A quantitative assay to characterize responses to thermal pulses of different amplitudes
To characterize the noxious temperature range of C. elegans, we transiently raised the local temperature around a forward-moving animal by defined amounts and quantified multiple aspects of the resulting avoidance response. Heating was produced by infrared laser pulses of 0.5-s duration. Using thermal imaging, we determined that the increases in temperature above baseline (ΔT) were directly proportional to the laser power, and that the heated area encompassed the entire animal (Figure 1). We were thus able to systematically deliver thermal pulses with a ΔT between 0.4°C to 9.1°C. We recorded the behavior of each animal for 15 s in response to the thermal stimulus. From these images we measured basic features of shape such as the worm's 'skeleton', center-of-mass, head-to-tail distance, and used these measures to calculate the animal's speed and metrics of different behavioral states (see Methods).
N2 animals exhibit dose-dependent changes in multiple aspects of the avoidance response elicited by thermal stimuli
N2 animals responded to a thermal pulse corresponding to a ΔT = 0.4°C with a stereotypical avoidance behavior that reorients the worm away from noxious stimuli. Typically, a thermal pulse applied to a forward-moving animal elicited a sequence of four behavioral states: a pause, reversal, an omega turn, and forward movement (Additional files 1 and 2, Movies S1 and S2). Increasing the amplitude of the thermal pulse did not induce any gross qualitative changes in the sequence (Figure 2). However, the duration and the speed of locomotion during specific behaviors changed proportionately as a function of the ΔT (Figure 2). From the speed (Figure 2A) and ethograms (Figure 2B), we quantified multiple behavioral parameters, including various aspects of speed changes, characteristics of different behavioral states, and the probability of switching between states at each ΔT. We found that while some features of the behavior changed with the pulse amplitude, others were stimulus independent; that is, they remained constant over an approximately 25-fold change in the amplitude of the thermal pulse (Figure 3A-H). For example, the pause duration (Figure 3A), time to respond with an increase in speed (Figure 3F) decreased as we increased the pulse amplitude. Acceleration (Figure 3G), the probability of responding with a reversal (Figure 3H) and reversal duration (Figure 3B) increased with the stimulus amplitude. However, the duration of omega turns remained constant over the entire range of stimuli (Figure 3C). We also found that the peak speed with which the worms avoided the thermal stimulus increased with the intensity of the stimulus (Figure 3E). However, the speed at the beginning of the thermal pulse stimuli did not change with an increase in amplitude of the thermal pulse (Figure 3D). We were thus able to quantify multiple aspects of the noxious, thermally induced avoidance response of C. elegans and identify aspects of the behavior that changed proportionately with the intensity of stimulus. We chose ΔT = 0.4°C, 1.0°C, 4.8°C and 9.1°C for further analysis, as multiple behavioral features that scaled with temperature differed significantly among these thermal pulses.
Additional file 1: Movie S1. Escape behavior of a N2 animal to ΔT = 0.4°C. (MOV 565 KB)
Additional file 2: Movie S2. Escape behavior of a N2 animal to ΔT = 9.1°C. (MOV 568 KB)
Unique combinations of molecules define responses to different increases in temperature
The quantitative differences in the avoidance behavior that we observed with increasing intensity of the thermal stimuli can be explained by either distinct molecular mechanisms operating at different temperature ranges or a single mechanism that scales with the amplitude of the thermal pulse. If a similar set of genetically distinct strains shows defects across a broad range of ΔT, this would suggest a common molecular mechanism. On the other hand, if distinct sets of genetically different strains show defects at different temperature ranges, then this would suggest multiple molecular mechanisms. To distinguish between these possibilities, we recorded escape responses of 47 mutants in the N2 genetic background and extracted 8 behavioral features that changed with increasing intensity of the stimulus for each strain (Figure 3I). We normalized each feature, scored in different units, to a common scale. Thus at each thermal pulse, the responses of the strains were summarized as a 'phenotype barcode' of 8 features, generating a 49 strain × 8 feature matrix at each ΔT (Figure 3I). Hierarchical clustering of this strain x feature matrix identified strains that belonged to clusters different from N2 at each ΔT (Figure 4). To identify strains that are robustly different from N2, we bootstrapped the phenotype barcode data and identified strains different from N2 as ones that remained outside the N2 cluster in at least 90% of the bootstrap clusters (Figure 4, see also Methods).
