Analgesic effects of the progesterone metabolite, 3 alpha-hydroxy-5 alpha-pregnan-20-one, and possible modes of action in mice

The effects of intracerebroventricular (i.c.v.) administrations of the progesterone metabolite, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3A5P), on the nociceptive responses of male mice were examined. 3A5P elicited significant, dose-dependent (0.001-1.0 microgram) analgesia for 90-120 min after administration. These effects of 3A5P were significantly more potent than those of progesterone. The stereoisomer, 3 beta-hydroxy-5 alpha-pregnan-20 one (3B5P), failed to affect the nociceptive responses, indicating that the analgesic effect of 3A5P is stereospecific. The analgesic effects of 3A5P were blocked by peripheral administrations of the GABA antagonists, bicuculline and picrotoxin, and reduced by both the opiate and benzodiazepine antagonists, naloxone and Ro 15-788, respectively. The calcium channel antagonists, nifedipine and verapamil, enhanced 3A5P-induced analgesia but had no evident effects on the actions of 3B5P. These results suggest that the central analgesic effects of the progesterone metabolite, 3A5P, may arise via mechanisms involving calcium channels, the GABA-benzodiazepine-chloride complex and endogenous opioid systems.     

Reduction of predator odor-induced anxiety in mice by the neurosteroid 3α-hydroxy-4-pregnen-20-one (3αHP)

The effects of the centrally produced allylic neurosteroid, 3α-hydroxy-4-pregnen-20-one (3αHP), on the responses of male mice to an aversive, anxiety-inducing, predator (cat) odor were examined in an odor preference test. Control untreated mice displayed an anxiogenic response to the cat odor, spending a minimal amount of time in a Y-maze in the vicinity of the cat odor. Intracerebroventricular (i.c.v.) administrations of 3αHP had an anxiolytic action, resulting in significant dose-related (0.01–1.0 μg) increases in the amount of time spent in the proximity of the cat odor. These anxiolytic effects of 3αHP were stereospecific, with the stereoisomer, 3β-hydroxy-4-pregnen-20-one (3βHP) having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the response to cat odor indicating that the anxiolytic actions of 3αHP were unlikely to be related to any analgesic effects. The effects of 3αHP were significantly reduced by peripheral administrations of the GABA_A antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results indicate that the allylic neurosteroid 3αHP has anxiolytic actions involving interactions with the GABA_A receptor.     

Male preference for the odors of estrous female mice is enhanced by the neurosteroid 3α-hydroxy-4-pregnen-20-one (3αHP)

The effects of the centrally produced allylic neurosteroid, 3α-hydroxy-4-pregnen-20-one (3αHP), on the responses of male mice to the odors of estrous female mice were examined in an odor preference test. Control untreated mice displayed a significant preference for the odors of an estrous female, spending more time in a Y-maze in the vicinity of the odors of an estrous than a non-estrous female. Intracerebroventricular (i.c.v.) administrations of 3αHP enhanced male preference for the odors of estrous females, causing a significant dose-related (0.01–1.0 μg) increase in the amount of time spent in the proximity of the odors of the estrous female, while having no significant effect on the responses to the non-estrous female odors. These effects of 3αHP were stereospecific, with the stereoisomer, 3β-hydroxy-4-pregnen-20-one (3βHP), having no significant effects on odor preferences. The analgesic, morphine, also had no significant effects on the responses to female odors suggesting that the enhanced preference for estrous female odors were unlikely to be directly due to any analgesic effects of 3αHP. The effects of 3αHP were significantly reduced by peripheral administrations of the GABA_A antagonists, bicuculline and picrotoxin, but were unaffected by either the benzodiazepine antagonist, Ro 15-1788, or the opiate antagonist, naloxone. These results suggest that the neurosteroid 3αHP has facilitatory effects on olfactory mediated male sexual interest or motivation that involve interactions with the GABA_A receptor.     

