Final Morphine Injection Research Articles

Neonatal Opioid Withdrawal Syndrome in Mice: Acute Molecular and Long‐Term Behavioral Consequences

Populations affected by the ongoing opioid epidemic include pregnant women and their offspring. Infants exposed to opioids in utero are at risk of developing Neonatal Opioid Withdrawal Syndrome (NOWS), a combination of somatic withdrawal symptoms including high pitched crying, sleeplessness, irritability, and potentially seizures. Confounding environmental factors and difficulty in conducting longitudinal clinical studies have made it challenging to elucidate the long‐term consequences of NOWS. In order to investigate the molecular mechanisms and long‐term effects of NOWS, we have developed a mouse model of opioid exposure and withdrawal that encompasses the developmental equivalent of all three trimesters of the human pregnancy. Female mice are implanted with morphine minipumps (10 mg/kg/day) before mating, ensuring that pups are exposed to morphine in utero for the entirety of embryonic development. Mice are then given twice daily morphine injections (10 mg/kg/day, SC) for post‐natal days 1‐14. Mice are tested for developmental milestones during this period, including eye opening, extinguishing pivoting, surface righting, and forelimb grasp. Following the final morphine injection withdrawal behaviors were measured by ultrasonic vocalizations, 55°C hotplate analgesia latency, and somatic withdrawal signs. After reaching adulthood, mice were tested for changes in affective behavior (elevated zero maze, tail suspension test and learned helplessness), opioid response (conditioned place preference and intravenous self‐administration), social behavior (three‐chamber social behavior test), and sleep patterns. Mice exposed to this paradigm show delayed latency to reach developmental milestones and acute withdrawal phenotypes. In adulthood, social behavior and sleep patterns were affected in a sex‐dependent manner, while affective behavior and opioid response was unchanged. Bulk RNA sequencing of mice at post‐natal day 1 revealed changes in gene expression related to synapse development and function, the GABAergic system, and mitochondrial function. These transcriptome changes may underlie the behavioral long‐term effects of NOWS.

15727 KATP channel prodrugs as therapeutics for chronic pain and substance abuse disorders

ABSTRACT IMPACT: Pharmacological activation of KATP channels may provide analgesia and attenuate opioid tolerance and withdrawal OBJECTIVES/GOALS: Our long term goal is to develop therapeutics for the treatment of the overuse of opioids. The objective of this application is to test novel KATP channel-targeting prodrugs in rodent models of neuropathic and inflammatory pain in addition to opioid tolerance after chronic morphine administration. METHODS/STUDY POPULATION: In one study, two different measures for chronic pain were implemented in mice. Male and female mice (n=10) were subjected to spinal nerve ligation (SNL) or intraplantar injection of Complete Freund’s Adjuvant (CFA) to induce neuropathic and inflammatory pain, respectively. Administration of KATP channel prodrugs (60ug, it) attenuated mechanical hypersensitivity after SNL or CFA compared to vehicle (saline). In a separate study, changes in mechanical hypersensitivity were tested while mice undergo chronic morphine treatment (15mg/kg, 2x, 5 days) with administration of the prodrugs. Tolerance was measured as the loss of antinociception, and withdrawal is measured ˜24 hours after the final morphine injection. RESULTS/ANTICIPATED RESULTS: Intrathecal administration of either KATP channel prodrugs significantly attenuated mechanical hypersensitivity after SNL and significantly attenuated mechanical hypersensitivity after CFA in mice. We predict that intrathecal administration of these prodrugs will also attenuate morphine tolerance and withdrawal in mice. This hypothesis is based off our previous data indicating non-water soluble KATP channel agonists produce analgesia and attenuate morphine tolerance in mice. DISCUSSION/SIGNIFICANCE OF FINDINGS: Pharmaceutical strategies to utilize KATP channels for therapeutics have been hindered due to the low solubility and low ability to cross the neurovascular unit. Newly developed, water-soluble KATP channel openers could be useful pharmaceutical strategy to reduce chronic pain, opioid tolerance, and withdrawal in human populations.

