Central injections of nocistatin or its C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-maze test

. Nocistatin (NST) antagonizes several actions of nociceptin/orphanin FQ (N/OFQ), but acts on distinct receptors. As N/OFQ exerts anxiolytic-like actions in various tests, its behavioural actions in the elevated plus-maze (EPM) test were compared with those of bovine NST. 2. Five minutes after i.c.v. treatment, mice were placed on the EPM for 5 min and entries into and time spent on open and closed arms were recorded alongside other parameters. 3. NST (0.1 - 3 pmol) reduced percentages of entries into (control 39.6+/-3.1%, peak effect at 1 pmol NST 8.5+/-2.9%) and time spent on open arms (control 30.8+/-2.3%, NST 2.7+/-1.5%). The C-terminal hexapeptide of NST (NST-C6; 0.01 - 10 pmol) closely mimicked these actions of NST, with peak effects at 0.1 pmol. 4. N/OFQ (1 - 100 pmol) increased percentages of entries into (control 38.5+/-3.4%; peak effect at 10 pmol N/OFQ 67.9+/-4.9%) and time spent on open arms (control 32.0+/-3.8%; N/OFQ 74.9+/-5.8%). Closed arm entries, an index of locomotor activity, were unchanged by all peptides. 5. Effects of NST or NST-C6, but not N/OFQ, were still detectable 15 min after injection. Behaviour of animals co-injected with NST (1 pmol) or NST-C6 (0.1 pmol) plus N/OFQ (10 pmol) was indistinguishable from that of controls. 6. These results reveal potent anxiogenic-like actions of NST and NST-C6, and confirm the anxiolytic-like properties of N/OFQ. As NST and N/OFQ both derive from preproN/OF, anxiety may be modulated in opposing directions depending on how this precursor is processed.     

Tonic inhibition by orphanin FQ/nociceptin of noradrenaline neurotransmission in the amygdala

The present microdialysis study investigated whether nociceptin/orphanin FQ exerts a tonic inhibition of the release of noradrenaline in the basolateral nucleus of the amygdala in awake rats. The non-peptide competitive nociceptin/orphanin FQ (N/OFQ) peptide receptor antagonist J-113397 (20 mg/kg i.p.) induced an increase in the release of noradrenaline to about 150-200%. The increase was strongly suppressed by local infusion of an endogenous N/OFQ peptide receptor agonist, nociceptin/orphanin FQ (1 microM) via retrograde microdialysis, into the basolateral nucleus of the amygdala. Local infusion of nociceptin/orphanin FQ (1 microM) itself reduced noradrenaline release in the basolateral nucleus of the amygdala to about 70% of basal levels. These results indicate that a large part of basal release of noradrenaline in the basolateral nucleus of the amygdala is under tonic inhibitory control by endogenous nociceptin/orphanin FQ through the N/OFQ peptide receptors localized within the basolateral nucleus of the amygdala.     

Antidepressant-like effects of the nociceptin/orphanin FQ receptor antagonist UFP-101: new evidence from rats and mice

Receptor antagonist and knockout studies have demonstrated that blockade of signalling via nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) has antidepressant-like effects in mice submitted to the forced swimming test (FST). The aim of the present study was to explore further the antidepressant-like properties of the NOP antagonist UFP-101 in different species (mouse and rat) and using different assays [FST and tail suspension test (TST)], and to investigate the mechanism(s) involved in its actions.UFP-101 (10 nmol i.c.v.) reduced immobility time of Swiss mice in the TST (mean±SEM) from 179±11 to 111±10 s. N/OFQ (1 nmol i.c.v.) was without effect per se, but fully prevented the effect of UFP-101. The spontaneous immobility time of NOP^–/– CD1-C57BL/6J-129 mice in the TST was much lower than that of wild-type (NOP^+/+) littermates (75±11 vs. 144±17 s) or of Swiss mice. UFP-101 (10 nmol i.c.v.) decreased immobility time (–65%) and increased climbing time (71%) in rats submitted to the FST. In rat brain slices, N/OFQ (100 nM) triggered robust K⁺-dependent hyperpolarizing currents in locus coeruleus and dorsal raphe neurons. UFP-101 (3 µM) fully prevented N/OFQ-induced currents, but was inactive per se. Fluoxetine, desipramine (both 30 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) reduced immobility time of mice in the FST. The serotonin synthesis inhibitor p-chlorophenylalanine methylester (PCPA, 4×100 mg/kg per day i.p.) prevented the antidepressant-like effects of fluoxetine and UFP-101 (but not desipramine), whereas N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, neurotoxic for noradrenergic neurons; 50 mg/kg i.p., 7 days beforehand), suppressed only the effect of desipramine. Neither pretreatment affected spontaneous immobility time per se.Thus, UFP-101 exhibits pronounced antidepressant-like effects in different species and animal models, possibly by preventing the inhibitory effects of endogenous N/OFQ on brain monoaminergic (in particular serotonergic) neurotransmission. Participation of the N/OFQ-NOP receptor system in mood modulation sets new potential targets for antidepressant drug development.     

