Morphine-induced mu opioid receptor trafficking enhances reward yet prevents compulsive drug use

Morphine, heroin and other commonly abused opioids induce little mu opioid receptor (MOR) trafficking compared to endogenous opioids. We utilized knock-in mice expressing a mutant recycling MOR (RMOR) that desensitizes and is internalized in response to morphine to show that facilitating MOR trafficking not only enhances morphine reward but, despite this, reduces the development of addiction-like behaviours. To demonstrate this, we developed a novel model of the transition from controlled to compulsive drug use that recapitulates many features of human addiction, including persistent drug seeking despite adverse consequences and a decreased preference for alternative rewards. These behaviours emerged spontaneously in wild-type but not RMOR mice, and their intensity predicted the reinstatement of morphine seeking after extended abstinence, while prior morphine intake did not. These results confirm previous findings in the rat that addiction can be dissociated from both reward and consumption. Most importantly, these results demonstrate that one can simultaneously reduce the ‘addictiveness’ of morphine and enhance its desirable effects by promoting agonist-induced MOR trafficking.


Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) 1st Editorial Decision 20 January 2011 Thank you for the submission of your manuscript "Morphine-Induced Mu Opioid Receptor Trafficking Enhances Reward Yet Prevents Compulsive Drug Use" to EMBO Molecular Medicine.
We have now heard back from the two referees whom we asked to evaluate your manuscript. You will see that they find the topic of your manuscript potentially interesting. However, they also raise significant concerns about the study, which should be addressed in a major revision of the manuscript.
You will see that reviewer #1 is positive about the study, while reviewer #2 is more reserved. In particular, reviewer #2 feels that it is crucial to investigate whether the decreased compulsive behavior in RMOR mice reflects a lack of tolerance development. This reviewer also notes that the significance of the manuscript could be further increased by experiments in wt mice, which address the effect of altered/manipulated receptor recycling on drug seeking behavior. In addition, both reviewers highlight that the presence of LL and HH groups among wt mice should be more extensively explained.
Given the balance of these evaluations, we feel that we can consider a revision of your manuscript if you can convincingly address the issues that have been raised within the space and time constraints outlined below.
Revised manuscripts should be submitted within three months of a request for revision. They will otherwise be treated as new submissions, unless arranged otherwise with the editor.
Mu opioid receptor internalization impacts on both acute and long-term effects of opioids, and this has been established since a long time by a number of authors (including the authors) in several approaches. This notion has important implications clinically, both in the treatment of chronic pain and the development of addiction, and is a hot area of investigation. The present authors have pioneered a mouse knock-in approach where a morphine-internalizing mu opioid receptor (RMOR) is expressed instead of the native mu receptor, notoriously known to be weakly regulated by morphine (low or no internalization, variable desensitization) compared to endogenous agonists or methadone. In a first paper, the authors showed that these animals show enhanced morphine analgesia and reduced tolerance and dependence in response to morphine (Kim et al. Current Biology 2008). In this paper, Berger et al. continue analyzing the mutant mice and address the importance of mu receptor internalization in morphine reward and the development of addictive behaviors. To this aim, the authors establish a novel mouse model of voluntary oral morphine consumption, which examines several features of addiction including motivation to consume the drug, loss-of-control in drug intake, consumption despite adverse consequences, preference for the drug rather than natural reward and relapse to drug taking. Data show that morphine-induced mu opioid receptor internalization leads to enhanced morphine reward, but protects the animal against the development of addictive behaviors. This is an extremely interesting result that has been awaited in the field since many years. Beyond the particularly appropriate and unique knock-in approach to address the issue, a main innovative aspect of this study is the successful establishment of a longitudinal behavioral mouse model of addiction. Similar models have been developed in the rat by several research teams, however translation in the mouse has been difficult, hampering any possibility to address molecular mechanisms governing behaviors. The positive correlation between individual compulsivity scores and reinstatement in wild-type mice is particularly compelling. Next, the study is very wellconducted, every possible control is provided and mutant mice phenotypes are convincing. There is no doubt that this paper is of great interest for the broad community of neuroscientists and G protein coupled researchers.
