Stimulating RMG inhibited spontaneous reversals and induced rapid and persistent forward movement for as long as blue light was on (Figure 1E,F). Stimulating RMG neurons is thus sufficient to confer a highly active locomotory state on npr-1 animals kept at 7% O₂.

In npr-1 animals, URX neurons respond in a graded manner to changes in O₂ between 7% and 21%, and evoke graded increases in locomotory rate according to final O₂ concentration (Cheung et al., 2005; Busch et al., 2012). To investigate the dynamic range of URX–RMG signaling, and to examine if graded RMG activity can evoke graded changes in the animal's speed, we stimulated RMG in animals kept at either 7% O₂ or 21% O₂. The effects of activating RMG using ChR2 summated with input from the O₂ sensory circuit: animals kept at 21% reached higher speeds and inhibited reversals more strongly than animals kept at 7% (Figure 1E,F). These data suggest that the dynamic range of the circuit permits higher levels of RMG activation to evoke qualitatively similar but quantitatively stronger behavioral responses.

RMG and URX are connected by gap junctions and by reciprocal chemical synapses. Stimulating RMG using ChR2 could therefore alter behavior by activating URX. To test this, we selectively activated RMG using ChR2 in animals ablated for URX (and AQR and PQR). Ablated animals robustly increased their speed and suppressed reversals upon light-activation of RMG, both at 7% and at 21% O₂ (Figure 1G,H). As expected if O₂ input was lost, the speed responses of the ablated animals were not influenced by the O₂ levels (Figure 1I). The ability of RMG stimulation to induce rapid movement and inhibit reversals in the absence of URX, AQR, and PQR neurons suggests that feed-forward signaling from RMG to other neurons evokes these behaviors.

Our results did not exclude that RMG influences URX activity. To investigate this possibility, we ablated RMG neurons in L1 larvae and imaged URX Ca²⁺ responses in young adults 2 days later. npr-1 animals lacking RMG showed a much smaller URX Ca²⁺ response to a 7–21% O₂ stimulus compared to mock ablated controls (Figure 1J). Thus, not only does URX activate RMG in response to rising O₂, but RMG input somehow regulates URX excitability. Although the gap junctions and synaptic connections between URX and RMG provide a direct route for such communication, we cannot exclude more indirect mechanisms. However, consistent with a role for synaptic input in sustaining URX excitability, partial loss-of-function mutants of unc-64 syntaxin, which have deficits in synaptic release, showed reduced Ca²⁺ levels in URX and RMG both at 7% and 21% O₂ (Figure 1D,K). URX neurons could still evoke Ca²⁺ responses in RMG in unc-64 mutants (Figure 1D). However, since the unc-64 allele we used is a partial loss-of-function (null mutants are dead), we cannot draw firm conclusions about the relative roles of gap junctions and synaptic transmission in mediating URX–RMG communication.

Excessive signaling from a hyperactive neuropeptide receptor, NPR-1 215V, prevents N2 (Bristol) animals from moving rapidly on food at 21% O₂ (de Bono and Bargmann, 1998; Cheung et al., 2005). The RMG neurons are one site of action for this receptor: expressing NPR-1 215V selectively in RMG reduces the locomotory activity of npr-1 animals (Macosko et al., 2009). Does NPR-1 215V expression in RMG inhibit responses evoked by a switch from 7% to 21% O₂? npr-1 animals that expressed NPR-1 215V in RMG did not exhibit long-lasting changes in locomotion in response to changes in O₂ (Figure 2, Figure 2—figure supplement 1A,B). However, they retained the transient bout of reversals and reorientation triggered by a sharp rise in O₂ (Figure 2—figure supplement 1C). Thus, NPR-1 215V receptor signaling in RMG selectively suppresses the sustained switch in locomotory behavior evoked by high and low O₂. A different circuit may act downstream of O₂ sensors to evoke transient avoidance responses, consistent with the observation that activating RMG using ChR2 failed to induce reversals.

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The FMRFamide neuropeptide encoded by the flp-21 gene is an in vivo ligand for NPR-1 (Rogers et al., 2003). flp-21 is expressed in several neurons, including RMG itself (Kim and Li, 2004; Macosko et al., 2009). FLP-21 peptide release could therefore serve to limit the O₂-evoked behavioral state switch by activating NPR-1 signaling in RMG. Consistent with this, deleting flp-21 caused animals expressing the natural npr-1 215F or derived npr-1 215V allele, but not npr-1(null) mutants, to increase the amplitude of their O₂-evoked behavioral switch (Figure 2A–C).

How does the NPR-1 neuropeptide receptor inhibit RMG function? Using the YC2.60 sensor, we compared the Ca²⁺ responses evoked in RMG by a 7–21–7% O₂ stimulus train in N2 and npr-1 mutants. N2 animals exhibited reduced Ca²⁺ responses in RMG compared to npr-1 mutants (Figure 2D), which would be consistent with NPR-1 215V inhibiting RMG Ca²⁺ signaling. However, the effects, although statistically significant, were surprisingly small compared to the strong inhibitory effects of NPR-1 215V signaling on O₂-evoked behaviors. More importantly, the promoter combination targeting YC2.60 expression to RMG (pncs-1::cre and pflp-21::LoxP-STOP-LoxP::YC2.60) drove lower expression in N2 compared to npr-1 animals (Figure 2—figure supplement 2A). Lower sensor expression correlated with lower YFP/CFP ratio at 21% O₂ (Figure 2—figure supplement 2B), suggesting that sensor expression differences could account for much of the YFP/CFP ratio differences between N2 and npr-1 animals (see below for an explanation of why pflp-21 drove lower RMG expression in N2 compared to npr-1 animals).

These observations led us to speculate that the main inhibitory role of NPR-1 was downstream of Ca²⁺ influx. To test this, we compared the effects of stimulating RMG neurons with ChR2 in N2 and npr-1 animals. If NPR-1's inhibitory role is predominantly downstream of Ca²⁺ entry, ChR2-activation of RMG should have little effect in N2 animals. Consistent with this, light activation of RMG triggered only a small increase in N2 locomotory activity, regardless of whether animals were kept at 21% or 7% O₂, and in marked contrast to its effects in npr-1 mutants (Figure 2E). Most compellingly, although npr-1 mutants kept at 7% O₂ had much lower Ca²⁺ in RMG compared to N2 animals kept at 21% O₂ (Figure 2D), they responded to RMG ChR2 activation significantly more strongly (Figure 2E). These results suggest that the inhibitory effects of npr-1 215V occur predominantly downstream of the Ca²⁺ responses in RMG, possibly at the presynaptic level.

A network of G protein pathways modulates neurotransmission presynaptically: G_q and G_s signaling stimulates neurotransmitter release whereas G_o/i signaling inhibits it, possibly by negatively regulating G_s and G_q signaling (Miller et al., 1999; Nurrish et al., 1999). In vitro work suggests that the NPR-1 215V and NPR-1 215F receptors can each couple to G_o/i signaling following stimulation by FLP-21 (Rogers et al., 2003). If NPR-1 215V inhibits neurotransmission by activating G_o, then mutations that promote G_q/G_s signaling over G_o signaling should restore O₂ control of locomotory state and mimic npr-1 mutants. The RGS (regulator of G protein signaling) protein EAT-16 terminates G_q signaling by activating intrinsic G_q GTPase activity (Hajdu-Cronin et al., 1999); RSBP-1 (R7 binding protein 1 homolog) interacts with and is required for EAT-16 activity (Porter and Koelle, 2010). Disrupting either eat-16 or rsbp-1 enabled N2 animals, which express npr-1 215V, to behave like npr-1 mutants in response to a change in O₂ (Figure 2F–H). Together, these results suggest that a change in the balance of G_o/G_q signaling in a subset of neurons explains how the NPR-1 215V neuropeptide receptor controls behavior.

