A Glial Hub-and-Spoke Circuitry in C. elegans orchestrates bidirectional thermosensation

Domain: neuroscience
Published: 2026-01-21
Editorial outcome: major_revision

Abstract
Thermosensation is evolutionarily conserved for survival, yet the roles of glia in temperature coding and circuit dynamics remain poorly understood. Here, we identify C. elegans AMsh glia as dual-mode thermosensory hubs that autonomously detect temperature fluctuations by co-expressing the heat receptor GCY-28 (guanylate cyclase) and cold receptor GLR-3 (ionotropic glutamate receptor). Thermal changes induce spatiotemporal calcium dynamics in glia, driving GABA release to bidirectionally modulate neural circuits: enhancing AFD-mediated warmth detection through the excitatory receptor EXP-1 and suppressing ASH-dependent cold avoidance via the inhibitory receptor LGC-38. This GABAergic hub-and-spoke architecture regulates a broad range of thermal behaviors, including thermal avoidance, thermal resistance, and thermal preference. These findings establish glia as critical interpreters of environmental cues, highlighting their dual roles as sensors and modulators in sensory processing and providing a paradigm for understanding conserved glial mechanisms in neural circuit dynamics and behavioral plasticity.

Introduction
The ability to interpret environmental thermal gradients, termed thermosensation, represents an evolutionarily conserved survival compass, enabling organisms to navigate thermal fluctuations through coordinated metabolic and behavioral recalibration1–3. Although molecular entities such as transient receptor potential (TRP) channels, guanylyl cyclases (GCs), and ionotropic glutamate receptors (e.g., GLR-3/GLUK2) have been identified as conserved thermosensory transducers1–5, the circuit-level mechanisms by which thermal signals are transduced into adaptive behavioral outputs remain in incompletely characterized.

As a main component of the nervous system and brain, glial cells are increasingly recognized for their active involvement in neural computation, as well as in the modulation of synaptic plasticity, network dynamics, and sensory information processing6–15. However, the extent to which glia directly mediate sensory perception and how they orchestrate behavioral responses remain largely unknown.

The nematode Caenorhabditis elegans, adapted to habitats characterized by steep thermal gradients and variable temperatures, has evolved a highly sensitive thermosensory system capable of detecting minute temperature fluctuations (<0.01°C)16, thereby driving preferential orientation toward thermally optimal microenvironments while evading noxious extremes1,17,18. Central to this capability is a diverse set of thermosensory neurons that respond to distinct temperature ranges, spanning noxious cold, ambient living temperatures, and noxious heat. Activated by either warming or cooling stimuli, these neurons regulate temperature-dependent behaviors and physiological responses, such as cold/hot avoidance, temperature preference, and tolerance to extreme temperatures1,18,19.

The first identified thermosensory neuron in C. elegans is the AFD neuron, located in the head amphid. It perceives heat via guanylyl cyclases and stands as the most critical heat-sensing neuron, participating in behaviors including temperature memory, temperature preference, and heat tolerance20–24. The polymodal amphid sensory neuron ASH was shown to play a central role in cold avoidance responses25. Among other head amphid sensory neurons, AWC, ASI, and ASJ were identified relatively early as temperature-sensitive neurons. ADL and ASG respond to warming and contribute to cold tolerance, while ASER detects cold through the glutamate receptor GLR-31,19. In addition, other neurons such as PHC, PVD, FLP, and OLL are also involved in temperature sensation19,26.

Central to the amphid sensory architecture are AMsh glial cells, which form ensheathing processes around the dendrites of all amphid sensory neurons11,15,27. Interestingly, the expression of ver-1 in AMsh glia has been previously reported to be modulated by temperature, suggesting these glial cells are sensitive to temperature28,29. Despite their strategic localization at the interface of sensory transduction and their established association with thermal modulation30–32, how AMsh glia contribute to the thermotransduction and the downstream circuit coding mechanism remains largely unexplored.

Here, we demonstrate that AMsh glia autonomously detect bidirectional thermal cues through co-expression of complementary thermoreceptors: GCY-28, a heat-activated guanylyl cyclase mediating warm stimulus detection, and GLR-3, a cold-sensitive ionotropic glutamate receptor transducing noxious cold signals. This cellular ensemble constitutes a GABAergic “hub-and-spoke” microcircuit, enabling differential modulation of downstream neural pathways: excitatory signaling via the EXP-1 GABA receptor potentiates AFD-mediated warm detection, whereas inhibitory signaling through the LGC-38 receptor attenuates ASH-dependent cold aversion. These findings establish a parsimonious mechanistic paradigm, wherein glial cells, in collaboration with neurons, jointly detect environmental thermal cues and serve as integrative hubs to synthesize sensory information, orchestrating adaptive behavioral outputs that optimize organismal fitness.

Results
AMsh glia are bona fide thermal receptor cells that respond to both warm and cold stimuli
Consistent with previous studies, we observed calcium responses in AFD neurons induced by warmth and in ASH neurons triggered by cold, using transgenically expressed GCaMP5 as the calcium indicator25,33 (Fig. 1a,b). Interestingly, calcium increases were also detected in AMsh glia, the sheath glial cells surrounding the receptor endings of 12 pairs of amphid sensory neurons, including AFD and ASH27, in response to both warm and cold stimuli (Fig. 1a-d). This suggests that these glial cells may actively participate in thermosensation.

We then asked whether temperature-induced calcium responses in AMsh glia are triggered by the activation of neighboring sensory neurons. AMsh glia retained both cold- and warm-induced calcium responses in unc-13 and unc-31 mutant animals, in which neurotransmitter and neuropeptide release are deficient (Fig. 1c,d)34–36. This finding suggests that the observed responses in AMsh glia are likely cell-autonomous. The thermosensory responses of AMsh glia were even more robust in unc-13 mutants than in wild type, accompanied by altered response kinetics. These observations indicate that AMsh glial activity is mildly suppressed by circuit inputs, which are likely mediated by surrounding neurons (Fig. 1c,d).

It has been reported that AMsh glia actively engulf fragments of AFD neurons and extracellular vesicles (EVs) from ciliated amphid neurons15,37,38. Thus, it is plausible that thermosensory neurons modulate the temperature response of AMsh glia via an UNC-13/UNC-31-independent pathway. To test this possibility, we transgenically expressed TRPV1 in ASH and AFD neurons, respectively. While capsaicin reliably activated ASH and AFD in these transgenic lines9,39, no corresponding calcium responses were detected in AMsh glia (Supplementary Fig. 1). These results reinforce the notion that AMsh glia may detect temperature in a cell-autonomous manner. In our previously reported work, we demonstrated that isolated single AMsh glial cells respond to odorant stimulation9. Building on this, we recorded calcium responses elicited by temperature stimuli in individual isolated AMsh glial cells. To rule out intercellular crosstalk, dissociated cells were seeded at a low plating density. Resemblance to those recorded in the intact organism, we observed in vitro cultured AMsh glial cells exhibited pronounced calcium elevations when subjected to either cold or heat stimuli (Fig. 1e–g).

Collectively, these results support that AMsh glia detect temperature in a cell-autonomous manner.

GCY-28 acts as a heat sensor in AMsh glia
We next investigated how temperature fluctuations activate AMsh glia. AFD neurons detect heat through membrane-bound guanylyl cyclases GCY-8, GCY-18, and GCY-23, which synthesize cGMP, activating cyclic nucleotide-gated (CNG) channels to mediate temperature-induced depolarization. Phosphodiesterases (PDEs) subsequently hydrolyze cGMP, lowering its concentration and thereby closing the CNG channels22,33. To investigate the role of cGMP in AMsh glia, we utilized two distinct AMsh glia-targeting promoters (vap-1 and R11D1.3)40,41 to transgenically express a blue light-inducible guanylyl cyclase (BlgC) alongside GCaMP5.042,43. Blue light induction of BlgC resulted in robust calcium increases in AMsh glia, suggesting that the increase of cGMP level can activate AMsh glia (Fig. 2a, b). Several amphid neurons respond to blue light through the light receptors LITE-1 and GUR-344,45. We found that blue light-induced calcium elevations in BlgC-expressing AMsh glia remained intact in lite-1;gur-3 double mutants (Fig. 2a,b). This indicates that blue light stimulation activates AMsh glia specifically via BlgC.

In AFD neurons, CNG channel subunits TAX-2 and TAX-4 act downstream of cGMP22. Six CNG channel subunits are present in C. elegans: TAX-2, TAX-4, and CNG-1 through CNG-446,47. BlgC induced calcium increases in AMsh glia were slightly reduced in either tax-4 mutants or cng-1, cng-3 double mutants, and were completely abolished in cng-1, cng-3, tax-4 triple mutant background, but were rescued by restoring tax-4 expression in AMsh glia (Fig. 2a,b). Consistently, heat responses in AMsh glia were abolished in cng-1, cng-3, tax-4 triple mutant worms and rescued by AMsh glial expression of tax-4 (Fig. 2c-e). These results suggest that the CNG channels CNG-1, CNG-3, and TAX-4 act downstream of cGMP in AMsh glia. C. elegans genome encodes six PDE genes. Of these PDEs, PDE-4 and PDE-6 are cAMP-specific, while the other four PDEs can hydrolyze both cAMP and cGMP46. We found warm-induced calcium responses in AMsh glia remained intact in pde-4, pde-6 double mutants but were enhanced in pde-1, pde-2, pde-3, pde-5 quadruple mutants (Fig. 2c–e). Moreover, heat-induced calcium responses in isolated single AMsh glial cells were augmented in the pde quadruple mutants (Fig. 1e, g). These results are consistent with a role for cGMP in regulating AMsh glial thermosensory responses.

We then sought to identify the membrane GC proteins responsible for temperature sensing in AMsh glia. In gcy-8, gcy-18, gcy-23 triple mutants, the warm-induced calcium responses in AMsh glia were not reduced compared to wild-type worms (Fig. 2e–g). These findings imply that other guanylyl cyclases might be responsible for sensing heat in AMsh glia. Through single-cell RNA sequencing, the expression of several membrane GCs such as GCY-28, GCY-12, and GCY-18 has been implicated in AMsh glia48. We revealed that the absence of GCY-28 abolished warm responses in AMsh glia. Restoration of GCY-28 expression in AMsh glia fully rescued these responses, indicating that GCY-28 may function as a heat sensor in these glial cells (Fig. 2f–h).

To gain more evidence that GCY-28 is a heat sensor, we ectopically expressed GCY-28 in the chemosensory AWB neurons as a previous study described33. Intriguingly, warm induced a calcium decline in AWB neurons. Nevertheless, AWB neurons with ectopic GCY-28 expression exhibited warm-induced calcium increases (Fig. 2i).

We next performed heterologous expression of GCY-28 in HEK293T cells and quantified intracellular cGMP levels using a commercial ELISA kit. Compared to empty vector-transfected controls, GCY-28-expressing cells showed markedly elevated intracellular cGMP levels under two distinct thermal stimuli: either a direct heat challenge from 37°C (the standard culture temperature for HEK293T cells) to 45°C for 30 min, or a two-step protocol involving precooling from 37°C to 20°C for 2h (mimicking the nematode’s culture temperature) followed by heating to 28°C for 30 min (Fig. 2k).

Using 7×GFP knock-in and transcriptional reporter lines, we confirmed that cng-1, cng-3, tax-4, and gcy-28 are all endogenously expressed in AMsh glia (Fig. 2l, m). Taken together, these findings suggest that GCY-28 acts as a heat sensor in AMsh glia, mediating warm responses by increasing intracellular cGMP levels. The elevated cGMP then activates the CNG channels CNG-1, CNG-3, and TAX-4, leading to calcium influx and triggering downstream responses in AMsh glia.

GLR-3 functions as a cold sensor in AMsh glia
It has been implicated that GCY-20 and the TRPA-1 channel transduce cold signals in C. elegans42,49,50, but we observed no defects in the cold responses of AMsh glia in either gcy-20 or trpa-1 mutants. A previous study identified GLR-3 as a cold receptor within the ASER sensory neuron, playing a crucial role in normal cold avoidance behaviors51. Similarly, the mammalian homolog of GLR-3, GluK2, is essential for detecting noxious cold temperatures and facilitating cold nociception in mice4. Remarkably, the loss of GLR-3 significantly reduced cold-induced calcium responses in AMsh glia and this deficit was rescued by restoring glr-3 expression in AMsh glia (Fig. 3a, b, e). Supporting this, AMsh glia-specific knockdown of glr-3 also resulted in reduced cold-induced calcium responses in these glia (Fig. 3a, b, e).

GLR-3 and GluK2 act as cold receptors through a noncanonical metabotropic mechanism, rather than through their ion channel function4,51. Our previous study demonstrated that odorant-induced calcium responses in AMsh glia involve inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium release from the endoplasmic reticulum (ER)9. We observed that cold-induced responses in AMsh glia were diminished in both itr-1 mutants and worms with AMsh glial itr-1 RNAi, indicating that IP3R-mediated ER calcium release is involved in cold sensing by AMsh glia (Fig. 3c, e). Notably, glr-3;itr-1 double mutants exhibited defects comparable to those of the respective single mutants (Fig. 3c, e). Additionally, cold-induced calcium responses in AMsh glia were unaltered both in worms expressing the tetanus toxin light chain (TeTx) in ASER neurons (to block their synaptic transmission)25,42 and in che-2 mutants, in which animals have extremely short cilia and abnormal posterior projections of amphid sensory neurons and exhibit defects in behaviors mediated by these neurons52 (Fig. 3d, e). Collectively, these results indicate that cold stimulation activates GLR-3 in AMsh glia, a process that trigger the release of calcium ions from the ER or the influx of extracellular calcium ions.