Here are a number of concerns that should be addressed: 1.Abstract, last sentence. That "addiction can be dissociated from both reward consumption" has been extensively shown earlier in the rat. The authors should indicate that this is now also established in the mouse. 2.The introduction section presents an extremely long and extensive review of the literature, explaining molecular events that may link receptor internalization with acute morphine effects, tolerance and dependence. The present study, however, addresses a very different question, and the development of addiction-like behaviors differs greatly from physical opioid dependence and analgesic tolerance. The reader is disappointed that almost no discussion addresses possible molecular/circuitry mechanisms underlying data presented in this paper. One may expect a much shorter introduction (contract the first 5 paragraphs) and a more extensive discussion -even if speculative-on possible molecular events linking mu receptor internalization and the several aspects of addictive-related behaviors examined here.
3. The last sentence if the introduction is unclear ("Moreover, we provide evidence...abstinence). 4.Methods. Animal cohorts used for each experiment should be detailed. Whether one large or several cohorts were used across experiments is unclear. 5. Fig 2C. Could the authors explain why morphine intake decreases at week 9? 6.The observation of two WT groups (WT-HH and WT-LL, Fig 5G), and the fact that about half of WT individuals behave as RMOR animals in reinstatement is intriguing. However a possible link between receptor internalization and the fact that some WT individuals are less prone to reinstate their morphine consumption is not discussed. Could there be a gene polymorphisms affecting receptor internalization in mice (and possibly in humans?), which may be predictive of vulnerability to opiate abuse? The observation may deserve some discussion.
7. Fig 6A. Whether this experiment is a morphine place preference experiment with naloxone reversal, or a naloxone place aversion experiment is unclear. 8. Altogether, the phenotype of RMOR animals is puzzling, but emphasis of the discussion is on the mouse behavioral model and its validity, rather than the role of mu receptor internalization. The link between receptor internalization and the very distinct behaviors -and their combination-studied here remains unclear, and certainly is highly complex, and clear hypotheses may be provided in the end.
Referee #2 Comments on Novelty/Model system: The authors have developed a mouse line to test their hypothesis that µ-opioid receptor recycling and resensitization have impact on the chronic morphine effect. They have reported such mouse line (RMOR) is more sensitive to morphine and developed less tolerance and dependence to morphine. Now they are testing whether addiction to morphine, or reward driven drug seeking behavior, is also modified by the recycling of the receptor. Surprising results were obtained to indicate that morphine addictive liability in RMOR mice are less than that observed in wild type mice even though these mice are more sensitive to morphine, both in the initial drug seeking behavior and in subsequent morphine-induced reinforcing paradigm during withdrawal and abstinence.
The novelty of the studies lies with the mouse model, and the development of drinking as drug selfadministered model in mice, though such approach has been used successfully in rats.
Although there are obvious difference in CPP and lever press studies between WT and RMOR mice, whether such differences can be attributed to receptor recycling can not be concluded. The current studies could be of greater significance if the authors could demonstrate by manipulating the ability of morphine to induce receptor recycling in WT mice, as suggested by their previous publications, that the drug seeking behavior in the WT animals can be altered. At the current state, the manuscript described the feasibility of the model, reported differences in different mouse lines, with minimal mechanistic implication.
Referee #2 Other Remarks : The key to the studies is the hypothesis that addictive behavior to morphine can be altered by the recycling of µ-opioid receptor. The RMOR mouse line appears to support such conclusion. However, due to the increase sensitivity and less tolerance developed toward morphine in the RMOR mice, whether decrease in the compulsive behavior tested in current paradigms observed with RMOR mice reflects the lack of tolerance development, such as in the control release of dopamine, need to be addressed. The reported behavioral studies can be strengthened with some neuropharmacological data. One issue that the authors need to resolve is the presence of LR and HR groups in WT animals. If receptor recycling is the key for the observed effects in RMOR, then the authors should provide some explanation for the LR and HR groups, in which some behavioral tests of LR group resembled that of RMOR. In some of behavioral experiments, i.e., the CPP and locomotor activities, detailed descriptions are lacking. From the reading of the manuscript, it appears that the authors only conditioned the animals to the drug-paired chamber only once before testing. Robust CPP was observed. Such paradigm deviates from many reported studies in which multiple conditioning sessions are needed for conditioned place preference response. Could the difference in paradigms result in the current observed difference in RMOR and WT mice? As for the locomotor test, it is confusing to see that the distance travel (in cm) reached a plateau vs time. Does it mean that the mice stop running after a certain period of time?
