Chronic pain is a common neurological disease involving lasting, multifaceted maladaptations from gene modulations to synaptic malfunctions and to emotional disorders. Sustained pathological stimuli in many diseases alter output activities of certain genes through epigenetic modifications, but it is unclear how epigenetic mechanisms operate in the development of chronic pain. We demonstrate here that, in the rat brainstem nucleus raphe magnus, which is important for central mechanisms of chronic pain, persistent inflammatory and neuropathic pain epigenetically suppresses gad65 activities through histone deacetylase (HDAC)-mediated histone hypoacetylation, resulting in impaired GABA synaptic inhibition. gad65 knockout mice display similar sensitized pain behavior and impaired GABA synaptic function in the brainstem neurons. HDAC inhibitors overwhelmingly increase gad65 activities, restore GABA synaptic function and relieve the sensitized pain behavior, but not in gad65 knockout mice. These findings suggest GAD65 and HDACs as potential therapeutic targets in an epigenetic approach to the treatment of chronic pain.

The analgesic effect of clinically used exogenous opioids, such as morphine, is mediated primarily through mu-opioid receptors, but the function of the kappa-receptor in opioid analgesia is unclear. Although kappa-receptor agonists can produce analgesia, behavioural studies indicate that kappa agonists applied intravenously or locally into the spinal cord antagonize morphine analgesia. As morphine, a primary mu agonist, also binds to kappa-receptors and the analgesic effectiveness of morphine decreases with repeated use (tolerance), it is important to understand the mechanism for the functional interaction between kappa- and mu-opioid receptors in the central nervous system. Here we present in vitro electrophysiological and in vivo behavioural evidence that activation of the kappa-receptor specifically antagonizes mu-receptor-mediated analgesia. We show that in slice preparations of a rat brainstem nucleus, which is critical for the action of opioids in controlling pain, functional kappa- and mu-receptors are each localized on physiologically different types of neuron. Activation of the kappa-receptor hyperpolarizes neurons that are activated indirectly by the mu-receptor. In rats, kappa-receptor activation in this brainstem nucleus significantly attenuates local mu-receptor-mediated analgesia. Our findings suggest a new cellular mechanism for the potentially ubiquitous opposing interaction between mu- and kappa-opioid receptors and may help in the design of treatments for pain.

SUMMARY1. Intracellular recordings were made from neurons of the nucleus raphe magnus (NRM) from rat (n = 128) and guinea-pig (n = 115). Two types of cells were found in each, primary (103 in rat, 27 in guinea-pig) and secondary cells (25 in rat, 88 in guinea-pig).2. Primary cells had input resistances of 186 + 9 MQ (n = 9) in rat and 255 + 50 MQ (n = 11) in guinea-pig. The action potential in each was about 1-5 ms in duration. Synaptic potentials were evoked by focal electrical stimulation and consisted of both y-aminobutyric acid (GABA) and excitatory amino acid components.3. Morphine, [Met5]enkephalin (ME) and [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) depressed the amplitude of the GABA-mediated synaptic potential by a maximum of 50 65 % and had little effect on the excitatory amino acid-mediated synaptic potential. There was no effect of these opioids on the resting membrane potential or input resistance of primary cells in rat or guinea-pig.4. Secondary cells had short duration action potentials (<1Ims) and an input resistance of 354 + 47 MQ in rat (n = 6) and 290 + 40 MQ in guinea-pig (n = 15). The synaptic potential observed in the cells of this group was mediated by activation of only excitatory amino acid receptors.5. ME hyperpolarized and/or abolished the spontaneous firing in sixteen out of twenty-four neurons in the secondary group from rat and eighty out of eighty-four neurons from guinea-pig. ME induced an outward current at -60 mV that reversed polarity at potentials more negative than -92 + 3 mV in rat (n = 6) and -98 + 2 mV in guinea-pig (n = 18). The reversal potential of the opioid current was shifted to less negative potentials when the external potassium concentration was increased, as predicted by the Nernst equation.

Intracellular recordings were made from neurons in rat dorsal raphe in the slice preparation maintained at 37 degrees C. The single-electrode voltage-clamp method was used to measure membrane currents at potentials more negative than rest (-60 mV). Three types of inward rectification were observed: 2 in the absence of any drugs and the third induced by 5-HT 1 and GABA-B receptor agonists. In the absence of any drugs, an inward current activated over 1–2 sec when the membrane potential was stepped to potentials more negative than -70 mV. This current was blocked by cesium (2 mM) and resembles IQ or IH. A second inward current (IIR) occurred at membrane potentials near the potassium equilibrium potential (EK). This inward current activated within the settling time of the clamp and was abolished by both barium (10–100 microM) and cesium (2 mM). 5-HT 1 agonists activated a potassium conductance that hyperpolarized the cells at rest. This potassium conductance was about 2 nS at -60 mV and increased linearly with membrane hyperpolarization to about 4 nS at -120 mV. Baclofen activated a potassium conductance identical in amplitude and voltage dependence to that induced by 5-HT 1 agonists. Both the baclofen- and 5-HT-induced currents were nearly abolished in animals pretreated with pertussis toxin. The results indicate that a common potassium conductance is increased by 5-HT acting on 5-HT 1 receptors and baclofen acting on GABA-B receptors. This potassium conductance rectifies inwardly and is distinct from the Q-current. The ligand-activated potassium conductance also differs from the other form of inward rectification (IIR) in its voltage dependence and sensitivity to pertussis toxin.