Orphanin FQ/nociceptin but not Ro 65-6570 inhibits the expression of cocaine-induced conditioned place preference

The present study investigated the effect of orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like 1 (ORL-1) receptor on the expression of cocaine-induced conditioned place preference (CPP) in rats. To extend this study, the new non-peptidic compound Ro 65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one), with agonist activity at ORL-1 receptors, was examined. The influence of both compounds on cocaine-induced hyperactivity was also studied. Our experiments indicated that intracerebroventricular (i.c.v.) injection of OFQ/N, at doses of 10 and 20 microg/rat, significantly suppressed the expression of cocaine-induced place preference. Ro 65-6570 (3 and 6 mg/kg, i.p.) did not change the effect of cocaine, although its acute injection in control rats significantly increased the time spent in the drug-associated compartment of the CPP apparatus. The substances exhibited opposite effects on cocaine-induced hyperactivity (OFQ/N suppressed it but Ro 65-6570 increased it). Our results suggest that the effect of OFQ/N on the expression of cocaine-induced CPP may be a result of its influence on dopamine (DA) neurotransmission in mesolimbic structures. Ro 65-6570 does not share this effect with OFQ/N.     

Anxiety-induced antinociception in mice: effects of systemic and intra-amygdala administration of 8-OH-DPAT and midazolam

RATIONALE: Mice exhibit antinociception after a single experience in the elevated plus maze (EPM), an animal model of anxiety. OBJECTIVE: This study investigated the mechanisms involved in this form of anxiety-induced antinociception. METHODS: Nociception was evaluated by means of the writhing test in mice confined either to the open or enclosed arms of the EPM. The effects of systemic (naloxone, midazolam and 8-OH-DPAT) or intra-amygdala (8-OH-DPAT, NAN-190 and midazolam) drug infusions were investigated in mice previously treated i.p. with 0.6% acetic acid, an algic stimulus that induces abdominal contortions. The effects of these drugs on conventional measures of anxiety (% entries and % time in open arms) in a standard EPM test were also independently investigated. RESULTS: Open-arm confinement resulted in a high-magnitude antinociception (minimum 85%, maximum 450%) compared with enclosed arm confinement. The opiate antagonist naloxone (1 mg/kg and 10 mg/kg) neither blocked this open arm-induced antinociception (OAIA) nor modified indices of anxiety in EPM. Administration of midazolam (0.5-2 mg/kg, s.c.) increased OAIA and produced antinociception in enclosed confined animals, as well as attenuating anxiety in the EPM. The 5-HT(1A) receptor agonist 8-OH-DPAT (0.05-1 mg/kg, s.c.) had biphasic effects on OAIA, antagonising the response at the lowest dose and intensifying it at the highest dose. In addition, low doses of this agent reduced anxiety in the EPM. Although bilateral injections of 8-OH-DPAT (5.6 nmol/0.4 microl) or NAN-190 (5.6 nmol and 10 nmol/0.4 microl) into the amygdala did not alter OAIA, increased anxiety was observed in the EPM. In contrast, intra-amygdala administration of midazolam (10 nmol and 30 nmol/0.4 microl) blocked both OAIA and anxiety. CONCLUSIONS: These results with systemic and intracerebral drug infusion suggest that 5-HT(1A) receptors localised in the amygdala are not involved in the pain inhibitory processes that are "recruited" during aversive situations. However, activation of these receptors does phasically increase anxiety. Although the intrinsic antinociceptive properties of systemically administered midazolam confounded interpretation of its effects on OAIA, intra-amygdala injections of this compound suggest that benzodiazepine receptors in this brain region modulate both the antinociceptive and behavioural (anxiety) responses to the EPM.     