The manuscript is difficult to read, maybe due to the myriads of behavioral tests reported. Authors might want to consider putting some the details of the behavioral tests, such as different aspects of compulsivity into material and method section instead of result section. The major points of the manuscript can be distilled somewhat to assist the non-specialist to fully appreciate the impact of the studies.

Referee #1 Other Remarks :
This is an extremely interesting result that has been awaited in the field since many years. Beyond the particularly appropriate and unique knock-in approach to address the issue, a main innovative aspect of this study is the successful establishment of a longitudinal behavioral mouse model of addiction. Similar models have been developed in the rat by several research teams, however translation in the mouse has been difficult, hampering any possibility to address molecular mechanisms governing behaviors. The positive correlation between individual compulsivity scores and reinstatement in wild-type mice is particularly compelling. Next, the study is very wellconducted, every possible control is provided and mutant mice phenotypes are convincing. There is no doubt that this paper is of great interest for the broad community of neuroscientists and G protein coupled researchers.
Here are a number of concerns that should be addressed: 1. Abstract, last sentence. That "addiction can be dissociated from both reward consumption" has been extensively shown earlier in the rat. The authors should indicate that this is now also established in the mouse.
We now mention these findings in the abstract as well as in the discussion section.
2. The introduction section presents an extremely long and extensive review of the literature, explaining molecular events that may link receptor internalization with acute morphine effects, tolerance and dependence. The present study, however, addresses a very different question, and the development of addiction-like behaviors differs greatly from physical opioid dependence and analgesic tolerance. The reader is disappointed that almost no discussion addresses possible molecular/circuitry mechanisms underlying data presented in this paper. One may expect a much shorter introduction (contract the first 5 paragraphs) and a more extensive discussion -even if speculative-on possible molecular events linking mu receptor internalization and the several aspects of addictive-related behaviors examined here.
We have shortened the introduction significantly while hopefully still providing sufficient background for readers to understand why facilitating morphine-induced MOR recycling may enhance acute analgesia (and reward), reduce tolerance, and reduce physical (and affective) dependence. The relative contribution of each of these behaviors to morphine is complicated and highly controversial; however, each of them likely plays a role in establishing and/or maintaining compulsive drug use. We think that a slightly longer introduction is appropriate for EMBO molecular medicine in order to give both a basic science and clinical overview of the rational.
Please see below for a more detailed discussion of a possible mechanism for the specific phenotype reported here, which we have included in the discussion section of the revised manuscript.
3. The last sentence if the introduction is unclear ("Moreover, we provide evidence...abstinence).
We have clarified this sentence.
4. Methods. Animal cohorts used for each experiment should be detailed. Whether one large or several cohorts were used across experiments is unclear.
We have added these details to the methods section. A single large cohort of mice was used for all the self-administration data presented in this manuscript (after many iterations to refine the best approach).
5. Fig 2C. Could the authors explain why morphine intake decreases at week 9?
We do not have a good explanation for why morphine intake fell at week 9. As a similar decrease was observed in all groups, we do not believe this pattern is related to either genotype or the development of compulsive drug seeking. More likely, it reflects some shared external influence that acted similarly on all animals, i.e. construction noise in our animal facility, for example (which did coincide with week 9).
6. The observation of two WT groups (WT-HH and WT-LL, Fig 5G), and the fact that about half of WT individuals behave as RMOR animals in reinstatement is intriguing. However a possible link between receptor internalization and the fact that some WT individuals are less prone to reinstate their morphine consumption is not discussed. Could there be a gene polymorphisms affecting receptor internalization in mice (and possibly in humans?), which may be predictive of vulnerability to opiate abuse? The observation may deserve some discussion. 7. Fig 6A. Whether this experiment is a morphine place preference experiment with naloxone reversal, or a naloxone place aversion experiment is unclear.

