Abstract
Presynaptic plasticity is known to modulate the strength of synaptic transmission. However, it remains unknown whether regulation in presynaptic neurons alters the directionality –positive or negative-of postsynaptic responses. We report here that the C. elegans homologs of MAST kinase, Stomatin and Diacylglycerol kinase act in a thermosensory neuron to elicit in its postsynaptic neuron an excitatory or inhibitory response that correlates with the valence of thermal stimuli. By monitoring neural activity of the valence-coding interneuron in freely behaving animals, we show that the alteration between excitatory and inhibitory responses of the interneuron is mediated by controlling the balance of two opposing signals released from the presynaptic neuron. These alternative transmissions further generate opposing behavioral outputs necessary for the navigation on thermal gradients. Our findings reveal the previously unrecognized capability of presynaptic regulation to evoke bidirectional postsynaptic responses and suggest a molecular mechanism of determining stimulus valence.
Introduction
Sensory stimuli that predict the valence of reward or punishment are major drivers of animal behaviors. For example, odors associated with predators are detrimental to the animals and elicit fear responses1, while smells predicting the presence of food or potential mates evoke different behaviors such as feeding2 or mating3. Extracting the valence information of sensory stimulus – whether the stimulus is attractive or aversive- and manifesting appropriate behavioral responses are the most vital function of the nervous system. Deciphering the molecular and circuit mechanisms underlying the valence coding of sensory information is thus fundamental to understand the principles of how animal behaviors emerge from the nervous system.
The valences of innate odor responses are guided by intrinsic property of the responding neurons, in which neurons expressing specific odorant receptors are hardwired in a neural circuit that elicits attractive or aversive behavior4,5. A similar labeled-line circuit operation was also demonstrated for encoding and responding to tastes such as sweet and bitter6–8, wherein cells expressing specific taste receptors are embedded in a specialized neural circuit that promotes or inhibits feeding behaviors.
Contrary to these developmentally programmed, stereotyped behaviors, the valence associated with certain sensory stimuli can vary depending on the past experience, the current environmental context and the stimulus intensity9. For example, olfactory preferences to the same odorants can differ depending on the odorant concentration10. Studies of worms, flies and mammals suggested a common feature of neural mechanism underlying the change in these odorant valences, wherein different concentrations of the same odorants recruit distinct sets of olfactory neurons and consequently change the perception of the same odorants11–13. However, the extent to which the brain utilizes different encoding strategies for alternating stimulus valence remains largely unexplored. In particular, the molecular and circuit mechanisms underlying the perception of altering valence for other sensory modalities are not yet understood.
The compact nervous system of C. elegans consisting of only 302 neurons provides an excellent opportunity to explore these questions14. C. elegans exhibits thermotaxis behavior15, in which the valence of thermal information varies depending on the past experience, current temperature environment and feeding states. Specifically, the temperature preference of C. elegans is plastic and determined by the cultivation temperature, in which animals that are cultivated at a constant temperature with food migrate toward that cultivation temperature on a thermal gradient without food15. When animals were placed at the temperature below the cultivation temperature they migrate up the thermal gradient, while above the cultivation temperature they move down the gradient, indicating that the valence associated with thermal stimuli alternates in opposing manners depending on the current temperature.
Previous studies identified neurons involved in thermotaxis16–18. Of those neurons, the AFD thermosensory neurons are essential for thermotaxis and are required for migrating up and down the thermal gradient to reach the cultivation temperature17,19. Calcium imaging analyses revealed that the AFD neuron displayed increases in calcium concentration upon warming phases of a temperature ramp both below and above the cultivation temperature20,21. However, it remains to be determined how the AFD neuronal activities with similar calcium dynamics below and above the cultivation temperature are transformed to encode appropriate valence of temperature information and the consequent manifestation of opposing behavioral regulations.
