Search

Background and Purpose: 5-HT receptor agonists have variable nociceptive effects within the spinal cord. While there is some evidence for 5-HT(1A) spinally-mediated analgesia, the role of other 5-HT(1) receptor subtypes remains unclear. In the present study, we examined the spinal actions of a range of 5-HT(1) agonists, including sumatriptan, on acute pain, plus their effect on afferent-evoked synaptic transmission onto superficial dorsal horn neurons.Experimental Approach: For in vivo experiments, 5-HT agonists were injected via chronically implanted spinal catheters to examine their effects in acute mechanical and thermal pain assays using a paw pressure analgesymeter and a Hargreave's device. For in vitro experiments, whole-cell patch-clamp recordings of primary afferent-evoked glutamatergic EPSC were made from lamina II neurons in rat lumbar spinal slices.Key Results: Intrathecal (i.t.) delivery of the 5-HT(1A) agonist R ± 8-OH-DPAT (30-300 nmol) produced a dose-dependent thermal, but not mechanical, analgesia. Sumatriptan and the 5-HT(1B), 5-HT(1D), 5-HT(1F) agonists CP93129, PNU109291 and LY344864 (100 nmol) had no effect on either acute pain assay. R ± 8-OH-DPAT (1 µM) and sumatriptan (3 µM) both reduced the amplitude of the evoked EPSC. In contrast, CP93129, PNU109291 and LY344864 (0.3-3 µM) had no effect on the evoked EPSC. The actions of both R ± 8-OH-DPAT and sumatriptan were abolished by the 5-HT(1A) antagonist WAY100635 (3 µM).Conclusions and Implications: These findings indicate that the 5-HT(1A) receptor subtype predominantly mediates the acute antinociceptive and cellular actions of 5-HT(1) ligands within the rat superficial dorsal horn. [ABSTRACT FROM AUTHOR]

Background and Purpose: While arachidonyl ethanolamine (anandamide) produces pharmacological effects mediated by cannabinoid CB1 receptors, it is also an agonist at the transient receptor potential vanilloid type 1 (TRPV1) ion channel. This study examined the cellular actions of anandamide in the midbrain periaqueductal grey (PAG), a region implicated in the analgesic actions of cannabinoids, and which expresses both CB1 receptors and TRPV1.Experimental Approach: In vitro whole cell patch clamp recordings of glutamatergic excitatory postsynaptic currents (EPSCs) were made from rat and mouse PAG slices.Key Results: Capsaicin (1 µM) increased the rate, but not the amplitude of miniature EPSCs in subpopulations of neurons throughout the rat and mouse PAG. Capsaicin had no effect on miniature EPSCs in PAG neurons from TRPV1 knock-out mice. In mouse PAG neurons, anandamide (30 µM) had no effect on the rate of miniature EPSCs alone, or in the presence of either the CB1 antagonist AM251 (3 µM) or the TRPV1 antagonist iodoresiniferatoxin (300 nM). Anandamide produced a decrease in miniature EPSC rate in the presence of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 µM). By contrast, anandamide produced an increase in miniature EPSC rate in the presence of both URB597 and AM251, which was absent in TRPV1 knock-out mice.Conclusions and Implications: These results suggest that the actions of anandamide within PAG are limited by enzymatic degradation by FAAH. FAAH blockade unmasks both presynaptic inhibition and excitation of glutamatergic synaptic transmission which are mediated via CB1 receptors and TRPV1 respectively. [ABSTRACT FROM AUTHOR]

