Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling
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eLife assessment
This manuscript examines the inhibition of transmitter release induced by the activation of opioid receptors, both MOR and DOR, using a novel imaging method. The authors specifically examine how the inhibition of transmitter release is changed following prolonged exposure to saturating concentrations of agonists. They showed convincingly that there is a depletion of plasma membrane-associated receptors and suggest that the decline in receptors at the plasma membrane underlies presynaptic tolerance. This work addresses a long-standing question about how tolerance develops at the presynaptic level and indicates that the location of receptors is critically important in the development of tolerance. This work is fundamental and a game changer in the understanding of tolerance at the cellular level.
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Abstract
Opioid tolerance is well-described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here, we delineate a cellular mechanism underlying this longer-term form of opioid tolerance in cultured rat medium spiny neurons. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the μ-opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptor’s cytoplasmic tail, and that partial or biased agonist drugs with reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the δ-opioid receptor (DOR) conforms to the same general paradigm except that presynaptic endocytosis of DOR, in contrast to MOR, does not require phosphorylation of the receptor’s cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.
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Author Response
Reviewer #1 (Public Review):
This work addresses a long-standing question about how tolerance develops at the presynaptic level. That the number of receptors is unchanged following the treatment of animals with opioids was known since the early work using receptor binding assays. The conclusion was that receptor/effector coupling was disrupted was thought to be the primary mechanism underlying tolerance. This work indicates that the location of receptors is critically important in the development of tolerance. This work is groundbreaking and a game changer in the understanding of tolerance at the cellular level.
We appreciate that the Reviewer is positive about the potential impact of our study.
Reviewer #2 (Public Review):
Jullie et al addressed the long-standing question of how presynaptic desensitization of …
Author Response
Reviewer #1 (Public Review):
This work addresses a long-standing question about how tolerance develops at the presynaptic level. That the number of receptors is unchanged following the treatment of animals with opioids was known since the early work using receptor binding assays. The conclusion was that receptor/effector coupling was disrupted was thought to be the primary mechanism underlying tolerance. This work indicates that the location of receptors is critically important in the development of tolerance. This work is groundbreaking and a game changer in the understanding of tolerance at the cellular level.
We appreciate that the Reviewer is positive about the potential impact of our study.
Reviewer #2 (Public Review):
Jullie et al addressed the long-standing question of how presynaptic desensitization of opioid receptor signaling can occur on the timescale of hours despite the fact that it does not occur on the timescale of minutes. They also compared the mu and delta opioid receptors in this context and asked whether their desensitization occurs in a homologous or heterologous manner when co-expressed in the same neurons.
A major strength of the work is the use of a relatively high-volume imaging assay of synaptic transmission based on VAMP2-SEP to detect exocytosis of synaptic vesicles and its modulation by heterologously expressed opioid receptors in cultured neurons. This allowed for large data sets to be acquired and analyzed with good statistical power. It also reports on a validated metric of synaptic transmission.
A significant weakness arises from the need to overexpress opioid receptors in cultured striatal neurons in order to conduct the experiments with high reliability. Because the authors did not attempt to address receptor expression levels and relate overexpression to endogenous receptor expression levels in axons, the physiological significance of the findings remains, to some extent, in doubt.
Using heterologously expressed receptors, the primary finding that slow desensitization (of presynaptic suppression of neurotransmission) occurs via endocytosis of membrane-localized opioid receptors, is well supported by multiple lines of evidence. 1) Blocking receptor endocytosis, either via mutation of GRK2/3 phosphorylation sites or pharmacological block with compound 101 prevents slow desensitization of MOR. ) SEP-MOR and SEP-DOR fluorescence (indicative of plasma membrane localization) is reduced by chronic agonist treatment.
The secondary findings that MOR and DOR do not desensitize or undergo endocytosis in a heterologous manner, and that DOR-depletion from the plasma membrane is more facile than MOR and independent of C-terminus phosphorylation, are well supported by the data and analyses.
Despite the reliance on heterologously expressed opioid receptors, the findings are likely to have a high impact on the fields of GPCR trafficking and opioid signaling, as they address a major outstanding question with direct relevance to opioid drug tolerance and may generalize to other GPCRs.
The findings also evoke new questions that will spur further work in the field. For example, just focusing on DOR, by what mechanism does agonist-driven DOR endocytosis occur not via GRK2/3 phosphorylation? By extension, would G protein-biased DOR agonists be expected to produce less tolerance? To be clear these are not to be addressed in this manuscript.
We appreciate that this Reviewer found that the current manuscript addresses long standing questions in the field and that our results are well supported by the data, acknowledging the strength of the presented method. We agree that our methods and results do have some associated limitations, particularly with respect to linking the present mechanistic findings to true physiology, and that the question of receptor expression level is pertinent to this link. We have attempted to address this to the best of our ability in the revised manuscript, as summarized below. We agree with the Reviewer that there remain many interesting questions for further study, and have modified the Discussion to more clearly point this out.