Persistent effects of the orexin-1 receptor antagonist SB-334867 on naloxone precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine dependent rats

Aim of the study In this study, we investigated the effect of long-term administration of orexin receptor 1 (OXR1) antagonist on naloxone-precipitated morphine withdrawal symptoms and nociceptive behaviors in morphine-dependent rats. Materials and methods Wistar rats received subcutaneous (s.c.) injections of morphine (6, 16, 26, 36, 46, 56, and 66 mg/kg, 2 ml/kg) at an interval of 24 h for 7 days. In chronic groups, the OXR1 antagonist, SB-334867 (20 mg/kg, i.p.), or its vehicle, was injected repetitively from postnatal day 1 (PND1)-PND23 and then for the following seven days before each morphine injection. Meanwhile, in acute groups, SB-334867, or its vehicle, was administered before each morphine injection. In groups of rats that were designated for withdrawal experiments, naloxone (2.5 mg/kg, i.p.) was administered after the last injection of morphine. In the formalin-induced pain, the effect of OXR1 inhibition on the antinociceptive effects of morphine was measured by injecting formalin after the final morphine injection. Results Animals that received long-term SB-334867 administration before morphine injection demonstrated a significant reduction in chewing, defecation, diarrhea, grooming, teeth chattering, wet-dog shake, and writhing. Inhibiting OXR1 for a long time increased formalin-induced nociceptive behaviors in interphase and phase II of the formalin-induced pain. Conclusions Our results indicated that the inhibition of OXR1 significantly reduces the development of morphine dependence and behavioral signs elicited by the administration of naloxone in morphine-dependent rats. Furthermore, the prolonged blockade of OXR1 might be involved in formalin-induced nociceptive behaviors.

(251) A Time Course of the Development of Hyperalgesia during Spontaneous Withdrawal in NIH Swiss Mice

Pain patients may experience heightened sensitivity to pain while trying to discontinue opioid use which may contribute to resumption of opioid use in order to avoid more pain. However, most studies in animals that investigate hyperalgesia during opioid withdrawal focus on hyperalgesia during precipitated opioid withdrawal and include only male animals. The present study was conducted to assess hyperalgesia during a protracted withdrawal state in female and male NIH Swiss mice. Mice were rendered opioid-dependent by twice daily s.c. injections of morphine in escalating doses (50-125 mg/kg) or saline vehicle for four consecutive days; there was a final dose of morphine on day 5. Body weight was taken prior to injections as well as mornings and evenings prior to testing. Paw withdrawal thresholds to increasing paw pressures were recorded prior to the first injection and at 5, 10, 24, 34, 48, 58, 72, 82, 96 and 106 h after the final injection. Mice consistently lost weight during morphine administration; however, both sexes gained weight immediately following cessation of drug treatment. Morphine-dependent animals exhibited pronounced hyperalgesia starting at 10 h after the final morphine injection and subsiding at roughly 58 h for females and 82 h for males. A main effect of morphine administration showed that animals that received morphine had significantly reduced paw withdrawal thresholds in percentage of baseline. A main effect of sex indicates that males had generally lower scores over time than females in hyperalgesia data. These results indicate that spontaneous withdrawal causes hyperalgesia in NIH Swiss mice. This is significant because while it is known that withdrawal can cause hyperalgesia, most animal models that investigate this phenomenon use a precipitated withdrawal model, while our study demonstrated hyperalgesia also occurs in a spontaneous withdrawal model. This research was supported in part by State of Washington Initiative Measure 171. Pain patients may experience heightened sensitivity to pain while trying to discontinue opioid use which may contribute to resumption of opioid use in order to avoid more pain. However, most studies in animals that investigate hyperalgesia during opioid withdrawal focus on hyperalgesia during precipitated opioid withdrawal and include only male animals. The present study was conducted to assess hyperalgesia during a protracted withdrawal state in female and male NIH Swiss mice. Mice were rendered opioid-dependent by twice daily s.c. injections of morphine in escalating doses (50-125 mg/kg) or saline vehicle for four consecutive days; there was a final dose of morphine on day 5. Body weight was taken prior to injections as well as mornings and evenings prior to testing. Paw withdrawal thresholds to increasing paw pressures were recorded prior to the first injection and at 5, 10, 24, 34, 48, 58, 72, 82, 96 and 106 h after the final injection. Mice consistently lost weight during morphine administration; however, both sexes gained weight immediately following cessation of drug treatment. Morphine-dependent animals exhibited pronounced hyperalgesia starting at 10 h after the final morphine injection and subsiding at roughly 58 h for females and 82 h for males. A main effect of morphine administration showed that animals that received morphine had significantly reduced paw withdrawal thresholds in percentage of baseline. A main effect of sex indicates that males had generally lower scores over time than females in hyperalgesia data. These results indicate that spontaneous withdrawal causes hyperalgesia in NIH Swiss mice. This is significant because while it is known that withdrawal can cause hyperalgesia, most animal models that investigate this phenomenon use a precipitated withdrawal model, while our study demonstrated hyperalgesia also occurs in a spontaneous withdrawal model. This research was supported in part by State of Washington Initiative Measure 171.