Background and Purpose: While triptans are used to treat migraine, there is evidence that they also reduce inflammation-induced pain at the spinal level. The cellular mechanisms underlying this spinal enhancement are unknown. We examined whether inflammation alters sumatriptan modulation of synaptic transmission in the rat spinal dorsal horn., Experimental Approach: Three to four days following intraplantar injection of complete Freund's adjuvant (CFA) or saline, whole cell recordings of evoked glutamatergic EPSCs were made from lumbar lamina I-II dorsal horn neurons in rat spinal slices KEY RESULTS: In 2- to 3-week-old animals, sumatriptan reduced the amplitude of evoked EPSCs and this was greater in slices from CFA, compared to saline-injected rats. In CFA-injected animals, sumatriptan increased the paired pulse ratio of evoked EPSCs and reduced the rate of spontaneous miniature EPSCs. The 5-HT 1B and 5-HT 1D agonists CP9 3129 and PNU109291 both inhibited evoked EPSCs in CFA but not saline-injected rats. By contrast, the 5-HT 1A agonist R(+)-8-OH-DPAT inhibited evoked EPSCs in saline but not CFA-injected rats. In CFA-injected rats, the sumatriptan-induced inhibition of evoked EPSCs was reduced by the 5-HT 1B and 5-HT 1D antagonists NAS181 and BRL-15572. Intriguingly, the difference in sumatriptan inhibition between CFA and saline-injected animals was only observed in animals less than 4 weeks old., Conclusion and Implications: These findings indicate that inflammation induces a developmentally regulated 5-HT 1B/1D presynaptic inhibition of excitatory transmission into the rat superficial dorsal horn. Thus, triptans could potentially act as spinal analgesic agents for inflammatory pain in the juvenile setting., (© 2020 The British Pharmacological Society.)

Background and Purpose: The midbrain periaqueductal grey (PAG) plays a central role in modulating pain through a descending pathway that projects indirectly to the spinal cord via the rostroventral medial medulla (RVM). While opioids are potent analgesics that target the PAG, their cellular actions on descending projection neurons are unclear., Experimental Approach: Patch clamp recordings in voltage- and current-clamp mode were made from acutely prepared PAG slices from animals that received retrograde tracer injections into the RVM., Key Results: The μ-agonist DAMGO reduced GABAergic evoked inhibitory postsynaptic currents (IPSCs) in retro-labelled, RVM-projecting neurons to a greater extent than in unlabelled neurons. The κ-opioid agonist U69593 reduced evoked IPSCs to a similar extent in both neuronal groups, while the δ-opioid agonist deltorphin-II was without effect. DAMGO and U69593 both produced a reduction in the rate, but not amplitude of spontaneous miniature IPSCs and asynchronous evoked IPSCs in retro-labelled neurons. DAMGO and U69593 also suppressed glutamatergic EPSCs in retro-labelled and unlabelled neurons. The DAMGO inhibition of evoked EPSCs, however, was less than that for evoked IPSCs in retro-labelled, but not unlabelled neurons. In current clamp, DAMGO produced a depolarizing increase in evoked postsynaptic potentials in retro-labelled neurons, but directly inhibited unlabelled neurons., Conclusion and Implications: These findings suggest that μ-opioids activate the descending analgesic pathway from the midbrain PAG by a combination of presynaptic disinhibition of RVM-projecting neurons and postsynaptic inhibition of presumptive interneurons., (© 2020 The British Pharmacological Society.)

Background and Purpose: Clinical studies have reported that pan-cannabinoid receptor agonists may have efficacy in neuropathic pain states and that this might be enhanced by co-administration with opioids. While cannabinoid-opioid analgesic synergy has been demonstrated in animal models of acute pain, it has not been examined in neuropathic pain models. We examined the effect of combination treatment with cannabinoid and opioid receptor agonists on allodynia and side effects in a nerve injury-induced neuropathic pain model., Experimental Approach: C57BL/6 mice were subjected to chronic constriction injury (CCI) of the sciatic nerve. The effects of systemic administration of morphine and the pan-cannabinoid receptor agonist, WIN55212, on allodynia and side effects were examined at 7-10 days post-CCI surgery. Isobolographic analysis was used to determine whether the effects of the combination were synergistic., Key Results: The opioid agonist morphine reduced CCI-induced mechanical and cold allodynia and produced motor incoordination, in a dose-dependent manner. WIN55212 reduced CCI-induced allodynia and produced motor incoordination, catalepsy and sedation, in a dose-dependent manner, as we have observed previously. When administered together, WIN55212 and morphine reduced allodynia in a synergistic manner but had only an additive effect on motor incoordination., Conclusions and Implications: These findings indicate that administration of a combination of a non-selective opioid and cannabinoid receptor agonist synergistically reduces nerve injury-induced allodynia, while producing side effects in an additive manner. This suggests that this combination treatment has an improved anti-allodynic potency and therapeutic index in a neuropathic pain model., (© 2016 The British Pharmacological Society.)