Here we report that the AFD neuron evokes opposing neuronal responses in its postsynaptic partner AIY interneuron below or above the cultivation temperature: the activation of AFD neuron stimulates the AIY neuron below the cultivation temperature, while it inhibits AIY above that temperature. We identified molecular components important for this process and showed that this alteration of the AFD-AIY communication is regulated by presynaptic actions of the C. elegans homologs of MAST (Microtubule-Associated Serine-Threonine) kinase22, Stomatin23 and Diacylglycerol kinase24,25. Our results further suggest that the alteration of the AFD-AIY synaptic transmission is mediated by the balance of two opposing signals released from the AFD neuron, an excitatory peptidergic signaling and an inhibitory glutamatergic signaling. A high-throughput behavioral analysis revealed that these alternative modes of the AFD-AIY transmission generate opposing behavioral biases in the steering direction of animal locomotion to warmer or colder side of the temperature gradient, thereby driving the animals toward the cultivation temperature. Our results suggest that bidirectional responses in the valence-coding neurons are regulated by presynaptic mechanism whereby the evolutionarily conserved MAST kinase, Stomatin and Diacylglycerol kinase control the presynaptic release of opposing signaling molecules.
Results
kin-4, mec-2 and dgk-1 Regulate the C. elegans Thermotaxis Behavior
To elucidate the molecular and neural mechanisms underlying the valence coding of thermal stimuli during C. elegans thermotaxis behavior, we conducted forward genetic screens and sought mutants that displayed abnormal thermotaxis behavior. We found that mutations in three genes, kin-4, dgk-1 and mec-2, affected this behavior (Fig. 1). While the wild-type animals that had been cultivated at 20 °C preferred to stay around the cultivation temperature, loss-of-function mutants of kin-4, which encodes the C. elegans ortholog of the mammalian MAST (Microtubule Associated Serine Threonine) kinase, displayed a cryophilic phenotype and migrated toward a colder temperature region than the wild-type animals (Fig. 1b and Supplementary Fig. 1a). This defect was rescued by introduction of a kin-4 genomic clone (Fig. 1c), indicating that kin-4 is required for thermotaxis. Animals carrying mutations in the gene dgk-1, which encodes a homolog of Diacylglycerol kinase θ24,25, also preferred to migrate toward a colder temperature region, as was previously reported 26 (Fig. 1b and Supplementary Fig. 1a). The cryophilic phenotype of dgk-1(nj274) mutants was rescued by introduction of a dgk-1 genomic clone (Supplementary Fig. 1b), indicating that dgk-1 is required for thermotaxis. We also isolated a mutation in mec-2, which encodes a C. elegans homolog of Stomatin23. mec-2(nj89) animals harbored a mutation that is predicted to alter the glutamic acid 270 to a lysine (E270K, see Supplementary Fig. 1a), and displayed a thermophilic phenotype (Fig. 1b). Introduction of a mutant mec-2(E270K) clone into the wild-type animals phenocopied mec-2(nj89) mutants, while introduction of a wild-type mec-2 clone into mec-2(nj89) mutants did not rescue the thermophilic defect (Fig. 1d). We also generated a null allele of mec-2(nj251 Δ) (Supplementary Fig. 1a) and found that mec-2 null mutants were grossly normal in thermotaxis (Supplementary Fig. 1c), suggesting that some of the nine additional Stomatin genes encoded by the C. elegans genome could compensate the deficit of mec-2. These results indicate that mec-2(nj89) is a gain-of-function mutation and causes a thermophilic phenotype.
To address genetic interactions among these genes, we analyzed thermotaxis behaviors of double mutants. Animals carrying mutations in both kin-4 and mec-2 showed a thermophilic phenotype similar to that of mec-2 single mutants (Fig. 1e), suggesting that mec-2 acts downstream of or in parallel to kin-4. Similarly, dgk-1 mutations partially suppressed the thermophilic phenotype conferred by mec-2(nj89gf) mutation (Fig. 1f), suggesting that dgk-1 acts downstream of or in parallel to mec-2. We hereafter focused on kin-4(tm1049Δ), mec-2(nj89gf) and dgk-1(nj274Δ) mutants for further analyses.
