We developed a high-content thermal nociception assay with precise control of the location of the stimulus in order to spatially dissect the thermal noxious response in C. elegans
. By reading the “body language” of C. elegans
as a function of stimulus position, we uncovered a number of new features of thermal nociception, including a midbody response distinct from the known head and tail responses [1
]. Previous studies [1
] used a large change in temperature (>10°C) to elicit escape response, but here we showed that stimuli as small as a fraction of a degree can elicit a response if the change in temperature is fast. In addition the behavioral features of the worm’s response such as the reaction time and the escape angle changes in a way that might be favorable to the worm as the level of the noxious stimuli increases.
Our results also show that the worm can respond to thermal stimuli localized to the midbody. The pair of polymodal nociceptors PVD possess a dendritic arbor that covers most of the worm’s body [23
], and have been shown to sense aversive stimuli such as harsh touch and cold shock [6
]. Through genetic tools, laser ablation, and calcium imaging, we have confirmed PVD’s involvement in sensing an abrupt increase of heat at the midbody and tail.
Since PVD covers the majority of the worm’s body, it is expected that it would have a large receptive field. Using genetic and neuronal ablation, we were able to delineate the thermally-stimulated receptive field of PVD to the middle and tail regions of the worm. We also generated a behavioral receptive field map for PVD by analyzing the worm’s response to the thermal stimulus as a function of stimulus location along the worm’s body. In doing so, we discovered that C. elegans is able to discriminate stimuli at the midbody with a spatial sensitivity of at least 80 microns. This result implies that PVD could be used as a model nociceptor for the study of spatial differentiation of noxious stimuli by a single neuron.
In addition to PVD, we investigated the spatial sensitivity of the midbody response related to the differential synaptic outputs to command interneurons AVA and PVC. It has been recently suggested that relative synaptic inputs to command interneurons due to the position of the stimulus modulate the forward/backward locomotion of the worm [27
]. Our measure of the -PVC worm’s behavioral receptive field revealed an extension of AVA initiated reversals into the posterior region of the worm, as well as in increase of pausing in the posterior. In effect, the worm’s “sensory middle” is shifted towards its tail. With PVC not functioning there is no differential excitation of the command interneurons to initiate a spatially biased withdrawal behavior, and the increased pause state in the far posterior may be due to the dominating bias of AVA on the response.
We also discovered a defect in the midbody response of the mutant glr-1
. Glutamate receptors play an important role in polymodal nociceptors, as they may serve to select for different stimulation modalities [9
]. GLR-1 has also been implicated in long term memory formation [38
], as well as the control of locomotion in foraging [31
]. Our assay found a defect in the midbody response for the mutant glr-1(n2466)
(Figure a), and this allele is expressed in the command interneurons AVA and PVC [30
]. This suggests a role for glr-1
and glutamate in thermal nociception through PVD.
The utility in quantifying the noxious response goes beyond investigating the midbody thermal avoidance behavior in C. elegans
. The establishment of C. eleg
ans as a model organism for nociception requires a comprehensive quantitative analysis of its wild type behaviors to serve as a benchmark in screening for defects caused by genetic, neuronal and pharmacological factors. We generated a dose response and identified several features that scaled with stimulus amplitude and can be used as a measure of nociception. Of note, the maximum mean speed of the response, the probability distribution of the first behavioral state after the stimulus, the reaction time, and the escape angle are all correlated to the strength of the stimulus. Interestingly, we observed that the reaction time is proportional to the logarithm of the stimuli strength, which suggests so-called logarithmic sensing [39
] consistent with Weber-Fechner [40
] in the sensorimotor transformation for thermal stimuli in C. elegans
. Regarding the escape angle of the animal, the articulation of the omega turn in the head noxious response modulates the escape trajectory. On average, the omega turn happens several seconds after the stimulus is presented (Figure b). Yet, our results show that the worm’s measurement of the strength of the stimulus is incorporated in the omega turn (Figure d). In fact, no worms reorient themselves more than 170° when presented with a less harmful stimulus of 30 mA, while 76% of worms do so when the stimulus is more intense (150 mA). In addition the response time decreases and the reversal duration increases as the noxious level of the stimulus increases [15
]. Further, the wild type midbody pause state could possibly be explained as a behavioral strategy to allow the worm more time to accrue additional information about the location of the stimulus before breaking the symmetry, and to increase the chances of choosing the trajectory that effectively directs the worm from the danger. C. elegans
has evolved a complex, multi-faceted behavioral response to a noxious stimulus with multiple features that change in a coordinated way to produce an adaptive protective behavior.
A conceptually similar attempt to quantify pain in animal models is the recent generation of the Mouse Grimace Scale – a catalog of laboratory mouse facial expressions that are meant to quantify the amount of pain felt by acute stimuli [41
]. Studies such as these, while promising, have inherent limitations because mammalian behaviors are very complex and difficult to quantify. Furthermore, these animals have a long pre-stimulus history that integrates many environmental stimuli that may confound the pain response. C. elegans
can be quickly grown in identical conditions and environmental stimuli controlled, making it a desirable model organism for the study of nociception. Furthermore, there is a molecular similarity in thermal nociception or pain among vertebrates and invertebrates [13
]. An example of this overlap is the TRPV channel OCR-2 expressed in sensory neurons, which we suggest is required for thermal nociception in PVD. Even though there will be differences in vertebrate and invertebrate nociception, this work adds to the growing evidence that investigating thermal nociception in C. elegans
may help in understanding this sensorimotor transformation in higher organisms.