As an independent means of identifying mutants that behave differently from N2 in response to a thermal pulse, we performed dimensionality reduction of the avoidance response barcodes. Principal component analysis (PCA) of 8 phenotype features produced 6 principal components that together explained approximately 95% of the variance in the avoidance responses of the 49 strains at each ΔT (Additional file 3, Table S1). We projected the strains in the six dimensional feature space onto two dimensions for each ΔT (Figure 5A, see also Methods). At all ΔT, we identified the strains that were greater than a fixed Euclidean distance away from N2 in this two-dimensional space as behaving differently from N2 (Figure 5B, see also Methods). All but one of the strains deemed different from N2 at each ΔT by this method were also predicted by bootstrapping (Figure 5B). The only exception was strain unc-80 at ΔT = 4.8°C, which was predicted to be different from N2 only in the PCA feature space but not by the bootstrap criteria (Figure 5A, B). Finally, we used non-parametric analysis of variance (ANOVA) on the list of the mutants predicted by either of the above criteria (PCA and hierarchical clustering) to identify a final set of strains different from N2 (see Methods). A total of 31 out of the 48 strains we tested were significantly different at either 1 (ΔT = 0.4°C) or more ΔT (Figure 6A). At each ΔT, unique sets of strains were found to be different from N2, suggesting that distinct molecular mechanisms underlie responses to noxious stimuli for temperature ranges examined.
Two strains were significantly different from N2 at all ΔT (Figure 6A; Kruskal-Wallis test, Dunn's multiple comparison P < 0.001), consistent with the general nature of defects in their neuromuscular system. These strains, akIs11 [Pnmr-1::ICE] [12] and twk-18(cn110) [13], are known to be defective in locomotory command interneuron and muscle function, respectively. akIs11 is a transgenic strain expressing human caspase (ICE) driven by the promoter of nmr-1, which results in the death of all the command interneurons known to be involved in mediating reversals, as well as three additional pairs of neurons. These animals are unable to execute proper reversals, and were defective at all ΔT (Figure 6B). However, these animals frequently displayed an escape response that consisted solely of omega turns without any reversals (Additional file 4, Movie S3). twk-18(cn110) is a temperature-sensitive gain of function mutation in a two-pore potassium channel expressed primarily in the body wall muscles [13]. This allele induces constitutive membrane hyperpolarization [13], thereby reducing excitability of the muscle at room temperature [14]. Consistent with this, animals harboring twk-18(cn110) were unable to elicit a wild-type escape response at any ΔT (Figure 6B).
Additional file 4: Movie S3. Escape behavior of an akIs11 animal to ΔT = 9.1°C. (MOV 429 KB)
Thermotaxis and responses to noxious thermal pulses are genetically separable
Thermotaxis in C. elegans is well studied at the behavioral, neuronal and molecular level [5]. To determine whether the molecular mechanisms underlying thermotaxis are also employed during the thermal avoidance behavior, we exposed 14 strains defective in thermotaxis behavior (bold, Additional file 5, Table S2) to thermal pulses of different amplitudes. Seven of these strains (bold and underlined, Additional file 5, Table S2) were significantly impaired in responding to a ΔT = 0.4°C but exhibited normal responses to thermal pulses of larger amplitudes, suggesting that thermotaxis signals in response to small ΔT are measured through a separate pathway from thermal noxious signals in response to larger ΔT.
Loss of function of the homeodomain proteins, ttx-1 [15] and ttx-3 [16], required for proper development of the thermosensory neuron AFD and the interneuron AIY, respectively, results in constitutive cryophilic behavior. These mutants were defective in responses to ΔT = 0.4°C but displayed N2-like escape responses to thermal pulses of larger amplitudes (Figure 7A). We also examined the molecules in the cyclic GMP (cGMP) dependent signal transduction pathway, mutations in which results in animals with abnormal temperature preference. Loss of function mutations in the genes encoding cGMP gated channel subunits, tax-2 [17] and tax-4 [18], as well as in three genes encoding guanylate cyclases, gcy-18, gcy-8 and gcy-23 [19], exhibited impaired avoidance responses to ΔT = 0.4°C (Figure 7A, B). Additionally, strains harboring mutations in tax-2 were significantly different in their escape responses to ΔT = 1°C and 4.8°C but exhibited normal responses at ΔT = 9.1°C (Figure 7B; Kruskal-Wallis test, Dunn's multiple comparison P < 0.01). Animals harboring mutations in three guanylate cyclases exhibited a normal avoidance response at larger ΔT (Figure 7A), suggesting that different mechanisms may be involved in mediating responses to noxious temperature of higher magnitudes.
