Screening Nuclear Field Fluctuations in Quantum Dots for Indistinguishable Photon Generation.

A semiconductor quantum dot can generate highly coherent and indistinguishable single photons. However, intrinsic semiconductor dephasing mechanisms can reduce the visibility of two-photon interference. For an electron in a quantum dot, a fundamental dephasing process is the hyperfine interaction with the nuclear spin bath. Here, we directly probe the consequence of the fluctuating nuclear spins on the elastic and inelastic scattered photon spectra from a resident electron in a single dot. We find the in-plane component of the nuclear Overhauser field leads to detuned Raman scattered photons, broadened over experimental time scales by field fluctuations, which are distinguishable from both the elastic and incoherent components of the resonance fluorescence. This significantly reduces two-photon interference visibility. However, we demonstrate successful screening of the nuclear spin noise, which enables the generation of coherent single photons that exhibit high visibility two-photon interference.

Highly directional emission from a quantum emitter embedded in a hemispherical cavity.

We report the design of a solid-state, micron-sized hemispherical cavity that yields significantly enhanced extraction efficiency with modest Purcell enhancement from embedded quantum emitters. A simple analytical model provides a guideline for the design and optimization of the structure, while finite-difference time-domain simulations are used for full analysis of the optimum structure. Cavity modes with up to 90% extraction efficiency, a Purcell enhancement factor >2, and a quality factor of ≈50 are achieved. In addition, Gaussian-like far-field beam profiles with low divergence are exhibited for several modes. These monolithic cavities are promising for solid-state emitters buried in a high dielectric environment, such as self-assembled quantum dots and optically active defects in diamond.

Involvement of neuropeptide FF receptors in neuroadaptive responses to acute and chronic opiate treatments.

BACKGROUND AND PURPOSE Opiates remain the most effective compounds for alleviating severe pain across a wide range of conditions. However, their use is associated with significant side effects. Neuropeptide FF (NPFF) receptors have been implicated in several opiate-induced neuroadaptive changes including the development of tolerance. In this study, we investigated the consequences of NPFF receptor blockade on acute and chronic stimulation of opioid receptors in mice by using RF9, a potent and selective antagonist of NPFF receptors that can be administered systemically. EXPERIMENTAL APPROACH The effects of RF9 were investigated on opioid pharmacological responses including locomotor activity, antinociception, opioid-induced hyperalgesia, rewarding properties and physical dependence. KEY RESULTS RF9 had no effect on morphine-induced horizontal hyperlocomotion and slightly attenuated the decrease induced in vertical activity. Furthermore, RF9 dose-dependently blocked the long-lasting hyperalgesia produced by either acute fentanyl or chronic morphine administration. RF9 also potentiated opiate early analgesic effects and prevented the development of morphine tolerance. Finally, RF9 increased morphine-induced conditioned place preference without producing any rewarding effect by itself and decreased naltrexone-precipitated withdrawal syndrome following chronic morphine treatment. CONCLUSION AND IMPLICATIONS The NPFF system is involved in the development of two major undesirable effects: tolerance and dependence, which are clinically associated with prolonged exposure to opiates. Our findings suggest that NPFF receptors are interesting therapeutic targets to improve the analgesic efficacy of opiates by limiting the development of tolerance, and for the treatment of opioid dependence.

Decreased motivational properties of morphine in mouse models of cancerous- or inflammatory-chronic pain: implication of supraspinal neuropeptide FF(2) receptors.

Our main purpose was to evaluate the influence of cancer pain on the rewarding properties of morphine. Opioids are very addictive when used by healthy persons, conversely the occurrence of an opioid addiction seems very low when patients suffering from cancer are treated with morphine. We investigated the reinforcing properties of morphine in the place preference paradigm on a new model of mice suffering from a cancer pain induced by syngenic melanoma cells injected in the hind paw. These data were compared with mice suffering either from a short-term- or a chronic-inflammatory pain induced respectively by injection of carrageenan or complete Freund's adjuvant. Remarkably, mice suffering from cancer pain or chronic inflammatory pain did not develop any preference for the environment associated with the injection of morphine. In mice injected with melanoma cells, the specific binding of [(125)I]EYWSLAAPQRF-NH(2), an agonist of neuropeptide FF(2) receptors, was increased in several brain areas involved in the rewarding properties of opiates, including the shell of the nucleus accumbens, the major islands of Calleja, the ventral endopiriform nucleus and the amygdaloid area. Our study is the first to reveal a modification of morphine rewarding properties under cancer pain in rodents. We postulate that anti-opioid neuropeptides might contribute to the suppression of morphine rewarding effects in this murine model of cancer pain.

Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding.

By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides.

