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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Biol. Author manuscript; available in PMC May 8, 2013.
Published in final edited form as:
Curr Biol. May 8, 2012; 22(9): 743–752.
Published online Apr 5, 2012. doi:  10.1016/j.cub.2012.02.066
PMCID: PMC3350619

Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors



Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons. First, we analyzed the FLP/PVD network by optogenetics and studied integration of signals from these cells in downstream interneurons. Second, we investigated which genes modulate PVD function, based on prior single neuron mRNA profiling of PVD.


Selectively photoactivating PVD, FLP and downstream interneurons using Channelrhodopsin-2 (ChR2) enabled functionally dissecting this nociceptive network, without interfering signals by other mechanoreceptors. Forward or reverse escape behaviors were determined by PVD and FLP, via integration by command interneurons. To identify mediators of PVD function, acting downstream of primary nocisensor molecules, we knocked down PVD-specific transcripts by RNAi and quantified light-evoked PVD-dependent behavior. Cell-specific disruption of synaptobrevin or voltage-gated Ca2+-channels (VGCCs) showed that PVD signals chemically to command interneurons. Knocking down the DEG/ENaC channel ASIC-1 and the TRPM channel GTL-1 indicated that ASIC-1 may extend PVD’s dynamic range and that GTL-1 may amplify its signals. These channels act cell-autonomously in PVD, downstream of primary mechanosensory molecules.


Our work implicates TRPM channels in modifying excitability of, and DEG/ENaCs in potentiating signal output from a mechano-nociceptor neuron. ASIC-1 and GTL-1 homologues, if functionally conserved, may denote valid targets for novel analgesics.


Several protein classes are implicated in the primary sensory signal transduction machinery in nociceptive cells, e.g. transient receptor potential (TRP) channels, degenerins/epithelial Na+-channels (DEG/ENaCs), and two-pore domain K+-channels (K2P) [14]. Nociceptor neuron signaling is facilitated or modulated by further molecules like neurotransmitters and - peptides, eicosanoids, neurotrophins, cyto- and chemokines, voltage-gated Na+-, K+- and Ca2+-channels, opioid and purinergic receptors, as well as TRP-channels [1]. Dissection of neural circuits and identification of nociceptor modulators is challenging in higher animals, which possess myriads of contributing neurons. In contrast, only few neurons mediate nociception in C. elegans, which responds to aversive stimuli like touch, heat, odorants, toxins, and non-physiological osmolarity or pH [5]. Six touch receptor neurons (TRN) detect “gentle touch” (e.g. by an eyelash) to anterior or posterior regions, evoking forward or reverse withdrawal reflexes, respectively [6]. Nociceptive "harsh touch", e.g. prodding with a platinum wire, is sensed by four additional neurons, pairs of FLP and PVD. These cover head (FLP) or body (PVD) with extensive dendritic arbors (Figure 1A) and also respond to acute heat or cold, respectively [710]. The DEG/ENaCs MEC-10 and DEGT-1 were suggested as putative components of the nociceptive mechanotransduction channel in PVD (though MEC-10’s contribution was questioned [3,11]), whereas TRPA-1 confers cold responses, as indicated by Ca2+-imaging [7]. Additional neurons putatively contributing to harsh touch sensation were identified by laser ablation and scoring fractions of animals responding to harsh touch insults to different body regions [3].

Figure 1
Photostimulation of PVD results in forward escape behavior

PVD directs equal numbers of synapses to the command interneurons PVC and AVA, which control forward and backward movement, respectively, while FLP mainly synapses to the backward command neurons AVA and AVD [12] (Figure S1). Harsh mechanical stimuli agitate large portions of the body, likely co-activating FLP and PVD. Animals must integrate mechanosensory input to predict optimal escape behavior (e.g. from predators). Thus the relative activation of various sensory and command neurons likely determines locomotor output.

