Chronic very low dose naltrexone administration attenuates opioid withdrawal expression.

BACKGROUND: Different regimens of agonist and antagonist drugs have been used in opioid withdrawal management, with variable results. We examined whether administering extremely small quantities of opiate antagonists in the presence of opiate agonist drugs reduces withdrawal expression. METHODS: Forty-one male Sprague-Dawley rats were implanted with morphine or placebo pellets for eight days. Starting on day 3, some rats received naltrexone in their drinking water (5 mg/L), or unadulterated water. On day 8, rats were injected with saline or naltrexone (100 mg/kg) and evaluated for behavioral signs of withdrawal. Next, sections through the locus coeruleus (LC) and nucleus of the solitary tract (NTS), brainstem areas exhibiting cellular activation following opiate withdrawal, were processed for c-Fos to detect early gene expression. Finally, the same nuclei were examined for protein kinase A regulatory subunit II (PKA) and phosphorylated cyclic adenosine monophosphate response element binding protein (pCREB), using Western blot analysis. RESULTS: Withdrawal was attenuated and c-Fos, PKA, and pCREB expression was decreased in the NTS and LC of rats receiving chronic very low doses of naltrexone. CONCLUSIONS: Reduction of withdrawal upon chronic very low naltrexone administration may be due in part to decreased activation of brainstem noradrenergic neurons in morphine dependent rats.     

Opioid circuits originating from the nucleus paragigantocellularis and their potential role in opiate withdrawal

Neurons in the rat nucleus paragigantocellularis (PGi), located in the ventrolateral medulla, send collateral projections to the locus coeruleus (LC) and to the nucleus of the solitary tract (NTS). The present study examined whether neurons in the PGi that project to both the LC and NTS contain leucine(5)-enkephalin (ENK), and also whether opioid-containing neurons in the PGi are activated following withdrawal from opiates. Retrograde transport of Fluoro-Gold (FG) from the LC and transport of a protein-gold tracer from the NTS was combined with detection of an antibody directed against ENK in the PGi. Using fluorescence and brightfield microscopy, it was established that more than half of the neurons containing both FG and the protein-gold tracer, also exhibited immunolabeling for ENK. The most frequent location of triply labeled neurons was the retrofacial portion of the PGi. In a separate series, rats were chronically implanted with morphine or placebo pellets and, on the fifth day, were subjected to an intraperitoneal injection of naltrexone. Two hours following initiation of withdrawal, rat brains were obtained and processed for detection of c-fos and in situ hybridization labeling of preproenkephalin (PPE) mRNA. Naltrexone injections into morphine-dependent rats caused a dramatic increase in c-fos as compared to control rats. Approximately 66% of the c-fos-labeled neurons exhibited labeling for PPE mRNA. These were also enriched in the retrofacial portion of the PGi. Taken together, the present data indicate that withdrawal from opiates engages opioid neurons in the PGi, some of which may coordinate activity of neurons in both the NTS and the LC.     

Lack of effect of chronic morphine treatment and naloxone-precipitated withdrawal on tyrosine hydroxylase,galanin, and neuropeptide Y mRNA levels in the rat locus coeruleus

Morphine dependence was experimentally induced in rats by daily injection of increasing doses of morphine for seven days. Withdrawal was precipitated in half of the morphine-dependent rats by a single injection of naloxone on day 8. Behavioral signs of withdrawal weret evident in the morphine/naloxone group. Gene expression in locus coeruleus (LC) neurons was investigated using quantitative in situ hybridization analysis. Messenger RNA (mRNA) levels for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and for precursors to galanin (GAL) and neuropep tide Y (NPY), peptides that coexist with norepinephrine in LC neurons, were not altered by chronic morphine treatment or naloxone-precipitated withdrawal. In contrast, mRNA levels for c fos were dramatically elevated in the LC following naloxone-precipitated withdrawal. Chronic morphine treatment caused a small decrease in levels of mRNA encoding the precursor to corticotropin-releasing factor (CRF) in Barrington's nucleus. Although long-term adaptations of LC neurons have previously been implicated in the development of morphine tolerance, dependence, and withdrawal, alterations in the levels of TH, GAL, or NPY mRNA in the LC apparently do not underlie this process. © 1995 Wiley-Liss, Inc.     