kin-4, mec-2 and dgk-1 Function in the AFD Thermosensory Neuron to Regulate Thermotaxis
To ask where kin-4 and mec-2 are expressed, we conducted expression analysis. We generated a functional kin-4::gfp translational transgene capable of rescuing the cryophilic phenotype of kin-4 mutants (Supplementary Fig. 2a). This transgene was broadly expressed in the nervous system, and its expression was observed in neurons previously shown to be involved in thermotaxis16,17,27, including the AFD and AWC thermosensory neurons and the AIY and RIA interneurons (Figs. 2a and b, and Supplementary Fig. 2b). We also assessed expression of mec-2 and found that a Pmec-2c::gfp reporter was expressed in AFD and AWC (Fig. 2b). As previously reported23, expression in mechanosensory neurons was also observed when gfp was fused to a promoter for another mec-2 isoform, mec-2a (Supplementary Fig. 2c). A previous study also showed that dgk-1 was ubiquitously expressed in the nervous system25. These results suggest that kin-4, mec-2 and dgk-1 are expressed in neurons known to be involved in regulation of thermotaxis, including the AFD and AWC thermosensory neurons.
To identify the neuron(s) in which kin-4, mec-2 and dgk-1 act to regulate thermotaxis, we attempted to express each of these genes in single neurons and assessed their effects on the thermotaxis behavior. Expression of a kin-4 cDNA in AFD rescued the cryophilic phenotype of kin-4 mutants, whereas expression in AWC, AIY, AIZ or RIA did not (Fig. 2c), indicating that kin-4 functions in AFD to regulate thermotaxis. When mutant mec-2(E270K) was expressed in AFD, it phenocopied mec-2(nj89gf) mutants, while expression in AWC did not (Fig. 2d). Expression of a dgk-1 cDNA in AFD but not in AWC partially rescued the cryophilic phenotype of dgk-1 mutants (Fig. 2e). We also observed that simultaneous expression of dgk-1 in both AFD and AWC fully rescued the dgk-1 mutant phenotype. These results indicate that kin-4, mec-2 and dgk-1 function in AFD to regulate thermotaxis.
kin-4, mec-2 and dgk-1 Act Downstream of Calcium Influx in AFD
To assess whether kin-4, mec-2 and dgk-1 regulate temperature-evoked activity of the AFD thermosensory neuron, we conducted calcium imaging of AFD. We immobilized animals expressing the GCaMP3 calcium indicator in AFD and subjected the animals to a warming stimulus. As previously reported20,21, the AFD neuron showed increases in calcium concentration upon warming stimuli both below and above the cultivation temperature (Figs. 3a and b), and this response required three guanylate cyclase genes, gcy-8, gcy-18 and gcy-23 exclusively expressed in the AFD neurons28–30 (Fig. 3b). The temperature-evoked calcium responses of AFD in kin-4, mec-2 and dgk-1 mutants were almost indistinguishable from that of the wild-type animals (Fig. 3b-d). These results suggest that kin-4, mec-2 and dgk-1 regulate a process downstream of the calcium influx in AFD.
Valence of Thermal Information Is Encoded by Bidirectional AIY Response
To ask whether kin-4, mec-2 and dgk-1 regulate the activity of the AIY interneuron, the sole chemical postsynaptic partner of AFD14, we monitored calcium dynamics of both AFD and AIY. We generated the animals expressing a calcium indicator in both AFD and AIY and subjected them to warming stimuli while they were freely moving under the microscope with an automated tracking system (Fig. 4). We first subjected animals to a warming stimulus below the cultivation temperature, in which the temperature was increasing toward the cultivation temperature (Fig. 4a). When the wild-type animals were exposed to this warming stimulus, the AFD neuron showed an increase in calcium concentration, and the AIY neuron also displayed a rise in calcium concentration after a slight drop observed at the beginning of the warming stimulus (Fig. 4b). By contrast, when the wild-type animals were subjected to a warming stimulus above the cultivation temperature, where the temperature was increasing away from the cultivation temperature (Fig. 4e), the AIY neuron instead showed a decrease in calcium concentration, despite the increase of calcium level in AFD (Fig. 4f). These results indicate that the bidirectional response of the AIY neuron correlates with the valence of thermal stimuli, with temperature increase toward the cultivation temperature evoking excitatory response and temperature increase away from the cultivation temperature inhibitory response.
