The mechanisms underlying chronic stress-induced dysfunction of glutamatergic transmission that contribute to helplessness-associated depressive disorder are unknown. We investigated the relationship of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and stress, and the neuroplastic changes of stress-induced depression-like behavior in the ventrolateral periaqueductal gray (vlPAG). We conducted whole-cell patch-clamp electrophysiological recordings in the vlPAG neurons. Depression-like behavior was assayed using tail suspension test and sucrose preference test. Surface and cytosolic glutamate receptor 1 (GluR1) AMPA receptor expression was analyzed using western blotting. Phosphorylated GluR1 expression was quantified using western blotting and immunohistochemical analysis. Unpredictable inescapable foot shock stress caused reduction in glutamatergic transmission originating from both presynaptic and postsynaptic loci in the vlPAG that was associated with behavioral despair and anhedonia in chronic stress-induced depression. Pharmacological inhibition of GluR1 function in the vlPAG caused depression-like behavior. Diminished glutamatergic transmission was due to reduced glutamate release presynaptically and enhanced GluR1-endocytosis from the cell surface postsynaptically. Chronic stress-induced neuroplastic changes and maladaptive behavior were reversed and mimicked by administration of glucocorticoid receptor (GR) antagonist and agonist, respectively. However, chronic stress did not affect γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission in the vlPAG. These results demonstrate that depression-like behavior is associated with remarkable reduction in glutamatergic, but not GABAergic, transmission in the vlPAG. These neuroplastic changes and maladaptive behavior are attributed to GR-dependent mechanisms. As reduced GluR1-associated responses in the vlPAG contribute to chronic stress-induced neuroplastic changes, this cellular mechanism may be a critical component in the pathogenesis of stress-associated neuropsychiatric disorders.

Ten-eleven translocation methylcytosine dioxygenase 1 (Tet1) mediates the conversion of 5-methylcytosine (5 mC) to 5-hydroxymethylcytosine (5 hmC), hence promoting DNA demethylation. Although recent studies have linked the DNA demethylation of specific genes to pain hypersensitivity, the role of spinal Tet1-dependent DNA demethylation in nociception hypersensitivity development remains elusive. Here, we report correlated with behavioral allodynia, spinal nerve ligation (SNL) upregulated Tet1 expression in dorsal horn neurons that hydroxylate 5 mC to 5 hmC at CpG dinucleotides in the bdnf promoter to promote spinal BDNF expression at day 7 after operation. Focal knockdown of spinal Tet1 expression decreased Tet1 binding and 5 hmC enrichment, further increased 5 mC enrichment at CpG sites in the bdnf promoter and decreased spinal BDNF expression accompanied by the alleviation of the developed allodynia. Moreover, at day 7 after operation, SNL-enhanced Tet1 expression also inhibited the binding of DNA methyltransferases (DNMTs, i.e., DNMT1, DNMT3a, and DNMT3b) to the bdnf promoter, a requirement for transcriptional silencing by catalysing 5-cytosine (5C) to 5 mC. Together, these data suggest at CpG sites of the bdnf promoter, SNL-enhanced Tet1 expression promotes DNA demethylation both by converting 5 mC to 5 hmC and inhibiting DNMT binding to regulate spinal BDNF expression, hence contributing to behavioral allodynia development.