To confirm the expression of GLR-3 in AMsh glia, we generated a 7×GFP knock-in line, which demonstrated GLR-3 expression in these cells (Fig. 3f). A transcriptional reporter line containing a 2.9 kb glr-3 promoter driving fluorescent protein expression further confirmed the presence of GLR-3 in AMsh glia (Fig. 3g).

AMsh glia modulate thermal avoidance and tolerance
The observation that AMsh glia are activated by temperature fluctuations prompted us to investigate whether these glial cells contribute to thermal behaviors. To avoid the developmental deficits of neurites may be caused by ablation of glial cells, we transgenically expressed the histamine-gated chloride channel HisCl1 from Drosophila, which has been shown to be an effective chemogenetic tool for the inducible silencing of C. elegans neurons and other cells in intact animals42,53. By applying histamine, we were able to temporally inhibit the activity of AMsh glia within minutes (Supplementary Fig. 2).

A well-known behavior of animals in response to temperature changes is the avoidance of cold and hot stimuli. To assess temperature avoidance, we performed cold- and hot-plate assays. In a 6 cm diameter NGM plate, the central 1.5 cm circular region was temperature-controlled to either 10°C or 30°C using a plate temperature regulator (Fig. 4a). We placed the worms at the center of this area and, after 10 min, counted the number of worms remaining within the central region of this assay plate. Next, we transgenically expressed HisCl1 with mCherry in AMsh glia and used histamine to inhibit the activity of these cells. While the application of histamine (10 mM) to the assay plates did not affect control worms expressing only mCherry in AMsh glia, inhibition of AMsh glia via histamine significantly reduced the number of worms remaining in the cold circle (10°C) after 10 min (Fig. 4b,c). Conversely, histamine-mediated inhibition of AMsh glia increased the number of worms staying in the hot (30°C) circle (Fig. 4d). We observe some degree of difference in thermal behaviors between control worms and HisCl1-expressing worms. This is not unexpected, as ectopic expression of an ion channel may induce basal activity changes. In particular, ectopic expression of HisCl1 has been implicated in a weak chloride ion leak effect42. Nevertheless, HisCl1 has been established as a relatively stable method for the real-time manipulation of neural activity25,53. Since histamine application had no effect on the locomotion of control worms (Fig. 4b–d), the altered thermal avoidance observed in histamine-treated AMsh glial HisCl1 worms indicate that AMsh glia function to suppress cold avoidance while enhancing hot avoidance.

Tolerance to extreme cold and hot temperatures is essential for an animal’s survival. To investigate this, we placed the NGM plates in a 2°C incubator and counted the number of worms alive after 72 h. We observed that while histamine (2 mM) application did not affect the mortality rate of wild-type worms at 2°C, inhibition of HisCl1-expressing AMsh glia via histamine significantly reduced the mortality rate of the worms (Fig. 4e). We next placed NGM plates in a 37°C incubator and monitored the survival ratio of the worms every two hours. Histamine treatment slightly increased the mortality rate in wild-type worms without HisCl1 expression under these conditions, but significantly increased the mortality rate in worms with HisCl1 expression (Fig. 4f). We then examined whether the absence of GCY-28, which we identified as a heat receptor in AMsh glia, affected hot tolerance. Remarkably, loss of gcy-28 significantly reduced the worms’ tolerance to heat, but this defect could be significantly rescued by restoring gcy-28 expression specifically in AMsh glia (Fig. 4f). Consistent with the role of GCY-28 in warm sensing of AMsh glia, no difference in cold tolerance was observed between WT and gcy-28 mutants (Supplementary Fig. 3a). It has been reported that sensory-deficient mutants often exhibit extended lifespan54. However, as demonstrated in both the present study and previous work, GCY-28 displays a broad expression pattern beyond AMsh glia (Fig. 2m)55. Given this wide expression profile, it is not surprising that gcy-28 mutants, essential for Amsh glial thermosensation, show reduced survival in extreme heat conditions.

Taken together, these results suggest that AMsh glia play a role in inhibiting cold tolerance while enhancing hot tolerance.

AMsh glia modulate temperature preference
C. elegans exhibit thermotactic navigation (thermotaxis, TTX) by moving toward and maintaining their position near their cultivation temperature within thermal gradients, helping them stay within their adaptive thermal range while avoiding potentially harmful conditions. This behavior serves as a model for neural computation, integrating temperature sensing, adaptive memory, and locomotor control to reveal fundamental principles of how organisms respond to environmental temperature cues17,56. We then established a linear temperature gradient of 0.5°C/cm and monitored the migration of worms. The worms were cultured at 23°C and initially placed in the zero subzone (20°C) of the gradient plate. After one hour, we quantified the distribution of worms across the gradient subzones (Fig. 4g). Consistent with previous studies, worms preferentially migrated toward higher temperatures than that in the zero subzone. When AMsh glial HisCl1-expressing worms were transferred to a histamine (2 mM)-containing temperature gradient plate (TTX plate), they migrated toward their cultivation temperature, indicating that inhibition of AMsh glia did not block temperature memory retrieval. However, AMsh glial HisCl1-expressing worms in the histamine-containing plate exhibited a higher TTX index compared to those in a histamine-free plate, indicating that thermal preference was modulated by AMsh glia (Fig. 4h, i).

The steepness of the temperature gradient plays a crucial role in worm thermotaxis. When the gradient reaches approximately 1.0°C/cm or surpasses this threshold, worms cultured at low temperatures (15–17°C) tend to accumulate near their cultivation temperature. In contrast, worms cultured at high temperatures (23–25°C) exhibit an almost athermotactic phenotype, showing minimal or no preference for the thermal gradient. Some worms even become trapped in the cold region, displaying cryophilic behavior17,56,57. Consistently, we observed that worms cultured at 23°C displayed cryophilic behavior in a 1.0°C/cm gradient. However, fewer AMsh glial HisCl1-expressing worms were trapped in the cold region (12–14°C) on histamine-containing TTX plates compared to those on histamine-free plates (Fig. 4j,k). Additionally, AMsh glial HisCl1-expressing worms cultured at 17°C migrated toward their cultivation temperature in the histamine-containing TTX plates in a 1.0°C/cm gradient, and again showed a higher TTX index compared to those in the histamine-free plates (Fig. 4l,m). As a control, we cultured gcy-8, gcy-18, gcy-23 triple mutants in either 17°C or 23°C, and examined their distribution in TTX plates in a 1.0°C/cm or 0.5°C/cm gradient. These triple mutants failed to migrate to their cultured temperature as previously reported (Supplementary Fig. 3b)58. These findings suggest that AMsh glia are involved in modulating thermotactic behavior, influencing the worm’s ability to track and respond to thermal gradients.

AMsh glia differentially regulate thermosensory neurons to modulate thermal responses
To investigate whether AMsh glia modulate thermal behavior through interactions with thermosensory neurons, we focused on AFD and ASH neurons, which play distinct roles in temperature sensing. AFD neurons detect temperature changes within the nematode’s optimal living range, mediating thermotactic behavior and contributing to heat avoidance and thermal tolerance21,22, whereas ASH neurons primarily respond to cold stimuli and drive cold avoidance25. Using worms expressing HisCl1 specifically in AMsh glia, we found that histamine treatment significantly attenuated warm-induced calcium responses in AFD neurons (Fig. 5a–d).

To further validate AMsh glia’s role in AFD activation, we subjected these worms to a cold stimulus (10°C, 4 min), which activates AMsh glia in histamine-free but not histamine-containing solutions, followed by a heat stimulus. Histamine treatment strongly suppressed subsequent heat-induced AFD responses (Fig. 5e–g), confirming that AMsh glia enhance warm sensitivity in AFD neurons. Consistent with this, warm-induced AFD responses were significantly reduced in gcy-28 mutants, which can be rescued by restoring GCY-28 expression specifically in AMsh glia (Fig. 5h,i). Conversely, in worms expressing HisCl1 in AMsh glia, histamine treatment prolonged cold-induced calcium responses in ASH neurons compared to untreated controls (Fig. 5j–m). These findings demonstrate that AMsh glia bidirectionally regulate thermosensory neurons—potentiating warm responses in AFD while suppressing cold responses in ASH.

AMsh glia differentially regulate temperature-sensitive neurons through distinct GABA receptor pathways
How does histamine-mediated inhibition of HisCl1-expressing AMsh glia differentially regulate the activity of AFD and ASH neurons? One possibility is that the opening of HisCl1 channels disrupts chloride homeostasis in the glial-neuronal extracellular space, a perturbation that has been shown to impact AFD neuron activity8,32,33. Another potential mechanism is that AMsh glia release transmitters to modulate the activity of these neurons. Previous studies have demonstrated that AMsh glia release GABA in response to nose touch or aversive odorant exposure, activating the inhibitory GABA receptor LGC-38 in ASH neurons to promote olfactory and mechanical adaptation8,9,59. Extending these findings, we observed significantly impaired adaptation of cold-induced calcium responses in ASH neurons of both unc-25 mutants (GABA biosynthesis-deficient46 and lgc-38 mutants compared to wild-type animals (Fig. 6a,b). Importantly, these adaptation deficits were rescued by restoring unc-25 expression specifically in AMsh glia or by expressing lgc-38 in ASH neurons of the respective mutants. These results demonstrate that GABAergic signaling from AMsh glia is critical for proper cold adaptation in ASH neurons.

We next investigated how AMsh glia enhance the activity of AFD neurons. Warm responses in AFD neurons were reduced in unc-13 mutants and rescued by restoration of unc-13 in AMsh glia, suggesting a role for neurotransmitter(s) (Fig. 6c,d). Further screening of neurotransmitter pathway mutants revealed that unc-25 mutants also exhibited significantly weaker warm responses in AFD neurons compared to wild-type animals. Importantly, rescuing unc-25 expression specifically in AMsh glia restored these responses (Fig. 6c, d), indicating that GABA release from AMsh glia is required for AFD neuron activation. Consistent with this, exogenous GABA application enhanced warm responses in AFD neurons (Fig. 6c,d). These findings suggest that heat stimulation triggers GABA release from AMsh glia, which in turn potentiates AFD neuron activity.

To identify the GABA receptor mediating this effect, we screened GABA receptor mutants and found that exp-1 mutants exhibited reduced warm-induced responses in AFD neurons, and this defect was rescued by expressing exp-1 specifically in AFD neurons (Fig. 6e,f). EXP-1 is an excitatory cation channel previously implicated in enteric muscle contraction60,61. While EXP-1 is known to be expressed in intestinal muscles and some head neurons, our transcriptional reporter analysis confirmed its presence in AFD neurons (Fig. 6g). These findings demonstrate that AMsh glia differentially regulate temperature-sensitive neurons through distinct GABA receptor pathways: EXP-1 mediates excitatory modulation of AFD neurons, while LGC-38 mediates inhibitory regulation of ASH neurons.

Methods
C. elegans strains and media
All nematode strains were raised on nematode growth medium (NGM) plates seeded with E. coli OP50 at 20˚C following standard methods73. Day 1 (D1) adult hermaphrodites under well-fed conditions were utilized in all experiments. The Bristol N2 strain, obtained from the Caenorhabditis Genetics Center, was used as the wild-type. C. elegans strains used in this study are listed in the supplementary file.

Calcium Imaging
Worms were immobilized in a bath solution containing 145 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 5 mM CaCl2, 10 mM HEPES, and 20 mM glucose (pH 7.3, adjusted with NaOH). They were glued to a glass coverslip using medical-grade cyanoacrylate adhesive (Gluture Topical Tissue Adhesive, Abbott Laboratories). Intracellular calcium signals were monitored using the green fluorescent proteins GCaMP5.0. Fluorescent imaging was performed using an Olympus IX71 microscope fitted with a 20x water immersion objective lens and a Teledyne Photometrics Retiga E7 camera. Data were collected via Micro-Manager software. Fluorescence from GCaMP was excited with a ThorLabs blue LED lamp (emission wavelength, 460–480 nm) and collected at a sampling rate of either 1 Hz or 0.33 Hz using Micro-Manager software. For BLGC, blue light was delivered in a series of 100 ms pulses with 100 ms intervals, while GCaMP5.0 was sample data frequency of 5 Hz. Temperature control was achieved using the CL-100 Temperature Controller and the LCS-1 Liquid Cooling System (Warner Instruments).

Molecular biology
Promoters were amplified from N2 genomic DNA using PCR and subsequently recombined with specific donor vector fragments utilizing the In-Fusion PCR Cloning Kit (TaKaRa Inc.). The following promoter regions were analyzed to investigate expression patterns: for gcy-28, a 3.4 kb fragment located upstream of the start codon; for glr-3, a 2.9 kb fragment upstream of the start codon; for exp-1, a 4 kb fragment upstream of the start codon.