Activation of neuropeptide FF neurons in the brainstem nucleus tractus solitarius following cardiovascular challenge and opiate withdrawal

Neuropeptide FF (NPFF), a morphine modulatory peptide, is localized within discrete autonomic regions including the brainstem nucleus tractus solitarius (NTS) and the parabrachial nucleus (PBN). We investigated the activation of NPFF neurons in the NTS of rats induced by cardiovascular challenge and centrally generated opiate withdrawal. For hypotensive stimulation, we used systemic infusions of sodium nitroprusside (NP) or hemorrhage (HEM), and hypertension was achieved by intravenous phenylephrine (PHENYL) or angiotensin II (AII). In rats that received continuous intracerebroventricular injections of morphine, intraperitoneal injections of naloxone precipitated behavioural signs of opioid withdrawal. Activated NTS neurons were identified by using a combined immunohistochemistry for Fos and NPFF, and neurons projecting to the PBN were determined with a retrograde tracer. HEM, administration of vasoactive drugs, and opiate withdrawal produced a very robust activation of NTS neurons. In NP and HEM groups, 25.6 ± 3.2% and 7.6 ± 1.3 % of NPFF neurons were activated, respectively. Lesser numbers of NPFF neurons were activated in the PHENYL (4.6 ± 1.6%) and AII (2.4 ± 0.8%) groups. However, following opiate withdrawal, virtually no Fos expression was observed in NPFF neurons. NPFF neurons activated during NP infusion constituted the largest number of cells projecting to the PBN. This study shows that NPFF neurons in NTS that project to the PBN respond selectively to NP as opposed to other cardiovascular challenges or opiate withdrawal. These data support an emerging and important role for NPFF in the context of central cardiovascular regulation. J. Comp. Neurol. 402:210–221, 1998. © 1998 Wiley-Liss, Inc.     

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla

In urethane anesthetized rats, iontophoretic application of morphine or α-methylnoradrenaline (α-MNE) inhibited (80–100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of α-MNE was blocked selectively by theα₂-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75–100%) by low i.v. doses of clonidine (10–15 μg/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 ± 0.9 spikes/s to 12.3 ± 1.5 spikes/s;n = 8). During withdrawal these cells could still be inhibited (80–100%) by i.v. injection of clonidine (15 μg/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenregic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive forα₂A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate andα₂-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal, Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.     

Alterations of BDNF and NT-3 genes expression in the nucleus paragigantocellularis during morphine dependency and withdrawal

Locus coeruleus (LC) plays a key role in opioid dependence and withdrawal. Chronic morphine administration induces neurochemical adaptations in the noradrenergic system. The nature of signal responsible for opiate-induced adaptations of noradrenergic neurons in LC is not well defined. Neurotrophins-signaling pathways such as brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3) play a key role for regulating the noradrenergic response of LC neurons to opiates. The nucleus paragigantocellularis (PGi) is one of the two major afferents to LC. The present study was designed to evaluate the expression of BDNF and NT-3 in the context of opiate dependence and withdrawal in PGi. Such data are important because they could reveal the role of PGi as an additional source of BDNF and NT-3 in the neurochemical plasticity of LC neurons. Opiate dependence was induced by a progressive intraperitoneal treatment of morphine. In morphine dependent group PGi nucleus was extracted for gene expression assay 6h after the last injection of morphine. In spontaneous withdrawal, rats received the same chronic treatment as morphine group. PGi was extracted for gene expression assay 24, 48 and 72 h after the last injection of morphine. PGi nucleus was assayed for the expression of BDNF and NT-3 using semi-quantitative RT-PCR normalized to beta-actin gene expression. Results showed that chronic administration of morphine significantly increased BDNF and NT-3 gene expression in PGi. In spontaneous withdrawal, BDNF/NT-3 genes expression were high in comparison to control group. It seems that BDNF/NT-3 -signaling pathway originating from PGi is essential for opiate-induced adaptations of the LC neurons.     

Chronic selective activation of excitatory opioid receptor functions in sensory neurons results in opioid 'dependence' without tolerance.

