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Published in final edited form as: J Pain. 2011 Feb 26;12(6):667–676. doi: 10.1016/j.jpain.2010.12.007

Descending Facilitatory Pathways From the Rostroventromedial Medulla Mediate Naloxone-precipitated Withdrawal in Morphine-Dependent Rats

Louis P Vera-Portocarrero 1, Michael H Ossipov 1, Josephine Lai 1, Tamara King 1, Frank Porreca 1
PMCID: PMC4028695  NIHMSID: NIHMS261044  PMID: 21354865

Abstract

Opioids produce analgesic effects and extended use can produce physical dependence in both humans and animals. Dependence to opiates can be demonstrated by either termination of drug administration or through precipitation of the withdrawal syndrome by opiate antagonists. Key features of the opiate withdrawal syndrome include hyperalgesia, anxiety and autonomic signs such as diarrhea. The rostral ventromedial medulla (RVM) plays an important role in the modulation of pain and for this reason, may influence withdrawal-induced hyperalgesia. The mechanisms that drive opiate withdrawal-induced hyperalgesia have not been elucidated. Here, rats made dependent upon morphine received naloxone to precipitate withdrawal. RVM microinjection of lidocaine, kynurenic acid (excitatory amino acid antagonist) or YM022 (CCK2 receptor antagonist) blocked withdrawal-induced hyperalgesia. Additionally, these treatments reduced both somatic and autonomic signs of naloxone-induced withdrawal. Spinal application of ondansetron, a 5HT3 receptor antagonist thought to ultimately be engaged by descending pain facilitatory drive, also blocked hyperalgesia and somatic and autonomic features of the withdrawal syndrome. These results indicate that the RVM plays a critical role in mediating components of opioid withdrawal that may contribute to opioid dependence.

Perspective

Manipulations targeting these descending pathways from the RVM may diminish the consequences of prolonged opioid administration-induced dependence and be useful adjunct strategies in reducing the risk of opioid addiction.

Keywords: naloxone-induced withdrawal, hyperalgesia, RVM, descending facilitation, morphine dependence

1. Introduction

In recent years, the use of opioids for chronic non-malignant pain has increased substantially10. Clinical reports indicate that sustained administration of opioids in persistent pain states can elicit physical dependence, opioid induced hyperalgesia (OIH) and analgesic tolerance in some patients2, 3. After periods of extended use, removal of opiates can result in withdrawal characterized by physical and affective-motivational symptoms that may contribute to continued opiate use and drug seeking behavior53. The severity of opioid withdrawal syndrome is considered a measurement of dependence on opioid drugs. In pre-clinical models, this syndrome is commonly induced by injection of an opioid antagonist in opioid-dependent rats4, 24 resulting in a cluster of withdrawal behaviors including hyperalgesia, wet dog shakes, teeth chattering, writhing, yawning and grooming8. Additionally, autonomic signs of withdrawal are produced including, piloerection, diarrhea and weight loss6. Injection of naloxone in opioid dependent rats also produces aversion41 and withdrawal from opiates has been described as highly aversive in humans15.

The rostral ventromedial medulla (RVM) has a prominent role in the modulation of pain states. Many studies have focused on the descending inhibitory influences arising from this region68. Additionally, the RVM has been demonstrated to exert descending facilitatory influences in pain processing46, 64. Recent evidence has implicated the RVM as a critical component to the maintenance, but not the initiation of pain arising from nerve injury9, 46 and visceral inflammation69. The RVM also has a prominent role in the modulation of opioid-induced hypersensitivity. Microinjection of lidocaine or a cholescystokinin (CCK) receptor antagonist into the RVM can block opioid-induced hyperalgesia resulting from continuous exposure to the opioid67, 73. The RVM appears to also have a role in promoting hyperalgesia during opioid withdrawal. Lidocaine microinjected into the RVM attenuates the hyperalgesia observed during opioid withdrawal26. The hyperalgesia during opioid withdrawal seems to be mediated by activation of facilitatory cells in the RVM5 which utilize cAMP-mediated mechanisms to promote hyperalgesia during opioid withdrawal7. Other studies have implicated the RVM in other homoeostatic functions besides pain modulation12, 51, 57. Lesions in this area blocked the activation of the locus coeruleus (LC) during naloxone-precipitated withdrawal48 indicating its prominent role in activation of supraspinal areas during opioid withdrawal.

