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Mu opioid receptor (MOPr) agonists are well-known and frequently used clinical analgesics but are also rewarding due to their highly addictive and often abusive properties. This may lead to opioid use disorder (OUD) a disorder that effects millions of people worldwide. Therefore, novel compounds are urgently needed to treat OUD. As opioids are effective analgesics and OUD often occurs in conjunction with chronic pain, these novel compounds may be opioids, but they must have a low abuse liability. This could be mediated by diminishing or slowing blood-brain barrier transport, slowing target receptor binding kinetics, and showing a long half-life. NKTR-181 is a PEGylated oxycodol and a MOPr agonist that has slowed blood-brain barrier transport, a long half-life, and diminished likeability in clinical trials. In this study, we examined the signaling and behavioral profile of NKTR-181 in comparison with oxycodone to determine whether further therapeutic development of this compound may be warranted. For this preclinical study, we used a number of in vitro and in vivo assays. The signaling profile of NKTR-181 was determined by the electrophysiological assessment of MOPr-Ca 2+ channel inhibition in the nociceptive neurons of rodent dorsal root ganglia. Heterologous cell-based assays were used to assess biased agonism and receptor trafficking. Different rodent behavioral models were used to define the NKTR-181-induced relief of effective and reflexive nociception and drug-seeking behavior as assessed by an intravenous self-administration (IVSA) of NKTR-181. We found that NKTR-181 and oxycodone are partial agonists in G-protein signaling and Ca 2+ channel inhibition assays and promote limited MOPr desensitization. However, NKTR-181 inhibits Ca 2+ channels by a different mechanism than oxycodone and induces a different pattern of arrestin recruitment. In addition, NKTR-181 has a slower receptor on-rate and a slower rate of Ca 2+ channel coupling than oxycodone. This signaling profile is coupled with a slower onset of antinociception and limited drug-seeking behavior in comparison with oxycodone. Together with its known long half-life and slow blood-brain barrier transport, these data suggest that NKTR-181 could be further studied as a pharmacotherapeutic treatment modality for OUD., Competing Interests: KD was employed by company KiloDalton Consulting. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lee, Tiwari, Bishop, Matossian, Romaneschi, Miyazaki, VanderVeen, Zalevsky, DeFea, Cahill and Walwyn.)

μ-Opioid receptors (MORs) are densely expressed in different brain regions known to mediate reward. One such region is the striatum where MORs are densely expressed, yet the role of these MOR populations in modulating reward is relatively unknown. We have begun to address this question by using a series of genetically engineered mice based on the Cre recombinase/loxP system to selectively delete MORs from specific neurons enriched in the striatum: dopamine 1 (D1) receptors, D2 receptors, adenosine 2a (A2a) receptors, and choline acetyltransferase (ChAT). We first determined the effects of each deletion on opioid-induced locomotion, a striatal and dopamine-dependent behavior. We show that MOR deletion from D1 neurons reduced opioid (morphine and oxycodone)-induced hyperlocomotion, whereas deleting MORs from A2a neurons resulted in enhanced opioid-induced locomotion, and deleting MORs from D2 or ChAT neurons had no effect. We also present the effect of each deletion on opioid intravenous self-administration. We first assessed the acquisition of this behavior using remifentanil as the reinforcing opioid and found no effect of genotype. Mice were then transitioned to oxycodone as the reinforcer and maintained here for 9 d. Again, no genotype effect was found. However, when mice underwent 3 d of extinction training, during which the drug was not delivered, but all cues remained as during the maintenance phase, drug-seeking behavior was enhanced when MORs were deleted from A2a or ChAT neurons. These findings show that these selective MOR populations play specific roles in reward-associated behaviors., (Copyright © 2020 Severino et al.)

