Neuropeptide FF and related peptides attenuates warm-, but not cold-water swim stress-induced analgesia in mice

Neuropeptide FF (NPFF) belongs to a neuropeptide family including two receptors (NPFF(1) and NPFF(2)). NPFF system has been reported to play important roles in pain transmission. The aim of the present study was to investigate the roles of NPFF related peptides and their receptors in swim stress-induced analgesia (SIA). Nociceptive test was performed in mice stressed by forced swimming in water at 15 °C (cold water swimming) or 32 °C (warm water swimming). Warm water swimming produced a naloxone-mediated antinociceptive effect. This warm water swim SIA was dose-dependently antagonized by i.c.v. injection of NPFF and two related peptides (3-30 nmol), NPVF and dNPA, which exhibited the highest selectivities for NPFF(1) and NPFF(2) receptors, respectively. Moreover, the selective NPFF receptor antagonist RF9 (30 nmol) was inactive by itself, but prevented the effects of NPFF and related peptides. Cold-water swimming produced a wilder analgesic effect that was blocked by MK-801, but not naloxone. However, NPFF system failed to modify the cold water swim stress-induced analgesia. These findings demonstrated that NPFF and related peptides attenuated opioid-mediated form of SIA via NPFF receptors in the brain, but not non-opioid swim stress-induced analgesia. These data further support an anti-opioid character of NPFF system.     

Levorphanol and swim stress-induced analgesia in selectively bred mice: evidence for genetic commonalities

Two independent selective breeding programs have developed divergent lines of mice expressing either high and low swim stress-induced analgesia (HA/LA lines; Jastrzebiec, Poland) or high and low levorphanol analgesia (HAR/LAR lines; Portland, OR). In the present study, mice from both programs were tested for both levorphanol analgesia (2 mg/kg) and an opioid-mediated swim stress-induced analgesia (3 min swimming in 32°C water) in the hot-plate test. Mice selected for high and low levorphanol analgesia displayed high and low swim stress-induced analgesia, respectively; mice selected for high and low swim stress-induced analgesia displayed high and low levorphanol analgesia, respectively. This pattern of correlated responses suggests a high degree of common genetic determination in opiate and swim stress-induced analgesia. These findings also suggest that individual differences in analgesic responsiveness to opiate drugs result from genetically determined individual differences in endogenous pain inhibitory mechanisms.     

Evidence for opioid and non-opioid forms of stress-induced analgesia in the snail, Cepaea nemoralis

Exposure to either cold or warm stress increased the thermal nociceptive thresholds of the terrestrial snail, Cepaea nemoralis. The warm stress-induced 'analgesia' was blocked by the prototypic opiate antagonist, naloxone, and the delta-opiate antagonist, ICI 154,129, and was suppressed by a 24-h pretreatment with the irreversible opiate antagonist, beta-funaltrexamine (B-FNA). In contrast, cold stress-induced analgesia was unaffected by either naloxone, ICI 154,129 or B-FNA. These results indicate that this mollusc displays both opioid and non-opioid forms of stress-induced analgesia in a manner analogous to that reported for mammals. These findings suggest an early evolutionary development and phylogenetic continuity of opioid and non-opioid mediated stress responses to aversive environmental stimuli.     

Changes in in vivo [(3)H]-Ro15-4513 binding induced by forced swimming in mice

Mice were forced to swim for 5 min in water at a temperature of 12 degrees C (cold water swim stress) or 32 degrees C (warm water swim stress), and stress-induced analgesia (SIA) was measured using the tail-flick test. The cold water swim stress induced non-opioid SIA as well as hypothermia, whereas the warm water swim stress caused opioid SIA. The in vivo binding of [(3)H]-Ro15-4513 was measured in the stressed mice and compared with that in control mice. The specific binding of [(3)H]-Ro15-4513 in the cerebral cortex, hippocampus, and cerebellum was significantly altered by forced swimming in cold water. Apparent association and dissociation rate of [(3)H]-Ro15-4513 binding were decreased, and the change in the dissociation rate was most pronounced in the hippocampus. In contrast, no significant alterations were observed in in vitro binding. The hypothermia induced by the cold water swim stress seems to be the main reason for alterations in the specific binding of [(3)H]-Ro15-4513. The kinetics of a saturable amount of [(3)H]-Ro15-4513 in the blood and brain were also measured. The relative ratio of the radioactivity concentration in the brain to that in the blood was significantly decreased by forced swimming in cold water, indicating that the cold water swim stress induced changes in the nonspecific binding of [(3)H]-Ro15-4513 in the brain. These results together with previous reports suggested that non-opioid SIA induced by the cold water swim stress might be related to alterations in the rates of general ligand-receptor interactions including GABA(A)/benzodiazepine system. Changes in the nonspecific binding might be also involved in non-opioid SIA.     