The homologs of calcineurin (TAX-6) [20] and protein kinase C (PKC-1) [21] are thought to negatively regulate thermosensory function of AFD, and loss of function of these genes results in constitutive thermophilic behavior. Loss of function mutants in genes encoding these molecules were also defective at ΔT = 0.4°C (Figure 7A, B). Our analysis indicated that tax-6(lf) mutant animals were also significantly different from N2 at ΔT = 1°C and 4.8°C but not 9.1°C (Figure 7B, D; Kruskal-Wallis test, Dunn's multiple comparison P < 0.01). tax-6(lf) mutants had multiple defects (mean reversal duration, acceleration) in their escape behavior at the lower ΔTs (Figure 7D). All defects of tax-6 mutant animals in thermal avoidance behavior were rescued by expressing the normal version of the gene in sensory neurons and interneurons (Figure 7B).
Loss of function of the genes ncs-1, encoding a neuronal calcium sensor protein [22], cmk-1, encoding a Ca+2/calmodulin-dependent protein kinase I [23] and ttx-7, encoding a inositol monophosphatase, required for correct localization of synapses of the interneuron RIA [24], result in abnormal thermotaxis. All three strains exhibited wild-type avoidance response at higher ΔT (Figures 6A and 7A). However, while ncs-1 and ttx-7 mutants were defective at ΔT = 0.4°C (Figure 7A), cmk-1 mutants behaved normally at that thermal pulse intensity.
Mutants of genes required for proper sensory cilia development, namely osm-3 and osm-6 [25], as well as of a gene encoding a secreted protein required for integration of sensory information, hen-1 [26], were shown to exhibit normal thermotaxis behavior. Animals harboring mutations in these genes were significantly impaired in their avoidance response to a thermal pulse of ΔT = 0.4°C (Figure 7C; Kruskal-Wallis test, Dunn's multiple comparison P < 0.01). Moreover, whereas the cilia defective mutants osm-3(p802) and osm-6(p811) were significantly impaired only at ΔT = 0.4°C, hen-1(tm501) animals were also unable to elicit a wild-type escape response at ΔT = 1.0°C (Figure 7C; Kruskal-Wallis test, Dunn's multiple comparison P < 0.01). These animals behaved like wild-type at ΔT = 4.8°C and 9.1°C. We have identified molecules (osm-3, osm-6 and hen-1) that are involved in responses to noxious thermal stimuli but are not required for thermotaxis. We also identified strains that are not defective in avoidance of thermal pulses but exhibit abnormal thermotaxis behavior (for example, cmk-1). Thus, thermotaxis and the escape response to a thermal pulse of ΔT = 0.4°C are genetically separable.
TRP and two-pore K+ (TWK) channels are required for mediating normal avoidance responses to ΔT = 0.4°C
Although the major thermosensors of invertebrates are ion channels of the TRP family [2], such channels have a relatively mild role in mediating responses to thermal stimuli in C. elegans. For example, mutations in the TRPV (ion channels known to be activated by heat in mammals) homolog of C. elegans, osm-9 [27], have been shown to exhibit mild defects in the thermal avoidance response [7, 8]. However, the TRPA ion channel homolog of C. elegans has been shown to be required for acute cold sensation [28]. To test the function of TRP channels in sensing different intensities of noxious stimuli, we examined the avoidance response of nine strains with mutations in different TRP channels elicited by thermal pulses with ΔT of 0.4°C, 1.0°C, 4.8°C and 9.1°C. Three of these mutants, trpa-1 (TRPA), ocr-1;ocr-4 (TRPV) and trp-4 (TRPN) [29], were defective in avoidance responses to ΔT = 0.4°C, but their responses did not differ significantly from N2 at the higher ΔT (Figure 7E; Kruskal-Wallis test, Dunn's multiple comparison, P < 0.01). However, we did not detect any major defect in the behavior of animals harboring mutations in the gene encoding TRPV channel subunit homolog OSM-9, which is required for sensation of noxious thermal stimuli (approximately 43°C) in mammals.
TWK channels have been implicated in defining temperature thresholds and ranges of activation of thermosensory neurons in mammals [30]. In C. elegans, there are approximately 40 genes encoding TWK channels [31]. We examined the effect of loss-of-function mutations in four TWK channels on the thermally induced avoidance responses. Animals harboring loss of function mutations in either twk-7 or twk-37 were impaired in their avoidance response at ΔT = 0.4°C but not at higher ΔT (Figure 7E).
