Ewa Gibula-Bruzda

Modulation of neuropeptide FF (NPFF) receptors influences the expression of amphetamine-induced conditioned place preference and amphetamine withdrawal anxiety-like behavior in rats.

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.

Influence of new deltorphin analogues on reinstatement of cocaine-induced conditioned place preference in rats.

The aim of this study was to investigate whether the δ-opioid receptors are involved in the rewarding and reinstatement effect of cocaine in the conditioned place preference (CPP) test. Male Wistar rats were conditioned with cocaine (5 mg/kg) or saline in a biased CPP procedure. The intracerebroventricular (i.c.v.) administration of naltrindole (5 nmol), δ-opioid receptor antagonist but not β-funaltrexamine (5 nmol), or nor-binaltorphimine (10 nmol), μ-opioid and κ-opioid receptor antagonists, respectively reversed the expression of the cocaine CPP. The i.c.v. administration of new analogues of deltorphins with potent agonist activity at δ-opioid receptors, such as cyclo(Nδ, Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) at the dose of 10 and 20 nmol and deltorphin II N-(ureidoethyl)amide (DK-4) at the dose of 10 and 20 nmol reinstated the rewarding effect of cocaine after extinction sessions in the CPP test. Naltrindole (5 nmol, i.c.v.) abolished the reinstated effect of DK-4 (10 nmol). In addition, DEL-6 and DK-4 induce anxiolytic-like effects in the elevated plus-maze test. However, neither peptide given alone either produced a rewarding effect in the CPP test, or influenced the locomotor activity and motor coordination, thus suggesting that these effects of peptides did not influence the results obtained in the reinstatement procedure of CPP. In conclusion, our results show that δ-opioid receptors play a dominant role in cocaine reward and reinstatement of cocaine seeking behavior in the CPP test.

Enkephalin derivative, cyclo[Nε,Nβ-carbonyl-D-Lys2, Dap5] enkephalinamide (cUENK6), induces a highly potent antinociception in rats

The aim of the study was to evaluate whether the newly synthesized analog of enkephalin, cyclo[N(epsilon),N(beta)-carbonyl-D-Lys(2), Dap(5)] enkephalinamide (cUENK6), a highly potent mu- (guinea pig ileum assay) and delta-receptors (mouse vas deferens assay) ligand, induces an antinociceptive effect in the hot-plate test and tail-immersion test after intracerebroventricular administration. Our study indicated that this peptide at the dose of 0.25 nmol produced comparable but at the dose of 0.5 nmol stronger than morphine (13 nmol), antinociceptive effect in both tests. Furthermore, rats with developed tolerance to morphine indicated cross-tolerance to antinociceptive effects of cUENK6. The antinociceptive effects of cUENK6 and morphine were inhibited by non-selective opioid receptor antagonist--naloxone. More detailed study indicated that the delta-opioid receptor antagonist - naltrindole very strongly and, to the lower extent, mu-opioid antagonist - beta-funaltrexamine (beta-FNA), inhibited antinociceptive effect of cUENK6 in the tail-immersion test. Nor-binaltorphimine (nor-BNI), a kappa-opioid receptor antagonist, did not influence this effect. These data suggest the dominant role of delta-opioid receptors as compared with mu-receptors in mediation antinociceptive effect of cUENK6. Furthermore, we found that cUENK6 is much more effective in inhibiting pain in the hot-plate (ED(50)=0.0792 nmol) than in the tail-immersion (ED(50)=0.3526 nmol) test. However, cUENK6 at the antinociceptive doses induced hypolocomotion, and although this effect is observed after administration of opioid agonists in rats as a one phase of their biphasic action (inhibition followed by activation), in our study it was not naloxone-reversible. Therefore, our study suggests that not only opioid receptors may be involved in behavioral effects of cUENK6.

Antinociceptive effects of two deltorphins analogs in the tail-immersion test in rats.

The antinociceptive effects of analogs of deltorphins: cyclo(Nδ,Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) after intracerebroventricular (i.c.v.) administration were investigated in the tail-immersion test in rats. Morphine, the most commonly used μ-opioid receptors (MOR) agonist, was employed as a reference compound. The contribution of the MOR, δ-(DOR) and κ-opioid receptors (KOR) in antinociceptive effects of the deltorphins analogs was studies using selective antagonists of these receptors. The results indicated that DK-4 (5, 10 and 20 nmol) and DEL-6 (5, 10 and 20 nmol) were the most effective in alleviating thermal pain at the dose of 20 nmol. The antinociceptive potency of DEL-6 at the dose of 20 nmol was approximately equal but DK-4 at the dose of 20 nmol was less effective than morphine at the dose of 13 nmol. DOR antagonist - naltrindole (NTI, 5 nmol) very strongly and, to the lower extent MOR antagonist - β-funaltrexamine (β-FNA, 5 nmol), inhibited antinociceptive effect of DK-4 (20 nmol). In turn, β-FNA was more potent than NTI in inhibition of the antinociceptive effects of DEL-6. Co-administration of DEL-6 and morphine at doses of 5 nmol, which do not produce measurable antinociception, generated additive antinociceptive effect. Chronic intraperitoneal (i.p.) injection of morphine (9 days) displayed a marked analgesic tolerance to the challenge dose of morphine and a slight cross-tolerance to challenge doses of DEL-6 and DK-4, given i.c.v. These findings indicate that the new deltorphin analogs recruit DOR and MOR to attenuate the nociceptive response to acute thermal stimuli.

