Our ability to detect physical stimuli like temperature is crucial for survival. From the smallest insects to the largest mammals, this process―thermosensation―is highly conserved across species. It enables us to sense, respond to, and adapt to dynamic temperatures within our environment. At the core of this sensory process are thermosensitive channels with distinct activation thresholds that detect a wide range of ambient temperatures. These channels transduce sensory stimuli into electrochemical signals from the peripheral nervous system to interneurons in the central nervous system, eliciting appropriate responses. However, the underlying mechanisms governing this process remain elusive.

The nematode Caenorhabditis elegans, with its transparent body and simple yet well-characterized nervous system, represents a particularly attractive model system for studying thermosensation at the single neuron level. Although research on experience-dependent thermosensation in C. elegans dates to the 1970s (see Hedgecock and Russell and Kimata et al.), our understanding of how worms respond to rapid temperature changes is still limited. This challenge has been compounded by a lack of suitable experimental tools that can induce fast temperature changes. In this regard, microfluidic approaches have emerged as a promising avenue, offering precise manipulation of small biological samples like C. elegans to study various environmental cues (see Le et al. and Levine and Lee). Yet, their potential for delivering acute temperature stimuli to monitor C. elegans neuronal responses remains largely unexplored. In their study titled “Dynamic temperature control in microfluidics for in vivo imaging of cold-sensing in C. elegans”, Lee et al. address this gap by developing a microfluidic-based platform able to deliver hot and cold temperature stimuli with sub-second temporal resolution, while simultaneously enabling high-resolution monitoring of C. elegans at the single-neuron level.

Microfluidics for rapid temperature switching

The primary aim of this study was to develop a microfluidic-based system coupled with temperature-control units to deliver precise and repeatable temperature stimuli to C. elegans, achieving sub-second-scale accuracy while monitoring neuronal responses. Lu and colleagues repurposed a rapid flow selection module, originally designed for chemical reagents, to deliver acute temperature stimuli by adjusting pressure balance. By using deformable polydimethysiloxane actuators, they streamlined worm handling, facilitating tasks such as individual worm loading into the imaging zone, unloading, and subsequent loading for consecutive recordings (Figure 1). A notable advantage of this system lies in its capacity to prevent repeated exposure of worms to thermal stimuli, which can diminish their response to subsequent trials. By connecting the system to a custom-built pressure controller, their design ensures reproducibility and minimizes human error in experimental protocols.

 

Figure 1.Schematic of microfluidic device design (left) showing experimental procedure (right) enabling sequential loading, imaging, and unloading of worms. Adapted from Lee et al., 2024.

Testing the system for acute and fast-changing thermal stimuli

Lee and colleagues first used their platform to induce temperature shifts that should elicit neuronal responses in C. elegans, specifically between 20 and 15 °C. By using computational modeling to optimize the pressure configuration, they determined that an optimal pressure ratio of 1.5 would enable rapid and complete stream switching while minimizing backflow. By quantifying fluorescence intensity of the fluorescein dye in the warm stream, they also characterized the temporal dynamics of their system, measuring sub-second rise and fall times of 0.8 and 1 s, respectively.

Next, they confirmed the platform’s feasibility to achieve the desired temperature transitions by using numerical modeling and simulations. Notably, their dimensionless analysis also suggested that their system could reach a steady state within 0.02 s, indicating the possibility of creating abrupt temperature perturbations by rapid flow switching in their device.

Investigating sensory and interneuronal responses to temperature

To assess the system’s utility, the authors investigated cold-shock responses via thermosensitive transient receptor potential channels, comparing wild-type (WT) worms and trpa-1 mutant worms. By using a genetically encoded calcium indicator, GCaMP6, Schafer and colleagues compared responses of physical vapor deposition (PVD) neurons, known to respond to cold-shock stimuli. Whereas WT worms elicited calcium transients precisely timed to acute cold shock stimuli, trpa-1 mutants were insensitive to the temperature changes applied. These findings underscore the platform’s ability to deliver cold shocks and discriminate between WT and mutant organisms.

Lee et al. next sought to examine the transmission of cold shocks from PVD neurons to downstream interneurons. They observed that a subset of animals exhibited calcium transient responses in polyvinyl chloride (PVC) command interneurons, which have direct synaptic connections to PVD neurons. The observed probabilistic responses indicate interneuronal responses through temperature stimulation. However, the platform could not distinguish if PVC responses were evoked by PVD activity or were intrinsic PVC thermosensitivity.

Lastly, they explored habituation and sensitization in sensory neurons and interneurons. Habituation was explored by exposing worms to repeated stimuli (30 s per stimulus at 30-s intervals), resulting in a decreased response magnitude of PVD neurons over time, which indicates a habituated neural response. Sensitization was explored by subjecting worms to subthreshold thermal stimuli before subsequent thermal stimulation, revealing increased calcium responses to subsequent cold stimuli, indicative of a sensitization phenomenon.

Conclusions

In their study, Lee and colleagues tackle an important need in the field while also addressing important implications and challenges of this novel microfluidics-based system. Their study emphasizes the importance of timing and duration of stimulus delivery and demonstrates the precision of their platform. Lee et al. acknowledge the need for further refinement to achieve a more symmetrical temperature profile, albeit at the cost of increased design complexity. Notably, they envision their system and findings as catalysts for further studies of molecular and cellular mechanisms underlying thermosensation in nervous systems.