Assistant Professor, Anesthesia, Neurosciences and (by courtesy) Molecular and Cellular Physiology, Stanford University (2012 - Present)
Honors & Awards
International Trainee Fellowship funded by the Scan|Design Foundation BY INGER & JENS BRUUN, International Association for the Study of Pain (2009)
K99/R00 Pathway to Independence Award, National Institutes of Health - National Institute on Drug Abuse (2011)
Rita Allen Foundation Scholar, Rita Allen Foundation - American Pain Society (2014)
Neurosensory Research Award, Department of Defense (2014)
Postdoc. (2), Columbia University, MacDermott Laboratory, Spinal Cord Physiology (2012)
Postdoc. (1), UCSF, Basbaum Laboratory, Neurobiology of Pain (2009)
Ph.D., Louis Pasteur University of Strasbourg, Cellular and Molecular Biology (2005)
Pharm.D., Louis Pasteur University of Strasbourg, Pharmacology (2001)
Current Research and Scholarly Interests
Cellular and Molecular Mechanisms of Pain and its Control by Opioids
Our laboratory searches to resolve the identity of the neurons in the nerves, spinal cord and brain that participate in generating the sensation of pain, and to uncover the molecular mechanisms that regulate neural activity in pain circuits.
Pain is normally an acute, physiological sensation that we experience when our body is exposed to noxious and potentially damaging stimuli (e.g. noxious heat of an open flame). The unpleasantness of pain drives us to engage adaptive behaviors for avoiding these stimuli and favoring healing. However, when chronic, pain is a disease that severely affects the quality of life of many patients. Injuries or diseases (trauma, diabetes, arthritis, cancer, etc) can induce neuroplasticity in somatosensory circuits that leads to miscoding of sensory information: pain can then become spontaneous and be perceived in the absence of actual stimuli, and normally innocuous stimuli such as light touch or warmth can generate excruciating pain.
We want to understand how neural circuits are functionally organized to encode qualitatively and quantitatively distinct pain signals, and to allow discrimination of pain from other somatosensory experiences such as touch or itch. Our ultimate goal is to identify the changes in this organization that underlie pathological chronic pain and to discover new molecular targets to treat this disease. One of our approaches is to gain understanding of how our endogenous opioid system functions. Opioid receptors and peptides composing this system modulate pain threshold and underlie the effect of the oldest, but still most effective, pain killers, namely opium poppy-extracted morphine and its derivatives. We search to establish the mechanisms by which opioids generate analgesia and detrimental side effects (e.g. tolerance, addiction, hyperalgesia, etc) to develop more efficient and safer analgesic treatments for managing chronic pain. To reach this goal we combine a variety of experimental approaches including molecular and cellular biology, neuroanatomy, electrophysiology, optogenetics and behavior.
Independent Studies (8)
- Directed Reading in Anesthesiology
ANES 299 (Win, Spr)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr)
- Early Clinical Experience in Anesthesia
ANES 280 (Win, Spr)
- Graduate Research
ANES 399 (Win, Spr)
- Graduate Research
NEPR 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
ANES 370 (Win, Spr)
- Out-of-Department Graduate Research
BIO 300X (Aut, Win, Spr, Sum)
- Undergraduate Research
ANES 199 (Win, Spr)
- Directed Reading in Anesthesiology
Loss of μ opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia.
Opioid pain medications have detrimental side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). Tolerance and OIH counteract opioid analgesia and drive dose escalation. The cell types and receptors on which opioids act to initiate these maladaptive processes remain disputed, which has prevented the development of therapies to maximize and sustain opioid analgesic efficacy. We found that μ opioid receptors (MORs) expressed by primary afferent nociceptors initiate tolerance and OIH development. RNA sequencing and histological analysis revealed that MORs are expressed by nociceptors, but not by spinal microglia. Deletion of MORs specifically in nociceptors eliminated morphine tolerance, OIH and pronociceptive synaptic long-term potentiation without altering antinociception. Furthermore, we found that co-administration of methylnaltrexone bromide, a peripherally restricted MOR antagonist, was sufficient to abrogate morphine tolerance and OIH without diminishing antinociception in perioperative and chronic pain models. Collectively, our data support the idea that opioid agonists can be combined with peripheral MOR antagonists to limit analgesic tolerance and OIH.
