Novel assay for quantifying the noxious response and mapping the behavioral receptive field
To date, thermal avoidance assays have been useful in studying the molecular mechanisms of thermal nociception, but have lacked the ability to carefully control variable doses of heat along the body of the worm [2, 6, 14]. In previous studies where regions along the body of the worm were targeted [1, 14], the laser focus was presented for a time long enough (10 seconds) for the heat to diffuse well beyond the worm’s body. Therefore the spatial extent of the stimulus in these experiments is uncertain because the temperature profile in time and space was not clearly shown. In other experiments of thermal nociception, the main drawbacks are that either the whole worm is heated or a thermal barrier selects for sensory neurons in the head [2, 6, 15]; either case cannot spatially dissect the noxious response. We have addressed these limitations by designing a new thermal avoidance assay that localizes the heat from an IR laser pulse to small regions (approximately 1/5 of the worm’s body length) along the entire body of the worm, and records the behavioral responses to the noxious stimulus (Figure 1a and b). The thermal profile of the beam was carefully calibrated using a thermal camera (Figure 1d, Methods). The beam diameter was measured to be 220 μm (FWHM). This constrained the heating so that the temperature change 500 microns away from the center of the beam is only 2% of the peak, which ensured heating the midbody was not simultaneously heating the head and tail. The temperature of the IR pulse was further independently verified using ratiometric imaging of a temperature sensitive dye pair (rhodamines B and 110) (Methods) [16]. The centroid worm speed and changes in the worm body shape were used to determine the behavioral states of the worm (reverse, forward, omega turn, or pause) before and after the thermal stimuli (Figure 1c, Methods).
Multi-parameter, high-content phenotyping of N2 noxious response for the head, midbody, and tail
We characterized the reaction of N2 (wild type strain) to a range of temperature ramps along the entire body of the worm to measure the spatial nociceptive dose response. The change in the worm’s centroid speed over time is an informative measure of the thermal response because aspects of this time series change with the stimulus strength. We used this metric to quantify behavioral differences in response to variations in both the power and location of the stimulus (Figure 2a), similar to a previous study [15]. Several features in the dose response scale with power, most notably the maximum mean speed, and the deceleration from the maximum mean speed. The general shape of the mean speed versus time curves also changes in response to the position of the thermal stimulus along the worm body; this is because these speed curves are a product of the underlying locomotory states, some of which change with the position and power of the IR laser. In order to examine these behaviors and further characterize the wild type noxious response, we generated ethograms for the different stimulus laser powers and locations (Figure 2b) [17, 18]. At lower laser powers, the behavior is stochastic; as the power increases, the worm’s response becomes more deterministic. From the ethogram, we identified another metric that discriminates the stimuli both by its location and intensity, namely the first behavioral state the worm enters after the stimulus: forward, reverse, and pause. We calculated the probabilities of the first response states (first behavioral states after the stimulus), and measured changes in these probabilities in reaction to changes in the position and power of the stimulus (Figure 1b).
The stereotypical withdrawal response for a crawling C. elegans thermally stimulated at the head is a reversal, followed by an omega turn, then a recommencement of forward motion (Figure 1c). The likely purpose of this behavioral series is to make a three-point turn to reorient the worm away from the noxious stimulus. Arguably the worm’s chance to escape danger improves if it is able to respond more quickly to the threat, and reorient itself so that instead of moving towards the hazard it is moving in the opposite direction (180°). We investigated if the escape response improves as a function of the laser power, indicating that these avoidance behaviors changed appropriately for the noxious level of the stimulus. Our results show that the animal’s reaction time does in fact vary inversely with stimulus amplitude (Figure 2c) and that the escape angle increases towards 180° with increasing stimulus power (Figure 2d).
The noxious response is elicited by a temporal temperature gradient rather than a temperature threshold
Previous studies have used high temperatures in the range of 30°C-35°C to study the noxious response in C. elegans[1, 2, 6, 14]. In the context of studying the noxious response, the requirement for high temperature is expected since previous work on mammalian transient receptor potential (TRP) channels in sensory neurons show that a subset of TRPs -- the TRP vanilloid group in particular -- are gated by high temperatures generally > 43°C and have a steep temperature dependence [7, 19–21]. Remarkably, our dose response and temperature measurements show that the worm’s robust, stereotypical avoidance response to noxious stimulus at the head can be elicited by relatively small changes in temperature (≤1.4°C) (Figure 1d, Figure 2a). It appears that the temperature ramp rate as opposed to the temperature change above a threshold induces the avoidance response. The ramp rate for the highest temperature stimulus in our dose response is ~9.4°C/s, which is in the noxious range of previous experiments with higher absolute temperatures [15].
