K⁺-Cl^- cotransporter KCC2 is involved in long-term potentiation at trigeminal excitatory synapses

The K⁺-Cl^- cotransporter (KCC)2 is critical for maintaining low intracellular chloride levels and ensuring hyperpolarizing GABAergic inhibition. While KCC2 dysfunction is well established in spinal mechanisms of chronic pain, its role in trigeminal synaptic plasticity remains unclear. This study investigated whether KCC2 activity is required for high-frequency stimulation (HFS)-induced long-term potentiation (LTP) and group I metabotropic glutamate receptor (mGluR)-mediated plasticity at excitatory synapses in ascending trigeminal pathways from the spinal trigeminal subnucleus caudalis (Vc) to the interpolaris (Vi) and oralis (Vo) regions. Using whole-cell voltage-clamp recordings in rat brainstem slices, HFS of the deep layer of Vc reliably induced robust LTP in Vi neurons under control conditions. However, bath application of furosemide, an inhibitor for non‑selective cation-chloride cotransporters including KCC2, significantly reduced the magnitude of HFS-induced LTP; LTP was maintained in five out of nine neurons, whereas four of nine neurons exhibited long-term depression (LTD). Furthermore, plasticity induced by 3,5-dihydroxyphenylglycine (DHPG), a group I mGluR agonist, showed a polarity reversal during KCC2 blockade in Vo neurons. Under control conditions, DHPG induced LTP in six out of ten neurons, whereas KCC2 blockade with VU0240551 predominantly induced DHPG-LTD in six of eleven neurons. In addition, HFS-induced suppression of GABAergic inhibitory responses in Vi neurons was significantly enhanced in the presence of furosemide, resulting in an increased magnitude of LTD. These findings indicate that acute KCC2 blockade alters both the magnitude and direction of synaptic plasticity in excitatory and inhibitory transmission within the spinal trigeminal nucleus.