Despite the large comorbidity between PTSD and opioid use disorders, as well as the common treatment of physical injuries resulting from trauma with opioids, the ability of opioid treatments to subsequently modify PTSD-related behavior has not been well studied. Using the stress-enhanced fear learning (SEFL) model for PTSD, we characterized the impact of chronic opioid regimens on the sensitization of fear learning seen following traumatic stress in mice. We demonstrate for the first time that chronic opioid pretreatment is able to robustly augment associative fear learning. Highlighting aversive learning as the cognitive process mediating this behavioral outcome, these changes were observed after a considerable period of drug cessation, generalized to learning about multiple aversive stimuli, were not due to changes in stimulus sensitivity or basal anxiety, and correlated with a marker of synaptic plasticity within the basolateral amygdala. Additionally, these changes were not observed when opioids were given after the traumatic event. Moreover, we found that neither reducing the frequency of opioid administration nor bidirectional manipulation of acute withdrawal impacted the subsequent enhancement in fear learning seen. Given the fundamental role of associative fear learning in the generation and progression of PTSD, these findings are of direct translational relevance to the comorbidity between opioid dependence and PTSD, and they are also pertinent to the use of opioids for treating pain resulting from traumas involving physical injuries.

Opioids are highly addictive substances with a relapse rate of over 90%. While preclinical models of chronic opioid exposure exist for studying opioid dependence, none recapitulate the relapses observed in human opioid addiction. The mechanisms associated with opioid dependence, the accompanying withdrawal symptoms, and the relapses that are often observed months or years after opioid dependence are poorly understood. Therefore, we developed a novel model of chronic opioid exposure whereby the level of administration is self-directed with periods of behavior acquisition, maintenance, and then extinction alternating with reinstatement. This profile arguably mirrors that seen in humans, with initial opioid use followed by alternating periods of abstinence and relapse. Recent evidence suggests that dietary interventions that reduce inflammation, including omega-3 polyunsaturated fatty acids (n-3 PUFAs), may reduce substance misuse liability. Using the self-directed intake model, we characterize the observed profile of opioid use and demonstrate that an n-3-PUFA-enriched diet ameliorates oxycodone-seeking behaviors in the absence of drug availability and reduces anxiety. Guided by the major role gut microbiota have on brain function, neuropathology, and anxiety, we profile the microbiome composition and the effects of chronic opioid exposure and n-3 PUFA supplementation. We demonstrate that the withdrawal of opioids led to a significant depletion in specific microbiota genera, whereas n-3 PUFA supplementation increased microbial richness, phylogenetic diversity, and evenness. Lastly, we examined the activation state of microglia in the striatum and found that n-3 PUFA supplementation reduced the basal activation state of microglia. These preclinical data suggest that a diet enriched in n-3 PUFAs could be used as a treatment to alleviate anxiety induced opioid-seeking behavior and relapse in human opioid addiction.

Prescription opioid misuse is an ongoing and escalating epidemic. Although these pharmacological agents are highly effective analgesics prescribed for different types of pain, opioids also induce euphoria, leading to increasing diversion and misuse. Opioid use and related mortalities have developed in spite of initial claims that OxyContin, one of the first opioids prescribed in the USA, was not addictive in the presence of pain. These claims allayed the fears of clinicians and contributed to an increase in the number of prescriptions, quantity of drugs manufactured, and the unforeseen diversion of these drugs for non-medical uses. Understanding the history of opioid drug development, the widespread marketing campaign for opioids, the immense financial incentive behind the treatment of pain, and vulnerable socioeconomic and physical demographics for opioid misuse give perspective on the current epidemic as an American-born problem that has expanded to global significance. In light of the current worldwide opioid epidemic, it is imperative that novel opioids are developed to treat pain without inducing the euphoria that fosters physical dependence and addiction. We describe insights from preclinical findings on the properties of opioid drugs that offer insights into improving abuse-deterrent formulations. One finding is that the ability of some agonists to activate one pathway over another, or agonist bias, can predict whether several novel opioid compounds bear promise in treating pain without causing reward among other off-target effects. In addition, we outline how the pharmacokinetic profile of each opioid contributes to their potential for misuse and discuss the emergence of mixed agonists as a promising pipeline of opioid-based analgesics. These insights from preclinical findings can be used to more effectively identify opioids that treat pain without causing physical dependence and subsequent opioid abuse.