Playing possum, and: me and the king, and: Sul ponticello

playing possum, and: me and the king, and: Sul ponticello Alina Stefanescu playing possum after Bayard Rustin This is my mother newly dead, Mom says. She died without suffering. I fondle the photo of my maternal grandmother playing possum. A dead possum in a ditch is called roadkill. A possum who's just playing is not a carcass. The women in my family will play anything to make you wonder. I am one of those women who finds herself playing. Just to see what you'd say about me then. The woman in the photo is my namesake. The photo is black and white, taken and developed by my grandfather. He was a gangly husband, a polymath, her fellow physician. They married in med school. Stars of the show, those two. It was my tall reticent grandfather who administered the final morphine injection when the cancer took over her brain—when she was no longer the person she might recognize. He did this in secret without consulting the family. He did it in the living room of the remote Transylvanian chalet he designed and built by hand. Her green and white wooden dream house, a retreat for their retirement. He did it because she wanted to remain herself. She made him promise. She made him promise again. She said if you love me . . . He signed her request with a blue fountain pen from Czechoslovakia. The request lay among papers and photos he preserved, images of her naked breasts, a voile nightgown, [End Page 114] smiling sepia, Alina, winsome, hungry, amorous, and finally, finally, faceless. Two slender hands crossed in languor over her lower chest. An Orthodox cross poised above the v-shaped limbs. A flock of birds on their way to warmer shores. Above their wings, a cardboard icon. "I am an atheist now," he announced. But no one believed a man who hated God so much could ever come to disbelieve Himself. This was Ceausescu's communist Romania. I know hate is a kind of hope. These lines in my hand, a Balkan mutation. me and the king In a Transylvanian village they speak of a kingwho laughs with one eye and weeps with the other.He keeps me company in the valley between city buildings. Have you ever wept when losing your journal in Manhattan?I cry with one eye and lie with another. Hop the subway to Coney Island so a mermaid can recovermy story. At least one story in life needs a mermaid to endthings. And the sky is too much—the sky is a certaintinge of fire when the poet accuses,the podium weeps. Sul ponticello The verb for longing in Romanian is a mouthfulof moist mountain soil. [End Page 115] Our betrothal avertedlike eyes across a wooden pewwe divide what we must equal from the trothwhere swine sink their snouts. Has it been years oryards, fond ache-rage? A fathom measures the distance between a pair of outstretched arms.Still you hold me like Jell-Osul ponticello. On the porch,in the kitchen, near a sink sunset cannot overcome its habitual marvel:gilded tongue, scalded marrow I cannot linger Farbehind your palm's urge knowingany body is a bowtuned to tremble tangled limbs, mottledfruits, minor ninth. [End Page 116] Copyright © 2019 University of Nebraska Press

Evaluation of hyperbaric oxygen (HBO2) as a treatment for hyperalgesia associated with naloxone‐precipitated withdrawal in morphine‐dependent mice

