Effects of central application of naloxone on the skin temperature response in morphine-dependent rats

This study was designed to identify if the surge in tail skin temperature (TST) observed following systemic administration of naloxone to morphine-dependent rats is mediated by a specific brain locus. Female rats were fitted with cannula located either in the preoptic area-anterior hypothalamus, locus coeruleus, or the frontal cortex. TST was monitored every 5 min for 60 min following central administration of naloxone (1-40 micrograms/0.4 microliters), in morphine-dependent rats. Regardless of the central site of naloxone injection, TST was significantly increased 4-5 degrees C. A threshold dose of 20 micrograms/0.4 microliters (10 micrograms/0.2 microliters, bilaterally) of naloxone was identified to produce this surge in TST in all three brain regions. These results suggest that morphine dependency sensitizes several brain regions to administration of naloxone such that the narcotic antagonist produces a similar change in TST as is observed following its systemic administration. These data further support the use of our morphine-dependent rat model to study the central mechanisms of the menopausal hot flush, and provide additional evidence that the flush response is centrally mediated.     

Alpha-adrenergic mediation of the tail skin temperature response to naloxone in morphine-dependent rats

Studies were undertaken to evaluate the role of central noradrenergic neurons in the tail skin temperature (TST) surge that accompanies morphine withdrawal in the rat. A 5 degrees C increase in TST and a 1-2 degrees C decrease in rectal temperature (Tr) was observed following administration of a dose of naloxone HCl (NAL, 1 mg/kg, s.c.) which precipitated withdrawal in morphine-dependent rats. Intracerebroventricular (i.c.v.) injection of clonidine HCl, a partial alpha 2-adrenergic agonist did not alter TST in morphine-dependent animals. However, clonidine (10 or 50 micrograms/rat, i.c.v.) given 10 min prior to the administration of NAL completely blocked the TST response to the opiate antagonist in the morphine-dependent animals. Although NAL and clonidine reduced Tr to a similar extent in morphine-dependent rats, their effects were not additive when the drugs were administered sequentially. Treatment with the alpha-adrenergic antagonist phentolamine (11.9 or 60 micrograms/rat, i.c.v.) failed to alter TST when administered alone, but the highest dose significantly reduced the TST response to naloxone in the morphine-dependent rat. In addition, phentolamine, at high doses only, moderately reduced Tr, but the alpha-adrenergic antagonist failed to modify the decline in Tr associated with NAL-precipitated morphine withdrawal. Collectively, these data indicate that brain noradrenergic neurons play a role in the TST surge which accompanies NAL-precipitated morphine withdrawal, and that the TST and Tr responses can be dissociated in the morphine-dependent rat.     

The cloned μ, δ and κ receptors and their endogenous ligands: Evidence for two opioid peptide recognition cores

The opioid peptides are derived from three prohormone precursors referred to as proopiomelanocortin (POMC), proenkephalin (ProEnk) and prodynorphin (ProDyn). Following specific cleavage, several biologically active peptides are generated that can bind the μ, δ and κ receptors. The present study examines the receptor binding affinities of the POMC, ProEnk and ProDyn peptides to the cloned μ, δ and κ receptors expressed transiently in transfected COS-1 cells. Consistent with previous findings using brain homogenates, competition studies demonstrate that no opioid peptide family can be exclusively associated with a specific opioid receptor type. Short ProEnk peptides, such as Leu- and Met-enkephalin are selective for δ, but C-terminally extended peptides such as Met-Enk-Arg-Gly-Leu and Met-Enk-Arg-Phe have a high affinity to μ, δ and κ. Similarly, Peptide E, the BAM peptides, and metorphamide have a high affinity for all three opioid receptors types. While dynorphin A peptides and - and β-neoendorphin have a preference for κ, they also bind the cloned δ and μ receptors. Our findings do not easily fit a simple ‘message-address’ model where the Try-Gly-Gly-Phe core is extended and this gradually alters selectivity. Rather, the pattern appears more discontinuous, and would fit better with the idea of two similar but distinct cores; a Tyr-Gly-Gly-Phe Met- or Leu core that is necessary and sufficient for μ and δ but not κ and a Tyr-Gly-Gly-Phe-Met or Leu core with an Arg-X extension that is equally necessary and sufficient for κ.     

