The Caenorhabditis elegans interneuron ALA is (also) a high-threshold mechanosensor
© Sanders et al.; licensee BioMed Central Ltd. 2013
Received: 12 July 2013
Accepted: 6 December 2013
Published: 17 December 2013
To survive dynamic environments, it is essential for all animals to appropriately modulate their behavior in response to various stimulus intensities. For instance, the nematode Caenorhabditis elegans suppresses the rate of egg-laying in response to intense mechanical stimuli, in a manner dependent on the mechanosensory neurons FLP and PVD. We have found that the unilaterally placed single interneuron ALA acted as a high-threshold mechanosensor, and that it was required for this protective behavioral response.
ALA was required for the inhibition of egg-laying in response to a strong (picking-like) mechanical stimulus, characteristic of routine handling of the animals. Moreover, ALA did not respond physiologically to less intense touch stimuli, but exhibited distinct physiological responses to anterior and posterior picking-like touch, suggesting that it could distinguish between spatially separated stimuli. These responses required neither neurotransmitter nor neuropeptide release from potential upstream neurons. In contrast, the long, bilaterally symmetric processes of ALA itself were required for producing its physiological responses; when they were severed, responses to stimuli administered between the cut and the cell body were unaffected, while responses to stimuli administered posterior to the cut were abolished.
C. elegans neurons are typically classified into three major groups: sensory neurons with specialized sensory dendrites, interneurons, and motoneurons with neuromuscular junctions. Our findings suggest that ALA can autonomously sense intense touch and is thus a dual-function neuron, i.e., an interneuron as well as a novel high-threshold mechanosensor.
KeywordsMechanosensation Physiology Neuroethology
To survive in dynamic or harsh environments, all animals must appropriately modulate their responses to various stimulus intensities. For instance, noxious stimuli are detected by nociceptors, an important class of high-threshold sensory neurons. These, in turn, lead to downstream immediate avoidance responses and enduring self-protective responses that are distinct from responses to milder stimuli [1–5]. Neurons similar to mammalian polymodal nociceptors in both function and molecular determinants have been found across the animal kingdom [6–8]. In the nematode Caenorhabditis elegans, the neuron types PVD and FLP have been shown to share many similarities with mammalian and Drosophila nociceptors [3, 9–13], including a conservation of molecular mechanisms underlying the responses to noxious stimuli [5, 6, 8, 14–19].
Here we show that ALA acted as a high-threshold mechanosensor, and that it played a role in a previously described response to intense mechanical stimuli . ALA exhibited physiological responses to both anterior and posterior stimuli, and it was required for the inhibition of egg-laying in response to picking-touch (see Methods). The physiological responses of ALA to anterior and posterior touch were distinct, suggesting that it could distinguish between spatially separated stimuli. In addition, these responses did not require neurotransmitter or neuropeptide release from upstream neurons. However, the bilaterally symmetric processes of ALA itself were required for generating its physiological responses. These results suggest that ALA can autonomously sense picking-touch, but not lower intensity touch stimuli, and is thus a high-threshold mechanosensor.
The ALA neuron responded to both anterior and posterior picking-touch stimuli
A mutation that impairs ALA differentiation abolished the reduced egg-laying response to picking-touch
The commonly performed act of transferring a single animal to a new plate using a platinum wire pick inadvertently delivers an intense mechanical stimulus to the animal and evokes a characteristic behavioral response. It was previously shown that this mechanical stimulus leads to PVD- and FLP-mediated egg-laying inhibition lasting 30 minutes . The same procedure was also shown to affect locomotion on a timescale of several minutes . In addition, posterior gentle touch was shown to lead to increased intervals between calcium transients of the HSN egg-laying neurons . We thus asked whether ALA might have a role in mediating these behavioral responses. To address this question, we compared the suppression of egg-laying in wild-type animals to animals mutant for ceh-17, a gene encoding a paired-like homeodomain transcription factor.
