Introduction
In Drosophila larvae, Class III (CIII) primary sensory neurons detect nociceptive cold temperatures, with about half responding to rapid cooling with transient bursting (1,2). Cold responses have been linked to activation of thermosensitive TRP channels, including TRPM, PKD2, and NOMPC (1,3). We previously showed that lowering extracellular Cl⁻ enhances spiking and promotes bursting in CIII neurons, consistent with a depolarizing shift of the Cl⁻ reversal potential. Here, we test whether pharmacological or ionic perturbations that produce appropriate membrane depolarization are sufficient to create bursting mechanisms in CIII neurons at room temperature, revealing a mechanism that does not rely on TRP channel activation.
Methods
Intracellular recordings were obtained from CIII neurons in Drosophila larvae under pharmacological and ionic manipulations. Experimental conditions included reduced extracellular Cl⁻ (6 mM; 134 mM control), elevated extracellular K⁺ (15 mM; 3 mM control), Ca²⁺ removal, and tetrodotoxin (TTX, 20 nM) to block voltage-gated Na⁺ channels. Direct current injection was used to characterize transitions between silence, spiking, and bursting across conditions. In parallel, a biophysical computational model of the CIII neuron was developed and constrained by experimental measurements. Model parameters were tuned to reproduce passive electrical properties and validated by comparison with experimentally observed activity patterns.
Results
In control, current injection (5–20 pA) produced tonic spiking. In low-Cl⁻ saline, the same stimulation induced bursting in 90% of neurons (18/20). Elevated extracellular K⁺ promoted bursting in all neurons examined (6/6), indicating that global depolarization facilitates burst generation. Removal of extracellular Ca²⁺ did not eliminate bursting, suggesting that Ca²⁺ influx is not strictly required for burst generation under these conditions. In contrast, tetrodotoxin (20 nM) abolished both spikes and the underlying depolarizing potentials. Biophysical modeling reproduced these transitions and suggested that the voltage-gated Na⁺ current plays a prominent role in sustaining the depolarizing envelope supporting burst generation.
Discussion
These results demonstrate that CIII neurons can generate the full spectrum of activity patterns—silence, tonic spiking, and bursting—without activation of thermosensitive TRP channels. Depolarizing manipulations such as reduced extracellular Cl⁻, elevated K⁺, or current injection reliably promoted bursting. These findings suggest that practically all CIII neurons are intrinsically burst-capable when operating within an appropriate depolarized regime. Biophysical modeling reproduced the observed transitions and dissected the contributions of ionic gradients and membrane conductances, providing a mechanistic framework in which Na⁺ channel dynamics contribute prominently to the generation of bursting activity.
References
1. Turner, H. N., et al. (2016). The TRP Channels Pkd2, NompC, and Trpm Act in Cold-Sensing Neurons to Mediate Unique Aversive Behaviors to Noxious Cold in Drosophila. Current Biology, 26(23): 3116-3128. https://doi.org/10.1016/j.cub.2016.09.038
2. Maksymchuk, N., et al. (2022). Transient and Steady-State Properties of Drosophila Sensory Neurons Coding Noxious Cold Temperature. Frontiers in Cellular Neuroscience,16, 831803. https://doi.org/10.3389/fncel.2022.831803
3. Himmel, N. J., et al., (2023). Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila. eLife, 12, e76863. https://doi.org/10.7554/eLife.76863
Acknowledgement
NIH grant R01NS115209 to DNC and GSC.