We previously showed that mouse sensory dorsal root ganglion (DRG) neurons chronically exposed to 1 microM D-ala2-D-leu5-enkephalin (DADLE) or morphine for > 2-3 days in culture become tolerant to the usual opioid inhibitory receptor-mediated effects, i.e. shortening of the duration of the calcium-dependent component of the action potential (APD), and supersensitive to opioid excitatory APD-prolonging effects elicited by low opioid concentrations. Whereas nanomolar concentrations of dynorphin(1-13) or morphine are generally required to prolong the APD of naive DRG neurons (by activating excitatory opioid receptors), femtomolar levels become effective after chronic opioid treatment. Whereas 1-30 nM naloxone or diprenorphine prevent both excitatory and inhibitory opioid effects but do not alter the APD of native DRG neurons, both opioid antagonists unexpectedly prolong the APD of most of the chronic opioid-treated cells. In the present study, chronic exposure of DRG neurons to 1 microM DADLE together with cholera toxin-B subunit (which selectively blocks GM1 ganglioside-regulated opioid excitatory, but not inhibitory, receptor functions) prevented the development of opioid excitatory supersensitivity and markedly attenuated tolerance to opioid inhibitory effects. Conversely, sustained exposure of DRG neurons to 1 nM DADLE, which selectively activates excitatory opioid receptor functions, resulted in characteristic opioid excitatory supersensitivity but no tolerance. These results suggest that 'dependence'-like properties can be induced in chronic opioid-treated sensory neurons in the absence of tolerance. On the other hand, development of some components of tolerance in these cells may require sustained activation of both excitatory, as well as inhibitory, opioid receptor functions.     

Involvement of neuropeptide Y in the acute, chronic and withdrawal responses of morphine in nociception in neuropathic rats: Behavioral and neuroanatomical correlates

Although morphine is a potent antinociceptive agent, its chronic use developed tolerance in neuropathic pain (NP). Furthermore, opioid antagonist naloxone attenuated the antinociceptive effect of neuropeptide Y (NPY). The present study investigated the role of NPY and NPY Y1/Y5 receptors in acute and chronic actions of morphine in neuropathic rats using thermal paw withdrawal test and immunocytochemistry. In acute study, intracerebroventricular (icv) administration of morphine, NPY or NPY Y1/Y5 receptors agonist [Leu³¹, Pro³⁴]-NPY produced antinociception, whereas selective NPY Y1 receptors antagonist BIBP3226 caused hyperalgesia. While NPY or [Leu³¹, Pro³⁴]-NPY potentiated, BIBP3226 attenuated morphine induced antinociception. Chronic icv infusion of morphine via osmotic minipumps developed tolerance to its antinociceptive effect, and produced hyperalgesia following withdrawal. However, co-administration of NPY or [Leu³¹, Pro³⁴]-NPY prevented the development of tolerance and withdrawal hyperalgesia. Sciatic nerve ligation resulted in significant increase in the NPY-immunoreactive (NPY-ir) fibers in ventrolateral periaqueductal gray (VLPAG) and locus coeruleus (LC); fibers in the dorsal part of dorsal raphe nucleus (DRD) did not respond. While chronic morphine treatment significantly reduced NPY-ir fibers in VLPAG and DRD, morphine withdrawal triggered significant augmentation in NPY-immunoreactivity in the VLPAG. NPY-immunoreactivity profile of LC remained unchanged in all the morphine treatment conditions. Furthermore, removal of sciatic nerve ligation reversed the effects of NP, increased pain threshold and restored NPY-ir fiber population in VLPAG. NPY, perhaps acting via Y1/Y5 receptors, might profoundly influence the processing of NP information and interact with the endogenous opioid system primarily within the framework of the VLPAG.     