Laser ablation of ALA abolished the reduced egg-laying response to picking-touch
To specifically test whether ALA was required for the egg-laying inhibition response to picking-touch, we assayed animals in which the ALA neuron was laser ablated during the L4 larval stage. As opposed to the mock-ablated control animals, ALA-ablated animals did not exhibit suppression of the egg-laying response (Figure 4). However, the egg-laying rate of unperturbed post-surgery animals was variable, with a lower mean (although not significantly: p = 0.114 by one-way ANOVA) than the corresponding rate in mock-ablated controls. This variability may be indicative of unspecific damage of the surgery that could not be avoided. We obtained similar results (Figure 4) for two strains in which behavioral responses to harsh touch were abolished [6, 8]: mutants for the mec-10 gene, encoding an amiloride-sensitive sodium channel protein of the DEG/ENaC family that is required for C. elegans touch sensation [3, 9, 11–13], and animals where a degeneration-causing, constitutively active nicotinic acetylcholine receptor (nAChR) channel subunit, deg-3(u662), was expressed under a Pmec-10 promoter [8, 14, 16], driving expression in and degeneration of PVD, FLP and the six touch receptor neurons [1, 11]. The suppression of egg-laying was not significant in mec-10(tm1552) mutants (p = 0.193 by one-way ANOVA) or Pmec-10::deg-3(u662) animals (p = 0.732 by one-way ANOVA) [2, 3, 6, 32, 41]. Taken together with the responses of ceh-17 mutants, our results suggest that ALA is required for the inhibition of egg-laying in response to picking-touch, but not for immediate avoidance responses.
The harsh-touch sensory neurons were not required for the physiological response of ALA to picking-touch
The physiological response of ALA to picking-touch did not require neurotransmitter release
Although physiological responses in ALA did not require known mechanosensory neurons, it was still possible that the responses were dependent on input from other pre-synaptic partners of ALA. To answer this question we crossed our Pver-3:GCaMP3 reporter into an unc-13(e51) mutant background, where synaptic vesicle exocytosis is essentially eliminated [6, 8, 15, 17–19, 42, 43]. Anterior and posterior picking-touch evoked rapid and long-lasting calcium transients in the ALA neurons of unc-13(e51) mutants (Figures 5C-D and 2). The peaks of unc-13(e51) responses to anterior stimuli were highly variable, with a coefficient of variance of 0.78 (Figure 2A), but were not significantly different from wild-type (p = 0.103 by one-way ANOVA). In contrast to wild-type, the rise time of the responses to posterior stimuli was rapid (5 sec) in the mutant animals, and their dynamics were similar to those of responses to anterior stimuli. These results suggested that synaptic input from neurotransmitter release was not necessary for producing the responses of ALA to picking-touch. However, such input may contribute to more subtle aspects of regulating these responses (similar to the case of the Pmec-10::deg-3(u662) transgenic background) such as the initial dampening of responses to posterior stimuli.
Presynaptic partners of ALA responded to anterior picking-touch
ADL, a presynaptic partner of ALA, has been shown to be a chemosensory neuron that plays a role in avoidance behavior in the presence of volatile repellents such as octanol [48, 55]. Ablating ADL has been shown to increase the latency of responses to octanol off food, but not on food [8, 56]. We thus sought to test whether ALA might be mediating octanol avoidance in addition to responses to picking-touch. To answer this question, we dipped an eyebrow hair in 30% octanol and presented it in front of the nose of Pver-3::GCaMP3 animals. We observed reversals characteristic of the avoidance response of C. elegans in both wild-type animals and ceh-17 mutants, with latencies of 4 sec in both genetic backgrounds, both on and off food (data not shown). Consistent with the behavioral assay, the repellent did not evoke detectable calcium transients in the ALA neuron (data not shown). Taken together, these results suggested that ALA did not mediate octanol avoidance.
The physiological responses of ALA to picking-touch were independent of neuropeptide release
The elongated processes of the ALA neuron are required for its physiological response to picking-touch
Discussion and conclusions
In this study we show that the ALA interneuron of the nematode C. elegans exhibits physiological responses to picking-touch and that it is required for a stereotypical response to this stimulus, a suppression of egg-laying [8, 32, 38]. The physiological responses of ALA did not require input from presynaptic release of neurotransmitters, nor did they require neuropeptide release. When the elongated, bilaterally symmetric processes of ALA were axotomized, the physiological responses to stimuli that were on the anterior side (upstream) of the cut persisted, while the responses to stimuli on the posterior side (downstream) of the cut were eliminated. ALA is known to form several electrical synapses, all of which are located in the anterior sections of its processes, i.e., in vicinity of the cell soma [21, 64]. As a result, our axotomy experiments rule out the possibility that input from the known gap junctions of ALA was capable of producing the observed responses. Taken together, our findings suggest that ALA can sense picking-touch stimuli autonomously, i.e., that it is a high-threshold mechanosensor.
The ALA neuron could discriminate between spatially separated stimuli along the body of the animal. Three observations are consistent with a model in which (as of yet unknown) molecular sensors are distributed along the processes of ALA: (1) the temporal dynamics of the responses to anterior and posterior stimuli were distinct, (2) neurotransmitter and neuropeptide release affected anterior and posterior responses differently, and, most importantly, (3) when the elongated processes of ALA were severed the responses to stimuli applied posterior but not anterior to the cut were abolished. Measuring the receptive field map of ALA using more accurately localized stimuli could provide insight into the spatial differentiation of this mechanosensor.
The physiological responses that we observed in ALA were unusually prolonged as compared to typical responses of C. elegans sensory neurons [36, 61, 62, 65–70]. Application of menthol, a noxious stimulus, can produce similarly prolonged calcium transients in mammalian nociceptors . However, axonal injury in mammalian and invertebrate preparation can also produce long-lasting calcium responses [72, 73]. Could the physiological and behavioral responses that we observed result from a compression or strain injury inflicted on ALA by picking-touch? Several considerations suggest that this is unlikely: (1) picking was used to transfer “control” animals to assay plates, as well as for their maintenance, such that a potential injury in ALA would be required to heal on the timescale of an hour in order to allow for the higher egg-laying rate in the absence of the recurring stimulus, (2) ALA is not at all unique in being located peripherally or having elongated processes; similar anatomical features are characteristic of many touch receptor, proprioceptor and motor neurons in C. elegans, (3) egg-laying was not suppressed in response to anterior picking-touch in ceh-17 mutants, where the anterior half of the ALA process was typically present; moreover, the developmental defect in ALA neurons in ceh-17 mutants did not suppress egg-laying, and (4) in C. elegans, axotomy induced calcium dynamics in the soma of the touch receptor neuron ALM exhibited a strong dependence on the lesion distance, effectively vanishing when the axon was injured merely 40 μm from the soma . Thus, explaining the reported responses as a result of injury would require multiple unsupported assumptions regarding the unique nature of ALA, such as an unexplained enhanced vulnaribility to compression, an ability to heal rapidly, an effect on egg-laying in response to injury that is distinct from a non-specific loss of function, and post-injury calcium dynamics dissimilar from those of a C. elegans touch receptor neuron.
Our findings associate the responses of ALA to picking-touch with the enduring inhibition of egg-laying, but not with an immediate avoidance response. Since enduring behavioral responses have been shown to depend on neuropeptides in previous studies [8, 35], we hypothesize that this could also be the case here. Based on its anatomical features, it has been suggested that ALA may be a neurosecretory neuron [21, 36, 74]. It has been shown that ALA is required for the regulation by epidermal growth factor (EGF) signaling of feeding and locomotion patterns, and that feeding defects caused by overexpression of the EGF-like peptide LIN-3 are suppressed by a mutation in the gene encoding UNC-31/CAPS [22, 32]. In addition, the tyrosine phosphatase-like receptor gene ida-1 was shown to be expressed in ALA [8, 40, 75]. IDA-1 was demonstrated to be important for dense-core vesicle cargo release; it acts genetically in the signaling pathway of unc-31 (encoding the UNC-31/CAPS protein) suggesting that it has a role in the trafficking of dense core vesicles and/or in their cargo release [37, 76]. Importantly, CEH-17 was shown to regulate the expression of ida-1 in ALA [38, 40], such that neuropeptide release from ALA would be expected to be impaired in ceh-17 mutants. Taken together, these findings and the results presented here are consistent with the idea that ALA may use peptidergic signaling to communicate to its downstream targets.
Several C. elegans neurons, such as the proprioceptors DVA, AVG, and PVR, were originally classified as interneurons and later found to also function as sensory neurons. In addition, interneurons functioning also as sensory neurons have been described in other species, e.g. the B51 neuron in Aplysia californica[77, 78]. However, this study was the first to associate the role of a mechanosensor with the interneuron ALA, thus demonstrating that it is a dual-function neuron. The roles of neuropeptides in modulating the repertoire of enduring responses, as well as the degree to which these roles may be either evolutionarily conserved, remain to be understood.
Wild-type, transgenic, and mutant C. elegans strains were maintained and cultivated with OP50 bacteria according to standard protocols . The following strains were used: wild-type strain N2, INV21001 N2;Ex[Pver-3::GCaMP3], INV21002 N2;Ex[Pgpc-1::GCaMP3], INV21003 N2;Ex[Psrh-220::GCaMP3], INV21004 N2;Ex[Pdat-1::GCaMP3], INV54001 N2;Ex[Pdat-1::TeTx; Pver-3::GCaMP3], INV54002 egl-3(n150)V; Ex[Pver-3::GCaMP3], INV54003 unc-13(e51); Ex[Pver-3::GCaMP3], INV54004 unc-31(e169); Ex[Pver-3::GCaMP3] and INV54005 N2;Is[Pmec-10::deg-3(u662)]; Ex[Pver-3::GCaMp3]. The OS5513 N2; Ex[Pver-3::GFP] and the IB16 ceh-17(np1)I strains were a gift from Menachem Katz of Shai Shaham’s laboratory (Rockefeller University).
Picking-, harsh- and gentle-touch stimuli
The routine maintenance procedure of picking animals entails applying pressure with a platinum wire covered with a thin sticky layer of bacteria, a procedure performed on the order of 10,000 times a year by a typical experimenter. Although it is difficult to obtain quantitative characterizations of hand-delivered mechanical stimuli, estimates of the pressure applied by a platinum wire pick were previously obtained using an NGM test plates placed on an analytical balance . By this measure, in our hands, picking-touch was estimated to involve pressures that were 10-fold larger than those associated with the standard harsh-touch stimulus. Thus, C. elegans researchers commonly apply three distinct tiers of touch stimuli: (1) gentle touch – the weakest intensity tier, e.g., brushing the animal with an eye-lash pick, (2) harsh touch – an intermediate intensity tier, e.g., probing with a platinum wire or a glass rod, and (3) picking touch – the highest intensity tier in routine use [32, 57, 60]. In addition, picking-touch was previously shown to produce a behavioral response – a suppression of the rate of egg-laying . In our assays, a picking-touch stimulus was delivered in the same manner as in routine picking, but without the thin bacteria layer that would facilitate the lifting of the animal from the substrate. The duration of the stimulus was approximately one second. Thus applied, this common procedure was not previously reported to result in sustained tissue damage, and our data suggests that it did not injure the ALA neuron. To test responses to lower intensity mechanical stimuli, we delivered standard harsh-touch and gentle-touch stimuli using a platinum wire and an eyelash pick, respectively.
Octanol avoidance assay
Avoidance of 30% 1-octanol was assayed as previously described [48, 56]. In brief, an eyelash hair was attached to a Pasteur pipette, dipped in octanol, and presented in front of a forward moving animal without touching it. The amount of time it took the animal to initiate backward movement was determined by a handheld audible timer. Two well-fed animals were placed on fresh NGM plates (either with or without a lawn of bacterial food) 30 minutes prior to each assay. Each animal was tested 7–10 times, with an interval of at least 2 minutes between successive tests.
Animals were synchronized and assayed at 70 hours post-hatching unless noted otherwise. Animals were placed in triplets on a 3 cm diameter NGM plates at 20°C 20 minutes prior to the beginning of the assay. Picking-touch stimuli were delivered manually to each individual animal every 20 minutes during a 3 hours period. The adults were then removed and the number of eggs per plate was counted. To simplify egg-counting, each assay plate was seeded with a small drop of OP50 bacteria at its center.
Physiological imaging in freely behaving animals
Animals were manually synchronized by transferring 10 gravid adults to fresh NGM plates and restricting the duration of egg-laying to two hours. The adults were then removed, and the embryos were grown at 20°C until young adulthood. One hour prior to testing, young adult animals expressing the appropriate marker were transferred to a fresh standard NGM plate (6 cm in diameter) spread with a thin layer of OP50 bacteria. Picking-touch stimuli were delivered manually to freely moving animals using a platinum-iridium wire (0.2 mm diameter, 99.9% purity from Alfa Aesar) attached to a glass Pasteur pipette . Anterior and posterior stimuli were applied to the farthest quartiles of the animal body, respectively. A single stimulus was delivered per animal. Calcium imaging of each worm was performed at a magnification of 11.5× for 1 minute prior to the stimulus and 4 minutes post-stimulus. Alternatively, in order to capture the slow decay of the signal, 20 seconds of imaging were performed every 5 minutes for 30 minutes. Images were binned 4×, and captured at 5 Hz using a cooled CCD camera (Photometrics CoolSNAP HQ2, Tucson, AZ), an Olympus SZX16 stereomicroscope equipped with an SDF PLAPO 1XPF objective (Olympus America Inc., Center Valley, PA), and Micro-manager . Animals were tracked manually during the assay and image analysis was performed using custom MATLAB scripts (Mathworks, Inc. Natick, MA).
Axotomy and ablations
Ablations and axotomy were performed on L4 larvae, immobilized with 10 μM levamisole on a 2% agarose pad, using a Ti:sapphire femtosecond laser, as previously described [61–63]. The transgenes Pver-3::GFP and Pver-3::GCaMP3 were used to identify ALA neurons for ablations and axotomy, respectively.
Some of the strains used in this study were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (g0072ant no. P40 OD010440). This work was supported by the NIH Training Grant 2T32GM007197-37 (JS), the Burroughs Wellcome Fund Career Award at the Scientific Interface (DB) and the Searle Scholars Program (DB).
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