Knowing that expression of metabotropic glutamate 2 (mGlu2) receptors in the dorsal root ganglia is regulated by acetylation mechanisms, we examined the effect of two selective and chemically unrelated histone deacetylase (HDAC) inhibitors, N- (2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide (MS-275) and suberoylanilide hydroamic acid (SAHA), in a mouse model of persistent inflammatory pain. Although a single subcutaneous injection of MS-275 (3 mg/kg) or SAHA (5-50 mg/kg) was ineffective, a 5-day treatment with either of the two HDAC inhibitors substantially reduced the nociceptive response in the second phase of the formalin test, which reflects the development of central sensitization in the dorsal horn of the spinal cord. Analgesia was abrogated by a single injection of the mGlu2/3 receptor antagonist (␣S)-␣-amino-␣-[(1S,2S)-2-carboxycyclopropyl]-9H-xantine-9-propanoic acid (LY341495; 1 mg/kg, i.p.), which was inactive per se. Both MS-275 and SAHA upregulated the expression of mGlu2 receptors in the dorsal root ganglion (DRG) and spinal cord under conditions in which they caused analgesia, without changing the expression of mGlu1a, mGlu4, or mGlu5 receptors. Induction of DRG mGlu2 receptors in response to SAHA was associated with increased acetylation of p65/RelA on lysine 310, a process that enhances the transcriptional activity of p65/RelA at nuclear factor-B-regulated genes. Transcription of the mGlu2 receptor gene is known to be activated by p65/RelA in DRG neurons. We conclude that HDAC inhibition produces analgesia by up-regulating mGlu2 receptor expression in the DRG, an effect that results from the amplification of NF-B transcriptional activity. These data provide the first evidence that HDAC inhibitors cause analgesia and suggest that HDACs are potential targets for the epigenetic treatment of pain.Histone deacetylase (HDAC) inhibitors are known to modulate gene expression by increasing acetylation of histone proteins, thus remodeling chromatin structure (Strahl and Allis, 2000). Eighteen HDAC isoenzymes have been identified and divided into four classes based on their homology to yeast deacetylase proteins. Class I HDACs include the HDAC1, -2, -3, and -8 isoforms. These enzymes are ubiquitously expressed and have a predominant nuclear localization. Class II HDACs, which include HDAC4, -5, -6, -7, -9, and -10, are cytosolic enzymes and have a more restricted tissue pattern of expression. Class III HDACs include sirtuins, whereas HDAC11 is the only member of class IV HDAC and shares properties of both class I and II HDACs (Xu et al., 2007;Yang and Seto, 2007).In addition to histones, a number of nonhistone proteins, mainly transcription factors, are regulated by acetylation and are specifically targeted by HDACs (Spange et al., 2009). In particular, members of the nuclear factor-B (NF-B) family of transcription factors are known to be regulated by reversible acetylation (Chen et al., 2001). The NF-B/Rel family consists of p50, p52, p65/RelA, c-Rel, and RelB, which Arti...