Light-dependent effects of magnetic fields on nitric oxide activation in the land snail.

The attenuation of opioid analgaesia is a well established effect of extremely low frequency (ELF) magnetic fields with light having a modulatory role in these effects such that when the ELF exposures are carried out in the dark, the inhibitory effects on analgesia are reduced. Here, we considered the light dependency of the effects of exposure to an ELF magnetic field prior to the induction of opioid analgesia. Brief exposure to a 60 Hz field potentiated in a light dependent manner the inhibitory effects of the nitric oxide (NO) precursor L-arginine on the level of subsequent opioid-induced analgesia in the land snail Cepaea nemoralis. This suggests a general light dependency of the actions of magnetic fields and the involvement of NO in the mediation of the effects of magnetic fields.     

Opioid and non-opioid stress analgesia from cold water swim: importance of stress severity

We have previously reported that stress severity plays an important role in determining the neurochemical basis of stress-induced analgesia from inescapable footshock. Increasing severity (duration or intensity of continuous footshock) causes a shift in mediation of the resultant analgesia from opioid to non-opioid. In this study, we find that stress severity plays a similar role in analgesia from cold water swim. More severe swims (longer duration or lower water temperature) produce stress analgesia insensitive to the opiate antagonist, naltrexone, whereas less severe swims produce analgesia significantly attenuated by this drug.     

Cold water swim stress increases the expression of neurotensin mRNA in the lateral hypothalamus and medial preoptic regions of the rat brain

Stress-induced analgesia is a well-documented phenomenon that occurs in all mammalian species. Forced cold water swim produces a type of stress-induced analgesia that is independent of mu opioid receptors. The neuropeptide neurotensin (NT) has been implicated in mu opioid-independent analgesia (MOIA), but the circuitry of this system is largely unknown. The medial preoptic area (MPO) and lateral hypothalamus (LH) are two regions that are known to modulate pain processing. These two regions also contain neurotensinergic projections to the periaqueductal gray, a region that has been shown to produce MOIA upon injection of NT. The goal of this study was to determine if cold water swim (CWS) stress, which produces MOIA, activates the NT-ergic systems in these two regions. In situ hybridization results indicate that CWS increases the level of NT mRNA within neurons in the MPO and LH, suggesting that these two regions are activated during this process.     

Morphine history sensitizes postsynaptic GABA receptors on dorsal raphe serotonin neurons in a stress-induced relapse model in rats

The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Previous work has shown that the dorsal raphe nucleus (DR)-5-HT system is inhibited by swim stress via stimulation of GABA synaptic activity by the stress neurohormone corticotropin-releasing factor (CRF). Additionally, the DR 5-HT system is regulated by opioids. The present study tests the hypothesis that the DR 5-HT system regulates stress-induced opioid relapse. In the first experiment, electrophysiological recordings of GABA synaptic activity in 5-HT DR neurons were conducted in brain slices from Sprague-Dawley rats that were exposed to swim stress-induced reinstatement of previously extinguished morphine conditioned place preference (CPP). Behavioral data indicate that swim stress triggers reinstatement of morphine CPP. Electrophysiology data indicate that 5-HT neurons in the morphine-conditioned group exposed to stress had increased amplitude of inhibitory postsynaptic currents (IPSCs), which would indicate greater postsynaptic GABA receptor density and/or sensitivity, compared to saline controls exposed to stress. In the second experiment, rats were exposed to either morphine or saline CPP and extinction, and then 5-HT DR neurons from both groups were examined for sensitivity to CRF in vitro. CRF induced a greater inward current in 5-HT neurons from morphine-conditioned subjects compared to saline-conditioned subjects. These data indicate that morphine history sensitizes 5-HT DR neurons to the GABAergic inhibitory effects of stress as well as to some of the effects of CRF. These mechanisms may sensitize subjects with a morphine history to the dysphoric effects of stressors and ultimately confer an enhanced vulnerability to stress-induced opioid relapse.     