Activation of the nociceptin/orphanin FQ receptor reduces bronchoconstriction and microvascular leakage in a rabbit model of gastroesophageal reflux

1. Nociceptin/orphanin FQ (N/OFQ) is the endogenous peptide ligand for a specific G-protein coupled receptor, the N/OFQ peptide receptor (NOP). The N/OFQ-NOP receptor system has been reported to play an important role in pain, anxiety and appetite regulation. In airways, N/OFQ was found to inhibit the release of tachykinins and the bronchoconstriction and cough provoked by capsaicin. 2. Here we evaluated the effects of NOP receptor activation in bronchoconstriction and airway microvascular leakage induced by intraesophageal (i.oe.) hydrochloric acid (HCl) instillation in rabbits. We also tested the effects of NOP receptor activation in SP-induced plasma extravasation and bronchoconstriction. 3. In anesthetized New Zealand rabbits bronchopulmonary function (total lung resistance (R(L)) and dynamic compliance (C(dyn))) and airway microvascular leakage (extravasation of Evans blue dye) were evaluated. 4. Infusion of i.oe. HCl (1 N) led to a significant increase in bronchoconstriction and plasma extravasation in the main bronchi and trachea of rabbits pretreated with propranolol, atropine and phosphoramidon. 5. Bronchoconstriction and airway microvascular leakage were inhibited by N/OFQ (3-30 microg kg(-1) i.v.) in a dose-dependent manner. The NOP receptor agonist [Arg14,Lys15]N/OFQ mimicked the inhibitory effect of N/OFQ, being 10-fold more potent, UFP-101, a peptide selective NOP receptor antagonist, blocked the inhibitory effects of both agonists. 6. Under the same experimental conditions, N/OFQ and [Arg14,Lys15]N/OFQ did not counteract the bronchoconstriction and airway microvascular leakage induced by substance P. 7. These results suggest that bronchoconstriction and airway plasma extravasation induced by i.oe. HCl instillation are inhibited by activation of prejunctional NOP receptors.     

Orphanin FQ/nociceptin and [Phe(1)Psi(CH(2)-NH)Gly(2)] nociceptin(1-13)-NH(2) stimulate gastric motor function in anaesthetized rats

Orphanin FQ/nociceptin (OFQ/N) is a preferred endogenous ligand for the orphan opioid receptor-like-1 receptor. This peptide has been reported to increase intestinal, but not gastric, motor activity. In the present study, OFQ/N (0.6-60 nmol kg(-1) i.v.) increased intragastric pressure and antral contractility and, as expected, decreased blood pressure in anaesthetized rats. The gastric motor effects of OFQ/N (6 nmol kg(-1)) were not affected by inhibition of nitric oxide synthase or opioid receptor blockade. OFQ/N (6 nmol kg(-1)) evoked gastric motor increases and hypotension were not affected by prior administration of its derivative [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) unless the pseudopepotide was administered shortly (5 min) prior to OFQ/N. This putative antagonist (6-300 nmol kg(-1)) alone increased antral motility with approximately 100 fold lower potency than OFQ/N. Neither bilateral vagotomy nor spinal cord transection altered OFQ/N-evoked increases in intragastric pressure and antral contractility. In conclusion, OFQ/N induces gastric motor excitation in addition to its known effects to increase intestinal motility. The gastric responses to OFQ/N are not dependent on 'classical' opioid receptor activation or nitric oxide, similar to the case for the intestines. The primary site of action of OFQ/N on the stomach is probably via enteric nerves, since central descending vagal or sympathetic pathways are not necessary for OFQ/N to increase gastric motility. The gastric motor effects of the derivative [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)-NH(2) are similar to OFQ/N, although with lower potency. The effects of the derivative as a partial agonist or antagonist in different experimental paradigms may reflect tissue OFQ/N receptor reserve.     

