Christopher W. Vaughan

How opioids inhibit GABA-mediated neurotransmission

The midbrain region periaqueductal grey (PAG) is rich in opioid receptors and endogenous opioids and is a major target of analgesic action in the central nervous system. It has been proposed that the analgesic effect of opioids on the PAG works by suppressing the inhibitory influence of the neurotransmitter GABA (γ-aminobutyric acid) on neurons that form part of a descending antinociceptive pathway. Opioids inhibit GABA-mediated (GABAergic) synaptic transmission in the PAG and other brain regions by reducing the probability of presynaptic neurotransmitter release,, but the mechanisms involved remain uncertain. Here we report that opioid inhibition of GABAergic synaptic currents in the PAG is controlled by a presynaptic voltage-dependent potassium conductance. Opioid receptors of the μ type in GABAergic presynaptic terminals are specifically coupled to this potassium conductance by a pathway involving phospholipase A2, arachidonic acid and 12-lipoxygenase. Furthermore, opioid inhibition of GABAergic synaptic transmission is potentiated by inhibitors of the enzymes cyclooxygenase and 5-lipoxygenase, presumably because more arachidonic acid is available for conversion to 12-lipoxygenase products. These mechanisms account for the analgesic action of cyclooxygenase inhibitors in the PAG and their synergism with opioids.

Actions of the FAAH inhibitor URB597 in neuropathic and inflammatory chronic pain models

While cannabinoid receptor agonists have analgesic activity in chronic pain states, they produce a spectrum of central CB1 receptor‐mediated motor and psychotropic side effects. The actions of endocannabinoids, such as anandamide are terminated by removal from the extracellular space, then subsequent enzymatic degradation by fatty‐acid amide hydrolase (FAAH). In the present study, we compared the effect of a selective FAAH inhibitor, URB597, to that of a pan‐cannabinoid receptor agonist HU210 in rat models of chronic inflammatory and neuropathic pain. Systemic administration of URB597 (0.3 mg kg−1) and HU210 (0.03 mg kg−1) both reduced the mechanical allodynia and thermal hyperalgesia in the CFA model of inflammatory pain. In contrast, HU210, but not URB597, reduced mechanical allodynia in the partial sciatic nerve‐ligation model of neuropathic pain. HU210, but not URB597, produced a reduction in motor performance in unoperated rats. The effects of URB597 in the CFA model were dose dependent and were reduced by coadministration with the cannabinoid CB1 antagonist AM251 (1 mg kg−1), or the CB2 and SR144528 (1 mg kg−1). Coadministration with AM251 plus SR144528 completely reversed the effects of URB597. These findings suggest that the FAAH inhibitor URB597 produces cannabinoid CB1 and CB2 receptor‐mediated analgesia in inflammatory pain states, without causing the undesirable side effects associated with cannabinoid receptor activation.

Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurones.

The actions of the endogenous ORL1‐receptor (opioid receptor‐like1) ligand, nociceptin, on the membrane properties of rat dorsal raphe nucleus neurones were examined by use of whole‐cell patch clamp recording in brain slices. Nociceptin produced an outward current in all neurones tested, with an EC50 of 12 ± 2 nM. Dynorphin A (100 nM to 1 μm) produced little outward current. Outward currents reversed polarity near the predicted K+ equilibrium potential in both 2.5 mM (measured/predicted = −105 mV/ −104 mV) and 6.5 mM (measured/predicted = −80 mV/ −77 mV) extracellular K+. The conductance increase was larger between −120 and −130 mV than between −70 and −80 mV, demonstrating that the nociceptin‐induced K current was due to an increased inwardly rectifying K conductance. The outward current produced by nociceptin was similar to, and occluded by, high concentrations of baclofen, demonstrating actions on the same population of K channels. Naloxone (1 μm) failed to inhibit outward currents produced by nociceptin. These results are consistent with the reported high density of ORL1 receptor mRNA in dorsal raphe nucleus and with inhibitory actions of nociceptin in cells expressing ORL1.

Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro

1 In this study we have examined the effects of nociceptin, an endogenous ligand for the opioid‐like receptor ORL1, on the membrane properties of rat locus coeruleus (LC) neurones in vitro, using intracellular and whole cell patch clamp recording. 2 When locus coeruleus neurones were voltage clamped to −60 mV, application of nociceptin caused an outward current in all cells examined (n = 49), with an EC50 of 90 nM. Neither the potency nor the maximal effect of nociceptin was altered in the presence of the peptidase inhibitors, bestatin (20 μm) or thiorphan (2 μm). 3 The outward currents caused by nociceptin in 2.5 mM extracellular K+ reversed polarity at −123 mV, more negative than the predicted K+ reversal potential of −105 mV. Increasing extracellular K+ to 6.5 mM resulted in a shift of the reversal potential of +25 mV, a shift consistent with a K+ conductance. The conductance activated by nociceptin showed mild inward rectification. 4 Application of a high concentration of nociceptin (3 μm) occluded the current produced by simultaneous application of high concentrations of Met‐enkephalin (10 μm), (3 μm) somatostatin and UK 14304 (3 μm), indicating that nociceptin activated the same conductance as μ‐opioid and somatostatin receptors and α2‐adrenoceptors. 5 The actions of nociceptin were weakly antagonized by the opioid antagonist, naloxone, with pKb′s estimated from 2 cells of −4.23 and −4.33. The μ‐opioid antagonist, CTAP (D‐Phe‐Cys‐Tyr‐D‐Trp‐Arg‐Pen‐Thr‐NH2, 1 μm), the opioid antagonist, nalorphine (30 μm), or the somatostatin antagonist, CPP (cyclo(7‐aminoheptanoyl‐Phe‐D‐Tip‐Lys‐Thr[Bzl]) 3 μm) did not affect the nociceptin‐induced current. 6 Dynorphin A (3 μm), another putative endogenous ligand for ORL1, caused a robust outward current in locus coeruleus neurones that was, however, completely antagonized by moderate concentrations of naloxone (300 nM‐1 μm). 7 Continuous application of nociceptin (3 μm) resulted in a decrease of the outward current to a steady level of 70% of the maximum response with a t1/2 of 120s. Desensitization was largely homologous because simultaneous application of Met‐enkephalin (30 μm) during the desensitized period of the nociceptin response resulted in an outward current that was 92% of control responses to Met‐enkephalin in the same cells. Conversely, continuous application of Met‐enkephalin (30 μm) resulted in a decrease of Met‐enkephalin current to a steady level that was 54% of the initial current. During this desensitized period application of nociceptin (3 μm) resulted in a current that was 78% of the control responses to nociceptin in the same cells. 8 Thus nociceptin potently activates an inwardly rectifying K+ conductance in locus coeruleus neurones, with a pharmacological profile consistent with activation of the ORL1 receptor. Dynorphin A does not appear to be a ligand for ORL1 in rat locus coeruleus neurones.

Presynaptic inhibitory action of opioids on synaptic transmission in the rat periaqueductal grey in vitro.

1. The actions of opioids on synaptic transmission in rat periaqueductal grey (PAG) neurones were examined using whole‐cell patch‐clamp recordings in brain slices. 2. Methionine enkephalin (ME; 10 microM) inhibited evoked GABAergic inhibitory postsynaptic currents (IPSCs) by 57%, non‐NMDA excitatory postsynaptic currents (EPSCs) by 60%, and NMDA EPSCs by 43% in PAG neurones. This inhibition was associated with an increase in paired‐pulse facilitation, was mimicked by the mu‐agonist DAMGO (1‐3 microM) and abolished by naloxone (1 microM). Neither the kappa‐agonist U69593 (1‐3 microM), nor the delta‐agonist DPDPE (3‐10 microM) had any specific actions on evoked PSCs. 3. ME decreased the frequency of spontaneous miniature, action potential‐independent postsynaptic currents (mIPSCs by 65%, mEPSCs by 54%) in all PAG neurones, but had no effect on their amplitude distributions. The reduction in mIPSC frequency persisted in nominally Ca(2+)‐free, high‐Mg2+ (10 mM) solutions, which also contained Cd2+ (100 microM), or Ba2+ (10 mM). Opioid inhibition of mIPSC frequency is unlikely to be mediated by presynaptic Ca2+ or K+ conductances which are sensitive to extracellular Cd2+ or Ba2+. 4. In a subpopulation of PAG neurones, ME increased a Ba(2+)‐sensitive K+ conductance at potentials below ‐97 mV. Opioids inhibited both GABAergic and glutamatergic synaptic transmission in all PAG neurones, independent of any postsynaptic opioid sensitivity. 5. These observations are consistent with, but only partially support, the opioid disinhibition model of PAG‐induced analgesia. mu‐Opioids also have the potential to modulate the behavioural and autonomic functions of the PAG via modulation of both inhibitory and excitatory presynaptic mechanisms, as well as postsynaptic mechanisms.

μO-conotoxin MrVIB selectively blocks Nav1.8 sensory neuron specific sodium channels and chronic pain behavior without motor deficits

The tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Nav1.8 is expressed predominantly by damage-sensing primary afferent nerves and is important for the development and maintenance of persistent pain states. Here we demonstrate that μO-conotoxin MrVIB from Conus marmoreus displays substantial selectivity for Nav1.8 and inhibits pain behavior in models of persistent pain. In rat sensory neurons, submicromolar concentrations of MrVIB blocked tetrodotoxin-resistant current characteristic of Nav1.8 but not Nav1.9 or tetrodotoxin-sensitive VGSC currents. MrVIB blocked human Nav1.8 expressed in Xenopus oocytes with selectivity at least 10-fold greater than other VGSCs. In neuropathic and chronic inflammatory pain models, allodynia and hyperalgesia were both reduced by intrathecal infusion of MrVIB (0.03–3 nmol), whereas motor side effects occurred only at 30-fold higher doses. In contrast, the nonselective VGSC blocker lignocaine displayed no selectivity for allodynia and hyperalgesia versus motor side effects. The actions of MrVIB reveal that VGSC antagonists displaying selectivity toward Nav1.8 can alleviate chronic pain behavior with a greater therapeutic index than nonselective antagonists.

