Mechanotransduction in mammalian trigeminal sensory neurons

Resumo

Many physiological processes depend critically on the proper sensing of mechanical forces, including hearing, proprioception and touch. Great efforts have been undertaken to unveil the mechanisms and discover the molecular entities responsible for mechanotransduction. Accordingly, recent studies have led to the description of the specific properties of mechanically activated (MA) currents in different subpopulations of mechanosensitive (MS) neurons from the dorsal root ganglia (DRG) of mice, although the channels that mediate these currents are largely unknown. Trigeminal (TG) neurons are primary sensory neurons that detect mechanical forces originating in the face and the head. However, unlike DRG neurons, the mechanosensitive conductances of TG neurons have not been described in detail. The main aim of this study was to characterize mechanosensitive neurons in mice and to correlate their properties with neuronal type (non-nociceptive vs. nociceptive neurons) and mechanotransductor expression. I used Fura2-based fluorimetric calcium imaging and whole-cell patch-clamp recordings to characterize the responses of different populations of TG neurons from newborn and adult mice to a hypoosmotic solution (210 mOsml Kg-1), a stimulus that causes membrane stretching, and to static indentation produced by a glass pipette driven by a micromanipulator system. I classified the neurons according to established criteria, like firing pattern and action potential shape, sensitivity to tetrodotoxin and IB4 labelling, for nociceptive and non-nociceptive neurons. Using calcium imaging techniques I identified three mechanically sensitive populations of neurons that responded to hypoosmotic solution (18%), indentation (37%) or both stimuli (34%). The proportion of mechanosensitive neurons was comparable in newborn and adult animals. However, I observed a significant increase in the proportion of mechanical neurons with nociceptive properties in adult animals. In patch clamp recordings, mechanical stimulation activated three types of currents that could be distinguished by their inactivation kinetics: rapidly (RA, 35%), intermediate (IA, 24%) and slowly (SA, 41%) adaptive currents. Both nociceptive and non-nociceptive neurons showed similar proportions of the three types of mechanically gated currents. The correlation between the different subsets of mechanically activated neurons and the expression of specific ion channels proposed to be involved in mechanotransduction (e.g., TRPA1, TRPC5, Piezo2, TRPC3 and ASIC3) was further investigated using single cell reverse transcription polymerase chain reaction (RT-PCR). While I observed considerable heterogeneity in the expression of mechanotransducer channels in the different neuronal subpopulations, the expression of the TRPA1, TRPC5 and Piezo2 genes predominated in the majority of the cells tested, regardless of the subpopulation. Indeed, a series of candidate mechanotransduction genes have been proposed in mammalian cells, which includes TRPA1, encoding a channel predominantly expressed in mechanosensitive TG neurons. TRPA1 has been proposed to fulfil a fundamental role in mediating SA and IA currents in a subpopulation of adult DRG neurons. To investigate the role of TRPA1 in trigeminal neurons, I recorded mechanically-activated currents in newborn and adult mice lacking TRPA1. I found that genetic ablation of TRPA1 in adult mice produced a significant increase in the proportion of nociceptive neurons exhibiting RA currents and a significant decrease in the amplitude of IA currents. In conclusion, I have identified distinct subsets of TG neurons that respond to different mechanical stimuli, suggesting that specific mechanical stimuli provoke distinct physiological responses. I also characterized mechanosensitive currents in nociceptive and non-nociceptive MS TG neurons. Heterogeneity in the expression of specific channels in distinct subpopulations of mechanosensitive neurons suggests that mechanotransduction is not dependent on a single molecule rather, it could depend on the activation of different mechanotransducers. Finally, my results indicate that TRPA1 may contribute to mechanically-activated currents in nociceptive neurons from adult mice.