Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

Recently we have begun to investigate a novel role of EphB receptors in opiate-dependant analgesia. EphB2-β-galactosidase knockins demonstrate that EphB2 is persistently expressed within a number of neural pathways involved in MOR-mediated nociception in vivo and that EphB2 colocalizes with markers of the MOR at the cellular level in the spinal cord and dorsal root ganglia. Despite demonstrating wild-type levels of sensory and motor activity, EphB2 null mice exhibit a significantly altered analgesic response to repeated (but not naive) opiate exposure compared to controls. Investigation of EphB2 null mice and wild type animals revealed no differences in MOR protein levels or affinity. Analysis of this opiate-mediated tolerance suggests that associative phenomena play a substantial role in mediating the analgesic effects observed, possibly due to defeciencies in CA1-mediated learning. Therefore, loss of EphB2 may diminish context-dependent learning and that such learning plays a substantial role in regulating morphine-dependent tolerance.

The goal of this dissertation was two fold: 1) To relate dopamine responses in the ventral striatum to ethanol preference and consumption, and 2) to investigate the role of the mu opioid receptors in this ethanol induced dopamine release in the ventral striatum. First a two bottle choice experiment established that a substrain of C57BL/6 mice (C57BL/6NCrl) had significantly less preference for and consumption of ethanol than a second substrain of mouse based on the same background (C57BL6/J). The C57BL/6 strain has been extensively used in alcohol drinking studies and is well known for it’s propensity to consume alcohol over water. To determine if differences in ventral striatal dopamine response vii could contribute to this variability in drinking behavior, we characterized the dopamine response in both substrains of mice after intraperitoneal injections of 1.0, 2.0 or 3.0 g/kg ethanol or saline. We found that the acute intraperitoneal ethanol injections in naïve mice caused a significant elevation in dopamine in both substrains at all three doses with a significant difference between substrains at the two highest alcohol doses. Therefore, ethanol induced dopamine release in the ventral striatum may contribute to ethanol preference and consumption. Next, we investigated the effect of acute intraperitoneal ethanol injections on naïve mu opioid receptor knockout mice and in mice pretreated with a mu opioid receptor antagonist. The mice used were all established on the C57BL/6J background. We found that ventral striatal dopamine response was similar in these mice after 1.0, 2.0 and 3.0 g/kg intraperitoneal ethanol injections compared to appropriate controls. As both gene deletion and pharmacological blockade of the mu opioid receptor did not affect ethanol stimulated dopamine release, it points to the conclusion that this receptor may not play a significant role in ethanol induced ventral striatal dopamine release. / text

Thesis advisor: Alexa H. Veemena / Across species, the juvenile period is characterized by increased social interaction with peers and heightened novelty-seeking behavior, as compared to any other life stage. These behaviors are likely to be highly adaptive during this developmental phase. Still, an excessive novelty-seeking phenotype may predispose individuals to risk-taking and substance abuse, while too little social engagement and low novelty-seeking are characteristics of neuropsychiatry disorders such as autism. The over-arching aim of this dissertation research has been to elucidate the neural mechanisms underlying juvenile social novelty-seeking behavior. Central activation of oxytocin, vasopressin V1a, and µ-opioid receptors (OTR, V1aR, and MOR, respectively) have been implicated in the regulation of adult social behavior, but our understanding of the expression and function of OTR, V1aR, and MORs in the juvenile brain is incomplete. Therefore, in Studies 1 and 2, age differences in binding density of OTR, V1aR, and MOR throughout the rat brain were identified using receptor autoradiography. Next, in Study 3, I established the social novelty preference test, a new paradigm designed to assess the preference of juvenile rats to interact with either a novel or a familiar (cage mate) conspecific. Using this social novelty preference test, in Studies 3, 4, and 5, the functional involvement of OTR, V1aR, and MOR in the regulation of juvenile social novelty preference was characterized using both intracerebroventricular and local in-vivo pharmacological manipulations. The results of these experiments demonstrate that both OTR and MOR activation in the brain are involved in the regulation of juvenile social novelty preference, particularly acting within the nucleus accumbens. Finally, in Study 5, I investigated the impact of social isolation on juvenile social novelty preference. My findings show that social isolation potently reduces social novelty preference, which, in turn, can be restored by MOR activation in the nucleus accumbens. Taken together, this body of work significantly advances our understanding of the neural systems underlying juvenile social novelty preference, and suggests that both oxytocin and opioid systems in the brain may be potential clinical targets for restoring social novelty-seeking behavior in neurodevelopmental disorders, such as autism.