Analyzing behavioral effects of hitting a worm with a wire in a cell-specific and quantifiable manner is difficult; thus it is unknown how these multidendritic neurons orchestrate the prevailing behavioral response. Unlike prodding, optogenetic tools such as the light-gated cation channel ChR2 allow selective non-invasive photostimulation of neurons of interest without perturbing others, particularly in the thin, transparent worm. Also, photostimulation can be performed more controllably and consistently. This approach enabled triggering sensory neuron activity to phenocopy endogenous behaviors, e.g. in C. elegans TRNs [13,14], putative harsh touch cells [3], aversive chemosensory neurons [15], and in multidendritic neurons in Drosophila larvae [16]. Here, we optogenetically dissect an entire harsh touch neuronal network at the single neuron level. As ChR2 directly depolarizes PVD, thus bypassing the primary mechanotransduction channels, we could uncover genes required for nociceptor function within PVD, downstream of primary sensory molecules. PVD evokes behavior across just three synaptic layers, including the NMJ. Hence, quantifying escape velocity of knockdown or knockout lines provided an accurate readout for PVD functionality. We demonstrate that the TRPM channel GTL-1 likely amplifies PVD signals, while the DEG/ENaC ASIC-1 facilitates signal output from PVD and determines the promptness of the behavioral response.


Photoactivation of PVD evokes rapid forward escape responses

To study the PVD-associated neural network, we sought to stimulate PVD without concomitantly activating other mechanosensors. We co-expressed ChR2(H134R)::mCherry and GFP using the F49H12.4 promoter [17] (zxIs12; Figure 1A) in lite-1(ce314) mutants [18] to avoid photophobic responses. When raised in the presence of the ChR2 chromophore all-trans retinal (ATR), zxIs12 animals illuminated with blue-light pulses of 0.2, 1, or 5s (Figure 1B; Movie S1) showed rapid forward escape responses. In contrast, a previous study reported reversals upon midbody harsh touch [10]. This indicates differences in optical (i.e. PVD-specific) vs. mechanical harsh touch stimulus perception (likely involving additional mechanoreceptive neurons). The evoked behavior depended on developmental stage, possibly correlating with PVD size (increasing until adulthood) and branching (Figure 1C). Of note, PVD neurons are born in the L2 larval stage, 1° longitudinal branches extend during L3 and complete branching is achieved by the end of L4 [9,19].

In addition to PVD, the F49H12.4 promoter also expresses in a head neuron (identified as AQR) and a tail neuron (Figure 1A). To exclude contributions of these cells to the observed behavior, we illuminated predefined body segments of freely moving animals that were simultaneously tracked (Figure 2A–C, Movie S2). In a recent report, ablation of AQR reduced responses to anterior harsh touch, and concomitant photoactivation of AQR, SDQR and BDU neurons evoked reversals [3]. In contrast, we observed no escape behavior when selectively photoactivating AQR (indicating that BDU and SDQR are responsible for photoevoked behaviors reported by Li et al. [3]), while illuminating the region containing the PVD soma robustly evoked acceleration. We observed minor responses when illuminating the tail neuron, probably due to concomitant illumination of a small posterior area of PVD. Accelerations were also evoked by illuminating different small areas of PVDs anterior dendrites (Figure S2). Thus, the forward escape we observed is specifically evoked by PVD photoactivation.

Figure 2
Selective illumination of PVD cell body and of other cells expressing ChR2

Less habituation to harsh than gentle touch or photostimuli

Gentle touch is subject to substantial habituation in C. elegans [20]. However, as nociceptors detect potential threats and evoke withdrawal to avoid tissue damage, we wondered whether PVD showed any pronounced habituation to repeating or continuing noxious insults. To test this, we used a “slow” ChR2 variant (C128S mutant with much slower off-kinetics) [21,22]. A 0.2s light pulse resulted in continuous forward locomotion for minutes (Movie S3). For repeated stimuli, we again used ChR2(H134R), applied 20 light pulses of 0.5s and plotted the fractions of animals responding to each stimulus (Figure 3A). Compared to withdrawal responses of animals expressing ChR2 in TRNs using Pmec-4::ChR2(H134R), PVD-evoked escape responses habituated less. This was confirmed by mechanical touch assays (Figure 3B), showing that nociceptive harsh touch is less susceptible to habituation than gentle touch.