Ultrastructural changes in rat locus coeruleus induced by chronic opioids

The locus coeruleus (LC) is a major noradrenergic nucleus in the brain. The activity of the LC neurons is chronically regulated by opioids. So far, very little is known about the morphological changes induced by chronic treatment with opioids. In the present study, the effects of chronic treatment with morphine and dihydroetorphine, a new narcotic analgesic with lower physical dependence potential than morphine, were investigated on the ultrastructure of the rat LC. Rats received saline or increasing doses of morphine or dihydroetorphine for 5 days by twice daily subcutaneous injections. Withdrawal was precipitated in half of the opioid-treated rats by a single intraperitoneal injection of naloxone 4 h after the last injections of opioids. The ultrastructure of the LC was examined by electron microscopy. Results showed that chronic morphine treatment induced a marked injury to the LC neurons. The primary changes in the cell body were the indentation of nuclei, the fragmentation and degranulation of rough endoplasmic reticulum, as well as the disaggregation of polyribosomes. Myelinoid bodies were seen in the processes. An accumulation of presynaptic vesicles was observed in some of the terminals which formed synaptic junctions with the LC neurons as compared to the normal controls. Naloxone-precipitated withdrawal from morphine did not stop the morphine-induced injury on the LC neurons except that less accumulation of presynaptic vesicles occurred. Chronic dihydroetorphine treatment only induced a slight change in the ultrastructure of the LC neurons. These results indicate that the LC neurons are more vulnerable to chronic treatment with morphine than to that with dihydroetorphine. Received: 30 November 1995 / Revised: 11 January 1996 / Revised, accepted: 3 February 1997     

Locus ceruleus discharge characteristics of morphine-dependent rats: effects of naltrexone

Spontaneous and sensory-evoked discharge was recorded from locus ceruleus (LC) neurons of halothane-anesthetized rats that were chronically administered morphine. LC spontaneous discharge rates of morphine-treated rats were comparable to those of rats chronically administered saline. Administration of 1.0 micrograms morphine (i.c.v.), a dose which completely inhibits LC discharge of morphine-naive rats, had no effect on LC spontaneous discharge of morphine-treated rats, demonstrating that opiate tolerance had developed. Naltrexone, 0.3 and 1.0 microgram i.c.v., produced increases in LC spontaneous discharge rates that were 172 and 166% greater than baseline, respectively. Additionally, naltrexone disrupted LC discharge evoked by repeated sciatic nerve stimulation such that evoked discharge was decreased with respect to tonic discharge, and postactivation inhibition was attenuated. Naltrexone did not alter spontaneous or sensory-evoked LC discharge of rats chronically administered saline indicating that these neuronal effects are specific to opiate withdrawal. Pretreatment of rats with dexamethasone, or with an antagonist of corticotropin-releasing factor (CRF), alpha-helical CRF, did not attenuate the effects of naltrexone on LC discharge of morphine tolerant rats. The present study confirms other reports of LC activation associated with antagonist precipitated opiate withdrawal in vivo, and extends these observations by characterizing the disruptive effect of opiate withdrawal on the response of LC cells to phasically presented sensory stimuli, and demonstrating that the withdrawal response is not mediated by release of endogenous CRF.     

Low dose naltrexone administration in morphine dependent rats attenuates withdrawal-induced norepinephrine efflux in forebrain

The administration of low dose opioid antagonists has been explored as a potential means of detoxification in opiate dependence. Previous results from our laboratory have shown that concurrent administration of low dose naltrexone in the drinking water of rats implanted with subcutaneous morphine pellets attenuates behavioral and biochemical signs of withdrawal in brainstem noradrenergic nuclei. Noradrenergic projections originating from the nucleus tractus solitarius (NTS) and the locus coeruleus (LC) have previously been shown to be important neural substrates involved in the somatic expression of opiate withdrawal. The hypothesis that low dose naltrexone treatment attenuates noradrenergic hyperactivity typically associated with opiate withdrawal was examined in the present study by assessing norepinephrine tissue content and norepinephrine efflux using in vivo microdialysis coupled to high performance liquid chromatography (HPLC) with electrochemical detection (ED). The frontal cortex (FC), amygdala, bed nucleus of the stria terminalis (BNST) and cerebellum were analyzed for tissue content of norepinephrine following withdrawal in morphine dependent rats. Naltrexone-precipitated withdrawal elicited a significant decrease in tissue content of norepinephrine in the BNST and amygdala. This decrease was significantly attenuated in the BNST of rats that received low dose naltrexone pre-treatment compared to controls. No significant difference was observed in the other brain regions examined. In a separate group of rats, norepinephrine efflux was assessed with in vivo microdialysis in the BNST or the FC of morphine dependent rats or placebo treated rats subjected to naltrexone-precipitated withdrawal that received either naltrexone in their drinking water (5 mg/L) or unadulterated water. Following baseline dialysate collection, withdrawal was precipitated by injection of naltrexone and sample collection continued for an additional 4 h. At the end of the experiment, animals were transcardially perfused and the brains were removed for verification of probe placement. Low dose naltrexone pre-treatment significantly attenuated withdrawal-induced increases of extracellular norepinephrine in the BNST, with a smaller effect in the FC. These findings suggest that alterations in norepinephrine release associated with withdrawal may be attenuated in forebrain targets of noradrenergic brainstem neurons that may underlie reduced behavioral signs of withdrawal following low dose naltrexone administration.     