View details for DOI 10.1038/nm.4262
View details for PubMedID 28092666
A Brainstem-Spinal Cord Inhibitory Circuit for Mechanical Pain Modulation by GABA and Enkephalins.
2017; 93 (4): 822–39.e6
Pain thresholds are, in part, set as a function of emotional and internal states by descending modulation of nociceptive transmission in the spinal cord. Neurons of the rostral ventromedial medulla (RVM) are thought to critically contribute to this process; however, the neural circuits and synaptic mechanisms by which distinct populations of RVM neurons facilitate or diminish pain remain elusive. Here we used in vivo opto/chemogenetic manipulations and trans-synaptic tracing of genetically identified dorsal horn and RVM neurons to uncover an RVM-spinal cord-primary afferent circuit controlling pain thresholds. Unexpectedly, we found that RVM GABAergic neurons facilitate mechanical pain by inhibiting dorsal horn enkephalinergic/GABAergic interneurons. We further demonstrate that these interneurons gate sensory inputs and control pain through temporally coordinated enkephalin- and GABA-mediated presynaptic inhibition of somatosensory neurons. Our results uncover a descending disynaptic inhibitory circuit that facilitates mechanical pain, is engaged during stress, and could be targeted to establish higher pain thresholds. VIDEO ABSTRACT.
View details for DOI 10.1016/j.neuron.2017.01.008
View details for PubMedID 28162807
In Vivo Interrogation of Spinal Mechanosensory Circuits.
2016; 17 (6): 1699-1710
Spinal dorsal horn circuits receive, process, and transmit somatosensory information. To understand how specific components of these circuits contribute to behavior, it is critical to be able to directly modulate their activity in unanesthetized in vivo conditions. Here, we develop experimental tools that enable optogenetic control of spinal circuitry in freely moving mice using commonly available materials. We use these tools to examine mechanosensory processing in the spinal cord and observe that optogenetic activation of somatostatin-positive interneurons facilitates both mechanosensory and itch-related behavior, while reversible chemogenetic inhibition of these neurons suppresses mechanosensation. These results extend recent findings regarding the processing of mechanosensory information in the spinal cord and indicate the potential for activity-induced release of the somatostatin neuropeptide to affect processing of itch. The spinal implant approach we describe here is likely to enable a wide range of studies to elucidate spinal circuits underlying pain, touch, itch, and movement.
View details for DOI 10.1016/j.celrep.2016.10.010
View details for PubMedID 27806306
- Structure-based discovery of opioid analgesics with reduced side effects NATURE 2016; 537 (7619): 185-?
Enhanced dendritic integration by ih reduction in the anterior cingulate cortex increases nociception.
2015; 86 (1): 4-6
In this issue of Neuron, Santello and Nevian (2015) report HCN channel plasticity and increased temporal summation in layer 5 ACC neurons following nerve injury. They are able to restore HCN channel function and reduce behavioral hypersensitivity with selective serotonin receptor targeting.
View details for DOI 10.1016/j.neuron.2015.03.045
View details for PubMedID 25856476
- GINIP, a G(alpha i)-Interacting Protein, Functions as a Key Modulator of Peripheral GABA(B) Receptor-Mediated Analgesia NEURON 2014; 84 (1): 123-136
Delta Opioid Receptors Presynaptically Regulate Cutaneous Mechanosensory Neuron Input to the Spinal Cord Dorsal Horn
2014; 81 (6): 1312-1327
Cutaneous mechanosensory neurons detect mechanical stimuli that generate touch and pain sensation. Although opioids are generally associated only with the control of pain, here we report that the opioid system in fact broadly regulates cutaneous mechanosensation, including touch. This function is predominantly subserved by the delta opioid receptor (DOR), which is expressed by myelinated mechanoreceptors that form Meissner corpuscles, Merkel cell-neurite complexes, and circumferential hair follicle endings. These afferents also include a small population of CGRP-expressing myelinated nociceptors that we now identify as the somatosensory neurons that coexpress mu and delta opioid receptors. We further demonstrate that DOR activation at the central terminals of myelinated mechanoreceptors depresses synaptic input to the spinal dorsal horn, via the inhibition of voltage-gated calcium channels. Collectively our results uncover a molecular mechanism by which opioids modulate cutaneous mechanosensation and provide a rationale for targeting DOR to alleviate injury-induced mechanical hypersensitivity.