Opioid dependence has become a major problem in the United States. Withdrawal from opioid drugs is associated with hyperalgesia (increased sensitivity to a pain stimulus) which may contribute to relapse [Carcoba et al., J Addict Dis 30(3): 258–70, 2012]. Hyperbaric oxygen (HBO2) is 100% oxygen at higher than atmospheric pressure. HBO2 has recently been shown to be effective at decreasing physical signs of withdrawal in morphine‐dependent mice, and there is a growing body of research showing that HBO2 is effective at decreasing pain in clinical conditions and in preclinical modeling [Nicoara et al., Brain Res. 1648:434–7, 2016; Efrati et al., PLOS One 10(5)1–25, 2015; Gibbons at al., Brain Res. 1537:111–6, 2013]. The effectiveness of HBO2 to decrease hyperalgesia caused by opioid withdrawal in an animal model of drug dependence was evaluated in this study. Male and female NIH Swiss mice were made dependent upon morphine by injecting animals s.c. with twice‐daily escalating doses of morphine for 4 consecutive days. On the 5th day, animals were given a final injection of the highest dose of morphine and injected i.p. with 1.0 mg/kg naloxone 8 hours after the final morphine injection. Animals were treated with 30 min of HBO2 at 3.5 atmospheres absolute or room air 60 min prior to naloxone injection. Nociception was evaluated using the tail withdrawal, hot plate and paw pressure tests every 10 min for 30 min after naloxone injection. Baseline levels of nociception for each endpoint were taken prior to induction of morphine dependence. Male and female animals exhibited hyperalgesia in the hot plate and paw pressure tests but not the tail withdrawal test. HBO2 significantly decreased the amount of hyperalgesia in the hot plate and paw pressure tests in male animals. In female animals, HBO2 had no effect on hyperalgesia in the hot plate test but significantly decreased the level of hyperalgesia associated with the paw pressure test. While no hyperalgesia was seen in the tail withdrawal test, HBO2 did produce significant antinociception in male and female animals. These intriguing results show that HBO2 is capable of reducing hyperalgesia associated with opioid withdrawal and warrant further investigation. Furthermore, these results help support previous findings showing that HBO2 may be effective at decreasing symptoms of opioid withdrawal.Support or Funding InformationThis research was supported by the Marchionne 12‐month Fellowship from the Department of Psychology at Washington State University, NIH Grant AT‐007222, and the Honors College Distinguished Professorship from Washington State University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intracranial self-stimulation reward thresholds during morphine withdrawal in rats bred for high (HiS) and low (LoS) saccharin intake

RationaleSweet preference is a marker of vulnerability to substance use disorders, and rats selectively bred for high (HiS) vs. low saccharin (LoS) intake display potentiated drug-seeking behaviors. Recent work indicated that LoS rats were more responsive to the negative effects of drugs in several assays. ObjectiveThe current study used the intracranial self-stimulation (ICSS) procedure to investigate the anhedonic component of morphine withdrawal in male HiS and LoS rats. MethodsRats were administered morphine (10mg/kg) or saline for 8 days. To evaluate withdrawal effects, reward thresholds were measured 24 and 28h following the 8th morphine injection (spontaneous withdrawal) and again for 4 days following daily acute morphine and naloxone (1mg/kg) administration (precipitated withdrawal). Results24h following the final morphine injection, reward thresholds in LoS rats were significantly elevated compared to reward thresholds in LoS controls, indicating spontaneous withdrawal. This effect was not observed in HiS rats. LoS rats also showed greater elevations of reward thresholds on several days during naloxone-precipitated withdrawal compared to their HiS counterparts. ConclusionsLoS rats were more sensitive to morphine withdrawal-mediated elevations in ICSS thresholds than HiS rats. While these differences were generally modest, our data suggest that severity of the negative affective component of opiate withdrawal may be influenced by genotypes related to addiction vulnerability.

Social housing conditions influence morphine dependence and the extinction of morphine place preference in adolescent mice

BackgroundAdolescent opioid abuse is on the rise, and current treatments are not effective in reducing rates of relapse. Our previous studies demonstrated that social housing conditions alter the acquisition rate of morphine conditioned place preference (CPP) in adolescent mice. Specifically, the acquisition rate of morphine CPP is slower in morphine-treated animals housed with drug-naïve animals. Thus, here we tested the effect of social housing conditions on the development of morphine dependence and the extinction rate of an acquired morphine CPP. MethodsAdolescent male mice were group-housed in one of two housing conditions. They were injected for 6 days (PND 28-33) with 20mg/kg morphine. Morphine only mice are animals where all four mice in the cage received morphine. Morphine cage-mate mice are morphine-injected animals housed with drug-naïve animals. Mice were individually tested for spontaneous withdrawal signs by quantifying jumping behavior 4, 8, 24, and 48h after the final morphine injection. Then, mice were conditioned to acquire morphine CPP and were tested for the rate of extinction. ResultsMorphine cage-mates express less jumping behavior during morphine withdrawal as compared to morphine only mice. As expected, morphine cage-mate animals acquired morphine CPP more slowly than the morphine only animals. Additionally, morphine cage-mates extinguished morphine CPP more readily than morphine only mice. ConclusionsSocial housing conditions modulate morphine dependence and the extinction rate of morphine CPP. Extinction testing is relevant to human addiction because rehabilitations like extinction therapy may be used to aid human addicts in maintaining abstinence from drug use.