It is thought that frontostriatal circuits play an important role in mediating conditioned behavioral responses to environmental stimuli that were previously encountered during drug administration. However, the neural correlates of conditioned responses to drug-associated cues are not well understood at the level of large populations of simultaneously recorded neurons, or at the level of local field potential (LFP) synchrony in the frontostriatal network. Here we introduce a behavioral assay of conditioned arousal to cocaine cues involving pupillometry in awake head-restrained mice. After just 24 h of drug abstinence, brief exposures to olfactory stimuli previously paired with cocaine injections led to a transient dilation of the pupil, which was greater than the dilation effect to neutral cues. In contrast, there was no cue-selective change in locomotion, as measured by the rotation of a circular treadmill. The behavioral assay was combined with simultaneous recordings from dozens of electrophysiologically identified units in the medial prefrontal cortex (mPFC) and ventral striatum (VS). We found significant relationships between cocaine cue-evoked pupil dilation and the proportion of inhibited principal cells in the mPFC and VS. Additionally, LFP coherence analysis revealed a significant correlation between pupillary response and synchrony in the 25-45 Hz frequency band. Together, these results show that pupil dilation is sensitive to drug-associated cues during acute stages of abstinence, and that individual animal differences in this behavioral arousal response can be explained by two complementary measures of frontostriatal network activity.

Ligand-specific recruitment of arrestins facilitates functional selectivity of G-protein-coupled receptor signaling. Here, we describe agonist-selective recruitment of different arrestin isoforms to the delta opioid receptor in mice. A high-internalizing delta opioid receptor agonist (SNC80) preferentially recruited arrestin 2 and, in arrestin 2 knock-outs (KOs), we observed a significant increase in the potency of SNC80 to inhibit mechanical hyperalgesia and decreased acute tolerance. In contrast, the low-internalizing delta agonists (ARM390, JNJ20788560) preferentially recruited arrestin 3 with unaltered behavioral effects in arrestin 2 KOs. Surprisingly, arrestin 3 KO revealed an acute tolerance to these low-internalizing agonists, an effect never observed in wild-type animals. Furthermore, we examined delta opioid receptor-Ca(2+)channel coupling in dorsal root ganglia desensitized by ARM390 and the rate of resensitization was correspondingly decreased in arrestin 3 KOs. Live-cell imaging in HEK293 cells revealed that delta opioid receptors are in pre-engaged complexes with arrestin 3 at the cell membrane and that ARM390 strengthens this membrane interaction. The disruption of these complexes in arrestin 3 KOs likely accounts for the altered responses to low-internalizing agonists. Together, our results show agonist-selective recruitment of arrestin isoforms and reveal a novel endogenous role of arrestin 3 as a facilitator of resensitization and an inhibitor of tolerance mechanisms., Significance Statement: Agonists that bind to the same receptor can produce highly distinct signaling events and arrestins are a major mediator of this ligand bias. Here, we demonstrate that delta opioid receptor agonists differentially recruit arrestin isoforms. We found that the high-internalizing agonist SNC80 preferentially recruits arrestin 2 and knock-out (KO) of this protein results in increased efficacy of SNC80. In contrast, low-internalizing agonists (ARM390 and JNJ20788560) preferentially recruit arrestin 3 and, surprisingly, KO of arrestin 3 produces acute tolerance and impaired receptor resensitization to these agonists. Arrestin 3 is in pre-engaged complexes with the delta opioid receptor at the cell membrane and low-internalizing agonists promote this interaction. This study reveals a novel role for arrestin 3 as a facilitator of receptor resensitization., (Copyright © 2016 the authors 0270-6474/16/363541-11$15.00/0.)

Corticostriatal signaling participates in sensitized responses to drugs of abuse, where short-term increases in dopamine availability provoke persistent, yet reversible, changes in glutamate release. Prior studies in mice show that amphetamine withdrawal promotes a chronic presynaptic depression in glutamate release, whereas an amphetamine challenge reverses this depression by potentiating corticostriatal activity in direct pathway medium spiny neurons. This synaptic plasticity promotes corticostriatal activity and locomotor sensitization through upstream changes in the activity of tonically active cholinergic interneurons (ChIs). We used a model of operant drug-taking behaviors, in which mice self-administered amphetamine through an in-dwelling catheter. Mice acquired amphetamine self-administration under fixed and increasing schedules of reinforcement. Following a period of abstinence, we determined whether nicotinic acetylcholine receptors modified drug-seeking behavior and associated alterations in ChI firing and corticostriatal activity. Mice responding to conditioned reinforcement showed reduced ChI and corticostriatal activity ex vivo, which paradoxically increased following an amphetamine challenge. Nicotine, in a concentration that increases Ca(2+) influx and desensitizes α4β2*-type nicotinic receptors, reduced amphetamine-seeking behaviors following abstinence and amphetamine-induced locomotor sensitization. Nicotine blocked the depression of ChI firing and corticostriatal activity and the potentiating response to an amphetamine challenge. Together, these results demonstrate that nicotine reduces reward-associated behaviors following repeated amphetamine and modifies the changes in ChIs firing and corticostriatal activity. By returning glutamatergic activity in amphetamine self-administering mice to a more stable and normalized state, nicotine limits the depression of striatal activity in withdrawal and the increase in activity following abstinence and a subsequent drug challenge.