Enkephalin analog, cyclo[Nε,Nβ-carbonyl-D-Lys2,Dap5] enkephalinamide (cUENK6), inhibits the ethanol withdrawal-induced anxiety-like behavior in rats

An analog of enkephalin, cyclo[N(ε),N(β)-carbonyl-D-Lys(2),Dap(5)] enkephalinamide (cUENK6), is predominantly a functional agonist of μ-opioid receptors (MOPr) and, to a lesser extent, of δ-opioid receptors (DOPr) in vitro. The aim of the present study was to determine whether cUENK6 could affect ethanol withdrawal-induced anxiety-like behavior in the elevated plus maze (EPM) test in rats. An anxiety-like effect of withdrawal was predicted to occur in the EPM test 24 h after the last ethanol administration (2 g/kg, intraperitoneally [i.p.]; 15% w/v once daily for 9 days). Ethanol withdrawal decreased the percent of time spent by rats in the open arms and the percent of open-arms entries. cUENK6 (0.25 nmol), given by intracerebroventricular (i.c.v.) injection, significantly reversed these anxiety-like effects of ethanol withdrawal and elevated the percent of time spent by rats in the open arms and the percent of open-arms entries. These effects of cUENK6 were significantly inhibited by the DOPr antagonist naltrindole (NTI) (5 nmol, i.c.v.), but not by the MOPr antagonist β-funaltrexamine (β-FNA) (5 nmol, i.c.v.). The preferential DOPr agonist [Leu(5)]-enkephalin (LeuEnk) (2.7 and 5.4 nmol, i.c.v.) and the MOPr agonist morphine (6.5 and 13 nmol, i.c.v.) reduced the anxiety-like effects of ethanol withdrawal. cUENK6 at the dose of 0.25 nmol did not disturb locomotor activity in the EPM, in contrast to cUENK6 at the dose of 0.5 nmol, and morphine at 6.5 and 13 nmol. However, similarly to LeuEnk, cUENK6 induced the anxiolytic-like effects in naïve rats. Thus, our study suggests that cUENK6 reduced ethanol withdrawal-induced anxiety-like behavior by activation of δ-opioid receptors rather than μ-opioid receptors.

Cholinergic activation affects the acute and chronic antinociceptive effects of morphine

Current studies indicate that the cholinergic and opioid systems interact to modulate pain. In the present work, we investigated the influence of the cholinesterase inhibitors, donepezil (0.5; 1 or 3mg/kg, i.p.) and rivastigmine (0.03; 0.5 or 1mg/kg, i.p.), on the acute antinociceptive effects of morphine (5mg/kg, i.p.) in the hot plate test in mice. Herein, both inhibitors were found to enhance and prolong the analgesic effects of morphine without affecting latencies themselves. In an extension of this work, we determined which cholinergic receptors subtype mediates the enhancement of analgesic effects of morphine, following inhibition of cholinesterases. In this part of the study, scopolamine (0.5mg/kg, i.p.), a muscarinic cholinergic receptors antagonist, but not mecamylamine (3mg/kg, i.p.), a nicotinic cholinergic receptors antagonist, reversed the enhancing effects of donepezil (3mg/kg, i.p.) and rivastigmine (1mg/kg, i.p.) on the morphine antinociception. Moreover, both cholinesterase inhibitors attenuated the development of tolerance to the antinociceptive effects of morphine. In contrast, acute administration of donepezil (3mg/kg, i.p.) or rivastigmine (1mg/kg, i.p.) on the day of expression of tolerance, had no effect on the already developed morphine tolerance. What is more, in both set of experiments, rivastigmine was slightly more potent than donepezil due to the broader inhibitory spectrum of this drug on acetylcholine degradation. Thus, our results suggest that the cholinesterase inhibitors, donepezil and rivastigmine, may be administered with morphine in order to enhance the latters analgesic effects for the treatment of acute and chronic pain.

Acquisition and reinstatement of ethanol-induced conditioned place preference in rats: Effects of the cholinesterase inhibitors donepezil and rivastigmine.