To achieve AMsh glia-specific knockdown, sense or antisense sequences targeting an exon-rich genomic region were expressed under the control of a 1245 bp promoter from the R11D1.3 gene upstream. The RNAi construct for itr-1 was generated using a 1134 bp PCR fragment spanning exons 3 to 6 (primers F: CTTGAGCACACTTGGGTTGG and R: GACATGTCCGGCTTGAGTTGTGG), while the construct for glr-3 used a 787 bp fragment spanning exons 2 to 3 (primers F: CGATGAGGTGAATCCCGTCTTGG and R: TACAGTACCCCACACATTGAC).

Cell-type-specific rescue or expression was performed by expressing genes under cell-specific promoters: vap-1p (2 kb) in AMsh glia, R11D1.3p (1.245 kb) in AMsh glia, sra-6p (3.8 kb) in ASH neurons, gcy-8p (0.7 kb) in AFD neurons, str-1p (3.7 kb) in AWB and gcy-5p (3.1 kb) in ASER neurons. The following coding sequences were cloned: gcy-28 isoform b (1914 bp cDNA), glr-3 (2511 bp cDNA), exp-1 (1620 bp cDNA), unc-13 isoform e (5451 bp cDNA), unc-25 isoform a (1527 bp cDNA), and tax-4 (4016 bp genomic DNA).

Confocal microscopy
A 20 μL aliquot of 2–3% agarose solution was applied to a glass slide, which was then covered with another slide. After a few minutes, once the agarose had solidified, the slides were carefully separated to create an agar-coated surface. Next, 10 μL of M9 solution containing 200 mM sodium azide (NaN3) was added to the agar. An adult D1 worm was placed onto the agar surface, and a coverslip was gently placed on top. If the worm is damaged during this step, it should be discarded. The prepared slide was then placed under an inverted confocal laser microscope, and images were captured using FV10-ASW Viewer software (Olympus). For 7*GFP knock-in lines, a Leica STELLARIS high-resolution confocal microscope was employed to acquire images with improved signal-to-noise ratio.

Linear thermotaxis (TTX) assay
A linear thermal gradient was established on a rectangular aluminum platform (60 cm length × 2 cm thickness) using two plate thermal controllers set to distinct temperatures at either end. Ambient temperature and humidity were maintained at 20°C and 25–40%, respectively. Temperature gradients of 0.5°C/cm or 1.0°C/cm were generated by adjusting the thermoregulator settings. A rectangular TTX plate (14 × 6 × 2 cm) containing 11 mL of TTX medium (2% agar, 0.3% NaCl, 25 mM potassium phosphate buffer, pH 6.0) was placed on the aluminum platform, with gaps sealed using conductive adhesive to ensure thermal contact. The temperature of the subzone 0 of the TTX plate was set to 20°C, and the system was allowed to stabilize for 15 min prior to experiments (Fig. 4). A plastic cover was placed over the platform to minimize airflow interference and TTX evaporation.

Hermaphroditic worms were synchronized and cultured on histamine-free NGM plates seeded with E. coli OP50 at 17°C or 23°C until reaching day 1 adulthood. For TTX assays, worms were collected using ~5 mL of NG buffer (0.3% NaCl, 1 mM CaCl₂, 1 mM MgSO₄, 25 mM phosphate buffer, pH 6.0), pelleted in a conical tube, and washed twice with ~2 mL of NG buffer. All steps were performed in a 20°C water bath with pre-equilibrated NG buffer. Approximately 250–500 worms were transferred to subzone #0 (20°C) of the TTX plate. Excess buffer was carefully removed using tissue paper. After 1 h of incubation, worm distribution was quantified via two approaches: (1) calculating the percentage of worms in each subzone; (2) determining the thermotaxis (TTX) index, which is defined by the following equation:

TTX index = (Σ ni Si) / N

where Si denotes the score of individual i, and N represents the total number of animals on the test plate.

We measured the actual temperature of each zone on individual assay plates prior to worm counting. Due to slight environmental fluctuations and plate-specific variations, the real temperature range of each plate deviated slightly from the theoretical gradient. Additionally, to more clearly and intuitively visualize the distribution range of worms, the temperature ranges showed in Fig. 4 were based on the actual worm distribution.

In histamine experiments, freshly made histamine stock was added to both the NG buffer and TTX medium to a final concentration of 2 mM. Previous studies have showed that 10 mM histamine induces paralysis within two minutes in worms with pan-neuronal expression of HisCl19,53. To minimize potential adverse effects of long-term histamine exposure, we employed 2 mM histamine in these specific assays.

Thermal avoidance assay
Cold- and hot-plate assays were performed to assess thermal avoidance. A 6 cm diameter NGM assay plate (0.2 cm thickness) was prepared with a 1.5 cm diameter circular central region temperature-controlled to either 10°C (cold plate assay) or 30°C (hot plate assay) using a plate thermal controller. The gap between central region of the assay plate and the plate of the thermal controller was sealed using conductive adhesive to ensure thermal contact. Room temperature and humidity were maintained at 20°C and 25–40%, respectively. We calibrated the set surface temperature of the temperature-controlled plate to 10°C, 20°C, or 28°C. However, during the actual avoidance behavior assay, the worms crawl on the surface of the NGM layer (where the surrounding air temperature is room temperature, 20°C). Additionally, due to systematic factors, including the thermal conductive adhesive, the bottom of the plastic petri dish, and the NGM layer, that separate the worms from the temperature-controlled plate, the actual temperature perceived by the worms may slightly differ from the set temperature. For each experiment, 10 well-fed day 1 adult hermaphroditic worms were placed at the center of the temperature-controlled region. After 10 minutes, the number of worms remaining within the central zone was quantified. Histamine-containing assay plates were prepared by adding freshly made histamine stock to the sterilized, molten NGM medium when it had cooled to approximately 55°C, achieving a final concentration of 10 mM.

Cold and heat tolerance assays
To evaluate cold tolerance, 30 well-fed day 1 adult hermaphroditic worms were transferred to an NGM plate seeded with E. coli OP50. Plates were incubated at 2°C for 72 hours in a temperature-regulated chamber. Following cold exposure, plates were transferred to a 20°C environment for a 1-hour recovery period to restore motility. Survival rates were quantified by counting motile worms responsive to gentle tactile stimulation. For heat tolerance assessment, 30 well-fed day 1 adult hermaphroditic worms were placed on an E. coli OP50-seeded NGM plate and exposed to 37°C in a calibrated incubator. Survival was monitored at 2-hour intervals for up to 12 h. At each time point, live worms were identified by spontaneous movement or response to mechanical prodding, and survival ratios were calculated as the percentage of live worms relative to the initial population. Histamine-containing NGM plate were made by addition of freshly made histamine stock to the NGM plate to a final concentration of 2 mM.

HEK293T cell culture and transfection
HEK293T cells (ATCC CRL-3216) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified 5% CO2 atmosphere. Upon reaching approximately 80% confluence in 6-well plates, cells were transfected with either an empty vector or a GCY-28 expression plasmid using Lipofectamine 2000, following the manufacturer’s protocol.

Thermal stimulation and sample preparation
At 40 h post-transfection, cells were subjected to cGMP induction using one of two protocols. Protocol 1: Cells were switched to DMEM supplemented with 1 mM IBMX (3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor) and immediately incubated at either 37°C (the standard cell culture temperature) or 45°C for 30 min. Protocol 2: Cells were first pre-incubated at 20°C for 2 h (mimicking the nematode’s living temperature) prior to medium replacement with IBMX-containing DMEM. Stimulation was then conducted at 30°C for 30 min. Following stimulation, cells were immediately washed with PBS, scraped, and pelleted. To generate sufficient material for each biological replicate, cell pellets from three wells per condition were pooled and lysed in 300 μL of ice-cold lysis buffer (supplemented with protease and phosphatase inhibitors). Lysates were centrifuged at 12,000 × g for 15 min, and the clarified supernatants were collected for subsequent cGMP analysis.

cGMP quantification by ELISA
The concentration of cGMP in the cell lysates was determined using a commercial human cGMP ELISA kit (mlbio). Briefly, samples and standards were acetylated to enhance assay sensitivity, then incubated in antibody-coated wells. Following a washing step, a horseradish peroxidase (HRP)-conjugated tracer was added. Color development was initiated with tetramethylbenzidine (TMB) substrate and stopped with acid. The absorbance was measured at 450 nm using a multi-mode microplate reader (MD). A four-parameter logistic (4-PL) regression model was used to generate the standard curve for each assay according to the manufacturer’s instructions. All standard curves exhibited a coefficient of determination (R² ≥ 0.99), ensuring reliable quantification. Data are derived from at least three independent biological replicates.

Isolated AMsh glial cell culture
Isolation and culture of AMsh glia were performed based on a previously established primary C. elegans cell culture protocol9,74. Gravid worms were maintained on 8X peptone plates with E. coli OP50, and enriched eggs were collected for cell dissociation. Dissociated embryonic cells were plated at low density on peanut lectin-coated cover glasses. L-15 medium was changed daily to remove secretions. After 3 days in culture at 20°C, isolated mature AMsh glia were recognized by co-expression of Pvap-1::mCherry and Pvap-1::GCaMP5.0.

CRISPR/Cas9-engineered knock-in combined with issue-specific Split SfGFP
For tissue-specific tissue-specific Split SfGFP experiments, knock-in (KI) strains containing a tandem array of seven copies of the GFP11 β-strand (GFP11×7) inserted into the endogenous genomic loci of cng-1, cng-3, tax-4, or glr-3 were generated by the SunyBiotech company using CRISPR/Cas9 technology. We then independently microinjected these GFP11×7 knock-in strains with our constructed plasmid expressing the complementary split-GFP fragment (spGFP1-10) under the control of the vap-1 promoter. This approach enables specific labeling of the respective endogenous proteins in AMsh glia within their native cellular context9. Additionally, CeBFP driven by the vap-1 promoter was used as a control for labeling AMsh glia.

