Cold-restraint stress reduces [H]etorphine binding to rat brain membranes: Influence of acute and chronic morphine and naloxone

[³H]Etorphine binding was characterized in rat brain homogenates depleted of endogenous opioids from animals acutely and chronically treated with morphine or naloxone and either unstressed or subjected to a 3-h restraint period in the cold. There was significant reduction in the number of high-affinity opiate binding sites in brain tissue from stressed as compared to unstressed animals. Despite the fact that the opiate drug regimens used produce marked behavioral and physiological effects, stress-induced opiate receptor depletion was not influenced by the drugs or by withdrawal. The various drug treatments also failed to produce significant changes in opiate receptor site densities or affinities in either stressed or unstressed animals. We propose that persistent activation of opiate receptors by endogenous opioids released during restraint stress leads to receptor ‘down-regulation’.     

Prenatal stress has long-term effects on brain opiate receptors

Prenatal stress has been associated with a number of behavioral consequences including altered sensitivity to exogenous opiates. In the present study, mu opiate receptors were compared in the 42-day-old offspring from females stressed on days 15-22 of gestation and from females who were unstressed controls. Membrane homogenates from the prenatal stress group showed less binding of the mu opiate receptor ligand, [3H]DAGO in striatum but not in several other brain regions. Saturation studies suggest this difference is due to fewer striatal mu opiate receptors in offspring of prenatally stressed females. Using in vitro receptor autoradiography, the decreased binding in striatum was found mostly in the rostral striatum, extending into the nucleus accumbens with conservation of the normal anatomic distribution of receptor rich patches.     

Opiate receptor subtype binding in gerbil hippocampus is altered by forebrain ischemia

A role for endogenous opioids in trauma-induced brain injury has been supported by pharmacological studies. The present series of experiments were initiated to extend these observations by measuring opiate receptor subtype binding in gerbil hippocampus following 7 days recovery from a 10 min ischemic insult. Quantitative in vitro autoradiography was utilized to measure mu [( 3H]DAGO), kappa [( 3H]bremazocine + 10 microM morphiceptin + 100 nM DSLET), delta [( 3H]DSLET + 10 microM morphiceptin) and lambda [( 3H]naloxone + 300 nM diprenorphine) binding. While ischemic tissue samples at the level of the dorsal hippocampus showed complete loss of CA1 pyramidal cells, we observed no significant alterations in mu or delta binding suggesting a non-pyramidal cell localization of these receptors. Kappa binding decreased significantly to 88% of control in the CA1 and CA3 regions while lambda binding in the stratum lucidum (CA3) increased to 165% of control. Our results show that opiate receptor subtypes are differentially affected by an ischemic insult.     

Rat brain PCP receptors: alterations in binding parameters following chronic administration of opiate agonists and antagonists

Phencyclidine (PCP) is a widely abused drug of the arylcyclohexylamine class which is capable of producing symptoms of acute psychosis in man. PCP interacts with a specific CNS receptor, for which a putative endogenous peptide ligand has been identified. We have investigated whether PCP receptor binding parameters are modulated by activity in central opiate pathways. We have found that chronic administration of both an opiate agonist (etonitazene) and an opiate antagonist (naloxone) are able to decrease the affinity of the PCP receptor for TCP, a thienyl derivative of PCP. Furthermore, naloxone, but not etonitazene, resulted in a significant increase in the Bmax of TCP binding to the PCP receptor. These results suggest that neural activity mediated by CNS opioids systems is capable of affecting the binding parameters of the PCP receptor.     

Preoptic area opioids and opiate receptors increase during pregnancy and decrease during lactation