Reviewer #3 (Public Review):
The studies in the manuscript "Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling" use a novel approach to assess the signaling of presynaptic opioid receptors that inhibit the release of neurotransmitters. Historically, studies have used whole-cell patch-clamp electrophysiology studies of spontaneous and evoked neurotransmitter release to measure the presynaptic effects of opioid receptors. Since the recordings were made in postsynaptic cells that expressed receptors for the released neurotransmitter, the electrophysiological measurements are indirect with respect to the presynaptic receptors under study. The technique used in this manuscript is based on a pHlorin-based unquenching assay that is a measure of synaptic vesicle exocytosis. In this case, the super-ecliptic pHluorin (SEP) is a pH-sensitive GFP that increases fluorescence as the synaptic vesicle protein that it is attached to (VAMP2-SEP) relocates from the acidic synaptic vesicle to the plasma membrane. Opioid agonists inhibit this activity with acute administration and this inhibition is reduced with prolonged, or chronic administration (hours), demonstrating tolerance. The SEP protein can also be conjugated to opioid receptors and used to measure the proportion of receptors on the plasma membrane compared to internalized receptors. The studies show that agonist activation of mu-opioid receptors (MORs) induces endocytosis that is dependent on phosphorylation of the C-terminus and that the development of tolerance is correlated with the loss of MORs at the surface. The results are different for the delta-opioid receptor (DOR) which is also internalized with acute agonist administration but that loss of receptors on the membrane occurs more rapidly and is not dependent on phosphorylation of the C-terminus.
The results in the studies are clearly presented and clearly substantiate the prior work using electrophysiology to show the late development of tolerance of presynaptic opioid receptor signaling. The studies extend prior work by showing that endocytosis of both MOR and DOR occurs in presynaptic locations but that the cellular mechanisms underlying the maintenance of these receptors on the plasma membrane differ. The imaging results show convincing effect sizes, even with genetic and pharmacological manipulations, that will allow for even further investigation into the cellular mechanisms underlying the development of tolerance. Since these studies transfected the cultured striatal neurons with both the opioid receptors and the VAMP2-SEP, one question that remains is whether imaging of the VAMP2-SEP has the resolution to detect inhibition of endocytosis by endogenous opioid receptors. Since the authors make the point that this technique has advantages over traditional electrophysiological approaches, it is important that the technique allows for the measurement of endogenous levels of receptors. There are minor questions about the statistics used in some of the graphs, and the utility of the presentation of p values on the right-hand axis but these concerns do not alter the overall significance of the studies, which are high impact.
We are pleased that this Reviewer found our results generally convincing and impactful. We are grateful for the critical comments and suggestions, particularly with regard to improving the statistical analysis and simplifying / removing speculation from our model. We have done our best to address both important aspects in the revised manuscript, as detailed below.
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eLife assessment
This manuscript examines the inhibition of transmitter release induced by the activation of opioid receptors, both MOR and DOR, using a novel imaging method. The authors specifically examine how the inhibition of transmitter release is changed following prolonged exposure to saturating concentrations of agonists. They showed convincingly that there is a depletion of plasma membrane-associated receptors and suggest that the decline in receptors at the plasma membrane underlies presynaptic tolerance. This work addresses a long-standing question about how tolerance develops at the presynaptic level and indicates that the location of receptors is critically important in the development of tolerance. This work is fundamental and a game changer in the understanding of tolerance at the cellular level.
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Reviewer #1 (Public Review):
This work addresses a long-standing question about how tolerance develops at the presynaptic level. That the number of receptors is unchanged following the treatment of animals with opioids was known since the early work using receptor binding assays. The conclusion was that receptor/effector coupling was disrupted was thought to be the primary mechanism underlying tolerance. This work indicates that the location of receptors is critically important in the development of tolerance. This work is groundbreaking and a game changer in the understanding of tolerance at the cellular level.
-
Reviewer #2 (Public Review):
Jullie et al addressed the long-standing question of how presynaptic desensitization of opioid receptor signaling can occur on the timescale of hours despite the fact that it does not occur on the timescale of minutes. They also compared the mu and delta opioid receptors in this context and asked whether their desensitization occurs in a homologous or heterologous manner when co-expressed in the same neurons.
A major strength of the work is the use of a relatively high-volume imaging assay of synaptic transmission based on VAMP2-SEP to detect exocytosis of synaptic vesicles and its modulation by heterologously expressed opioid receptors in cultured neurons. This allowed for large data sets to be acquired and analyzed with good statistical power. It also reports on a validated metric of synaptic transmission.
Reviewer #2 (Public Review):
Jullie et al addressed the long-standing question of how presynaptic desensitization of opioid receptor signaling can occur on the timescale of hours despite the fact that it does not occur on the timescale of minutes. They also compared the mu and delta opioid receptors in this context and asked whether their desensitization occurs in a homologous or heterologous manner when co-expressed in the same neurons.