L-acetylcarnitine (LAC), a drug utilized for the treatment of neuropathic pain in humans, has been shown to induce analgesia in rodents by up-regulating the expression of metabotropic glutamate receptor 2 (mGlu2) in dorsal root ganglia (DRG). We now report that LAC-induced upregulation of mGlu2 expression in DRG cultures involves transcriptional activation mediated by nuclear factor-kappaB (NF-κB). A single application of LAC (250 μM) to DRG cultures induced a transient increase in mGlu2 mRNA, which was observable after 1 hour and was no longer detectable after 1 to 4 days. In contrast, LAC treatment had no effect on mGlu3 mRNA expression. Pharmacological inhibition of NF-κB binding to DNA by caffeic acid phenethyl ester (CAPE) (2.5 μg/ml for 30 minutes) reduced the constitutive expression of mGlu2 and mGlu3 mRNA after 1–4 days and reduced the constitutive expression of mGlu2/3 protein at 4 days. This evidence combined with the expression of p65/RelA and c-Rel in DRG neurons indicated that expression of mGlu2 and mGlu3 is endogenously regulated by the NF-κB family of transcription factors. Consistent with this idea, the transient increase in mGlu2 mRNA induced by LAC after 1 hour was completely suppressed by CAPE. Furthermore, LAC induced an increase in the acetylation of p65/RelA, a process that enhances the transcriptional activity of p65/RelA. These results are consistent with the hypothesis that LAC selectively induces the expression of mGlu2 by acting as a donor of acetyl groups, thus enhancing the activity of the NF-κB family of transcription factors. Accordingly, we show that carnitine, which has no effect on pain thresholds, had no effect on p65/RelA acetylation and did not enhance mGlu2 expression. Taken together, these results demonstrate that expression of mGlu2 and mGlu3 mRNA is regulated by the NF-κB transcriptional machinery, and that agents that increase acetylation and activation of NF-κB transcription factors might induce analgesia via upregulation of mGlu2 in DRG neurons.

Activation of extracellular signal-regulated kinases (ERK) 1/2 in dorsal horn neurons is important for the development of somatic hypersensitivity and spinal central sensitization after peripheral inflammation. However, data regarding the roles of spinal ERK1/2 in the development of visceral hyperalgesia are sparse. Here we studied the activation of ERK1/2 in the lumbosacral spinal cord following innocuous and noxious distention of the inflamed (cyclophosphamide- treated) and non-inflamed urinary bladder in mice. We also correlated the spinal ERK1/2 activation to distention-evoked bladder nociception as quantified by the abdominal visceromotor response (VMR). Cyclophosphamide treatment (bladder inflammation) evoked increased bladder hyperalgesia and allodynia to bladder distention, as evident from an upward and leftward shift of the VMR stimulus-response curve compared to that of non-inflamed mice. Development of bladder hyperalgesia was associated with robust enhancement of ERK1/2 activation in the dorsal horn and deeper laminae bilaterally in the L6-S1 spinal cord. Functional blockade of spinal ERK1/2 activity via intrathecal administration of the upstream MEK inhibitor U0126 attenuated distention-evoked bladder nociception, and caused a significant downward shift of the VMR stimulus-response curve. In summary, we have provided functional and immunohistochemical evidence that activation of lumbosacral spinal ERK1/2 is associated with the development of primary visceral (bladder) hyperalgesia. Our results suggest that aberrant processing of visceral nociceptive information at the level of the lumbosacral spinal cord via activation of ERK1/2 signaling may contribute to chronic bladder pain in the context of inflammation.

Removal of a small segment of tail at weaning is a common method used to obtain tissue for the isolation of genomic DNA to identify genetically modified mice. When genetically manipulated mice are used for pain research, this practice could result in confounding changes to the animals' responses to noxious stimuli. In this study, we sought to systematically investigate whether tail biopsy representative of that used in standard genotyping methods affects behavioral responses to a battery of tests of nociception. Wild-type littermate C57BL/6J and 129S6 female and male mice received either tail biopsies or control procedural handling at Day 21 after birth and were then tested at 6–9 weeks for mechanical and thermal sensitivity. C57BL/6J mice were also tested in the formalin model of inflammatory pain. In all tests performed (von Frey, Hargreaves, modified Randall Selitto, and formalin), C57BL/6J tail-biopsied animals' behavioral responses were not significantly different from control animals. In 129S6 animals, tail biopsy did not have a significant effect on behavioral responses in either sex to the von Frey and the modified Randall-Selitto tests of mechanical sensitivity. Interestingly, however, both sexes exhibited small but significant differences between tail biopsied and control responses to a radiant heat stimulus. These results indicate that tail biopsy for genotyping purposes has no effect on nocifensive behavioral responses of C57BL/6J mice, and in 129S6 mice, causes only a minor alteration in response to a radiant heat stimulus while other nocifensive behavioral responses are unchanged. The small effect seen is modality- and strain-specific.

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