Background and Purpose: While cannabinoids have been proposed as a potential treatment for neuropathic pain, they have limitations. Cannabinoid receptor agonists have good efficacy in animal models of neuropathic pain; they have a poor therapeutic window. Conversely, selective fatty acid amide hydrolase (FAAH) inhibitors that enhance the endocannabinoid system have a better therapeutic window, but lesser efficacy. We examined whether JZL195, a dual inhibitor of FAAH and monacylglycerol lipase (MAGL), could overcome these limitations., Experimental Approach: C57BL/6 mice underwent the chronic constriction injury (CCI) model of neuropathic pain. Mechanical and cold allodynia, plus cannabinoid side effects, were assessed in response to systemic drug application., Key Results: JZL195 and the cannabinoid receptor agonist WIN55212 produced dose-dependent reductions in CCI-induced mechanical and cold allodynia, plus side effects including motor incoordination, catalepsy and sedation. JZL195 reduced allodynia with an ED50 at least four times less than that at which it produced side effects. By contrast, WIN55212 reduced allodynia and produce side effects with similar ED50s. The maximal anti-allodynic effect of JZL195 was greater than that produced by selective FAAH, or MAGL inhibitors. The JZL195-induced anti-allodynia was maintained during repeated treatment., Conclusions and Implications: These findings suggest that JZL195 has greater anti-allodynic efficacy than selective FAAH, or MAGL inhibitors, plus a greater therapeutic window than a cannabinoid receptor agonist. Thus, dual FAAH/MAGL inhibition may have greater potential in alleviating neuropathic pain, compared with selective FAAH and MAGL inhibitors, or cannabinoid receptor agonists., (© 2015 The British Pharmacological Society.)

Background and Purpose: Cannabinoids and opioids produce antinociception by modulating GABAergic synaptic transmission in a descending analgesic pathway from the midbrain periaqueductal grey (PAG). While chronic opioid treatment produces opioid tolerance, it has recently been shown to enhance cannabinoid-induced antinociception within the PAG. This study examined the effect of repeated opioid treatment on opioid and cannabinoid presynaptic modulation of GABAergic synaptic transmission in PAG., Experimental Approach: Midbrain PAG slices were prepared from untreated rats, and rats that had undergone repeated morphine or saline pretreatment. Whole-cell voltage-clamp recordings were made from neurons within the ventrolateral PAG., Key Results: In slices from untreated animals, the cannabinoid receptor agonist WIN55212 and the μ receptor agonist DAMGO inhibited electrically evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) IPSCs in PAG neurons, with IC50 s of 30 and 100 nM respectively. The inhibition of evoked IPSCs produced by WIN55212 (30 nM) and DAMGO (100 nM) was similar in PAG neurons from morphine- and saline-treated animals. The cannabinoid CB1 receptor antagonist AM251 increased the frequency of spontaneous miniature IPSCs in PAG neurons from repeated morphine-, but not saline-treated animals. DAMGO inhibition of evoked IPSCs was enhanced in the presence of AM251 in morphine-, but not saline-treated animals., Conclusions and Implications: These results indicate that the efficiency of agonist-induced inhibition of GABAergic synaptic transmission is enhanced by morphine treatment, although this is dampened by endocannabinoid-mediated tonic inhibition. Thus, endocannabinoid modulation of synaptic transmission could provide an alternative analgesic approach in a morphine-tolerant state., Linked Articles: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2., (© 2014 The British Pharmacological Society.)

Background and Purpose: Endogenous cannabinoids (endocannabinoids) in the periaqueductal grey (PAG) play a vital role in mediating stress-induced analgesia. This analgesic effect of endocannabinoids is enhanced by pharmacological inhibition of their degradative enzymes. However, the specific effects of endocannabinoids and the inhibitors of their degradation are largely unknown within this pain-modulating region., Experimental Approach: In vitro electrophysiological recordings were conducted from PAG neurons in rat midbrain slices. The effects of the major endocannabinoids and their degradation inhibitors on inhibitory GABAergic synaptic transmission were examined., Key Results: Exogenous application of the endocannabinoid, anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), produced a reduction in inhibitory GABAergic transmission in PAG neurons. This AEA-induced suppression of inhibition was enhanced by the fatty acid amide hydrolase (FAAH) inhibitor, URB597, whereas a 2-AG-induced suppression of inhibition was unmasked by the monoacylglycerol lipase (MGL) inhibitor, JZL184. In addition, application of the CB1 receptor antagonist, AM251, facilitated the basal GABAergic transmission in the presence of URB597 and JZL184, which was further enhanced by the dual FAAH/MGL inhibitor, JZL195., Conclusions and Implications: Our results indicate that AEA and 2-AG act via disinhibition within the PAG, a cellular action consistent with analgesia. These actions of AEA and 2-AG are tightly regulated by their respective degradative enzymes, FAAH and MGL. Furthermore, individual or combined inhibition of FAAH and/or MGL enhanced tonic disinhibition within the PAG. Therefore, the current findings support the therapeutic potential of FAAH and MGL inhibitors as a novel pharmacotherapy for pain., (© 2014 The British Pharmacological Society.)

