Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K+ channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. Here we show that nerve injury increased H3K9me2 occupancy at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect DNA methylation levels of these genes in DRGs. Nerve injury increased activity of G9a, histone deacetylases and EZH2, but only G9a inhibition consistently restored K+ channel expression. Selective G9a knockout in DRG neurons completely blocked K+ channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced K+ channel genes but also normalized 638 genes down- or up-regulated by nerve injury. Thus G9a plays a dominant role in transcriptional repression of K+ channels and in acute-to-chronic pain transition after nerve injury.

This study identifies by microautoradiography activated microglia/macrophages as the main cell type expressing the peripheral benzodiazepine binding site (PBBS) at sites of active CNS pathology. Quantitative measurements of PBBS expression in vivo obtained by PET and [(11)C](R)-PK11195 are shown to correspond to animal experimental and human post-mortem data on the distribution pattern of activated microglia in inflammatory brain disease. Film autoradiography with [(3)H](R)-PK11195, a specific ligand for the PBBS, showed minimal binding in normal control CNS, whereas maximal binding to mononuclear cells was found in multiple sclerosis plaques. However, there was also significantly increased [(3)H](R)-PK11195 binding on activated microglia outside the histopathologically defined borders of multiple sclerosis plaques and in areas, such as the cerebral central grey matter, that are not normally reported as sites of pathology in multiple sclerosis. A similar pattern of [(3)H](R)-PK11195 binding in areas containing activated microglia was seen in the CNS of animals with experimental allergic encephalomyelitis (EAE). In areas without identifiable focal pathology, immunocytochemical staining combined with high-resolution emulsion autoradiography demonstrated that the cellular source of [(3)H](R)-PK11195 binding is activated microglia, which frequently retains a ramified morphology. Furthermore, in vitro radioligand binding studies confirmed that microglial activation leads to a rise in the number of PBBS and not a change in binding affinity. Quantitative [(11)C](R)-PK11195 PET in multiple sclerosis patients demonstrated increased PBBS expression in areas of focal pathology identified by T(1)- and T(2)-weighted MRI and, importantly, also in normal-appearing anatomical structures, including cerebral central grey matter. The additional binding frequently delineated neuronal projection areas, such as the lateral geniculate bodies in patients with a history of optic neuritis. In summary, [(11)C](R)-PK11195 PET provides a cellular marker of disease activity in vivo in the human brain.

Inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive (damage-sensing) dorsal root ganglion (DRG) neurons innervating inflamed tissue. However, most of the evidence for this is derived from experiments using acute inflammatory states. Herein, we used several approaches to examine the impact of chronic or persistent inflammation on the excitability of nociceptive DRG neurons and on their expression of I(h) and the underlying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which regulate neuronal excitability. Using in vivo intracellular recordings of somatic action potentials from L4/L5 DRG neurons in normal rats and rats with hindlimb inflammation induced by complete Freund's adjuvant (CFA), we demonstrate increased excitability of C- but not Aδ-nociceptors, 5 to 7 days after CFA. This included an afterdischarge response to noxious pinch, which may contribute to inflammatory mechanohyperalgesia, and increased incidence of spontaneous activity (SA) and decreased electrical thresholds, which are likely to contribute to spontaneous pain and nociceptor sensitization, respectively. We also show, using voltage clamp in vivo, immunohistochemistry and behavioral assays that (1) the inflammation-induced nociceptor hyperexcitability is associated, in C- but not Aδ-nociceptors, with increases in the mean I(h) amplitude/density and in the proportion of I(h) expressing neurons, (2) increased proportion of small DRG neurons (mainly IB4-negative) expressing HCN2 but not HCN1 or HCN3 channel protein, (3) increased HCN2- immunoreactivity in the spinal dorsal horn, and (4) attenuation of inflammatory mechanoallodynia with the selective I(h) antagonist, ZD7288. Taken together, the findings suggest that C- but not Aδ-nociceptors sustain chronic inflammatory pain and that I(h)/HCN2 channels contribute to inflammation-induced C-nociceptor hyperexcitability.