Opiate receptor and endogenous opioid content were determined in pregnant, lactating, ovariectomized, and ovariectomized and subsequently estradiol- and progesterone-treated adult female rats. Levels of estradiol and progesterone produced by Silastic capsules implanted in animals of the ovariectomized, hormone-treated group were similar to natural levels of those hormones induced during pregnancy. Quantitative receptor autoradiography and radioimmunoassay were used to determine [3H]naloxone binding density and immunoreactive beta-endorphin content, respectively, in the preoptic area of the hypothalamus. Both opiate receptor binding density and beta-endorphin content in the preoptic area varied in the same direction in all experimental groups. The highest levels of both were observed during pregnancy and the lowest levels during lactation. Ovariectomy without subsequent hormone treatment produced intermediate levels of both opiate receptor and beta-endorphin. Ovariectomy with experimentally-induced estradiol and progesterone levels similar to those of pregnancy produced opiate receptor density and beta-endorphin content similar to those observed in pregnant animals. These data suggest that gonadal steroids are capable of altering function of the endogenous opiate system in the preoptic area. Moreover, preoptic area levels of opioids and opiate receptors are normally elevated during pregnancy and reduced during lactation. Since opiates are known to disrupt ongoing maternal behavior, a reduction of preoptic opiate function during lactation may be required to promote normal maternal behavior. The specific preoptic region involved in opiate regulation of maternal behavior may be illustrated by the zone of opiate receptor alteration observed herein.     

Opiate receptor binding in the brain of the seizure sensitive Mongolian gerbil (Meriones unguiculatus)

Opiate receptor binding was studied in seizure sensitive (SS) and seizure resistant (SR) strains of the Mongolian gerbil. Cryostat sections of the brain were labeled with [3H]-dihydromorphine, subjected to autoradiography and analysed by microdensitometry. SS gerbils, prior to seizure induction, demonstrated overall greater brain opiate binding when compared to SR animals. Immediately following a seizure, binding in the interpeduncular nucleus fell to levels found in SR animals. The increased opiate binding in the SS (pre-seizure) compared to SR gerbils could reflect a deficit of endogenous ligand which could underlie the seizure diathesis in the gerbil.     

Differential impact of naltrexone on luteinizing hormone release during single versus repetitive exposure to restraint stress.

The objective of the present study was to determine the impact of repeated exposure to the same stressor on opiate receptor-mediated inhibition of basal and stress-related alterations of pituitary LH release. Groups of intact adult male rats were exposed to 8 hr of restraint stress for either 1 or 14 consecutive days. Animals in each group were injected intravenously with the specific opioid receptor antagonist naltrexone (NALT, 2 mg/kg bodyweight) or the vehicle saline (SAL) prior to the final scheduled stress episode. Rats pretreated with SAL prior to the single exposure to stress exhibited an increase in plasma LH over the 1st hr of stress, followed by a decline in hormone levels, which reached significance between 3 and 7 hr after initiation of the stress. NALT pretreatment of rats prior to restraint significantly blunted the suppressive effect of stress on circulating LH. Rats repeatedly exposed to stress did not show any significant alteration in plasma LH levels from prestress values at any time during their final stress episode. Pretreatment of chronically stressed rats with NALT before reexposure to stress resulted in plasma hormone levels that were not different from those in animals pretreated with SAL. When the opioid antagonist was administered to animals 24 hr after termination of single or multiple exposures to restraint, NALT-induced increases in basal plasma LH were significantly attenuated in the chronically stressed rats compared to animals subjected to stress only once or not at all.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of morphine on noradrenaline release in brain regions of stressed and non-stressed rats

Effects of morphine on noradrenaline (NA) turnover in the 8 brain regions were investigated in non-stressed and stressed rats. Morphine at 3 mg/kg and 6 mg/kg caused dose-dependent increases in levels of 3-methoxy-4-hydroxyphenylethyleneglycol sulfate (MHPG-SO₄), the major metabolite of brain NA, in the hypothalamus, amygdala, thalamus, hippocampus and midbrain and decreases in NA levels in the first 4 of these regions. In contrast to these enhancing effects of morphine on NA release in non-stressed rats, pretreatment with morphine at 6 mg/kg significantly attenuated immobilization-stress-induced increases in MHPG-SO₄ levels in the above regions. The morphine effects in both states, non-stressed and stressed, were reversed by naloxone at 0.5 mg/kg and 5 mg/kg in the hypothalamus, amygdala and thalamus. These neurochemical changes are apparently related to the distress-evoked hyperemotionality. Behavioral changes observed during the restraint stress such as struggling, vocalization and defecation were attenuated by morphine at 6 mg/kg and enhanced by naloxone at 5 mg/kg, and this action of morphine was also reversed by naloxone at 5 mg/kg. These results suggest that morphine acts to attenuate stress-induced increases in NA release in the hypothalamus, amygdala and thalamus via opiate receptors, although the drug facilitates NA release in these regions in non-stressed rats. Together with previous findings that naloxone enhances stress-induced increases in NA release selectively in these regions, it is further suggested that endogenous opioids released during stress might act to inhibit NA release in these specific brain areas and that these decreased noradrenergic activities might be closely related to the relief of the distress-evoked hyperemotionality in animals.     