Evidence for the involvement of protein kinase C in the modulation of morphine-induced 'analgesia' and the inhibitory effects of exposure to 60-Hz magnetic fields in the snail, Cepaea nemoralis

There is substantial evidence that magnetic fields can reduce opiate-induced analgesia, with alterations in calcium channel function and/or calcium ion flux being implicated in the mediation of these inhibitory effects. The present experiments were designed to examine the effects of protein kinase C (PKC), a calcium/diacylglycerol/phospholipid-dependent protein kinase, on opiate-induced analgesia and its involvement in mediating the inhibitory effects of exposure to magnetic fields. We observed that morphine-induced antinociception, or 'analgesia', in the land snail, Cepaea nemoralis, as measured by the enhanced latency of response to a thermal (38.5 degrees C) stimulus, was reduced in dose-related manner by the PKC activator, SC-9. Exposure of snails for 2 h to a low intensity (1.0 gauss rms) 60-Hz magnetic field also reduced morphine-induced analgesia. The inhibitory effects of the 60-Hz magnetic field on morphine-induced analgesia were significantly reduced by the PKC inhibitors, H-7 and H-9, and significantly enhanced by the PKC activator, SC-9. The non-specific protein kinase inhibitor, HA-1004, and the preferential calmodulin inhibitor, W-7, had no significant effects on either morphine-induced analgesia or the inhibitory actions of exposure to the magnetic fields. These results suggest that: (1) PKC has antagonistic effects on opiate-mediated analgesia in the snail, Cepaea, and (2) that the inhibitory effects of magnetic fields on opiate-induced analgesia involve alterations in PKC.     

Antagonism of the non-opioid component of ethanol-induced analgesia by the NMDA receptor antagonist MK-801

Recent evidence from our laboratory suggests that the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) selectively antagonizes non-opioid (i.e. naloxone-insensitive) mechanisms of stress-induced analgesia in mice. For example, we have recently demonstrated that a low dose of MK-801 (0.075 mg/kg, i.p.) antagonizes the non-opioid component of a mixed opioid/non-opioid swim stress-induced analgesia (SSIA) resulting from forced swimming for 3 min in 20°C water. Since ethanol-induced analgesia (EIA) has been found to be only partially attenuated by naloxone, we hypothesized that MK-801 would similarly block the non-opioid component of EIA. The effects of MK-801 and of the opioid receptor antagonist naloxone (10 mg/kg, i.p.) on analgesia produced by ethanol (2.5 g/kg in 20% vol/vol, i.p.) were studied in control mice and in mice selectively bred for high (HA) or low (LA) SSIA. HA mice showed significantly more, and LA mice significantly less, EIA than controls. Naloxone and MK-801 significantly attenuated EIA in control and HA mice, and in these lines the combined administration of both antagonists blocked EIA completely. In LA mice, which displayed very little EIA, naloxone but not MK-801 reversed EIA completely. These findings provide additional evidence for the role of the NMDA receptor in non-opioid mechanisms of analgesia. The finding that mice selectively bred for high and low SSIA also display high and low EIA suggests common mediation of the effects of stress and ethanol on antinociceptive processes.     

Magnetic fields as environmental specific cues for morphine-induced analgesia: Interactions with tolerance development

1. It has been previously demonstrated that weak fluctuating magnetic fields can act as environmental specific cues for the development of tolerance to morphine-induced analgesia in mice. During the course of this tolerance development the basal nociceptive response of the animals preexposed to magnetic fields are also increased.

2. The magnetic field-induced increase in the basal noiciceptive response can be blocked by the opiate antagonist, naloxone, suggesting an opioid-mediated stress effect of the magnetic stimuli.

3. It is suggested that part of the actions of the magnetic stimuli as environmental specific stimuli may arise through their actions as Stressors during the tolerance acquisition phase, as well as from their effects on opioid systems.     

Reduction of stress-induced analgesia but not of exogenous opioid effects in mice lacking CB1 receptors

CB1 cannabinoid receptors are widely distributed in the central nervous system where they mediate most of the cannabinoid-induced responses. Here we have evaluated the interactions between the CB1 cannabinoid receptors and the endogenous opioid system by assaying a number of well-characterized opioid responses, e.g. antinociception and stress-mediated effects, on mutant mice in which the CB1 receptor gene was invalidated. The spontaneous responses to various nociceptive stimuli (thermal, mechanical and visceral pain) were not changed in mutant CB1 mice. Furthermore, the absence of the CB1 cannabinoid receptor did not modify the antinociceptive effects induced by different opioid agonists: morphine (preferential mu opioid agonist), D-Pen2-D-Pen5-enkephalin (DPDPE) and deltorphin II (selective delta opioid agonists), and U-50,488H (selective kappa opioid agonist) in the hot-plate and tail-immersion tests. In contrast, the stress-induced opioid mediated responses were modified in CB1 mutants. Indeed, these mutants did not exhibit antinociception following a forced swim in water at 34 degrees C and presented a decrease in the immobility induced by the previous exposure to electric footshock. However, the antinociception induced by a forced swim in water at 10 degrees C was preserved in CB1 mutants. These results indicate that CB1 receptors are not involved in the antinociceptive responses to exogenous opioids, but that a physiological interaction between the opioid and cannabinoid systems is necessary to allow the development of opioid-mediated responses to stress.     