kin-4, mec-2 and dgk-1 Regulate Bidirectional AIY Response
We next examined the AIY responses in kin-4, dgk-1 and mec-2 mutants. The AIY neuron in kin-4 mutants exhibited a decrease in calcium concentration even below the cultivation temperature, the condition in which the wild-type AIY neuron would normally increase its calcium level (Figs. 4b and c). Consistent with the calcium imaging analysis of AFD in immobilized animals (Fig. 3), the AFD neuron of kin-4 mutants showed increases in calcium concentration both below and above the cultivation temperature (Figs. 4b and c). The defect in the AIY response of kin-4 mutants was partially rescued by expression of a kin-4 cDNA solely in the AFD neuron (Figs. 4b and d), indicating that the AIY calcium response is indeed modulated by it presynaptic partner AFD. A similar response profile was also observed in the cryophilic dgk-1 mutants: the AIY calcium level dropped upon warming stimuli both below and above the cultivation temperature, while the AFD neuron responded to the warming stimuli by increasing the calcium concentration (Figs. 4b, c, f and g). Furthermore, the thermophilic mec-2 mutants showed an abnormal increase in the AIY calcium concentration even above the cultivation temperature (Figs. 4f and g). Similarly in the wild type, the AFD neurons in mec-2 mutants showed increases in calcium concentration under both conditions tested (Figs. 4b and f). These results indicate that the valence-coding bidirectional AIY activity is regulated by kin-4, mec-2 and dgk-1. Given that kin-4 expression only in AFD restored the defect of the AIY response in kin-4 mutants, these results further suggest that presynaptic regulation is important for determining the bidirectional responses of the AIY neuron.
A recent study reported that the difference in the AIY calcium responses below or above the cultivation temperature was represented by a difference in the fraction of animals in which AIY showed an increase in calcium concentration upon warming stimulus under the experimental condition where the animals were immobilized31. Our results further suggest that in freely behaving animals, the AFD-AIY transmission can alter between excitatory and inhibitory communication below or above the cultivation temperature.
Alteration of the AFD-AIY Synaptic Transmission Requires Components Essential for Neuropeptide and Glutamate Release
We next asked how the presynaptic regulation in AFD evokes opposing neuronal responses in the AIY postsynaptic neuron. Previous studies indicated that AFD employs two kinds of signaling molecules to communicate with AIY27,32: one is neuropeptide, and the other is glutamate. The peptidergic signaling was shown to be excitatory32, whereas the glutamatergic input is inhibitory due to a glutamate-gated chloride channel acting on AIY27. We hypothesized that the balance of these two opposing signals released from AFD might be modulated, thereby inducing an excitatory or inhibitory postsynaptic response. To test this possibility, we monitored the AIY calcium response in mutants for the gene unc-31, which encodes the C. elegans ortholog of calcium-dependent activator protein required for secretion of neuropeptides33. The AIY neurons of unc-31 mutants failed to increase the calcium level and instead showed a decrease even below the cultivation temperature, while the AFD neuron showed increases in calcium concentration under both conditions tested (Figs. 5a-c). This defect in the bidirectional AIY response was partially rescued by expression of an unc-31 cDNA only in AFD (Figs. 5b and d), indicating that the peptidergic signals from AFD is required for the bidirectional AIY response.
We also examined the AIY response in mutants for the eat-4 gene, which encodes a C. elegans homolog of vesicular glutamate transporter required for transporting glutamate into synaptic vesicles34. In eat-4 mutants, the AIY neurons displayed abnormal increase in the calcium concentration above the cultivation temperature (Figs. 5e-g), and this defect was rescued by expression of eat-4 only in the AFD neurons (Figs. 5f and h). Like in unc-31 mutants, the AFD neurons in eat-4 mutants showed increase in the calcium level under both conditions tested. These results suggest that alteration of the positive and negative modes of the AFD-AIY communication is mediated by the presynaptic control of the glutamatergic and peptidergic outputs.