Cerebral glucose metabolism during opioid withdrawal following methylnaloxonium injection into the locus coeruleus

Previous studies have demonstrated a widespread stimulation of regional cerebral metabolic rate(s) for glucose (rCMRglc) in morphine-dependent rats subjected to opioid withdrawal precipitated by systemic injection of naloxone. Nonetheless, many of the behavioral signs of opioid withdrawal are produced by intracerebral injections of an opioid antagonist, methylnaloxonium (MN), into the locus coeruleus (LC). The purpose of the present work was to determine the extent to which cerebral metabolic alterations in opioid withdrawal could be initiated by a local action in LC. Intracerebral injections of MN into LC increased rCMRglc in morphine-dependent rats, and the anatomical distribution of this effect was similar to that produced by systemic injections of naloxone. The present data support the view that LC is a major substrate of opioid withdrawal in the brain, and they suggest that LC plays an important role in changing rCMRglc during opioid withdrawal induced by systemic naloxone administration.     

Opposing effects of morphine and the neurotrophins, NT-3, NT-4, and BDNF, on locus coeruleus neurons in vitro

The development of noradrenergic locus coeruleus (LC) neurons is subject to regulation by multiple epigenetic signals. To examine the potential regulation of LC ontogeny by opiates and neurotrophins, we studied the effects of morphine and NT-3, NT-4, and BDNF on the survival and differentiation of LC neurons from prenatal rats in dissociated cell culture. Noradrenergic cells were identified and counted following tyrosine hydroxylase (TH) immunocytochemistry, and their state of differentiation was assessed by measuring norepinephrine (NE) uptake. Treating LC cultures with morphine starting on day 1 after plating resulted in a 20% decrease in NE uptake and a small (12%) but significant decrease in the number of TH-immunoreactive (TH + ) cells. Application of morphine on day 4 after plating had the same effect on NE uptake without influencing TH + cell number. This effect of morphine was blocked by concomitant exposure to naloxone (an opioid receptor antagonist), and mimicked by exposure to opioid peptides. Treatment of cultures with the neurotrophins, NT-3 or NT-4, increased NE uptake and TH + cell number, as reported previously. Moreover, we show for the first time that brain-derived neurotrophic factor (BDNF) exerts similar effects, with a large (110%) increase in NE uptake and a modest (20%) increase in TH + cell number. Cotreatment of LC cultures with morphine and NT-3 resulted in an attenuation of the NT-3 effect on both NE uptake and the number of TH + cells. In contrast, cotreatment of LC cultures with morphine and NT-4 or BDNF attenuated the neurotrophin effect on TH + cell number but not on NE uptake. Our results raise the possibility that opioid peptides may modulate the influence of neurotrophins on LC neuronal survival and differentiation.     

Prolonged reversal of morphine tolerance with no reversal of dependence by protein kinase C inhibitors