Analgesia for formalin-induced pain by lateral hypothalamic stimulation.

Few studies of analgesia induced by electrical brain stimulation have examined the effects of brain stimulation on responses to tonic pain stimuli. Recent evidence suggests that analgesia for tonic and phasic pain may involve different neural substrates. The present study examined the effects of lateral hypothalamic (LH) stimulation on responses to tonic pain induced by subcutaneous formalin. Our findings demonstrate that LH stimulation produced analgesia for tonic pain and that the effect is primary and not related to stress-induced analgesia. The results are discussed in relation to hypotheses regarding the different neural substrates involved in analgesia for tonic and phasic pain.     

Calcium channel blockers inhibit the antagonistic effects of PheMetArgPhe-amide (FMRFamide) on morphine- and stress-induced analgesia in mice

Determinations were made of the effects of the calcium channel blockers, nifedipine and verapamil, on the antagonistic effects of FMRFamide (PheMetArgPheNH₂) and naloxone on morphine- and immobilization-induced opioid analgesia in mice. Intraperitoneal (i.p.) administrations of the calcium channel antagonists significantly reduced the inhibitory effects of intracerebroventricular (i.c.v.) FMRFamide, but had no effects on i.p. or i.c.v. naloxone-mediated inhibition of either morphine- or immobilization-induced analgesia. These results suggest that the antagonistic effects of FMRFamide, (or other endogenous FMRFamide-like peptides) on both opiate- and opioid-mediated analgesia in mice may involve alterations in the functioning of calcium channels.     

Evidence for the involvement of nitric oxide and nitric oxide synthase in the modulation of opioid-induced antinociception and the inhibitory effects of exposure to 60-Hz magnetic fields in the land snail

The attenuation of opioid peptide-mediated antinociception is a well-established effect of extremely low frequency (ELF) electromagnetic fields with alterations in calcium channel function and/or calcium ion flux and protein kinase C activity being implicated in the mediation of these effects. The present study was designed to examine the effects of nitric oxide (NO) and calcium ion/calmodulin-dependent nitric oxide synthase (NOS) on opioid-induced antinociception and their involvement in mediating the inhibitory effects of exposure to ELF magnetic fields. We observed that enkephalinase (SCH 34826)-induced, and likely enkephalin-mediated, antinociception in the land snail, Cepaea nemoralis, as measured by the enhanced latency of a foot withdrawal response to a thermal (40°C) stimulus, was reduced by the NO releasing agent, S-nitro-N-acetylpenicillamide (SNP), and enhanced by the NO synthase inhibitor, N^G-nitro- -arginine methyl ester ( -NAME). Exposure of snails to an ELF magnetic field (15 min, 60 Hz, 141 μT peak) also reduced the enkephalinase-induced antinociception. The inhibitory effects of the 60-Hz magnetic field were significantly reduced by the NO synthase inhibitor, -NAME, and significantly enhanced by the NO releasing agent, SNP, at dosages which by themselves had no evident effects on nociceptive sensitivity. These results suggest that: (1) NO and NO synthase have antagonistic effects on opioid-induced analgesia in the snail, Cepaea and (2) the inhibitory effects of ELF magnetic fields on opioid analgesia involve alteration in NO and NO synthase activity.     

Morphine-induced analgesia and exposure to low-intensity 60-Hz magnetic fields: inhibition of nocturnal analgesia in mice is a function of magnetic field intensity

In 2 experiments male CF-1 mice were exposed for 60 min, during the mid-dark period of the day-night cycle, to low-intensity (0.5-1.5 gauss, rms) 60-Hz magnetic fields and then tested for levels of analgesia induced by morphine (10 mg/kg) injections. The magnetic field exposures inhibited the degree of morphine-induced analgesia in a field intensity-dependent manner in both experiments (P less than 0.01) with the largest inhibitory effect after exposure to the 1.5-gauss field. Analysis of the combined data from the two experiments revealed a significant (P less than 0.001) linear relationship between level of analgesia and magnetic field intensity. Thus, these data demonstrated a functional relationship between the behavioral effects of morphine in mice and the strength of the 60-Hz magnetic field. Possible mechanisms underlying these effects are discussed.     