Thermal sensitivity as a measure of spontaneous morphine withdrawal in mice

IntroductionOpioid withdrawal syndrome is a critical component of opioid abuse and consists of a wide array of symptoms including increases in pain sensitivity (hyperalgesia). A reliable preclinical model of hyperalgesia during opioid withdrawal is needed to evaluate possible interventions to alleviate withdrawal. The following study describes a method for assessing increases in thermal sensitivity on the hotplate in a mouse model of spontaneous morphine withdrawal. MethodsC57BL/6J mice received 5.5days of 30, 56, or 100mg/kg morphine or saline (s.c., twice daily). In Experiment I, thermal sensitivity data were collected at baseline and at 8, 24, 32, 48h and 1week following the final injection. Thermal sensitivity was assessed by examining latency to respond on a hotplate across a range of temperatures (50, 52, 54, and 56°C). In Experiment II, 0.01mg/kg buprenorphine was administered 30min prior to each testing session during the withdrawal period. In Experiment III, jumping during a 30min period was assessed at baseline and at 0, 8, 24, 32, and 48h following the final morphine injection. ResultsDuring the withdrawal period, thermal sensitivity increased significantly in all morphine-treated mice as compared to saline-treated mice. Thermal sensitivity was greater in mice treated with 56mg/kg morphine compared to 30mg/kg and peaked earlier than in mice treated with 100mg/kg (32h v 1wk). The increase in thermal sensitivity following 56mg/kg morphine was attenuated by a dose of buprenorphine that did not produce antinociception alone (i.e., 0.01mg/kg). In general, the results of the jumping experiment paralleled those obtained in Experiment I. DiscussionResponse latency on the hotplate is a reliable and sensitive measure of spontaneous morphine withdrawal in mice, making it an ideal behavior for assessing the potential of medications and environmental interventions to alleviate opioid withdrawal.

Alleviation of Morphine Withdrawal Signs but Not Tolerance by the Essential Oil ofKelussia odoratissimaMozaff.

The aim of the present study was to assess the effects of chronic and acute treatment of the essential oil (EO) of Kelussia odoratissima Mozaff. on the development of morphine tolerance and dependence in mice. Mice were rendered tolerant to and dependent on morphine by subcutaneous injection of morphine over a period of 5 days. Tolerance was assessed using the tail-pinch test and withdrawal signs of morphine were precipitated by injecting naloxone 2 h after the final morphine injection. Repeated injection of the EO of K. odoratissima (5 and 10 mg/kg) for 4 days significantly suppressed morphine-withdrawal jumps, a sign of the development of dependence to opiate as assessed by naloxone precipitation withdrawal on day 5 of testing. A single injection (25, 50, 100 mg/kg) of the EO on day 5, 1 h prior to morphine failed to produce any significant change in morphine withdrawal signs. Neither the acute nor the chronic administration of EO of the K. odoratissima did significantly influence the development of tolerance to the analgesic effect of morphine. Alleviation in morphine signs of withdrawal after chronic injection with K. odoratissima is indicative of reversal of neuronal adaptation that takes place during morphine presence in the brain.