Many chronic pain disorders alternate between bouts of pain and periods of remission. The latent sensitization model reproduces this in rodents by showing that the apparent recovery ("remission") from inflammatory or neuropathic pain can be reversed by opioid antagonists. Therefore, this remission represents an opioid receptor-mediated suppression of a sustained hyperalgesic state. To identify the receptors involved, we induced latent sensitization in mice and rats by injecting complete Freund's adjuvant (CFA) in the hindpaw. In WT mice, responses to mechanical stimulation returned to baseline 3 weeks after CFA. In μ-opioid receptor (MOR) knock-out (KO) mice, responses did not return to baseline but partially recovered from peak hyperalgesia. Antagonists of α2A-adrenergic and δ-opioid receptors reinstated hyperalgesia in WT mice and abolished the partial recovery from hyperalgesia in MOR KO mice. In rats, antagonists of α2A adrenergic and μ-, δ-, and κ-opioid receptors reinstated hyperalgesia during remission from CFA-induced hyperalgesia. Therefore, these four receptors suppress hyperalgesia in latent sensitization. We further demonstrated that suppression of hyperalgesia by MORs was due to their constitutive activity because of the following: (1) CFA-induced hyperalgesia was reinstated by the MOR inverse agonist naltrexone (NTX), but not by its neutral antagonist 6β-naltrexol; (2) pro-enkephalin, pro-opiomelanocortin, and pro-dynorphin KO mice showed recovery from hyperalgesia and reinstatement by NTX; (3) there was no MOR internalization during remission; (4) MORs immunoprecipitated from the spinal cord during remission had increased Ser(375) phosphorylation; and (5) electrophysiology recordings from dorsal root ganglion neurons collected during remission showed constitutive MOR inhibition of calcium channels., Significance Statement: Chronic pain causes extreme suffering to millions of people, but its mechanisms remain to be unraveled. Latent sensitization is a phenomenon studied in rodents that has many key features of chronic pain: it is initiated by a variety of noxious stimuli, has indefinite duration, and pain appears in episodes that can be triggered by stress. Here, we show that, during latent sensitization, there is a sustained state of pain hypersensitivity that is continuously suppressed by the activation of μ-, δ-, and κ-opioid receptors and by adrenergic α2A receptors in the spinal cord. Furthermore, we show that the activation of μ-opioid receptors is not due to the release of endogenous opioids, but rather to its ligand-independent constitutive activity., (Copyright © 2016 the authors 0270-6474/16/360204-18$15.00/0.)

μ-opioid receptors (MORs) 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 new conditional bacterial artificial chromosome rescue strategy to show, in mice, that targeted MOR expression in a subpopulation of striatal direct-pathway neurons enriched in the striosome and nucleus accumbens, in an otherwise MOR-null background, restores opiate reward and opiate-induced striatal dopamine release and partially restores motivation to self administer an opiate. However, these mice lack opiate analgesia or withdrawal. We used Cre-mediated deletion of the rescued MOR transgene to establish that expression of the MOR transgene in the striatum, rather than in extrastriatal sites, is needed for the restoration of opiate reward. Our study demonstrates that a subpopulation of striatal direct-pathway neurons is sufficient to support opiate reward-driven behaviors and provides a new intersectional genetic approach to dissecting neurocircuit-specific gene function in vivo.

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.