The phosphatidylinositol (PI) cascade plays a pivotal role in mediating behavioral tolerance to the antinociceptive effects of morphine. Earlier we reported that antinociceptive tolerance was completely reversed 30 min after the administration of inhibitors of each step in the PI cascade. The aim of this study was to determine whether injection of a single dose of protein kinase C (PKC) inhibitor would elicit a prolonged reversal of morphine tolerance for up to 24 h. Three days after implantation of placebo- or 75-mg morphine pellets, mice received intracerebroventricular (i.c.v.) injections of vehicle or PKC inhibitor drug. Morphine challenge doses were then administered 4, 8 and 24 h later to test for tolerance reversal. In non-tolerant mice, G?-7874 and sangivamycin had no effect on the potency of morphine. However, G?-7874 and sangivamycin significantly reversed morphine tolerance at 4, 8 and 24 h. In addition, the role of PKC in morphine physical dependence was determined. G?-7874 and sangivamycin by themselves did not precipitate spontaneous morphine withdrawal. Therefore, experiments were conducted to determine whether the PKC inhibitors would block naloxone-precipitated withdrawal. However, neither a 30-min nor a 24-h pretreatment with G?-7874 or sangivamycin blocked naloxone withdrawal. Our results along with other publications indicate that PKC is a pivotal kinase essential for maintaining animals in an opioid tolerant state. Finally, the use of persistent PKC inhibitors that lasted for 24 h demonstrated that the neuronal systems in these animals did not adapt by increasing the activity of other protein kinase cascades to re-establish morphine tolerance.     

GABA and opioid mechanisms of the central amygdala underlie the withdrawal-potentiated startle from acute morphine

Anxiety is an affective symptom common to withdrawal from acute or chronic opiate treatment. Although the potentiation of the acoustic startle reflex has been proposed as an index of increased anxiety, there are variable effects of the opiate withdrawal on the startle reflex in chronic dependence models. On the other hand, withdrawal from acute morphine treatment consistently potentiates the acoustic startle reflex, a response that seems to be mediated by the central nucleus of the amygdala (CeA). However, the underlying neurochemical mechanisms have not been elucidated yet. In the present study, we firstly made a comparison between the effects of the withdrawal from both acute and chronic treatments with morphine on the motor activity and the anxiety-like behavior of rats tested in two experimental models, the acoustic startle reflex and the open-field tests. Our second objective was to investigate the role of GABAergic and opioid mechanisms of the CeA in the modulation of the withdrawal-potentiated startle as a measure of anxiety induced by morphine withdrawal. For the production of chronic dependence, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of this treatment, independent groups were probed in the startle reflex and open-field tests. For the acute dependence model, groups of rats were tested in the open field and startle tests under control conditions and under withdrawal from a single injection of morphine (10 mg/kg; s.c.) precipitated by naltrexone injections (0.1 mg/kg; s.c.). The results obtained showed that withdrawal from chronic and acute morphine treatments produced anxiety-like behavior in the open field test, although the anxiogenic-like effects could not be dissociated from the motor effects in the acute dependence model. On the other hand, only the withdrawal from acute morphine treatment significantly potentiated the startle response. Next, we examined the effects of intra-CeA microinjections of muscimol—a GABA_A receptors agonist—and DAMGO—a μ-opioid receptors agonist—on the potentiated startle induced by acute morphine withdrawal. The results obtained showed that intra-CeA injections of muscimol (1 nmol) and DAMGO (0.5 and 1 nmol) significantly inhibited this response. These findings suggest that the acute dependence model is more suitable to study the aversive effects of morphine withdrawal on the acoustic startle response than the chronic opiate dependence model. Besides, mechanisms mediated by μ- and GABA_A-receptors in the CeA appear to exert an inhibitory influence on the anxiety-like behavior induced by withdrawal from acute morphine treatment.     

Ultra-low doses of naltrexone or etorphine increase morphine's antinociceptive potency and attenuate tolerance/dependence in mice