Animals integrate information across multiple sensory modalities to respond appropriately to changing environments. The ASK sensory neurons respond to pheromones and food (Macosko et al., 2009; Wakabayashi et al., 2009), and, like the URX O₂ sensors, make gap junctions with RMG (White et al., 1986; wormwiring.org; Figure 1—figure supplement 1). Previous work suggested that cGMP signaling in ASK is required for npr-1 animals to move rapidly and to aggregate (Tremain, 2004; Macosko et al., 2009), leading to the hypothesis that ASK neurons are a major output of the RMG hub-and-spoke circuit (Macosko et al., 2009). The ASH and ADL nociceptive neurons also make gap junctions with RMG (Figure 1—figure supplement 1). Previous work suggested that TRPV signaling in ASH and ADL promotes aggregation behavior and avoidance of high O₂ (de Bono et al., 2002; Chang et al., 2006; Rogers et al., 2006). Together, these results suggested that gap-junctional communication across the RMG circuit integrates multiple sensory cues and is necessary for npr-1 animals to move rapidly at 21% O₂ and to aggregate (Macosko et al., 2009).

To investigate this model in the context of O₂-evoked responses, we used the YC3.60 reporter to examine if changing [O₂] altered [Ca²⁺] in ASK or ASH neurons in npr-1 animals. A switch from 7% to 21% O₂ elicited a small increase in YFP/CFP FRET in ASK and ASH, indicating a rise in [Ca²⁺] (Figure 3A,B). The responses were sustained while animals were at 21% O₂. Deleting gcy-35 abolished the O₂-evoked Ca²⁺ responses in ASK and ASH (Figure 3A,B), consistent with these responses being driven by URX, and therefore potentially via RMG. These results indicate that O₂ input can indeed modify ASK and ASH Ca²⁺ levels, but the effect is small—at least at the cell body, where we made our measurements.

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To ask if ASK neurons contributed to O₂-evoked behavioral states, we ablated ASK in npr-1 animals by targeted expression of the egl-1 cell death gene (Wakabayashi et al., 2004). ASK ablation was confirmed by dye filling and using a psra-9::YC3.60 fiduciary marker. npr-1 animals lacking ASK neurons responded to a 7–21–7% O₂ regime indistinguishably from control npr-1 animals (Figure 3C). Optogenetic inhibition of ASK, in an npr-1 strain expressing halorhodopsin (NpHR) specifically in ASK, had no effect on speed (Figure 3D). Ablating ASK neurons in npr-1 animals also did not impair aggregation behavior (Figure 3—figure supplement 1). Aggregation is highly sensitive to O₂ circuit function and is disrupted in gcy-35 mutants (Cheung et al., 2004; Gray et al., 2004; Rogers et al., 2006). These data suggest that ASK neurons are not necessary for O₂-evoked behavioral responses, or for aggregation.

To investigate if ASH and ADL are required for the O₂-controlled behavioral state switch, we ablated these neurons in npr-1 animals expressing channelrhodopsin in RMG, using targeted expression of a miniSOG gene and light-induced ablation (Qi et al., 2012). The ASH and ADL ablated animals responded to high and low O₂ and to current injection into RMG like npr-1 controls (Figure 3E). Tetanus toxin in the ASH and ADL neurons also did not disrupt O₂ responses (Figure 3F). To monitor toxin expression, we used a polycistronic construct that also expressed RFP, and we confirmed that tetanus toxin disrupted synaptic release from ASH and ADL by monitoring reversals in response to a 3 mM Cu²⁺ drop (Figure 3F, inset). Thus, ablating ASK alone, or ASH and ADL together, does not disrupt relay of O₂-modulated RMG activity to downstream circuits that promote rapid movement.

The ASH and ADL neurons express the TRPV1 homolog ocr-2, and disrupting ocr-2, or its partner subunit osm-9, attenuates animals' ability to navigate spatial O₂ gradients and to aggregate (de Bono et al., 2002; Chang et al., 2006; Rogers et al., 2006). ocr-2; npr-1 animals only weakly modulated their locomotory activity when switched between 7% and 21% O₂ (Figure 4A). Expressing ocr-2 cDNA in ASH or ADL or the serotonergic ADF neurons increased the speed of ocr-2; npr-1 animals at 21% O₂ and, as expected, rescued the bordering and aggregation phenotype (Figure 4A, Figure 4—figure supplement 1A) (de Bono et al., 2002; Chang et al., 2006). The ability of ocr-2 expression in ASH or ADL neurons to rescue the speed response and aggregation behavior of ocr-2; npr-1 animals was retained when neurotransmission from these neurons was blocked using tetanus toxin (Figure 4B,C, Figure 4—figure supplement 1B). These data suggest that ocr-2 TRPV activity in ASH and ADL is unlikely to promote O₂ responses by facilitating these neurons' neurosecretory activity, but may instead influence their gap junctional communication, for example, with RMG.

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The OCR-2/OSM-9 TRPV channel is required for the ASH and ADL neurons to respond to sensory inputs (Hilliard et al., 2004). If disrupting ocr-2 reduces ASH and ADL tonic or chronic activity, it could decrease RMG activity by reducing current input into RMG, or by shunting current away from RMG through the ASH–RMG and ADL–RMG gap junctions (Figure 1—figure supplement 1). If this model is correct, injecting current into ASH or ADL using ChR2 should restore to ocr-2; npr-1 mutants rapid movement at 21% O₂.

To test this, we expressed ChR2 specifically in ASH by using the FLP/FRT system and by monitoring expression using YFP-tagged ChR2 (see ‘Materials and methods’). We exposed transgenic animals to constant blue light of different intensities, ranging from 0.005 to 0.05 mW/mm². As expected, control animals not exposed to blue light did not strongly modulate their speed when switched between 7% and 21% O₂ (Figure 4D). By contrast, animals exposed to 0.005, 0.015 or 0.05 mW/mm² of continuous blue light modulated their locomotory state according to O₂ levels (Figure 4D). The amplitude of the O₂-evoked change in locomotory activity was similar to that obtained when we selectively restored ocr-2 expression to ASH in ocr-2; npr-1 animals (compare Figure 4C,D). Thus, injecting a constant Ca²⁺/Na⁺ current into ASH neurons is sufficient to restore O₂ modulation to the circuit in ocr-2; npr-1 animals. This suggests that the effects of the ocr-2 mutation are not developmental, as they can be rescued in adult animals. Our results support a model in which tonic or chronic ASH (and ADL) activity facilitates O₂-evoked behavioral switches either by injecting current into RMG or by reducing shunting from the RMG circuit.