Tianeptine reduces morphine antinociceptive tolerance and physical dependence

Long-term use of morphine can cause neuronal dystrophic changes in specific areas of the brain. These changes may underlie the mechanism for developing morphine antinociceptive tolerance and physical dependence. We evaluated the effect of tianeptine, an antidepressant with prominent neuroprotective and neuroplastic properties, on the development of morphine antinociceptive tolerance and physical dependence. Male C57BL/6 mice were rendered tolerant to or dependent on morphine by subcutaneously injecting them with morphine (10 mg/kg) and intraperitoneally with saline or tianeptine (1, 3, or 5 mg/kg) twice daily for 6 days. The mice were given a daily tail-flick test 1 h after the first morphine injection to evaluate the development of their tolerance to morphine antinociception. To evaluate their physical dependence on morphine, 3 h after the final morphine injection on day 6, naloxone-HCl-precipitated (2 mg/kg, intraperitoneally) withdrawal symptoms were counted for 30 min, and body weight was checked 1 h after the naloxone injection. Tianeptine per se produced no antinociception, neither did it modify the antinociception produced by morphine, nor did it evoke the behavioral responses different from those in the saline controls. The combination of tianeptine with morphine significantly reduced the development of morphine antinociceptive tolerance and suppressed the incidence of naloxone-precipitated withdrawal symptoms. We conclude that tianeptine is an effective inhibitor of morphine-induced antinociceptive tolerance and physical dependence in mice. Our results would imply that comedication with tianeptine and morphine might benefit those who need long-term morphine treatment.

Effects of chronic administration of calcium-magnesium soft gels on morphine tolerance and dependence in mice

The aim of present study was to assess the effects of chronic administration of calcium-magnesium soft gels on the development of morphine tolerance and dependence in mice. Tolerance was assessed using the tail-pinch test and withdrawal signs of morphine were precipitated by injecting naloxone 2 h after the final morphine injection. CalMag capsules were given in final doses of 50/25, 25/12.5 and 12.5/6.25 mg/kg based on calcium/magnesium ratio. Similar doses of Ca and/or Mg were prepared, separately. CalMag at 50/25, 25/12.5 mg/kg and the mixture of calcium and magnesium (Ca + Mg) at 50/25, 25/12.5, 12.5/6.25 mg/kg and calcium at 50, 25 mg/kg significantly reduced the number of jumps. The number of standings was only reduced after the administration of CalMag at 50/25 mg/kg and Ca + Mg at 25/12.5 mg/kg. The development of morphine tolerance was prevented in all drug-treated groups, except the one which received 6.25 mg/kg Mg. The data suggested that combination of calcium and magnesium at 50/25 and 25/12.5 mg/kg prevented the development of tolerance and dependence. It seems that other ingredients of CalMag capsules do not have an important effect on preventing tolerance and withdrawal signs. Compared to the acute effects, chronic administration of Cal-Mag allowed the effective dose to be reduced. Unlike the acute treatment, chronic administration of calcium alone was effective in reducing morphine tolerance and dependence, and magnesium had no significant effect on withdrawal signs, suggestive of some pharmacological adaptations.

Rapid neuroadaptation in the nucleus accumbens and bed nucleus of the stria terminalis mediates suppression of operant responding during withdrawal from acute opioid dependence

Single injections of morphine induce a state of acute opioid dependence in humans and animals, measured as precipitated withdrawal when an antagonist is administered 4–24 h after morphine. Additional morphine exposure at daily or weekly intervals results in further increases in withdrawal severity, suggesting that acute opioid dependence reflects the early stages in the development of a chronic state of dependence. The current study evaluated the role of the nucleus accumbens (NAC), bed nucleus of stria terminalis (BNST), interstitial nucleus of posterior limb of the anterior commissure (IPAC), and central amygdala (CeA) in the expression of antagonist-precipitated suppression of operant responding for food as a measure of withdrawal from acute opioid dependence. Rats trained on a fixed-ratio 15 schedule received one or four daily injections of morphine, with the lipophobic opioid antagonist methylnaloxonium (16–2000 ng) infused into one of the brain regions or the lateral ventricle (i.c.v.) 4 h after the final morphine injection. After acute morphine methylnaloxonium was more potent upon infusion into the NAC (17.9-fold potency shift), BNST (6.8-fold) and CeA (5.5-fold) than it was upon i.c.v. administration. Following repeat morphine the NAC and BNST but not CeA continued to show greater sensitivity relative to i.c.v. infusion (12.9-, 8.7-, and 3.2-fold potency shifts, respectively). The IPAC was insensitive to methylnaloxonium after acute or repeat morphine at doses that reliably suppressed responding upon i.c.v. infusion (125–500 ng). Thus, among the components of extended amygdala examined in this study, rapid neuroadaptation within the nucleus accumbens and bed nucleus of the stria terminalis appear to play the most prominent role in antagonist-precipitated suppression of operant responding during the early stages in the development of opioid dependence.