In previous studies we showed that low (pM) concentrations of naloxone (NLX), naltrexone (NTX) or etorphine selectively antagonize excitatory, but not inhibitory, opioid receptor-mediated functions in nociceptive types of sensory neurons in culture. Cotreatment of these neurons with pM NTX or etorphine not only results in marked enhancement of the inhibitory potency of acutely applied nM morphine [or other bimodally-acting (inhibitory/excitatory) opioid agonists], but also prevents development of cellular manifestations of tolerance and dependence during chronic exposure to μM morphine. These in vitro studies were confirmed in vivo by demonstrating that acute cotreatment of mice with morphine plus a remarkably low dose of NTX (ca. 10 ng/kg) does, in fact, enhance the antinociceptive potency of morphine, as measured by hot-water tail-flick assays. Furthermore, chronic cotreatment of mice with morphine plus low doses of NTX markedly attenuates development of naloxone-precipitated withdrawal-jumping in physical dependence assays. The present study provides systematic dose-response analyses indicating that NTX elicited optimal enhancement of morphine's antinociceptive potency in mice when co-administered (i.p.) at about 100 ng/kg together with morphine (3 mg/kg). Doses of NTX as low as 1 ng/kg or as high as 1 μg/kg were still effective, but to a lesser degree. Oral administration of NTX in the drinking water of mice was equally effective as i.p. injections in enhancing the antinociceptive potency of acute morphine injections and even more effective in attenuating development of tolerance and NLX-precipitated withdrawal-jumping during chronic cotreatment. Cotreatment with a subanalgesic dose of etorphine (10 ng/kg) was equally effective as NTX in enhancing morphine's antinociceptive potency and attenuating withdrawal-jumping after chronic exposure. These studies provide a rationale for the clinical use of ultra-low-dose NTX or etorphine so as to increase the antinociceptive potency while attenuating the tolerance/dependence liability of morphine or other conventional bimodally-acting opioid analgesics.     

Effects of calcium channel antagonists on the motivational effects of nicotine and morphine in conditioned place aversion paradigm

The motivational component of drug withdrawal may contribute to drug seeking and relapse through the negative reinforcement-related process; thus, it is important to understand the mechanisms that mediate affective withdrawal behaviors. The present study was undertaken to examine the calcium-dependent mechanism of negative motivational symptoms of nicotine and morphine withdrawal using the conditioned place aversion (CPA) paradigm. Rats were chronically treated with nicotine (1.168 mg/kg, free base, s.c., 11 days, three times daily) or morphine (10 mg/kg, s.c., 11 days, twice daily). Then, during conditioning, rats pre-treated with nicotine or morphine received a nicotinic receptor antagonist mecamylamine (3.5 mg/kg) or an opioid receptor antagonist naloxone (1 mg/kg) to precipitate withdrawal in their initially preferred compartment, or saline in their non-preferred compartment. Our results demonstrated that after three conditioning sessions, mecamylamine induced a clear place aversion in rats that had previously received nicotine injections, and naloxone induced a significant place aversion in rats that had previously received morphine injections. Further, the major findings showed that calcium channel antagonists, i.e., nimodipine, verapamil and flunarizine (5 and 10 mg/kg, i.p.), injected before the administration of mecamylamine or naloxone, attenuated nicotine or morphine place aversion.As an outcome, these findings support the hypothesis that similar calcium-dependent mechanisms are involved in aversive motivational component associated with nicotine a morphine withdrawal. We can suggest that calcium channel blockers have potential for alleviating nicotine and morphine addiction by selectively decreasing the incentive motivational properties of both drugs, and may be beneficial as smoking cessation or opioid dependence pharmacotherapies.     

Opiate withdrawal and the rat locus coeruleus: behavioral, electrophysiological, and biochemical correlates

We have compared the time course of the behavioral manifestations of opiate withdrawal to the in vivo activity of locus coeruleus (LC) neurons and to increases in the levels of G-proteins, adenylate cyclase, and cAMP-dependent protein kinase known to occur in the LC in opiate-dependent animals. Rats were given morphine by daily subcutaneous implantation of morphine pellets for 5 d. On the sixth day, morphine withdrawal was induced by subcutaneous administration of naltrexone, an opiate receptor antagonist, with additional doses given 6 and 24 hr later, conditions that resulted in sustained, maximal levels of withdrawal over the duration of the experiment. We found a striking parallel between the time courses of the behavioral signs and the increased activity of LC neurons during withdrawal, both of which appeared to follow 2 phases. There was an early, rapid phase, during which withdrawal signs and increased LC activity became most pronounced within 15-30 min after naltrexone administration, and then recovered rapidly by over 50% within 4 hr of withdrawal. Subsequently, there was a slower phase, during which the persisting withdrawal signs and elevated LC activity remained roughly constant from 4 to 24 hr and did not recover completely until after 72 hr of continuous withdrawal. Adenylate cyclase and cAMP-dependent protein kinase activities in isolated LC subcellular fractions, both elevated in dependent animals, recovered to control levels after 6 hr of withdrawal, in parallel with the rapid phase of withdrawal. Levels of G1 and Go, also elevated in dependent animals, remained only slightly elevated at 6 hr and returned to normal by 24 hr. Taken together, these data suggest that increased neuronal activity in the LC is associated temporally with the behavioral morphine withdrawal syndrome and that increased levels of G-proteins and an up-regulated cAMP system may contribute to the early withdrawal activation of these neurons.     