A rise in O₂ evoked a small increase in ASH Ca²⁺ (Figure 3B), suggesting that depolarizing current flows from RMG to ASH in npr-1 animals at 21% O₂. To test explicitly if current can flow in the reverse direction, from ASH to RMG, we asked if strongly activating ASH using a noxious stimulus evoked a rise in RMG Ca²⁺. As expected, a 10 mM Cu²⁺ stimulus resulted in a large, sharp rise in ASH Ca²⁺ that was easily detectable using YC3.60 (Hilliard et al., 2004) (Figure 4E). By contrast, this stimulus failed to evoke any Ca²⁺ responses in the RMG cell body that could be measured with YC2.60 (Figure 4F). These results suggest that the anatomically defined gap junctions may not allow significant Ca²⁺ current to flow from the ASH to the RMG cell body under our imaging conditions. How, then, does OCR-2 promote RMG activity? An alternative model is that by tonically/chronically depolarizing ASH and ADL neurons, OCR-2 reduces the current flowing from RMG to ASH and ADL via gap junctions. In ocr-2; npr-1 mutants, a more negative membrane potential in ASH and ADL leads to more current being shunted from RMG to the nociceptive neurons, reducing RMG activity. To test this hypothesis, we compared O₂-evoked Ca²⁺ responses in URX and RMG in npr-1 and ocr-2; npr-1 animals (Figure 4G,H). Surprisingly, the steady state Ca²⁺ levels in the URX or RMG cell bodies were not significantly affected by the ocr-2 mutation. Since the tetanus toxin experiments suggested that rescue of the ocr-2; npr-1 phenotype by a pASH::ocr-2 transgene did not require synaptic transmission, we speculate that the Ca²⁺ effects of disrupting ocr-2 are local to the gap junctions and not visible at the RMG cell body. Alternatively, ASH and ADL have the potential to leach away some other excitatory factor from RMG via gap junctions, and OCR-2 activity in ASH and ADL can attenuate this.

If loss of the OCR-2 TRPV channel somehow reduced the functionality of RMG, injecting current directly into RMG should rescue the phenotype of ocr-2; npr-1 animals. Consistent with this, ChR2-activation of RMG in ocr-2; npr-1 animals stimulated rapid movement, and inhibited reversals, both at 21% and 7% O₂ (Figure 4I,J and data not shown). This contrasts with the failure of ChR2-driven RMG activation to drive tonic changes in behavioral state in N2 animals (which express the NPR-1 215V receptor) (Figure 2F). These results suggest that the OCR-2-expressing neurons can facilitate RMG signaling in a way that is upstream of NPR-1 signaling.

Genetically encoded Ca²⁺ sensors are blind to signaling mechanisms that modulate neurosecretion without altering Ca²⁺ (Miller et al., 1999; Nurrish et al., 1999; Rhee et al., 2002; Oda et al., 2011). This limitation prompted us to seek a way to monitor enduring changes in neurosecretion as readouts of different global states. In vertebrates, several studies have reported that increased neural activity is associated with increased neuropeptide gene transcription (Uhl and Nishimori, 1990). In pancreatic β cells, feedback mechanisms couple insulin production and release (Borgonovo et al., 2006). If transcription of most neuropeptide genes is coupled to peptide release, promoter::GFP reporters for such genes should provide a readout of peptidergic circuit activity. To test this, we first studied a pflp-11::GFP reporter expressed in the URX, AUA, and SAB neurons (Kim and Li, 2004). URX and AUA both show tonically elevated Ca²⁺ at 21% O₂ compared to 7% O₂ (Busch et al., 2012). Cultivating animals in 7% O₂, or deleting gcy-35, reduced pflp-11::gfp expression in both URX and AUA (Figure 5A). By contrast, knocking out ocr-2 did not, suggesting that TRPV signaling did not alter URX or AUA peptide expression (Figure 5A). The genotype at the npr-1 locus did not affect pflp-11::gfp expression in URX, but expression in the AUA neurons was reduced in animals encoding the npr-1 215V allele compared to npr-1 null mutants (Figure 5A). pflp-11::gfp expression in SAB, a neuron not known to be modulated by O₂, was not affected either by O₂ experience or the genotypes tested, providing an internal control. These data are consistent with pflp-11 expression reporting neural activity.

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To test more explicitly if modulation of pflp-11::gfp expression levels was related to neuron depolarization state, we inhibited URX by expressing a constitutively active K⁺ channel related to Drosophila ether-a-go, EGL-2(GF) (Weinshenker et al., 1999). This transgene abolishes behavioral responses evoked by 21% O₂ (Cheung et al., 2005). EGL-2(GF) expression strongly reduced pflp-11::gfp expression in URX (Figure 5B). We next tested if feedback control of pflp-11::gfp expression was coupled to peptide release downstream of Ca²⁺. To selectively block neurosecretion from URX, we cell-specifically expressed tetanus toxin, which cleaves synaptobrevin (Schiavo et al., 2000). Tetanus toxin expression strongly reduced pflp-11::gfp expression in URX, and the effect was stronger when toxin expression was higher (Figure 5C). These results suggest that pflp-11::gfp expression is coupled to peptide release in URX, and that peptide gene reporters may be useful surrogates to monitor long-term neurosecretory activity.

To test this hypothesis further, we examined a pflp-5::GFP transgene expressed in the RMG, ASE, and M4 neurons (Kim and Li, 2004). pflp-5::gfp expression in RMG was higher in npr-1 animals grown at 21% O₂ than in npr-1 animals grown at 7% O₂ or gcy-35; npr-1 mutants grown at 21% O₂ (Figure 5D). These data suggest that pflp-5::gfp reporter expression is also coupled to neural activity. In contrast, expression of pflp-5::GFP in the M4 pharyngeal neuron, which is not known to respond to O₂, was not altered by O₂ experience or by disrupting gcy-35. To explore if pflp-5 expression in RMG, like pflp-11 expression in URX, was coupled to peptide release, we blocked RMG neurosecretion by cell-specifically expressing tetanus toxin. Animals expressing tetanus toxin in RMG showed significantly less pflp-5 expression in RMG than non-expressing siblings (Figure 5E), consistent with neurosecretion feeding back to stimulate pflp-5 transcription.

Expression of pflp-5::gfp in RMG was reduced in N2 animals compared to npr-1 mutants, both at 21% and 7% O₂. Since at 21% O₂ RMG Ca²⁺ levels were not strikingly different between N2 and npr-1 mutants (Figure 2E), our data support a model in which NPR-1 215V signaling acts either downstream of Ca²⁺ or locally to inhibit RMG neurosecretion. Interestingly, ocr-2; npr-1 animals kept at 21% O₂ also showed reduced pflp-5::gfp expression compared to npr-1 worms kept at the same O₂ levels (Figure 5D), suggesting that disrupting ocr-2 reduced RMG neurosecretion in npr-1 animals.

We next studied expression from a long version of the flp-21 promoter that drives GFP expression in URA, M4, M2, ASJ, RMH, and RMG (Macosko et al., 2009). The RMH interneurons make gap junctions with RMG (White et al., 1986; wormwiring.org). Expression of the long pflp-21::GFP transgene in RMG and RMH was highest in npr-1 animals cultivated at 21% O₂, and reduced in gcy-35; npr-1 and ocr-2; npr-1 animals grown at 21% O₂, as well as npr-1 animals kept overnight at 7% O₂ (Figure 5F). pflp-21::GFP transgene expression in RMG and RMH was reduced in N2 animals compared to npr-1 mutants, both at 21% and 7% O₂, supporting a model in which NPR-1 215V signaling inhibits neurosecretion from these neurons (Figure 5F). Thus, expression in RMG neurons driven from the long flp-21 promoter and the flp-5 promoter is regulated very similarly by O₂ experience and genotype at the npr-1 and ocr-2 loci. Together, our data suggest that neurosecretion from RMG and RMH is tonically modulated by O₂ levels and TRPV signaling.

A shorter version of the flp-21 neuropeptide gene promoter drives GFP expression in the ADL, ASH, ASJ, URA, M4, and M2 neurons (Kim and Li, 2004). We wondered if this promoter could provide readouts of ASH and ADL signaling. Disrupting the ocr-2 TRP channel subunit strongly reduced expression of the short pflp-21::gfp transgene in ASH and ADL in npr-1 animals (Figure 5G). For ASH, we showed that expressing tetanus toxin also reduced pflp-21::gfp expression in this neuron (Figure 5H). These results are consistent with tonic/chronic OCR-2-dependent neurosecretory activity in these neurons driving a feedback loop to promote transcription of neuropeptides. Changing O₂ levels in npr-1 animals did not significantly alter expression of this shorter pflp-21::gfp in ASH and ADL, suggesting that O₂ modulation of ASH and ADL secretion, if it occurs, is minor (Figure 5G). Note that npr-1 is expressed in ASH neurons (Figure 1—figure supplement 1), which may account for differences in pflp-21::gfp expression in this neuron between npr-1 and N2 animals. These data are consistent with the ablation studies and the Ca²⁺ imaging data for ASH, which suggest that ASH neurons exhibit only weak O₂-evoked changes in Ca²⁺ and are dispensable for the O₂-evoked behavioral switch.