Characterization of remifentanil‐induced heart rate and blood pressure changes in the rat

The cardiovascular effects of mu-opioid agonists are a primary concern when administered as both anesthetics and analgesics. Opiates have been shown to decrease blood pressure (BP) and heart rate (HR) in both primates and rodents, and tolerance to this effect has been observed in rats chronically treated with methadone or morphine. To build upon these observations, the bradycardic effect of remifentanil (R), a fast-acting mu-opioid agonist frequently used for post-operative care, was characterized in rodents. Using a telemetry system (Data Sciences International, St. Paul, MN), HR and BP were measured in both morphine-naïve and –experienced rats. Changes in cardiovascular activity induced by non-contingent infusions of R (1.0, 3.0, 10.0, and 30.0 ug/kg) or sterile water were recorded and analyzed. In morphine-naïve rats, R dose-dependently decreased HR; the lowest effective dose (3ug/kg) decreased HR by 26.9+/−13.1 beats per minute, and the highest dose tested (30 ug/kg) decreased HR by 154.34+/−20.7 beats per minute. The duration of effect was dose-related, such that HR returned to baseline 30 seconds after 3 ug/kg, 1 minute after 10 ug/kg, and about 2.5 minutes after 30 ug/kg infusions. Naltrexone (0.01, 0.1, and 1.0 mg/kg, s.c.) or sterile water was administered 15 minutes prior to agonist treatment in order to confirm mu-opioid mediation of bradycardia This pretreatment dose-dependently reduced the potency of R in morphine-naïve rats. Rats treated with increasing doses of morphine twice-daily demonstrated complete tolerance to R, when tested 24 hours after the final morphine injection. The hemodynamic effects of contingent (self-administered) R were also compared between morphine-naïve and –experienced rats. Research supported by USPHS NIDA Grants DA00254 and DA14349

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 h for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis, however these were in CRH negative neurons.

Effect of naloxone-precipitated morphine withdrawal on c- fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 h for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis, however these were in CRH negative neurons.

Elevated D3 dopamine receptor mRNA in dopaminergic and dopaminoceptive regions of the rat brain in response to morphine

As opiates increase dopamine transmission, we measured the effects of morphine on dopamine-related genes using a real-time optic PCR assay that reliably detects small differences in mRNA in discrete brain regions. Tissue from dopaminoceptive and dopaminergic brain regions was collected from rats injected twice daily for 7 days with saline or increasing doses of morphine. Tissues were assayed for D1, D2 and D3 dopamine receptor mRNAs (D1R, D2R and D3R), as well as for mRNAs for tyrosine hydroxylase (TH) and the dopamine transporter (DAT). The neuron-associated mRNAs for SNAP-25 and synaptophysin, as well as the glial-associated mRNA for S100-β and three ‘housekeeping’ mRNAs, were also measured. As reported previously by others, there was no alteration in D1R mRNA and a 25% decrease in D2R mRNA in the caudate-putamen, 2 h after the final morphine injection. Importantly, in the same RNA extracts, D3R mRNA showed significant increases of 85% in the caudate-putamen and 165% in the ventral midbrain, including the substantia nigra and ventral tegmental area. There were no other significant morphine effects. Mapping of brain regions in saline control rats agreed with previous studies, including showing the presence of low abundance TH mRNA and the absence of DAT mRNA in the caudate-putamen. The finding that chronic, intermittent injections of morphine caused an increase in D3R mRNA extends our understanding of the ability of D3R agonists to reduce the effects of morphine.

Differential responsivity of the hypothalamic-pituitary-adrenal axis to glucocorticoid negative-feedback and corticotropin releasing hormone in rats undergoing morphine withdrawal: possible mechanisms involved in facilitated and attenuated stress responses.