We agree that this is an extremely important question and are interested in identifying molecular and behavioral biomarkers that predict which WT animals will become WT-LL and WT
The purpose of this experiment was to measure the motivational effect of withdrawing morphine from RMOR and WT mice that had received chronic morphine treatment. We would argue that in the RMOR mice, naloxone merely reversed morphine reward; whereas, in the WT mice, naloxone not only reversed morphine reward but also revealed an aversive effect of withdrawal. We have clarified this in the text.
8. Altogether, the phenotype of RMOR animals is puzzling, but emphasis of the discussion is on the mouse behavioral model and its validity, rather than the role of mu receptor internalization. The link between receptor internalization and the very distinct behaviors -and their combination -studied here remains unclear, and certainly is highly complex, and clear hypotheses may be provided in the end.

On p. 16-19 and 23-24 of the manuscript, we have suggested that the prevention of compulsive morphine seeking in RMOR mice could be due to a reduction in affective dependence and reward sensitization. One possible mechanism for this (that we have added to the discussion) is the prevention of a cAMP-dependent increase in GABA release onto dopamine neurons in the ventral tegmental area (VTA). Specifically, we have previously shown that both cAMP superactivation and
the cAMP-dependent increase in GABA release caused by chronic morphine are prevented in RMOR mice. We have added a discussion of this possibility to the discussion, and otherwise shortened the discussion for better flow.
We have also shown that other biochemical "markers" of morphine dependence are prevented in RMOR mice. For example, chronic morphine induces brain region-specific increases in NMDA receptor and glucocorticoid receptor expression in WT but not RMOR mice, and these changes have been hypothesized to play roles in withdrawal-related hyperalgesia and stress-induced craving, respectively. We have added this to the discussion as well.
Referee #2 Comments on Novelty/Model system: The authors have developed a mouse line to test their hypothesis that mu opioid receptor recycling and resensitization have impact on the chronic morphine effect. They have reported such mouse line (RMOR) is more sensitive to morphine and developed less tolerance and dependence to morphine. Now they are testing whether addiction to morphine, or reward driven drug seeking behavior, is also modified by the recycling of the receptor. Surprising results were obtained to indicate that morphine addictive liability in RMOR mice are less than that observed in wild type mice even though these mice are more sensitive to morphine, both in the initial drug seeking behavior and in subsequent morphine-induced reinforcing paradigm during withdrawal and abstinence. The novelty of the studies lies with the mouse model, and the development of drinking as drug self-administered model in mice, though such approach has been used successfully in rats.
1. Although there are obvious differences in CPP and lever press studies between WT and RMOR mice, whether such differences can be attributed to receptor recycling can not be concluded. The current studies could be of greater significance if the authors could demonstrate by manipulating the ability of morphine to induce receptor recycling in WT mice, as suggested by their previous publications, that the drug seeking behavior in the WT animals can be altered. At the current state, the manuscript described the feasibility of the model, reported differences in different mouse lines, with minimal mechanistic implication.
The referee grants that our results are "surprising" and "novel" but is not convinced that the genotypic difference in addiction liability can be attributed to a difference in MOR recycling. In short, the inability to conclusively test our hypothesis by comparing different ligands is what led us to create the RMOR receptor and the RMOR knock in mouse. We believe our experiment comparing morphine in the RMOR and WT mice is much more elegant and much "cleaner" than any study in which two drugs with many varying properties were compared.

2.
The key to the studies is the hypothesis that addictive behavior to morphine can be altered by the recycling of mu opioid receptor. The RMOR mouse line appears to support such conclusion. However, due to the increase sensitivity and less tolerance developed toward morphine in the RMOR mice, whether decrease in the compulsive behavior tested in current paradigms observed with RMOR mice reflects the lack of tolerance development, such as in the control release of dopamine, need to be addressed. The reported behavioral studies can be strengthened with some neuropharmacological data.
The referee hypothesizes that the decrease in compulsive behavior observed in the RMOR mice is due to a decrease in tolerance to the rewarding effects of morphine -specifically, a decrease in tolerance to morphine-induced dopamine release in the nucleus accumbens. The referee proposes neuropharmacological experiments to test this hypothesis.