Background and Purpose: Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices., Experimental Approach: Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents., Key Results: Menthol (150-750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn(2+) (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM)., Conclusions and Implications: These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA -mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA -related pharmacotherapies., (© 2014 The British Pharmacological Society.)

Background and Purpose: Antagonists of the N-type voltage gated calcium channel (VGCC), Cav 2.2, have a potentially important role in the treatment of chronic neuropathic pain. ω-conotoxins, such MVIIA and CVID are effective in neuropathic pain models. CVID is reported to have a greater therapeutic index than MVIIA in neuropathic pain models, and it has been suggested that this is due to faster reversibility of binding, but it is not known whether this can be improved further., Experimental Approach: We examined the potency of CVID, MVIIA and two intermediate hybrids ([K10R]CVID and [R10K]MVIIA) to reverse signs of neuropathic pain in a rat nerve ligation model in parallel with production of side effects. We also examined the potency and reversibility to inhibit primary afferent synaptic neurotransmission in rat spinal cord slices., Key Results: All ω-conotoxins produced dose-dependent reduction in mechanical allodynia. They also produced side effects on the rotarod test and in a visual side-effect score. CVID displayed a marginally better therapeutic index than MVIIA. The hybrids had a lesser effect in the rotarod test than either of their parent peptides. Finally, the conotoxins all presynaptically inhibited excitatory synaptic neurotransmission into the dorsal horn and displayed recovery that was largely dependent upon the magnitude of inhibition and not the conotoxin type., Conclusions and Implications: These findings indicate that CVID provides only a marginal improvement over MVIIA in a preclinical model of neuropathic pain, which appears to be unrelated to reversibility from binding. Hybrids of these conotoxins might provide viable alternative treatments., (© 2013 The British Pharmacological Society.)

Background and Purpose: The arachidonyl-amino acid N-arachidonyl-glycine (NAGly) is an endogenous lipid, generated within the spinal cord and producing spinally mediated analgesia via non-cannabinoid mechanisms. In this study we examined the actions of NAGly on neurons within the superficial dorsal horn, a key site for the actions of many analgesic agents., Experimental Approach: Whole cell patch clamp recordings were made from lamina II neurons in rat spinal cord slices to examine the effect of NAGly on glycinergic and NMDA-mediated synaptic transmission., Key Results: N-arachidonyl-glycine prolonged the decay of glycine, but not β-alanine induced inward currents and decreased the amplitude of currents induced by both glycine and β-alanine. NAGly and ALX-1393 (inhibitor of the glycine transporter, GLYT2), but not the GLYT1 inhibitor, ALX-5407, produced a strychnine-sensitive inward current. ALX-5407 and ALX-1393, but not NAGly prolonged the decay phase of glycine receptor-mediated miniature inhibitory postsynaptic currents (IPSCs). NAGly prolonged the decay phase of evoked IPSCs, although to a lesser extent than ALX-5407 and ALX-1393. In the presence of ALX-1393, NAGly shortened the decay phase of evoked IPSCs. ALX-5407 increased and NAGly decreased the amplitude of evoked NMDA-mediated excitatory postsynaptic currents., Conclusions and Implications: Our results suggest that NAGly enhanced inhibitory glycinergic synaptic transmission within the superficial dorsal horn by blocking glycine uptake via GLYT2. In addition, NAGly decreased excitatory NMDA-mediated synaptic transmission. Together, these findings provide a cellular explanation for the spinal analgesic actions of NAGly., (© 2010 The Authors. British Journal of Pharmacology © 2010 The British Pharmacological Society.)