The influence of the benzodiazepine receptor antagonist flumazenil on the anxiolytic-like effects of CGP 37849 and ACPC in rats

In this paper we examined the effect of flumazenil (Ro 15-1788, 10 mg/kg), a benzodiazepine receptor antagonist, on the anticonflict activity of DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, and 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at glycine(B) receptors, in the Vogel conflict drinking test in rats. The effect of flumazenil on the anxiolytic-like (in the plus-maze test) and the anticonvulsant (in the maximal electroshock-induced seizures) activities of CGP 37849 in rats was also studied. Diazepam was used as a reference drug. CGP 37849 (2. 5-5 mg/kg), ACPC (50-200 mg/kg) and diazepam (2.5-5 mg/kg) significantly and dose-dependently increased the number of shocks accepted during experimental sessions in the conflict drinking test. Flumazenil partly but significantly reduced the anticonflict effect of CGP 37849, and it fully blocked the anticonflict effect of ACPC and diazepam. CGP 37849 (2.5-5 mg/kg) and diazepam (2.5-5 mg/kg) were also active in the plus-maze test, as they significantly increased the percentage of the time spent in and entries into the open arms of the plus-maze, both those effects having been antagonized by flumazenil. Flumazenil alone was inactive in both the conflict drinking and the plus-maze tests. In the maximal electroshock-induced seizures, both CGP 37849 (2.5-5 mg/kg) and diazepam (5-10 mg/kg) produced anticonvulsant effects, of which only that of diazepam was antagonized by flumazenil. The results of the present study showing antagonism of flumazenil towards the anxiolytic-like effects of CGP 37849 and ACPC suggest involvement of benzodiazepine receptors in such an activity of the NMDA and glycine(B) receptor ligands, respectively, which may be due to a possible interaction between NMDA and GABA/benzodiazepine systems. The lack of effect of the benzodiazepine antagonist on the anticonvulsant activity of CGP 37849 indicates that involvement of benzodiazepine receptors in the pharmacological action of the NMDA antagonist is not a general phenomenon.     

Nociceptin/orphanin FQ blocks the antinociception induced by mu, kappa and delta opioid agonists on the cold water tail-flick test

Nociceptin/orphanin FQ (N/OFQ), a 17-amino-acid peptide, is an endogenous agonist whose receptor is similar in sequence to mu, delta and kappa opioid receptors. It has been reported that N/OFQ can block antinociceptive effects induced by opioid receptor agonists in the radiant heat tail-flick test and warm water tail-withdrawal test. The present study was designed to see the effect of N/OFQ on antinociception induced by opioid receptor agonists in the cold water tail-flick (CWT) test, which measures a different type of pain. In adult male Sprague-Dawley (S-D) rats given subcutaneous (s.c.) injections of saline or morphine (8 mg/kg), intracerebroventricular (i.c.v.) injection of N/OFQ (18 microg) 15 min later produced a significant reversal of morphine antinociception (P<0.01, ANOVA followed by Duncan's test), compared to the corresponding saline control group. Saline (t=+15 min, i.c.v.) had no effect on s.c. morphine antinociception (P>0.01), compared to the corresponding saline control group. When the kappa opioid receptor agonist spiradoline (80 mg/kg, s.c.) was used instead of morphine, similar results were observed. In another series of experiments, it was found that i.c.v. injection of N/OFQ (18 microg) reversed the antinociception induced by i.c.v. injection of the selective mu opioid agonist PL017 (2 microg), delta opioid agonist DPDPE (50 ng) and kappa opioid agonist dynorphin (21.5 microg), respectively. These results indicate that N/OFQ may be an endogenous anti-opioid peptide in the brain of rats in the CWT test.     

The ORL-1 (NOP1) receptor ligand nociceptin/orphanin FQ (N/OFQ) inhibits neurogenic dural vasodilatation in the rat