Inhibition of morphine tolerance and dependence by diazepam and its relation to μ-opioid receptors in the rat brain and spinal cord

We have recently observed that concomitant administration of diazepam to morphine pellet implanted rats results in the inhibition of the development of morphine tolerance and dependence. We have now analyzed μ-opioid receptors in rats treated with morphine and diazepam for 5 days by using -DAMGO for binding studies. Male Sprague–Dawley rats were made tolerant and dependent by subcutaneous (s.c.) implantation of six morphine pellets (two pellets on the first day, and four on the second day). Diazepam (0.25 mg/kg b.wt) was injected once daily intraperitoneally (i.p.) for 5 days. Control rats were implanted with placebo pellets and injected once daily with saline or diazepam (i.p.). Animals were administered s.c. naloxone (10 mg/kg) to induce naloxone-precipitated withdrawal syndrome on the final day of the experiment (day 5). There was an up-regulation of μ-receptor (B_max increased) in the spinal cord of morphine tolerant (+139%) and dependent (+155%) rats compared to saline treated animals. Diazepam treatment abolished the up-regulation of μ-receptors in spinal cord of morphine treated rats. In the cortex, B_max was not affected in morphine tolerant or dependent rats but it decreased by 38% in morphine tolerant and 65% in morphine dependent rats treated with diazepam. The K_d of μ-receptors increased in the cortex, striatum and hypothalamus of morphine dependent rats. Diazepam treatment decreased the K_d of μ-receptors in the cortex of morphine tolerant and hypothalamus of morphine-dependent rats. These results suggest that diazepam treatment antagonizes the up-regulation of CNS μ-receptors observed in morphine tolerant rats. In addition, morphine tolerance and dependence may be associated with conversion of μ-opioid receptors to μ*-constitutive opioid receptors that are less active, and this conversion is prevented in the brain of animals treated with diazepam.     

N-Acylethanolamine Acid Amidase Inhibition Potentiates Morphine Analgesia and Delays the Development of Tolerance

Opioids are essential drugs for pain management, although long-term use is accompanied by tolerance, necessitating dose escalation, and dependence. Pharmacological treatments that enhance opioid analgesic effects and/or attenuate the development of tolerance (with a desirable opioid-sparing effect in treating pain) are actively sought. Among them, N-palmitoylethanolamide (PEA), an endogenous lipid neuromodulator with anti-inflammatory and neuroprotective properties, was shown to exert anti-hyperalgesic effects and to delay the emergence of morphine tolerance. A selective augmentation in endogenous PEA levels can be achieved by inhibiting N-acylethanolamine acid amidase (NAAA), one of its primary hydrolyzing enzymes. This study aimed to test the hypothesis that NAAA inhibition, with the novel brain permeable NAAA inhibitor AM11095, modulates morphine’s antinociceptive effects and attenuates the development of morphine tolerance in rats. We tested this hypothesis by measuring the pain threshold to noxious mechanical stimuli and, as a neural correlate, we conducted in vivo electrophysiological recordings from pain-sensitive locus coeruleus (LC) noradrenergic neurons in anesthetized rats. AM11095 dose-dependently (3–30 mg/kg) enhanced the antinociceptive effects of morphine and delayed the development of tolerance to chronic morphine in behaving rats. Consistently, AM11095 enhanced morphine-induced attenuation of the response of LC neurons to foot-shocks and prevented the attenuation of morphine effects following chronic treatment. Behavioral and electrophysiological effects of AM11095 on chronic morphine were paralleled by a decrease in glial activation in the spinal cord, an index of opioid-induced neuroinflammation. NAAA inhibition might represent a potential novel therapeutic approach to increase the analgesic effects of opioids and delay the development of tolerance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01116-4.