Norman Cousins Lecture. Glia as the "bad guys": implications for improving clinical pain control and the clinical utility of opioids

Within the past decade, there has been increasing recognition that glia are far more than simply "housekeepers" for neurons. This review explores two recently recognized roles of glia (microglia and astrocytes) in: (a) creating and maintaining enhanced pain states such as neuropathic pain, and (b) compromising the efficacy of morphine and other opioids for pain control. While glia have little-to-no role in pain under basal conditions, pain is amplified when glia become activated, inducing the release of proinflammatory products, especially proinflammatory cytokines. How glia are triggered to become activated is a key issue, and appears to involve a number of neuron-to-glia signals including neuronal chemokines, neurotransmitters, and substances released by damaged, dying and dead neurons. In addition, glia become increasingly activated in response to repeated administration of opioids. Products of activated glia increase neuronal excitability via numerous mechanisms, including direct receptor-mediated actions, upregulation of excitatory amino acid receptor function, downregulation of GABA receptor function, and so on. These downstream effects of glial activation amplify pain, suppress acute opioid analgesia, contribute to the apparent loss of opioid analgesia upon repeated opioid administration (tolerance), and contribute to the development of opioid dependence. The potential implications of such glial regulation of pain and opioid actions are vast, suggestive that targeting glia and their proinflammatory products may provide a novel and effective therapy for controlling clinical pain syndromes and increasing the clinical utility of analgesic drugs.     

Increased GABA binding in mouse brain following acute swim stress

Acute swim stress of mice produces increases in the density of high and low affinity binding sites in the brain for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), together with analgesia as measured by an increase in tail flick latency. Apparent tolerance develops in repeated swimming with analgesia and GABA binding returning towards control levels. The time course of analgesia and increases GABA binding following a single swim are also similar. Acute swim stress does not alter diazepam binding. GABA systems may be important in analgesia and in responses to environmental stress.     

Analgesia and impact induced by anticipation stress: involvement of the endogenous opioid peptide system

Anticipation of an unavoidable painful stimulation, i.e. anticipation stress, induces analgesia. This analgesia may result from activation of the endogenous opioid peptide system as it is blocked by naloxone. The present paper characterizes the anticipation stress further by following the kinetics of the analgesia rise, the impact of the stress on the stress-sensitive organs and the involvement of the endogenous opioid peptide system in the anticipation stress effects. Adult male rats exposed repeatedly to placing into a conditioning box followed by a painful stimulation develop analgesia almost immediately after a transfer to the box. Anticipation acts as a specific trigger, no other side stressor is effective. Anticipation stress has a significant impact on stress-sensitive organs: weights of the adrenals increase while those of the thymus and spleen decrease. These changes are associated with a significant increase of plasma corticosterone. Blockade of the endogenous opioid system by naloxone before the exposure to anticipation stress potentiates these stress-induced impacts, especially the decrease of weight of the thymus. Plasma corticosterone levels are not affected by naloxone. During the anticipation stress, the amount of opioid receptors, i.e. of [3H]naloxone binding sites, in the hypothalamus, but not in the striatum, decreases. The possible biological role of stress-induced activation of endogenous opioid system, namely the maintenance of the intensity of stress reaction within certain limits and thus the prevention of the self-destructive effects of stress reactions, is discussed.     

Stress reduces morphine's antinociceptive potency: dependence upon spinal cholecystokinin processes

The antinociceptive potency of opioids is altered by stress. We have shown that repetitive exposure of rats to noxious heat produced stress-induced analgesia as detected by the tail-flick test, but decreased the potency of the opioid beta-endorphin in the periaqueductal gray region of the midbrain (PAG). In this study, we examined the effects of this same stressor on the antinociceptive actions of the alkaloid narcotic, morphine, following either i.p. or intracerebral administration. Regardless of the route of administration, a significant reduction in the narcotic's ability to produce antinociception during stress was observed. The stress-induced reduction in morphine's potency was reversed by the intrathecal administration of the cholecystokinin (CCK) receptor antagonist L-365,260 (0.1 ng per rat), suggesting that spinal CCK-dependent 'anti-analgesic' processes are involved. Since stress influences the potency of narcotics, it may be an important physiological component to be considered in the clinical management of pain. Moreover, CCK receptor antagonists may improve the reliability of narcotic therapy.