Sex differences in the inhibitory effects of the NMDA antagonist, MK-801, on morphine and stress-induced analgesia

We examined the effects of intraperitoneal administrations of the noncompetitive NMDA receptor antagonist, (+) MK-801, its inactive enantiomer, (-) MK-801, and the prototypic opiate antagonist, naloxone, on restraint- and morphine-induced analgesia in male and female deer mice, Peromyscus maniculatus. Both restraint (30 min) and morphine (1.0 mg/kg) induced significant analgesic responses with male mice displaying significantly greater levels of opioid-induced analgesia than female animals. These analgesic responses were completely blocked by, naloxone (1.0 mg/kg), significantly reduced by (+) MK-801 (0.25 mg/kg) and unaffected by (-) MK-801 (0.25 mg/kg) pretreatments. There were significant male-female differences in the inhibitory effects of (+) MK-801; the higher levels of morphine- and restraint-induced analgesia of the males were completely blocked, while the lower level analgesic responses of the females were significantly reduced, but not blocked, by (+) MK-801. These observations provide further evidence that NMDA receptors are involved in the mediation of endogenous and exogenous opioid analgesia and show that there are significant male-female differences in the inhibitory effects of (+) MK-801 on opioid-mediated analgesia.     

Different opioid systems may participate in post-electro-convulsive shock (ECS) analgesia and catalepsy

Administration of electroconvulsive shock (ECS) to rats results in post-ictal analgesia and catalepsy both of which can be partially reversed by the opiate antagonists, naloxone and naltrexone. Tolerance to both phenomena develops following daily ECS administrations for 10 days. However, qualitatively different patterns of tolerance development of analgesia and catalepsy are seen. Naloxone treatment prior to ECS provides partial protection against the development of tolerance to ECS-induced catalepsy but does not prevent the tolerance to post-ECS analgesia. In contrast, the long-lasting opiate antagonist, naltrexone, blocked the development of tolerance to ECS analgesia. Furthermore, the same animals that showed tolerance to the analgesic effects of repeated ECS failed to show analgesia following the administration of 10 mg/kg of morphine while naltrexone, but not naloxone, treatment prior to ECS blocked the development of cross-tolerance to morphine analgesia. A dose-response investigation of morphine's action (5, 10, 15 and 20 mg/kg) in additional animals receiving 10 daily administrations of ECS reveals that a greater tolerance to morphine's motor inhibitory effect than to its analgesic effect results from repeated ECS administration. Finally, animals receiving daily injections of either a low (10 mg/kg) or a high (100 mg/kg) dose of morphine for 10 days showed markedly attenuated post-ECS analgesia and catalepsy. However, whereas similar effects of opiate antagonists and the chronic administration of both doses of morphine were observed with post-ECS catalepsy, analgesia was least affected by naloxone (50% of control) and most affected by the chronic high dose of morphine (14% of control). Furthermore, a similar degree of tolerance to post-ECS analgesia was seen following either repeated ECS in drug-naive animals or the chronic administration of the high dose of morphine. Thus, the partial naloxone blockade of ECS analgesia and the more substantial attenuation of post-ECS analgesia seen in morphine-tolerant animals provide different estimates of opioid involvement in these phenomena. It is proposed that these results may demonstrate the involvement of different opioid systems in analgesia and catalepsy and it is suggested that more than one opioid system may also be involved in post-ECS analgesia.     

Cholecystokinin antagonists selectively potentiate analgesia induced by endogenous opiates

We have recently observed that exogenous sulfated cholecystokinin octapeptide (CCK) can antagonize various forms of opiate analgesia and that the CCK receptor blocker proglumide potentiates morphine analgesia. These observations, plus the similarity in the distribution of CCK and opiate systems, suggest that endogenous CCK may act as a physiological opiate antagonist. We have extended these initial studies by examining the effect of CCK antagonists on opiate analgesia produced by release of endogenous opiates (front paw footshock induced analgesia) and by intrathecal administration of D-Ala-methionine enkephalinamide, a stable analogue of an endogenous opiate. Additionally, the specificity of proglumide's effect was examined by testing the effect of this drug on various forms of non-opiate analgesia. This series of experiments demonstrate that CCK antagonists can markedly potentiate analgesia induced by endogenous opiates and provide strong support for the hypothesis that endogenous CCK systems can oppose the analgesic effects of opiates. Potentiation of analgesia by CCK receptor blockers appears to be selective for opiate systems since proglumide typically attenuated or had no effect on various forms of non-opiate analgesia. These data suggest that CCK blockers may be clinically useful for enhancing the analgesic effects of procedures such as acupuncture, which may be mediated by release of endogenous opiates.     