kin-4, mec-2 and dgk-1 Regulate Curving Bias During Thermotaxis
To address how the alteration in the modes of the AFD-AIY communication contributes to the regulation of thermotaxis behavior, we undertook multi-worm tracking analysis35. We classified the animal behavior into several behavioral components discernable during C. elegans locomotion, such as forward locomotion, turns and reversals36. We sought the behavioral components that were oppositely biased below or above the cultivation temperature in the wild-type animals, and were oppositely affected by the cryophilic (kin-4 and dgk-1) or thermophilic (mec-2) mutations. Among the behavioral components we have examined (Fig. 6), we found that the curve, change in the moving direction during forward locomotion, was one such component (Fig. 6a): the wild-type animals behaving below the cultivation temperature showed curving bias toward warmer temperature when migrating up the thermal gradient, while they curve toward colder temperature above the cultivation temperature36 (Fig. 6a), suggesting that this regulation of the curve would drive the animals toward the cultivation temperature. By contrast, the curving rates of the two cryophilic mutants, kin-4 and dgk-1, below the cultivation temperature were abnormally biased toward the colder side (Fig. 6a). Furthermore, mec-2 mutants behaving above the cultivation temperature failed to show a curving bias toward colder temperature (Fig. 6a). These results indicate that kin-4, mec-2 and dgk-1 regulate the curve during thermotaxis and suggest that the alteration of the AFD-AIY synaptic transmission generates opposing curving biases that drive the animals toward the cultivation temperature.
Discussion
When animals encounter environmental stimuli, they need to quickly assess whether the stimuli are beneficial or detrimental. How the brain determines whether the valence of sensory information is attractive or aversive has been a fundamental question in neurobiology. A number of previous studies indicated that the opposing valences of sensory stimuli are encoded by two separate populations of neurons, each of which represents either positive or negative valence of the stimuli37,38. By contrast, recent studies proposed an alternative strategy, wherein a single population of neurons responds to appetitive or aversive stimuli and represents the positive or negative valence of the stimuli by increasing or decreasing neuronal activity39,40. Specifically, CRF (corticotropin-releasing factor)-releasing neurons in the paraventricular nucleus of the hypothalamus are activated by aversive stimuli and inhibited by appetitive stimuli40. Likewise, in C. elegans, experience-dependent modulation enables a single set of interneurons to elicit bidirectional responses to carbon dioxide, which can be either attractive or aversive, depending on prior experience39. Thus, these observations indicate a previously unrecognized mechanism of valence coding for even a single modality of stimulus, in which the bidirectional activity in a single population of neurons can be modulated by prior experience and environment to represent stimulus valence. However, little was known about the molecular mechanism and circuit logics for such modulation of valence-coding activity. In this study, we report the molecular components important for determining a valence-coding activity and show that MAST kinase, Stomatin and Diacylglycerol kinase control the activity of the AIY neuron that correlates with the valence of thermal stimuli. Our results also reveal a circuit principle of such valence coding, in which a presynaptic neuron modulates its neuronal outputs and evokes an excitatory or inhibitory postsynaptic response.
We showed that kin-4, mec-2 and dgk-1 regulate the curving bias during thermotaxis behavior (Fig. 6). A previous study also showed that optogenetic manipulations of the AIY activity evoked biases in the curve: stimulation of AIY caused the animals to turn in the direction in which the head of the animal was bent at the time of the AIY excitation, while inhibition of AIY induced the animals to turn in the opposite direction41. Given this observation, our results suggest that below the cultivation temperature, a warming stimulus activates both AFD and AIY, leading to curve toward the warmer side of the temperature gradient, while above the cultivation temperature, a warming stimulus activates AFD, which in turn inhibits AIY, resulting in curving toward the colder side (Supplementary Fig. 3). Thus, the alteration of the AFD-AIY transmission mode would generate the curving bias that drives the animals toward the cultivation temperature.
Recent investigation of the global brain dynamics in C. elegans revealed that most interneuron layers represent the motor states of the animals42. Indeed, representation of the motor states is pervasive even in the first-layer interneurons that receive direct inputs from sensory neurons. The activity of the AIB interneuron, which is directly innervated by gustatory sensory neurons ASE and olfactory neurons AWC, is correlated with reversals43, and the AIY neuron can represent multiple motor states41,43,44. Because calcium dynamics within the AFD, ASE and AWC neurons represent the respective sensory stimuli20,21,45–47, these observations suggest that transformation of sensory information into motor representation occurs early in the neural circuit and underlies between the sensory neurons and the first-layer interneurons.