Encouraged by our results, we looked for other neuropeptide genes that are reported to be expressed in neurons connected to the O₂-sensing circuit by gap junctions or synapses. One such gene encodes the C. elegans ortholog of pigment dispersal factor, pdf-1 (Barrios et al., 2012; Meelkop et al., 2012). A ppdf-1::gfp transgene is expressed in PVP and AVB, two neurons heavily connected with O₂-sensing neurons: PVP has gap junctions with AQR and PQR, whereas AVB has gap junctions with AQR and receives synaptic input from AQR, URX, and RMG. Expression of ppdf-1::gfp in both PVP and AVB was upregulated at 21% O₂ compared to 7% O₂, suggesting that the activity of these neurons is also modulated by O₂ (Figure 5I and see below). The expression of ppdf-1::gfp in RMG from the transgene was too weak and variable to be assayed.

Finally, we studied the expression of two additional peptide genes to demonstrate that not all neuropeptide promoters are stimulated by exposure to 21% O₂. The neuropeptide FLP-17 is expressed by BAG neurons and released when BAG is depolarized in vitro (Ringstad and Horvitz, 2008; Smith et al., 2013). BAG neurons are stimulated by low O₂ concentrations (Zimmer et al., 2009). As expected, pflp-17::gfp expression in BAG was stimulated at 7% O₂ compared to 21% O₂ (Figure 5J). The neuropeptide FLP-3 is expressed in the IL1 neurons (Kim and Li, 2004), which are part of a touch-responsive circuit, are poorly connected to known O₂-modulated neurons, and do not express NPR-1. pflp-3::gfp expression in IL1 was not regulated by O₂ or npr-1 (Figure 5K).

Together, our data suggest that O₂ levels regulate flp-11, flp-5, flp-21, flp-17, and pdf-1 peptide gene expression and peptide release in multiple neurons, including URX, AUA, RMG, RMH, BAG, PVP, and AVB, but not URA, SAB, IL1, M2, and M4. They also suggest that peptide release from RMG is stimulated by O₂-sensing neurons, depends on OCR-2 TRPV signaling, and is inhibited by NPR-1 215V signaling.

Previous work has shown that RMG activity is necessary for high locomotory activity at 21% O₂ (Macosko et al., 2009; Busch et al., 2012). We have shown that RMG activity is sufficient to drive high locomotory activity, and that ASH, ADL, and ASK are each dispensable for these effects. Does peptidergic signaling from RMG contribute to driving high locomotory activity at high O₂? We first tested the effects of blocking all neurosecretion from RMG using cell-specific expression of a tetanus toxin transgene in npr-1. To monitor expression, we used a polycistronic construct that co-expressed the toxin with RFP. Animals expressing the transgene lost any tonic response to high O₂ and remained very poorly active at all O₂ concentrations (Figure 6A,B). This was expected from previous work (Macosko et al., 2009), although that study did not explicitly examine O₂-evoked responses. We next tested more specifically if neuropeptides secreted from RMG were required, using cell-specific RNAi to knock down the carboxypeptidase E (CPE) egl-21 in RMG. Carboxypeptidase E removes C-terminal lysine and arginine residues from pro-peptides during maturation, and analysis of peptide extracts from egl-21 mutants shows a deficit in the maturation of most C. elegans neuropeptides (Husson et al., 2007). npr-1 animals with selective RNAi knockdown of egl-21 in RMG moved appropriately slowly in 7% O₂, but sped up much less than non-transgenic siblings when switched to 21% O₂ (Figure 6C) and reversed more frequently (Figure 6D). These results indicate that neuropeptide release from the RMG neurons plays a major role in evoking the highly active state of feeding C. elegans at 21% O₂.

Figure 6

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Ultimately, changes in O₂ levels modify behavioral state by altering motor circuits. We envisioned three ways by which O₂ circuit output might alter downstream circuits. In one model, the downstream circuits would exhibit O₂-evoked tonic changes in Ca²⁺ levels, much as we observe in the URX, AQR, PQR, AUA, and RMG neurons (Busch et al., 2012). In a different model, behavioral state would be encoded across multiple neurons whose activity does not faithfully track O₂ concentration, but which on average show O₂-evoked changes in Ca²⁺ levels. To investigate these possibilities, we used Ca²⁺ indicators to image the activity of motoneurons, and of interneurons in layers upstream of motoneurons, at different O₂ environments. The third possibility would not involve regulation of Ca²⁺ levels, but rather presynaptic effects that would only be observed using other reporters, such as the peptide promoter assay.

We focused our studies on four sets of neurons: the A and B motoneurons, which are thought to mediate reverse and forward movement, respectively; the AVA ‘command’ interneurons, which promote backward movement by ensuring that the A motoneurons are more active than the B motoneurons; the AVB ‘command’ interneurons which promote forward movement by ensuring that the B motoneurons are more active than the A motoneurons; and the AIY interneurons which do not have anatomically defined connections with URX or RMG but are post-synaptic to multiple other sensory neurons (White et al., 1976, 1986; Chalfie et al., 1985; Kawano et al., 2011; wormwiring.org; Figure 1—figure supplement 1). As a control, we imaged the RMG neurons.

We first measured spontaneous (Figure 7A,B) and O₂-evoked (Figure 7C,D) Ca²⁺ changes in animals immobilized with 3 mM levamisole; levamisole immobilizes C. elegans by activating nicotinic acetylcholine receptors expressed in body wall muscle (Lewis et al., 1980). As expected (Kawano et al., 2011), Ca²⁺ levels in AVA and AVB neurons, and in the A and B neurons, were anti-correlated. As observed previously (Schrödel et al., 2013), in unstimulated animals the bouts of activity in which neurons promoting backward movement (the AVA and VA neurons) were more active than neurons promoting forward movement (the AVB and VB neurons) often lasted for more than a minute before switching stochastically (Figure 7A,B). This fictive behavior contrasted with the real behavior of animals moving on an agar plate, which only reverse for a few seconds. Nevertheless, the fictive behavior evoked by a rise in O₂ recapitulated some of the expected response: the backward module (VA neurons) was reliably activated by O₂ upsteps from 7% to 21% O₂ (Figure 7C). Moreover, when we exposed animals to a graded O₂ series of 21–14–7–21%, we found that, despite frequent switches in activity, the average activity in AVB and AIY was modulated by O₂ levels, with higher Ca²⁺ at 21% compared to 7% in both neurons (Figure 7D).

Figure 7

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The AVA and AVB interneurons, and their downstream targets the A and B motoneurons, respond to a variety of sensory cues transmitted by upstream circuits to control C. elegans' direction of movement. These upstream circuits include highly connected interneurons called AIA, AIB, and AIY that are post-synaptic to multiple sensory neurons (White et al., 1986; wormwiring.org). We speculated that the highly variable Ca²⁺ baseline we observed in AVA, AVB, and AIY reflected these diverse inputs, and that modulation by O₂ levels acts on top of these other inputs. In levamisole-treated animals, the frequent switches observed in AVA, AVB, and AIY might reflect attempts by the animals to change their direction of movement.