The effects of the ORL-1 (NOP(1)) receptor ligand nociceptin (N/OFQ) and the nociceptin antagonists [Nphe(1)]N/OFQ-(1-13)-NH(2) (Nphe) and nocistatin (NST) on neurogenic dural vasodilatation (NDV) in the rat dura mater evoked by electrical stimulation of a closed cranial window were studied. The middle meningeal artery was visualised using intravital microscopy, and the vessel diameter analysed using a video dimension analyser. N/OFQ (1, 10, 100 nmol kg(-1); i.v., n=10) significantly and dose-dependently suppressed NDV maximally by 65% (P<0.01). Neither Nphe (100 nmol kg(-1); n=5) nor NST (100 nmol kg(-1); n=4) alone had an effect on NDV (P>0.05). Baseline vessel diameter was not significantly affected by application of N/OFQ, NST or Nphe. Application of the selective N/OFQ antagonist Nphe (10, 100 nmol kg(-1) i.v., n=8) dose-dependently and significantly (P<0.01) reversed the inhibition of NDV induced by application of N/OFQ (10 nmol kg(-1)). NST (10, 100 nmol kg(-1); n=7) failed to reverse the effects elicited by N/OFQ. Application of N/OFQ elicited a dose-dependent transient decrease in arterial blood pressure (P<0.01). Nphe dose-dependently reversed the cardiovascular effects induced by application of N/OFQ (10 nmol kg(-1)) (P<0.01),while NST did not alter the blood pressure reaction elicited by N/OFQ. The results show that N/OFQ inhibits NDV, an effect which is antagonised by Nphe, but not by NST. ORL-1 (NOP(1)) receptors located on trigeminal sensory fibres may be involved in the regulation of dural vessel diameter and hence may play a role in migraine pathophysiology.     

Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat

The current studies further investigated the effects, in animal models of anxiety, of novel putative anxiolytic and anxiogenic compounds believed to induce their effects by actions at the GABA-benzodiazepine receptor complex. It was expected that the results would also provide further validation for a novel test of anxiety based on the ratio of open to closed arm entries in an elevated plus maze in the rat. The novel putative anxiolytics CL 218,872 (10-20 mg/kg) and tracazolate (5 mg/kg) significantly elevated the percentage of time spent on the open arms of an elevated plus-maze, consistent with their anxiolytic activity in several other animal tests. Also consistent with results from other animal tests, no anxiolytic activity was observed for the phenylquinoline PK 8165 (10-25 mg/kg), the 3,4-benzodiazepine tofisopam (25-50 mg/kg), or buspirone (0.5-20 mg/kg). The benzodiazepine receptor inverse agonists FG 7142 (1-5 mg/kg) and CGS 8216 (3-10 mg/kg) had anxiogenic activity in this test, as did the atypical benzodiazepine Ro 5-4864 (1-5 mg/kg). Interestingly, however, the benzodiazepine receptor antagonists Ro 15-1788 (10-20 mg/kg) and ZK 93426 (5-10 mg/kg) had no anxiogenic activity in this test.     

Chronic treatment with antidepressants affects glycine/NMDA receptor function: behavioral evidence

Like the clinically effective benzodiazepine anxiolytic, chlordiazepoxide, the glycine/NMDA receptor antagonist L-701,324 (3, 7.5 and 10 mg/kg), produces dose-related increases in the percentage of time spent in the open arms and the percentage of entries into the open arms of an elevated plus maze in mice. Consistent with its proposed mechanism of action, these anxiolytic effects of L-701,324 (7.5 mg/kg) are reversed by pretreatment with glycine (500 and 800 mg/kg). Chronic treatment with citalopram (20 mg/kg for 21 days), imipramine (15 mg/kg for 21 days) and electroconvulsive shock (ECS, for 8 days), produced a reduction in the anxiolytic-like actions of L-701,324 (7.5 mg/kg) such that they could not be reversed by glycine. In contrast, the anxiolytic effects of L-701,324 and reversal of these effects by glycine were unaffected by acute treatment with imipramine, chronic administration with placebo or the neuroleptic chlorpromazine, or sham ECS. Further, imipramine administered for 21 days did not affect the anxiolytic effect of 5 mg/kg of chlordiazepoxide. The apparent reduction in the anxiolytic-like actions of a specific glycine/NMDA receptor antagonist following chronic treatment with a variety of antidepressants is consistent with previous neurochemical and molecular studies indicating that chronic antidepressant treatment can affect NMDA receptor function.     