Figure 3
Both mechanical and optical nociceptive harsh touch stimuli elicit less habituation than gentle touch stimuli

Optogenetic dissection of the harsh touch nociceptive neural network

We mainly observed forward locomotion when PVD was photostimulated, while midbody harsh touch reportedly causes reversals [12], potentially through recruitment of additional mechanosensory neurons. We thus asked whether reversals may be triggered by concomitant photoactivation of such candidate cells. When PVD somata were photostimulated with a fixed light intensity, co-activation of anterior TRNs (ALM, AVM) with increasing light intensities gradually shifted escape from forward to reverse (Figure 4A, Figure S3A–I). Moreover, reversals prevailed when PVD soma stimulation was reduced, while TRN stimulation was kept maximal (i.e. mimicking a more anteriorly presented harsh touch). Thus, relative stimulus strengths that different mechanosensory neurons receive determine the probability of forward or backward escape responses, likely via integration by downstream command interneurons. Interestingly, PVD synapses equally onto forward and backward command neurons PVC and AVA (28 and 27 synapses, respectively) [12] (Figure 4B, Figure S1). The exact nature of these synapses is unknown, i.e. whether they are all excitatory or if some are inhibitory. Mechanical harsh touch was shown to induce Ca2+-increases in PVC, which were higher than those evoked by gentle touch [3]. First, we directly examined whether PVD activates PVC. We phototriggered PVD and measured Ca2+-responses in PVC using a novel, red-fluorescent, genetically encoded Ca2+-sensor, RCaMP (Akerboom et al., in preparation), which requires yellow-light excitation and thus can be used without unwanted co-activation of ChR2. PVD photostimulation evoked robust Ca2+-increases in PVC (Figure 4C, Movie S4). Second, we photoactivated PVD in a deg-1(d) mutant background in which PVC neurons degenerate, thus limiting PVD output to AVA. Animals now moved backward (Figure 4D, E; Movie S5), as confirmed by mechanical touch assays. Thus, PVD-AVA synapses are functional and (net) excitatory; however, in sum, PVD-PVC synapses must be more excitatory than PVD-AVA synapses.

Figure 4
Functional analysis of the PVD and FLP harsh touch nociceptor network

Interestingly, mec-4(d) mutants lacking TRNs responded with accelerations when we mechanically stimulated the tail (likely due to PVD activation) and reversed when anteriorly poked (Figure 4D). This may be explained by the action of additional mechanosensory neurons; possible candidates are the branched FLP neurons that envelop the anterior region and mainly synapse to AVA. Indeed, selective FLP photoactivation resulted in instantaneous reversals (Figure 4F; Movie S6). As command interneurons must integrate PVD and FLP harsh touch signals, behaviors evoked by the sensory neurons may be mimicked via photoactivation of command cells. In animals expressing Pglr-1::ChR2::mCherry (in command neurons and other cells), selective tail illumination, activating only PVC, triggered forward movement, while head illumination, activating AVD, AVA, AVB (and other) neurons, evoked robust reversals. Illuminating all command neurons caused mild reversals (Figure S3J–L; Movie S7). In sum, PVDs and FLPs constitute a nociceptive network responding to harsh mechanical insults and orchestrate forward vs. backward escape reflexes via the command neurons.

An RNAi screen with optogenetic readout identifies genes required for PVD function

Optical stimulation, together with genetic tools in C. elegans, allowed us to unravel factors required for PVD function, acting downstream of the primary sensory proteins; Figure 5A describes our strategy. Previously, FLAG-tagged poly-A binding protein was expressed specifically in PVD (and OLL) neurons and used to pull down mRNAs, which were identified by microarray profiling [19]. We chose candidate genes (Table S1) from this data set and knocked them down systemically, by feeding bacteria containing dsRNA targeting the respective gene [23] to animals sensitized for RNAi in neurons (nre-1; lin-15b; zxIs12) [24]. To test gene knockdown efficiency, we monitored mRNA levels of several targeted genes by RT-PCR and further confirmed feasibility of RNAi in PVD by effective GFP knockdown (Figure S4). Adverse effects of gene knockdown on PVD function were monitored by calculating the fraction of animals responding to photostimulation (Figure 5B).

Figure 5
Optogenetics-assisted functional RNAi screen of PVD-enriched genes

We first targeted genes known to affect PVD development: in mec-3 or unc-86 (encoding transcription factors) knockdown animals, PVD neurons lacked all but the primary branches (Figure S4B) [19,25]. This loss also impaired phototriggered escape responses (Figure 5B). Thus, only full branching of PVD may permit efficient ChR2-mediated behavior, e.g. as more PVD plasma membrane may accommodate more ChR2. Alternatively, MEC-3 and UNC-86 may regulate genes required for PVD signaling. The EFF-1 fusogen is implicated in ordered branching of PVD and is required for mechanical harsh touch responses [9]. Yet, eff-1 knockdown did not reduce photoevoked PVD-dependent escape behavior, although these animals had malformed dendrites (Figure S4B). This suggests that PVD synapses are not affected by eff-1 and that branching of PVD is crucial for its sensory function, but not for its ability to evoke neurotransmission.

