Expression of Mechanosensitive Ion Channels in Mesencephalic Trigeminal Nucleus Neurons

Objectives: Proprioceptors on the primary afferent neurons associated with the mesencephalic trigeminal nucleus (MesV) in the periodontal ligament are sensors for occlusal force. However, the detailed mechanosensitive processes of the MesV neurons innervating the periodontal ligament require clarification. We examined the protein expression of the mechanosensitive ion channels and measured the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in isolated MesV neurons using Fura-2 calcium fluorescence indicator.
Methods: We injected the neuronal anterograde tracer cholera toxin subunit B (CTB) into the periodontal ligament of the bilateral maxillary first molars in P15 rats. Two days after administration, brainstem slices (400-µm thick) containing the MesV cells were prepared from P17 rats. Following deep anesthesia with isoflurane, the rats were decapitated and the brainstem was immediately isolated. Slice preparations were obtained using a microslicer in ice-cold (4 °C) carbogenated (95% O₂ and 5% CO₂) sucrose Ringer solution. After sectioning, the slices were incubated in sucrose Ringer at 34 °C, followed by incubation at room temperature (27 °C) in normal glucose Ringer solution. We then punched out the MesV cells from the slices and acutely dissociated and isolated them using papain.
Results: Immunofluorescence analysis revealed the expression of peripherin and POU domain class 4 transcription factor 1 (Brn3a), suggesting that isolated MesV cells to be peripheral sensory neurons. MesV cells also expressed the Piezo1 channels, indicating their involvement in mechanosensitive processes. The [Ca²⁺]_i in MesV neurons increased transiently in extracellular high K⁺ (50 mM) solution, whtch induced depolarization in isolated CTB-positive MesV cells. This showed that the obtained cells had neurological properties. Mechanical stimuli induced [Ca²⁺]_i increases in CTB-positive MesV neurons.
Conclusions: These results suggest that CTB-positive MesV neurons have mechanosensitivity that may be involved in proprioceptive function in the periodontal ligament.