Analgesia produced by low doses of the opiate antagonist naloxone in arthritic rats is reduced in morphine-tolerant animals

In a model of experimental chronic pain (adjuvant-induced arthritic rats), low doses of the opiate antagonist naloxone produced a profound analgesia. Maximum analgesia was seen with 3 micrograms/kg (i.v.). In contrast, hyperalgesia was obtained with much higher doses (1-3 mg/kg, i.v.). The hyperalgesic effects were not affected in arthritic animals rendered tolerant to morphine, but the paradoxical analgesic effects were significantly reduced. This decrease suggests that naloxone analgesia involves interaction with opiate receptors and that the operation of endorphinergic systems differs in normal animals and animals which experience persistent pain.     

The nature of conditioned anti-analgesia: spinal cord opiate and anti-opiate neurochemistry

The central nervous system contains circuitry that inhibits pain sensitivity (analgesia), as well as circuitry that opposes pain inhibition (anti-analgesia). Activation of analgesia systems and anti-analgesia systems can each be brought under environmental control using classical conditioning procedures. Analgesia can be produced by cues present before and during aversive events such as electric shock, while active inhibition of analgesia comes to be produced by cues never present immediately before or during shock and therefore signal safety. We have recently reported that these analgesia and anti-analgesia systems interact at the level of the spinal cord. A series of 3 experiments were performed to examine how such interactions occur. First, potential opioid mediation of conditioned analgesia was investigated using systemic and intrathecal (i.t.) delivery of opiate antagonists. Conditioned analgesia was found to be mediated by activation of spinal μ and δ opiate receptors. Second, analgesia produced by each of these receptor subtypes was challenged by environmental signals for safety. Analgesias produced by μ and δ opiate agonists were each abolished by safety signals. Third, antagonists/antisera directed against several putative anti-opiate neurotransmitters were tested i.t. to identify which mediate conditioned anti-analgesia at the level of the spinal cord. A cholecystokinin antagonist abolished conditioned anti-analgesia. In contrast, neuropeptide FF antiserum and a κ opiate antagonist were without effect.     

Dissociation of tolerance and dependence to morphine: a possible role for cholecystokinin

Since cholecystokinin (CCK) has been suggested to be an endogenous opiate antagonist, we tried to evaluate if this peptide could be involved in the development of tolerance to morphine. Naive rats were chronically administered morphine, either alone or concomitantly with proglumide or benzotript, two putative CCK receptor antagonists. Chronic treatments with both CCK antagonists alone were also established. Drugs were administered by the oral route, dissolved in the drinking water. At the end of the chronic treatments, the development of tolerance to morphine was assessed by an evaluation of the analgesic responses evoked by graded doses of acutely injected morphine in the tail-flick and hot plate tests. Proglumide and benzotript were able to inhibit the shift to the right of the dose-response curve for morphine, i.e. they prevented the development of tolerance to morphine-induced analgesia. Chronically given alone, the two CCK antagonists never modified the responses to the acute challenge with morphine. We also determined the development of physical dependence by looking at the withdrawal syndrome precipitated by graded doses of acutely injected naloxone. In these experiments the concomitant treatment with morphine and proglumide or benzotript did not modify the occurrence of dependence. These observations are consistent with the hypothesis of CCK being an endogenous opiate antagonist, involved in the development of tolerance to morphine-induced analgesia but not of dependence. Moreover, tolerance to and dependence on morphine can be pharmacologically dissociated.     

Delta opiate receptors mediate tail-shock induced antinociception at supraspinal levels.