Our results indicate that the initial step of information processing that transforms thermal information into stimulus valence occurs within the AFD sensory neuron and suggest that information processing for this transformation resides in cellular processes between calcium influx and neurotransmitter release. These observations underscore neural computation at axonal regions in a single neuron. In addition to the current view that neural computations take place at the synaptic communication and dendritic computations, our results highlight the importance of axonal computation in information processing in the nervous system. These observations, in turn, raise a future challenge in neuroscience. Although electrophysiological and calcium imaging analyses undoubtedly reveal certain aspects of neuronal properties, understanding the axonal computation requires the development of methods to quantitatively measure the release of the signaling molecules, including small neurotransmitters, neuropeptides and biogenic amines48,49, preferably in behaving animals with high spatiotemporal resolution. Utilization of a pH-sensitive green fluorescent protein, pHluorin50 has been successfully used to monitor the release and recycle of synaptic vesicles51–54. However, since synaptic vesicle neurotransmitter content can be dynamically regulated in both vertebrates and invertebrates55,56, direct measurements of signaling molecules rather than that of vesicle release are required to fully understand the neuronal output. Development of such methods would unveil the dynamics of axonal computations and help dissect the mechanisms by which neurons execute this type of neural computation.
Neurons expressing multiple neurotransmitters are present in virtually all animal species. Co-transmission of multiple transmitters from single neurons can influence the same target neurons (convergent actions) or different targets (divergent actions), thereby providing additional flexibility to the circuit functions57,58. A few studies reported co-transmission of multiple transmitters with opposing actions59–62. In mammals, orexin and dynorphin neuropeptides exert opposing actions on excitability of ventral tegmental area dopamine neurons59,60, and in Aplysia neuromuscular junction, multiple co-transmitted peptides exert antagonistic actions, with one peptide promoting muscle contraction and the other increasing relaxation rate61,62. In these cases, multiple transmitters are co-packaged and co-released in a fixed ratio. The apparently antagonistic actions of multiple transmitters likely facilitate rhythmic muscle contraction, thereby providing temporal flexibility in the circuit function61,62.
Our results suggest that by altering the balance of multiple transmitters with opposing actions, a single presynaptic neuron can evoke excitatory and inhibitory postsynaptic responses, convey the valence information of sensory stimulus to the circuit, and induce an appropriate behavior. Our observations provide a sharp contrast to current views of presynaptic plasticity, in which presynaptic regulation facilitates or depresses the strength of the postsynaptic response63,64 but does not change the mode of transmission from an excitatory (inhibitory) to an inhibitory (excitatory) communication. We speculate that such mechanism of presynaptic control might function in other systems65 and be particularly effective in assigning the stimulus valence over a range of stimulus intensity. Since previous studies suggested that dgk-1 regulates synaptic transmission at neuromuscular junction in C. elegans24,25,66, kin-4, mec-2 and dgk-1 might also regulate the release of glutamate and/or neuropeptide from AFD to control the bidirectional AIY response. Thus, presynaptic control of multi-transmitter release could be a fundamental mechanism of generating plasticity without extensive structural modification on the defined neural circuitry, thereby extending the computational repertoire employed by the nervous system.