PVD uses chemical transmission that requires VGCC subunits UNC-2 and CCB-1

Although PVD has no known electrical synapses in the ventral nerve cord [12], the innexin mRNA inx-19 was highly enriched in PVD (and/or OLL) neurons, possibly forming gap junctions in the branches. However, the inx-19(ky634) mutation did not affect PVD-dependent behaviors and neither did knockdown of INX-19 or UNC-7 innexins (Figure 5B), despite clear reduction of their transcripts (Figure S4A). In contrast, PVD-specific disruption of chemical synaptic transmission by expressing Tetanus toxin light-chain (TeTx) abolished PVD-mediated escape behavior (Figure 6A, B). Importantly, expression of TeTx in TRNs did not affect PVD-dependent behavior. Among PVD-enriched transcripts possibly acting in neurotransmission were the VGCC α-subunits egl-19 and unc-2 and the β-subunit ccb-1. Knockdown of unc-2 and ccb-1 (but not egl-19) essentially eliminated photoevoked reactions (Figure 5B). To assess whether these channel subunits act in nociception cell-autonomously in PVD, we expressed sense and antisense RNAs [26] of unc-2 or ccb-1 from the F49H12.4 promoter (Figure S5A–C). The resulting PVD-specific RNAi strains responded significantly less to PVD photoactivation (Figure 6C, D), yet remained responsive to gentle touch (Figure S5D). Thus, CCB-1 and UNC-2 are likely essential for PVD chemical transmission; alternatively, they may enhance PVD excitability. Notably, our approach allows identifying genes acting in nociception within PVD, downstream of primary sensor molecules.

Figure 6
Inhibition of synaptic signaling and cell-specific knockdown of unc-2 and ccb-1 impair PVD function

The TRPM channel GTL-1 may amplify signals within PVD, while the DEG/ENaC channel ASIC-1 may extend PVD’s dynamic range and accelerate signal output

TRP and DEG/ENaC channels were previously implicated in nociception in several organisms, either as sensors, signal facilitators, or enhancing neurotransmission [14,27,28]. Among transcripts enriched in PVD/OLL were four DEG/ENaCs, mec-10, degt-1, del-1 and asic-1, and three TRP channels, trp-2, gon-2 and gtl-1. Light-responses were unaffected by mec-10, del-1, degt-1 and gon-2 knockdown or introduction of the trp-2(gk298) allele (Figure 5B). gtl-1 knockdown moderately reduced reactions to PVD photoactivation, and these effects were significant in gtl-1(ok375) mutants. Likewise, asic-1(ok415) mutants were significantly affected. We investigated potential cell-autonomous roles of asic-1 and gtl-1 in PVD-specific knockdown lines asic-1 RNAi(PVD) and gtl-1 RNAi(PVD). We used different light intensities and whole-body illumination, measuring fractions of animals responding (Figure 7A). Regression analysis of fitted Boltzmann sigmoidals showed reduced maximal fractions of animals reacting for both PVD-specific RNAi lines, and reduced responsiveness at all light intensities was observed for gtl-1 RNAi(PVD) animals, reflected by a significantly right-shifted sigmoidal.

Figure 7
ASIC-1 and GTL-1 accentuate and amplify ChR2-evoked signals in PVD

We also analyzed how escape velocities evolved over time as a sensitive readout for promptness and amplitude of PVD output. When selectively illuminating PVD cell bodies and tracking the animals, loss of ASIC-1 caused a ~50% delay to reach the maximal speed, while GTL-1 deficiency did not affect the immediateness of escape (Figure 7B). Maximal velocities for asic-1 RNAi(PVD) animals differed from wild type only for the highest light intensity used (Figure 7C, Figure S6). However, gtl-1 RNAi(PVD) animals showed reduced maximal velocities over a much broader range of intensities. Consistent with Figure 7A, this also caused an overall right-shift of stimulus intensity vs. fraction responding (Figure 7D). Importantly, these effects were not due to variations in ChR2::mCherry expression in PVD in knockdown or knockout lines (Figure S5E). In sum, ASIC-1 may expand the dynamic range of PVD signal output, particularly for strong noxious stimuli where a boost of synaptic transmission could allow faster escape, while GTL-1 may generally amplify signals within PVD, i.e. at all stimulus intensities.