The serotonergic system is highly plastic, capable of adapting to changing afferent information in diverse mammalian systems. We hypothesized that removing supraspinal and/or peripheral input would play an important role in defining the distribution of one of the most prevalent serotonergic receptors, the 5-HT(1A) receptor (R), in the spinal cord. We investigated the distribution of this receptor in response to a complete thoracic (T7-T8) spinal cord transection (eliminating supraspinal input), or to spinal cord isolation (eliminating both supraspinal and peripheral input) in adult rats. Using two antibodies raised against either the second extracellular region (ECL(2)) or the third intracellular region (ICL(3)) of the 5-HT(1A)R, we compared the 5-HT(1A)R levels and distributions in specific laminae of the L3-L5 segments among the control, spinal cord-transected, and spinal cord-isolated groups. Each antibody labeled different populations of 5-HT(1A)R: ECL(2) labeled receptors in the axon hillock, whereas ICL(3) labeled receptors predominantly throughout the soma and proximal dendrites. Spinal cord transection increased the number of ECL(2)-positive cells in the medial region of laminae III-IV and lamina VII, and the mean length of the labeled axon hillocks in lamina IX. The number of ICL(3)-labeled cells was higher in lamina VII and in both the medial and lateral regions of lamina IX in the spinal cord-transected compared to the control group. In contrast, the length and number of ECL(2)-immunolabeled processes and ICL(3)-immunolabeled cells were similar in the spinal cord-isolated and control groups. Combined, these data demonstrate that the upregulation in 5-HT(1A)R that occurs with spinal cord transection alone is dependent on the presence of sensory input.

Formation of receptor complexes between micro-opioid and alpha2A-adrenergic receptors has been demonstrated in transfected cells. The functional significance and underlying mechanisms of such receptor interactions remain to be determined in neuronal systems. We examined functional interactions between endogenous micro and alpha2A receptors in mouse dorsal root ganglion neurons. Acute application of the micro agonist [D-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) or the alpha2 agonist clonidine inhibited voltage-gated Ca2+ currents in these neurons. Prolonged treatment with either DAMGO or clonidine induced a mutual cross-desensitization between micro and alpha2A receptor-mediated current inhibition. The cross-desensitization was closely associated with simultaneous internalization of micro and alpha2A receptors. Morphine, a mu agonist triggering little mu receptor endocytosis, induced neither cross-desensitization nor internalization of alpha2A receptors. Furthermore, inhibition of p38 MAPK prevented the cross-desensitization as well as cointernalization of micro and alpha2A receptors. Changes in receptor trafficking profiles suggested that p38 MAPK activity was required for initiating micro receptor internalization and maintaining possible micro-alpha2A association during their cointernalization. Finally, the micro-alpha2A cross-desensitization was absent in dorsal root ganglion neurons lacking beta-arrestin 2. These findings demonstrated p38 MAPK- and beta-arrestin 2-dependent cross-regulation between neuronal micro and alpha2A receptors. By promoting receptor cross-desensitization and cointernalization, such functional interactions may serve as negative feedback mechanisms triggered by prolonged agonist exposure to modulate the signaling of functionally related G protein-coupled receptors.

Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely mechanism of morphine tolerance in vivo. In contrast to the muOR, the mu/partial differentialOR showed equivalent desensitization whether morphine or DAMGO treated, but internalized further with DAMGO than morphine. Such ligand-independent desensitization could be a result of the observed higher rate of synthesis and degradation of this chimeric receptor.

A previous phase III clinical trial failed to show significant therapeutic benefit of repeated subcutaneous nerve growth factor (NGF) administration in the treatment of diabetic neuropathy. Animal studies have since shown that site-specific viral-mediated expression of NGF in the lumbar dorsal root ganglia prevents peripheral nerve dysfunction associated with chemically induced neuropathy. Using a Herpes simplex virus expression vector, we have investigated the effect of localized NGF expression in a genetic mouse model of progressive diabetic neuropathy, the +/+ Leprdb mouse. We found that site-specific delivery of NGF initially delayed the appearance of hypoalgesia, assessed by the Hargreaves test, by 1 month and effectively attenuated this deficit for 2 months over the approximately 10 months normal life-span of these animals. Once the disease progressed into its more severe stages, NGF, although still capable of altering the electrophysiological profile of the sensory A- and C-fibers and influencing the expression of p75 and substance P in the dorsal root ganglia, could no longer maintain normal nociception. These data suggest that maximal therapeutic benefit in future NGF-based gene therapy trials will be gained from early applications of such viral-mediated neurotrophin delivery.

Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.