The present studies explored the mechanisms that drive opiate-induced hyperalgesia and also determined whether these mechanisms are relevant to other components of the withdrawal syndrome. Activation of descending facilitation from the RVM is believed to promote hyperalgesia by ultimately engaging spinal 5HT3 receptors59, 70. For this reason, we have also used the 5HT3 receptor antagonist ondansetron to investigate whether this pathway is active during opioid-induced withdrawal.

2. Methods

2.1. Experimental Design

For evaluation of hyperalgesia and somatic/autonomic signs of withdrawal, rats were implanted subcutaneously with osmotic minipumps containing either morphine (64 mg/ml) or saline. On day 6 after implantation of minipumps, baseline measurements were taken of hot plate latency and weight. After the baseline measurements, naloxone hydrochloride was injected subcutaneously in a dose of 3 mg/kg, animals were placed in individual observation chambers and withdrawal signs were observed for the following 30 min. At the end of the observation period, measurements of hot plate latency and weight were again taken. Drugs were microinjected into the RVM before systemic naloxone injection at a time so as to coincide with their peak effect. Ondansetron was injected spinally 20 min before naloxone injection. Groups of animals were microinjected in sites outside the RVM as off-site controls. To control for any effect the act of injection may have, animals were microinjected with the vehicle for the respective drugs. To control for the effects of the injection of naloxone, animals were injected systemically with saline. All procedures were approved by the University of Arizona Animal Care and Use committee and conform to the guidelines for the use of laboratory animals of the National Institutes of Health.

2.2. Animals

Male, Sprague-Dawley rats (Harlan-Sprague-Dawley, Indianapolis) were 250–300 g at the time of testing. Animals were maintained in a climate-controlled room on a 12-h light/dark cycle and were allowed to have food and water ad libitum.

2.3. Drug Administration

2.3.1 RVM Drug Administration

All animals were prepared for bilateral microinjections into the RVM as described previously9. The rats were anesthetized with a mixture of intraperitoneal ketamine/xylazine (10/100 mg/kg of ketamine/xylazine, respectively (Sigma-Aldrich, St Louis, MO), and positioned in a stereotaxic head holder. The skull was exposed and two 26 ga guide cannulae separated by 1.2 mm (Plastics One Inc. Roanoke, VA, USA) were directed toward the lateral portions of the RVM (−11.0 mm anteroposterior from Bregma; ± 0.6 mm lateral from midline; −8.5 mm dorsoventral from the cranium, Paxinos and Watson, 1986). The guide cannulas were secured to the skull, and the animals were allowed to recover for 5 days after surgery before osmotic pump implantation. Drug administration into the RVM was performed by slowly expelling 0.5 µl of drug solution through a 33 ga injection cannula inserted through the guide cannula and protruding an additional 1 mm into fresh brain tissue to prevent backflow of the drug into the injection cannula. For off-site control injections, separate groups of animals were implanted with guide cannulae with the following coordinates: −11.0 mm from bregma; ±0.6 lateral and −6.5 mm from the cranium (2 mm above target, Figs 1E, 2E, 3E, X symbols). Other groups of animals were implanted with guide cannulae with the following coordinates: −9.0 mm from bregma, ±0.6 lateral; −8.5 mm from the cranium. The cannulae were left in place to be able to confirm the microinjection sites at the end of the experiments.

Figure 1.

Figure 1

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A) Microinjection of lidocaine (4% in 0.5µl bilaterally) attenuated the naloxone-precipitated withdrawal behaviors in morphine dependent rats. Off-site microinjection of lidocaine did not alter naloxone-precipitated withdrawal behaviors. B). Lidocaine also prevented the reduction in hot plate latency observed 30 minutes after naloxone injection. Off-site microinjection of lidocaine failed to block morphine-induced thermal hypersensitivity or naloxone induced enhanced thermal hypersensitivity. C). Lidocaine reduced the number of animals demonstrating naloxone precipitated diarrhea. Off-site microinjection of lidocaine failed to block naloxone induced diarrhea. D) RVM lidocaine, but not off-site injections, also attenuated the weight loss observed after naloxone injection. (E) Diagram showing site of injection within the RVM (0) and off-site injections (X). Data in all graphs are expressed as mean ± SEM, n=6–12 per group, * p<0.05 compared to RVM saline/systemic naloxone; # p<0.05 compared to RVM saline/systemic saline (control group).

Figure 2.