Using an optogenetic approach, we defined the function of PVD and FLP harsh touch nociceptors and downstream interneurons in a network mediating escape behavior. Furthermore, we identified factors required for transmitter output from PVD and/or enhanced depolarization. We found that the TRPM channel GTL-1 may amplify signals within PVD, whereas the DEG/ENaC ASIC-1 potentiates PVD output at high stimulus regimes, thus expanding the dynamic range of PVD and enabling faster nocifensive behaviors.

Optogenetic deconstruction of a harsh touch nociceptive network

The multidendritic FLP and PVD neurons were previously reported to sense harsh touch. Recently, additional cells (BDU, SDQR, AQR, ADE, PDE, PHA and PHB) contributing to harsh touch sensation were identified by laser ablation, although it is unclear whether these cells are sensors or participate indirectly [3]. When we photoactivated AQR, we observed no acute behavior. Similarly, photostimulation of PDE failed to evoke an escape response ([3] and data not shown). Here, we focused on escape behavior induced by FLP and PVD, and characterized their integration into the downstream command interneuron network. Studying the behavioral output of the mechanosensory modality of PVD or FLP cells was previously complicated as harsh touch agitates the whole animal, co-evoking contributions from TRNs or other mechanosensors. Hence, we selectively photoactivated PVD or FLP, causing forward or reverse escape responses, respectively.

Equivalent numbers of synapses link PVD to forward and backward command neurons, PVC and AVA [12]. Forward escape depended on PVC, in which Ca2+-transients were evoked by PVD photostimulation. Upon PVC ablation, PVD photoactivation evoked backward escape via PVD-AVA synapses. Direction-specific responses were mimicked by selective PVC photoactivation, resulting in forward movement, whereas illumination of backward command neurons AVA, AVD, AVE (and one of the two forward command neurons, AVB) evoked reversals. When PVC was co-activated, backward responses prevailed (although decreased), suggesting that behavioral “output” depends on the strength of synaptic inputs (number, synaptic weight) that individual interneurons receive. Also, the relative input that different sensory neurons receive (e.g. by touch at different positions, as mimicked by co-activation of PVD and anterior TRNs using different light intensities), determines the probability of forward vs. backward escape. In sum, PVD, FLP and other mechanoreceptors like TRNs signal to command neurons, which integrate this information, coordinating the prevailing escape response.

Combining mRNA profiling, RNAi and optogenetics to identify nociceptor genes

We combined quantifying optogenetically triggered behavior and RNAi screening, to identify and characterize genes critical for PVD-mediated nociceptive behavior. Here, we focused on ion channels and genes acting in neurotransmission, among transcripts we found to be enriched in PVD by microarray profiling [19]. Following RNAi (or in knockouts), we identified genes affecting PVD-specific, light-induced escape behavior. Further assays showed cell-autonomous functions for several genes, yielding information on the nature of PVD signaling defects. Thus, we revealed genes acting downstream of the primary PVD mechanosensory cation channels, which were bypassed by photodepolarization.

VGCC subunits UNC-2 and CCB-1 are crucial for PVD function

VGCCs mediate Ca2+-influx after membrane depolarization, triggering neurotransmission, and/or causing further depolarization. Two VGCC α- (EGL-19, UNC-2) and one β-subunit (CCB-1) are enriched in PVD. EGL-19 L-type VGCCs are critical particularly for muscle function [29], yet knocking down egl-19 did not reduce the frequency of animals responding to PVD stimulation. UNC-2, homologous to mammalian CACNA1A, functions mainly in neurons [30]. Phototriggered, PVD-mediated escape behavior was almost completely suppressed by PVD-specific unc-2 and ccb-1 knockdown, demonstrating cell-autonomous roles in PVD excitatory output. PVD signals mainly, if not exclusively, by chemical transmission, as PVD-specific expression of TeTx completely abolished escape responses and knockdown of innexins expressed in PVD had no effect. CCB-1 and UNC-2 specifically localize to pre-synaptic terminals ([31] and K. Shen, personal communication), consistent with their apparent function in chemical neurotransmission.