L’utilisation des opioïdes est à l’origine de phénomènes de tolérance et d’hyperalgésie induite (HIO) aussi bien chez l’animal qu’en utilisation clinique. Ces phénomènes surviennent avec tous les opioïdes de manière dose-dépendante. Les mécanismes qui les sous-tendent sont complexes et imparfaitement connus. Le rémifentanil et le sufentanil sont les opioïdes les plus utilisés en France en anesthésie-réanimation. Leur utilisation s’accompagne d’une HIO qui majore la douleur postopératoire et peut être responsable de la persistance de la douleur à long terme. La perception des stimuli nociceptifs chez un patient sous anesthésie générale n’est pas aisée et repose encore sur des signes cliniques indirects d’activation du système sympathique. Ces signes peu sensibles et peu spécifiques conduisent à sous doser ou sur-doser les patients en opioïdes. Récemment, un nouvel outil de monitorage de la nociception est apparu, l’analgesia nociception index (ANI). L’ANI reflète le tonus parasympathique et de ce fait permettrait aux anesthésistes de mieux adapter le dosage des opioïdes. Dans cette thèse, nous avons d’abord évalué la sensibilité et la spécificité de l’ANI à détecter les stimuli nociceptifs, puis montré qu’elles étaient supérieures à celles des signes cliniques, et supérieures à d’autres indices de monitorage proposés. Ensuite nous avons validé la capacité de l’ANI à guider l’analgésie peropératoire du rémifentanil dans différentes situations.Sur le plan expérimental, nous avons exploré, après une exposition courte ou prolongée à différentes doses de rémifentanil et de sufentanil, les mécanismes associés à l’hyperalgésie thermique in vivo, chez la souris, et ex vivo, sur la voie des MAP kinases ERK1/2 et sur le trafic membranaire des récepteurs opioïdes de type µ (MOR) dans différentes cultures cellulaires. Chez la souris, nous avons mis en évidence une hyperalgésie précoce au saut sur plaque chaude, après exposition aux doses les plus élevées de rémifentanil, mais pas avec le sufentanil. De plus, nous n’avons pas observé d’HIO sur le léchage des pattes.Sur les cultures cellulaires, le rémifentanil comme le sufentanil activent la voie des MAPK ERK1/2 lors d’une exposition courte, avec apparition d’une désensibilisation lorsque l’exposition se prolonge. Le rémifentanil comme le sufentanil induisent une internalisation précoce et progressive des récepteurs MOR. / The use of opioids is associated with tolerance and induced hyperalgesia (OIH). Tolerance and OIH occur with all opioids and have been demonstrated both, in animals and in humans and are likely to be dose-dependent. The underlying mechanisms are complex and partially known. Remifentanil and sufentanil are the most used opioids in France in anesthesia and intensive care. Their use is associated with OIH that increases postoperative pain and may be responsible for persistent pain. In anesthetized patients, nociceptive stimuli are still detected according to clinical signs of sympathetic activation. These signs lack sensitivity and specificity and lead to underdosing or overdosing opioids. Recently, the analgesia nociception index (ANI), has been proposed as surrogate marker of nociception. The ANI reflects the parasympathetic tone and thus may allow anesthetists to better adapt the opioid dosage. In this thesis, we first evaluated the sensitivity and specificity of ANI to detect nociceptive stimuli, and showed that it better detects them than do clinical signs or than other currently available monitoring tools. Subsequently, we validated the ability of the ANI to adequately guide the intraoperative dosing of remifentanil in different clinical setting.After acute and sustained exposure to different doses of remifentanil and sufentanil we investigated, in vivo, the mechanisms associated with thermal hyperalgesia in mice, and ex vivo, the effect on the MAP kinase ERK1/2 pathway and the μ-type opioid receptor (MOR) membrane trafficking in human neuroblastoma and embryonic kidney cell cultures. In mice, high-dose remifentanil induced early hyperalgesia assessed by the jumping latency in a hot-plate test, but not the sufentanil. We did not observe OIH for the hind paw licking test. On cell cultures, after short exposure, both remifentanil and sufentanil produced activation of the MAP kinase ERK1/2 pathway, and rapid desensitization and internalization of the MOR.