Interaction of ethanol and stress with the GABA/BZ receptor in LS and SS mice

The interaction of stress and ethanol with the GABA/BZ receptor system was evaluated in LS and SS mice. The effects of two separate in vivo treatments, a 2.5 g/kg injection of ethanol or a behavioral stressor, on GABA-enhanced [3H]-FNZ binding were nearly identical in both lines of mice. A 2.5 g/kg ethanol- or stress-pretreatment resulted in increased enhancement in SS cortex, but not LS. In cerebellum, treatment effects were demonstrated in both SS and LS mice. Intraperitoneal injections of increasing doses of ethanol produced biphasic stimulation of GABA-enhanced [3H]-FNZ binding in LS brain regions, but not SS. Adrenalectomies performed one week prior to ethanol administration produced a loss of ethanol enhancement in cerebellum of both lines. However, in cortex, removal of the adrenals had no effect. The in vitro addition of 30 mM ethanol to brain preparations incubated at 37 degrees C from stressed and unstressed animals resulted in greater enhancement of binding in cortex, but not cerebellum of stressed mice. Differences in the degree of enhancement between the lines of mice were lost if the animals were stressed prior to sacrifice or if membrane preparations were incubated at 4 degrees C. The results of this study suggest that the interaction between ethanol and stress is mediated by the GABAergic system, but responses vary dependent on brain region, dose of ethanol, and degree of ethanol sensitivity.     

The sex hormone-dependent development of opiate receptors in the rat medial preoptic area

The opiate receptor content of the sexually dimorphic medial preoptic area (MPOA) was examined in newborn and 5-day-old (D6) male and female rats. A significant increase of [³H]naloxone binding was observed in and around the sexually dimorphic nucleus of the preoptic area (SDN-POA) in D6 female rats, relative to newborn females. Opiate receptor labeling did not increase over this period in males, nor was labeling different between males and females at birth. This dramatic alteration of MPOA opiate receptor content was observed to occur in either sex in the absence of testosterone postnatally; that is, neonatally-castrated males exhibited the same increase of labeling by D6 as did normal females. Conversely, daily postnatal testosterone treatment of females from birth to D6 resulted in the development of male-like MPOA opiate receptor pattern. The sex hormone-dependence of MPOA opiate receptor development is discussed in relation to the sex hormone-dependent ontogeny of SDN-POA structure. The overlap of critical periods for the development of these structural and chemical sexual dimorphisms suggests a role for endogenous opioids in modulating MPOA development.     

Involvement of the nigrotectal and nigrothalamic pathways in kappa opioid-induced circling.

The relationship between kappa opioid-induced movement and output stations of the substantia nigra pars reticulata (SNpr) was examined using the rodent circling model. Contralateral rotation produced by intranigral microinjection of the kappa opiate U50,488 was lower in animals with ibotenic acid lesions of either the ipsilateral ventromedial thalamus or superior colliculus than in control animals without lesions. These results suggest that endogenous kappa opioids in the SNpr may influence movement through actions on the nigrothalamic and nigrotectal pathways. In contrast, animals with ipsilateral lesions of the striatum showed an increase in circling relative to controls, possibly due to kappa receptor supersensitivity in the SNpr.     

Autoradiographic visualization of sex differences in the pattern and density of opiate receptors in hamster hypothalamus