Mapping the entire frequency bandwidth of brain electrophysiological signals is of paramount importance for understanding physiological and pathological states. The ability to record simultaneously DC-shifts, infraslow oscillations (<0.1Hz), typical LFP signals (0.1-80 Hz) and higher frequencies (80-600 Hz) using the same recording site would particularly benefit preclinical epilepsy research and could provide clinical biomarkers for improved seizure onset zone delineation. However, commonly used metal microelectrode technology suffers from instabilities that hamper the high-fidelity of DC-coupled recordings, which are needed to access signals of very low frequency. Here, we use flexible graphene depth neural probes (gDNP), consisting of a linear array of graphene microtransistors, to concurrently record DC-shifts and high frequency neuronal activity in awake rodents. We show that gDNPs can reliably record and map with high spatial resolution seizures, pre-ictal DC-shifts and seizure associated spreading depolarizations together with higher frequencies through the cortical laminae to the hippocampus in a mouse model of chemically-induced seizures. Moreover, we demonstrate functionality of chronically implanted devices over 10 weeks by recording with high fidelity spontaneous spike-wave discharges and associated infraslow oscillations in a rat model of absence epilepsy. Altogether, our work highlights the suitability of this technology for in vivo electrophysiology research, and in particular epilepsy research, by allowing stable and chronic DC-coupled recordings.

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Diabetic peripheral neuropathy (DPN) is the most incapacitating complication of diabetes mellitus. Up to 50% of patients with DPN develop peripheral neuropathic pain (PNP). The underlying ionic and molecular mechanisms of diabetic PNP (DPNP) are poorly understood. However, voltage gated potassium (K v 7) channels which have been implicated in the pathogenesis of other types of PNP are likely to be involved. Here we examined, in the streptozotocin (STZ) rat model of DPNP, whether activating the Kv7 channels with a potent activator retigabine (ezogabine) would reverse/attenuate behavioural signs of DPNP. STZ rats exhibited behavioural indices of mechanical and heat hypersensitivity, but not cold hypersensitivity or spontaneous pain, 35 days after STZ injection. Retigabine given at a dose of 15 mg/kg (but not at 7.5 mg/ kg, i.p.) significantly attenuated mechanical, but not heat hypersensitivity in DPNP rats, and was as effective as the positive control gabapentin. This analgesic effect of retigabine was completely reversed by the K v 7/M channel blocker XE991 (3 mg/kg, i.p.) indicating that the anti-allodynic effects of retigabine were mediated by K v 7 channels. In conclusion, the findings suggest that Kv7 channels are involved in DPNP pathogenesis, and that strategies that target their activation may prove to be effective in treating DPNP.

Peripheral neuropathic pain associated with partial nerve injury is believed to be driven partly by aberrant spontaneous activity (SA) in both injured and uninjured dorsal root ganglion (DRG) neurons. The underlying ionic mechanisms are not fully understood, but hyperpolarization-activated cyclic nucleotide-gated (HCN) channels which underlie the excitatory Ih current have been implicated in SA generation in axotomized A-fiber neurons after L5-spinal nerve ligation/axotomy (SNL/SNA). Here, using a modified model of SNA (mSNA) which involves, in addition to L5-SNA, loose ligation of the L4-spinal nerve with neuroinflammation-inducing chromic gut, we examined whether HCN channels also contribute to SA in the adjacent L4-neurons. Intracellular recordings from L4-DRG neurons in control rats, and L4-DRG neurons in mSNA rats were made using in vivo voltage- and current-clamp techniques. Compared with control, L4 C-nociceptors and Aβ-low-threshold mechanoreceptors (LTMs) exhibited SA 7 days after mSNA. This was accompanied, in C-nociceptors, by a significant increase in Ih amplitude, the percentage of Ih-expressing neurons, and Ih activation rate. Hyperpolarization-activated cyclic nucleotide-gated channel blockade with ZD7288 (10 mg/kg, intravenously) suppressed SA in C-nociceptors, but not Aβ-LTMs, and caused in C-nociceptors, membrane hyperpolarization and a decrease in Ih activation rate. Furthermore, intraplantar injection of ZD7288 (100 μM) was found to be as effective as gabapentin (positive control) in attenuating cold hypersensitivity in mSNA rats. These findings suggest that HCN channels contribute to nerve injury-induced SA in L4 C-nociceptors, but not Aβ-LTMs, and that ZD7288 exerts its analgesic effects by altering Ih activation properties and/or causing membrane hyperpolarization in L4 C-nociceptors.

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