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.

Buprenorphine reduces somatic withdrawal in a mouse model of early-life morphine exposure.

The rising prevalence of early-life opioid exposure has become a pressing public health issue in the U.S. Neonates exposed to opioids in utero are at risk of experiencing a constellation of postpartum withdrawal symptoms commonly referred to as neonatal opioid withdrawal syndrome (NOWS). Buprenorphine (BPN), a partial agonist at the mu-opioid receptor (MOR) and antagonist at the kappa-opioid receptor (KOR), is currently approved to treat opioid use disorder in adult populations. Recent research suggests that BPN may also be effective in reducing withdrawal symptoms in neonates who were exposed to opioids in utero. We sought to determine whether BPN attenuates somatic withdrawal in a mouse model of NOWS. Our findings indicate that the administration of morphine (10mg/kg, s.c.) from postnatal day (PND) 1-14 results in increased somatic symptoms upon naloxone-precipitated (1mg/kg, s.c.) withdrawal. Co-administration of BPN (0.3mg/kg, s.c.) from PND 12-14 attenuated symptoms in morphine-treated mice. On PND 15, 24h following naloxone-precipitated withdrawal, a subset of mice was examined for thermal sensitivity in the hot plate test. BPN treatment significantly increased response latency in morphine-exposed mice. Lastly, neonatal morphine exposure elevated mRNA expression of KOR, and reduced mRNA expression of corticotropin-releasing hormone (CRH) in the periaqueductal gray when measured on PND 14. Altogether, this data provides support for the therapeutic effects of acute low-dose buprenorphine treatment in a mouse model of neonatal opioid exposure and withdrawal.

Neurobehavioral effects of neonatal opioid exposure in mice: Influence of the OPRM1 SNP.

Opioid use among pregnant women is a growing public health concern in the United States. Infants exposed to opioids in utero are at risk of exhibiting neonatal opioid withdrawal syndrome (NOWS). The biological mechanisms underlying short and long-term consequences of in utero opioid exposure and NOWS are unknown. A potential genetic factor is a single-nucleotide polymorphism (SNP) in the mu-opioid receptor gene (OPRM1 A118G). Opioid exposed infants with the G-allele spend less time in hospitals after birth. To determine whether this SNP modulates the neurobehavioral effects of neonatal opioid exposure and withdrawal, we used mice possessing the equivalent Oprm1 SNP (A112G). Pups were treated chronically with saline or morphine from postnatal days (PNDs) 1 to 14, a developmental period equivalent to the third trimester of a human pregnancy and a sensitive period for opioid exposure in rodents. Morphine treatment produced significant developmental delays regardless of genotype and increased total ultrasonic vocalizations in males during spontaneous withdrawal. Animals were aged and tested for anxiety and drug response during adolescence and adulthood, respectively. AA morphine-treated animals showed reduced activity in the marble burying task compared with saline controls; however, this effect was absent in AG and GG animals. As adults, AA males exposed to morphine from PNDs 1 to 14 exhibited enhanced development of locomotor sensitization to morphine, whereas females showed reduced locomotor sensitization. These data suggest the involvement of the Oprm1 SNP for certain outcomes of neonatal opioid exposure and highlight the importance of considering sex and genetic variability for the prognosis of NOWS.

Buprenorphine prevents stress-induced blunting of nucleus accumbens dopamine response and approach behavior to food reward in mice.

Alterations to the mesolimbic dopamine (DA) system are thought to underlie dysfunctional reward processing in stress-related psychiatric disorders. Using in vivio microdialysis in awake freely moving mice, we assessed the effects of stress on the motivational and neurochemical correlates underlying conditioned approach behavior for palatable food in the non-deprived mouse. Mice trained to approach and consume food in a familiar environment exhibited a 30% increase in nucleus accumbens shell (AcbSh) extracellular dopamine levels coincident with approach towards and consumption of the food reward. This effect was not observed in mice that were presented with the food in an unfamiliar environment or were exposed for the first time and were region specific. The addition of an acute environmental stressor (bright light and novel scent) during food exposure decreased DA release and delayed approach to the food. The disruptive impact of acute novelty stress on DA levels and approach behavior was reversed in animals pretreated with buprenorphine, an opioid drug with antidepressant-like and anxiolytic effects. Together, these data indicate that exposure to mild stress reduces incentive drive to approach palatable food via alterations in AcbSh dopamine responsiveness to food reward. Moreover, they implicate the brain opioid system as a potential pharmacological target for counteracting behavioral and neurochemical elements associated with stress.

Reversal of Stress-Induced Social Interaction Deficits by Buprenorphine.