As shown in Figure 6, neither RMOR nor WT mice developed tolerance to the rewarding effects of morphine, even when WT mice were tolerant to the analgesic effects. In fact, after a period of abstinence, WT mice became the opposite of tolerant, more sensitive, to morphine reward.
We believe that the decrease in compulsive behavior seen in the RMOR mice is more likely related to decreases in affective dependence and its sequelae, as discussed on p. 16-19 and 23-24 of the manuscript.
Indeed, we have previously shown that neuropharmacological correlates of dependence, i.e. increased GABA release onto dopamine neurons in the ventral tegmental area, are decreased in RMOR mice (see Madhavan et al, 2010).
3. One issue that the authors need to resolve is the presence of LR and HR groups in WT animals. If receptor recycling is the key for the observed effects in RMOR, then the authors should provide some explanation for the LR and HR groups, in which some behavioral tests of LR group resembled that of RMOR.
The referee would like a mechanistic explanation of the individual variation observed among WT mice. We have now received the enclosed reports from the referees whom we asked to re-assess it.
As you will see, both Reviewers acknowledge that the manuscript was significantly improved during revision and Reviewer #1 indicates that it is suitable for publication. However, Reviewer #2 still raises concerns. His/her main concern is the different genetic background of mice used in CCP/CPA compared to morphine drinking experiments, which could influence the outcome.
We agree that it would be ideal to perform the suggested experiment, however, we realize that the addition of more in vivo data would be time-consuming. Should you be able to provide data from CPP/CPA experiments with C57B1/6 mice, we would encourage you to include them into the present study. Otherwise, we would strongly encourage you to include a very brief discussion of this issue into the manuscript.
Revised manuscripts should be submitted within one month of a request for revision; they will otherwise be treated as new submissions, unless arranged differently with the editor.
I look forward to reading a new revised version of your manuscript as soon as possible.

REFEREE REPORTS:
Referee #1 Comments on Novelty/Model system: A highly sophisticated behavioral model of addiction in mice for the first time. A highly debated issue in the opioid field (receptor intrenalization and addiction) and an elegant knock-in mouse to address the issue.
Referee #1 Other Remarks : The authors have adequately addressed all the cricisms Referee #2 Comments on Novelty/Model system: The generation of a mouse line to test the hypothesis that recycling of µ-opioid receptor could influence the morphine chronic effects, in this case drug reward behavior. The use of drinking for self-administration in mice is novel, and the results appear to support the authors' conclusion. However, it is difficult to evaluate the medical impact because there is no data on alteration of drug seeking and reward behavior.
Referee #2 Other Remarks : The manuscript has improved,and it is easier to follow this time around. The authors have convincingly argued their points in their replies to previous review. However, the authors should consider removing the part on the description of the components of each operant session from the result section and incorporate those in the method section. Although using drinking as self administration is novel, the various aspects of drug self administration on drug reward and addiction have been debated in numerous articles. Current description in the text distracts from the flow of the results.
One minor concern that was not raised during last review is the difference in the mouse genetic background between CPP/CPA and the morphine drinking studies. Probably there should not be any difference between the mixed background and the C57/B6 background in CPP/CPA responses, but literature has reported other morphine responses are different with different background. Since the authors could observe CPP with 1 training session, it would be informative if those studies are repeated with the mice having the same background as the drinking studies. Enclosed is a revised version of our manuscript "Morphine-Induced Mu Opioid Receptor Trafficking Enhances Reward Yet Prevents Compulsive Drug Use".
We have included a discussion of the genetic background of the mice in the revised version. Although, it would have been preferable to use the congenic strain for all studies, the CPP/CPA experiments were completed long before the congenic animals were available (and repeating all these experiments with the full dose responses would take several months and considerable resources). Nevertheless, this difference should have little impact on the overall direction or interpretation of the presented results, as each experiment was internally controlled for genotype and answered an independent research question. In addition, we have previously shown that RMOR mice have a similar reduction in analgesic tolerance and physical dependence compared to their WT littermates, regardless of their background composition (see Kim et al, 2008;Madhavan et al, 2010).
We thank you for the opportunity to resubmit the manuscript, and hope that you find the revised version suitable for publication in EMBO Molecular Medicine.