We tested our hypothesis that the ramp rate and not the absolute temperature jump is what produces the thermal nociceptive response by using thermal stimuli with a constant ramp rate but with different ∆Ts (~5.9°C/s, ∆T = 0.22, 0.41, and 0.67°C) (Figure 3). When we stimulated the worm with these short duration, small amplitude thermal pulses, we were able to robustly elicit nearly identical noxious responses (Figure 3). When the ramp rate is lowered and the animal is stimulated with a similar temperature jump (∆T/t ~1.5°C/s, ∆T = 0.2°C), the response is noticeably lower and statistically different compared to the noxious response elicited by the higher ramp rate but same ∆T (Figure 3; Kruskal-Wallis test, Dunn’s multiple comparison p < 0.05). This indicates that the avoidance response is dependent on a rate of change in temperature, rather than a crossing of a thermal threshold. Previously reported experiments also stimulated worms with an abrupt change in temperature [1, 2, 6, 14], but our results show that extreme heat is not required to initiate a noxious response if the ∆T/t is above some threshold. For our experiments we stimulated the worm for a fixed duration (133 ms) at different laser powers to produce a range of ∆Ts and ramp rates.
Spatial sensitivity of the midbody response
The midbody behavior is distinct from the head and tail responses (Figure 1b; p < 0.001, Fisher’s exact test). At the two extremities of a forward moving worm, the transition to the first state after the stimulus is deterministic—the worm will reverse if stimulated in the head, and will accelerate its forward motion if stimulated in the tail. At the midbody, however, the response is probabilistic as the worm enters a reversal, a forward, or a pause state (Figure 1b). The forward or reverse bias of this behavioral response is strongly correlated with the anterior/posterior position of the stimulus. For example, a laser pulse directed to the anterior middle region closer to the head of the worm will cause a reversal the majority of the time, whereas a laser pulse targeted at the posterior middle has a higher probability to elicit a forward response (Figure 4). We uncovered the “sensory middle” of the worm--a region where the worm may move forward, move backward, or enter a pause state, roughly with equal probability. The brief midbody pause state could arise as a behavioral strategy when there is insufficient asymmetry in the signal, in order to give the worm another opportunity to accrue additional anterior/posterior information about the stimulus before initiating its escape. As the stimulating beam is moved a small distance around this “sensory middle” we can measure a change in the probability of the behavioral response. A statistically significant change in behavior suggests that the worm perceives a difference in the stimuli location. Using this measure we found that statistically the worm has the ability to spatially differentiate the location of our constrained thermal stimuli by as little as 80 microns (Figure 4; p < 0.05, Fisher’s exact test).
Mutant behavioral analyses identify neurons involved in the midbody and tail responses
Mutations in the gene mec-3 affect the development of the bilaterally symmetric pair of nociceptors PVD, such that the neurons lack all but the primary dendritic branching [11, 22–24]. We found that mec-3(gk1126) had a pronounced defect in the midbody and tail response compared to N2 (Figure 5a; p < 0.01, Fisher’s exact test), but the head response showed only a very minor defect (Figure 5a; p > 0.05, Fisher’s exact test). PVD has been shown to be the nociceptor for harsh midbody touch [6, 10, 11], and these results strongly suggest that PVD is also the nociceptor for the midbody thermal avoidance response. Since a mutation in mec-3 also affects the touch receptor neurons (ALM, AVM, PLM, PVM), we tested the touch resistant mec-4(e1339) mutant strain [25] to ensure that the touch neurons were not involved. Our behavioral and speed data show that the mec-4 mutant response is statistically similar to wild type (Figure 5a; p > 0.05, Fisher’s exact test). Since the touch neurons are not involved in transducing the response this leaves PVD as the primary candidate for thermal nociception at the midbody.
Furthermore, PVC has been identified as a command interneuron for the forward tail noxious heat response, being a main synaptic output to the PHC neuron [14]. PVC is also postsynaptic to PVD [26, 27]. Our definition of the tail region (Materials and Methods) includes the posterior branching of PVD. Our results show a severe defect in deg-1(u38)--a mutant where PVC is degenerated along with four other cell types--in the tail response (Figure 5b; Kruskal-Wallis test, Dunn’s multiple comparison p < 0.0001). The tail defect seen in the mec-3(gk1126) result (Figure 5a) implicates PVD as a possible nociceptor for the tail response (Figure 5a), suggesting that PVC is acting as the command interneuron in the thermal avoidance circuit in the tail as a postsynaptic target to both PVD and PHC.
PVD chemically synapses equally to AVA (27 synapses) and PVC (28 synapses) [26]. Recent optogenetic analysis of PVD has shown that the probability of backward versus forward movement is determined by the relative synaptic input to the command interneurons as a result of the location of the stimulus along the body of the worm [27]. We investigated this further analyzing deg-1(u38) (−PVC) upon noxious stimulation along the body, compared to the wild type response. The loss of functionality of the forward command interneuron effectively shifts the wild type midbody response to the posterior of the worm, and increases the probability of entering a pause state (Figure 5c). This result suggests that the worm’s nociceptive “sensory middle” may be determined by the balance of the synaptic inputs to the command interneurons, and elicits spatially sensitive behavior accordingly (reversal for anterior stimulation, forward for posterior stimulation, and an increased pause state when the signal is symmetric). When the forward command interneuron PVC is not functional, the anterior response (reversal) extends to the posterior, since the PVD-AVA activity dominates.