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

Functional consequences of neuropeptide FF receptors stimulation in mouse: a cerebral glucose uptake study.

The brain substrates involved in the pharmacological effects of neuropeptide FF (NPFF, Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) including interactions with opioid systems, were investigated with the [14C]-2-deoxyglucose ([14C]-2-DG) autoradiography technique in mouse. The changes in cerebral activity were mapped after i.p. administration of 1DMe ([D-Tyr1,(NMe)Phe3]NPFF; 70 mg/kg), a neuropeptide FF analogue partially resistant to peptidases, alone or in combination with morphine (15 mg/kg). 1DMe induced a rapid decrease in the cerebral activity in the thalamus, the pontine reticular nuclei and the cerebellar cortex, brain regions involved in the control of motor activity and/or the processing of sensory data. This decrease, observed when 1DMe was administered 5 min before [14C]-2-DG, was reversed by morphine, which was devoid of significant effect at this time. When administered 30 min before the radioisotope, 1DMe was without effect, whereas morphine induced a significant increase in cerebral glucose utilization in the caudate putamen, the primary somatosensory cortex, the thalamus, the superior colliculus, the pontine reticular nuclei and the spinal cord. The association of morphine and 1DMe significantly increased cerebral glucose utilization in the same regions as morphine alone and also in three additional regions: the auditory cortex, the inferior colliculus and the dorsomedial periaqueductal gray. Following systemic administration, 1DMe and morphine modulated cerebral activity in brain regions involved in pain transmission and motor control, but their effects were temporally shifted, as were their effects on horizontal locomotor activity. However, neuropeptide FF-induced changes in brain activity were modulated in part by opioid receptors activation.

Rodent strain differences in the NPFF1 and NPFF2 receptor distribution and density in the central nervous system.

The present study evaluates the putative differences between NPFF1 and NPFF2 receptor distribution and density throughout the central nervous system between rat and mouse strains by using in vitro quantitative autoradiography. The binding of [125I]YVP ([125I]YVPNLPQRF-NH2) and [125I]EYF ([125I]EYWSLAAPQRF-NH2), used to label NPFF1 and NPFF2 receptors, respectively, was compared between Sprague-Dawley and Wistar rats and between Swiss and C57BL/6-SV129 mice. In contrast to Wistar, Sprague-Dawley brains contained NPFF1 binding sites in the cortical and spinal cord areas, the accumbens nucleus, the anterodorsal thalamic nucleus, the parafascicular thalamic nucleus, the inferior colliculus and the nucleus of the solitary tract. The distribution of NPFF2 binding sites was also different between the two strains of rats. As compared to Swiss, C57BL/6-SV129 mice showed higher basal NPFF2 receptor levels in cortical areas, telencephalon and some other regions. In contrast, they showed lower amounts in thalamic structures, except the reuniens nucleus, and in mesencephalic and rhombencephalic regions. In the cervical spinal cord the levels of NPFF2 receptors were similar. The NPFF1 binding levels were nearly the same in telencephalic structures while distinct in the forebrain. Differences in amount of NPFF receptor subtypes among these strains of rats or mice could lead to differences in NPFF control of opioid nociception.

Effects of neuropeptide SF and related peptides on acid sensing ion channel 3 and sensory neuron excitability.

Acid sensing ion channel 3 (ASIC3) is a cation channel gated by extracellular protons. It is highly expressed in sensory neurons, including small nociceptive neurons and has been proposed to participate in pain perception associated with tissue acidosis and in mechanoperception. Neuropeptide FF (NPFF) and FMRFamide have been shown to potentiate proton-gated currents from cultured sensory neurons and acid sensing ion channel (ASIC) cDNA transfected cells. In this study, we report that another mammalian peptide neuropeptide SF (NPSF), derived from the same precursor, also considerably increases the amplitude of the sustained current of heterologously expressed ASIC3 (12-fold vs. 19- and nine-fold for FMRFamide and NPFF, respectively) with an EC(50) of approximately 50 microM. Similar effects were also observed on endogenous ASIC3-like sustained current recorded from DRG neurons although of smaller amplitudes (two-, three- and seven-fold increase for NPSF, NPFF and FMRFamide, respectively), and essentially related to a slowing down of the inactivation rate. Importantly, this modulation induced changes in neuronal excitability in response to an electrical stimulus applied during extracellular acidification. ASIC3-mediated sustained depolarisation, and its regulation by neuropeptides, could thus be important in regulating polymodal neuron excitability particularly under inflammatory conditions where the expression levels of both NPFF precursor and ASIC3 are increased.

Quantitative autoradiographic distribution of NPFF1 neuropeptide FF receptor in the rat brain and comparison with NPFF2 receptor by using [125I]YVP and [(125I]EYF as selective radioligands.