View details for DOI 10.1016/j.neuron.2014.01.044
View details for Web of Science ID 000333326000012
Cellular and Molecular Mechanisms of Pain
2009; 139 (2): 267-284
The nervous system detects and interprets a wide range of thermal and mechanical stimuli, as well as environmental and endogenous chemical irritants. When intense, these stimuli generate acute pain, and in the setting of persistent injury, both peripheral and central nervous system components of the pain transmission pathway exhibit tremendous plasticity, enhancing pain signals and producing hypersensitivity. When plasticity facilitates protective reflexes, it can be beneficial, but when the changes persist, a chronic pain condition may result. Genetic, electrophysiological, and pharmacological studies are elucidating the molecular mechanisms that underlie detection, coding, and modulation of noxious stimuli that generate pain.
View details for DOI 10.1016/j.cell.2009.09.028
View details for Web of Science ID 000270857500012
View details for PubMedID 19837031
Dissociation of the Opioid Receptor Mechanisms that Control Mechanical and Heat Pain
2009; 137 (6): 1148-1159
Delta and mu opioid receptors (DORs and MORs) are inhibitory G protein-coupled receptors that reportedly cooperatively regulate the transmission of pain messages by substance P and TRPV1-expressing pain fibers. Using a DOReGFP reporter mouse we now show that the DOR and MOR are, in fact, expressed by different subsets of primary afferents. The MOR is expressed in peptidergic pain fibers, the DOR in myelinated and nonpeptidergic afferents. Contrary to the prevailing view, we demonstrate that the DOR is trafficked to the cell surface under resting conditions, independently of substance P, and internalized following activation by DOR agonists. Finally, we show that the segregated DOR and MOR distribution is paralleled by a remarkably selective functional contribution of the two receptors to the control of mechanical and heat pain, respectively. These results demonstrate that behaviorally relevant pain modalities can be selectively regulated through the targeting of distinct subsets of primary afferent pain fibers.
View details for DOI 10.1016/j.cell.2009.04.019
View details for Web of Science ID 000266916400025
View details for PubMedID 19524516
Inhibition Mediated by Glycinergic and GABAergic Receptors on Excitatory Neurons in Mouse Superficial Dorsal Horn Is Location-Specific but Modified by Inflammation.
The Journal of neuroscience : the official journal of the Society for Neuroscience
2017; 37 (9): 2336–48
The superficial dorsal horn is the synaptic termination site for many peripheral sensory fibers of the somatosensory system. A wide range of sensory modalities are represented by these fibers, including pain, itch, and temperature. Because the involvement of local inhibition in the dorsal horn, specifically that mediated by the inhibitory amino acids GABA and glycine, is so important in signal processing, we investigated regional inhibitory control of excitatory interneurons under control conditions and peripheral inflammation-induced mechanical allodynia. We found that excitatory interneurons and projection neurons in lamina I and IIo are dominantly inhibited by GABA while those in lamina IIi and III are dominantly inhibited by glycine. This was true of identified neuronal subpopulations: neurokinin 1 receptor-expressing (NK1R+) neurons in lamina I were GABA-dominant while protein kinase C gamma-expressing (PKCγ+) neurons at the lamina IIi-III border were glycine-dominant. We found this pattern of synaptic inhibition to be consistent with the distribution of GABAergic and glycinergic neurons identified by immunohistochemistry. Following complete Freund's adjuvant injection into mouse hindpaw, the frequency of spontaneous excitatory synaptic activity increased and inhibitory synaptic activity decreased. Surprisingly, these changes were accompanied by an increase in GABA dominance in lamina IIi. Because this shift in inhibitory dominance was not accompanied by a change in the number of inhibitory synapses or the overall postsynaptic expression of glycine receptor α1 subunits, we propose that the dominance shift is due to glycine receptor modulation and the depressed function of glycine receptors is partially compensated by GABAergic inhibition.SIGNIFICANCE STATEMENT Pain associated with inflammation is a sensation we would all like to minimize. Persistent inflammation leads to cellular and molecular changes in the spinal cord dorsal horn, including diminished inhibition, which may be responsible for enhance excitability. Investigating inhibition in the dorsal horn following peripheral inflammation is essential for development of improved ways to control the associated pain. In this study, we have elucidated regional differences in inhibition of excitatory interneurons in mouse dorsal horn. We have also discovered that the dominating inhibitory neurotransmission within specific regions of dorsal horn switches following peripheral inflammation and the accompanying hypersensitivity to thermal and mechanical stimuli. Our novel findings contribute to a more complete understanding of inflammatory pain.