The myriad functions of lipids as signalling molecules is one of the most interesting fields in contemporary pharmacology, with a host of compounds recognized as mediators of communication within and between cells. The N-acyl conjugates of amino acids and neurotransmitters (NAANs) have recently come to prominence because of their potential roles in the nervous system, vasculature and the immune system. NAAN are compounds such as glycine, GABA or dopamine conjugated with long chain fatty acids. More than 70 endogenous NAAN have been reported although their physiological role remains uncertain, with various NAAN interacting with a low affinity at G protein coupled receptors (GPCR) and ion channels. Regardless of their potential physiological function, NAAN are of great interest to pharmacologists because of their potential as flexible tools to probe new sites on GPCRs, transporters and ion channels. NAANs are amphipathic molecules, with a wide variety of potential fatty acid and headgroup moieties, a combination which provides a rich source of potential ligands engaging novel binding sites and mechanisms for modulation of membrane proteins such as GPCRs, ion channels and transporters. The unique actions of subsets of NAAN on voltage-gated calcium channels and glycine transporters indicate that the wide variety of NAAN may provide a readily exploitable resource for defining new pharmacological targets. Investigation of the physiological roles and pharmacological potential of these simple lipid conjugates is in its infancy, and we believe that there is much to be learnt from their careful study.

Background and Purpose: The transient receptor potential (TRP) channels, transient receptor potential melastatin-1 (TRPM8) and transient receptor potential ankyrin-1 (TRPA1), are expressed in subpopulations of sensory neurones and have been proposed to mediate innocuous and noxious cold sensation respectively. The aim of this study was to compare TRPM8 and TRPA1 modulation of glutamatergic afferent transmission within the spinal dorsal horn., Experimental Approach: Whole cell patch clamp recordings were made from rat spinal cord slices in vitro to examine the effect of TRP agonists and temperature on glutamatergic excitatory postsynaptic currents (EPSCs)., Key Results: Icilin (3 or 100 micromol.L(-1)), menthol (200 micromol.L(-1)) and capsaicin (1 micromol.L(-1)) reduced the amplitude of primary afferent evoked EPSCs in subpopulations of lamina I and II neurones. In a subpopulation of superficial neurones, innocuous cold (threshold 29 degrees C), 3 micromol.L(-1) icilin (EC50 1.5 micromol.L(-1)) and menthol (EC50 263 micromol.L(-1)) increased the rate of spontaneous miniature EPSCs. In the majority of lamina I and II neurones, 100 micromol.L(-1) icilin (EC50 79 micromol.L(-1)), allyl isothiocyanate (EC50 226 micromol.L(-1)), cinnamaldehyde (EC50 38 micromol.L(-1)) and capsaicin (1 micromol.L(-1)) increased miniature EPSC rate. The response to 100 micromol.L(-1), but not 3 micromol.L(-1) icilin, was abolished by ruthenium red, while neither was affected by iodoresiniferatoxin. Responsiveness to 3 micromol.L(-1), but not to 100 micromol.L(-1) icilin, was highly predictive of innocuous cold responsiveness. Neurones responding to 3 micromol.L(-1) icilin and innocuous cold were located more superficially than those responding to 100 micromol.L(-1) icilin., Conclusions and Implications: Activation of TRPM8 and TRPA1 presynaptically modulated glutamatergic transmission onto partially overlapping but distinct populations of superficial dorsal horn neurones. Spinal TRPM8 and TRPA1 channels may therefore provide therapeutic targets in cold hyperesthesia.

While cannabinoid receptor agonists have analgesic activity in chronic pain states, they produce a spectrum of central CB(1) 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.

Bradykinin receptor activation plays an important role in pain arising following tissue inflammation, and recent studies have suggested that bradykinin B1 receptors in particular may be important in chronic pain related to arthritis and various neuropathies. The investigation of the function of the B1 receptors in vivo has been hampered by the lack of nonpeptide antagonists, and the development of such compounds made more difficult by the considerable species variation between human and rodent B1 receptors. In this issue, Fox and co-workers report the creation of a mouse that has had the human B1 gene inserted into the corresponding mouse locus, and they exploit this animal to study the effects of a novel, nonpeptide B1 receptor antagonist on measures of acute nociception and nociception following inflammation. By creating a platform that allows the study of human B1 receptors in vivo, these investigators have provided a tool to significantly advance the understanding of the kallikrein-kinin system in physiological and pathophysiological states.