ASIC-1 and GTL-1 act within PVD for signal output and amplification

DEG/ENaC and TRP channels function in mechanosensation, forming mechanotransduction channels, or by contributing indirectly to mechanoreceptor potentials [1]. Precise roles of these channels often remain elusive. The DEG/ENaCs MEC-4 and MEC-10 form heteromeric mechanoelectrical transduction channels in TRNs [4]. Impairing mec-10 and degt-1 eliminated Ca2+-transients in PVD upon harsh touch, thus their gene products were suggested to form the mechanotransduction channel in PVD [7]. However, mec-10(tm1552) mutants retained mechanosensitive currents in PVD [3], and similar findings were made in TRNs [11]. Possibly, additional channels contribute to the primary sensation, as shown for nose touch perception in ASH neurons [32]. Knockdown of mec-10 and degt-1 did not perturb the ChR2-mediated escape response, emphasizing that they do not determine general PVD physiology or excitability.

In mice, removal of Brain Na+-Channel 1 (BNC1/ASIC2), homologous to MEC-4/-10, reduces sensitivity of low-threshold mechanoreceptors [33]. Disruption of the related DRASIC/ASIC3 sensitized gentle touch mechanoreceptors, but reduced sensitivity of nociceptors responding to noxious pinch, heat or acid [34]. Thus, only modulatory effects were observed for these vertebrate ASICs, suggesting roles as signal facilitators. Similarly, PVD-specific knockdown of ASIC-1 reduced ChR2-mediated escape behavior and delayed animals in reaching maximal velocity, thus ASIC-1 may modulate PVD output. Acidification of the synaptic cleft, resulting from synaptic vesicle fusion, may trigger this putative proton-gated Na+-channel, increasing presynaptic depolarization and neurotransmission, as suggested for dopaminergic neurons in which ASIC-1 facilitated associative learning [28]. Likewise, vesicular protons were proposed to enhance neurotransmission via ASIC to modulate synaptic plasticity in hippocampal neurons and to affect learning and memory [35]. We suggest that ASIC-1 might augment PVD depolarization, thereby expanding its dynamic range and speeding up the escape reaction.

Many TRP channels mediate noxious stimulus detection, yet, it is often unclear if they directly transduce mechanical stimuli or if they function in downstream signaling. In Drosophila, the Painless TRP channel [36] was suggested to amplify mechanically gated currents mediated by the Pickpocket DEG/ENaC [37]. Similarly, the TRPN channel NompC was assumed to act downstream of the TRPVs Nanchung and Inactive as a potential amplifier [38,39], while a study on amplification of sound-evoked vibrations of the fly hearing organ led to opposite conclusions [27]. C. elegans contains at least 24 TRP channels, in each of the subdivisions identified in mammals [40]. TRP-4 was shown to be a mechanotransduction channel, as mutations in the pore altered mechanoreceptor current ion-selectivity [2]. TRPA-1 is required for nose-touch responses [41] and for cold sensation in PVD [7]. OSM-9/OCR-2 TRPV channels function in the polymodal neuron ASH to mediate different aversive stimuli including noxious mechanosensation [42]; yet, they do not act as mechanoelectrical transduction channels in ASH, and thus may function in signal encoding or transmission [32]. Our results suggest similar functions for the TRPM channel GTL-1. PVD-specific GTL-1 knockdown rendered fewer animals responsive to PVD photoactivation, and resulted in significantly lower velocity increases at all but the smallest stimulus amplitudes. As ChR2 bypasses natural nociceptive stimuli, we suggest that GTL-1 may cell-autonomously enhance PVD depolarization as an amplifier, potentially by increasing membrane Ca2+-permeability.

In conclusion, we analyzed a nociceptive circuit at single neuron level and combined mRNA profiling of single neurons with an RNAi screen using optogenetics-induced nocifensive behavior as readout for single neuron function. Our approach may become a general workflow for similar analyses, potentially uncovering valid targets for pharmacological intervention in human (chronic) pain conditions.

Experimental Procedures


Strains used in this work are described in the Supplemental Experimental Procedures. C. elegans culture was according to standard procedures. Transgene zxEx609 was generated by injecting pSH103(pF49H12.4::ChR2::mCherry) and pF49H12.4::GFP into wild type (N2) animals, and integrated by UV-irradiation, yielding zxIs12. The resulting strain (ZX679) was outcrossed to N2 (6x), and further crossed into different genetic backgrounds.