The present study examined the influence of the cholinesterase inhibitors donepezil (a selective inhibitor of acetylcholinesterase) and rivastigmine (also an inhibitor of butyrylcholinesterase) on the acquisition and reinstatement of ethanol-induced conditioned place preference (CPP) in rats. Before the CPP procedure, animals received a single injection of ethanol (0.5 g/kg, 10% w/v, intraperitoneally [i.p.]) for 15 days. The ethanol-induced CPP (biased method) was developed by four injections of ethanol (0.5 g/kg, 10% w/v, i.p.) every second day. Control rats received saline instead of ethanol. Donepezil (0.5, 1 or 3 mg/kg, i.p.) or rivastigmine (0.03, 0.5 or 1 mg/kg, i.p.) were administered before ethanol during conditioning or before the reinstatement of ethanol-induced CPP. The cholinesterase inhibitors were equally effective in increasing (dose dependently) the acquisition of ethanol-induced CPP. Furthermore, priming injections of both inhibitors reinstated (cross-reinstatement) the ethanol-induced CPP with similar efficacy. These effects of both cholinesterase inhibitors were reversed by mecamylamine (3 mg/kg, i.p.), a nicotinic acetylcholine receptor antagonist, but not by scopolamine (0.5 mg/kg, i.p.), a muscarinic acetylcholine receptor antagonist. Thus, our results show that the cholinergic system is involved in the reinforcing properties of ethanol, and nicotinic acetylcholine receptors play an important role in the relapse to ethanol-seeking behaviour.

The influence of the new enkephalin derivative, cyclo[Nε,Nβ-carbonyl-d-Lys2,Dap5] enkephalinamide (cUENK6), on reinstatement of ethanol-induced conditioned place preference in rats

The aim of the present study was to determine whether a new cyclic analog of enkephalin, cyclo[N(ε),N(β)-carbonyl-d-Lys(2),Dap(5)] enkephalinamide (cUENK6), a preferential μ-(MORs), and, to a lower extent, a δ-opioid receptor (DORs) agonist in vitro, could reinstate ethanol-induced conditioned place preference (CPP). In our work, male Wistar rats were first conditioned either with ethanol (10% w/v, 0.5g/kg, intraperitoneally (i.p.)) or 0.9% NaCl in a biased CPP procedure. The intracerebroventricular (i.c.v.) administration of DORs antagonist (naltrindole, 2.5 and 5nmol) or MORs antagonist (β-funaltrexamine, 5 and 10nmol), but not the κ opioid receptor (KORs) antagonist (norbinaltorphimine, 5 and 10nmol) was then administered and inhibited the expression of ethanol-induced CPP. After the extinction session, i.c.v. administration of cUENK6 at the dose of 0.125, 0.25 and 0.5nmol occurred, and was found to reinstate the ethanol-induced CPP similar to that of the priming injection of ethanol. However, the reinstated effect of cUENK6 (0.25nmol) was strongly abolished by administration of naltrindole and, to lesser extent, by β-funaltrexamine. Furthermore, the preferential MORs agonist-morphine (13nmol, i.c.v.) and the DORs agonist-[Leu(5)]-enkephalin (2.7 and 5.4nmol, i.c.v.) also reinstated the ethanol-induced CPP. cUENK6 given alone at the dose of 0.25nmol before the testing phase had no effect in animals that received 0.9% NaCl during the conditioning phase and also did not influence their locomotor activity. These data suggest that the effects of cUENK6 did not have an impact on the results obtained in the reinstatement procedure of CPP. Overall, the data support the idea that both MORs and DORs are normally involved in the expression and reinstatement of ethanol conditioned seeking behavior - as indexed by CPP in rats.

Involvement of delta and mu opioid receptors in the acute and sensitized locomotor action of cocaine in mice

Analogs of deltorphins, such as cyclo(Nδ, Nδ-carbonyl-d-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) are functional agonists predominantly for the delta opioid receptors (DOR) in the guinea-pig ileum and mouse vas deferens bioassays. The purpose of this study was to examine an influence of these peptides (5, 10 or 20 nmol, i.c.v.) on the acute cocaine-induced (10mg/kg, i.p.) locomotor activity and the expression of sensitization to cocaine locomotor effect. Sensitization to locomotor effect of cocaine was developed by five injections of cocaine at the dose of 10mg/kg, i.p. every 3 days. Our results indicated that DK-4 and DEL-6 differently affected the acute and sensitized cocaine locomotion. Co-administration of DEL-6 with cocaine enhanced acute cocaine locomotion only at the dose of 10 nmol, with minimal effects at the doses 5 and 20 nmol, whereas co-administration of DK-4 with cocaine enhanced acute cocaine-induced locomotion in a dose-dependent manner. Similarly to the acute effects, DEL-6 only at the dose of 10 nmol but DK-4 dose-dependently enhanced the expression of cocaine sensitization. Pre-treatment with DOR antagonist - naltrindole (5 nmol, i.c.v.) and mu opioid receptor (MOR) antagonist, β-funaltrexamine abolished the ability of both peptides to potentiate the effects of cocaine. Our study suggests that MOR and DOR are involved in the interactions between cocaine and both deltorphins analogs. A distinct dose-response effects of these peptides on cocaine locomotion probably arise from differential functional activation (targeting) of the DOR and MOR by both deltorphins analogs.

Crypteins derived from the mouse neuropeptide FF (NPFF)A precursor display NPFF-like effects in nociceptive tests in mice.

NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests.