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1.  Does the kappa opioid receptor system contribute to pain aversion? 
The kappa opioid receptor (KOR) and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.
doi:10.3389/fphar.2014.00253
PMCID: PMC4233910  PMID: 25452729
kappa opioid receptor; pain; aversion; reward system ventral tegmental area; dopamine; negative reinforcement
2.  Opioid self-administration results in cell-type specific adaptations of striatal medium spiny neurons 
Behavioural brain research  2013;256:10.1016/j.bbr.2013.08.009.
Medium-sized spiny neurons (MSNs), the predominant neuronal population of the striatum, are an integral component of the many cortical and limbic pathways associated with reward-related behaviors. A differential role of the D1 receptor-enriched (D1) MSNs of the striatonigral direct pathway, as compared with the D2 receptor-enriched (D2) MSNs of the striatopallidal indirect pathway, in mediating the addictive behaviors associated with cocaine is beginning to emerge. However, whether opioids, well-known analgesics with euphoric properties, similarly induce dissociable signaling adaptations in these neurons remains unclear. Transgenic mice expressing Green Fluorescent Protein (GFP)-labeled D1 or D2 neurons were implanted with intravenous jugular catheters and trained to self-administer the opioid remifentanil. D1- and D2-GFP mice learned to self-administer 0.1 mg/kg/infusion remifentanil during 2hr sessions over 13 contiguous days. Thereafter, the electrophysiological properties of D1 and D2 MSNs in the shell region of the nucleus accumbens (NAc) were assessed. We found that prior opioid exposure did not alter the basic membrane properties nor the kinetics or amplitude of miniature excitatory postsynaptic currents (mEPSCs). However, when challenged with the mu opioid receptor (µOR) agonist DAMGO, the characteristic inhibitory profile of this receptor was altered. DAMGO inhibited the frequency of mEPSCs in D1-MSNs from control mice receiving saline and in D2-MSNs from mice exposed to remifentanil or saline, but this inhibitory profile was reduced in D1-MSNs from mice receiving remifentanil. Remifentanil exposure also altered the probability of glutamate release onto D1-, but not D2-MSNs. Together these results suggest a D1-pathway specific effect associated with the acquisition of opioid-seeking behaviors.
doi:10.1016/j.bbr.2013.08.009
PMCID: PMC3815993  PMID: 23968589
striatum; medium spiny neurons; intravenous self-administration; mu opioid receptor; electrophysiology; mice
3.  Targeted Expression of Mu-Opioid Receptors in a Subset of Striatal Direct-Pathway Neurons Restores Opiate Reward 
Nature neuroscience  2014;17(2):254-261.
SUMMARY
Mu-Opioid Receptors (MOR) are necessary for the analgesic and addictive effects of opioids such as morphine, but the MOR-expressing neuronal populations that mediate the distinct opiate effects remain elusive. Here we devised a novel conditional BAC rescue strategy to show that mice with targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restore opiate reward, opiate-induced striatal dopamine release, and partially restore motivation to self-administer opiates. However, they lack opiate analgesia or withdrawal. Importantly, we used Cre-mediated deletion of the rescued MOR transgene to establish that striatal, rather than a few extrastriatal sites of MOR transgene expression, is needed for the restoration of opiate reward. Together, our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a novel intersectional genetic approach to dissect neurocircuit-specific gene function in vivo.
doi:10.1038/nn.3622
PMCID: PMC4008330  PMID: 24413699
4.  Sequestration of the δ Opioid Receptor 
The Journal of biological chemistry  1996;271(46):29279-29285.
The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type δ opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process.
PMCID: PMC3856721  PMID: 8910588
6.  Drug-primed reinstatement of cocaine seeking in mice: increased excitability of medium-sized spiny neurons in the nucleus accumbens 
ASN NEURO  2013;5(4):e00122.
To examine the mechanisms of drug relapse, we first established a model for cocaine IVSA (intravenous self-administration) in mice, and subsequently examined electrophysiological alterations of MSNs (medium-sized spiny neurons) in the NAc (nucleus accumbens) before and after acute application of cocaine in slices. Three groups were included: master mice trained by AL (active lever) pressings followed by IV (intravenous) cocaine delivery, yoked mice that received passive IV cocaine administration initiated by paired master mice, and saline controls. MSNs recorded in the NAc shell in master mice exhibited higher membrane input resistances but lower frequencies and smaller amplitudes of sEPSCs (spontaneous excitatory postsynaptic currents) compared with neurons recorded from saline control mice, whereas cells in the NAc core had higher sEPSCs frequencies and larger amplitudes. Furthermore, sEPSCs in MSNs of the shell compartment displayed longer decay times, suggesting that both pre- and postsynaptic mechanisms were involved. After acute re-exposure to a low-dose of cocaine in vitro, an AP (action potential)-dependent, persistent increase in sEPSC frequency was observed in both NAc shell and core MSNs from master, but not yoked or saline control mice. Furthermore, re-exposure to cocaine induced membrane hyperpolarization, but concomitantly increased excitability of MSNs from master mice, as evidenced by increased membrane input resistance, decreased depolarizing current to generate APs, and a more negative Thr (threshold) for firing. These data demonstrate functional differences in NAc MSNs after chronic contingent versus non-contingent IV cocaine administration in mice, as well as synaptic adaptations of MSNs before and after acute re-exposure to cocaine. Reversing these functional alterations in NAc could represent a rational target for the treatment of some reward-related behaviors, including drug addiction.
doi:10.1042/AN20130015
PMCID: PMC3789142  PMID: 24000958
intravenous self-administration; nucleus accumbens; relapse; synaptic transmission; AHP, afterhyperpolarization; AL, active lever; BIC, bicuculline; Coc, cocaine; CsMeth, Cs-Methanesulfonate; FR, fixed ratio; GABA, γ-aminobutyric acid; IAL, inactive lever; IC, current clamp; IVSA, intravenous self-administration; KGluc, K-gluconate internal solution; mEPSCs, miniature excitatory postsynaptic currents; MSNs, medium-sized spiny neurons; NAc, nucleus accumbens; Rin, resistance; rm, repeated measure; RMP, resting membrane potential; sEPSCs, spontaneous excitatory postsynaptic currents; sIPSCs, spontaneous inhibitory postsynaptic currents; Thr, threshold; TTX, tetrodotoxin; VC, voltage clamp
7.  Linguistic validation of translation of the self-assessment goal achievement (saga) questionnaire from English 
Background
A linguistic validation of the Self-Assessment Goal Achievement (SAGA) questionnaire was conducted for 12 European languages, documenting that each translation adequately captures the concepts of the original English-language version of the questionnaire and is readily understood by subjects in the target population.
Methods
Native-speaking residents of the target countries who reported urinary problems/lower urinary tract problems were asked to review a translation of the SAGA questionnaire, which was harmonized among 12 languages: Danish, Dutch, English (UK), Finnish, French, German, Greek, Icelandic, Italian, Norwegian, Spanish, and Swedish. During a cognitive debriefing interview, participants were asked to identify any words that were difficult to understand and explain in their own words the meaning of each sentence in the questionnaire. The qualitative analysis was conducted by local linguistic validation teams (original translators, back translator, project manager, interviewer, and survey research expert).
Results
Translations of the SAGA questionnaire from English to 12 European languages were well understood by the participants with an overall comprehension rate across language of 98.9%. In addition, the translations retained the original meaning of the SAGA items and instructions. Comprehension difficulties were identified, and after review by the translation team, minor changes were made to 7 of the 12 translations to improve clarity and comprehension.
Conclusions
Conceptual, semantic, and cultural equivalence of each translation of the SAGA questionnaire was achieved thus confirming linguistic validation.
doi:10.1186/1477-7525-10-40
PMCID: PMC3349573  PMID: 22525050
SAGA; Validation; Lower urinary tract symptoms; Overactive bladder; Goal achievement; Patient-reported questionnaire
8.  Project Brainstorm: Using Neuroscience to Connect College Students with Local Schools 
PLoS Biology  2012;10(4):e1001310.
Neuroscience can be used as a tool to inspire an interest in science in school children as well as to provide teaching experience to college students.
doi:10.1371/journal.pbio.1001310
PMCID: PMC3328426  PMID: 22529746
9.  Regional and cell-type-specific effects of DAMGO on striatal D1 and D2 dopamine receptor-expressing medium-sized spiny neurons 
ASN NEURO  2012;4(2):e00077.
The striatum can be divided into the DLS (dorsolateral striatum) and the VMS (ventromedial striatum), which includes NAcC (nucleus accumbens core) and NAcS (nucleus accumbens shell). Here, we examined differences in electrophysiological properties of MSSNs (medium-sized spiny neurons) based on their location, expression of DA (dopamine) D1/D2 receptors and responses to the μ-opioid receptor agonist, DAMGO {[D-Ala2-MePhe4-Gly(ol)5]enkephalin}. The main differences in morphological and biophysical membrane properties occurred among striatal sub-regions. MSSNs in the DLS were larger, had higher membrane capacitances and lower Rin (input resistances) compared with cells in the VMS. RMPs (resting membrane potentials) were similar among regions except for D2 cells in the NAcC, which displayed a significantly more depolarized RMP. In contrast, differences in frequency of spontaneous excitatory synaptic inputs were more prominent between cell types, with D2 cells receiving significantly more excitatory inputs than D1 cells, particularly in the VMS. Inhibitory inputs were not different between D1 and D2 cells. However, MSSNs in the VMS received more inhibitory inputs than those in the DLS. Acute application of DAMGO reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents, but the effect was greater in the VMS, in particular in the NAcS, where excitatory currents from D2 cells and inhibitory currents from D1 cells were inhibited by the largest amount. DAMGO also increased cellular excitability in the VMS, as shown by reduced threshold for evoking APs (action potentials). Together the present findings help elucidate the regional and cell-type-specific substrate of opioid actions in the striatum and point to the VMS as a critical mediator of DAMGO effects.
doi:10.1042/AN20110063
PMCID: PMC3297119  PMID: 22273000
D1/D2 receptors; electrophysiology; nucleus accumbens; opioid receptors; striatum; ACSF, artificial cerebrospinal fluid; AHP, after hyperpolarization; AP, action potential; AP-5, dl-2-amino-5-phosphonovaleric acid; BIC, bicuculline; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CsMeth, Cs-methanesulfonate; DA, dopamine; DAMGO, [d-Ala2-MePhe4-Gly(ol)5]enkephalin; DLS, dorsolateral striatum; EPSC, excitatory postsynaptic current; EGFP, enhanced green fluorescent protein; IPSC, inhibitory postsynaptic current; KGluc, K-gluconate; mEPSC, miniature EPSC; mIPSC, miniature IPSC; MSSN, medium-sized spiny neuron; NAcC, nucleus accumbens core; NAcS, nucleus accumbens shell; Rin, input resistance; RMP, resting membrane potential; sEPSC, spontaneous EPSC; sIPSC, spontaneous IPSC; TBST, TBS containing 0.1% Tween 20; TTX, tetrodotoxin; UCLA, University of California at Los Angeles; VMS, ventromedial striatum; VTA, ventral tegmental area
10.  Pavlovian conditioning of multiple opioid-like responses in mice 
Drug and alcohol dependence  2009;103(1-2):74-83.
Conditional responses in rodents such as locomotion have been reported for drugs of abuse and similar to the placebo response in humans, may be associated with the expectation of reward. We examined several conditional opioid-like responses and the influence of drug expectation on conditioned place preference and concomitant conditional locomotion. Male C57BL/6J mice were conditioned with the selective mu opioid receptor agonist fentanyl (0.2 mg/kg, i.p.) in a novel context and subsequently given a vehicle injection. In separate experiments, locomotor activity, Straub tail, hot plate sensitivity, and conditioned place preference (CPP) were measured. Mice exhibited multiple conditional opioid-like responses including conditional hyperlocomotion, a conditional pattern of opioid-like locomotion, Straub tail, analgesia, and place preference. Modulating drug expectation via administration of fentanyl to “demonstrator” mice in the home cage did not affect the expression of conditioned place preference or the concomitant locomotor activity in “observer” mice. In summary, Pavlovian conditioning of an opioid in a novel context induced multiple conditional opioid-like behaviors and provides a model for studying the neurobiological mechanisms of the placebo response in mice.
doi:10.1016/j.drugalcdep.2009.03.016
PMCID: PMC3085957  PMID: 19419821
Associative learning; Context-dependent; Opiate; Expectancy; Pain; Open field; Cocaine
11.  Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2 
Molecular Pain  2011;7:24.
Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs) over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2) augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.
doi:10.1186/1744-8069-7-24
PMCID: PMC3090352  PMID: 21486473
12.  Opioid pharmaceuticals and addiction: The issues, and research directions seeking solutions 
Drug and alcohol dependence  2010;108(3):156-165.
There are few pharmaceuticals superior to opiates for the treatment of pain. However, with concerns of addiction, withdrawal and questionable efficacy for all types of pain, these compounds are far from a magical panacea for pain-relief. As it is unlikely that other classes of compounds will supersede the opioids in the very near future, it is important to both optimize current opioid therapies and curb the astounding diversion of opioids from their intended analgesic use to non-medical abuse. In optimizing opioid therapeutics it is necessary to enhance the clinical awareness of the benefits of treating pain and combine this with aggressive strategies to reduce diversion for non-medical use. At the heart of the issue of opioid misuse is the role of opioid systems in the reward circuitry, and the adaptive processes associated with repetitive opioid use that manifest during withdrawal. Emerging pharmacological insights of opioid receptors will be reviewed that provide future hope for developing opioid-based analgesics with reduced addictive properties and perhaps, reduced opponent processes. In addition, with the increased understanding of nociceptive circuitry and the molecules involved in transmitting pain, new therapeutic targets have become evident that may result in effective analgesics either alone or in combination with current opioid therapies.
doi:10.1016/j.drugalcdep.2010.01.001
PMCID: PMC3072810  PMID: 20188495
13.  A Potential Role for Shed Soluble Major Histocompatibility Class I Molecules as Modulators of Neurite Outgrowth 
PLoS ONE  2011;6(3):e18439.
The neurobiological activities of classical major histocompatibility class I (MHCI) molecules are just beginning to be explored. To further examine MHCI's actions during the formation of neuronal connections, we cultured embryonic mouse retina explants a short distance from wildtype thalamic explants, or thalami from transgenic mice (termed “NSE-Db”) whose neurons express higher levels of MHCI. While retina neurites extended to form connections with wildtype thalami, we were surprised to find that retina neurite outgrowth was very stunted in regions proximal to NSE-Db thalamic explants, suggesting that a diffusible factor from these thalami inhibited retina neurite outgrowth. It has been long known that MHCI-expressing cells release soluble forms of MHCI (sMHCI) due to the shedding of intact MHCI molecules, as well as the alternative exon splicing of its heavy chain or the action proteases which cleave off it's transmembrane anchor. We show that the diffusible inhibitory factor from the NSE-Db thalami is sMHCI. We also show that COS cells programmed to express murine MHCI release sMHCI that inhibits neurite outgrowth from nearby neurons in vitro. The neuroinhibitory effect of sMHCI could be blocked by lowering cAMP levels, suggesting that the neuronal MHCI receptor's signaling mechanism involves a cyclic nucleotide-dependent pathway. Our results suggest that MHCI may not only have neurobiological activity in its membrane-bound form, it may also influence local neurons as a soluble molecule. We discuss the involvement of complement proteins in generating sMHCI and new theoretical models of MHCI's biological activities in the nervous system.
doi:10.1371/journal.pone.0018439
PMCID: PMC3069096  PMID: 21483793
14.  Neurons Preferentially Respond to Self-MHC Class I Allele Products Regardless of Peptide Presented 
Studies of mice lacking MHC class I (MHC I)-associated proteins have demonstrated a role for MHC I in neurodevelopment. A central question arising from these observations is whether neuronal recognition of MHC I has specificity for the MHC I allele product and the peptide presented. Using a well-established embryonic retina explant system, we observed that picomolar levels of a recombinant self-MHC I molecule inhibited neurite outgrowth. We then assessed the neurobiological activity of a panel of recombinant soluble MHC Is, consisting of different MHC I heavy chains with a defined self- or nonself-peptide presented, on cultured embryonic retinas from mice with different MHC I haplotypes. We observed that self-MHC I allele products had greater inhibitory neuroactivity than nonself-MHC I molecules, regardless of the nature of the peptide presented, a pattern akin to MHC I recognition by some innate immune system receptors. However, self-MHC I molecules had no effect on retinas from MHC I-deficient mice. These observations suggest that neuronal recognition of MHC I may be coordinated with the inherited MHC I alleles, as occurs in the innate immune system. Consistent with this notion, we show that MHC I and MHC I receptors are coexpressed by precursor cells at the earliest stages of retina development, which could enable such coordination.
doi:10.4049/jimmunol.0902159
PMCID: PMC2997386  PMID: 20018625
15.  Opioid receptors: from binding sites to visible molecules in vivo 
Neuropharmacology  2008;56(Suppl 1):205-212.
Opioid drugs such as heroin interact directly with opioid receptors whilst other addictive drugs, including marijuana, alcohol and nicotine indirectly activate endogenous opioid systems to contribute to their rewarding properties. The opioid system therefore plays a key role in addiction neurobiology and continues to be a primary focus for NIDA-supported research. Opioid receptors and their peptide ligands, the endorphins and enkephalins, form an extensive heterogeneous network throughout the central and peripheral nervous system. In addition to reward, opioid drugs regulate many functions such that opioid receptors are targets of choice in several physiological, neurological and psychiatric disorders. Because of the multiplicity and diversity of ligands and receptors, opioid receptors have served as an optimal model for G protein coupled receptor (GPCR) research. The isolation of opioid receptor genes opened the way to molecular manipulations of the receptors, both in artificial systems and in vivo, contributing to our current understanding of the diversity of opioid receptor biology at the behavioral, cellular and molecular levels. This review will briefly summarize some aspects of current knowledge that has accumulated since the very early characterization of opioid receptor genes. Importantly, we will identify a number of research directions that are likely to develop during the next decade.
doi:10.1016/j.neuropharm.2008.07.033
PMCID: PMC2950281  PMID: 18718480
16.  Neurokinin1 receptors regulate morphine-induced endocytosis and desensitization of mu opioid receptors in CNS neurons 
Mu opioid receptors (MORs) are G protein-coupled receptors (GPCRs) that mediate the physiological effects of endogenous opioid neuropeptides and opiate drugs such as morphine. MORs are co-expressed with neurokinin 1 receptors (NK1Rs) in several regions of the central nervous system (CNS) that control opioid dependence and reward. NK1R activation affects opioid reward specifically, however, and the cellular basis for this specificity is unknown. We found that ligand-induced activation of NK1Rs produces a cell autonomous and non-reciprocal inhibition of MOR endocytosis induced by diverse opioids. Studies using epitope-tagged receptors expressed in cultured striatal neurons and a neuroblastoma cell model indicated that this heterologous regulation is mediated by NK1R-dependent sequestration of arrestins on endosome membranes. First, endocytic inhibition mediated by wild type NK1Rs was overcome in cells over-expressing β-arrestin2, a major arrestin isoform expressed in striatum. Second, NK1R activation promoted sequestration of β-arrestin2 on endosomes, whereas MOR activation did not. Third, heterologous inhibition of MOR endocytosis was prevented by mutational disruption of β-arrestin2 sequestration by NK1Rs. NK1R-mediated regulation of MOR trafficking was associated with reduced opioid-induced desensitization of adenylyl cyclase signaling in striatal neurons. Further, heterologous regulation of MOR trafficking was observed in both amygdala and locus coeruleus neurons that naturally co-express these receptors. These results identify a cell autonomous mechanism that may underlie the highly specific effects of NK1R on opioid signaling and suggest, more generally, that receptor-specific trafficking of arrestins may represent a fundamental mechanism for coordinating distinct GPCR-mediated signals at the level of individual CNS neurons.
doi:10.1523/JNEUROSCI.4315-08.2009
PMCID: PMC2775560  PMID: 19129399
trafficking; opioid; arrestin; morphine; endocytosis; neurokinin
17.  Sex Chromosome Complement Affects Nociception in Tests of Acute and Chronic Exposure to Morphine in Mice 
Hormones and behavior  2007;53(1):124-130.
We tested the role of sex chromosome complement and gonadal hormones in sex differences in several different paradigms measuring nociception and opioid analgesia using “four core genotypes” C57BL/6J mice. The genotypes include XX and XY gonadal males, and XX and XY gonadal females. Adult mice were gonadectomized and tested 3–4 weeks later, so that differences between sexes (mice with testes vs. ovaries) were attributable mainly to organizational effects of gonadal hormones, whereas differences between XX and XY mice were attributable to their complement of sex chromosomes. In experiment 1 (hotplate test of acute morphine analgesia), XX mice of both gonadal sexes had significantly shorter hotplate baseline latencies prior to morphine than XY mice. In experiment 2, (test of development of tolerance to morphine), mice were injected twice daily with 10mg/kg morphine or saline for 6 days. Saline or the competitive NMDA antagonist CPP [3-]2-carboxypiperazin-4yl)propyl-1-phospionic acid] (10mg/kg) was co-injected. On day 7, mice were tested for hotplate latencies before and after administration of a challenge dose of morphine (10mg/kg). XX mice showed shorter hotplate latencies than XY mice at baseline, and the XX-XY difference was greater following morphine. In experiment 3, mice were injected with morphine (10mg/Kg) or saline,15 minutes before intraplantar injection of formalin (5%/25µl). XX mice licked their hindpaw more than XY mice within 5 minutes of formalin injection. The results indicate that X- or Y-linked genes have direct effects, not mediated by gonadal secretions, on sex differences in two different types of acute nociception.
doi:10.1016/j.yhbeh.2007.09.003
PMCID: PMC2713052  PMID: 17956759
X chromosome; Y chromosome; pain; sex difference; hotplate; sex chromosomes
18.  Morphine analgesic tolerance in 129P3/J and 129S6/SvEv mice 
Morphine analgesic tolerance is heritable in both humans and rodents, with some individuals and strains exhibiting little and others exhibiting robust tolerance. 129S6/SvEv and 129P3/J mice reportedly do not demonstrate tolerance to morphine analgesia. Using our laboratory's standard morphine tolerance regimen and a between-subjects design, tolerance developed in the hot plate and tail withdrawal assays as indicated by a change in analgesic efficacy following a morphine challenge dose. Furthermore, the non-competitive NMDA receptor antagonist MK-801 (dizocilipine) blocked morphine tolerance in 129S6/SvEv and CD-1 mice in the hot plate assay. As previously reported, when a within-subjects design and cumulative dosing was employed, no tolerance was observed in the 129P3/J strain. However, using the same morphine regimen and a between-subjects design, comparable tolerance developed between 129P3/J and C57BL/6J strains following a single challenge dose of morphine. Spontaneous hyperalgesia was observed in the tail withdrawal assay following chronic morphine in C57BL/6J, but not 129P3/J mice. Additionally, morphine-tolerant C57BL/6J mice, but not 129P3/J mice, exhibited a large increase in the frequency of tail flicks during the first second following the baseline nociceptive response which may facilitate detection of the response during the tolerant state. We conclude that the method of tolerance assessment affects the ability to detect tolerance and thus, may affect the degree and pattern of heritability of this trait and this could have implications for gene mapping studies.
doi:10.1016/j.pbb.2006.11.012
PMCID: PMC1905890  PMID: 17196637
19.  Morphine Induces Desensitization of Insulin Receptor Signaling 
Molecular and Cellular Biology  2003;23(17):6255-6266.
Morphine analgesia is mediated principally by the μ-opioid receptor (MOR). Since morphine and other opiates have been shown to influence glucose homeostasis, we investigated the hypothesis of direct cross talk between the MOR and the insulin receptor (IR) signaling cascades. We show that prolonged morphine exposure of cell lines expressing endogenous or transfected MOR, IR, and the insulin substrate 1 (IRS-1) protein specifically desensitizes IR signaling to Akt and ERK cascades. Morphine caused serine phosphorylation of the IR and impaired the formation of the signaling complex among the IR, Shc, and Grb2. Morphine also resulted in IRS-1 phosphorylation at serine 612 and reduced tyrosine phosphorylation at the YMXM p85-binding motifs, weakening the association of the IRS-1/p85 phosphatidylinositol 3-kinase complex. However, the IRS-1/Grb2 complex was unaffected by chronic morphine treatment. These results suggest that morphine attenuates IR signaling to Akt by disrupting the IRS-1-p85 interaction but inhibits signaling to ERK by disruption of the complex among the IR, Shc, and Grb2. Finally, we show that systemic morphine induced IRS-1 phosphorylation at Ser612 in the hypothalamus and hippocampus of wild type, but not MOR knockout, mice. Our results demonstrate that opiates can inhibit insulin signaling through direct cross talk between the downstream signaling pathways of the MOR and the IR.
doi:10.1128/MCB.23.17.6255-6266.2003
PMCID: PMC180943  PMID: 12917346

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