To examine this possibility without the caveats associated with levamisole treatment, and in a way that allows us to record information about the animals' behavior, we imaged neural activity in animals moving freely on food under a layer of PDMS, at 7% or 21% O₂. Under these conditions, animals modulated their speed of movement in response to O₂ levels, although with reduced amplitude, but did not exhibit the high reversal state normally observed at 7% O₂, perhaps because of the effects of being under a PDMS layer (Figure 8A; Figure 8—figure supplement 1).

Figure 8 with 1 supplement see all

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As expected from our imaging of immobilized animals, RMG interneurons responded to a 7–21% increase in O₂ with a large tonic rise in Ca²⁺ that lasted for as long as animals were at 21% O₂ (Figure 8B). Ca²⁺ levels in RMG did not change when animals reversed or stopped moving forward. By contrast, the AVB, AVA, AIY, AIA, and AIB interneurons each displayed frequent, large but brief changes in Ca²⁺ levels, at both 21% and 7% O₂ (Figure 8C–G). Superficially, these Ca²⁺ changes appeared to occur stochastically, as in immobilized animals, but careful analysis showed that they usually coincided with a switch in the direction of travel, and never lasted more than a few seconds (Figure 8C–G).

We hypothesized that the large changes in Ca²⁺ levels associated with executing reversal behaviors would obscure any smaller Ca²⁺ changes in these neurons that were evoked by a change in O₂ concentration. To examine this possibility, we registered Ca²⁺ traces to the initiation of a reversal, and averaged responses across multiple animals kept at either 7% or 21% O₂. We then compared Ca²⁺ levels in each neuron as animals executed reversals at 7% or 21% O₂ (Figure 9A). As expected, we observed changes in Ca²⁺ associated with the reversal behavior sequence. Superimposed on this, our analysis revealed that Ca²⁺ levels in AVB and AIY, but not in AIA, AIB, and AVA neurons, differed significantly between animals kept at 21% and 7% O₂ (Figure 9B–F). AVB showed higher Ca²⁺ levels during forward locomotion at 21% O₂ than at 7% O₂ (Figure 9B). AIY also showed significantly higher Ca²⁺ levels at 21% O₂ than 7% O₂ during forward locomotion (Figure 9C), and only showed changes in activity correlating with reversal behavior at 7% O₂. As expected, the RMG interneurons showed strong modulation by O₂ but no change in activity correlating with reversal state (Figure 9G). Together, our data predict that modulation of AVB and AIY downstream of RMG mediates behavioral changes induced by different O₂ environments.

Figure 9

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Our data also suggest a sequence of changes in neural activity associated with the initiation and the termination of reversals at 7% and 21% O₂. In particular, we observed a fall in AIA Ca²⁺ coincident with a rise in AIB Ca²⁺ that immediately preceded reversal initiations, and was followed by a drop in AVB Ca²⁺ and a rise in AVA Ca²⁺ around the time reversals begin (Figure 9H). Termination of reversals was preceded by a fall in AVA Ca²⁺, and associated with a rise in Ca²⁺ in AVB and AIA, and a drop in Ca²⁺ in AIB (Figure 9I).

To test for physiological roles of AIY, AVB, and AIA interneurons in O₂-evoked changes in behavioral state we turned to optogenetics and ablation experiments. We first expressed halorhodopsin (eNpHR2) in the AIY, AVB, and AIA interneurons, or in AIA alone, in an npr-1 background, and used light to inhibit these neurons. At 7% O₂, acute inhibition of AIY, AVB and AIA together, or of AIA alone, only had minor effects on behavior (Figure 10A). When the light was turned off, we observed a transient decrease in the rate of reversals that probably reflected disinhibition of AIA (Figure 10A, compare AIA alone with AIA, AIY, and AVB).

Figure 10

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When animals were kept at 21% O₂, acute inhibition of AIA caused a sustained increase in reversal rate compared to control animals, but did not significantly alter their speed of movement (Figure 10B). Inhibition of AIA, AIY and AVB together at 21% O₂ caused a sustained decrease in the speed of movement together with an increased rate of reversals compared to control animals (Figure 10B, compare AIA alone with AIA, AIY, and AVB). Together, our data suggest that although we do not observe regulation of AIA Ca²⁺ by O₂ (Figure 9D), tonic AIA activity plays a role in inhibiting reversal rate at 21% O₂, while AIY and AVB promote rapid forward movement.

Since inhibiting AIA at 21% O₂ promoted reversal behavior (and disinhibiting AIA suppressed reversals at 7% O₂), we examined how altering AIA activity modulated downstream circuits. We imaged Ca²⁺ in the AVA interneurons of freely moving npr-1 animals kept at 21% O₂ while acutely inhibiting or disinhibiting AIA with light. Inhibiting AIA evoked a rise in AVA Ca²⁺ (Figure 10E), while disinhibiting AIA caused a rapid fall in AVA Ca²⁺ (Figure 10F). These results suggest that AIA has inhibitory control of AVA activity, and is consistent with AIA activity preceding AVA activity during spontaneous reversals.

We next expressed ChR2 selectively in AIY in an npr-1 background, and used light to activate this interneuron at low and high O₂ concentrations (Figure 10C,D). Activating AIY caused a sustained decrease in reversal rate and an increase in speed, both at 7% and 21% O₂. Thus, consistent with the predictions made from our Ca²⁺ imaging experiments, optogenetics suggest that increasing AIY activity can increase the speed of locomotion and decrease reversal frequency.

To test further for a role of AIY in the O₂-evoked behavioral switch, we examined the O₂ responses of npr-1 ttx-3 mutants. ttx-3 encodes a LIM homeodomain transcription factor required for specification of AIY (Hobert et al., 1997). ttx-3 npr-1 animals sped up less than npr-1 animals upon being switched from 7% to 21% O₂, but inhibited reversal behavior to a similar extent (Figure 10G,H). Ablating AIA interneurons in ttx-3 npr-1 animals did not alter speed further, but significantly increased reversal rate at 21% O₂ (Figure 10I,J). These results are consistent with AIY playing a physiological role in promoting rapid movement at 21% O₂, and suggest that AIA acts with AIY to inhibit reversal behavior at 21% O₂, although neither neuron appears to be essential.

To delineate long-term changes in the activity of neural populations, we sought simple readouts of neurosecretory activity that do not depend on Ca²⁺ sensors. Ca²⁺ imaging approaches, although powerful, are blind to signaling mechanisms that alter synaptic and neurosecretory activity without affecting Ca²⁺ levels. We were inspired by sporadic reports suggesting a positive correlation between neural activity and neuropeptide gene transcription in the mammalian brain. By studying multiple neuropeptide genes expressed in URX, RMG, and other neurons, we showed that transcription of these genes is positively coupled to the secretory activity of the neurons expressing them. The feedback loop appears to act downstream of Ca²⁺, and to be linked to neurosecretion itself, since it can be disrupted by expressing tetanus toxin, which inhibits synaptic release by cleaving synaptobrevin. The effects of tetanus toxin on neuropeptide expression recapitulate the effects of manipulating circuit activity by disrupting signaling molecules or by altering O₂ levels. If our observations are generalizable, neuropeptide transcriptional reporters provide a way to dissect tonic modulation of secretory activity in neural networks.