Effects of morphine and naloxone on hippocampal CA3 field potentials following systemic administration in the freely-moving rat

The effect of IV morphine, 2, 6 and 15 mg/kg, on hilar-evoked CA3 field potentials was studied to determine if this area would be more sensitive to mu-type opiate agonists than the CA1 or dentate regions. In addition, the effect of IV naloxone, 2 and 25 mg/kg, on the same responses was studied to determine if endogenous opiates reported to be present in the mossy fibers are released by electrical stimulation of this pathway. Neither morphine nor naloxone had an effect on CA3 field potentials at any dose used. The CA1 region of the hippocampus is the area most sensitive to morphine, and this effect of morphine correlates best, anatomically, with the localization of mu-receptors identified by the binding of dihydromorphine. Physiological release of endogenous opiates from the hippocampus remains to be shown.     

Possible involvement of the hypothalamic pro-opiomelanocortin gene and β-endorphin expression on acute morphine withdrawal development

We studied the effects of supraspinally administered morphine on the expression of the hypothalamic pro-opiomelanocortin (POMC) gene and β-endorphin. Mice were administered morphine intracerebroventricularly (i.c.v.) either once or 5 times for 5 days (once/day). A single morphine administration significantly increased the hypothalamic POMC gene and β-endorphin expression at 2 h after application in dose-dependent fashion; however, repeated morphine administration had no effect on the hypothalamic POMC gene and β-endorphin expression. In the immunoblot and immunohistochemical study, the increase of β-endorphin was observed in the arcuate nucleus of the hypothalamus. Moreover, the expressions of c-Fos, phosphorylated calcium/calmodulin-dependent protein kinase-IIα (pCaMK-IIα), and phosphorylated cAMP response element-binding protein (pCREB) were increased by a single i.c.v. morphine injection at various time points, but the expressions of phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2) and phosphorylated IκB (pIκB) were not. We also found that the expressions of c-Fos, pCaMKIIα, and pCREB were co-localized with the POMC expression. Meanwhile, naloxone as well as muscimol and baclofen significantly attenuated the increases of the POMC gene expression induced by a single morphine administration. Furthermore, the pretreatment of muscimol and baclofen 10 min before morphine injection robustly attenuated the withdrawal behavior induced by a single morphine administration. These results imply that the hypothalamic POMC gene and β-endorphin expression may play an important role in the development of an acute physical dependency of morphine. In that, GABAergic neurotransmission appear to be involved in the regulation of the hypothalamic POMC gene expression induced by supraspinal morphine administration.     

Catecholaminergic mediation of morphine-induced activation of pituitary-adrenocortical axis in the rat: implication of α- and β-adrenoceptors

The present study investigates the role of hypothalamic catecholamines in the effects of morphine on hypothalamo-pituitary-adrenocortical (HPA) axis. Acutely administered morphine (30 mg/kg i.p) increased plasma corticosterone and reduced the hypothalamic noradrenaline (NA) content but it did not change either the dopamine (DA) concentration or the ratio DOPAC/DA. After reserpine administration the hypothalamic contents of NA and DA were drastically reduced without changing plasma corticosterone concentrations. The increase in plasma corticosterone induced by morphine was significantly reduced by the pretreatment with reserpine. Theα₁-andα₂-antagonists prazosin and yohimbine, respectively, significantly antagonized the effect of morphine on plasma corticosterone. The β-antagonist propranolol also significantly attenuated the increase of corticosterone secretion induced by morphine. The results suggest that the action of the opiate on HPA axis activity may be dependent on stimulatory catecholaminergic systems which utilizeα₁-, α₂-andβ-adrenoceptors.     