Previous work has demonstrated that 3 pharmacologically and neuroanatomically distinct analgesia systems can be sequentially activated by increasing numbers of transcutaneous tail-shock. To date, the categorization of the early (after 2 tail-shocks) and late (after 80-100 tail-shocks) analgesias as opiate-mediated has been based on the ability of systemic naltrexone and morphine tolerance to block these effects. In contrast, the analgesia observed after 5-40 tail-shocks is unaffected by these manipulations, leading to its categorization as non-opiate. The preceding companion paper and the present work were aimed at identifying the neuroanatomical loci at which opiates exert their analgesic effects in this tail-shock paradigm and, further, to identify which opiate receptor subtypes are involved. The 8 experiments included in the present paper examined the effect of microinjecting either naltrexone (a relatively non-selective opiate receptor antagonist), binaltorphimine (kappa receptor antagonist), Cys2-Tyr3-Orn5-Pen7-amide (CTOP) (mu receptor antagonist), or naltrindole (delta receptor antagonist) either into the third ventricle or over the frontal cortex. Taken together, these experiments demonstrate that the late (80-100 shock) opiate analgesia is mediated by delta opiate receptors located within subcortical structures rostral to the 4th ventricle. No evidence for supraspinal opiate involvement in the early (2 shock) opiate analgesia was found.     

Analgesic effects of μ antagonists after naloxone non-reversible stress-induced analgesia

Three antagonists at the mu opiate receptor site: naloxone, naltrexone and diprenorphine, and one agonist-antagonist compound nalorphine, at doses usually not analgesic elicited analgesia in rats when administered after non-naloxone-reversible shock-induced analgesia had disappeared. The chi receptor antagonist, MR 2266, and the delta antagonist, ICI 154129, were all ineffective. This effect was no longer present when non-naloxone-reversible shock-induced analgesia was inhibited by the administration of the chi receptor antagonist, MR 2266. These results suggest that the mu opiate receptor may change its conformation under particular conditions such as continuous inescapable shock.     

Rats with decreased brain cholecystokinin levels show increased responsiveness to peripheral electrical stimulation-induced analgesia

Using the P77PMC strain of rat, which is genetically prone to audiogenic seizures, and also has decreased levels of cholecystokinin (CCK), we examined the analgesic response to peripheral electrical stimulation, which is, in part, opiate-mediated. A number of studies have suggested that CCK may function as an antagonist to endogenous opiate effects. Therefore, we hypothesized that the P77PMC animals would show an enhanced analgesic response based on their decreased CCK levels producing a diminished endogenous opiate antagonism. We found that the analgesic effect on tail flick latency produced by 100 Hz peripheral electrical stimulation was more potent and longer lasting in P77PMC rats than in control rats. Moreover, the potency of the stimulation-produced analgesia correlated with the vulnerability to audiogenic seizures in these rats. We were able to block the peripheral electrical stimulation-induced analgesia (PSIA) using a cholecystokinin octapeptide (CCK-8) administered parenterally. Radioimmunoassay showed that the content of CCK-8 in cerebral cortex, hippocampus and periaqueductal gray was much lower in P77PMC rat than in controls. These results suggest that low CCK-8 content in the central nervous system of the P77PMC rats may be related to the high analgesic response to peripheral electrical stimulation, and further support the notion that CCK may be an endogenous opiate antagonist.     

Inhibitory influences of FMRFamide and PLG on stress-induced opioid analgesia and activity

The effects of i.c.v. administrations of the peptide FMRFamide (Phe-Met-Arg-Phe-NH₂), as well as i.p. injections of PLG (Pro-Leu-Gly-NH₂) and the opiate antagonist, naloxone, on immobilization-induced analgesia and locomotor activity were examined in CF-1 and C57BL strains of mice. Both naloxone (1.0 mg/kg) and FMRFamide (0.10–1.0 μg) blocked the experimentally induced analgesia and activity, whereas PLG (0.10–10 mg/kg) suppressed only analgesia. These results indicate that FMRFamide (or FMRFamide-like neuropeptides) and PLG may function as differential antagonists of the behavioral and physiological consequences of endogenous opioid activation.     