The ability of RMG interneurons to alter behavioral state according to ambient O₂ is inhibited by an NPY/RFamide-like neuropeptide receptor, NPR-1. The npr-1 allele found in the N2 standard C. elegans laboratory strain, npr-1 215V, essentially abolishes RMG-mediated escape responses in feeding animals. Our data suggest npr-1 acts by inhibiting neurosecretion from RMG downstream of Ca²⁺. First, we do not observe a striking difference in O₂-evoked Ca²⁺ responses in RMG between npr-1 and npr-1 215V animals. Second, whereas ChR2 activation of RMG can induce npr-1 animals kept at 7% O₂ to switch to rapid movement, it has little effect on animals encoding the NPR-1 215V receptor, even at 21% O₂ when RMG Ca²⁺ levels are high. Third, transcriptional reporters of neuropeptide genes suggest that NPR-1 215V inhibits neurosecretion from RMG and other neurons. NPR-1 may inhibit synaptic release by altering the balance between G_o and G_q signaling in favor of G_o. Increasing G_q activity, by disrupting the EAT-16 RGS protein or its binding protein RSBP-1, phenocopies the effects of disrupting npr-1. EAT-16 and RSBP-1 activate the G_q GTPase.

RMG lies at the center of an anatomically defined hub-and-spoke network connected by gap junctions that include the ASK, ADL, ASH, and URX sensory neurons (Figure 11). Previous work suggested that the RMG hub redistributes inputs across the spoke sensory neurons through gap junctions to direct behavioral responses (Macosko et al., 2009). The importance of gap junctions in the circuit was inferred from the anatomy and genetic manipulations. Whether RMG could propagate electrical signals or Ca²⁺ across the network, and if so in which direction, or if gap junctions were rectifying or passive gates was unknown. Our data revise our understanding of how this circuit works, as detailed below.

ASK are ‘OFF’ neurons that act analogously to vertebrate photoreceptors, responding to ascaroside pheromones or food stimuli with a decrease in Ca²⁺ (Macosko et al., 2009; Wakabayashi et al., 2009). ASK neurons were proposed to be a major output of the RMG hub-and-spoke network, inducing rapid movement at 21% O₂ and promoting aggregation in response to pheromones (Macosko et al., 2009). In this model, RMG stimulates ASK excitability via gap junctions in aggregating strains, but in solitary strains this is prevented because NPR-1 215V directly or indirectly inhibits gap junctional signaling in the network. We find that ablating ASK neurons or reducing their activity using halorhodopsin does not disrupt either O₂-evoked changes in locomotory activity, or aggregation behavior. These data suggest ASK is not required for either of these responses, although we cannot exclude that it acts redundantly with other neurons to promote these behaviors.

The ASK ablation result contrasts with both our own data (Tremain, 2004) and those of others (Macosko et al., 2009). Both these studies show that npr-1 animals defective in the cGMP gated ion channel TAX-4 fail to aggregate or to move rapidly in normoxia, and that these behaviors can be restored by expressing TAX-4 in URX and ASK (Tremain, 2004; Macosko et al., 2009). Why does ablating ASK have different behavioral consequences from disrupting cGMP signaling in this neuron? One possibility is that removing tax-4 reduces the basal excitability of ASK (and other TAX-4-expressing neurons) and shunts current from RMG, compromising its ability to direct O₂ responses. However, since tax-4 mutations disrupt many sensory modalities (de Bono and Maricq, 2005), other explanations are also possible.

Disrupting the OCR-2 TRPV-like cation channel attenuates the switch in locomotory state evoked in npr-1 animals by 21% O₂. Restoring ocr-2 to any one of the ASH, ADL, or ADF neurons restores O₂ modulation of locomotion to ocr-2; npr-1 animals. Injecting current in ASH neurons using channelrhodopsin also restores strong modulation of locomotion by O₂ to ocr-2; npr-1 mutants, consistent with ocr-2 mutations chronically reducing ASH activity. The neuroanatomy suggests that ASH and ADL are gap-junctionally connected to RMG. Although a rise in O₂ evokes an increase in ASH Ca²⁺, several findings argue against a simple ‘hub-to-spoke’ model in which ASH and ADL spokes are necessary to relay RMG hub activity outwards to evoke behavioral responses. The Ca²⁺ increase in ASH upon switch to 21% O₂ is very small. Ablating ASH and ADL, or expressing tetanus toxin in these neurons, does not disrupt the O₂ regulation of locomotion. Moreover, selective expression of ocr-2 in ADF neurons, which are not gap-junctionally connected to RMG, can partially rescue the ocr-2; npr-1 phenotype. Nevertheless, OCR-2 currents in ASH or ADL can influence the O₂-evoked changes in locomotion. How? One model is that tonic/chronic OCR-2 channel activity in ASH and ADL keeps these neurons depolarized and limits the current shunted locally from RMG to these neurons. Several observations support this possibility. First, expression of neuropeptide reporters in RMG is downregulated in ocr-2; npr-1 animals to levels found in npr-1 animals kept at 7% O₂. This suggests that loss of TRPV activity reduces neurosecretion from RMG. Second, the ability of ocr-2 expression in ASH or ADL neurons to rescue the ocr-2; npr-1 phenotype does not require neurotransmission, suggesting gap junctions are involved. Third, injecting current into ASH can restore to ocr-2; npr-1 animals the O₂ regulated switch in high locomotory activity. Fourth, injecting current directly into RMG using channelrhodopsin can also rescue the ocr-2; npr-1 phenotype, indicating that increasing RMG Ca²⁺ is sufficient to bypass the lack of OCR-2-dependent activity. Unexpectedly, deleting ocr-2 did not affect the steady state O₂-evoked Ca²⁺ responses in the RMG cell body, suggesting that any effects on RMG Ca²⁺ dynamics are local. The simplest model that explains our data is that OCR-2 signaling in ASH and ADL prevents shunting of current, or loss of a Ca²⁺-dependent second messenger, from RMG to ASH and ADL through gap junctions. Membrane potential and Ca²⁺ dynamics are not necessarily coupled, and a local current leak through gap junctions can be functionally important without necessarily altering Ca²⁺ at the soma.

We show that none of the spoke neurons of the RMG ‘hub and spoke’ are individually necessary for RMG to alter the behavioral state in response to changes in O₂ in npr-1 animals. This contrasts with the analysis of an anatomically similar circuit that mediates nose touch perception in C. elegans (Chatzigeorgiou and Schafer, 2011). In that circuit, OLQ and CEP mechanoreceptors that are coupled by gap junctions to each other and to the RIH hub interneuron act as coincidence detectors, pooling information through RIH, which in turn enables the high threshold FLP mechanoreceptors, also connected to RIH via gap junctions, to evoke a response to a gentle nose touch. In the RMG circuit, reducing RMG neurosecretory output by cell-selective expression of tetanus toxin or RNAi knockdown of the carboxypeptidase E egl-21 strongly reduces transmission of the high O₂ information, suggesting that peptidergic release from RMG is a major output of the URX and RMG couple.

Optogenetic experiments coupled with imaging experiments in freely moving animals provide insights into the information carried by different neurons in the O₂ circuit. RMG tracks O₂ levels, becoming more active at higher O₂ levels. Increased RMG activity drives faster forward movement and inhibits short reversals. However, RMG activity does not change when animals slow down or execute a reversal. Thus, RMG provides modulatory input but is not actually part of the circuit executing the behavior. Like RMG, AVB and AIY have, on average, increased Ca²⁺ at 21% O₂. However, unlike RMG, these neurons do not show continuously high Ca²⁺ levels. Instead, they show switches in Ca²⁺ levels that correlate with reversal behavior. Most likely, the connectivity within the reversal modules (AIA, AIB, AIY, AIZ, AVA, and AVB) shapes a sequence of neuronal activation triggering/terminating the reversals. Modulation of the AVB and AIY interneurons by O₂ acts on top of that patterned activity to control overall locomotory pattern. AIA, AIB, and AVA neurons do not appear to be modulated by O₂ levels, and are involved in the reversal behavioral sequence irrespective of O₂ concentration (Figure 9D–F).