Figure 2

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A) Microinjection of kynurenic acid (1 nmol in 0.5µl bilaterally) attenuated the withdrawal behaviors observed after systemic injection of naloxone in morphine-dependent rats. B). Kynurenic acid also prevented the reduction in hot plate latency observed 30 minutes after naloxone injection C). RVM kynurenic acid reduced the number of animals demonstrating diarrhea and D) also attenuated the weight loss observed after naloxone injection. (E) Diagram showing site of injection within the RVM (0) and off-site injections (X). Data are expressed as mean ± SEM, n=6–10 per group, * p<0.05 compared to RVM saline/systemic naloxone; # p<0.05 compared to RVM saline/systemic saline (control group).

Figure 3.

Figure 3

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A) Microinjection of YM022 (0.25 ng in 0.5µl bilaterally) attenuated the withdrawal behaviors observed after systemic injection of naloxone in morphine dependent rats. B). YM022 also prevented the reduction in hot plate latency observed 30 minutes after naloxone injection C). YM022 reduced the number of animals demonstrating diarrhea and D) also attenuated the weight loss observed after naloxone injection. (E) Diagram showing site of injection within the RVM (0) and off-site injections (X). Data are expressed as mean ± SEM, n=6–10 per group, * p<0.05 compared to RVM saline/systemic naloxone; # p<0.05 compared to RVM saline/systemic saline (control group).

2.3.2 Spinal Drug Administration

Rats were anesthetized with the use of intraperitoneal ketamine/xylazine (Sigma, 10/100 mg/kg, respectively) and placed in a stereotaxic holder. The cisternum magnum was exposed, and an 7.8 cm catheter (PE-10) was implanted, terminating in the lumbar region of the spinal cord74. Catheters were sutured into place with the tubing externalized at the back of the neck. Animals with signs of motor weakness or paralysis after surgery were not used (~25%). Animals were allowed to recover for 7 days prior to osmotic pump implantation.

2.3.3. Drugs and Chemicals

Morphine, a generous gift from the National Institute on Drug Abuse Drug Supply Program, was dissolved to 64 mg/kg to fill the osmotic minipumps (Alzet, Mountain View, CA) which were surgically implanted subcutaneously under isofluorane anesthesia. Naloxone (RBI Chemicals, Boston, MA) was dissolved in physiological saline to 3 mg/kg and was given through intraperitoneal injection4. Ondansetron (Zofran purchased from the University Medical Center Pharmacy) was dissolved to 10 µg in 5µl of physiological saline and delivered through the intrathecal cathether59. Kynurenic acid (Tocris, Ellisville, MO) was dissolved to 1 nmol in 0.5µl of physiological saline for bilateral microinjection into the RVM22. YM022 (Tocris, Ellisville, MO) was dissolved in 0.25 ng in 0.5µl of physiological saline for microinjection into the RVM. Lidocaine hydrochloride (4% w/v; Sigma-Aldrich, St Louis, MO) was given by bilateral microinjection (0.5 µl/side) into the RVM. Pilot studies were carried out to determine the best dose of naloxone to induce the opioid withdrawal. The doses used for all drugs either microinjected in the RVM or applied intrathecally are doses used before in previous studies in our laboratory. Injections of all drug either into the RVM or spinally took place 15 minutes prior systemic naloxone.

2.4. Behavioral Measurements

2.4.1. Hot plate latency

Before any drug administrations, rats were placed in the hot plate apparatus set at 52°C to attain baseline latency. The hot plate response latency was measured as the time at which the rat withdraws and shakes either hindpaw from the plate surface. The latency was measured with a timer and approximated to the tenth of second. A cut-off latency of 30-s was used. After baseline, rats received an injection of naloxone and observed for withdrawal behaviors during a 30 min period. At the end of the observation period, rats underwent a final measurement of hot plate latency testing.

2.4.2. Physical signs of morphine withdrawal

Rats were monitored for physical signs of morphine withdrawal as previously described4, 24. Briefly, rats were placed in Plexiglas cylinders (radius 7 cm; height 24 cm) covered with saw dust bedding. Before any drug administrations, rats were observed for a period of 30 min for spontaneous signs of morphine withdrawal. After this observation period, rats were separated in different groups receiving a subcutaneous injection of saline or naloxone (3 mg/kg) and placed back on the cylinders and the frequency of naloxone-induced morphine withdrawal signs such as wet dog shakes, teeth chattering, writhing, yawning and grooming were scored. Frequency of behaviors was quantified instead of duration of behaviors as this methodology has been reported in the literature4, 24, 33. A single observer was trained in the methodology and blinded to the treatment that each rat had received as previously reported4, 24, 32, 34, 44, 49, 50, 54, 76, 77. Additionally, autonomic signs of withdrawal were recorded such as diarrhea (yes/no) and weight loss (weight was recorded before and then 30 min after naloxone injection).