Statistical analysis
Calcium imaging data were acquired using Macro-Manager software and analyzed with ImageJ. Statistical analysis was performed in GraphPad Prism 8, and results are presented as mean ± standard error of the mean (S.E.M.). An unpaired two-tailed t-test was used to compare two groups of data. For non-normally distributed data within a group, the Wilcoxon test was applied. Two-way ANOVA was used for comparisons of heat resistance. A p-value of less than 0.05 was considered statistically significant, indicated by an asterisk (*), while p-values less than 0.01 and 0.001 were denoted by ** and ***, respectively. Sample sizes were chosen based on the reproducibility of the experiments and are consistent with standard practices in the field.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Zhu and colleagues investigate the role of Amsh glia in thermosensation in C. elegans, focusing on their functional interactions with two neuronal pairs: the thermosensory AFD neurons and the nociceptive ASH neurons. Using calcium imaging, behavioral assays, and fluorescence microscopy, the authors demonstrate that Amsh glia respond to both heat and cold stimuli through two distinct molecular pathways: one involving the guanylyl cyclase receptor GCY-28 for heat, and the other involving the glutamate receptor GLR-3 for cold. Activation of these pathways leads to GABA release from Amsh glia, which in turn activates distinct downstream receptors in the AFD and ASH neurons. Importantly, these glia-to-neuron signaling mechanisms significantly influence behavioral responses to temperature. The authors propose a “Hub-and-Spoke” model, positioning glial cells as central regulators in thermosensory circuits. This study expands our understanding of glial functions within neuronal networks and contributes new insights into the mechanisms of thermosensation. Overall, the data are generally well controlled, but several key points need to be addressed.\n\nMajor Points:\n1. Calcium Transient Dynamics:\nThe manuscript shows that cooling triggers a single calcium transient, while heating induces multiple transients. Greater clarity is needed here.\na. How many calcium transients are typically induced by heat?\nb. Do different mutants show variations in the number of transients?\nc. Are these peaks random, or do they follow a defined temporal pattern?\n2. PDE Quadruple Mutant Effects:\nThe PDE quadruple mutant shows only a modest effect on heat-induced calcium transients, with no statistically significant difference in peak amplitude compared to wild-type. Given that only 7 cells were tested, can the authors increase the sample size? Additionally, would it be possible to test pde-4, since some cGMP-gated channels can also be regulated by cAMP?\n3. Decay Time in osm-9 Mutants:\nThe decay time of calcium transients in osm-9 mutants should be quantified and compared to wild-type to better understand the kinetics of the response.\n4. Role of osm-9 in Heat Response:\nIs there any evidence that osm-9 contributes to heat-induced responses?\n5. Histamine Concentration Usage:\nThe rationale behind using 2 mM histamine in some experiments and 10 mM in others should be clarified.\n6. Survival vs. Sensory Impairment:\nFigure 2F shows that gcy-28 is required in Amsh glia for thermosensation, and Figure 4F shows that gcy-28 mutants have reduced survival. However, previous studies (e.g., PMID: 10617200) indicate that sensory-deficient mutants often live longer. The authors should address this apparent discrepancy in the discussion.\n\nMinor Points:\n1. Please comment on the observation that expression of HisCl in Amsh glia has an effect on its own (Figure 4B).\n2. Figure 4J (right panel): The x-axis labels “24” and “26” are positioned above the axis line and should be corrected.\n3. Line 38: Change “acts a homeostatic gain control mechanism” to “acts as a homeostatic gain control mechanism.”\n4. Line 152: Change “guanylyl cyclase might be responsible” to “guanylyl cyclases might be responsible.”\n5. Line 160: Change “as previous study described” to “as a previous study described.”\n6. Figures 2C, F, and H show signs of photobleaching. It may be helpful to apply photobleaching correction to improve signal clarity.", "reviewer_2": "This manuscript investigates a novel and potentially transformative concept: that glial cells could function as dual-mode thermosensors and modulators of temperature-related neural circuit activity in C. elegans. The authors propose that AMsh glia are capable of autonomous thermosensation via co-expression of GCY-28 (a heat receptor) and GLR-3 (a cold receptor), and that they release GABA to bidirectionally regulate warmth and cold-responsive neurons, namely AFD and ASH, respectively.\n\nIf fully supported by robust experimental evidence, the central message—that glia act as both sensors and modulators in thermosensory circuits—would represent an exciting advance for the field. However, at present, many of the key mechanistic claims, particularly those regarding cell-autonomous glial thermosensation and downstream circuit interactions, are only partially supported by the data presented. Many of the conclusions rely on correlative or indirect evidence, and essential cell-specific controls or pathway-dissection experiments are missing.\n\nThe claims regarding cellular autonomy, sensor identity, and signaling pathways remain preliminary, and require substantial additional experimentation to be convincingly established. Therefore, without substantial work to address the concerns below, I cannot recommend publication at this stage.\n\nMajor Points:\n1) The current experimental design does not conclusively demonstrate cell-autonomous temperature sensing by AMsh glia. The observed delay in heat-evoked calcium responses raises the possibility that these are secondary to inputs from neighboring thermosensory neurons. The recording in unc-13 and unc-31 mutants are not sufficient. Transmission of thermosensory ‘signals’ could indeed be caused e.g. either directly via innexins (in particular hemichannels) transmitting chemical messenger or indirectly via a modulation of the ionic composition of the extracellular medium connecting AMsh glia and a variety of thermosensory neurons: AFD, ASH, but also others like AWC and ASER (which intriguingly express GCY-28 and GLR-3, respectively, and are actually ideally placed to trigger response in nearby glial cells). Suggested experiment: Examine AMsh responses in conditions lacking thermosensory neuron functon(e.g., broadly-acting che mutation), or, better, in isolated conditions ex vivo.\n2) The significant expression of vap-1 in AWC, even if lower than in glial cells and even potentially undetectable with fluorescent reporter, raises a concern that the observed rescue may be attributed to leaky expression in this neighboring neuron (potentially others). The observed rescue could potentially indeed arise from off-target expression in adjacent neurons. This impact all the experiments relying on the use of the vap-1 promoter for glial-specific expression. Suggested solution: Employ a promoter with no detectable expression in neighboring sensory neurons to validate AMsh-specific rescue.\n3) Missing data (in Fig. 2): Traces for gcy-8/18/23 triple mutants are not shown, making it difficult to interpret their role.\n4) The cell-specific expression of gcy-28 in AWB, intended to establish GCY-28’s role as a thermosensor, is nice but too weak to make a strong case. A key surprising point is that the thermal upstep triggers a AWB calcium decline. This decline is prevented when GCY-28 is expressed in AWB. This is not really a case where GCY-28 can “confer thermo-sensitivity”. Instead, the data just show that GCY-28 overexpression block the cool evoked calcium decline in AWB. And this might be interpreted very differently. Indeed, it could be that the constitutive elevation of cGMP production (via over-expression of a protein that is not normally their and whose activity might be out of control), might cause cGMP saturation to a level where it is very difficult to close CNG channels and then prevent the natural heat-evoked calcium reduction in AWB. Therefore, these results can be explained even if GCY-28 is not a molecular thermosensor. Suggested experiment: Express GCY-28 in cell or tissue that do not react to temperature, e.g., in cell line as used in previous studies (PMC6743979).\n5) No data supports that GLR-3 act as a cold sensor in AMsh, despite being a main claim of the paper. Suggested experiments: Perform a glr-3 cell-specific rescue in AMsh to test for functional sufficiency. Perform cell-specific knock-down. Verify cold-responsiveness of AMsh in ASER-ablated animals.\n6) Likewise, we have strictly no reason to believe that ITR-1 works cell-autonomously in glial cells, nor is the downstream relationship between GLR-3 and ITR-1 adequately established. Suggested experiment: Conduct epistasis analyses using glr-3; itr-1 double mutants to test for genetic interaction. Conduct AMsh-specific knock out of ITR-1 (e.g. using existing floxed alleles of ITR-1 (PMC11087772)\n7) Missing data in Figure 4: The manuscript lacks a heat tolerance assay under conditions where AMsh activity is suppressed, analogous to the cold tolerance assays shown.\n8) The calcium responses in AFD neurons with AMsh-specific gcy-28 rescue appear unusually elevated in Figure 5, raising concerns about sensor calibration. Suggested control: Include an internal RFP control to normalize GCaMP and validate calcium imaging signals.\n9) The reported ASH response defects in unc-25 and lgc-38 mutants (in Fig. 6) do not directly link to AMsh function. Suggested experiments: To fully substantiate the proposed model, checking the ASH response of animals with cell specific rescue of unc-25 and lgc-38 in AMsh and ASH respectively would be necessary.\n10) Similarly, to properly support the proposed model, the authors should also assess the AFD response in animals with AFD-specific rescue of exp-1 to validate the proposed excitatory glia-to-neuron signaling.\n11) One general weakness of the behavioral analyses is that it is hard to relate the behavioral phenotypes (often extremely mild, e.g, in thermal gradient) with the proposed inter-cellular mechanisms. A much deeper discussion is needed here, before one can correctly judge of their relevance.\n12) The introduction focuses on the key cellular players under study. However, this seems too focused and cursory. It is important for the large audience of this journal to have a broader view of what is known in C. elegans. In particular, additional thermosensory neurons must be presented in more details, e.g. ASER, AWC, because ultimately, they might be the ones triggering the apparent thermal responsiveness of AMsh glia. Likewise, a better introduction of the molecular thermosensors is needed.\n\nConclusion:\nThis study proposes an intriguing and potentially field-shifting role for glia in thermosensory processing. However, the current data fall short of definitively demonstrating most of the manuscript’s key mechanistic claims. Strengthening the experimental evidence—particularly through rigorous cell-type-specific manipulations, genetic dissection, and assessment of the new proposed thermosensor will be essential for the work to become convincing. I encourage the authors to pursue these additional experiments, which will help clarify and validate the central hypothesis and significantly enhance the manuscript’s contribution to the field.", "reviewer_3": "C. elegans AMsh glia associate with multiple sensory neurons, including the thermosensory AFD, and modulate multiple sensory behaviors and sensory neuron properties. Building on this prior research, Zhu et al aim to define how glia do so. They report that AMsh glia co-express GCY-28 (which acts as a heat receptor) and GLR-3 (which acts as a cold receptor), and have calcium changes in response to noxious thermal changes. Through functional imaging and behavior studies, authors propose that glial calcium activity drives ER calcium and GABA release, which bidirectionally impacts AFD and ASH neurons in dictating animal thermal preferences. They conclude that this GABAergic hub-and-spoke architecture allows homeostatic weighting of environmental temperature inputs.\n\nOverall, this study tackles an exciting and open question – glial roles in neural circuit processing. However, while the results and model are intriguing, significant technical and logical concerns are noted. Thus, major revisions are recommended before this manuscript can be supported for publication.\n\nMajor comments:\n1. A key conclusion is that AMsh glial cGMP triggers calcium dynamics, partly based on blue-light triggered guanylyl cyclase BlgC (Fig 2). This is tricky because multiple amphid neurons respond to blue light (PMID: 25640076). Additional controls are needed to strengthen this conclusion. At minimum:\na. key experiments should be replicated in lite-1; gur-3 mutants (see for e.g. PMID: 38194919, 27459302)\nb. traces with blue light stimulation without BlgC need to be documented.\nc. AFD should be chronically activated, and glial responses recorded.\n2. The authors use vap-1 promoter for AMsh glial studies. (PMID: 18974354) noted faint expression in AFD, a relevant cell-type for this study’s conclusions. To address concerns for the central conclusions of this study, authors should\na. detail which promoter fragment was used (in Methods)\nb. verify with another promoter (esp. BlgC & HisCl baseline, gcy-28 rescue).\n3. The authors report identifying GCY-28 from single cell sequencing (Line 153). Data for this key conclusion should be either reported or cited.\n4. Many edits are needed for prior published literature to be cited, discussed or interpreted correctly. Some examples:\na. Line 90 and Line 332. Authors state that AMsh glia were “hypothesized to serve primarily..”. However, multiple prior studies (see PMID: 38565269 for recent review), including work cited here (Ray et al) from multiple labs including the authors has already demonstrated that AMsh glia are not passive support, but active participants in different sensory processing. This can be stated without detracting from the current study.\nb. Line 197-199. ..t is incorrect that the studies referenced reported “embryonic or larval stages” or “developmental deficits”. This is incorrect because these studies imply post-developmental effect of glia on sensory neuron properties and animal behavior. In fact, one of the few embryonic AMsh glial promoters (Pvap-1) PMID: 15935778) is used in THIS study (Line 200-202), but not the others referenced!\nc. Line 332. Studies in (PMID: 22298710, 21350017) have shown AMsh temperature-dependent expression of ver-1 is independent of AFD or sensory neuron activity i.e. AMsh glia are thermosensors. These should be referenced.\nd. AMsh glia engulf gcy- laden AFD membranes, and ciliated amphid neuron EVs (PMID: 33759761, 34533135); and AFD engulfment was shown to impact thermosensory animal behavior (PMID: 33759761). Given that this study invokes AMsh glial gcy function, this should be at least discussed as cGMP/gcy source.\n5. Figs. 1 & 2 do not adequately support the conclusion that glia independently detect temperature. To demonstrate this conclusively, authors need to report on AMsh glial calcium dynamics in response to temperature in AFD and ASH ablated animals. This is because it is also possible that glia have secondary bystander response to neuron activity, with AFD/ASH as the sensors (see also PMID: 16837592).