Reserpine impairs spatial working memory performance in monkeys: reversal by the α2-adrenergic agonist clonidine

Repeated daily treatment with the catecholamine-depleting agent, reserpine, dramatically reduced performance on the delayed response task, a test of spatial working memory that depends upon the integrity of the prefrontal cortex. Delayed response performance fell from an average of 27.2/30 trials correct before reserpine treatment to an average of 20.4/30 trials correct after repeated response performance fell from an average of α₂-adrenergic agonist, clonidine (0.0001–0.05 mg/kg), to chronic reserpine-treated monkeys significantly restored performance on the delayed response task; performance after an optimal dose averaged 27.8/30 trials correct. Clonidine's beneficial effects on delayed response performance were longlasting; monkeys remained improved for more than 24 h after a single clonidine injection. The finding that clonidine is efficacious in reserpinized animals supports the hypothesis that α₂-adrenergic agonists improve cognitive function through actions at postsynaptic, α₂-adrenergic receptors on non-adrenergic cells. In contrast to the delayed response task, reserpine had little effect on performance of a visual discrimination task, a reference memory task which does not depend on the prefrontal cortex. These results emphasize the importance of postsynaptic, α₂-adrenergic mechanisms in the regulation of working memory.     

Brain β-endorphin-like immunoreactivity in adult rats given β-endorphin neonatally

β-endorphin-like immunoreactivity was measured by radioimmunoassay in the brains of adult rats treated neonatally with β-endorphin, naloxone, or vehicle. After treatment with β-endorphin, the decreases observed in β-endorphin-like immunoreactivity in the hypothalamus, pineal, midbrain, pons-medulla, hippocampus, striatum, frontal cortex, occipital cortex, and posterior cortex were highly significant but the 23% decrease in the thalamus was not significantly different from that of control rats. Neonatal administration of naloxone only resulted in a significant decrease in β-endorphin-like immunoreactivity in the hypothalamus. In contrast, no differences were discernible in content of either β-endorphin-like immunoreactivity or ACTH-like immunoreactivity in the pituitary of rats treated with β-endorphin, naloxone, or vehicle in the neonatal period. These same rats had shown an increased threshold to painful thermal stimulation by the tail-flick test after administration of either β-endorphin or naloxone at birth. The results suggest that neonatally injected β-endorphin may alter the levels of β-endorphin-like immunoreactivity in rat brain as well as the response to pain.     

Involvement of α- and β-adrenoreceptors in the central action of norepinephrine on temperature, metabolism, heart and respiratory rates of the conscious primate

The effect of the central blockade of alpha- and beta-adrenoreceptors on the action of norepinephrine (NE) infused by the intracerebroventricular (ICV) route was examined in the unanesthetized primate. After cannulae for ICV infusion were implanted sterotaxically, each monkey (Macaca fasicularis) was placed in a restraint chair fitted with a transparent dome for measurement of O2 uptake. During an experiment, the following responses were monitored simultaneously on a polygraph: skin and colonic temperatures, heart and respiratory rates, and metabolic rates as reflected by O2 uptake. Following an ICV infusion of 150 micrograms NE, colonic temperature, heart and metabolic rates declined while skin temperature increased and respiratory rate remained unchanged. Pretreatment with ICV phentolamine (100 micrograms), an alpha-adrenoreceptor antagonist, attenuated the changes in temperature, heart and metabolic rates and reduced respiratory rate. Similar ICV infusion of 100 micrograms propranolol, a beta-adrenoreceptor antagonist, tended also to attenuate the responses induced by NE, but overall was much less efficacious than phentolamine. Our results indicate that although catecholaminergic neurons mediate these vital physiological processes principally by central alpha-adrenoreceptors, a substantial beta-adrenoreceptor component also participates in the mechanism responsible for specific functional changes.     