Functional studies indicate that the midbrain periaqueductal grey (PAG) is involved in the analgesic actions of somatostatin; however, the cellular actions of somatostatin in this brain region are unknown. In the present study, whole-cell patch clamp recordings were made from rat PAG neurons in vitro. In 93% of acutely isolated neurons, somatostatin inhibited Ca(2+)-channel currents. This effect was mimicked by the sst-2 selective agonist BIM-23027, but not by the sst-1 and sst-5 selective agonists CH-275 and L-362855. In brain slices, 81% of neurons responded to somatostatin (300 nm) with an increase in K(+) conductance that reversed polarity at -114 mV. A greater proportion of somatostatin-sensitive neurons (93%) than somatostatin-insensitive neurons (53%) responded to the opioid agonist met-enkephalin (10 microm). Somatostatin also reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). The actions of somatostatin in brain slices were mimicked by BIM-23027, but not by CH-275. Somatostatin had a variable effect on the rate of spontaneous miniature IPSCs in normal external potassium solutions. In high external potassium solutions, somatostatin reduced the rate of miniature IPSCs in all neurons, and this inhibition was abolished by addition of Cd(2+) (30 microm). Somatostatin had no effect on the amplitude of miniature IPSCs. These results indicate that somatostatin acts via sst-2 receptors to directly inhibit a subpopulation of PAG neurons by activating a potassium conductance and inhibits GABA release within PAG via a presynaptic Ca(2+)-dependent mechanism. Thus, like opioids, somatostatin has the potential to exert pre- and postsynaptic disinhibitory effects within the PAG.

1 Patch clamp recordings were made from periaqueductal grey (PAG) neurons in vitro to investigate the cellular actions of opioids in wild-type C57B16/J mice and mutant mice lacking the first exon of the micro -opioid (MOP) receptor. 2 In wild-type mice, the kappa-(KOP) agonist U-69593 (300 nM) and the mixed micro /delta-opioid agonist met-enkephalin (10 micro M), but not the delta-(DOP) agonist deltorphin (300 nM), reduced the amplitude of evoked GABA(A)-mediated inhibitory postsynaptic currents (IPSCs). Met-enkephalin and U-69593 also reduced the rate of spontaneous miniature IPSCs, but had no effect on their amplitude and kinetics. In micro -receptor-deleted mice, only U-69593 (300 nM) reduced the amplitude of evoked IPSCs. 3 In wild-type mice, the MOP agonist DAMGO (3 micro M) produced an outward current in 76% of the neurons. Deltorphin and U-69593 produced outward currents in 24 and 32% of the neurons, respectively. In micro -receptor-deleted mice, deltorphin and U-69593 produced similar outward currents in 32 and 27% of the neurons, respectively, while DAMGO was without effect. All neurons in both the wild-type and micro -receptor-deleted mice responded with similar outward currents to either the GABA(B) receptor agonist baclofen (10 micro M), or the opioid-like receptor ORL1 (NOP) agonist nociceptin (300 nM). 4 The DAMGO-, deltorphin-, U-69593-, baclofen- and nociceptin-induced currents displayed inward rectification and reversed polarity at -109 to -116 mV. 5 These findings indicate that micro -, delta- and kappa-opioid receptor activation has complex pre- and postsynaptic actions within the mouse PAG. This differs to the rat PAG where only micro -opioid receptor actions have been observed.