Retromer, which crucially contributes to endosomal sorting machinery through the retrieval and recycling of signaling receptors away from degradation, has been identified as a critical element for glutamatergic-receptor-dependent neural plasticity at excitatory synapses. We observed it accompanied by behavioral allodynia; neuropathic injury time-dependently enhanced VPS26A and SNX27 expression; VPS26A–SNX27 coprecipitation; and VPS26A-positive, SNX27-positive, and VPS26A–SNX27 double-labeled immunoreactivity in the dorsal horn of Sprague Dawley rats that were all sufficiently ameliorated through the focal knock-down of spinal VPS26A expression. Although the knock-down of spinal SNX27 expression exhibited similar effects, spinal nerve ligation (SNL)-enhanced VPS26A expression remained unaffected. Moreover, SNL also increased membrane-bound and total mGluR5 abundance, VPS26A-bound SNX27 and mGluR5 and mGluR5-bound VPS26A and SNX27 coprecipitation, and mGluR5-positive and VPS26A/SNX27/mGluR5 triple-labeled immunoreactivity in the dorsal horn, and these effects were all attenuated through the focal knock-down of spinal VPS26A and SNX27 expression. Although administration with MPEP adequately ameliorated SNL-associated allodynia, mGluR5 expression, and membrane insertion, SNL-enhanced VPS26A and SNX27 expression were unaffected. Together, these results suggested a role of spinal VPS26A–SNX27-dependent mGluR5 recycling in the development of neuropathic pain. This is the first study that links retromer-associated sorting machinery with the spinal plasticity underlying pain hypersensitivity and proposes the possible pathophysiological relevance of endocytic recycling in pain pathophysiology through the modification of glutamatergic mGluR5 recycling.SIGNIFICANCE STATEMENTVPS26A–SNX27-dependent mGluR5 recycling plays a role in the development of neuropathic pain. The regulation of the VPS26A–SNX27 interaction that modifies mGluR5 trafficking and expression in the dorsal horn provides a novel therapeutic strategy for pain relief.

In a rat model of neuropathic pain, histone deacetylases 4 phosphorylation led to its cytoplasmic retention due to phosphorylation-dependent interaction with 14-3-3β. Inhibition of histone deacetylases phosphorylation reduced allodynia and prevented its cytoplasmic translocation, suggesting a novel therapeutic target for neuropathic pain. Background: The histone deacetylases (HDACs) have been implicated in pain hypersensitivity. This study investigated the potential involvement of an HDAC4-related mechanism in the spinal nerve ligation (SNL)-induced nociceptive hypersensitivity. Methods: The left L5 to L6 spinal nerves of 627 adult male Sprague–Dawley rats were surgically ligated. The withdrawal threshold of hind paws and the abundances, cellular location, and interactions of proteins in the dorsal horn were assayed before and after surgery. The 14-3-3β-targeting small-interfering RNA, a serum- and glucocorticoid-inducible kinase 1 (SGK1) antagonist, or an HDAC inhibitor was spinally injected to elucidate the role of 14-3-3β, SGK1, and HDAC4. Results: Without affecting the HDAC4 level, SNL provoked SGK1 phosphorylation (mean ± SEM from 0.24 ± 0.02 to 0.78 ± 0.06 at day 7, n = 6), HDAC4 phosphorylation (from 0.38 ± 0.03 to 0.72 ± 0.06 at day 7, n = 6), 14-3-3β expression (from 0.53 ± 0.09 to 0.88 ± 0.09 at day 7, n = 6), cytoplasmic HDAC4 retention (from 1.18 ± 0.16 to 1.92 ± 0.11 at day 7, n = 6), and HDAC4-14-3-3β coupling (approximately 2.4-fold) in the ipsilateral dorsal horn in association with behavioral allodynia. Knockdown of spinal 14-3-3β expression prevented the SNL-provoked HDAC4 retention (from 1.89 ± 0.15 to 1.32 ± 0.08 at day 7, n = 6), HDAC4-14-3-3β coupling (approximately 0.6-fold above SNL 7D), and behavioral allodynia (from 0.16 ± 0.3 to 6 ± 1.78 at day 7, n = 7), but not SGK1 (from 0.78 ± 0.06 to 0.71 ± 0.04 at day 7, n = 6) or HDAC4 (from 0.75 ± 0.15 to 0.68 ± 0.11 at day 7, n = 6) phosphorylation. Conclusion: Neuropathic pain maintenance involves the spinal SGK1 activation–dependent HDAC4 phosphorylation and its subsequent association with 14-3-3β that promotes cytoplasmic HDAC4 retention in dorsal horn neurons.