Paradoxical analgesia produced by naloxone in diabetic mice is attributable to supersensitivity of δ-opioid receptors

The effects of naloxone on the analgesic response were examined using the tail-flick test, in mice with streptozotocin-induced diabetes. Subcutaneous injection of naloxone (5 mg/kg, s.c.) produced a marked analgesia in diabetic mice but not in age-matched non-diabetic mice. Naloxone-induced analgesia in diabetic mice was significantly reduced by pretreatment with naltrindole (0.1 mg/kg, s.c.), a selective antagonist of δ-opioid receptors. By contrast, no significant naloxone-induced increase in tail-flick latency in diabetic mice was observed after chronic treatment with naloxone (5 mg/kg, s.c.) for 5 days. However, the tail-flick latency was significantly increased by chronic treatment with naloxone in non-diabetic mice. Furthermore, the significant naloxone-induced increase in tail-flick latency in non-diabetic mice that had been chronically treated with naloxone was also antagonized by pretreatment with naltrindole. Chronic pretreatment with 5 mg/kg of naloxone for 5 days markedly attenuated the analgesic effect of the δ-agonist DPDPE in diabetic mice, whereas this pretreatment significantly enhanced the effect of DPDPE in non-diabetic mice. These results suggest that naloxone-induced ‘paradoxical’ analgesia in mice may be mediated predominantly by δ-opioid receptors.     

Differential effects of specific δ and κ opioid receptor antagonists on the bidirectional dose-dependent effect of systemic naloxone in arthritic rats, an experimental model of persistent pain

In an attempt to determine the opioid receptor class(es) which underly the two opposing effects of naloxone in models of persistent pain, we tested the action of the selective δ antagonist naltrindole, and that of the κ antagonist MR-2266 on the bidirectional effect of systemic naloxone in arthritic rats. As a nociceptive test, we used the measure of the vocalization thresholds to paw pressure. The antagonists were administered at a dose (1 mg/kg i.v. naltrindole, 0.2 mg/kg i.v. MR-2266), without action per se, but which prevents the analgesic effect of the δ agonist DTLET (3 mg/kg, i.v.) or the κ agonist U-69, 593 (1.5 mg/kg, i.v.) respectively, and does not influence the effect of morphine (1 mg/kg i.v.) or the μ agonist DAMGO (2 mg/kg, i.v.) in these animals. In arthritic rats injected with the δ antagonist, the paradoxical antinociceptive effect produced by 3 μg/kg i.v. naloxone was not significantly modified (maximal vocalization thresholds (% of control) were 146 ± 9% versus 161 ± 7% in the control group). By contrast, the hyperalgesic effect produced by 1 mg/kg i.v. naloxone was significantly reduced (maximal vocalization thresholds were87 ± 4% versus 69 ± 5% in the control group). In rats injected with the κ antagonist, the antinociceptive effect of the low dose of naloxone was almost abolished (mean vocalization thresholds were 115 ± 3% versuss 169 ± 7%) whereas the hyperalgesic effect of naloxone 1 mg/kg i.v. was not significantly modified (mean vocalization thresholds =70 ± 3% and 65 ± 3%, respectively). Based on these results, the possible role of each receptor subtype in the putative control exerted by endogenous opioid substances on nociceptive messages in pathological conditions is discussed.     

α1-Adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [¹²⁵I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70–85% specific. Various adrenergic agonists and antagonists competed for [¹²⁵I]HEAT binding to the potencies of prazosin >, yohimbine , clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showedB_max of 209 fmol/mg protein and a K_d of 184 pM for [¹²⁵I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [¹²⁵I]HEAT binding sites with a maximal response observed after 8 h at 100 μM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 μM NE.Clonidine expressed differential effects on α₁-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 μM clonidine caused a 40% decrease in the B_max of [¹²⁵I]HEAT binding without influencing the K_d value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [¹²⁵I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

α-Adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro

Extracellular recordings from 141 rat supraoptic nucleus neurons maintained in vitro in a perfused hypothalamic explant indicated that the excitability of 85% of cells was enhanced by the addition of 10–200 μM norepinephrine (NE) and the α-agonist methoxamine (MOXY), but not by isoproterenol, to the perfusion medium or by pressure application. Similar responses were observed from 7 cells maintained in synaptic isolation in medium containgng 15 mM Mg²⁺. At the lowest effective concentration (10 μM), NE and MOXY induced bursting activity without an overall increase in firing frequency; at higher concentrations an initial increase in firing frequency was followed by the appearance of phasic activity patterns. The actions of NE and MOXY were reversibly blocked by administration of the α-adrenergic antagonists phenoxybenzamine (1–10 μM), yohimbine (> 10 μM) and irreversibly blocked by prazosin (10 nM−1 μM). These observations suggest that NE has a predominatly facilitatory role to enhance the excitability and promote bursting activity in supraoptic neurosecretory neurons through an α-1 adrenoreceptor mechanism.     