View details for DOI 10.1523/JNEUROSCI.2354-16.2017
View details for PubMedID 28130358
Structure-based discovery of opioid analgesics with reduced side effects.
2016; 537 (7619): 185-190
Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by μ-opioid-receptor (μOR) signalling through the β-arrestin pathway or by actions at other receptors. Conversely, G-protein μOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the μOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for μOR and minimal β-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle μOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.
View details for DOI 10.1038/nature19112
View details for PubMedID 27533032
Ensuring transparency and minimization of methodologic bias in preclinical pain research: PPRECISE considerations
2016; 157 (4): 901-909
There is growing concern about lack of scientific rigor and transparent reporting across many preclinical fields of biological research. Poor experimental design and lack of transparent reporting can result in conscious or unconscious experimental bias, producing results that are not replicable. The Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION) public-private partnership with the U.S. Food and Drug Administration sponsored a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and Efficacy (PPRECISE) Working Group. International participants from universities, funding agencies, government agencies, industry, and a patient advocacy organization attended. Reduction of publication bias, increasing the ability of others to faithfully repeat experimental methods, and increased transparency of data reporting were specifically discussed. Parameters deemed essential to increase confidence in the published literature were clear, specific reporting of an a priori hypothesis and definition of primary outcome measure. Power calculations and whether measurement of minimal meaningful effect size to determine these should be a core component of the preclinical research effort provoked considerable discussion, with many but not all agreeing. Greater transparency of reporting should be driven by scientists, journal editors, reviewers, and grant funders. The conduct of high-quality science that is fully reported should not preclude novelty and innovation in preclinical pain research, and indeed, any efforts that curtail such innovation would be misguided. We believe that to achieve the goal of finding effective new treatments for patients with pain, the pain field needs to deal with these challenging issues.
View details for DOI 10.1097/j.pain.0000000000000458
View details for Web of Science ID 000378261600017
View details for PubMedID 26683237
- Knock-In Mice with NOP-eGFP Receptors Identify Receptor Cellular and Regional Localization JOURNAL OF NEUROSCIENCE 2015; 35 (33): 11682-11693
A mu-delta opioid receptor brain atlas reveals neuronal co-occurrence in subcortical networks
BRAIN STRUCTURE & FUNCTION
2015; 220 (2): 677-702
Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.
View details for DOI 10.1007/s00429-014-0717-9
View details for Web of Science ID 000350350300006
View details for PubMedID 24623156
A novel anxiogenic role for the delta opioid receptor expressed in GABAergic forebrain neurons.
2015; 77 (4): 404-415
The delta opioid receptor (DOR) is broadly expressed throughout the nervous system; it regulates chronic pain, emotional responses, motivation, and memory. Neural circuits underlying DOR activities have been poorly explored by genetic approaches. We used conditional mouse mutagenesis to elucidate receptor function in GABAergic neurons of the forebrain.We characterized DOR distribution in the brain of Dlx5/6-CreXOprd1(fl/fl) (Dlx-DOR) mice and tested main central DOR functions through behavioral testing.The DOR proteins were strongly deleted in olfactory bulb and striatum and remained intact in cortex and basolateral amygdala. Olfactory perception, circadian activity, and despair-like behaviors were unchanged. In contrast, locomotor stimulant effects of SNC80 (DOR agonist) and SKF81297 (D1 agonist) were abolished and increased, respectively. The Dlx-DOR mice showed lower levels of anxiety in the elevated plus maze, opposing the known high anxiety in constitutive DOR knockout animals. Also, Dlx-DOR mice reached the food more rapidly in a novelty suppressed feeding task, despite their lower motivation for food reward observed in an operant paradigm. Finally, c-fos protein staining after novelty suppressed feeding was strongly reduced in amygdala, concordant with the low anxiety phenotype of Dlx-DOR mice.We demonstrate that DORs expressed in the forebrain mediate the described locomotor effect of SNC80 and inhibit D1-stimulated hyperactivity. Our data also reveal an unanticipated anxiogenic role for this particular DOR subpopulation, with a potential novel adaptive role. In emotional responses, DORs exert dual anxiolytic and anxiogenic roles, both of which may have implications in the area of anxiety disorders.
