Molecular and long-term behavioral consequences of neonatal opioid exposure and withdrawal in mice.

Introduction: Infants exposed to opioids in utero are at high risk of exhibiting Neonatal Opioid Withdrawal Syndrome (NOWS), a combination of somatic withdrawal symptoms including high pitched crying, sleeplessness, irritability, gastrointestinal distress, and in the worst cases, seizures. The heterogeneity of in utero opioid exposure, particularly exposure to polypharmacy, makes it difficult to investigate the underlying molecular mechanisms that could inform early diagnosis and treatment of NOWS, and challenging to investigate consequences later in life. Methods: To address these issues, we developed a mouse model of NOWS that includes gestational and post-natal morphine exposure that encompasses the developmental equivalent of all three human trimesters and assessed both behavior and transcriptome alterations. Results: Opioid exposure throughout all three human equivalent trimesters delayed developmental milestones and produced acute withdrawal phenotypes in mice reminiscent of those observed in infants. We also uncovered different patterns of gene expression depending on the duration and timing of opioid exposure (3-trimesters, in utero only, or the last trimester equivalent only). Opioid exposure and subsequent withdrawal affected social behavior and sleep in adulthood in a sex-dependent manner but did not affect adult behaviors related to anxiety, depression, or opioid response. Discussion: Despite marked withdrawal and delays in development, long-term deficits in behaviors typically associated with substance use disorders were modest. Remarkably, transcriptomic analysis revealed an enrichment for genes with altered expression in published datasets for Autism Spectrum Disorders, which correlate well with the deficits in social affiliation seen in our model. The number of differentially expressed genes between the NOWS and saline groups varied markedly based on exposure protocol and sex, but common pathways included synapse development, the GABAergic and myelin systems, and mitochondrial function.

Mu-opioid receptor-expressing neurons in the paraventricular thalamus modulate chronic morphine-induced wake alterations.

Disrupted sleep is a symptom of many psychiatric disorders, including substance use disorders. Most drugs of abuse, including opioids, disrupt sleep. However, the extent and consequence of opioid-induced sleep disturbance, especially during chronic drug exposure, is understudied. We have previously shown that sleep disturbance alters voluntary morphine intake. Here, we examine the effects of acute and chronic morphine exposure on sleep. Using an oral self-administration paradigm, we show that morphine disrupts sleep, most significantly during the dark cycle in chronic morphine, with a concomitant sustained increase in neural activity in the Paraventricular Nucleus of the Thalamus (PVT). Morphine binds primarily to Mu Opioid Receptors (MORs), which are highly expressed in the PVT. Translating Ribosome Affinity Purification (TRAP)-Sequencing of PVT neurons that express MORs showed significant enrichment of the circadian entrainment pathway. To determine whether MOR + cells in the PVT mediate morphine-induced sleep/wake properties, we inhibited these neurons during the dark cycle while mice were self-administering morphine. This inhibition decreased morphine-induced wakefulness but not general wakefulness, indicating that MORs in the PVT contribute to opioid-specific wake alterations. Overall, our results suggest an important role for PVT neurons that express MORs in mediating morphine-induced sleep disturbance.

A novel Oprm1-Cre mouse maintains endogenous expression, function and enables detailed molecular characterization of µ-opioid receptor cells.

Key targets of both the therapeutic and abused properties of opioids are µ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary. Therefore, we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3'UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. The breadth of utility of this new tool will greatly facilitate the study of opioid biology under varying conditions.

Modulation of cocaine-related behaviors by low doses of the potent KOR agonist nalfurafine in male C57BL6 mice.

RATIONALE: Agonists of the kappa opioid receptor (KOR) have been shown to block the rewarding effects of drugs of abuse, but with negative side effects. The antipruritic drug nalfurafine, approved in Japan in 2009, is a potent, selective KOR agonist that does not cause significant side effects in humans. Nalfurafine has not been extensively tested for its effect on drug reward and reinforcement in preclinical models. OBJECTIVES: The goal of this study was to compare the effects of nalfurafine and a reference KOR agonist for a variety of KOR-mediated endpoints in male C57BL6 mice. Specifically, we aimed to evaluate the "therapeutic window"-doses of agonists lower than those eliciting negative side effects, while still effective for desired therapeutic effects. METHODS: In this study, several low doses of nalfurafine and U50,488 were tested for serum prolactin release, rotarod-mediated sedation, and place-conditioning in male C57BL6 mice. These agonists were also tested for effects on intravenous cocaine self-administration, both on an FR1 schedule and on a progressive ratio schedule for 0.5 mg/kg/infusion cocaine. RESULTS: Serum prolactin levels increased following doses of both nalfurafine (3 µg/kg and 10 µg/kg) and U50,488 (3 mg/kg). These doses did not cause sedation in the rotarod assay or aversion in a place-conditioning assay, but blocked conditioned place preference for cocaine. Immediate pretreatment of mice with 10 µg/kg nalfurafine and 3 mg/kg U50,488, however, potentiated cocaine self-administration. Further 10 µg/kg nalfurafine was also observed to potentiate cocaine-seeking behavior as demonstrated by increased progressive ratio break point. CONCLUSIONS: Both nalfurafine and U50,488 showed a separation of negative side effects and the modulation of cocaine reward, suggesting this effect of KOR agonists at low doses may be characteristic of the KOR system in general. At higher doses, nalfurafine had similar effects to traditional KOR agonists like U50,488, indicating that its relative potency, rather than differences in KOR signaling, may be responsible for its unique effects in humans.