For behavioral effectiveness, each module state should enhance a specific behavior while suppressing incompatible behaviors. Our results suggest a direct role for AVB and AIY in both promoting high speed and suppressing reversals at high [O₂]. Such functions have been proposed for these neurons in other contexts (Kawano et al., 2011). Interestingly, AIY was also proposed to be involved in the control of lifespan and metabolism, something also modulated by O₂ (Shen et al., 2010). Potentially, information about O₂ concentration could flow from URX–RMG to AVB and AIY in part through neuropeptide secretion.

Persistent changes in behavior and physiology require persistent changes in neural network activity. We identified several layers of neurons that display sustained changes in activity according to ambient O₂. However, the logic of the activity of different layers differs. In the O₂-sensing neurons, URX, AQR, and PQR, Ca²⁺ sensors reported O₂ levels, as expected (Busch et al., 2012). In the first interneuronal layer, including the RMG hub interneuron and potentially the PVP interneurons, Ca²⁺ levels also tracked O₂ concentration, although these neurons probably relay other information besides O₂ concentration, such as activity of the TRPV expressing neurons. Moreover, the activity level of RMG controls behavioral state rather than directly commanding a specific action: RMG activity remains high at 21% O₂ regardless of the animal's speed or direction of travel. By contrast, the second and third layers of interneurons modulated by O₂ appear to be directly involved in generating specific behaviors. Ca²⁺ changes in these neurons anticipate or report the animal's behavior. The activity of some but not all of these neurons, including AVB and AIY, is modulated but not set by O₂ input. These downstream interneurons are probably not dedicated to a small subset of sensory inputs, as RMG or PVP might be, but instead simultaneously translate multiple streams of information into the appropriate behaviors.

Changing RMG activity is sufficient to evoke the behavioral states associated with high and low O₂. Downstream of URX–RMG we find widespread modulation of neurons and neuropeptide expression/secretion, highlighting the complexity of even a simple contextual cue, O₂. Neuropeptide signaling appears to be key to generating the behavioral switch. Similar to motivated states, organismic states re-organize the salience of different sensory cues and change the physiology of the animal. It is tempting to propose that peptides whose release is modulated by O₂ re-organize sensory responses and modify the physiology of C. elegans. Studies of cross-modulation of CO₂ avoidance by the O₂ circuit support this scenario (Carrillo et al., 2013; Kodama-Namba et al., 2013). A similar example of how activation of a neuron and secretion of its associated peptides can coordinate behavioral and physiological responses is provided by mammalian tonic nociceptive neurons. In addition to amplifying the nociceptive signal in the spinal cord, the release of tachykinin and CGRP peptides from the nociceptive neurons alters gene expression in the surrounding tissues, producing neurogenic inflammation (Carlton, 2014).

Behavioral states and emotions are under intense investigation in mammalian systems, but the circuits engaged in implementing these states are only partially mapped. Changes in the global state of mammals are proposed to involve altered activities in multiple brain areas. Here, we outline how a global organismic state is encoded in a system that can be more easily circumscribed and comprehensively dissected.

C. elegans was grown under standard conditions unless otherwise indicated (Sulston and Hodgkin, 1988). To cultivate animals in defined O₂ environments, we used a Coy O₂ control glove box (Coy, Grass Lake, Michigan, USA).

Sample preparation: To prevent aggregation behavior, which could confound our comparisons (Andersen et al., 2014), we grew animals at very low density on a thin lawn of Escherichia coli OP50 spread to the edges of a 6 cm petri dish. To obtain a thin lawn, we grew bacteria on NGM containing only 5% of the regular amount of peptone. We picked five gravid hermaphrodites onto the lawn, let them lay eggs for 2–3 hr, and then removed them. The ∼40 eggs were allowed to hatch and grow to adult worms at the indicated O₂ concentration. Animals from 10 such plates were rinsed off in M9 buffer and their RNA was extracted using RNeasy Mini Kits (Qiagen, Germany). cDNA libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) and sequenced on an Illumina HiSeq 2500 platform. We generated independent libraries of biological replicates as follows: npr-1, four libraries for 21% O₂ and six for 7% O₂; gcy-35; npr-1, seven libraries for 21% O₂ and seven for 7% O₂. Approximately 30 million 50 bp single-end reads were produced for each sample.

RNA-seq data analysis: Reads were output in FASTQ format and their quality assessed using FastQC v0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). A small portion (3–4%) of reads containing over-represented sequences (e.g., Illumina adapters) identified by FastQC were removed with Trimmomatic v0.30 (Bolger et al., 2014). The remaining reads were aligned to the C. elegans genome (WBcel235) with TopHat v2.0.13 (Kim et al., 2013). TopHat was run using default parameters with the following exceptions: coverage search was disabled using --no-coverage-search; library-type was changed to fr-firststrand; and the WBcel235 transcriptome annotations were provided via --transcriptome-index. In addition, since C. elegans has comparatively short introns (Steijger et al., 2013), --min-intron-length and --min-segment-intron were both reduced to 30. The aligned reads were then processed using the Cufflinks suite v2.2.1 (Trapnell et al., 2012) to assemble transcripts and ultimately compute differential gene expression between conditions. Transcripts were assembled for each sample with the Cufflinks tool (Trapnell et al., 2010), again lowering --min-intron-length, as well as --overlap-radius, to 30. The sample transcript assemblies were then merged with reference annotations (WBcel235) using Cuffmerge to generate a single, overall transcript assembly. The Cuffquant tool, using this merged assembly, was then used to compute gene and transcript abundances for each sample. Finally, sample abundances were integrated with the merged assembly by Cuffdiff (Trapnell et al., 2013) to test for differential expression between all condition pairs. All the Cufflinks tools, aside from Cuffmerge, were used with the --library-type parameter again set to fr-firststrand and both --frag-bias-correct (Roberts et al., 2011) and --multi-read-correct enabled. A gene was considered as differentially expressed between two conditions if the q-value (p-value after Benjamini-Hochberg correction for multiple testing) for the given comparison was <0.05.

The gene-level differential expression output from Cuffdiff was used to generate the presented excel files, which were annotated with WormBase IDs (WBcel235) and InterPro domains using custom Perl scripts.

DNA cloning was carried out using standard methods (Green and Sambrook, 2012). Promoters used in this work included: gcy-32 (Yu et al., 1997), sra-9 (Troemel et al., 1995), pdf-1 (Barrios et al., 2012), flp-8 (Kim and Li, 2004), flp-21 short (Kim and Li, 2004), flp-21 long (Macosko et al., 2009), gpa-11 (Jansen et al., 1999), srh-220 ADL (F47C12.5) (Troemel et al., 1995), srh-142 ADF (T08G3.3) (Troemel et al., 1995), ins-1 (Tomioka et al., 2006), gcy-28 (Tsunozaki et al., 2008), sra-11 (Troemel et al., 1995), ttx-3 (AIY fragment) (Wenick and Hobert, 2004), and ncs-1 (Macosko et al., 2009). Promoters were cloned into position 1 of the Multisite Gateway system (Invitrogen). Expression clones used in the course of this work are listed in supplemental data.

Microfluidic chambers for Ca²⁺ imaging and behavioral assays were cast as described previously (Busch et al., 2012).

For each assay, 20–25 adult hermaphrodites were transferred onto NGM plates seeded 12–14 hr earlier with 40 μl of E. coli OP50. To control O₂ levels experienced by the worms, we placed a 200 μm deep square PDMS chamber on top of the agar pad, with inlets connected to a PHD 2000 Infusion syringe pump (Harvard Apparatus) delivering humidified gas mixtures at a flow rate of 2.5 ml/min. We began pumping 5 min prior to the start of the recording to ensure that the initial environment was in a steady state. Movies were recorded using FlyCapture on a Leica M165FC dissecting microscope with a Point Grey Grasshopper camera running at two frames per second.