View details for DOI 10.1016/j.biopsych.2014.07.033
View details for PubMedID 25444168
- A Novel Anxiogenic Role for the Delta Opioid Receptor Expressed in GABAergic Forebrain Neurons BIOLOGICAL PSYCHIATRY 2015; 77 (4): 404-415
- Delta opioid receptors expressed in forebrain GABAergic neurons are responsible for SNC80-induced seizures BEHAVIOURAL BRAIN RESEARCH 2015; 278: 429-434
Input- and Cell-Type-Specific Endocannabinoid-Dependent LTD in the Striatum
2015; 10 (1): 75-87
Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD) is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs; however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN) subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1) receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.
View details for DOI 10.1016/j.celrep.2014.12.005
View details for Web of Science ID 000347465600008
View details for PubMedID 25543142
In vivo techniques to investigate the internalization profile of opioid receptors.
Methods in molecular biology (Clifton, N.J.)
2015; 1230: 87-104
G-protein-coupled receptors (GPCRs) regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Receptor internalization is commonly observed following agonist binding and activation. Receptor trafficking events have been well characterized in cell systems, but the in vivo significance of GPCR internalization is still poorly understood. To address this issue, we have developed an innovative knock-in mouse model, where an opioid receptor is directly visible in vivo. These knockin mice express functional fluorescent delta opioid receptors (DOR-eGFP) in place of the endogenous receptor, and these receptors are expressed at physiological levels within their native environment. DOR-eGFP mice have proven to be an extraordinary tool in studying receptor neuroanatomy, real-time receptor trafficking in live neurons, and in vivo receptor internalization. We have used this animal model to determine the relationship between receptor trafficking in neurons and receptor function at a behavioral level. Here, we describe in detail the construction and characterization of this knockin mouse. We also outline how to use these mice to examine the behavioral consequences of agonist-specific trafficking at the delta opioid receptor. These techniques are potentially applicable to any GPCR, and highlight the powerful nature of this imaging tool.
View details for DOI 10.1007/978-1-4939-1708-2_7
View details for PubMedID 25293318
Sensory biology: it takes Piezo2 to tango.
2014; 24 (12): R566-9
A trio of papers has resolved an outstanding controversy regarding the function of Merkel cells and their afferent nerve fiber partners. Merkel cells sense mechanical stimuli (through Piezo2), fire action potentials, and are sufficient to activate downstream sensory neurons.
View details for DOI 10.1016/j.cub.2014.05.011
View details for PubMedID 24937283
- The Netrin-1 receptor DCC is a regulator of maladaptive responses to chronic morphine administration BMC GENOMICS 2014; 15
Delta opioid receptors expressed in forebrain GABAergic neurons are responsible for SNC80-induced seizures.
Behavioural brain research
2014; 278C: 429-434
The delta opioid receptor (DOR) has raised much interest for the development of new therapeutic drugs, particularly to treat patients suffering from mood disorders and chronic pain. Unfortunately, the prototypal DOR agonist SNC80 induces mild epileptic seizures in rodents. Although recently developed agonists do not seem to show convulsant properties, mechanisms and neuronal circuits that support DOR-mediated epileptic seizures remain to be clarified. DORs are expressed throughout the nervous system. In this study we tested the hypothesis that SNC80-evoked seizures stem from DOR activity at the level of forebrain GABAergic transmission, whose inhibition is known to facilitate the development of epileptic seizures. We generated a conditional DOR knockout mouse line, targeting the receptor gene specifically in GABAergic neurons of the forebrain (Dlx-DOR). We measured effects of SNC80 (4.5, 9, 13.5 and 32mg/kg), ARM390 (10, 30 and 60mg/kg) or ADL5859 (30, 100 and 300mg/kg) administration on electroencephalograms (EEGs) recorded in Dlx-DOR mice and their control littermates (Ctrl mice). SNC80 produced dose-dependent seizure events in Ctrl mice, but these effects were not detected in Dlx-DOR mice. As expected, ARM390 and ADL5859 did not trigger any detectable change in mice from both genotypes. These results demonstrate for the first time that SNC80-induced DOR activation induces epileptic seizures via direct inhibition of GABAergic forebrain neurons, and supports the notion of differential activities between first and second-generation DOR agonists.