2.5. Data Analysis

Group differences were determined using a 2 way ANOVA, with factors being 1) saline vs. morphine treatment and 2) drug vs vehicle treatment. Post-hoc analysis of selected pairings was done using the Student t test with Bonferroni’s correction. Differences were considered significant if p<0.05. A Fisher’s exact test was run to determine group differences for the diarrhea measure, differences were considered significant if p<0.05.

3. Results

3.1. Inactivation of RVM neuronal transmission blocks somatic signs of naloxone-induced withdrawal

Systemic injection of naloxone (3 mg/kg, s.c.) induced a robust spectrum of withdrawal behaviors including grooming, yawning, teeth chattering, wet dog shakes and writhing behaviors in morphine treated rats. Microinjection of lidocaine into the RVM reduced the incidence of all naloxone-precipitated withdrawal behaviors except for grooming behavior (Fig. 1a). Of note, there was low incidence of these behaviors in saline-treated rats irrespective of RVM treatment, with no yawning, teeth-chattering, wet dog shakes and few instances (< 2) of grooming and writhing behaviors following this dose of naloxone (data not shown).

In rats treated with systemic morphine infusion, pre-lidocaine hot plate latencies were significantly reduced compared to rats treated with systemic saline infusion, consistent with the development of opioid-induced hyperalgesia (OIH) (Fig 1b, #p<0.05). Microinjection of lidocaine reversed the thermal hyperalgesia in the morphine dependent animals, returning hot plate latencies to saline control values. Systemic injection of naloxone further reduced hot plate latencies in morphine-treated rats (Fig 1b, *p < 0.05). Administration of lidocaine 15 minutes prior to systemic naloxone prevented the naloxone-induced reduction in hot plate response latencies, resulting in latencies equivalent to saline-treated control animals (Fig 1b). Administration of lidocaine into the RVM did not alter hot-plate latencies of rats treated with saline infusion (Fig. 1b). Administration of saline into the RVM did not alter hot-plate latencies of saline- or morphine-infused animals prior to systemic administration of naloxone (data not shown).

Naloxone also precipitated autonomic signs of withdrawal, measured as the number of rats that had diarrhea (Fig. 1c) and rapid weight loss within 30 min in morphine-treated rats (Fig. 1d). RVM microinjection of lidocaine significantly reduced the number of rats with naloxone-induced diarrhea and the amount of weight loss in morphine-treated rats (Figs 1c and 1d, respectively). Rats treated with saline infusion did not develop diarrhea or show weight loss following administration of naloxone irrespective of microinjection of saline or lidocaine into the RVM (data not shown).

The effects of lidocaine were specific for the RVM (Fig. 1e). Lidocaine microinjections outside the RVM (Fig 1e, “X” symbols) did not alter the naloxone-induced withdrawal syndrome (Fig. 1A) or hyperalgesia (Fig. 1B).

3.2. Glutamatergic antagonist into the RVM blocks naloxone-induced withdrawal syndrome

It has been shown previously that facilitatory cells in the RVM express glutamate receptors and antagonizing these receptors blocks facilitation of pain transmission20, 22. To determine the role of glutamatergic transmission in the RVM in naloxone-induced withdrawal, the non-selective excitatory amino acid receptor antagonist kynurenic acid was microinjected into the RVM, at a non-neurotoxic dose72 used in electrophysiological and behavioral studies within the RVM18, 19, 22, 15 min prior to systemic naloxone. As described above, saline microinjection into the RVM did not prevent naloxone-induced withdrawal behaviors of grooming, yawning, teeth-chattering, wet dog shakes, or writhing (Fig 2a). Microinjection of kynurenic acid (1 nmol in 0.5 µl, bilaterally) into the RVM prior to systemic injection of naloxone significantly inhibited naloxone-precipitated yawning, teeth-chattering, wet-dog shakes, and writhing in morphine dependent rats (*p < 0.05), but did not alter naloxone precipitated grooming (Fig 2a).

