Transcriptional changes in microglial Kcnk2 during fear memory reconsolidation and TREK-1-associated regulation of TNF-α invitro.

Fear memory is associated with microglial tumor necrosis factor-alpha (TNF-α) production; however, the potassium (K+) channel-related mechanisms underlying microglial TNF-α regulation remain incompletely understood. Using in silico reanalysis of a whole-brain microglial microarray dataset obtained after contextual fear conditioning, we identified 20 K+ channel-related transcripts altered during fear memory reconsolidation, including Tnf. Promoter motif enrichment analysis revealed a shared GC-rich regulatory signature among these genes. During reconsolidation, Tnf and Kcnk2 (encoding the two-pore domain K+ channel TREK-1) transcripts were significantly increased in microglia isolated from the prefrontal cortex and hippocampus, accompanied by reduced microglial K+ levels. Furthermore, hippocampal microglial Tnf expression was positively correlated with Kcnk2 expression during reconsolidation. To investigate the potential association between TREK-1-related signaling and microglial TNF-α responses, in vitro mechanistic analyses were performed. TREK-1 knockdown significantly reduced TNF-α production in primary CD11b+ microglia. Pharmacological inhibition of TREK-1 with Spadin decreased basal and lipopolysaccharide-induced TNF-α production, whereas activation with LPS2336 increased TNF-α levels in BV-2 microglial cells. Reduction of extracellular K+ concentrations enhanced TNF-α production in BV-2 cells, an effect attenuated by TREK-1 inhibition. In contrast, comparable TREK-1-associated regulation was not observed in J774.1 macrophages, suggesting a microglia-specific mechanism under the present in vitro conditions. Collectively, these findings identify coordinated alterations in microglial Kcnk2, Tnf, and K+ levels during fear memory reconsolidation and demonstrate TREK-1-associated regulation of TNF-α production in microglia in vitro.