Capsaicin activation of glutamatergic synaptic transmission in the rat locus coeruleus in vitro

The vanilloid receptor protein (VR1) is a well‐characterised integrator of noxious stimuli in peripheral sensory neurones. There is evidence for the presence of VR1 in the central nervous system, but little information as to its role there. In this study we have examined the actions of agonists for VR1 receptors in the rat locus coeruleus (LC), using whole‐cell patch‐clamp recordings from acutely isolated neurones and neurones in slices. Superfusion with capsaicin resulted in a concentration‐dependent increase in the frequency of isolated miniature excitatory postsynaptic currents (mEPSCs) in LC neurones. The mean amplitude of the mEPSCs was not affected by capsaicin. The effects of capsaicin (1 μM) were abolished by the VR1 receptor antagonists capsazepine (10 μM) and iodoresiniferatoxin (300 nm). Removal of extracellular Ca2+ abolished the capsaicin‐induced increase in frequency of mEPSCs. Capsaicin superfusion had no consistent effects on evoked excitatory postsynaptic currents. Capsaicin superfusion also resulted in the release of an adrenoceptor agonist in the LC but did not affect the membrane currents of acutely isolated LC neurones. These data demonstrate that the VR1 receptor appears to be located presynaptically on afferents to the LC, and that activation of VR1 may serve to potentiate the release of glutamate and adrenaline/noradrenaline in this brain region.

Cannabinoid receptor activation inhibits GABAergic neurotransmission in rostral ventromedial medulla neurons in vitro

The rostral ventromedial medulla (RVM) is thought to play a crucial role in the antinociceptive actions of cannabinoids. This study examined the actions of the cannabinoid receptor agonist, WIN55,212‐2, on membrane properties and GABAergic synaptic transmission in RVM neurons using whole cell patch clamp recordings in brain slices. WIN55,212‐2 (3 μM) had no effect on membrane K+ conductance of primary or secondary RVM neurons. Primary neurons responded to the κ‐opioid receptor agonist U69,593 (300 nM–1 μM). Secondary neurons responded to the μ,δ‐opioid receptor agonist met‐enkephalin (10 μM). WIN55,212‐2 reduced the amplitude of electrically evoked (GABAergic) inhibitory postsynaptic currents (IPSCs) in all neurons (58%, pEC50=6.2±0.1). The inhibition was reversed by the CB1 receptor selective antagonist, SR141716 (3 μM). WIN55,212‐2 also produced relative facilitation of the second IPSC to paired evoked IPSCs. WIN55,212‐2 and met‐enkephalin reduced the rate of spontaneous miniature IPSCs in all cells (44 and 53%), but had no effect on their amplitude distributions or kinetics. These results suggest that the antinociceptive actions of cannabinoids within RVM are primarily due to presynaptic inhibition of GABAergic neurotransmission. The neuronal substrates of cannabinoid actions in RVM therefore differ from those of opioids, which have both pre‐ and postsynaptic inhibitory actions.

Opioid inhibition of rat periaqueductal grey neurones with identified projections to rostral ventromedial medulla in vitro.

1. Rat caudal periaqueductal grey (PAG) output neurones containing rhodamine microspheres, retrogradely transported from an injection site in the rostral ventromedial medulla (RVM), were visualized in brain slices and recorded from using whole‐cell patch clamp techniques. 2. The specific GABAB receptor agonist baclofen (10 microM) produced an outward current or hyperpolarization in fifty out of fifty‐six caudal PAG output neurones. In 44% of these baclofen‐sensitive neurones, the opioid agonist methionine enkephalin (30 microM) also produced an outward current or hyperpolarization. The opioid current reversed polarity at ‐104 mV and could also be produced by DAMGO, an agonist selective for the mu‐subtype of opioid receptor. 3. Opioid‐responding output neurones were not distributed uniformly in the caudal PAG. In horizontal slices containing lateral PAG, 56% of output neurones were inhibited by opioids, as compared with only 14% of the output neurones in slices containing ventrolateral PAG. 4. These observations are consistent with opioid disinhibition of ventrolateral PAG neurones projecting to the RVM as the predominant mechanism underlying opioid‐induced analgesia in the PAG. The role of opioid receptors found on a major proportion of the output neurones in the lateral PAG remains to be established, but is assumed not be related to modulation of nociceptive function.

Cellular Actions Of Opioids And Other Analgesics: Implications For Synergism In Pain Relief

1. μ‐Opioid receptor agonists mediate their central analgesic effects by actions on neurons within brain regions such as the mid‐brain periaqueductal grey (PAG). Within the PAG, μ‐opioid receptor‐mediated analgesia results from inhibition of GABAergic influences on output projection neurons. We have established that μ‐opioid receptor activation in the PAG causes a presynaptic inhibition of GABA release that is mediated by activation of a voltage‐dependent K+ channel via 12‐lipoxygenase (LOX) metabolites of arachidonic acid.