Signaling Properties of Structurally Diverse Kappa Opioid Receptor Ligands: Toward in Vitro Models of in Vivo Responses.

Biased ligands preferentially activate certain signaling pathways downstream of their target receptor, leading to differential physiological or behavioral responses downstream. The kappa opioid receptor (KOR) is a drug target for diseases involving mood and reward, such as depression and addiction. Biased KOR ligands offer the potential to overcome negative side effects that have previously hampered the therapeutic development of KOR agonists by preferentially activating certain signaling pathways. Understanding relationships between ligand bias and behavior is difficult, however, because differences in cellular context and bias quantification methods lead to variation between studies. Here, a set of 21 structurally diverse KOR ligands were tested in parallel, to systematically quantify ligand bias at the KOR. Compounds included the endogenous peptide ligand Dynorphin A(1-17), two novel compounds synthesized for our research, and 18 additional compounds of different structural classes, including morphinans and the natural product Salvinorin A. Compounds were tested for their activity in early KOR signaling pathways (G-protein and ß-arrestin recruitment) in KOR-expressing U2OS cells, and ligand bias was calculated. A subset of compounds was tested for sedative properties in the rotarod assay in mice. We found that rotarod sedation significantly correlated with ß-arrestin signaling in this system, indicating that this in vitro system can be used to accurately describe this in vivo behavior caused by KOR agonists. Additionally, downstream signaling pathways ERK1/2 and mTOR were evaluated, and we determined that signaling via both of these pathways could diverge from KOR-mediated G-protein and arrestin signaling in this system.

Structurally Related Kappa Opioid Receptor Agonists with Substantial Differential Signaling Bias: Neuroendocrine and Behavioral Effects in C57BL6 Mice.

Background: The kappa opioid receptor system has been revealed as a potential pharmacotherapeutic target for the treatment of addictions to substances of abuse. Kappa opioid receptor agonists have been shown to block the rewarding and dopamine-releasing effects of psychostimulants. Recent investigations have profiled the in vivo effects of compounds biased towards G-protein-mediated signaling, with less potent arrestin-mediated signaling. The compounds studied here derive from a series of trialkylamines: N-substituted-N- phenylethyl-N-3-hydroxyphenylethyl-amine, with N-substituents including n-butyl (BPHA), methylcyclobutyl (MCBPHA), and methylcyclopentyl (MCPPHA). Methods: BPHA, MCBPHA, and MCPPHA were characterized in vitro in a kappa opioid receptor-expressing cell line in binding assays and functional assays. We also tested the compounds in C57BL6 mice, assaying incoordination with rotarod, as well as circulating levels of the neuroendocrine kappa opioid receptor biomarker, prolactin. Results: BPHA, MCBPHA, and MCPPHA showed full kappa opioid receptor agonism for G-protein coupling compared with the reference compound U69,593. BPHA showed no measurable ß-arrestin-2 recruitment, indicating that it is extremely G-protein biased. MCBPHA and MCPPHA, however, showed submaximal efficacy for recruiting ß-arrestin-2. Studies in C57BL6 mice reveal that all compounds stimulate release of prolactin, consistent with dependence on G-protein signaling. MCBPHA and MCPPHA result in rotarod incoordination, whereas BPHA does not, consistent with the reported requirement of intact kappa opioid receptor/ß-arrestin-2 mediated coupling for kappa opioid receptor agonist-induced rotarod incoordination. Conclusions: BPHA, MCBPHA, and MCPPHA are thus novel differentially G-protein-biased kappa opioid receptor agonists. They can be used to investigate how signaling pathways mediate kappa opioid receptor effects in vitro and in vivo and to explore the effects of candidate kappa opioid receptor-targeted pharmacotherapeutics.

A Robust Workflow for Native Mass Spectrometric Analysis of Affinity-Isolated Endogenous Protein Assemblies.

The central players in most cellular events are assemblies of macromolecules. Structural and functional characterization of these assemblies requires knowledge of their subunit stoichiometry and intersubunit connectivity. One of the most direct means for acquiring such information is so-called "native mass spectrometry (MS)", wherein the masses of the intact assemblies and parts thereof are accurately determined. It is of particular interest to apply native MS to the study of endogenous protein assemblies-i.e., those wherein the component proteins are expressed at endogenous levels in their natural functional states, rather than the overexpressed (sometimes partial) constructs commonly employed in classical structural studies, whose assembly can introduce stoichiometry artifacts and other unwanted effects. To date, the application of native MS to the elucidation of endogenous protein complexes has been limited by the difficulty in obtaining pristine cell-derived assemblies at sufficiently high concentrations for effective analysis. Here, to address this challenge, we present a robust workflow that couples rapid and efficient affinity isolation of endogenous protein complexes with a sensitive native MS readout. The resulting workflow has the potential to provide a wealth of data on the stoichiometry and intersubunit connectivity of endogenous protein assemblies-information that is key to successful integrative structural elucidation of biological systems.