1. The actions of the neuropeptide nociceptin, the putative nociceptin receptor antagonist [Phe1psi(CH(2)-NH)Gly(2)]-nociceptin-(1 - 13)NH(2) (Phe(1)psi-nociceptin(1 - 13)) and the putative nociceptin precursor products nocistatin (rat prepronociceptin(125 - 132)) and rat prepronociceptin(154 - 181) were examined on membrane properties of rat locus coeruleus (LC) neurons using whole cell patch clamp techniques. 2. Nociceptin inhibited I(Ba) in all LC neurons, (pD(2) of 8.9, maximum inhibition 50%). The inhibition of I(Ba) by nociceptin was associated with slowing of the activation of I(Ba) and could be significantly reversed by a strong depolarizing prepulse. Phe(1)psi-nociceptin(1 - 13) also inhibited I(Ba) in LC neurons (notional pD(2) of 7.6, maximum inhibition 18%). Application of Phe(1)psi-nociceptin(1 - 13) (1 microM) significantly occluded the subsequent effects of a co-application of nociceptin (3 nM) on I(Ba). 3. As previously reported for nociceptin, Phe(1)psi-nociceptin(1 - 13) caused an outward current in LC neurons voltage clamped at -60 mV (pD(2) of 7.1, maximum current 50% of that of methionine enkephalin, 10 microM). The Phe(1)psi-nociceptin(1 - 13) induced current reversed polarity at -112 mV and exhibited pronounced inward rectification. Phe(1)psi-nociceptin(1 - 13) (1 microM) reversibly inhibited the current caused by nociceptin (300 nM) by 30%. 4. Neither nocistatin nor rat prepronociceptin(154 - 181) inhibited I(Ba) in LC neurons, or prevented the subsequent inhibition by nociceptin. Neither nocistatin or prepronociceptin(154 - 181) affected the membrane properties of LC neurons. 5. This study demonstrates that nociceptin modulates somatic I(Ba) in rat LC neurons. The putative ORL1 antagonist Phe(1)psi-nociceptin(1 - 13) exhibited partial agonist activity at inhibiting I(Ba) and opening K(+) channels in LC. Other putative nociceptin precursor products were without effect on LC cells.

1. 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. 2. WIN55,212-2 (3 microM) had no effect on membrane K+ conductance of primary or secondary RVM neurons. Primary neurons responded to the kappa-opioid receptor agonist U69,593 (300 nM - 1 microM). Secondary neurons responded to the mu,delta-opioid receptor agonist met-enkephalin (10 microM). 3. 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 microM). WIN55,212-2 also produced relative facilitation of the second IPSC to paired evoked IPSCs. 4. 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. 5. 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.

Cyclo-oxygenase (COX) inhibitors potentiate opioid inhibition of GABAergic synaptic transmission in rat periaqueductal grey (PAG) (Vaughan et al., 1997). In the present study, the relative contribution of cyclo-oxygenase-1 (COX-1) and COX-2 inhibition to this phenomenon was examined by use of whole-cell patch clamp recordings in brain slices. The mu-receptor partial agonist morphine (10 microM) had little effect on GABAergic synaptic transmission. Morphine reduced the frequency of spontaneous miniature inhibitory postsynaptic currents (m.i.p.s.cs) by 13%. The nonselective COX inhibitor, indomethacin, produced a dose-dependent potentiation of the morpine inhibition of m.i.p.s.c. frequency (maximum inhibition 42%, IC50=6 nM). More selective COX-2 inhibitors produced a similar potentiation of the morphine inhibition of m.i.p.s.c. frequency; however, at greater concentrations (IC50=57 nM piroxicam, 1.7 microM DFU). Maintaining slices in the protein synthesis inhibitor cycloheximide (1 microM), to prevent COX-2 induction, had no effect on the potentiation action of DFU (10 microM). These results demonstrate that the potentiation of opioid inhibition of GABAergic synaptic transmission in PAG is largely a result of inhibition of COX-1 activity. These findings suggest that COX-1, rather than COX-2 inhibition, mediates the synergistic analgesic actions of opioids and non-steroidal anti-inflammatory drugs (NSAIDs) in the midbrain PAG.

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 to 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 microM) or thiorphan (2 microM). 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 microM) occluded the current produced by simultaneous application of high concentrations of Met-enkephalin (10 microM), (3 microM) somatostatin and UK 14304 (3 microM), indicating that nociceptin activated the same conductance as mu-opioid and somatostatin receptors and alpha 2-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 mu-opioid antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2, 1 microM), the opioid antagonist, nalorphine (30 microM) or the somatostatin antagonist, CPP (cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bz1]) 3 microM) did not affect the nociceptin-induced current. 6. Dynorphin A (microM), 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 microM). 7. Continuous application of nociceptin (3 microM) 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 microM) 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 microM) 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 microM) 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.

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 microM) 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, 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 microM) 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.