As with lidocaine microinjection, microinjection of kynurenic acid into the RVM reversed morphine induced hyperalgesia, elevating hot-plate latencies to saline control values. Moreover, microinjection of kynurenic acid into the RVM prevented the naloxone-induced enhanced reduction in hot plate latencies in morphine-treated rats with paw withdrawal latencies equivalent to saline control values (Fig 2b).

Autonomic signs of naloxone-induced withdrawal were also prevented by microinjection of kynurenic acid into the RVM. Microinjection of kynurenic acid into the RVM reduced the number of animals demonstrating naloxone precipitated diarrhea (Fig. 2c) and reduced the weight loss in morphine-dependent rats (Fig. 2d). As with lidocaine injections, the effects of kynurenic acid were specific for the RVM, as off-site injections (Fig 2e, “X” symbols) did not have any effects on naloxone-induced withdrawal (data not shown). No signs of neurotoxicity were observed in any animals that had received kynurenic acid injections. Additionally, no signs of neuronal cell loss were observed in histological sections taken from randomized selected rats that had received kynurenic acid.

3.3. CCK receptor antagonist in the RVM blocks naloxone-induced withdrawal syndrome

It has been shown previously that CCK plays a role in the RVM to engage descending facilitation promoting opioid-induced hyperalgesia21, 62. The present study investigated the role of CCK transmission in the RVM in the naloxone-induced withdrawal syndrome. Microinjection of the specific CCK2 receptor antagonist YM022 (0.25 ng in 0.5 µl, bilaterally) 15 min before systemic naloxone injection significantly inhibited naloxone-precipitated yawning, teeth chattering, wet dog shakes, and writhing, but failed to alter grooming (Fig. 3a).

As previously reported62, antagonizing CCK2 receptors in the RVM reversed sustained morphine-induced hyperalgesia, with hot-plate response latencies equivalent to saline control values. Moreover, YM022 microinjection into the RVM prevented the naloxone-induced enhanced reduction in hot plate latencies observed in morphine treated rats, with response latencies returning to saline control values (Fig 3b).

Autonomic signs of naloxone-induced withdrawal were also prevented by microinjection of YM022 into the RVM. Animals receiving control microinjections of saline into the RVM demonstrated signs of diarrhea and greater weight loss than animals receiving YM022 (Figs 3c and 3d, respectively). The effects of YM022 were specific for the RVM, as off-site injections (Fig 3e) did not have any effects on naloxone-induced withdrawal (data not shown).

3.4. Antagonizing 5HT3 receptors in the spinal cord blocks naloxone-induced withdrawal syndrome

It has been shown previously that serotonin acting at the 5HT3 receptor can facilitate pain transmission at the spinal cord level58, 59, 61. The present study investigated the role of spinal 5-HT3 receptors in naloxone-induced withdrawal syndrome by spinal application of the 5HT3 receptor antagonist ondansetron (10 µg) 15 min prior to systemic administration of naloxone. Spinal ondansetron attenuated naloxone-induced yawning, teeth chattering, wet dog shakes, and writhing, but did not alter naloxone-induced grooming behavior in morphine dependent rats (Fig. 4a).

Figure 4.

Figure 4

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A) Spinal application of ondansetron (30µg in 5µl) attenuated the withdrawal behaviors observed after systemic injection of naloxone in morphine dependent rats B). Ondansetron also prevented the reduction in hot plate latency observed 30 minutes after naloxone injection C). Ondansetron reduced the number of animals demonstrating diarrhea and D) also attenuated the weight loss observed after naloxone injection. Data are expressed as mean ± SEM, n=6–10 per group, * p<0.05 compared to RVM saline/systemic naloxone; # p<0.05 compared to RVM saline/systemic saline (control group).

As previously reported70, spinal administration of ondansetron reversed sustained morphine-induced thermal hypersensitivity, with response latencies equivalent to saline control values (Fig 4b). Administration of naloxone further reduced response latencies in morphine-treated rats given intrathecal saline. Administration of spinal ondansetron prevented the naloxone-precipitated reduction in hot plate latency in morphine-treated rats, with response latencies equivalent to saline-treated animals (i.e. control values). Animals receiving control saline intrathecal injections demonstrated lower hot plate latencies when compared with animals receiving ondansetron (p<0.05).