Chronic morphine treatment produces profound and long-lasting changes in the pituitary-adrenal responses to stressful stimuli. The purpose of the present study was to explore the mechanisms involved in these altered stress responses. Chronic morphine administration increased basal plasma concentrations of corticosterone and adrenocorticotropic hormone (ACTH), which peaked at 36 h after the final morphine injection and returned to normal levels within 84-h. Whole brain glucocorticoid receptor protein expression was reduced (approximately 70%) in morphine-treated rats 4-h after the final morphine injection and these levels recovered within 16-h. Twelve hours following morphine withdrawal, rats displayed normal ACTH, but potentiated and prolonged corticosterone responses to restraint stress. Both the ACTH and corticosterone responses to restraint in acutely withdrawn rats were insensitive to dexamethasone. Furthermore, acutely withdrawn rats displayed reduced ACTH but prolonged corticosterone responses to peripheral corticotropin releasing hormone (CRH) administration. These findings suggest that the normal ACTH and enhanced corticosterone responses to stress in acutely withdrawn rats involved decreased sensitivity of negative-feedback systems to glucocorticoids, reduced pituitary responsivity to CRH, and enhanced sensitivity of the adrenals to ACTH. Eight days following morphine withdrawal, rats displayed dramatically reduced ACTH, but normal corticosterone responses to restraint stress. These rats displayed enhanced sensitivity to dexamethasone and normal pituitary-adrenal responses to CRH. These data suggest that the reduced ACTH responses to stress in 8-day withdrawal rats involved increased sensitivity of negative-feedback systems to glucocorticoids as well as reduced CRH and/or AVP function in response to stress. Taken together, the results of this study illustrate some of the mechanisms mediating altered stress responsivity in rats that have received chronic morphine treatment.

Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats

We have previously reported the increase of preproenkephalin (PPE) mRNA in the caudal periaqueductal gray (PAG) of rats during morphine withdrawal. In this study, it was further evidenced that PPE mRNA in the caudal PAG was not increased by various kinds of stressor, suggesting that the increase in PPE mRNA in the caudal PAG is specific to morphine withdrawal. In order to investigate the physiological significance of the increase of PPE mRNA in the caudal PAG, we compared the time course of the increase of PPE mRNA in the caudal PAG with that of naloxone-precipitated or spontaneous morphine withdrawal signs. The increase of plasma corticosterone (PCS: 52 and 52 μg/100 ml; control group, 18 and 15 μg/100 ml) and body weight loss (−6 and −9%; control group, 0 and −1%) were observed but PPE mRNA increase was not detected 1 and 2 h after naloxone in morphine treated rats. PPE mRNA increased by 37 to 56%, while PCS elevation and body weight loss gradually diminished 4 h to 2 days after naloxone. A total of 12 h after spontaneous withdrawal, PCS was prominently increased (51 μg/100 ml; control group, 12 μg/100 ml), but body weight and PPE mRNA were not affected. One day after spontaneous withdrawal, PCS elevation (38 μg/100 ml; control group, 8 μg/100 ml) and body weight loss (−5%; control group, +3%) were observed and PPE mRNA also increased by 42%. Two to 3 days after the final morphine injection, PCS recovered to control level and body weight loss gradually disappeared, while PPE mRNA was still increased by 74 to 46%. These results suggest that PPE gene expression in the caudal PAG is stimulated in the recuperative phase of these morphine withdrawal signs.

Effects of Peptidase Inhibitors, [D-Ala2, Met5]-Enkephalinamide and Antiserum to Methionine-Enkephalin Microinjected Into the Caudal Periaqueductal Gray on Morphine Withdrawal in Rats

We examined the involvement of enkephalins in the caudal periaqueductal gray (cPAG) in morphine withdrawal in rats. Rats were treated with increasing doses of morphine (20–30 mg/kg/day, s.c., for 5 days) to develop morphine dependence. Morphine withdrawal was induced by naloxone (5 mg/kg, s.c.) 24 hr after the final morphine injection. The level of preproenkephalin (PPE) mRNA in the cPAG was estimated by quantitative in situ hybridization. PPE mRNA in the cPAG was increased 4–24 hr after naloxone in morphine-treated rats. A mixture of peptidase inhibitors (0.5 μl of a solution of amastatin, captopril and phosphoramidon, 3 × 10−3 M each) microinjected into the cPAG suppressed morphine withdrawal (a decrease in the number of jumping, chin rubbing, paw rubbing and teeth chattering). Antiserum to methionine-enkephalin (1:10 dilution) microinjected into the cPAG did not significantly aggravate morphine withdrawal with or without the mixture of peptidase inhibitors. However, [D-Ala2, Met5]-enkephalinamide (20 nmol), an enkephalin analog, injected into the cPAG decreased the number of jumping without any influence on the other withdrawal signs. These results suggest that the increase in enkephalins in the cPAG may participate in the alleviation of morphine withdrawal (jumping behavior).