\n6. unc-13 does not affect AMsh glial calcium dynamics (Fig 1) but reduces AFD response (Fig 6). As stated, it seems the authors imply that glial GABA release is via UNC-13 regulated neurotransmitters (Line 310-312). For this conclusion, authors need to evaluate glial and pan-neuronal UNC-13 rescue. Also discuss altered AMsh glial response kinetics in these mutants, which suggests neuronal input is relevant.\n7. Glial gcy-28 rescue does not phenocopy wild type (Fig 4, Line 234), and alters AWB responses i.e. alters response property of cell expressed in. To bolster conclusion that it acts in glia, authors should\na. show rescue of gcy-28 from neurons.\nb. Examine a second allele, preferably a genetic null\nc. Show the region used for expression analyses in xx, and perform either endogenous tag or recombineered fosmid expression analysis\nd. Note which cDNA isoform was used for rescue, given that the lesion used is possibly only truncating one isoform. Showing the gene and protein domain structure with lesions and promoters noted will aid evaluation.\ne. Include wild type traces in Fig 5g-j.\nf. Optional: will help to see gcy-28 cold mortality rates (Fig 4e) for completeness\n8. Studies from multiple labs have reported roles for chloride homeostasis, some of which are referenced here (Singhvi et al, Yoshida et al, Ray et al); but also Wang et al. The authors need to discuss how this may impact interpretation of HisCl chloride channel experiments because one possibility is that altering Cl in the glia-neuron inter-cellular space impacts AFD activity.\n9. The model suggests autonomous glial responses, but for most genes no cell-specificity is evaluated. Specific examples are noted below, with experiments suggested to enable the conclusion as noted:\na. Responses are lost in CNG channel mutants (Fig 2c,d), and the authors conclude that CNG channels are downstream of cGMP in AMsh glia. To make this conclusion, authors need to demonstrate that (i) CNG channels are expressed in glia (ii) function in glia but not neurons (iii) do not impact cold responses, per their model (Extended Fig 2).\nb. Diminished responses in itr-1 mutants (Fig 3a-c) does not imply ER store release from AMsh glia (Lines 185, 187). This needs to be rephrased or cell-specific requirement of ITR-1 in AMsh glia shown. Independently, rationale for examining ITR-1, above other possible mechanisms, is unclear and should be detailed.\nc. Effect of osm-9 as a glial modulator (Fig 3a-c, Line 193) is over-interpreted.\nd. Similarly, lgc-38 results cannot be concluded as stated without cell-specific evidence. It is also expressed in many downstream interneurons.\ne. Likewise, it is unclear if the pde genes act in neurons or glia, or both.\n10. Behavior assays:\na. Line 219. This assay is not standard in the field. It needs to be confirmed that the HisCl array does not cause locomotion defects by showing speed/movement traces. Further, >1 array should be reported, with promoter-only controls. It should also be noted if animals leave the field with delay (>10-20 mins)\nb. Conclusions in Fig. 4j-m are unclear from data provided with single-array study\nc. A control mutant will be helpful (e.g. triple gcy-8/18/23 or ttx-1).\n11. To conclude “homeostatic gain..recalibrate preference” (Abstract), the authors need to demonstrate this via the following directed studies:\na. AFD, ASH responses in wild type, gcy-28 and unc-25 (glia-degron) animals.\nb. Chronically activate ASH and evaluate the pathway by imaging, behavior or both.\nc. Demonstrate if this is cold-specific or any noxious stimuli from polymodal ASH\n\nGeneral and minor comments:\n1. A colorblind safe palette is recommended. Also, it is advised to be consistent with color scheme (e.g. Fig 1: glia text and unc-13 are similar colors, but the color denotations change in subsequent figures; Fig 5: color for AFD neuron text and histamine trace is both red, and color for ASH is same as GCY-28 rescue, Fig 4f: plot color title and trace color same; Fig 6d: unc-25 and rescue lines are hard to differentiate, which can be confusing)\n2. Statistical tests used should be stated in Figure Legend. Also,\na. if “not significant” is inferred from a plot, the comparison done should also be noted on the plot (e.g. Fig 2b).\nb. Also, for some panels (e.g. Fig 4b) it is unclear what statistics was done.\nc. Finally, for rescue experiments, comparison should be done also with WT, and reported with statistical comparison and discussion (e.g. Fig 2f-g).\nd. Fig 4f: ANOVA and comparison between wild type and rescue seems appropriate\n3. Information missing but needed for the studies presented:\na. Information on biological replicates should be noted for the different assays.\nb. Information on what promoter used for AWB, with relevant citations to lend confidence to cell-specificity\nc. Since both GCaMP5 and GCaMP6s are used interchangeably, it will be helpful to have functional dynamic traces for both with temperature reported side-by-side\nd. For all Ex arrays, per best practice, >1 array should be reported.\ne. For Ex arrays, per best practice, over-expression in wildtype should be reported. This is especially important for studies like Fig 2f-g, where the rescuing array alters AMsh responses beyond wild type.\nf. Promoter only control in AWB will strengthen the argument (Fig 2h)\ng. Extended Fig. 1: controls should be included. Again, text and trace colors are same, but do not imply correlation.\nh. Basis for skew of range toward cold is unclear (i.e. 20C not in center of plate).\ni. Line 219-220: show data for higher numbers in central region for 10C and 20C\nj. Check references – for e.g. missing in Line 75, 376.\n4. Fig 2j, 3d, and 6h: close-up of cell-body is needed. Also,\na. Fig. 2j: glial cell (point of study) is out of focus\nb. Fig. 3d: why does the cytosolic fluorophore not fill the glial process.\n5. Confusing phrasing, consider reword:\na. Line 200: “avoid this..”. It is unclear what authors mean “this” refers to.\nb. Line 260-267: “exceeds 1C/cm” stated and “in a 1C/cm” used.\nc. Line 337: “single cellular compartment” implies cell region, which is not evaluated. “single cell” is more appropriate.\nd. Suggest moving Line 244 (Strongyloides..) to discussion.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We deeply appreciate the constructive feedback provided by the reviewers. All raised points have been addressed in the revised manuscript, which integrates the requested experiments alongside updated text and figures. The added experiments conducted as suggested by the reviewers not only validate the interpretations presented in our original submission but also strengthen the study by providing substantial additional experimental data and deeper mechanistic insights.\nBelow is our point-by-point response to the reviewers' comments:\nReviewer #1\n1. Calcium Transient Dynamics: The manuscript shows that cooling triggers a single calcium transient, while heating induces multiple transients. Greater clarity is needed here. a. How many calcium transients are typically induced by heat? b. Do different mutants show variations in the number of transients? c. Are these peaks random, or do they follow a defined temporal pattern?\nResponse: We appreciate the reviewer’s comment. While cooling triggers a single calcium transient, our results as shown in Fig. 1b, 1d and 2e-h demonstrate that heating also induces only a single calcium transient in most AMsh glial cells.\n2. PDE Quadruple Mutant Effects: The PDE quadruple mutant shows only a modest effect on heat-induced calcium transients, with no statistically significant difference in peak amplitude compared to wild-type. Given that only 7 cells were tested, can the authors increase the sample size? Additionally, would it be possible to test pde-4, since some cGMP-gated channels can also be regulated by cAMP?\nResponse: As suggested by the reviewer, we expanded the sample size for heat-induced AMsh glial calcium transient analyses in PDE quadruple mutant worms (Fig. 2c-e). Our data demonstrate that the peak amplitude of these transients in the quadruple mutants was significantly higher than in wild-type, suggesting cGMP regulates heat-induced calcium transients in AMsh glia. We also tested pde-4;pde-6 double mutants (genes required for C. elegans cAMP hydrolysis) and found no increase in the peak amplitude of heat-induced calcium transients in AMsh glia (Fig. 2c-e). These results align with our model that cGMP signaling is involved in AMsh glial heat sensing.\n3. Decay Time in osm-9 Mutants: The decay time of calcium transients in osm-9 mutants should be quantified and compared to wild-type to better understand the kinetics of the response.\n4. Role of osm-9 in Heat Response: Is there any evidence that osm-9 contributes to heat-induced responses?\nResponse: We have carefully considered potential explanations for the kinetic differences between osm-9 mutants and wild-type (WT). However, we noticed that the data pertaining to osm-9 has relatively low relevance to other experiments in the study and thus stands as an isolated component. Therefore, we have decided to remove this section in the revised manuscript. This adjustment aims to better streamline the narrative and ensure the focus of the study remains concentrated on its core themes.\n5. Histamine Concentration Usage: The rationale behind using 2 mM histamine in some experiments and 10 mM in others should be clarified.\nResponse: As previously reported by Navin et al. (PNAS, 2014; PMID: 24550306) and in our prior study (Duan et al., Neuron, 2020; PMID:32970991), 10 mM histamine induces paralysis within two minutes in worms with pan-neuronal expression of HisCl1. Therefore, for most experiments, where each worm is typically exposed for 5–15 minutes, we used 10 mM histamine to achieve optimal effects. However, in TTX experiments and cold/heat tolerances assays, worms require prolonged exposure to histamine. The total duration, from washing to distribution on TTX plates, is approximately 70 minutes for TTX experiments, and ranges from several hours to 72 hours for cold/heat tolerances assays. To minimize potential adverse effects of long-term histamine exposure, we employed 2 mM histamine in these specific assays. This detail has been clarified in the revised manuscript for accuracy (Line 577).\n6. Survival vs. Sensory Impairment: Figure 2F shows that gcy-28 is required in Amsh glia for thermosensation, and Figure 4F shows that gcy-28 mutants have reduced survival. However, previous studies (e.g., PMID: 10617200) indicate that sensory-deficient mutants often live longer. The authors should address this apparent discrepancy in the discussion.\nResponse: We appreciate this valuable suggestion. We have accordingly clarified and discussed this issue in the revised manuscript (line 301-307).\nMinor Points:\n1. Please comment on the observation that expression of HisCl in Amsh glia has an effect on its own (Figure 4B).\nResponse: Thanks for this insightful comment. We propose several potential explanations for this observation. First, it is not uncommon for the heterologous overexpression of proteins, particularly ion channels, to exert some degree of non-specific side effects in host cells. Second, prior studies have indicated that overexpressed HisCl1 may exhibit small basal (histamine-independent) leak chloride currents (Li et al, Cell Reports 2024, PMID: 38294902), which could account for the observed effect in the absence of exogenous histamine. In this study, we deliberately selected HisCl1 over HisCl2, as the former has a well-documented lower basal activity and an overall safety profile in numerous published reports. Our data confirm that the overall calcium responses of AMsh glia, ASH and AFD to temperature stimuli remained intact in worms with Amsh glial-specific expression of HisCl1 (Fig. 5). Importantly, our conclusions rely on the relative phenotypic differences between histamine-treated and untreated conditions. This comparative approach not only enables real-time and precise manipulation of AMsh glial activity but also ensures the reliability of our core observations and conclusions.\n2. Figure 4J (right panel): The x-axis labels “24” and “26” are positioned above the axis line and should be corrected.\nResponse: We have corrected this in the revised manuscript.\n3. Line 38: Change “acts a homeostatic gain control mechanism” to “acts as a homeostatic gain control mechanism.”.\nResponse: Thank you for pointing this out.We have corrected this in the revised manuscript.\n4. Line 152: Change “guanylyl cyclase might be responsible” to “guanylyl cyclases might be responsible.”.\nResponse: We have corrected this in the revised manuscript.\n5. Line 160: Change “as previous study described” to “as a previous study described.”\nResponse: We have corrected this in the revised manuscript.\n6. Figures 2C, F, and H show signs of photobleaching. It may be helpful to apply photobleaching correction to improve signal clarity.\nResponse: Thanks for the nice suggestion. We have applied photobleaching correction to improve signal clarity in the revised figures.\nReviewer #2\n1. The current experimental design does not conclusively demonstrate cell-autonomous temperature sensing by AMsh glia. The observed delay in heat-evoked calcium responses raises the possibility that these are secondary to inputs from neighboring thermosensory neurons. The recording in unc-13 and unc-31 mutants are not sufficient. Transmission of thermosensory ‘signals’ could indeed be caused e.g. either directly via innexins (in particular hemichannels) transmitting chemical messenger or indirectly via a modulation of the ionic composition of the extracellular medium connecting AMsh glia and a variety of thermosensory neurons: AFD, ASH, but also others like AWC and ASER (which intriguingly express GCY-28 and GLR-3, respectively, and are actually ideally placed to trigger response in nearby glial cells). Suggested experiment: Examine AMsh responses in conditions lacking thermosensory neuron functon(e.g., broadly-acting che mutation), or, better, in isolated conditions ex vivo.\nResponse: Thank you for this perceptive comment. As suggested, we first examined cold- and heat-induced calcium responses in che-2 mutants and observed no differences compared to wild-type (Fig. 2d,e; 3d,e). Furthermore, we isolated and cultured single AMsh glial cells expressing GCaMP5 as described in our prior study (Duan et al., Neuron 2020), and found that these isolated cells exhibited cold- and heat-induced calcium responses that were identical to those observed in vivo (Fig. 1e-g). Utilizing 7×GFP knock-in and transcriptional reporter lines, our data further demonstrate that the core molecular machinery underlying thermal sensing, including of cng-1, cng-3, tax-4, gcy-28, and glr-3, is all endogenously expressed in AMsh glia (Fig. 2l,m; Fig. 3f,g). Importantly, the thermal sensing defects of AMsh glia in each of these mutants were rescued by the restoration of the corresponding cDNAs under the control of AMsh glial-specific promoters (vap-1 and R11D1.3)(Fig.2a-h; 3b,e). Together with our observations in unc-13 and unc-31 mutants (Fig. 1c,d), these independent lines of evidence provide strong support for the conclusion that AMsh glia respond to temperature stimuli in a cell-autonomous manner.