A major strength of the work is the use of a relatively high-volume imaging assay of synaptic transmission based on VAMP2-SEP to detect exocytosis of synaptic vesicles and its modulation by heterologously expressed opioid receptors in cultured neurons. This allowed for large data sets to be acquired and analyzed with good statistical power. It also reports on a validated metric of synaptic transmission.
A significant weakness arises from the need to overexpress opioid receptors in cultured striatal neurons in order to conduct the experiments with high reliability. Because the authors did not attempt to address receptor expression levels and relate overexpression to endogenous receptor expression levels in axons, the physiological significance of the findings remains, to some extent, in doubt.
Using heterologously expressed receptors, the primary finding that slow desensitization (of presynaptic suppression of neurotransmission) occurs via endocytosis of membrane-localized opioid receptors, is well supported by multiple lines of evidence. 1) Blocking receptor endocytosis, either via mutation of GRK2/3 phosphorylation sites or pharmacological block with compound 101 prevents slow desensitization of MOR. ) SEP-MOR and SEP-DOR fluorescence (indicative of plasma membrane localization) is reduced by chronic agonist treatment.
The secondary findings that MOR and DOR do not desensitize or undergo endocytosis in a heterologous manner, and that DOR-depletion from the plasma membrane is more facile than MOR and independent of C-terminus phosphorylation, are well supported by the data and analyses.
Despite the reliance on heterologously expressed opioid receptors, the findings are likely to have a high impact on the fields of GPCR trafficking and opioid signaling, as they address a major outstanding question with direct relevance to opioid drug tolerance and may generalize to other GPCRs.
The findings also evoke new questions that will spur further work in the field. For example, just focusing on DOR, by what mechanism does agonist-driven DOR endocytosis occur not via GRK2/3 phosphorylation? By extension, would G protein-biased DOR agonists be expected to produce less tolerance? To be clear these are not to be addressed in this manuscript.
-
Reviewer #3 (Public Review):
The studies in the manuscript "Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling" use a novel approach to assess the signaling of presynaptic opioid receptors that inhibit the release of neurotransmitters. Historically, studies have used whole-cell patch-clamp electrophysiology studies of spontaneous and evoked neurotransmitter release to measure the presynaptic effects of opioid receptors. Since the recordings were made in postsynaptic cells that expressed receptors for the released neurotransmitter, the electrophysiological measurements are indirect with respect to the presynaptic receptors under study. The technique used in this manuscript is based on a pHlorin-based unquenching assay that is a measure of synaptic vesicle exocytosis. In this case, the super-ecliptic …
Reviewer #3 (Public Review):
The studies in the manuscript "Endocytic trafficking determines cellular tolerance of presynaptic opioid signaling" use a novel approach to assess the signaling of presynaptic opioid receptors that inhibit the release of neurotransmitters. Historically, studies have used whole-cell patch-clamp electrophysiology studies of spontaneous and evoked neurotransmitter release to measure the presynaptic effects of opioid receptors. Since the recordings were made in postsynaptic cells that expressed receptors for the released neurotransmitter, the electrophysiological measurements are indirect with respect to the presynaptic receptors under study. The technique used in this manuscript is based on a pHlorin-based unquenching assay that is a measure of synaptic vesicle exocytosis. In this case, the super-ecliptic pHluorin (SEP) is a pH-sensitive GFP that increases fluorescence as the synaptic vesicle protein that it is attached to (VAMP2-SEP) relocates from the acidic synaptic vesicle to the plasma membrane. Opioid agonists inhibit this activity with acute administration and this inhibition is reduced with prolonged, or chronic administration (hours), demonstrating tolerance. The SEP protein can also be conjugated to opioid receptors and used to measure the proportion of receptors on the plasma membrane compared to internalized receptors. The studies show that agonist activation of mu-opioid receptors (MORs) induces endocytosis that is dependent on phosphorylation of the C-terminus and that the development of tolerance is correlated with the loss of MORs at the surface. The results are different for the delta-opioid receptor (DOR) which is also internalized with acute agonist administration but that loss of receptors on the membrane occurs more rapidly and is not dependent on phosphorylation of the C-terminus.
The results in the studies are clearly presented and clearly substantiate the prior work using electrophysiology to show the late development of tolerance of presynaptic opioid receptor signaling. The studies extend prior work by showing that endocytosis of both MOR and DOR occurs in presynaptic locations but that the cellular mechanisms underlying the maintenance of these receptors on the plasma membrane differ. The imaging results show convincing effect sizes, even with genetic and pharmacological manipulations, that will allow for even further investigation into the cellular mechanisms underlying the development of tolerance. Since these studies transfected the cultured striatal neurons with both the opioid receptors and the VAMP2-SEP, one question that remains is whether imaging of the VAMP2-SEP has the resolution to detect inhibition of endocytosis by endogenous opioid receptors. Since the authors make the point that this technique has advantages over traditional electrophysiological approaches, it is important that the technique allows for the measurement of endogenous levels of receptors. There are minor questions about the statistics used in some of the graphs, and the utility of the presentation of p values on the right-hand axis but these concerns do not alter the overall significance of the studies, which are high impact.
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