Methods
C. elegans strains
All C. elegans strains were cultivated at 20 °C on nematode growth medium plates seeded with E. coli OP50 bacteria67. N2 (Bristol) was the wild-type strain. The following mutations, extrachromosomal arrays and integrated transgenes were generated or described previously. LGI: njIs24[gcy-8p::GCaMP3, gcy-8::TagRFP]68. LG III: eat-4(ky5)34. LG IV: kin-4(tm1049Δ), kin-4(nj170Δ), unc-31(e928)67,69. LG V: njIs110[gcy-8p::4xNLS::YCX1.6, AIYp::YCX1.6]. LG X: mec-2(nj89gf), mec-2(nj251Δ), dgk-1(nj271), dgk-1(nj274Δ). Extrachromosomal arrays: njEx682[kin-4(+), ges-1p::gfp], njEx683[kin-4::gfp, ges-1p::TagRFP], njEx753[gcy-8p::kin-4d cDNA, ges-1p::gfp] (used for rescuing the cryophilic phenotype of kin-4(tm1049Δ), njEx759[ttx-3p::kin-4d cDNA, ges-1p::gfp], njEx760[lin-11p::kin-4d cDNA, ges-1p::gfp], njEx763[ceh-36p::kin-4d cDNA, ges-1p::gfp], njEx764[glr-3p::kin-4d cDNA, ges-1p::gfp], njEx1029[mec-2(E270K), ges-1p::gfp], njEx1035[mec-2(+), ges-1p::gfp], njEx1158[Pmec-2a::gfp, ges-1p::TagRFP], njEx1159[Pmec-2c::gfp, ges-1p::TagRFP], njEx1220[ceh-36p::mec-2a cDNA (E270K), ceh-36p::mec-2c cDNA (E195K), ges-1p::gfp], njEx1231[gcy-8p::mec-2a cDNA (E270K), gcy-8p::mec-2c cDNA (E195K), ges-1p::gfp], njEx1317[gcy-8p::dgk-1b cDNA, ges-1p::gfp], njEx1319[odr-1p::dgk-1b cDNA, ges-1p::gfp], njEx1330[dgk-1(+), ges-1p::gfp], njEx1331[gcy-8p::dgk-1b cDNA, odr-1::dgk-1b cDNA, ges-1p::gfp], njEx1435[gcy-8p::kin-4d, ges-1p::TagRFP] (used for rescuing AIY calcium response), njEx1438[gcy-8p::unc-31 cDNA, ges-1p::TagRFP], njEx1439[gcy-8p::eat-4 cDNA, ges-1p::TagRFP]. We used following promoters for cell-specific expression of transgenes and cDNAs: gcy-8 promoter for AFD; ttx-3 promoter for AIY; ceh-36 or odr-1 promoters for AWC; lin-11 promoter for AIZ; glr-3 promoter for RIA; ges-1 promoter for intestinal cells.
Thermotaxis behavioral tests
Thermotaxis assays were performed as previously described68. Animals that had been cultivated at 20 °C were placed on the center of thermotaxis assay plate with a temperature gradient ranging from 17 °C to 23 °C. The steepness of the temperature gradient was set at 0.45 ~ 0.5 °C / cm. Animals were allowed to freely move on the plate for 1 hour. The thermotaxis assay plate was divided into 8 sections along the temperature gradient, and the number of adult animals in each section was counted.
Expression analysis of kin-4 and mec-2
To construct kin-4::gfp, we modified the fosmid containing the kin-4 locus, WRM0635dF07. We inserted a gfp coding sequence immediately before the stop codon of kin-4 using bacterial recombineering. To construct Pmec-2a::gfp and Pmec-2c::gfp, a 3.5 kb and a 3.2 kb fragments upstream of the start codon of mec-2a and mec-2c were cloned into the gfp expression vector pPD95.75, respectively. Animals carrying kin-4::gfp, Pmec-2a::gfp or Pmec-2c::gfp were anesthetized with 50 mM sodium azide and were observed under Nomarski optics equipped with epifluorescence. Identification of the AFD, ALM, AVM, AWC, AIY, PLM, PVM and RIA neurons was conducted by observing the positions and sizes of the nuclei and the patterns of neuronal processes.
Multi worm tracking analysis
Multi worm tracking analysis was performed as described36. Animals were cultivated at 20 °C and were placed on the thermal gradient with the center temperature of 18.5 °C or 21.5 °C to monitor their behaviors below or above the cultivation temperature, respectively. The behaviors during the first 10 minutes of the thermotaxis assay were captured by a CMOS camera and were analyzed by multi worm tracker to obtain the coordinates of animal’s centroids and spines35. The data were further analyzed by a custom-written program in MATLAB to classify the behaviors into behavioral components.
Calcium imaging of AFD in immobilized animals
Calcium imaging of the AFD neuron was performed as described68. Animals expressing GCaMP3 and TagRFP in AFD were cultivated at 20 °C and placed on a 5 – 10 % agarose pad with polystyrene beads to immobilize the animals. Animals were subjected to a warming stimulus, and the fluorescence intensities from the AFD cell body were captured and analyzed by the MetaMorph software. The ratio of the fluorescence intensity (GCaMP3/TagRFP) was used to calculate ratio change, which was defined as (ratio – baseline ratio)/baseline ratio, where baseline ratio was the mean of the ratio values during the first 30 seconds of the experiment. The response temperature was previously defined68 as the temperature at which the ratio change first exceeded 1.