\n2. The significant expression of vap-1 in AWC, even if lower than in glial cells and even potentially undetectable with fluorescent reporter, raises a concern that the observed rescue may be attributed to leaky expression in this neighboring neuron (potentially others). The observed rescue could potentially indeed arise from off-target expression in adjacent neurons. This impact all the experiments relying on the use of the vap-1 promoter for glial-specific expression. Suggested solution: Employ a promoter with no detectable expression in neighboring sensory neurons to validate AMsh-specific rescue.\nResponse: Thank you for this valuable suggestion. To address concerns about potential off-target expression, we utilized the R11D1.3 promoter—previously validated to drive expression specifically in AMsh glia (head) and PHsh glia (tail) with no detectable activity in neighboring sensory neurons. Importantly, rescue experiments using the R11D1.3 promoter yielded results indistinguishable from those obtained with the vap-1 promoter (Fig.2a-h;3b,e;4b-e;5c,d,h,i,l,m;6a-d). This consistency across two distinct glial-specific promoters strengthens our conclusion that the observed rescue is attributable to AMsh glial expression rather than leaky activity in adjacent neurons. We have included these new data in the revised manuscript.\n3. Missing data (in Fig. 2): Traces for gcy-8/18/23 triple mutants are not shown, making it difficult to interpret their role.\nResponse: We have added the traces in the revised figure (Fig. 2g,h).\n4. The cell-specific expression of gcy-28 in AWB, intended to establish GCY-28’s role as a thermosensor, is nice but too weak to make a strong case. A key surprising point is that the thermal upstep triggers a AWB calcium decline. This decline is prevented when GCY-28 is expressed in AWB. This is not really a case where GCY-28 can “confer thermo-sensitivity”. Instead, the data just show that GCY-28 overexpression block the cool evoked calcium decline in AWB. And this might be interpreted very differently. Indeed, it could be that the constitutive elevation of cGMP production (via over-expression of a protein that is not normally their and whose activity might be out of control), might cause cGMP saturation to a level where it is very difficult to close CNG channels and then prevent the natural heat-evoked calcium reduction in AWB. Therefore, these results can be explained even if GCY-28 is not a molecular thermosensor. Suggested experiment: Express GCY-28 in cell or tissue that do not react to temperature, e.g., in cell line as used in previous studies (PMC6743979).\nResponse: The heat-evoked calcium decline in AWB neurons was unexpected, given their well-established role in olfaction. However, our data clearly demonstrate that overexpression of GCY-28 converts AWB’s heat response from suppression to activation, implying that GCY-28 itself is heat-responsive (Fig.2i). To confirm this hypothesis, we expressed GCY-28 in HEK293T cells, and quantified intracellular cGMP levels using a commercial ELISA kit. GCY-28-transfected cells exhibited markedly elevated intracellular cGMP levels relative to empty vector-transfected controls under two distinct thermal challenges: a direct heat-up from 37°C (the culture temperature of HEK293T cells) to 45°C for 30 minutes, or a precooling step (from 37°C to 20°C for 2 hours) followed by heating to 28°C for 30 minutes. These newly generated data (Fig. 2k), together with our observation that GCY-28 is indispensable for heat-evoked responses in AMsh glia, collectively indicate that GCY-28 is a heat-activated protein.\n5. No data supports that GLR-3 act as a cold sensor in AMsh, despite being a main claim of the paper. Suggested experiments: Perform a glr-3 cell-specific rescue in AMsh to test for functional sufficiency. Perform cell-specific knock-down. Verify cold-responsiveness of AMsh in ASER-ablated animals.\nResponse: Thanks for the valuable comment. As suggested, we first restored glr-3 expression in AMsh glia using two distinct AMsh glial promoters, vap-1 and R11D1.3, respectively, and successfully rescued the cold-induced defect in AMsh glia of glr-3 mutants (Fig. 3b,e). Conversely, RNAi-mediated knockdown of glr-3 in AMsh glia led to a significant reduction in their cold responses (Fig. 3c,e). Furthermore, we detected no significant difference in the cold responses of AMsh glia between ASER::TeTx worms and wild-type controls (Fig. 3d,e). This finding, which indicates that ASER neurons are not required for AMsh glial cold responses, aligns with our complementary observations from both isolated AMsh glia and unc-13/-31 mutants (Fig. 1). Additionally, our 7×GFP knock-in line and transcriptional reporter line both confirmed the endogenous expression of GLR-3 in AMsh glia (Fig. 3f,g). Taken together with our calcium imaging and behavioral data, as well as prior reports identifying GLR-3/GluK2 as a cold sensor in both nematodes and mice, these findings collectively demonstrate that GLR-3 functions as a cold sensor in AMsh glia. Notably, since cold-induced calcium responses in AMsh glia were not completely abolished in glr-3 mutants, our results do not exclude the possibility that additional proteins may also act as cold sensors in AMsh glia.\n6. Likewise, we have strictly no reason to believe that ITR-1 works cell-autonomously in glial cells, nor is the downstream relationship between GLR-3 and ITR-1 adequately established. Suggested experiment: Conduct epistasis analyses using glr-3; itr-1 double mutants to test for genetic interaction. Conduct AMsh-specific knock out of ITR-1 (e.g. using existing floxed alleles of ITR-1 (PMC11087772).\nResponse: Our prior study demonstrated that ITR-1/IP3R is essential for odorant-induced calcium elevations in AMsh glia (Duan et al., Neuron 2020). Moreover, IP3R represents one of the most prominent calcium sources for astrocytic calcium responses in mice. Our results from itr-1 mutants and AMsh glial itr-1 RNAi lines suggest that ITR-1 is involved in the cold responses of AMsh glia. We further tested glr-3; itr-1 double mutants, and observed no additional defects compared to either itr-1 or glr-3 single mutants (Fig. 3c,e). These findings indicate that ITR-1 may act downstream of GLR-3 in mediating cold responses in AMsh glia. This observation aligns with prior studies showing that GLR-3 detects cold through its non-channel function, which requires G proteins as downstream signaling molecules to active calcium increases in both nematode ASER neurons and mouse DRG neurons. Nevertheless, we fully agree with the reviewer's comment that this evidence may not definitively confirm the relationship between GLR-3 and ITR-1 in AMsh glia. We do not intend to elaborate on the detailed mechanisms; instead, we believe further research would be valuable to dissect how GLR-3 activates calcium increases in AMsh glia. We have included the newly generated experimental data and discussed this issue in the revised manuscript to ensure clarity and accuracy (Line 253).\n7. Missing data in Figure 4: The manuscript lacks a heat tolerance assay under conditions where AMsh activity is suppressed, analogous to the cold tolerance assays shown.\nResponse: In the revised manuscript, we have added a heat tolerance assay examining the effects of histamine treatment on both AMsh glial HisCl1-expressing worms and wild-type (Fig. 4f). Our observations revealed that 2 mM histamine exerted minimal effects on WT worms, whereas it significantly impaired heat tolerance in AMsh glia::HisCl1 worms. Together with our finding that AMsh glial GCY-28 promotes heat tolerance, these results demonstrate that AMsh glia is functionally involved in regulating heat tolerance.\n8. The calcium responses in AFD neurons with AMsh-specific gcy-28 rescue appear unusually elevated in Figure 5, raising concerns about sensor calibration. Suggested control: Include an internal RFP control to normalize GCaMP and validate calcium imaging signals.\nResponse: We sincerely apologize for the confusion this may have caused. In our initial work, calcium responses in AFD neurons were measured in gcy-28 mutants and the gcy-28 rescue line using GCaMP6s. We recognized that GCaMP6s has distinct kinetic properties and sensitivity relative to GCaMP5, which was the calcium indicator employed for all other AFD neuron recordings. To resolve this inconsistency and ensure data comparability, we have constructed AFD-specific GCaMP5-expressing strains for the revised manuscript. Using these standardized strains, we systematically compared warm-induced calcium changes (detected by GCaMP5) in AFD neurons between wild-type, gcy-28 mutant, and gcy-28 rescue animals. These updated results (Fig. 5h,i) provide additional confirmation that GCY-28 expressed in AMsh glia regulates the warm-responsive calcium signals of AFD neurons.\n9. The reported ASH response defects in unc-25 and lgc-38 mutants (in Fig. 6) do not directly link to AMsh function. Suggested experiments: To fully substantiate the proposed model, checking the ASH response of animals with cell specific rescue of unc-25 and lgc-38 in AMsh and ASH respectively would be necessary.\nResponse: We agree with the reviewer’s suggestion. Our prior publication established a functional connection wherein odorant-evoked GABA release from AMsh glia acts on LGC-38 receptors in ASH neurons to downregulate ASH activity. To explicitly test and validate our current model, we have performed the recommended cell-specific rescue experiments. Specifically, we expressed unc-25 selectively in AMsh glia of unc-25 mutants and lgc-38 selectively in ASH neurons of lgc-38 mutants. Importantly, both targeted rescue strategies successfully restored the ASH response to control levels (Fig. 6a,b).\n10. Similarly, to properly support the proposed model, the authors should also assess the AFD response in animals with AFD-specific rescue of exp-1 to validate the proposed excitatory glia-to-neuron signaling.\nResponse: As recommended, we conducted cell-specific rescue experiments. Restoring exp-1 in AFD neurons of exp-1 mutants effectively rescued their warm response defect (Fig. 6e).\n11. One general weakness of the behavioral analyses is that it is hard to relate the behavioral phenotypes (often extremely mild, e.g, in thermal gradient) with the proposed inter-cellular mechanisms. A much deeper discussion is needed here, before one can correctly judge of their relevance.\nResponse: We appreciate the reviewer’s insightful comment. We acknowledge that linking behavioral phenotypes (which can be mild, as observed in thermal gradient assays) to the proposed intercellular mechanisms requires careful consideration. This complexity arises, in part, from the unique properties of AMsh glia and their broad regulatory scope. Unlike specialized thermosensory neurons that typically detect only cold or warm stimuli, AMsh glia are bi-directionally responsive to both thermal stimuli. Furthermore, AMsh glia ensheath the receptive endings of twelve pairs of amphid sensory neurons, among which at least AFD, ASH, AWC, ASI, ASE, ASJ, and ASG are known to be thermosensitive. This widespread association suggests that AMsh glia exert a coordinated, yet complex, regulatory influence over multiple thermosensory pathways. For these reasons, we did not expect manipulating AMsh glial activity would generate behavioral phenotypes as robust as those seen when perturbing individual, specialized thermosensory neurons like AFD. Our study focuses on defining a representative model of glial regulation of thermosensation, exemplified by our findings that AMsh glia differentially modulate the activity of warm-sensitive AFD and cold-sensitive ASH neurons. We fully agree that additional research is invaluable to fully dissect the complex interactions and mechanisms between glia and the wider thermosensory neuron network, and to thereby clarify how glial function translates to behavior. Accordingly, we have added a more detailed discussion in the revised manuscript to clarify these points and improve accuracy (Line 92; 456).\n12. The introduction focuses on the key cellular players under study. However, this seems too focused and cursory. It is important for the large audience of this journal to have a broader view of what is known in C. elegans. In particular, additional thermosensory neurons must be presented in more details, e.g. ASER, AWC, because ultimately, they might be the ones triggering the apparent thermal responsiveness of AMsh glia. Likewise, a better introduction of the molecular thermosensors is needed.\nResponse: Thank you for this helpful suggestion. We acknowledge the need to broaden the Introduction for the journal’s wide audience by contextualizing C. elegans thermosensation. In the revision, we have added detailed introduction to additional thermosensory neurons and key molecular thermosensors (Line 79-97).\nReviewer #3:\n1. A key conclusion is that AMsh glial cGMP triggers calcium dynamics, partly based on blue-light triggered guanylyl cyclase BlgC (Fig 2). This is tricky because multiple amphid neurons respond to blue light (PMID: 25640076). Additional controls are needed to strengthen this conclusion. At minimum:\na. key experiments should be replicated in lite-1; gur-3 mutants (see for e.g. PMID: 38194919, 27459302)\nb. traces with blue light stimulation without BlgC need to be documented.\nc. AFD should be chronically activated, and glial responses recorded.\nResponse: Thanks for the invaluable suggestion. We expressed of BlgC in AMsh glia using two distinct AMsh glial-specific promoters, vap-1 and R11D1.3. We observed blue light-induced calcium elevations in AMsh glia in both the wild-type and lite-1; gur-3 double mutant backgrounds. In contrast, no such calcium increases were detected in control lines where only mCherry was expressed in AMsh glia. These calcium response traces have been included in the revised manuscript (Fig. 2a,b). To further address the concern, we performed transgenetic experiments where we expressed TRPV1 in AFD and ASH neurons under the control of the gcy-8 and sra-6 promoters, respectively. While treatment with two concentrations of capsaicin (100 μM and 400 μM) reliably induced calcium increases in AFD and ASH neurons, no corresponding calcium elevations were observed in AMsh glia (Extended Fig. 1). Collectively, these new data strengthen our conclusion that light activation of BlgC in AMsh glia triggers glial calcium dynamics.\n2. The authors use vap-1 promoter for AMsh glial studies. (PMID: 18974354) noted faint expression in AFD, a relevant cell-type for this study’s conclusions. To address concerns for the central conclusions of this study, authors should\na. detail which promoter fragment was used (in Methods)\nb. verify with another promoter (esp. BlgC & HisCl baseline, gcy-28 rescue).\nResponse: We have included details of the vap-1 promoter fragment used in the revised Methods section (Line 532). To further validate our conclusions, we conducted key experiments—including those involving BlgC, HisCl1, and gcy-28, glr-3 and unc-25 rescue—using the R11D1.3 promoter, which is specifically expressed in AMsh glia in the head and PHsh glia in the tail (Fig.2a-h;3b,e;4b-e;5c,d,h,i,l,m;6a-d). These experiments yielded identical phenotypes to those obtained with the vap-1 promoter, reinforcing the robustness of our findings.