Slide-mounted brain sections were used to visualize the distribution of opiate receptors in the hypothalamus of male and female hamsters using the vitro film autoradiography. Sex differences were found in the binding density and patterns of [3H]naloxone-labeled receptors. The distribution and density of [3H][D-Ala2,D-Leu5]enkephalin-labeled delta-receptors in adjacent brain sections were similar in males and females. Male hamsters showed a U-shaped pattern of [3H]naloxone binding in the sexually dimorphic nuclear complex with 28% and 34% greater labeling of the sexually dimorphic nucleus (SDN) and bed nucleus/stria terminalis (BNST), respectively, than periovulatory estrous females. Estrous and diestrous females showed a V-shaped pattern of [3H]naloxone binding in the same region, but binding density was higher at diestrus. Greater specific [3H]naloxone binding in diestrous females was also evident following extensive prewashing of slide-mounted tissue sections indicating that residual endogenous opioids were not occupying receptors, and thus, reducing the labeling of receptors in tissue from estrous females. An estrous-linked change in the affinity of hypothalamic opiate receptors was suggested by findings that [3H]naloxone binding density was greater in tissue from diestrous females when incubations were conducted in the presence of a 1-nM, but not a 10-nM, concentration of the labeled antagonist. Finally, a dense are of [3H]naloxone binding was discovered in the dorso-suprachiasmatic region of the hypothalamus. These data provide evidence for a sexual dimorphism in the distribution and density of opiate receptors in hamsters. The data suggest that mu- or kappa-receptors are more likely than delta-receptors to be involved in mediating hypothalamic effects of endogenous and exogenous opioids on reproductive functions in this species.     

The effect of neonatal exposure to chronic footshock on pain-responsiveness and sensitivity to morphine after maturation in the rat

Rat pups in 3 groups respectively were given daily footshock, exposure to a footshock apparatus without shock, or no handling from birth to 21 days of age and reared with no manipulation afterwards. After maturation (90-100 days of age), they were assessed for hot-plate paw-lick latency, morphine-induced analgesia and opiate receptor binding assay. In footshocked animals, a significant increase was found in paw-lick latency and in antinociceptive effects of morphine (1.25, 2.5, and 5.0 mg/kg) in comparison with two control groups. The antinociceptive effect of morphine in all 3 groups was antagonized by pretreatment with naloxone (2.0 mg/kg). No significant difference was found in binding activities (Bmax and Kd) for both [3H]naloxone and [3H]Dala2, D-Leu5-enkephalin between the 3 groups. These results suggest that exposure to footshock stress in the preweanling period has a long-term effect on the sensitivity of rats to painful events, probably due to chronic functional changes in endogenous opiate systems at presynaptic level rather than in postsynaptic opiate receptor binding activity.     

Naloxone alters the local metabolic rate for glucose in discrete brain regions associated with opiate withdrawal

The current 2-deoxy-d-[1-¹⁴C]glucose investigation was performed to test the hypothesis that endogenous opioids influence basal synaptic activity within discrete brain regions. To examine this hypothesis, the effects of naloxone (1.0 mg/kg s.c.) on local cerebral metabolic rate for glucose (LCMR_glu) in 84 brain regions were compared to saline controls. The specificity of naloxone's effects for opioid receptors was assessed by the coadministration of the opiate agonist morphine in a separate group. In naloxone-treated rats, there was a significant decrease in LCMR_glu in the locus coeruleus (LC) and an increase in the central nucleus of the amygdala (CAMY), supporting a tonic influence of endogenous opioids on these regions. These metabolic changes were reversed by coadministered morphine, indicating that naloxone's metabolic actions are specific for opioid receptors. Based on the role of the LC and CAMY in opiate withdrawal, the present results suggest a subthreshold naloxone precipitated withdrawal from endogenous opioids. Although morphine administered alone significantly reduced LCMR_glu in 16 brain regions, these did not include the LC or the CAMY. These results identify brain regions in which synaptic activity is under tonic modulation by endogenous opioids.     

A test of the opponent-process theory of motivation using lesions that selectively block morphine reward

The opponent-process theory of motivation postulates that motivational stimuli activate a rewarding process that is followed by an opposed aversive process in a homeostatic control mechanism. Thus, an acute injection of morphine in nondependent animals should evoke an acute rewarding response, followed by a later aversive response. Indeed, the tegmental pedunculopontine nucleus (TPP) mediates the rewarding effects of opiates in previously morphine-naive animals, but not other unconditioned effects of opiates, or learning ability. The aversive opponent process for acute morphine reward was revealed using a place-conditioning paradigm. The conditioned place aversion induced by 16-h spontaneous morphine withdrawal from an acute morphine injection in nondependent rats was abolished by TPP lesions performed prior to drug experience. However, TPP-lesioned rats did show conditioned aversions for an environment paired with the acute administration of the opioid antagonist naloxone, which blocks endogenous opioids. The results show that blocking the rewarding effects of morphine with TPP lesions also blocked the opponent aversive effects of acute morphine withdrawal in nondependent animals. Thus, this spontaneous withdrawal aversion (the opponent process) is induced by the acute rewarding effects of morphine and not by other unconditioned effects of morphine, the pharmacological effects of morphine or endogenous opioids being displaced from opiate receptors.     