Calcium imaging of AIY in freely moving animals
We generated animals expressing the ratiometric calcium probe YCX1.6 in AFD and AIY. YCX1.6 in AFD was localized to the nucleus to separate the fluorescence signals from these neurons. Animals were cultivated at 20 °C and placed on a 2 – 2.5 % agarose pad on a cover glass. The sample was covered by another cover glass and was placed onto a transparent temperature-controlled device (TOKAI HIT Co. Ltd., Fujinomiya). This device was installed onto a motorized stage (HawkVision Inc., Fujisawa) that keeps the target image of animals in the field of view. Controlling the stage movement was achieved by real-time analysis of transmitted infrared light images. The fluorescence images were captured twice a second and split into YFP and CFP channels by W-VIEW GEMINI (Hamamatsu photonics K.K., Hamamatsu). The YFP and CFP fluorescence intensities were obtained from the cell body of AFD and an axonal region of AIY68. The fluorescent images were analyzed by a custom-written program in MATLAB with manual inspection of region of interest in every frame, and the fluorescent intensities of YFP and CFP were determined. We eliminated from the analysis the trials in which the temperature program failed to activate AFD. For this purpose, we applied a low-pass filter to the YFP/CFP ratio with the cut-off frequency at 0.05 Hz, and the resulting ratio data were used to calculate the ratio change. We eliminated from the analysis the trails in which the maximum ratio changes were smaller than 0.25 for recordings below the cultivation temperature or 0.22 for above the cultivation temperature. These threshold values were determined from experiments in which the temperature was kept constant. For the reminder of the trials, the ratio of fluorescence intensities (YFP/CFP) was used to calculate the standardized ratio change of AFD and AIY, which was defined as (ratio – minimum ratio)/(maximum ratio – minimum ratio). The baseline standardized ratio, which was the mean of the standardized ratio values of 5 consecutive frames immediately before the onset of warming stimulus, was subtracted from the standardized ratio change of each frame. For comparison of the peak standardized ratio change, the maximum standardized ratio change (positive or negative) in a 2-second time window centered at the peak of the mean control standardized ratio change was used.
Statistics
Normality of the data was assessed by Shapiro-Wilk test. Equal variance among data sets was assessed by F-test or Bartlett test. When both normality and equal variance were assumed for the data set, we used two-tailed student t-test for pairwise comparison and one-way analysis of variance (ANOVA) with Tukey-Kramer or Dunnett’s test for multiple comparisons. In other cases, we applied Wilcoxon rank sum test for pairwise comparison and Kruskal-Wallis rank sum test with Steel method for multiple comparisons.
Data availability
All raw images, source data and custom scripts are available from the authors upon reasonable request.
Author Contributions
S.N. and I.M. designed the experiments. S.N., R.A., A.S. and R.K. conducted experiments. S.N. and M.I. wrote custom codes for the analyses. Y.T., X.F. and K.H. developed and set up the tracking system for calcium imaging of freely moving animals. T. S., K. I. and T. H. conducted whole genome sequencing of mutants isolated in this study. S.N. and I. M. wrote the manuscript.
Competing Interests
The authors declare no competing interests.
Materials & Correspondence
Correspondence and requests for materials should be addressed to I.M. (email: m46920a{at}nucc.cc.nagoya-u.ac.jp)
Acknowledgments
We thank Y. Kohara, K. G. Miller for cDNAs; S. Mitani at National BioResouce for strains; K. Noma for comments on this manuscript; K. Ikegami, Y. Murakami, J. Okada, T. Sakaki, K. Sawayama, F. Takeshige for technical assistance. M.I. was supported by KAKENHI 16J05770. This work was supported by JSPS KAKENHI Grant Numbers 17K07499 (to S.N.), 18H05123 (to S.N.), 26560549 (to Y.T.), 16H06536 (to K.H.) 18H04693 (to I.M.), 16H01272 (to I.M.), 16H02516 (to I.M.) and by ERATO project (JPMJER1004 to TH) from JST.