Autonomic signs of naloxone-induced withdrawal were also prevented by spinal injection of ondansetron (Figs 4d and 4e). Animals, receiving control intrathecal injections of saline, demonstrated signs of diarrhea and greater weight loss than animals receiving ondansetron (p<0.05).

4. Discussion

Opiate use is associated with feelings of well-being and euphoria. The desire to recapture these pleasurable feelings may lead to craving and drug seeking behaviors. Opiates thus are positively reinforcing, which contributes to continued drug use and abuse. It has been suggested that dependence may form part of a continuum that stretches from no drug use through “controlled” drug use and finally to, in extreme cases, compulsive drug use. The transition from “controlled” drug use to compulsive use is considered the initiation phase of drug dependence. Transition to compulsive drug use likely depends, in part, on the ability of drug self-administration to prevent the consequences of drug abstinence. During continual drug use, neuroadaptive changes occur which underlie negative symptoms of withdrawal55. In humans, opiate withdrawal is characterized by somatic and autonomic signs such as pupil dilation, piloerection, shivering, sweating, lacrimation, tremors, muscle pain (myalgia), diarrhea, vomiting, insomnia, and yawning, and affective symptoms including dysphoria or anhedonia, hyper-irritability, anxiety, restlessness, tension, and depression17, 23, 25, 38, 55. Moreover, tolerance to the positive subjective effects of opioids (e.g., euphoria) has been demonstrated in humans, suggesting that a decrease in the positive reinforcing effects of the drug may occur concurrently with neuroadaptive changes that produce negative reinforcing effects55. Thus, it is possible that compulsive, uncontrolled drug use is driven in some individuals by a desire to avoid the negative consequences of opioid withdrawal rather than to derive the positive subjective effects originally associated with drug use during the initiation phase of drug dependence28, 31, 55. However, as some studies indicate that complete tolerance to the euphoric effects of opioids does not occur, pleasure-seeking and withdrawal-avoidance most likely exist as parallel motivational factors in continued drug use37, 55.

The present study demonstrates that the RVM plays an important role in many characteristic behaviors observed during naloxone-precipitated withdrawal syndrome in morphine-dependent rats. Disrupting descending facilitatory signaling from the RVM by microinjection of lidocaine, the EAA antagonist kynurenic acid, or the CCK2 receptor antagonist YM022 not only blocked naloxone-induced thermal hyperalgesia, but also blocked all other measured behavioral signs of the opioid-induced withdrawal syndrome with the exception of increased grooming behaviors. Spinal 5-HT3 receptors have been suggested to be critical in descending facilitatory signaling from the RVM58, 60, 61. We demonstrate that spinal administration of the 5HT3 receptor antagonist ondansetron also blocked all measured signs of naloxone-precipitated withdrawal except for grooming, in a similar manner to disrupting descending facilitatory signaling from the RVM. The present results indicate a prominent role of descending facilitatory signaling from the RVM in mediating naloxone-precipitated symptoms of opioid withdrawal.

A large body of literature describes the role of brainstem structures in the descending inhibition of pain68. In recent years, it has additionally been recognized that there is a descending facilitatory component to this brainstem pathway, which uses the RVM as its major output nuclei46, 64 The descending facilitation component is engaged especially in situations of tissue injury9, 43, 45, inflammation of somatic35, 63 and visceral tissues11, 63 and is likely important even in situations such as opioid infusion induced hyperalgesia, where there is no apparent tissue injury14, 67, 69. This body of evidence indicates that the RVM has a prominent role in maintaining sensitization of neural pathways responsible for pain transmission. Our findings expand the role of descending pathways from the RVM to other physical signs of opioid withdrawal. Interrupting neuronal transmission in the RVM prevented most signs of opioid withdrawal including hyperalgesia and autonomic signs. Previous studies have demonstrated that lidocaine microinjection into the RVM blocked the opioid withdrawal-induced hyperalgesia observed after acute injections of morphine and subsequent naloxone26. It was speculated that blocking of synaptic transmission of “ON” cells was responsible for the reduction in hyperalgesia, since “ON” cells were the only cells active during the hyperalgesic period5. There is evidence that neurons in this area mediate other homoeostatic functions besides pain12, 36, 51, 57, 66. Additionally, the RVM may serve as a relay output for effects of the locus coeruleus47, 52, 65, 75. Indeed, lesions of the locus coeruleus (LC) have been shown to decrease physical signs of opioid withdrawal33, and electrical stimulation of this nucleus or direct injection of opioid antagonists into the locus coeruleus precipitate the expression of opiate withdrawal16, 29. There are reciprocal connections between the RVM and the LC40, 56. Some of these connections are excitatory in nature as activation of LC neurons by nalaxone-precipitated withdrawal is blocked by lesions in the nucleus paragigantocellularis48, or by injection of EAA antagonists into the LC1. It would seem that the RVM, given its central role in modulating the antinociceptive effects of opioids and its multiple connections with other brain structures that have a role in the expression of opiate withdrawal, is a prime candidate to modulate multiple physical symptoms that occur during opioid withdrawal.