Review of receptor agonist and antagonist studies relevant to the opiate system in restless legs syndrome

BACKGROUND AND PURPOSE: Therapeutic studies of the opioids in restless legs syndrome (RLS) are reviewed. PATIENTS AND METHODS: Opioids appear to be a good short and long-term treatment for RLS as shown in open-label, double-blind and long-term follow-up studies. However, the number of studies is more limited than for other therapeutic modalities in RLS such as dopaminergic agents. Furthermore, the one long-term study on the therapeutic effect of opioids on RLS published in full paper form is retrospective; prospective long-term studies are needed. RESULTS: Little addiction and tolerance were noted in any of the studies. Sleep apnea was not induced or exacerbated in short-term studies, but this was not the case in the long-term study. We have recommended that RLS patients treated with chronic opioid therapy be monitored clinically for the development of sleep apnea. Studies consistently show that the opiate receptor blocker naloxone reverses the improvement in RLS induced by the opioids, implicating the endogenous opiate system with its endorphins and enkephalins in the pathogenesis of RLS. Studies are mostly consistent in showing that naloxone does not worsen RLS symptoms in patients not treated with opioids. This result may be related to the differential ability of naloxone to antagonize exogenous opioids as opposed to endogenous opioids at the site of the opiate receptor. Naloxone does not block the therapeutic effect of dopaminergic agents on RLS symptoms, but dopamine receptor blockers do block the therapeutic effects of opioids on RLS symptoms. This suggests that the endogenous opioids act upon dopamine systems to improve the symptoms of RLS. Some of these receptor blocking studies are blinded and, in some cases, the results are dramatic. However, with one exception, there were limited numbers of patients studied with receptor blocking agents. CONCLUSION: The results of such studies therefore need further verification. In addition, future studies should focus on other methodologies, such as opiate receptor PET scanning, that might further implicate the endogenous opiate system in the pathogenesis of RLS.     

Brain stimulation of the ventral tegmental area attenuates footshock escape: an in vivo autoradiographic analysis of opiate receptors

An in vivo autoradiographic technique was employed to visualize discrete neuroanatomical changes in opiate receptor binding as a result of aversive footshock (FS) and rewarding electrical brain stimulation (ICS). Footshock-induced escape responding was shown to be attenuated by the simultaneous presentation of non-contingent ICS. Rats were divided into 4 groups (n = 6) receiving ICS, FS, ICS + FS or neither stimulus in an escape paradigm. During the final behavioral test session, rats were injected with 0.002 mg/kg [3H]diprenorphine ( [3H]Dpr) and subsequently prepared for autoradiography. Results indicated two groups of brain areas distinguishable by their treatment-induced changes in [3H]Dpr binding. One group of areas included the nucleus accumbens, claustrum, claustrocortex, perirhinal cortex and ventral tegmental area. These structures showed increased binding due to both FS and ICS. The other group consisted of the diagonal band of Broca, bed nucleus of the stria terminalis, lateral hypothalamus-medial forebrain bundle and amygdala. In these regions, an increase in binding ipsilateral to the electrode was observed in animals receiving ICS with no apparent effect of FS. These results demonstrate that non-contingent ICS may not be strictly aversive and suggest an anatomic, opioid-sensitive basis for both a rewarding and aversive component of this stimulus. It appears, further, that ICS can inhibit the release of endogenous opioid peptides in areas along the mesotelencephalic dopamine pathways, possibly to regulate the activity of neurons conveying reward information. Finally, the observed changes in opiate receptor binding may indicate a mechanism for ICS to produce both drive and reward.     