View details for DOI 10.1016/j.bbr.2014.10.029
View details for PubMedID 25447299
- Impaired Hippocampus-Dependent and Facilitated Striatum-Dependent Behaviors in Mice Lacking the Delta Opioid Receptor NEUROPSYCHOPHARMACOLOGY 2013; 38 (6): 1050-1059
- Pre- and postsynaptic inhibitory control in the spinal cord dorsal horn NEURONS, CIRCUITRY, AND PLASTICITY IN THE SPINAL CORD AND BRAINSTEM 2013; 1279: 90-96
DISTRIBUTION OF DELTA OPIOID RECEPTOR-EXPRESSING NEURONS IN THE MOUSE HIPPOCAMPUS
2012; 221: 203-213
Delta opioid receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. We examined the distribution of delta receptor-expressing cells in the hippocampus using fluorescent knock-in mice that express a functional delta receptor fused at its carboxyterminus with the green fluorescent protein in place of the native receptor. Colocalization with markers for different neuronal populations was performed by immunohistochemical detection. Fine mapping in the dorsal hippocampus confirmed that delta opioid receptors are mainly present in GABAergic neurons. Indeed, they are mostly expressed in parvalbumin-immunopositive neurons both in the Ammon's horn and dentate gyrus. These receptors, therefore, most likely participate in the dynamic regulation of hippocampal activity.
View details for DOI 10.1016/j.neuroscience.2012.06.023
View details for Web of Science ID 000308628100020
View details for PubMedID 22750239
In Vivo Visualization of Delta Opioid Receptors upon Physiological Activation Uncovers a Distinct Internalization Profile
JOURNAL OF NEUROSCIENCE
2012; 32 (21): 7301-7310
G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.
View details for DOI 10.1523/JNEUROSCI.0185-12.2012
View details for Web of Science ID 000304421000022
View details for PubMedID 22623675
Localization and Regulation of Fluorescently Labeled Delta Opioid Receptor, Expressed in Enteric Neurons of Mice
2011; 141 (3): 982-U695
Opioids and opiates inhibit gastrointestinal functions via ?, ?, and ? receptors. Although agonists of the ? opioid receptor (DOR) suppress motility and secretion, little is known about the localization and regulation of DOR in the gastrointestinal tract.We studied mice in which the gene that encodes the enhanced green fluorescent protein (eGFP) was inserted into Oprd1, which encodes DOR, to express an approximately 80-kilodalton product (DOReGFP). We used these mice to localize DOR and to determine how agonists regulate the subcellular distribution of DOR.DOReGFP was expressed in all regions but was confined to enteric neurons and fibers within the muscularis externa. In the submucosal plexus, DOReGFP was detected in neuropeptide Y-positive secretomotor and vasodilator neurons of the small intestine, but rarely was observed in the large bowel. In the myenteric plexus of the small intestine, DOReGFP was present in similar proportions of excitatory motoneurons and interneurons that expressed choline acetyltransferase and substance P, and in inhibitory motoneurons and interneurons that contained nitric oxide synthase. DOReGFP was present mostly in nitrergic myenteric neurons of colon. DOReGFP and ? opioid receptors often were co-expressed. DOReGFP-expressing neurons were associated with enkephalin-containing varicosities, and enkephalin-induced clathrin- and dynamin-mediated endocytosis and lysosomal trafficking of DOReGFP. DOReGFP replenishment at the plasma membrane was slow, requiring de novo synthesis, rather than recycling.DOR localizes specifically to submucosal and myenteric neurons, which might account for the ability of DOR agonists to inhibit gastrointestinal secretion and motility. Sustained down-regulation of DOReGFP at the plasma membrane of activated neurons could induce long-lasting tolerance to DOR agonists.