Hyperalgesia mediated by peripheral opiate receptors in the rat

Behavioural experiments were undertaken to investigate the possible functional significance of opiate receptors located at peripheral endings of primary sensory neurons. The responses of animals to noxious chemical stimuli applied to the ear (ear scratch test) were measured after local pretreatment of these areas with etorphine. Local etorphine administration produced a low dose hyperalgesia and high dose analgesia. Local as opposed to systemic effects of etorphine were inferred from the absence of effects on the contralateral vehicle-treated ear. Systemic administration of naloxone or of a quaternary opiate antagonist (MRZ 2663-BR), which is relatively ineffective in crossing the blood-brain barrier, blocked the low dose hyperalgesic effect of etorphine in the ear scratch test. As a test for the putative hyperalgesic function of peripheral sensory nerve opiate receptors, neonatal rats were treated with capsaicin (50 mg/kg s.c.) to destroy specifically the subpopulation of primary sensory neurons on which the peripheral opiate receptors are thought to be located, without markedly altering pain thresholds. As adults, these neonatally treated rats showed potentiated analgesic responses to systemic morphine, as would be predicted by central ‘analgesic’ opiate receptors now acting without opposition from peripheral ‘hyperalgesic’ opiate receptors. These findings suggest that opiate receptors on primary sensory neurons may mediate hyperalgesic functions and that endogenous opioids might normally play a role in the peripheral induction of irritation, inflammation and pain reactions.     

Inhibition of baroreflex following microinjection of GABA or morphine into the nucleus tractus solitarii in rabbits

The possibility that putative transmitters may influence the aortic nerve stimulation-produced bradycardia and depressor responses was examined in urethane- and chloralose-anesthetized, paralyzed and artificially ventilated rabbits. The ipsilateral microinjection of gamma-aminobutyric acid (GABA, 2-4 micrograms) or morphine hydrochloride (2-4 micrograms) into the nucleus tractus solitarii (NTS) area could partially block the evoked bradycardia and depressor responses produced by stimulation of the aortic nerve without influencing the basal blood pressure and heart rate. This blocking effect of either GABA or morphine was dose-related. Pretreatment with GABA receptor antagonist bicuculline methiodide (0.15-0.20 micrograms) and opiate receptor antagonist naloxone hydrochloride (1-2 micrograms) into the same medullary area completely abolished the effect of GABA and morphine, respectively. Application of bicuculline also greatly antagonized the effect of morphine, but the blocking effect of GABA on the evoked bradycardia and depressor responses still existed following the pretreatment of naloxone. These results indicate that GABAergic and opiate systems present at the NTS exert an inhibitory influence on the evoked baroreflexes and inhibitory effect of the latter may be related to the activation of GABAergic receptor in this nucleus.     

Opposing actions of cimetidine on naloxone-sensitive and naloxone-insensitive forms of footshock-induced analgesia

The effects of the opiate antagonist naloxone (10 mg/kg) and the histamine H₂-antagonist cimetidine (100 mg/kg; both administered i.p.) were studied on the analgesia elicited by 3 currents of continuous-scrambled AC footshock (FSIA). Repeated analgesic measurements were made in each animal by use of the radiant heat tail-flick test. As shown by others, naloxone effectively inhibited the FSIA produced by 3 min of 2.0 mA, but had no effect on the responses elicited by higher currents (2.5 and 3.5 mA) of the same duration. Cimetidine significantly reduced the naloxone-insensitive FSIA after 3.5 mA, had no effect on that produced by 2.5 mA andpotentiated the naloxone-sensitive analgesia elicited by 2.0 mA. These findings add to existing data supporting a role for brain histamine as a mediator of naloxone-insensitive analgesia, and also suggest the possibility that histamine may mediate hyperalgesic responses.     

Potent Analgesia Induced in Rats by Combined Action at PCP and Polyamine Recognition Sites of the NMDA Receptor Complex

The present study was performed to examine the analgesic effects of the intrathecal administration of agents acting at various sites in the N -methyl- d -aspartic acid (NMDA) receptor complex on the nociceptive responses to s.c. formalin injection in rats. Both the competitive NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) and the non-competitive NMDA antagonist dizocilpine maleate (MK-801) produced dose-dependent analgesic effects in the late, but not the early, phase of the formalin test. The polyamine antagonist ifenprodil, and the strychnine-insensitive glycine antagonists DCQX and 7-chlorokynurenic acid, failed to produce any analgesic effects in either the early or the late phase of the formalin test. The analgesic effects of APV were enhanced slightly by combined administration with a non-analgesic dose of glycine, and the analgesic effects of MK-801 were dramatically potentiated by combined adminstration of a non-analgesic dose of the polyamine spermine. The results indicate that much more potent analgesia can be produced in the formalin test by a combination of open channel blockers (such as MK-801) with agonists acting at the polyamine site, than by a single treatment with antagonists to either glycine allosteric or polyamine sites within the NMDA receptor complex.     