The selectivity of two new radioligands, [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) and [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF(1) and CHO cells expressing hNPFF(2) receptors. Saturation binding and displacement experiments showed that [(125)I]YVP and [(125)I]EYF bound selectively with a very high affinity, K(D)=0.18 nM and 0.06 nM, to NPFF(1) and NPFF(2) receptors respectively. By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF(2) binding sites, NPFF(1) and NPFF(2) receptor distribution was analyzed throughout the rat CNS. The highest densities of [(125)I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [(125)I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF(1) receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord. The restricted distribution of NPFF(1) receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF(1) system is absent, there is no strict separation between NPFF(1) and NPFF(2) system at the supraspinal level.

The neuropeptide FF analogue, 1DMe, acts as a functional opioid autoreceptor antagonist in the rat spinal cord.

We assessed the possible influence of a neuropeptide FF analogue, 1DMe ([D-Tyr(1),(NMe)Phe(3)]neuropeptide FF), on the inhibitory action of endogenous and exogenous partial differential-opioid receptor agonists on K(+)-evoked [Met(5)]-enkephalin release from superfused rat spinal cord slices. 1DMe (0.1-10 microM) dose-dependently enhanced the increase in superfusate [Met(5)]-enkephalin content due to the peptidase inhibitors thiorphan (1 microM) and bestatin (20 microM), and prevented the reduction in [Met(5)]-enkephalin release due to stimulation of partial differential receptors by 1 microM deltorphin I. Because it had the same effects as partial differential-opioid receptor antagonists, 1DMe might act through the functional blockade of presynaptically located partial differential-opioid autoreceptors.

Effect of 1DMe, a neuropeptide FF analog, on acetylcholine release from myenteric plexus of guinea pig ileum.

Since neuropeptide FF (NPFF) is a putative neurotransmitter to exert anti-opioid activity, we examined the effects of [D-Tyr', (NMe)Phe3]neuropeptide FF (IDMe), a stable NPFF analog, on acetylcholine (ACh) release from a longitudinal muscle-myenteric plexus (LMMP) preparation of guinea pig ileum in which opioids were known to inhibit ACh release when muscarinic autoinhibition was not fully activated. In the presence of atropine, 1DMe increased spontaneous and electrical field stimulation (EFS)-evoked ACh release in a concentration-dependent manner. Naloxone also increased ACh release. The stimulatory effects of 1DMe and naloxone were not additive. In the absence of atropine, 1DMe did not affect ACh release. Morphine decreased spontaneous and EFS-evoked ACh release in the presence of 1 microM atropine. 1DMe as well as naloxone counteracted the inhibitory effects of morphine on EFS-evoked ACh release. The combination of 1DMe and naloxone was not more inhibitory than either drug alone. 1DMe had no appreciable effect on norepinephrine-induced inhibition of spontaneous and EFS-evoked ACh release. These results first demonstrated the effects of a NPFF analog on neurotransmitter release: 1DMe had a stimulatory effect on spontaneous and EFS-induced ACh release from the LMMP preparation of guinea pig ileum, probably by counteracting the inhibitory effect of endogenous opioids on ACh release.

Structure-activity relationships of neuropeptide FF: role of C-terminal regions.

A structure-activity study was carried out to determine the importance of the C-terminal amino acids of the octapeptide Neuropeptide FF (NPFF) in binding and agonistic activity. Affinities of NPFF analogues were tested toward NPFF receptors of the rat spinal cord and the human NPFF2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine7 by a lysine or D. Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe8 and Arg7 residues are also extremely critical for activation of anti-opioid effects on dorsal raphe neurons. The sequence of the C-terminal dipeptide seems also to be responsible for the high affinity and the activity on human NPFF2 receptors. The results support the view that a code messaging the molecular interaction toward NPFF-receptors is expressed in the C-terminal region of these peptides but the N-terminal segment is important to gain very high affinity.

Identification of neuropeptide FF-related peptides in rodent spinal cord.

Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.

[(125)I]EYF: a new high affinity radioligand to neuropeptide FF receptors.

[(125)I]EYF ([(125)I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [(125)I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K(D) = 0.041 and 0.019 nM, respectively). Competition studies showed that [(125)I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity. Autoradiographic studies demonstrated that [(125)I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [(125)I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn. Non specific binding reached 5-10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%. These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [(125)I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.

Neuropeptide FF receptors couple to a cholera toxin-sensitive G-protein in rat dorsal raphe neurones.