View details for DOI 10.1053/j.gastro.2011.05.042
View details for Web of Science ID 000294281200038
View details for PubMedID 21699782
Behavioral indices of ongoing pain are largely unchanged in male mice with tissue or nerve injury-induced mechanical hypersensitivity
2011; 152 (5): 990-1000
Despite the impact of chronic pain on the quality of life in patients, including changes to affective state and daily life activities, rodent preclinical models rarely address this aspect of chronic pain. To better understand the behavioral consequences of the tissue and nerve injuries typically used to model neuropathic and inflammatory pain in mice, we measured home cage and affective state behaviors in animals with spared nerve injury, chronic constriction injury (CCI), or intraplantar complete Freund's adjuvant. Mechanical hypersensitivity is prominent in each of these conditions and persists for many weeks. Home cage behavior was continuously monitored for 16 days in a system that measures locomotion, feeding, and drinking, and allows for precise analysis of circadian patterns. When monitored after injury, animals with spared nerve injury and complete Freund's adjuvant behaved no differently from controls in any aspect of daily life. Animals with CCI were initially less active, but the difference between CCI and controls disappeared by 2 weeks after injury. Further, in all pain models, there was no change in any measure of affective state. We conclude that in these standard models of persistent pain, despite the development of prolonged hypersensitivity, the mice do not have significantly altered "quality of life." As alteration in daily life activities is the feature that is so disrupted in patients with chronic pain, our results suggest that the models used here do not fully reflect the human conditions and point to a need for development of a murine chronic pain model in which lifestyle changes are manifest.
View details for DOI 10.1016/j.pain.2010.12.003
View details for Web of Science ID 000289507500010
View details for PubMedID 21256675
VGLUT2 expression in primary afferent neurons is essential for normal acute pain and injury-induced heat hypersensitivity
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (51): 22296-22301
Dorsal root ganglia (DRG) neurons, including the nociceptors that detect painful thermal, mechanical, and chemical stimuli, transmit information to spinal cord neurons via glutamatergic and peptidergic neurotransmitters. However, the specific contribution of glutamate to pain generated by distinct sensory modalities or injuries is not known. Here we generated mice in which the vesicular glutamate transporter 2 (VGLUT2) is ablated selectively from DRG neurons. We report that conditional knockout (cKO) of the Slc17a6 gene encoding VGLUT2 from the great majority of nociceptors profoundly decreased VGLUT2 mRNA and protein in these neurons, and reduced firing of lamina I spinal cord neurons in response to noxious heat and mechanical stimulation. In behavioral assays, cKO mice showed decreased responsiveness to acute noxious heat, mechanical, and chemical (capsaicin) stimuli, but responded normally to cold stimulation and in the formalin test. Strikingly, although tissue injury-induced heat hyperalgesia was lost in the cKO mice, mechanical hypersensitivity developed normally. In a model of nerve injury-induced neuropathic pain, the magnitude of heat hypersensitivity was diminished in cKO mice, but both the mechanical allodynia and the microgliosis generated by nerve injury were intact. These findings suggest that VGLUT2 expression in nociceptors is essential for normal perception of acute pain and heat hyperalgesia, and that heat and mechanical hypersensitivity induced by peripheral injury rely on distinct (VGLUT2 dependent and VGLUT2 independent, respectively) primary afferent mechanisms and pathways.
View details for DOI 10.1073/pnas.1013413108
View details for Web of Science ID 000285521800066
View details for PubMedID 21135246
In Vivo Delta Opioid Receptor Internalization Controls Behavioral Effects of Agonists
2009; 4 (5)
GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.