\n3. The authors report identifying GCY-28 from single cell sequencing (Line 153). Data for this key conclusion should be either reported or cited.\nResponse: The expression of gcy-28 in AMsh glia was implicated by RNA-seq data available in the CeNGENApp (https://cengen.shinyapps.io/adult/). Our own single-cell RNA-seq results revealed that the membrane GCYs potentially expressed in AMsh glia are highly consistent with those documented in the CeNGENApp. In particular, gcy-28 emerged as the membrane GCY with high putative expression level in AMsh glia in both datasets (left panel: our RNA-seq data; right panel: CeNGEN App data). Regrettably, our RNA-seq data, generated in 2013, has incomplete metadata/parameter records, making it probably not suitable for deposition in public RNA-seq repositories. Consequently, we have directly cited the findings from the CeNGEN App in the revised manuscript (Line 202). Importantly, our Pgcy-28::GFP reporter strain has clearly validated the expression of GCY-28 in AMsh glia (Fig. 2m), providing experimental confirmation of this expression pattern.\n4. Many edits are needed for prior published literature to be cited, discussed or interpreted correctly. Some examples:\na. Line 90 and Line 332. Authors state that AMsh glia were “hypothesized to serve primarily.”. However, multiple prior studies (see PMID: 38565269 for recent review), including work cited here (Ray et al) from multiple labs including the authors has already demonstrated that AMsh glia are not passive support, but active participants in different sensory processing. This can be stated without detracting from the current study.\nb. Line 197-199. ..t is incorrect that the studies referenced reported “embryonic or larval stages” or “developmental deficits”. This is incorrect because these studies imply post-developmental effect of glia on sensory neuron properties and animal behavior. In fact, one of the few embryonic AMsh glial promoters (Pvap-1) PMID: 15935778) is used in THIS study (Line 200-202), but not the others referenced!\nc. Line 332. Studies in (PMID: 22298710, 21350017) have shown AMsh temperature-dependent expression of ver-1 is independent of AFD or sensory neuron activity i.e. AMsh glia are thermosensors. These should be referenced.\nd. AMsh glia engulf gcy- laden AFD membranes, and ciliated amphid neuron EVs (PMID: 33759761, 34533135); and AFD engulfment was shown to impact thermosensory animal behavior (PMID: 33759761). Given that this study invokes AMsh glial gcy function, this should be at least discussed as cGMP/gcy source.\nResponse: We appreciate the reviewer’s careful attention to the literature. We have accordingly revised the manuscript to ensure the accurate citation, discussion, and interpretation of published work, while preserving the clarity of our current study’s contributions (Line 98-107, 141-145).\n5. Figs. 1 & 2 do not adequately support the conclusion that glia independently detect temperature. To demonstrate this conclusively, authors need to report on AMsh glial calcium dynamics in response to temperature in AFD and ASH ablated animals. This is because it is also possible that glia have secondary bystander response to neuron activity, with AFD/ASH as the sensors (see also PMID: 16837592).\nResponse: We appreciate the reviewer’s insightful comments. First, we examined cold- and heat-induced AMsh glial calcium responses in che-2 mutants, animals with extremely short cilia and abnormal posterior projections that exhibit defects in behaviors mediated by ciliated sensory neurons, and observed no differences compared to wild type (Fig. 2d,e; 3d,e). Furthermore, we isolated and cultured single AMsh glial cells expressing GCaMP5 as described in our prior study (Duan et al., Neuron 2020), and found that these isolated cells displayed cold- and heat-induced calcium responses identical to those observed in vivo (Fig. 1e-g). Thirdly, we transgenically expressed TRPV1 in AFD and ASH neurons, respectively. Our results showed that activating AFD or ASH with capsaicin did not trigger calcium increases in AMsh glia (Extended Data Fig. 1). Moreover, using 7×GFP knock-in and transcriptional reporter lines, our data further demonstrate that the core molecular machinery underlying thermal sensing, including cng-1, cng-3, tax-4, gcy-28, and glr-3, is all expressed in AMsh glia (Fig. 2l,m; 3f,g). Restoring the corresponding cDNAs under the control of AMsh glial promoters (vap-1 and R11D1.3) further rescued defects in AMsh glial thermosensation in the respective mutants (Fig. 2c-h; 3b,e). Together with our observations in unc-13 and unc-31 mutants (Fig. 1c,d), these independent lines of evidence provide strong support for the conclusion that glia detect temperature independently.\n6. unc-13 does not affect AMsh glial calcium dynamics (Fig 1) but reduces AFD response (Fig 6). As stated, it seems the authors imply that glial GABA release is via UNC-13 regulated neurotransmitters (Line 310-312). For this conclusion, authors need to evaluate glial and pan-neuronal UNC-13 rescue. Also discuss altered AMsh glial response kinetics in these mutants, which suggests neuronal input is relevant.\nResponse: We appreciate this insightful comment. A study from Dr. Andrew Dillin's group demonstrated that CEPsh (cephalic sheath) glia in nematodes can sense mitochondrial stress and utilize small clear vesicles (SCVs) as signaling vehicles (Bar-Ziv et al., 2023). This process depends on UNC-13/Munc-13, a protein essential for calcium-dependent secretion of synaptic vesicles (Speese et al., 2007), which enables signal transmission from CEPsh glia to neurons (Bar-Ziv et al., 2023). Our previous work also showed that calcium triggers vesicular GABA release from AMsh glia (Duan et al., Neuron 2020). To address the reviewer's concern, we have now performed additional experiments where we restored unc-13 in AMsh glia, which rescued the reduced AFD response (Fig. 6c,d). These support our conclusion that glial GABA release is mediated by UNC-13-regulated neurotransmitter release mechanisms. We agree that neuronal input may regulate AMsh glial responses, and this is indeed an observation we have made (Fig. 1c, d). However, this does not affect our conclusions that AMsh glia can independently sense both cold and heat, and that they bidirectionally regulate ASH and AFD through GABA signaling.\n7. Glial gcy-28 rescue does not phenocopy wild type (Fig 4, Line 234), and alters AWB responses i.e. alters response property of cell expressed in. To bolster conclusion that it acts in glia, authors should\na. show rescue of gcy-28 from neurons.\nb. Examine a second allele, preferably a genetic null\nc. Show the region used for expression analyses in xx, and perform either endogenous tag or recombineered fosmid expression analysis\nd. Note which cDNA isoform was used for rescue, given that the lesion used is possibly only truncating one isoform. Showing the gene and protein domain structure with lesions and promoters noted will aid evaluation.\ne. Include wild type traces in Fig 5g-j.\nf. Optional: will help to see gcy-28 cold mortality rates (Fig 4e) for completeness.\nResponse: We appreciate these constructive suggestions. To address them, we have performed additional experiments and revised our findings. Restoration of gcy-28 in AMsh glia using two distinct promoters (vap-1 and R11D1.3) rescued both the heat-evoked responses in AMsh glia and AFD neurons in gcy-28(tm3028) mutants (Fig. 2g,h; 5h,i). We examined a second allele, gcy-28(tm2411), and observed phenotypes identical to those of gcy-28(tm3028) (Fig. 2g,h; 5h,i). For heat-induced AFD responses monitored via GCaMP, we have standardized the calcium indicator by changing GCaMP6s to GCaMP5 (consistent with the GCaMP variant used in our other calcium imaging strains) and included traces for wild type, gcy-28(tm2411) mutants, gcy-28(tm3028) mutants, and gcy-28(tm3028) mutants with gcy-28 restored in AMsh glia using both promoters(Fig. 5h,i) . As suggested, we further examined the cold-induced mortality rates of gcy-28(tm3028) mutants and observed no difference from wild type (Extended Data Fig. 3a). Given that the previously reported neuronal expression of GCY-28, we acknowledge that it may also function in neurons. However, our results strongly support a cell-autonomous role for GCY-28 in AMsh glia. These new data and corresponding revisions have been incorporated into the revised manuscript.\n8. Studies from multiple labs have reported roles for chloride homeostasis, some of which are referenced here (Singhvi et al, Yoshida et al, Ray et al); but also Wang et al. The authors need to discuss how this may impact interpretation of HisCl chloride channel experiments because one possibility is that altering Cl in the glia-neuron inter-cellular space impacts AFD activity.\nResponse: We have added a discussion section addressing the critical role of chloride homeostasis, specifically highlighting the possibility that manipulating chloride levels via the HisCl1 chloride channel could alter chloride concentrations in the glia-neuron intercellular space, thereby impacting neuronal activity (Line 380).\n9. The model suggests autonomous glial responses, but for most genes no cell-specificity is evaluated. Specific examples are noted below, with experiments suggested to enable the conclusion as noted:\na. Responses are lost in CNG channel mutants (Fig 2c,d), and the authors conclude that CNG channels are downstream of cGMP in AMsh glia. To make this conclusion, authors need to demonstrate that (i) CNG channels are expressed in glia (ii) function in glia but not neurons (iii) do not impact cold responses, per their model (Extended Fig 2).\nResponse: To address this point, we performed three key experiments: First, we created 7×GFP knock-in lines and used a Leica STELLARIS high-resolution confocal microscope to definitively show that CNG-1, CNG-3, and TAX-4 are expressed in AMsh glia (Fig. 2l,m). Second, we restored tax-4 in AMsh glia (using vap-1 or R11D1.3 promoters), which rescued the heat-induced response defect in AMsh glia of cng-1,cng-3,tax-4 triple mutants (Fig. 2c-e). Third, consistent with our model, cold-induced responses in AMsh glia remained unchanged in cng-1,cng-3,tax-4 triple mutants (Fig. 3d,e). Together, these independent lines of evidence confirm that CNG channels function downstream of cGMP in AMsh glia to mediate heat sensing.\nb. Diminished responses in itr-1 mutants (Fig 3a-c) does not imply ER store release from AMsh glia (Lines 185, 187). This needs to be rephrased or cell-specific requirement of ITR-1 in AMsh glia shown. Independently, rationale for examining ITR-1, above other possible mechanisms, is unclear and should be detailed.\nResponse: Our prior study demonstrated that ITR-1/IP3R is essential for odorant-induced calcium elevations in AMsh glia (Duan et al., Neuron 2020). Moreover, IP3R represents one of the most prominent calcium sources for astrocytic calcium responses in mice. Our results from itr-1 mutants and AMsh glial itr-1 RNAi lines suggest that ITR-1 is involved in the cold responses of AMsh glia. We further tested glr-3; itr-1 double mutants, and observed no additional defects compared to either itr-1 or glr-3 single mutants (Fig. 3c,e). These findings indicate that ITR-1 likely acts downstream of GLR-3 in mediating cold responses in AMsh glia. This observation aligns with prior studies showing that GLR-3 detects cold through its non-channel function, which requires G proteins as downstream signaling molecules to active calcium increases in both nematode ASER neurons and mouse DRG neurons. Nevertheless, we fully agree with the reviewer's comment that this evidence may not definitively confirm the relationship between GLR-3 and ITR-1 in AMsh glia. We do not intend to elaborate on the detailed mechanisms; instead, we believe further research would be valuable to dissect how GLR-3 activates calcium increases in AMsh glia. We have included the newly generated experimental data and discussed this issue in the revised manuscript to ensure clarity and accuracy (Line 242-254).\nc. Effect of osm-9 as a glial modulator (Fig 3a-c, Line 193) is over-interpreted.\nResponse: We have carefully considered potential explanations for the kinetic differences between osm-9 mutants and wild-type (WT). However, we noticed that the data pertaining to osm-9 has relatively low relevance to other experiments in the study and thus stands as an isolated component. Therefore, we have decided to remove this section in the revised manuscript. This adjustment aims to better streamline the narrative and ensure the focus of the study remains concentrated on its core themes.\nd. Similarly, lgc-38 results cannot be concluded as stated without cell-specific evidence. It is also expressed in many downstream interneurons.\nResponse: As suggested, we performed rescue experiments by restoring lgc-38 expression specifically in ASH neurons. This fully rescued the defective calcium response of ASH neurons in lgc-38 mutants (Fig. 6a,b).\ne. Likewise, it is unclear if the pde genes act in neurons or glia, or both.\nResponse: Our experimental results demonstrate that AMsh glia sense heat via GCY-28 and subsequently activate cyclic nucleotide-gated (CNG) channels. Given that PDEs function to regulate intracellular cGMP homeostasis, it is plausible that these pde genes act in AMsh glia. This is further supported by our calcium imaging data collected from isolated AMsh glial cells (Fig. 1e-g). Notably, PDEs are also well-documented to play critical roles in neurons involving cGMP-CNG signaling pathways. Our current experimental results do not exclude the possibility of their functional involvement in both neurons and glia.\n10. Behavior assays:\na. Line 219. This assay is not standard in the field. It needs to be confirmed that the HisCl array does not cause locomotion defects by showing speed/movement traces. Further, >1 array should be reported, with promoter-only controls. It should also be noted if animals leave the field with delay (>10-20 mins)\nb. Conclusions in Fig. 4j-m are unclear from data provided with single-array study.\nc. A control mutant will be helpful (e.g. triple gcy-8/18/23 or ttx-1).\nResponse: a, In our thermal avoidance assays, the Pvap-1::HisCl1 line exhibited fewer worms remaining in the plate center at 20°C compared to controls, indicating that this line does not induce locomotion defects. In our newly added HisCl1 line (PR11D1.3::HisCl1), locomotion was even more comparable to controls (Fig. 4b). Both these two lines showed identical phenotypes across behavioral paradigms, including cold/heat avoidance and thermotolerance, as well as the effect on thermal responses of ASH and AFD (Fig. 4b-f). Importantly, our key conclusions regarding AMsh glial HisCl1 are primarily derived from within-line comparisons (presence vs. absence of histamine), which ensures the reliability of our results. For controls, we used the same promoters driving mCherry alone (matching the mCherry marker in HisCl1 transgenics) (Fig. 4b-f).\nb, In the TTX plate distribution assay, we employed two different cultivation temperatures and two temperature gradients, collectively demonstrating AMsh-mediated regulation of thermal distribution. TTX distribution may involve not only primary thermosensory neurons like AFD and ASH but also potentially other thermosensory neurons (e.g., AWC, ASE, ASJ). Additionally, AMsh glia ensheath 12 pairs of amphid sensory neurons, which may contribute to the complexity of its regulatory mode. Nevertheless, our results clearly show that AMsh glia can differentially modulate AFD and ASH, providing a compelling example of how glia regulate thermal sensory networks.