Effects of amperozide in two animal models of anxiety

The novel psychotropic agent amperozide (amp) was investigated in two different rat anxiety models, Vogel's conflict test (VT) and Montgomery's conflict test (MT). In the VT, amp in lower doses (0.2-0.6 mg/kg subcutaneously) increased the number of shocks accepted, as compared to controls. Pretreatment with the specific benzodiazepine receptor antagonist Ro 15-1788 (10.0 mg/kg orally) or the GABA-A receptor antagonist bicuculline (2.0 mg/kg intraperitoneally) antagonized the anticonflict effect of amp (0.4 mg/kg subcutaneously). At the highest dose tried (2.0 mg/kg subcutaneously) amp instead decreased the number of shocks accepted. Both 0.4 mg/kg and 2.0 mg/kg of amp raised the shock threshold, as compared to controls. The latter dose also decreased the motivation to drink. Pretreatment with Ro 15-1788 (10.0 mg/kg orally) did not significantly change the shock threshold or the drinking motivation, as compared to the group receiving amp alone. In the MT, amp (0.05-0.1 mg/kg subcutaneously) increased the percentage time spent in the open arms, while no changes were seen in the number of entries made into these arms. After higher doses (0.4-0.8 mg/kg subcutaneously) no differences, as compared to controls, were observed. Amp (1 nM-10microM) exhibited no affinity for 3H-flunitrazepam binding sites in mouse forebrain membranes in vitro. Taken together, the present data suggest that amp in low doses produces anticonflict (anxiolytic-like) effects. These effects appear to be mediated through an indirect (via 5-HT and/or DA systems?) activation of the GABA/benzodiazepine chloride ionophore receptor complex.     

Involvement of diazepam-insensitive benzodiazepine receptors in the suppression of DOI-induced head-twitch responses in diabetic mice

Rationale We previously reported that the head-twitch responses induced by the 5-HT₂ receptor agonist (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) (DOI-HTRs) were decreased in streptozotocin-induced diabetic mice.Objectives We examined the involvement of γ-aminobutyric acid (GABA)/benzodiazepine system on the suppression of DOI-HTRs in diabetic mice.Results The benzodiazepine receptor antagonist flumazenil (0.1–1 mg/kg, i.v.) dose-dependently and significantly increased DOI-HTRs in diabetic mice to the same levels as in nondiabetic mice. However, flumazenil (0.1–1 mg/kg, i.v.) did not affect DOI-HTRs in nondiabetic mice. The benzodiazepine receptor agonist diazepam (0.1–1 mg/kg, i.p.) had no effect on DOI-HTRs in either nondiabetic or diabetic mice. The GABA_A receptor antagonist bicuculline (0.1–1 mg/kg, i.p.) and the benzodiazepine receptor partial inverse agonist Ro 15-4513 (0.1–1 mg/kg, i.v.) dose-dependently and significantly suppressed DOI-HTRs in nondiabetic mice to the same levels as in diabetic mice. Ro 15-4513-induced reduction of DOI-HTRs in nondiabetic mice was completely antagonized by flumazenil (1 mg/kg, i.v.), but not diazepam (0.3 mg/kg, i.p.).Conclusions We suggest that the abnormal diazepam-insensitive benzodiazepine receptor function partly underlies the suppression of DOI-HTRs in diabetic mice.     

Benzodiazepine/GABA(A) receptors are involved in magnesium-induced anxiolytic-like behavior in mice

Behavioral studies have suggested an involvement of the glutamate pathway in the mechanism of action of anxiolytic drugs, including the NMDA receptor complex. It was shown that magnesium, an NMDA receptor inhibitor, exhibited anxiolytic-like activity in the elevated plus-maze test in mice. The purpose of the present study was to examine interaction between magnesium and benzodiazepine/GABA(A) receptors in producing anxiolytic-like activity. We examined behavior of mice treated with magnesium and benzodiazepine/GABA(A) receptor ligands, in the elevated plus maze. The anxiolytic-like effect of magnesium (20 mg/kg) was antagonized by flumazenil (10 mg/kg) (benzodiazepine receptor antagonist) while combined treatment with the non-effective doses of magnesium (10 mg/kg) and benzodiazepines (diazepam (0.5 mg/kg) or chlordiazepoxide (2 mg/kg)) produced synergistic interaction (increased time in open arms and number of open arm entries) in this test. The obtained data indicate that benzodiazepine receptors are nvolved in the anxiolytic-like effects of magnesium.     