Optogenetics and video analysis

Preparation of animals for optogenetic assays is described in the Supplemental Experimental Procedures, which also contain more extended descriptions of optogenetic methods. Whole-field illumination was done on a Leica MZ16F stereomicroscope equipped with a Sony XCD-SX90 camera (15fps) and an external light source (filtered 450–490nm) with built-in shutter. The speed of individual animals was calculated over two successive frames using a modified version of the parallel worm tracker [43] (http://www.biochem.uni-frankfurt.de/index.php?id=236). Selective illumination and tracking was described previously [14]. Briefly, we equipped an inverted microscope (Axiovert 35, Zeiss) with an LCD projector (Hitachi CP-X605) to illuminate individual segments of a freely moving nematode while tracking its position and quantifying evoked behavior, using custom-written software.

RNAi-optogenetics screen and cell-specific RNAi

Systemic knockdown was described previously [23]. RNAi efficiency was assessed by RT-PCR for a number of genes; experimental details are explained in Supplemental Experimental Procedures. Cell-specific RNAi was achieved by expressing sense- and antisense-constructs of genes of interest, driven by the F49H12.4 promoter (also expressing in AQR and one tail neuron) as described [26]; see Figure S5A for PCR fusion strategy.

Confocal and Ca2+-imaging

Adult animals were immobilized on agarose pads in M9 buffer with 30mM NaN3. Images in Figure 1 were obtained using a Marianas™ spinning-disk confocal (SDC) system (Intelligent Imaging Innovations – 3I) equipped with an Evolve EMCCD camera (Photometrics). All other pictures were obtained on an LSM510 (Zeiss). To image Ca2+-transients in PVC, we used RCaMP (Akerboom et al., in preparation), a red-fluorescent GCaMP-like Ca2+-indicator employing a circularly permuted mRuby protein [44], using a 561nm laser (i.e. without concomitant photoactivation of ChR2) on an Andor SDC system equipped with an iXon897 EMCCD camera. Z-stacks were obtained at 1Hz. PVD was photostimulated for 1s with blue light from a filtered HBO lamp (450–490nm).


  • -
    Photoactivation of nociceptors or downstream interneurons evoke escape behaviors
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    Optogenetics and cell-specific RNAi enables identifying mediators of nociceptor function
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    The DEG/ENaC channel ASIC-1 seems to extend PVD's dynamic range
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    The TRPM channel GTL-1 amplifies signal output from PVD

Supplementary Material










We thank the Caenorhabditis Genetics Centre (CGC; supported by the NIH – National Center for Research Resources), for providing strains. We are grateful to K. Preckel and B. Rummel for expert technical assistance, to H. Hutter, C. Bargmann and W. Schafer for sharing reagents, to M. Goodman for communicating results prior to publication, and to M. Goodman, W. Schafer and M. Zhen for comments on the manuscript. SJH was supported by the Human Frontiers Science Program Organization (HFSPO) and the Research Fund Flanders (FWO-Vlaanderen). This work was supported by grants from the US-Israel Binational Science Foundation Grant 2005036 (MT and DMM), NIH R01 NS26115, R21 NS06882 (DMM), NIH T32 MH64913 and F31 NS49743 (JDW), by NIH grants to Vanderbilt University: P30 CA68485, P60 DK20593, P30 DK58404, HD15052, P30 EY08126 and PO1 HL6744, as well as by grants from the DFG (SFB807, FOR1279, Cluster of Excellence Frankfurt – CEF-MC; EXC115/1) and the Schram foundation to AG.


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Author Contributions

SJH performed and analyzed most experiments. WSC wrote the interface for the parallel worm tracker, performed RT-PCR and analyzed data. SB performed Ca2+-imaging, RCaMP was constructed by JA and LLL. JNS and HL generated the selective illumination and tracking setup. WCS and MT constructed the mRNA tagging line for generating the PVD microarray data set. JDW generated the PVD microarray data set and helped with microarray data analysis. DMM and MT directed the effort to generate the PVD microarray data set and helped with microarray data analysis. SJH and AG wrote the manuscript with feedback by the co-authors.


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