Indiana University-Purdue University Indianapolis (IUPUI) / Mu opioid receptors (MORs) mediate the analgesic and rewarding effects of opioids. Most research has focused on MORs in GABAergic neurons; however, MORs are also in glutamatergic neurons and their role in opioid-related behaviors was unclear. Our lab previously showed that MORs inhibit glutamate transmission from vesicular glutamate transporter 2 (vGluT2)-expressing thalamostriatal synapses. The behavioral relevance of MORs in vGluT2-expressing neurons was unknown; therefore, I utilized a conditional MOR knockout mouse with MORs deleted in vGluT2-expressing neurons (MORflox-vGluT2cre). MORflox-vGluT2cre mice have disrupted opioid reward, locomotor stimulation, and withdrawal, compared to cre-recombinase negative littermate controls. However, other MOR-mediated behaviors, including opioid-induced antinociception, alcohol reward, and palatable substance consumption are intact. MORs are expressed in vGluT2 neurons in several reward-related brain regions, including the thalamus and lateral habenula (LHb). To determine whether MORs in these brain regions modulate opioid-related behaviors, an adeno-associated viral (AAV) vector encoding cre-recombinase was stereotaxically injected into the thalamus or LHb of MORflox mice to specifically delete MORs in these brain regions. Opioid reward and locomotor stimulation remained intact in both thalamic and LHb MOR knockout mice; however, basal locomotor activity was increased in LHb MOR knockout mice. Sucrose consumption was also intact in LHb MOR knockout mice. Interestingly, in LHb MOR KO mice opioid withdrawal-induced paw shakes were increased, while withdrawal-induced jumping was completely ablated. Our lab previously showed that MORs inhibit glutamate transmission from the anterior insular cortex (AIC), which is disrupted by in vivo alcohol exposure. To determine the role of AIC MORs, AIC MORs were deleted with AAV vectors. AIC MOR knockout mice had intact opioid, sucrose, and alcohol reward, but had increased basal locomotor activity. MORs in glutamatergic neurons are critical mediators of opioid reward; however, the specific glutamatergic neurons mediating the rewarding effects of opioids remains to be determined.

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Since the use of opioids started to emerge for analgesic reasons in the 19th century with the synthetization of morphine, opioids have been studied rigorously to better understand its effects on the brain. This thesis shows that both the analgesic effects and the reinforcing effects of opioids are mediated by the same receptor, the mu opioid receptor (MOR). MOR activity has been correlated to both primary and secondary reinforcers and should be considered to cause positive reinforcement together with increases in dopamine transmission for all drugs of abuse, and not only in relation to opioids. Opioid tolerance, dependence and even addiction are to some extent thought to relate to opioids’ acute effect of cyclic adenosine monophosphate (cAMP) superactivation. Based upon these findings, the allostasis theory of addiction is considered to be the most suitable in defining opioid addiction. The theory claims that the mesolimbic dopamine system becomes sensitized, increasing the attractiveness of opioids. This while counteradaptation increases the pleasurable tolerance of opioids, encouraging the user to increase its intake for the same initial reward. Furthermore the theory claims that cAMP superactivation is causing an unfolding effect of neurobiological and neurochemical expressions which leads to the disorder of addiction. cAMP superactivation is mediating the negatively reinforcing aspects of opioid addiction together with changes to corticotropin-releasing factor (CRF) in the brain stress system, such as the hypothalamic-pituitary-adrenal (HPA) axis and the extended amygdala.

Today, more Americans die each year because of drug overdoses than are killed in motor vehicle accidents. In fact, in 2015, more than 33,000 individuals died due to an overdose of heroin or prescription opioids. Sadly, 40-60 % of patients on current opioid addiction treatment medications relapse. Studies have shown that the addiction/abuse liability of opioids are abolished in mu opioid receptor (MOR) knock-out mice; this indicates that the addiction and abuse liability of opioids are mainly mediated through MOR. Utilizing the “message-address concept”, the our laboratory reported a novel non-peptide, reversible MOR selective ligand 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α (isoquinoline-3-carboxamido)morphinan (NAQ). Molecular modeling and mutagenesis studies revealed that the selectivity of NAQ for MOR is because of the π-π stacking of the isoquinoline ring of NAQ with W318. Therefore, other heterocyclic ring systems were explored to obtain a diverse library of compounds with similar or different molecular interactions and pharmacologic characteristics as NAQ. The newly designed compounds were indole analogs of 6α/β-naltrexamine. The compounds were synthesized and the affinity and selectivity for MOR determined using the radioligand binding assay while the functional activity at MOR was determined using the [35S]GTPγS binding assay. The indole analog of 6α-naltrexamine substituted at position 7 (compound 6) was found to be very potent and had the lowest efficacy in the [35S]GTPγS functional assay while the indole analog of 6β-naltrexamine substituted at position 2 (compound 10) was identified as a MOR agonist and had the greatest efficacy. In vivo studies were conducted using the warm-water immersion assay to find whether the synthesized compounds had antinociceptive effects and/or blocked the antinociceptive effects of morphine. Not surprisingly, compound 10 was identified as an opioid agonist while compound 6 almost completely blocked morphine’s antinociceptive effects. The opioid antagonist effect of compound 6 was found to be dose dependent with an AD50 of 2.39 mg/kg (0.46-12.47). An opioid withdrawal assay was conducted on compound 6 using morphine-pelleted mice. Compound 6 produced significantly less withdrawal symptoms at 50 mg/kg than naltrexone at 1 mg/kg. Therefore, compound 6 has the potential to be used in opioid addiction and withdrawal treatment.