Stimulation of sensory neuropeptide release by nociceptin/orphanin FQ leads to hyperaemia in acutely inflamed rat knees

The peripheral effect of the 'opioid-like' peptide nociceptin/orphanin FQ (N/OFQ) on joint blood flow was investigated in acutely inflamed rats. Sensory neuropeptide release from capsaicin-sensitive nerves and the involvement of synovial mast cells and leukocytes on these vasomotor responses were also studied. Blood flow measurements of exposed knee joints were performed in urethane-anaesthetised rats (2 mg kg(-1) intraperitoneal) using laser Doppler perfusion imaging. Topical administration of N/OFQ (10(-13)-10(-8) mol) to acutely inflamed joints caused a dose-dependent increase in synovial perfusion with an ED(50) of 4.0 x 10(-10) mol. This vasodilatatory response was blocked by the selective NOP receptor antagonist [Phe(1)-(CH(2)-NH)-Gly(2)]-Nociceptin(1-13)-NH(2) (10(-9) mol) (P<0.0001).Co-administration of N/OFQ with the neurokinin-1 (NK(1)) receptor antagonist [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-Substance P (10(-12) mol), the vasoactive intestinal peptide (VIP) receptor antagonist VIP(6-28) (10(-9) mol) or the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37) (10(-9) mol) all blocked the hyperaemic effect of N/OFQ (P<0.0001). Treatment of acutely inflamed knees with capsaicin (8-methyl-N-vanillyl-6-noneamide) to destroy unmyelinated joint afferents also inhibited N/OFQ vasomotor activity. Stabilisation of synovial mast cells with disodium cromoglycate (cromolyn) ameliorated N/OFQ responses, whereas inactivation of circulating leukocytes with the pan-selectin inhibitor fucoidin completely blocked N/OFQ-induced hyperaemia in these joints. These experiments show that in acutely inflamed knee joints, N/OFQ acts on NOP receptors located on synovial mast cells and leukocytes leading to the secondary release of proinflammatory mediators into the joint. These agents subsequently stimulate sensory neuropeptide release from capsaicin-sensitive nerves culminating in vasodilatation and increased articular blood flow.     

Participation of the GABA/benzodiazepine receptor and the NO-cyclicGMP pathway in the "antinociceptive-like effects" of diazepam

The mechanism of action underlying the "analgesic activity" of diazepam remains unclear. In this study, the possible participation of the GABA/benzodiazepine receptor and the nitric oxide-cyclic GMP (NO-cGMP) pathway was assessed utilizing the pain-induced functional impairment model in the rat (PIFIR). Nociception was induced by an intra-articular injection of 15% uric acid. Diazepam (1 and 2 mg/kg, i.p.) reversed the dysfunction induced by uric acid. Flumazenil (10 mg/kg, i.p.), a GABA/benzodiazepine receptor antagonist, abolished the "antinociceptive-like effect" of diazepam (at 2 mg/kg). The "antinociceptive-like effect" of diazepam (at 2 mg/kg) was antagonized by the non-selective nitric oxide synthase (NOS) inhibitor, N(omega)-l-nitro-arginine methyl ester hydrochloride (l-NAME, 5 mg/kg, s.c.) (but not by its inactive isomer), and by the selective neuronal NOS inhibitor, 7-nitroindazole (7-NI, 1 mg/kg, i.p). While, the NO precursor, l-arginine (125 mg/kg, s.c.), but not d-arginine (125 mg/kg, s.c.), increased the "antinociceptive-like effect" of a non-effective dose of diazepam (1 mg/kg). Methylene blue (10 mg/kg, i.p.), a guanylate cyclase inhibitor, also prevented the "antinociceptive-like effect" of diazepam (at 2 mg/kg). These results suggest that the GABA/benzodiazepine receptor and the NO-cGMP pathway participate in the "antinociceptive-like effect" of diazepam.     

Abnormal benzodiazepine receptor function in the depressive-like behavior of diabetic mice

We previously reported that streptozotocin (STZ)-induced diabetic mice exhibited depressive-like behavior in the tail suspension test. In this study, we examined the involvement of benzodiazepine receptor functions in this diabetes-induced depressive-like behavior in mice. STZ-induced diabetes significantly increased the duration of immobility without affecting spontaneous locomotor activity. This increase was dose-dependently and significantly suppressed by a benzodiazepine receptor antagonist, flumazenil (0.1-1 mg/kg, i.v.). However, flumazenil (0.1-1 mg/kg, i.v.) did not affect the duration of immobility in non-diabetic mice. Furthermore, flumazenil (1 mg/kg, i.v.) had no significant effect on spontaneous locomotor activity in either non-diabetic or diabetic mice. The benzodiazepine receptor inverse agonist methyl beta-carboline-3-carboxylate (beta-CCM; 0.03-0.3 mg/kg, i.v.) dose-dependently and significantly increased the duration of immobility in non-diabetic mice, but not in diabetic mice. beta-CCM (0.3 mg/kg, i.v.) significantly suppressed spontaneous locomotor activity in non-diabetic mice, but not in diabetic mice. These results indicate that diabetic mice may have enhanced negative allosteric modulation by benzodiazepine receptor ligands, such as diazepam binding inhibitors, under stressful conditions, but not free-moving conditions, and this abnormal function of benzodiazepine receptors may cause, at least in part, the expression of depressive-like behavior in diabetic mice.     