Anxiolytic-like effect induced by chronic stress is reversed by naloxone pretreatment

The present study assesses the influence of different restraint schedules on behavioral parameters determined by a conflict test, namely the light-dark transitions (LDT) as well as the opiate modulation on the behavioral consequences induced by chronic restraint. Finally, another group of animals that received naloxone (NAL) and/or chronic stress was either exposed to a single foot shock session or administered a single dose of the β-carboline FG 7142 (N′-methyl-β-carboline-3-carboxamide) immediately prior to the LDT test. We observed that a single restraint session (2 h) induced a decrease of LOT and time spent in the lit box, while chronic restraint (2 h per day for up to 7 days) induced a significant increase in both parameters. However, this increasing effect was blocked by a NAL administration (2 mg/kg IP) prior to each of the seven restraint events. A single foot shock or FG administration produced a clear anxiogenic response, an effect that was absent in animals previously submitted to chronic stress. In addition, NAL pretreatment abolished the chronic stress-induced attenuating effect on the behavioral suppression induced after either foot shock or FG administration. Therefore, these findings demonstrate that a previous history of chronic stress, leading to adaptation, induced an anxiolytic-like effect, and attenuated the behavioral supresslon produced by acute stressors. There seems to be an endogenous opiate mechanism involved in the behavioral influence induced by chronic stress.     

Anticonvulsant effects of acute and repeated fluoxetine treatment in unstressed and stressed mice

Comorbidity of epilepsy and depression is not rare. Stress can affect both depression and seizures. Therefore, it is important to know whether an antidepressant drug has pro- or anticonvulsant properties and whether these properties will be modified by stress. We tested the effects of the antidepressant drug fluoxetine on the seizure threshold for picrotoxin in unstressed and swim-stressed mice. The mice were, prior to exposure to swim stress and the intravenous infusion of picrotoxin (a non-competitive GABA(A) receptor antagonist), pretreated with fluoxetine (a selective serotonin reuptake inhibitor), either acutely or repeatedly (5 days), and the latency to the onset of two convulsant signs and death was registered. The convulsant signs were running/bouncing clonus and tonic hindlimb extension. As expected, swim stress enhanced the seizure threshold for picrotoxin. Fluoxetine (20 mg/kg ip) given acutely increased in unstressed and swim-stressed mice the dose of picrotoxin producing tonic hindlimb extension and in unstressed mice the dose of picrotoxin producing death. Neither 10 nor 20 mg/kg of fluoxetine affected doses of picrotoxin needed to produce running bouncing/clonus. Repeated treatment with fluoxetine (20 mg/kg ip) enhanced significantly in unstressed and swim-stressed mice doses of picrotoxin needed to produce tonic hindlimb extension and death, and in stressed mice also the dose of picrotoxin producing running/bouncing clonus. The results demonstrate that the antidepressant drug fluoxetine, given acutely or repeatedly, shows anticonvulsant properties against convulsions induced in unstressed and swim-stressed mice by antagonist of GABA(A) receptors, picrotoxin. Swim stress failed to modify the anticonvulsant properties of fluoxetine.     

Stress alters caffeine action on investigatory behaviour and behavioural inhibition in the mouse

A lot of studies have demonstrated that the physiological action of drugs can be modified by stressors. The present study investigates the effects of stressors on caffeine action on investigatory behaviour and behavioural inhibition in C57Bl6/J mice. For 6 consecutive days, the mice were subjected to one stressful procedure each day consisting on days 1 and 3 of immersion in cold water for 10 periods of 10s each, on days 2 and 5 of a restraint stress for 2 periods of 5 min each, and on days 3 and 6 of placing the animals in a lit openfield for a 10-min period. Saline or caffeine (30, 60 or 120 mgkg-1) were i.p. administered immediately after the last stressful experience, and the animals were tested behaviourally 10 min later. Their behaviour was compared to that of unstressed mice injected with either saline or caffeine. The results show that: (1) in saline-treated mice, stress decreased the investigatory behaviour and increased the behavioural inhibition; (2) in unstressed mice, caffeine decreased the investigatory behaviour in a dose-dependent manner; moreover, behavioural disinhibition, which appeared at low doses of the drug, did not at higher doses; (3) in stressed mice, the dose-dependent action of caffeine was almost abolished and the drug elicited, whatever the dose, a slight increase of the investigatory behaviour and a strong behavioural disinhibition. It is concluded that stress antagonizes the inhibitory action of caffeine on the investigatory behaviour and potentiates its action on behavioural disinhibition. The results are discussed in terms of interaction of stress and caffeine on the dopaminergic system.