Glutamatergic neurotransmission is essential for responses to ΔT = 0.4°C and 1.0°C
Glutamatergic neurotransmission has been reported to be essential for normal thermotaxis [32], as well as for avoidance responses induced by noxious thermal stimuli [7]. Thus it was likely that glutamate would play a role in the thermal avoidance response in our thermal pulse assays. To test this hypothesis, we examined three loss-of-function alleles of the gene encoding the vesicular glutamate transporter (eat-4) that concentrates glutamate onto synaptic vesicles. All three alleles were defective in eliciting a wild-type response to a thermal pulse corresponding to ΔT = 0.4°C and 1°C, but were not significantly different from N2 at larger ΔT (Figure 8A). Transgenic expression of functional EAT-4, driven by the odr-3 promoter, failed to rescue the defects of eat-4(ky5) at ΔT = 0.4°C and 1.0°C and induced a significantly impaired escape response at ΔT = 4.8°C (Figure 8A; Kruskal-Wallis test, Dunn's multiple comparison P < 0.01). odr-3 drives expression in a subset of neurons (AWA, AWB, AWC and ASH) in which EAT-4 is normally expressed. Our results suggest that additional neurons beyond the ones defined by the expression driven by odr-3 promoter are required to elicit a proper escape response at ΔT = 0.4°C and 1.0°C. The abnormal phenotype at ΔT = 4.8°C and N2-like avoidance response at ΔT = 9.1°C of this strain suggest involvement of distinct sets of neurons contributing to avoidance responses at these thermal pulse amplitudes.
We also analyzed the thermally induced escape responses of animals harboring mutations in the genes encoding the 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) and N-methyl-D-aspartate (NMDA) classes of glutamate receptors, as well as glutamate-gated chloride channels. We detected significantly impaired escape responses induced by ΔT = 0.4°C in strains harboring mutations in the glutamate-receptor subunit glr-2 and the glutamate-gated chloride channel glc-3 (Kruskal-Wallis test followed by Dunn's multiple comparison). Escape responses of these strains to thermal pulses resulting in larger ΔT were indistinguishable from N2 (Figure 8B). However, we observed that eat-4 mutants were defective in their avoidance response at ΔT = 1.0°C (Figure 8A) suggesting that different combinations of glutamate-gated channels were responsible for mediating effects of glutamatergic neurotransmission at ΔT = 1.0°C. Additionally, defects in catecholaminergic neurotransmission due to a mutation in the vesicular monoamine transporter, cat-1, resulted in abnormal avoidance responses only at ΔT = 0.4°C (Figure 8D). Thus response to a thermal stimulus of ΔT = 0.4°C requires participation of both glutamatergic and catecholaminergic neurotransmission.
Neuropeptides define escape responses to a subset of thermal pulse stimuli
Neuropeptide signaling was shown to be required for avoidance of noxious thermal stimuli [7, 8]. We examined thermally induced avoidance responses of mutants harboring loss of function of genes encoding proprotein convertase (egl-3) and carboxypeptidase (egl-21) [33] that are required for efficient neuropeptide processing. We found that animals carrying loss-of-function alleles of these genes were defective in responding to a ΔT = 0.4°C. Interestingly, all neuropeptide-processing-impaired strains were able to elicit an avoidance response similar to N2 at ΔT = 1.0°C. However, animals harboring loss-of-function mutations in egl-21 and egl-3 were defective at ΔT = 4.8°C and ΔT = 9.1°C, respectively. This difference probably reflects the penetrance of the different alleles of egl-21 and egl-3 with respect to the thermal avoidance behavior (Figure 8C). We conclude that whereas glutamate and neuropeptide signaling is required for responses to ΔT = 0.4°C, neuropeptides but not glutamate are dispensable for responses at ΔT = 1.0°C. For higher ΔT, we found neuropeptide but not glutamate involvement in mediating different aspects of the thermal avoidance behavior. Taken together, these results suggest that distinct combinations of glutamatergic, catecholaminergic and peptidergic signaling shape avoidance responses at different intensities of noxious thermal stimuli. However, the lack of consistent responses for multiple alleles of the same gene precludes us from a definitive conclusion about the involvement of neuropeptides at higher ΔT. Nevertheless, taken together, these data suggest that distinct combinations of neurotransmitter systems operate at ΔT = 0.4°C, 1.0°C, 4.8°C and 9.1°C to give rise to a qualitatively similar stereotypical behavioral response.