Central administration of interleukin-1β increases norepinephrine turnover in the spleen

We have previously shown that intracerebroventricular(ICV) injection of interleukin-1β (IL-1β) suppressed splenic macrophage function. Sympathetic noradrenergic innervation of the spleen was implicated as a mediator of this IL-1β signal as surgical sympathectomy ablated the macrophage suppression. In this study, we have determined whether ICV administration of IL-1β has an effect on sympathetic outflow and norepinephrine (NE) turnover in the spleen. Adult male rats were injected with 5 ng of IL-1 or saline, and NE turnover in the spleen was determined using the rate of decline of NE content in the spleen after synthesis inhibition. The splenic NE turnover rate was increased significantly from 69.52 ng/g/h in saline-treated animals to 111.05 ng/g/h in IL-1-treated animals. In addition, serum corticosterone and ACTH were significantly elevated in IL-1β-treated animals 4 h postinjection. These data indicate that central administration of IL-1β increases both sympathetic outflow to the spleen and activates the hypothalamic-pituitary-adrenal axis during the period when IL-1β induces immunosuppression.     

α-Adrenergic receptor-mediated depolarization of rat neocortical astrocytes in primary culture

The membrane potentials of rat neocortical astrocytes growing in primary cultures (mean resting potential; −79 mV at [K⁺]_o = 4.5 nM) were depolarized by up to 30 mV by 10⁻⁵ M norepinephrine added to the medium, or up to 11 mV by norepinephrine or phenylephrine applied by ionophoresis. This depolarization could be inhibited by the α-adrenergic receptor antagonist phentolamine (10⁻⁵ M) but not by the β-adrenergic antagonist propranolol (10⁻⁵ M). These results suggest that the norepinephrine-evoked depolarizations seen in these cells may be mediated through an α-adrenergic receptor.     

Suppression of morphine withdrawal by electroacupuncture in rats: dynorphin and κ-opioid receptor implicated

Our previous work has demonstrated that 100-Hz electroacupuncture (EA) or 100-Hz transcutaneous electrical nerve stimulation (TENS) was very effective in ameliorating the morphine withdrawal syndrome in rats and humans. The mechanism was obscure. (1) Rats were made dependent on morphine by repeated morphine injections (5–140 mg/kg, s.c., twice a day) for eight days. They were then given 100-Hz EA for 30 min 24 h after the last injection of morphine. A marked increase in tail flick latency (TFL) was observed. This effect of 100-Hz EA could be blocked by naloxone (NX) at 20 mg/kg, but not at 1 mg/kg, suggesting that 100-Hz EA-induced analgesia observed in morphine-dependent rats is mediated by κ-opioid receptors. (2) A significant decrease of the concentration of dynorphin A (1–17) immunoreactivity (-ir) was observed in the spinal perfusate in morphine-dependent rats, that could be brought back to normal level by 100-Hz EA. (3) 100-Hz EA was very effective in suppressing NX-precipitated morphine withdrawal syndrome. This effect of EA could be prevented by intrathecal administration of nor-BNI (2.5 μg/20 μl), a κ-opioid receptor antagonist, or dynorphin A (1–13) antibodies (25 μg/20 μl) administered 10 min prior to EA. In conclusion, while the steady-state spinal dynorphin release is low in morphine-dependent rats, it can be activated by 100-Hz EA stimulation, which may be responsible for eliciting an analgesic effect and ameliorating morphine withdrawal syndrome, most probably via interacting with κ-opioid receptor at spinal level.     