In rat dorsal raphe neurones, nociceptin (300 nM) reduced the peak [Ca(2+)](i) transient, triggered by depolarization, by 36.7+/-1.8% (n=46). This effect of nociceptin decreased to 16.7+/-2.9% (n=18) after pre-treatment of the neurones with pertussis toxin (5 microg/ml, 2-6 h) but was unchanged (37.4+/-2.1%, n=44) after pre-incubation with cholera toxin (5 microg/ml, 2-6 h). This suggests that, in dorsal raphe neurones, the ORL1 receptor couples to inhibitory (G(i/o)) G-proteins. The neuropeptide FF analogue, [D-Tyr1, (N-Me)Phe(3)]neuropeptide FF (10, 100, 1000 nM), acted as an anti-opioid and reduced the effect of nociceptin (300 nM, 30 s) by 62.0+/-3.3% (n=28). Following pre-incubation with cholera toxin (5 microg/ml, 2-6 h) [D-Tyr1, (N-Me)Phe3] neuropeptide FF was unable, at the three concentrations tested, to block nociceptin activity. We conclude that, in rat dorsal raphe neurones, neuropeptide FF receptors couple to stimulatory G-proteins (Gs).

Agonist and antagonist activities on human NPFF(2) receptors of the NPY ligands GR231118 and BIBP3226.

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non-peptide or peptide NPFF-antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF(2) receptors. NPY Y(1) antagonist BIBP3226 and mixed Y(1) antagonist/Y(4) agonist GR231118 are able to displace with low affinity, 50 -- 100 nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y(2), Y(4) or Y(5) receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin-stimulated cyclic AMP production mediated by NPFF(2) receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

Functional characterization of a human receptor for neuropeptide FF and related peptides.

1. Neuropeptides FF (NPFF) and AF (NPAF) are involved in pain modulation and opioid tolerance. These peptides were known to act through uncharacterized G protein-coupled receptors (GPCR). We describe here, using an aequorin-based assay as screening tool, that an orphan GPCR, previously designated HLWAR77, is a functional high affinity receptor for NPFF and related peptides. This receptor is further designated as NPFFR. 2. Binding experiments were performed with a new radioiodinated probe, [(125)I]-EYF, derived from the EFW-NPSF sequence of the rat NPFF precursor. Chinese hamster ovary (CHO) cell membranes expressing NPFFR bound [(125)I]-EYF with a K(d) of 0.06 nM. Various NPFF analogues and related peptides inhibited [(125)I]-EYF specific binding with the following rank order (K(i)): human NPAF (0.22 nM), SQA-NPFF (0.29 nM), NPFF (0.30 nM), 1DMe (0.31 nM), EYW-NPSF (0.32 nM), QFW-NPSF (0.35 nM), 3D (1.12 nM), Met-enk-RF-NH(2) (3.25 nM), FMRF-NH(2) (10.5 nM) and NPSF (12.1 nM). 3. The stimulatory activity of the same set of peptides was measured by a functional assay based on the co-expression of NPFFR, G(alpha 16) and apoaequorin. The rank order of potency was consistent with the results of the binding assay. 4. Membranes from NPFFR expressing CHO cells bound GTP gamma[(35)S] in the presence of SQA-NPFF. This functional response was prevented by pertussis toxin treatment, demonstrating the involvement of G(i) family members. 5. SQA-NPFF inhibited forskolin induced cyclic AMP accumulation in recombinant CHO cells in a dose dependent manner. This response was abolished as well by pertussis toxin pre-treatment. 6. RT -- PCR analysis of human tissues mRNA revealed that expression of NPFFR was mainly detected in placenta, thymus and at lower levels in pituitary gland, spleen and testis.

Cholera and pertussis toxins inhibit differently hypothermic and anti-opioid effects of neuropeptide FF.

In mice pretreated intracerebroventricularly (i.c.v.) with pertussis or cholera toxins, effects of neuropeptide FF (NPFF), on hypothermia and morphine-induced analgesia, were assessed. NPFF and a potent NPFF agonist, 1DMe (0.005-22 nmol) injected into the lateral ventricle decreased morphine analgesia and produced naloxone (2.5 mg x kg(-1), s.c.)-resistant hypothermia after administration into the third ventricle. Cholera toxin (CTX 1 microg, i.c.v.) pretreatment (24 or 96 h before) inhibited the effect of 1DMe on body temperature, but failed to reverse its anti-opioid activity in the tail-flick test. CTX reduced hypothermia induced by a high dose of morphine (8 nmol, i.c.v.) but not the analgesic effect due to 3 nmol morphine. Pertussis toxin (PTX) pretreatment inhibited both morphine-hypothermia and -analgesia but did not modify hypothermia induced by 1DMe. The present results suggest that NPFF-induced hypothermia depends on the stimulation of Gs (but not Gi) proteins. In contrast, anti-opioid effects resulting from NPFF-receptor stimulation do not involve a cholera toxin-sensitive transducer protein.