PVD is required for the midbody and tail thermal noxious response
Our mec-3 and mec-4 results suggest that PVD is the sensory neuron underlying the midbody noxious response. In order to confirm its involvement, we eliminated the pair of PVD neurons using laser ablation microsurgery [28]. We generated a transgenic strain expressing cameleon in PVD (Materials and Methods), and ablated both PVDL and PVDR in the late L2 stage. We then tested the response of PVD-ablated young adults to localized thermal stimuli. Our results show the head response is consistent with the mock-ablated response in our transgenic strain, but the midbody and tail responses are severely reduced (Figure 6, left column; Kruskal-Wallis test, Dunn’s multiple comparison p < 0.001). We also tested a transgenic strain, ser-2prom3:DEG-3-N293I, where PVD is specifically eliminated [24]. These genetic ablation results confirm our laser ablation results – the head response remains unaffected, but we found a clear decrease in the midbody and tail compared to the wild type response (Figure 6, right column; Kruskal-Wallis test, Dunn’s multiple comparison p < 0.0001). This demonstrates that the PVD neurons are required for the sensation of noxious heat at the midbody and tail.
C. elegans possesses more motor neuron commissures on the right side compared to the left side and accordingly there are more fasciculations with motor neuron commissures from the secondary branches of the PVDR neuron compared to the PVDL neuron [23, 26]. This left/right asymmetry led us to investigate the single neuron contribution to the midbody thermal noxious response. Using the same method as the double neuron ablation, we ablated either PVDL or PVDR and tested the head, midbody, and tail responses to our noxious thermal stimulus. The speed versus time analysis suggests that either PVDL or PVDR alone is sufficient since the single neuron ablation data at the midbody and tail respond to the noxious stimulus – although slightly less robustly – compared with mock ablation data (Figure 6, left column).
PVD responds differently to spatially localized heat pulses targeted at different locations near the midbody
To show PVD senses localized noxious heat at the midbody, and that it can differentiate the location of the stimuli, we used a G-GECO 1.2 calcium indicator coexpressed with a reference DsRed2 chromophore in the nuclei to measure the influx of calcium into PVD when the midbody is heated with an IR laser pulse at two distinct locations (Figure 7a, 7b). The IR stimulus used for calcium imaging was nearly identical to the one used in the behavior measurements (Materials and Methods). Previous studies applied heat to the whole worm [6, 14], which may have selected for a head response and a calcium influx in PVD may not be seen as a result. We measured calcium transients in PVD when stimulated with a 133 ms pulse of heat at two locations, namely anterior to the cell body and posterior to the cell body (Figure 7b). The thermal maxima of the anterior and posterior pulses were approximately 200 μm apart, and fall within the spatially distinct behavioral responses at the midbody that we found in Figure 4. Our calcium transients confirm PVD’s role in transducing the midbody avoidance response. Furthermore, the difference in the signal due to changing the location of the stimulus demonstrates the neuron’s ability to detect a difference between anterior versus posterior stimulation (Figure 7b).
Mutant strains show defective noxious behavior suggesting molecules involved in sensing heat at the midbody
Our quantitative analysis of 21 mutant strains revealed previously reported results for molecules involved in thermal avoidance for the head and tail [1, 14] (Additional file 1: Table S1). Here we focus on those that affect the midbody thermal avoidance response.
The GLR-1 glutamate receptor has been shown to mediate mechanosensory signaling in interneurons postsynaptic to the polymodal nociceptor ASH, and discriminate between sensory inputs [9, 29, 30]. In particular, glr-1 mutants are defective to nose touch but not to osmotic shock, even though both modalities are primarily sensed by ASH. GLR-1 is expressed in interneurons controlling locomotion [31], including PVC and AVA which are both direct postsynaptic outputs to PVD. Our results show that a mutation in glr-1(n2466) produces a strong midbody behavioral defect (Figure 8a). In particular, the probability of forward locomotion is reduced from 0.18 to 0, the probability of backward motion is reduced from 0.65 to 0.46, and the probability of the pause state dramatically increases from 0.17 to 0.54, relative to the wildtype response. This defective behavior suggests that glutamate could be the transmitter for PVD in the midbody thermal noxious response. This is consistent with the finding that PVD expresses the vesicular glutamate transporter EAT-4, which is required for glutamatergic transmission [32].
The TRPV1 subfamily channels are involved in noxious heat perception in humans and mice [20, 21, 33]. Recently TRPV channels have been found to contribute to the thermal avoidance response in the head and the tail of C. elegans[2, 14], and so we investigated their involvement in the midbody thermal noxious response. OCR-2 and OSM-9 are homologues of the mammalian TRPV channel genes in C. elegans, and are coexpressed in sensory neurons [34]. Both are expressed in PVD [35–37]. Our results suggest that ocr-2 is required for noxious heat sensation at the midbody, but osm-9 is not (Figure 8b). Therefore, it is possible that the OCR-2/OSM-9 heteromer does not function in PVD to control the noxious heat response, as only ocr-2 produces a behavioral defect.