Movies were analyzed using Zentracker, a custom-written Matlab software (available at https://github.com/wormtracker/zentracker). Speed was calculated as instantaneous worm centroid displacement between successive frames. Frames in which the shape of the worm had an eccentricity value (ratio of the two axes of an ellipse with the same second moments) smaller than 0.8 or a compactness value (perimeter²/area) smaller than 30, combined with a solidity value (area/convex hull) greater than 0.575 were identified as omega-turns. To detect reversals, sharp (greater than 60°) changes in direction were first identified. Each potential behavior pattern that could be assigned to each track was then examined using a heuristic algorithm based on penalizing any occurrences of continuous movements lasting for more than 7.5 s in the same direction that do not correspond to forward movements, any time an omega-turn is not followed by forward movement, and cases where a worm does not spend more time in forward than in backward movement. Using this information, directions of movement were determined for each worm in each frame according to the prospective behavioral pattern with the lowest overall penalty score.

ChR2 codon-optimized for C. elegans and C-terminally tagged with mCitrine, or halorhodopsin tagged with mCherry, were expressed from the promoters of interest. Worms were grown on plates pre-seeded with 200 μl of the E. coli OP50, with 15 μl of 5 mM all trans-Retinal (Sigma) dissolved in 100% ethanol added to the bacterial lawn prior to picking the worms onto the plates. Control worms were grown in parallel on seeded plates onto which 15 μl of 100% ethanol was added without the all trans-Retinal. Worms were preselected for fluorescence in the neurons of interest, and then assayed as described above. Light stimuli were delivered using a Leica EL6000 mercury lamp filtered for ChR2 or NpHR excitation using a Leica GFP2 or ET DSR filter, respectively. To avoid unwanted light-activation of the optogenetic actuators, we filtered the trans-illumination light using a 595 nm long-pass red optical cast plastic filter (Edmund Optics) for ChR2, and a 705 nm long-pass colored glass filter (Thorlabs) for NpHR.

We imaged immobilized animals as described previously (Busch et al., 2012). The experimental setup to image freely moving animals on an agar pad is shown in Figure 8—figure supplement 1. Emission light filtered for CFP and YFP wavelengths (460–495 nm and 525–580 nm, respectively), separated using the Tu-Cam system (Andor) with a 510 nm longpass, was relayed onto two identical Photometrics Cascade II 1024 EMCCD cameras running in frame-transfer modes with 60 ms exposure times. The imaged worm was kept in the field of view by moving the Prior stage manually using a joystick, with the acceleration rate set to its lowest value so as to disturb the worm as little as possible. To reconstruct the speed and trajectory of the worm, the actual stage position was continuously logged while acquiring image stacks using Micromanger (Edelstein et al., 2010) or custom-written software.

To deliver gas stimuli to the worm, we placed a square PDMS chamber on top of the agar pad, with inlets connected to a PHD 2000 Infusion syringe pump (Harvard Apparatus) running at a flow rate of 50 μl/s. An electronic valve system placed between the syringes and the PDMS chamber allowed us to switch between two different gas mixtures in a controlled manner at pre-specified frames. Using a spot Optode (PreSens), we monitored the O₂ concentration within the chamber while switching between mixtures containing 7% and 21% O₂, and found that O₂ levels could be switched reliably using this configuration (Figure 8—figure supplement 1).

We used Neuron Analyzer, a custom-written Matlab program, to analyze the resulting image stacks (available at https://github.com/neuronanalyser/neuronanalyser). In movies in which the neuron of interest provided the brightest signal in the field of view, we simply tracked the brightest points in each channel. Where this was not a viable option due to the complexity of the expression pattern, we used a semi-supervised heuristic tracking approach in which in each frame the area within a 20-by-20 pixel square centered around the location of the centroid of the region of interest (ROI) in the previous frame was examined for potential matches. Regions whose intensity differed by less than 1/3 from that of the ROI identified in the previous frame, and whose centroids were located within a 10-pixel radius of the ROI location in the previous frame, were identified as candidates for the ROI in the current frame. If no such candidates were detected, user input was requested in identifying the ROI in the current frame. If exactly one such candidate was detected, it was identified as the ROI in the current frame. If more than one such candidate was detected, match quality scores were calculated for each candidate based on their differences in intensity and centroid location from the ROI in the previous frame. If one candidate had a better quality score in both respects than all others, it was identified as the ROI in the current frame; user input was otherwise requested. In cases where the heuristic tracking method struggled to identify the ROI reliably in an automatic way, we used a user-supervised tracking method, in which a custom-sized moveable region was specified using the GUI, restricting the tracking of the brightest point within this regon of interest.

Once the centroid of the neuron of interest was identified, we calculated the mean of the 20 brightest pixels within a 10-pixel radius of the centroids for both channels independently, and subtracted from this the median pixel intensity of the respective channels as the background. We then corrected for the crosstalk by subtracting 0.6 from the ratio values.

To extract the behavior of the worm during the recording, first a more accurate measure of worm position was obtained based on not only the stage position, but also the location, within the field of view, of a successfully tracked neuron, effectively calculating the position, in stage coordinates, of the centroid of the ROI. The path was then smoothed using a moving average filter, and potential short gaps in the position data were identified, which may reflect the neuron temporarily leaving the field of view due to unexpected worm movements during the recordings. Gaps shorter than 30 frames were then filled using a spline interpolant, while gaps longer than this were excluded from the analysis.

In order to identify reversals, the direction of movement, within the coordinate system of the stage, was calculated for each frame using a central differences method, and changes in direction between successive frames were obtained. Intervals in which the worm moved slower than 10 μm/s were flagged as stationary behavior, and were excluded from further analysis. For the remaining frames, the relative changes in direction between successive frames were then examined, and periods containing changes no larger than 7.5° were classified as consistent movement. Consistent movement lasting longer than 90 s was flagged automatically as forward movements. For consistent intervals lasting shorter than this, user input was requested about the direction of the movement.

Statistics for both Ca²⁺ imaging and behavioral assays used the Mann–Whitney U-test. Where we sought to compare steady state values, we chose time intervals where we expected the fluorescence ratios, or behavioral parameters, to have plateaued, that is, with a delay with respect to the timing of the switch in O₂ concentration or the presentation of blue or green light.

Ca²⁺ imaging: when presenting time series over the duration of an experiment, n refers to the number of animals imaged. When presenting event-triggered averages (e.g., time relative to the onset of a reversal), n indicates the number of events. In this case, each animal was still only imaged once, but each animal could contribute to multiple events.

Behavior: for the intervals of interest, we extracted independent per-subject means deriving from worms flagged as continuously valid for at least 10 s during the interval. A worm was considered valid at a time point if it was not in contact with another animal, it was on the food lawn, and it was located at least half a worm-length from the border. Following these criteria, each worm was sampled at most once per interval; n indicates the minimum number of samples obtained per interval for the two intervals being compared.

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

We thank Dr Yuichi Iino, Dr Chris Li, Dr P Sengupta, Dr R Shingai, the C. elegans Knockout Consortium, the National BioResource Project (Japan) and the Caenorhabditis Genetics Centre for strains, and de Bono and Schafer lab members for comments and advice. We are grateful to James Hadfield and Michelle Pugh at the Cambridge Institute Genomics Core for the RNA sequencing. ZS was supported by a Studentship from the Medical Research Council. MdB acknowledges support from the European Research Council (Advanced ERC Grant 269058).

© 2015, Laurent et al.

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