View details for DOI 10.1371/journal.pone.0005425
View details for Web of Science ID 000265688200012
View details for PubMedID 19412545
Dense transient receptor potential cation channel, vanilloid family, type 2 (TRPV2) immunoreactivity defines a subset of motoneurons in the dorsal lateral nucleus of the spinal cord, the nucleus ambiguus and the trigeminal motor nucleus in rat
2008; 151 (1): 164-173
The transient receptor potential cation channel, vanilloid family, type 2 (TRPV2) is a member of the TRPV family of proteins and is a homologue of the capsaicin/vanilloid receptor (transient receptor potential cation channel, vanilloid family, type 1, TRPV1). Like TRPV1, TRPV2 is expressed in a subset of dorsal root ganglia (DRG) neurons that project to superficial laminae of the spinal cord dorsal horn. Because noxious heat (>52 degrees C) activates TRPV2 in transfected cells this channel has been implicated in the processing of high intensity thermal pain messages in vivo. In contrast to TRPV1, however, which is restricted to small diameter DRG neurons, there is significant TRPV2 immunoreactivity in a variety of CNS regions. The present report focuses on a subset of neurons in the brainstem and spinal cord of the rat including the dorsal lateral nucleus (DLN) of the spinal cord, the nucleus ambiguus, and the motor trigeminal nucleus. Double label immunocytochemistry with markers of motoneurons, combined with retrograde labeling, established that these cells are, in fact, motoneurons. With the exception of their smaller diameter, these cells did not differ from other motoneurons, which are only lightly TRPV2-immunoreactive. As for the majority of DLN neurons, the densely-labeled populations co-express androgen receptor and follow normal DLN ontogeny. The functional significance of the very intense TRPV2 expression in these three distinct spinal cord and brainstem motoneurons groups remains to be determined.
View details for DOI 10.1016/j.neuroscience.2007.09.073
View details for Web of Science ID 000252608800017
View details for PubMedID 18063314
Knockin mice expressing fluorescent delta-opioid receptors uncover G protein-coupled receptor dynamics in vivo
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2006; 103 (25): 9691-9696
The combination of fluorescent genetically encoded proteins with mouse engineering provides a fascinating means to study dynamic biological processes in mammals. At present, green fluorescent protein (GFP) mice were mainly developed to study gene expression patterns or cell morphology and migration. Here we used enhanced GFP (EGFP) to achieve functional imaging of a G protein-coupled receptor (GPCR) in vivo. We created mice where the delta-opioid receptor (DOR) is replaced by an active DOR-EGFP fusion. Confocal imaging revealed detailed receptor neuroanatomy throughout the nervous system of knock-in mice. Real-time imaging in primary neurons allowed dynamic visualization of drug-induced receptor trafficking. In DOR-EGFP animals, drug treatment triggered receptor endocytosis that correlated with the behavioral response. Mice with internalized receptors were insensitive to subsequent agonist administration, providing evidence that receptor sequestration limits drug efficacy in vivo. Direct receptor visualization in mice is a unique approach to receptor biology and drug design.
View details for DOI 10.1073/pnas.0603359103
View details for Web of Science ID 000238660400055
View details for PubMedID 16766653
The delta agonists DPDPE and deltorphin II recruit predominantly mu receptors to produce thermal analgesia: a parallel study of mu, delta and combinatorial opioid receptor knockout mice
EUROPEAN JOURNAL OF NEUROSCIENCE
2004; 19 (8): 2239-2248
Delta-selective agonists have been developed to produce potent analgesic compounds with limited side-effects. DPDPE and deltorphin II are considered prototypes, but their delta-selectivity in vivo and the true ability of delta receptors to produce analgesia remain to be demonstrated. Here we have performed a parallel analysis of mu, delta and combinatorial opioid receptor knockout mice, in which we found no obvious alteration of G-protein coupling for remaining opioid receptors. We compared behavioural responses in two models of acute thermal pain following DPDPE and deltorphin II administration by intracerebroventricular route. In the tail-immersion test, both compounds were fully analgesic in delta knockout mice and totally inactive in mu knockout mice. In the hotplate test, the two compounds again produced full analgesia in delta knockout mice. In mu knockout mice, there was significant, although much lower, analgesia. Furthermore, DPDPE analgesia in the delta knockout mice was fully reversed by the mu selective antagonist CTOP in both tests. Together, this suggests that mu rather than delta receptors are recruited by the two agonists for the tail withdrawal and the hotplate responses. Finally, deltorphin II slightly prolonged jump latencies in double mu/kappa knockout mice (delta receptors only) and this response was abolished in the triple knockout mice, demonstrating that the activation of delta receptors alone can produce weak but significant mu-independent thermal antinociception.
View details for DOI 10.1111/j.1460-9568.2004.03339.x
View details for Web of Science ID 000221197600023
View details for PubMedID 15090050