Background: Pentobarbital decreases the gain of the baroreceptor reflex on the order of 50%, and this blunting is caused nearly entirely by decreasing cardioinhibitory parasympathetic activity. The most likely site of action of pentobarbital is the gamma-aminobutyric acid type A (GABA(A)) receptor. The authors tested whether pentobarbital augments the inhibitory GABAergic neurotransmission to cardiac parasympathetic neurons, and whether expression of the GABA(A) epsilon subunit prevents this facilitation., Methods: The authors used a novel approach to study the effect of pentobarbital on identified cardiac parasympathetic preganglionic neurons in rat brainstem slices. The cardiac parasympathetic neurons in the nucleus ambiguus were retrogradely prelabeled with a fluorescent tracer and were visually identified for patch clamp recording. The effects of pentobarbital on spontaneous GABAergic synaptic events were tested. An adenovirus was used to express the epsilon subunit of the GABA(A) receptor in cardiac parasympathetic neurons to examine whether this transfection alters pentobarbital-mediated changes in GABAergic neurotransmission., Results: Pentobarbital increased the duration but not the frequency or amplitude of spontaneous GABAergic currents in cardiac parasympathetic neurons. Transfection of cardiac parasympathetic neurons with the epsilon subunit of the GABA(A) receptor prevented the pentobarbital-evoked facilitation of GABAergic currents., Conclusions: Pentobarbital, at clinically relevant concentrations, prolongs the duration of spontaneous inhibitory postsynaptic currents that impinge on cardiac parasympathetic neurons. This action would augment the inhibition of cardiac parasympathetic neurons, reduce parasympathetic cardioinhibitory activity, and increase heart rate. Expression of the GABA(A) receptor epsilon subunit in cardiac parasympathetic neurons renders the GABA receptors insensitive to pentobarbital.

There is growing evidence that tonic activity of the opioid system may be important in the modulation of affective state. Naloxone produces a conditioned place aversion in rodents, an effect that is centrally mediated. Previous pharmacological data using antagonists with preferential actions at mu-, delta-, and kappa-opioid receptors indicate the importance of the mu-opioid receptor in mediating this effect. We sought to test the mu-opioid receptor selectivity of naloxone aversion using mu-opioid receptor knock-out mice. mu-Opioid receptor knock-out and wild-type mice were tested for naloxone (10 mg/kg, s.c.) aversion using a place conditioning paradigm. As a positive control for associative learning, knock-out mice were tested for conditioned place aversion to a kappa agonist, U50,488H (2 mg/kg, s.c.). Naloxone produced a significant place aversion in wild-type mice, but failed to have any effect in mu-opioid receptor knock-out mice. On the other hand, both knock-out and wild-type mice treated with U50,488H spent significantly less time in the drug-paired chamber compared to their respective vehicle controls. We conclude that the mu-opioid receptor is crucial for the acquisition of naloxone-induced conditioned place aversion. Furthermore, in a separate experiment using C57BL/6 mice, the delta-selective antagonist naltrindole (10 or 30 mg/kg, s.c.) failed to produce conditioned place aversion.Taken together, these data further support the notion that naloxone produces aversion by antagonizing tonic opioid activity at the mu-opioid receptor.

The capacity to reestablish locomotor function after complete spinal cord transection in the adult mammal is now well documented. Further studies have shown different neurotransmitters to be involved in the initiation and maintenance of these locomotor patterns. However, there has been no in vivo evidence of the changes in glutamate or any other neurotransmitter in the extracellular space of the dorsal horn during an alternating motor pattern such as hindlimb stepping. This study describes an in vivo microdialysis technique to measure extracellular glutamate in the dorsal horn of the spinal cord in the fully awake intact rat. A concentric microdialysis probe was placed in the dorsal horn at L5, and 18 h later dialysate samples were collected at 20-min intervals before, during, and after 20 min of hindlimb stepping. During stepping, extracellular glutamate rose 150% above resting levels and returned to resting levels 40 min later. This increase may have occurred either as a result of primary afferent depolarization or modulation by the descending and ascending supraspinal pathways. In another series of experiments extracellular glutamate was, therefore, measured in the dorsal horn of the chronic spinally transected rat during 20 min of hindlimb stepping. Although the spinal group did not take as many steps as the intact group, those taking more than 40 steps showed a significant rise in extracellular glutamate, and the number of steps taken by the individual spinal rats correlated positively with the individual values of extracellular glutamate (r2 = 0.63). These results are consistent with glutamate being an important neurotransmitter in the spinal cord in normal locomotion.