\nc, Following this suggestion, we have supplemented our study with TTX experiments performed on the gcy-8/18/23 triple mutants (Extended Data Fig. 3b).\n11. To conclude “homeostatic gain..recalibrate preference” (Abstract), the authors need to demonstrate this via the following directed studies:\na. AFD, ASH responses in wild type, gcy-28 and unc-25 (glia-degron) animals.\nb. Chronically activate ASH and evaluate the pathway by imaging, behavior or both.\nc. Demonstrate if this is cold-specific or any noxious stimuli from polymodal ASH\nResponse: We appreciate the reviewer’s insightful suggestion. Given the complexity of the thermal sensory circuit, including prior reports of cross-regulation between cold- and heat-sensing neuronal pathways, and the anatomical association of AMsh glia with 12 pairs of sensory neurons (encompassing multiple temperature-sensing neurons such as AFD, ASH, AWC, ASI, and ASJ), the overall regulatory paradigm is highly intricate. Our core findings demonstrate that AMsh glia sense temperature stimuli via distinct cold- and heat-responsive receptors, and differentially regulate the activity of cold-sensing ASH neurons and heat-sensing AFD neurons through GABA signaling, thereby modulating thermosensory behaviors. Notably, downstream pathways of ASH-mediated cold sensing have been identified in previous study, and activation of ASH or AFD via TRPV1-capsaicin in our newly added experiments did not elicit calcium responses in AMsh glia (Extended Data Fig. 1). Building on our previous work and the current findings, we have demonstrated that AMsh glia modulate both olfactory and thermosensory responses in ASH. Nevertheless, we recognize that the precise regulatory mechanisms underlying thermosensory circuits, along with their interplay with other modalities, demand further rigorous investigation. To align with our current data, we have revised the phrase \"homeostatic gain..recalibrate preference\" to \"This GABAergic hub-and-spoke architecture regulates a broad range of thermal behaviors, including thermal avoidance, thermal resistance, and thermal preference\" in the Abstract (Line 38). Additionally, we have supplemented new experiments, including AMsh glia-specific unc-25 rescue assays for AFD and ASH responses, AMsh glia-specific gcy-28 rescue experiments, as well as TRPV1-capsaicin experiments (Fig. 6a-d; Extended Data Fig. 1). These revisions and additions have been incorporated into the revised manuscript.\nGeneral and minor comments:\n1. A colorblind safe palette is recommended. Also, it is advised to be consistent with color scheme (e.g. Fig 1: glia text and unc-13 are similar colors, but the color denotations change in subsequent figures; Fig 5: color for AFD neuron text and histamine trace is both red, and color for ASH is same as GCY-28 rescue, Fig 4f: plot color title and trace color same; Fig 6d: unc-25 and rescue lines are hard to differentiate, which can be confusing).\nResponse: Thank you for the recommendation. We have revised the figures with a colorblind-safe color palette and ensured consistent color coding throughout to avoid confusion.\n2. Statistical tests used should be stated in Figure Legend. Also,\na. if “not significant” is inferred from a plot, the comparison done should also be noted on the plot (e.g. Fig 2b).\nb. Also, for some panels (e.g. Fig 4b) it is unclear what statistics was done.\nc. Finally, for rescue experiments, comparison should be done also with WT, and reported with statistical comparison and discussion (e.g. Fig 2f-g).\nd. Fig 4f: ANOVA and comparison between wild type and rescue seems appropriate.\nResponse: Thank you for pointing out these statistical issues. The recommended revisions regarding statistics have been implemented in the revised manuscript.\n3. Information missing but needed for the studies presented:\na. Information on biological replicates should be noted for the different assays.\nResponse: We have noted the number of biological replicates for the different assays as requested.\nb. Information on what promoter used for AWB, with relevant citations to lend confidence to cell-specificity\nResponse: We have noted the promoter used for AWB (str-1p) and provided relevant citations to confirm its cell specificity (Line 534). To obtain definitive evidence that GCY-28 acts as a heat receptor, we performed heterologous expression in HEK293T cells. Heat activation of these cells resulted in a significant increase in intracellular cGMP, as quantified by ELISA. This assay further confirms GCY-28’s role as a heat sensor (Fig. 2k).\nc. Since both GCaMP5 and GCaMP6s are used interchangeably, it will be helpful to have functional dynamic traces for both with temperature reported side-by-side\nResponse: We apologize for the confusion. We have standardized the calcium indicator for heat-induced AFD response measurements by switching from GCaMP6s to GCaMP5, ensuring consistency with the GCaMP variant used in our other calcium imaging experiments. We have now re-examined and included AFD heat response traces for wild type, gcy-28(tm2411) mutants, gcy-28(tm3028) mutants, and gcy-28(tm3028) mutants with gcy-28 restored in AMsh glia (using both promoters) using this standardized GCaMP5 indicator (Fig. 6h,i).\nd. For all Ex arrays, per best practice, >1 array should be reported.\ne. For Ex arrays, per best practice, over-expression in wildtype should be reported. This is especially important for studies like Fig 2f-g, where the rescuing array alters AMsh responses beyond wild type.\nResponse: We agree that using multiple Ex arrays and reporting wild-type overexpression are best practices. We have therefore included data from additional independent Ex arrays for critical experiments, such as those involving HisCl1, gcy-28, and rescue/RNAi lines each driven by the two AMsh glial promoters (Fig. 2a-h;3b-f;5a-m;6a-d). These newly supplemented experiments yielded identical results and conclusions to our original arrays, thereby validating the robustness and accuracy of our findings.\nf. Promoter only control in AWB will strengthen the argument (Fig 2h)\ng. Extended Fig. 1: controls should be included. Again, text and trace colors are same, but do not imply correlation.\nResponse: The control line contains Pstr-1::mCherry, serving as the control for Pstr-1::gcy-28::sl2::mCherry. For Extended Fig. 1, we have included all required controls and adjusted the color scheme to ensure text and trace colors are distinct and non-overlapping (now Extended Fig. 2 in the revision).\nh. Basis for skew of range toward cold is unclear (i.e. 20C not in center of plate).\nResponse: The skew of the temperature range toward cold is due to adjustments made to the TTX plate based on the distribution range of the worms, which allows for better visualization and demonstration of the results.\ni. Line 219-220: show data for higher numbers in central region for 10C and 20C\nj. Check references – for e.g. missing in Line 75, 376.\nResponse: Quantitative analysis of Fig. 4b,c reveals that the number of worms localized in the central region is marginally higher at 10°C compared to 20°C. However, this observed difference is not statistically significant. In response to point j: We acknowledge the oversight of missing references. We have thoroughly checked the manuscript and supplemented the relevant references.\n4. Fig 2j, 3d, and 6h: close-up of cell-body is needed. Also,\na. Fig. 2j: glial cell (point of study) is out of focus\nb. Fig. 3d: why does the cytosolic fluorophore not fill the glial process.\nResponse: We appreciate this constructive feedback. For Fig. 2j, we have replaced the original image with a new one where the AMsh glial cell is more clearly in focus. We have added close-up views of the cell bodies in Fig. 6h to enhance clarity. Regarding the concern about Fig. 3d, the cytosolic fluorophore appears incomplete in filling the glial processes due to relatively dim GFP signal from the transcriptional reporter line used. To address this and rigorously confirm GLR-3 expression in AMsh glia, we have generated a 7×GFP knock-in line, which provides definitive evidence of GLR-3 localization in these glial cells (Fig. 3f). These revisions have been incorporated into the revised manuscript.\n5.Confusing phrasing, consider reword:\na. Line 200: “avoid this..”. It is unclear what authors mean “this” refers to.\nb. Line 260-267: “exceeds 1C/cm” stated and “in a 1C/cm” used.\nc. Line 337: “single cellular compartment” implies cell region, which is not evaluated. “single cell” is more appropriate.\nd. Suggest moving Line 244 (Strongyloides..) to discussion..\nResponse: We appreciate you bringing these confusing phrasing issues to our attention. We have carefully revised the manuscript to clarify these points as suggested.\nWe deeply appreciate the constructive feedback and enthusiastic support the reviewers have provided for our manuscript. Every comment and suggestion raised has been thoroughly addressed in the revised version of the manuscript. Below, we present our detailed point-by-point response to each of the reviewers’ comments:\nReviewer #1 (Remarks to the Author):\nI am satisfied with how the authors addressed my concerns through new experiments, additional data analyses, and revisions to the manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors have made remarkable effort in thoroughly revising the manuscript, including substantial new data acquisition and systematically addressing the concerns raised in the previous review. The conclusions are now much more robust, in support of a significant conceptual advance regarding the role of glial cells in thermosensory processing.\nFour minor points would still warrant attention and correction:\n1. Line 290: The terms “cold nociception” and “hot nociception” may be misleading. It is somewhat excessive to refer to nociception when the behavioral assay merely quantifies the fraction of animals leaving a hot or cold region. The observed effect could reflect the execution of the behavior or general mobility, rather than nociceptive processing (controls at non-noxious temperatures are not informative in this regard). It would be clearer and more precise to refer to “cold avoidance” and “heat avoidance”.\nResponse: As suggested, we have changed the terms “nociception” to “avoidance” in the revised manuscript.\n2) Line 488: The phrase “molecular innovation” is ambiguous in this context. The term “innovation” carries evolutionary implications, which are not supported by the data presented. A different phrasing is needed.\nResponse: We appreciate this insightful comment. We have changed the phrase “molecular innovation” to “molecular machinery” in the revised manuscript.\n3) Figure 2 legend: The description of the HEK cell data corresponding to the newly added panel k is missing and should be included.\nResponse: We apologize for the oversight. We have now added the description of the HEK cell data corresponding to Fig. 2k in the revised manuscript.\n4) Figure 4: The word “temperature” is misspelled in the six x-axis labels presented in panels h, j, and l.\nResponse: We have corrected the misspelling in the revised Figure 4.\nReviewer #3 (Remarks to the Author):\nWe commend and appreciate the impressive efforts of Zhu et al to address reviewer comments to such exhaustive detail. They have done so to our satisfaction, and the revised manuscript is significantly improved and well-reasoned. This will be a strong contribution to our understanding of glial roles in circuit function and sensory behaviors. We have no further concerns on the experiments or conclusions and are happy to recommend it for acceptance.\nBelow, we suggest minor text revisions/clarification points for the authors to consider for final publication.\n1.We think PMID: 18660808 is appropriate for Line 77. Takeishi 2022 (cited in Line 77) is more suited for Line 89/90.\nResponse: Thank you for the inputs. We have followed the suggestion in the revised manuscript.\n2.Typo error: Fig 1g: 38°C not 28°C.\nResponse: We have corrected the typo error in the revised Fig 1g.\n3.Fig 3e: adding a gap between genotypes or breaking into smaller panels will help with readability\nResponse: We have added gaps between genotypes in the revised Fig 3e.\n4. Line 193: Did the authors mean to reference Fig 3d,e? Their text refers to cold-response which is Fig 3.\nFurther, we note that che-2 was also reported for warm-induced (Fig 2d,e) and no response was seen. The authors do not comment on this data, but it actually makes sense with prior report that CHE-2 is absent from AFD (PMID: 10518500). The authors may decide if they want to address this in text (or consider removing che-2 result from Fig 2d, e).\nResponse: Thank you for your careful observation and valuable suggestion.\nOur data show that calcium increases in AMsh glia in response to warm and cold stimuli were intact, referring to Fig 2d,e (warm) and Fig 3d,e (cold), respectively. We have removed the che-2 results from Fig 2d,e (warm) as suggested.\n5.The different time points to end the data are confusing, e.g. Fig 1c vs 3b, or 1a vs 1c. Keeping this consistent will help the reader to follow.\nResponse: We have standardized the time points for data termination of the figures.\n6.Fig 4: We were confused that for an assay plate of 14 cm with 0.5°C/cm gradient, plot data was 18°C - 23°C (and not 25°C). Similarly, for 1°C/cm gradient, the range was shown as 11°C - 27°C (and not 25°C). Finally, the range was different between Fig 4j and l. A note in Methods on these will be helpful.\nResponse: Thank you for this valuable comment. We have provided detailed explanations for the temperature range-related issues in Fig. 4 and supplemented corresponding notes in the Methods section: (1) For the 14 cm assay plate with a 0.5°C/cm gradient: The plotted temperature range is 18–23°C (instead of reaching 25°C) because rare worms were detected in the subzones above 24°C during the experiment, so these data were not included in the figures. (2) For the 1°C/cm gradient: The temperature range is shown as 11–27°C (rather than a fixed 25°C) because we measured the actual temperature of each zone on individual assay plates prior to worm counting. Due to slight environmental fluctuations and plate-specific variations, the real temperature range of each plate deviated slightly from the theoretical gradient, leading to the observed 11–27°C range. (3) Regarding the different temperature ranges between Fig. 4j and 4l: In Fig. 4l, the 23–26°C range was intentionally omitted to more clearly visualize the migration behavior of worms cultured at 17°C—specifically, their preference for lower-temperature zones when initially placed in the 20°C zone. To more intuitively present the distribution ranges of worms under the two different conditions, we have adjusted both Fig. 4j and 4l in the revised manuscript to display the temperature interval of 11–24°C. As there was essentially little worm distribution in the higher temperature ranges, these are not shown.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}]

Decision letter