Nicotine-induced anxiogenic-like behaviours of rats in the elevated plus-maze: possible role of NMDA receptors of the central amygdala

The objective of the present study was to investigate the possible role of the N-methyl-D-aspartate (NMDA) receptor system of the central amygdala (CeA) in the anxiogenic-like effect of nicotine. Male Wistar rats with cannulas aimed to the CeA were submitted to the elevated plus-maze (EPM). Intraperitoneal (i.p.) injections of nicotine (0.6 and 0.8 mg/kg) decreased percentage open arm time spent (%OAT) and percentage open arm entries (%OAE), but not locomotor activity, indicating an anxiogenic-like response. Bilateral intra-CeA microinjection of NMDA (0.005-0.1 μ g/rat) decreased %OAT, but not %OAE and locomotor activity. Moreover, intra-CeA microinjection of NMDA (0.05 μ g) with an ineffective dose of nicotine (0.4 mg/kg, i.p.) reduced %OAT and %OAE without effect on locomotor activity. On the other hand, intra-CeA microinjection of the NMDA receptor antagonist D-AP5 (0.05-0.5 μ g/rat) increased both %OAT and %OAE, showing an anxiolytic-like effect of the drug. Co-administration of the same doses of D-AP5 with nicotine (0.6 mg/kg, i.p.) increased %OAT and %OAE, but not locomotor activity. Intra-CeA microinjection of D-AP5 reversed the response induced by NMDA (0.1 μ g/rat) in the EPM. The results may support the possible involvement of glutamate transmission, through NMDA receptors of central amygdala in the anxiogenic-like effect of nicotine in the EPM task.     

Interaction between cannabinoid compounds and diazepam on anxiety-like behaviour of mice

Previous studies have suggested that cannabinoidergic system is involved in anxiety. However, a complete picture of cannabinoid association in the anxiety is still lacking. In the present study, we investigated the possible interaction between cannabinoidergic and GABAergic systems in the anxiety-like behaviour of mice. Intraperitoneal (i.p.) administration of the cannabinoid receptor agonist WIN55212-2 (0.25-5 mg/kg), the endocannabinoid transport inhibitor AM404 (0.25-2 mg/kg) and diazepam (0.25-8 mg/kg) dose dependently exhibited an anxiolytic effect evaluated in terms of increase in the percentage of time spent in the open arms in the elevated plus maze (EPM) test. Administration of certain fixed-ratio combinations (3:1 and 1:1) of WIN55212-2 and diazepam produced a synergistic anxiolytic effect, while the 1:3 combination produced an additive effect. In hole-board test, administration of certain ratios of WIN55212-2-diazepam combination significantly altered the animal behaviour compared to groups that received each drug alone. Co-administration of AM404 (1 and 2 mg/kg) and diazepam (0.5 mg/kg) abolished the anxiolytic effect of the former drug in EPM and the latter in hole-board test, respectively. The combination of an ineffective dose of the fatty acid amide hydrolase (FAAH) inhibitor, URB597 (0.3 mg/kg, i.p.) on anxiety-related responses with an ineffective dose of diazepam (0.25 mg/kg, i.p.) led to a synergistic effect. Co-administration of the CB1 receptor antagonist, AM251 (5 mg/kg) and an effective dose of diazepam (2 mg/kg, i.p.) attenuated diazepam-induced elevation of percentage of time spent in open arm, while lower dose of AM251 (0.5 mg/kg) failed to inhibit diazepam-induced anxiolytic effect. Taken together, the present study showed that co-administration of exogenous cannabinoids and diazepam produce additive or synergistic effect at different combinations. Moreover, it has been shown that enhancement of the function of endocannabinoids could increase the anxiolytic effect of diazepam.