α2-Adrenoceptor-mediated restraint of norepinephrine synthesis, release, and turnover during immobilization in rats

Stress-related release of norepinephrine (NE) in the brain and periphery probably underlies several neuroendocrine and neurocirculatory responses. NE might influence its own synthesis, release, and turnover, by negative feedback regulation via α₂-adrenoceptors. We examined central and peripheral noradrenergic function by measuring concentrations of NE, dihydroxyphenylglycol (DHPG), and dihydroxyphenylacetic acid (DOPAC) in hypothalamic paraventricular nucleus (PVN) microdialysate and arterial plasma simultaneously during immobilization (IMMO) in conscious rats. The α₂-adrenoceptor antagonist yohimbine (YOH) was injected i.p. or perfused locally into the PVN via the microdialysis probe. The i.p. YOH increased plasma NE, epinephrine (EPI), DHPG, dihydroxyphenylalanine, and DOPAC levels by 4.3, 7.3, 2.5, 0.6 and 1.8-fold and PVN microdialysate NE, DHPG, and DOPAC by 1.2, 0.6 and 0.5-fold. The i.p. YOH also enhanced effects of IMMO on plasma and microdialysate NE, DHPG, and DOPAC. YOH delivered via the PVN microdialysis probe did not affect microdialysate or plasma levels of the analytes at baseline and only slightly augmented microdialysate NE responses to IMMO. The results indicate that α₂-adrenoceptors tonically restrain NE synthesis, release, and turnover in sympathetic nerves and limit IMMO-induced peripheral noradrenergic activation. In the PVN, α₂-adrenoceptors do not appear to contribute to these processes tonically and exert relatively little restraint on IMMO-induced local noradrenergic activation.     

α-Adrenergic regulation of androgen receptor concentration in the preoptic area of the rat

We examined the effect of the pharmacological disruption of the catecholaminergic system on the concentration of nuclear androgen receptor, as measured by the in vitro binding of methyltrienolone ([³H]R1881) to salt extracts of anterior pituitary (AP), preoptic are (POA) and medial basal hypothalamus (MBH). Treatment of gonadectomized male and female rats with the dopamine-ß-hydroxylase inhibitor, diethyldithiocarbamate (400 mg/kg b. wt.), 30 min before treatment with dihydrotestosterone (1 mg/animal) produced a decrease in the number of nuclear androgen receptor compared with saline-treated controls (P<0.05). This effect was specific for the POA and was not present 15 h after DHT treatment. There was no effect on cytosolic androgen receptor nor was there a drug effect on the apparent dissociation constant (K_d) of [³H]R1881 binding to hypothalamus-preoptic area cytosols. Treatment of intact males and castrated, testosterone-treated males with thea₁- anda₂-adrenergic antagonists, prazosin (5 mg/kg b. wt.) and yohimbine (2 mg/kg b. wt.), respectively, resulted in a significant decrease in the number of nuclear AR 2 h following drug treatment (P<0.05). There was no effect of the ß-adrenergic receptor antagonist propranolol (10 mg/kg b. wt.) when given to intact animals, nor was there an effect of idazoxan (5 mg/kg) when given to testosterone-treated animals. The effects of yohimbine and prazosin were restricted to the POA. None of the drugs competed with the binding of [³H]R1881 for the androgen receptor nor did they alter theK_d of cytosol or nuclear androgen receptor. These data provide evidence for an adrenergic interaction with the POA androgen receptor and suggest a role for catecholamines in modulating androgen sensitivity in the rat brain.     

Endogenous opioids and ATP-sensitive potassium channels are involved in the mediation of apomorphine-induced antinociception at the spinal level: a behavioral study in rats

The effects of intrathecally (i.t.) administered glibenclamide, a blocker of adenosine triphosphate-sensitive potassium (K_ATP) channels, or naloxone on the antinociception produced by i.t. apomorphine or morphine were observed and analyzed in rats by tail-flick (TF) test. The results showed that: (1) i.t. apomorphine produced a significant and dose-dependent antinociception, (2) the antinociception produced by i.t. apomorphine could be blocked dose-dependently by i.t. glibenclamide or naloxone, (3) the antinociception produced by i.t. morphine could also be blocked dose-dependently by i.t. glibenclamide. The results suggest that endogenous opioids and ATP-sensitive potassium channels might be involved in the mediation of apomorphine-induced antinociception at the spinal level.