Kappa opiate receptors mediate tail-shock induced antinociception at spinal levels.

Previous work has demonstrated that 3 pharmacologically and neuroanatomically distinct analgesia systems can be sequentially activated by increasing numbers of transcutaneous tail-shock. To date, the categorization of the early (after 2 tail-shocks) and late (after 80-100 tail-shocks) analgesias as opiate-mediated has been based on the ability of systemic naltrexone and morphine tolerance to block these effects. In contrast, the analgesia observed after 5-40 tail-shocks is unaffected by these manipulations, leading to its categorization as non-opiate. The present work and the following companion paper were aimed at identifying the neuroanatomical loci at which endogenous opiates exert their analgesic effects in this tail-shock paradigm and, further, to identify which opiate receptor subtypes are involved. The 3 experiments included in the present paper focus on the role of spinal opiates in tail-shock induced analgesia. The first experiment demonstrates that the tail-shock parameters used do not directly activate pain suppressive circuitry within the spinal cord, but rather activate centrifugal pain modulation circuitry originating within the brain. The last two experiments examine the effect of intrathecal microinjection of either naltrexone (a relatively non-selective opiate receptor antagonist), binaltorphimine (kappa receptor antagonist), Cys2-Tyr3-Orn5-Pen7-amide (CTOP) (mu receptor antagonist), or naltrindole (delta receptor antagonist). Taken together, these latter 2 experiments demonstrate that both the early (after 2 shocks) and late (after 80-100 shocks) opiate analgesias are mediated by kappa opiate receptors within the spinal cord.     

NMDA-mediated social learning of fear-induced conditioned analgesia to biting flies

Although fear conditioning has received extensive neurobiological attention little is known about social learning whereby one individual may learn and acquire the fear responses of another. A 30 min exposure to intact biting flies (stable fly, Stomoxys colcitrans L.) elicits in individual fly-naive mice analgesia and active self burying responses to avoid the flies. Fly-naive observer mice that witnessed other demonstrator mice being attacked by biting flies exhibited analgesia and self-burying to avoid flies when exposed 24 h later to altered flies whose biting mouth parts were removed. The opiate antagonist naloxone, while reducing the analgesic responses elicited by exposure to a fly-stressed demonstrator, did not affect either the subsequent conditioned analgesia or self-burying. However, the specific NMDA receptor antagonist NPC 12626, given to observers prior to, but not after, presentation of fly attacked demonstrators blocked the socially determined conditioned analgesia and self burying avoidance. This supports NMDA involvement in the mediation of the social transmission and long-term (24h) retention of conditioned analgesia and fear.     

Opiate, enkephalin, and endorphin analgesia: relations to a single subpopulation of opiate receptors

Differences in the receptor mechanisms of opiate analgesia and respiratory depression have been studied with three novel irreversible opiates. A single injection of the irreversible agonist oxymorphazone produces analgesia in mice, lasting over 24 hours. Conversely, the irreversible antagonist naloxazone dramatically reduces the analgesic effectiveness of a variety of opiate alkaloids and enkephalin analogs for over a day. Despite this marked reduction in analgesia after naloxazone treatment, morphine lethality (LD50) is unchanged in similarly treated mice. Receptor binding studies show that naloxazone irreversibly and selectively blocks a subpopulation of opiate receptors (the mu1 sites) to which all classes of opiates and enkephalins bind with highest affinity, whereas the drug has little to no effect on their lower-affinity sites (mu, and delta). The return of high-affinity receptor (mu1) binding to normal levels corresponds closely to the return of analgesic sensitivity and possibly represents receptor turnover in the central nervous system. These studies suggest that both opiate and opioid peptide analgesia is mediated through a single receptor subpopulation distinct from those involved with respiratory depression, and raise the possibility of specific opiate analgesics without respiratory depression.