In recent years, there has been an effort to identify the neurochemical signature of the pain facilitatory (i.e., presumably “ON”) cells in the RVM. Electrophysiological experiments have elucidated that these cells may express glutamate receptors since microinjection of glutamate antagonists reduces the firing of this specific population of cells20, 22. Recent studies have also suggested that these cells likely express CCK2 receptors. CKK can act as a physiological “anti-opioid” which can work against the antinociceptive effects of opioids71. Injection of CCK into the RVM facilitates nociception13, 30, and this effect can be attenuated by CCK2 antagonists73. CCK levels are elevated in the RVM after spinal nerve injury and sustained exposure to morphine leading to hypersensitivity30, 73. In the present study, antagonizing EAA receptors with the broad EAA antagonist kynurenic acid or blocking CCK2 receptors with the antagonist YM022 was able to reduce behavioral signs of morphine withdrawal. These data suggest that EAA transmission in the RVM is important in the output from the RVM involved with the manifestation of behavioral signs of withdrawal. CCK2 receptors in the RVM seem to be also important in the facilitation of the withdrawal behavior. These data support the possibility that modulation of facilitatory output from the RVM requires activation of EAA and CCK2 receptors to promote features of the opiate withdrawal syndrome.

Previous studies by Suzuki, Dickenson and colleagues have determined that descending facilitation from the RVM uses 5HT acting at 5HT3 receptors to enhance nociceptive transmission at the spinal cord level58, 59, 61. In our study we have taken a similar approach and used the specific 5HT3 receptor antagonist ondansetron in the spinal cord to block the effects of descending facilitation. Ondansetron had similar effects on behavioral signs of opioid withdrawal as the manipulations done in the RVM. These data suggest that serotonin acting at the 5HT3 receptor in the spinal cord ultimately mediate many of the behavioral signs of opioid withdrawal. It should be noted that systemic injection of ondansetron has also been demonstrated to inhibit behavioral signs of opioid withdrawal44 raising the possibility of other sites within the nervous system where 5HT3 receptors may participate36. However, it is not likely that spinal administered ondansetron exerted its effects in supraspinal sites. Several lines of evidence suggest that spinally applied ondansetron does not exert its effects through supraspinal sites including: 1) consistent with previous studies69, the effects of ondansetron are very rapid and last no more than one hour supporting a local (spinal) effect of spinally-administered ondansetron; 2) antagonizing 5HT3 receptors in the medulla blocks the effects of opiate analgesia27 suggesting that supraspinal ondansetron would have a net pro-nociceptive effect, opposite to that observed here; and 3) i.c.v. injection of ondansetron did not produce any changes in baseline thresholds in naïve rat42 suggesting that the effects of spinal administration are likely to be mediated locally.

The RVM thus has a prominent role in the behavioral syndrome observed after injection of naloxone in morphine-dependent rats. This study provides new insights into the mechanisms that drive descending facilitation to elicit opiate-induced withdrawal hyperalgesia and additionally demonstrates a critical role for this circuit in other withdrawal behaviors. It should be noted that throughout our studies, the manipulations made to interfere with descending output from the RVM were effective in reducing multiple aspects of naloxone-precipitated withdrawal but consistently failed to affect grooming behaviors. Grooming has been associated with increased states of anxiety and may reflect the activation of neural circuits that are rostral to the RVM39. As the withdrawal syndrome is known to be aversive, understanding the mechanisms that promote withdrawal through the descending facilitation circuit may be useful in developing adjunct strategies that can diminish drug seeking in order to prevent this unpleasant state. Whether mechanisms that drive descending facilitation also mediate aversiveness is unknown and requires further study.

Acknowledgement

This work was funded by P01 DA 06284.

Footnotes

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