Background: Patients with post-traumatic stress disorder frequently report persistent problems with social interactions, emerging after a traumatic experience. Chronic social defeat stress is a widely used rodent model of stress that produces robust and sustained social avoidance behavior. The avoidance of other rodents can be reversed by 28 days of treatment with selective serotonin reuptake inhibitors, the only pharmaceutical class approved by the U.S. Food and Drug Administration for treating post-traumatic stress disorder. In this study, the sensitivity of social interaction deficits evoked by 10 days of chronic social defeat stress to prospective treatments for post-traumatic stress disorder was examined. Methods: The effects of acute and repeated treatment with a low dose of buprenorphine (0.25 mg/kg/d) on social interaction deficits in male C57BL/6 mice by chronic social defeat stress were studied. Another cohort of mice was used to determine the effects of the selective serotonin reuptake inhibitor fluoxetine (10 mg/kg/d), the NMDA antagonist ketamine (10 mg/kg/d), and the selective kappa opioid receptor antagonist CERC-501 (1 mg/kg/d). Changes in mRNA expression of Oprm1 and Oprk1 were assessed in a separate cohort. Results: Buprenorphine significantly reversed social interaction deficits produced by chronic social defeat stress following 7 days of administration, but not after acute injection. Treatment with fluoxetine for 7 days, but not 24 hours, also reinstated social interaction behavior in mice that were susceptible to chronic social defeat. In contrast, CERC-501 and ketamine failed to reverse social avoidance. Gene expression analysis found: (1) Oprm1 mRNA expression was reduced in the hippocampus and increased in the frontal cortex of susceptible mice and (2) Oprk1 mRNA expression was reduced in the amygdala and increased in the frontal cortex of susceptible mice compared to non-stressed controls and stress-resilient mice. Conclusions: Short-term treatment with buprenorphine and fluoxetine normalized social interaction after chronic social defeat stress. In concert with the changes in opioid receptor expression produced by chronic social defeat stress, we speculate that buprenorphine's efficacy in this model of post-traumatic stress disorder may be associated with the ability of this compound to engage multiple opioid receptors.

A role for the mu opioid receptor in the antidepressant effects of buprenorphine.

Buprenorphine (BPN), a mixed opioid drug with high affinity for mu (MOR) and kappa (KOR) opioid receptors, has been shown to produce behavioral responses in rodents that are similar to those of antidepressant and anxiolytic drugs. Although recent studies have identified KORs as a primary mediator of BPN's effects in rodent models of depressive-like behavior, the role of MORs in BPN's behavioral effects has not been as well explored. The current studies investigated the role of MORs in mediating conditioned approach behavior in the novelty-induced hypophagia (NIH) test, a behavioral measure previously shown to be sensitive to chronic treatment with antidepressant drugs. The effects of BPN were evaluated in the NIH test 24h post-administration in mice with genetic deletion of the MOR (Oprm1-/-) or KOR (Oprk1-/-), or after pharmacological blockade with the non-selective opioid receptor antagonist naltrexone and selective MOR antagonist cyprodime. We found that behavioral responses to BPN in the NIH test were blocked in Oprm1-/- mice, but not in Oprk1-/- mice. Both cyprodime and naltrexone significantly reduced approach latency at doses experimentally proven to antagonize the MOR. In contrast the selective MOR agonist morphine and the selective KOR antagonist nor-BNI were both ineffective. Moreover, antinociceptive studies revealed persistence of the MOR antagonist properties of BPN at 24h post-administration, the period of behavioral reactivity. These data support modulation of MOR activity as a key component of BPN's antidepressant-like effects in the NIH paradigm.

Corticosterone exposure augments sensitivity to the behavioral and neuroplastic effects of fluoxetine in C57BL/6 mice.

Both genetic background and pre-existing stress play critical roles in the effects of antidepressant drugs. The current studies showed this principal by demonstrating that exposure to the stress hormone corticosterone (CORT) allowed behavioral and neurogenic effects to emerge following chronic treatment with fluoxetine of C57BL/6 mice, a strain ordinarily resistant to these effects. Adult male mice were implanted subcutaneously with 21-day slow-release CORT pellets (10 mg) or placebo and then co-treated with 5 mg/kg fluoxetine (b.i.d., i.p.) or saline for 14 days. Animals were then assessed for approach behavior in the novelty-induced hypophagia (NIH) test, hippocampal cell proliferation, corticosteroid receptor expression, and CORT plasma levels. Co-treatment of CORT with fluoxetine significantly reduced approach behavior in the novel environment of the NIH test and increased hippocampal cell proliferation whereas fluoxetine given alone was ineffective. CORT given alone did not alter approach behavior in the novel environment and caused a smaller increase of cell proliferation. The CORT effect was blocked by adrenalectomy and was likely due to increased adrenal feedback. Cell proliferation in CORT-treated animals was associated with reduced mineralocorticoid, but not glucocorticoid, receptor mRNA expression. Although the pellets were advertised to release CORT for 21 days, plasma CORT levels were increased at 1 day after implantation but were not sustained when measured at 7 days or longer intervals. Nevertheless, the transient CORT increase was sufficient to induce long-lasting behavioral and molecular changes when followed by fluoxetine treatment. These studies warrant further investigation into the role of glucocorticoids and environmental stress as adjunctive facilitators of the response to antidepressants, especially for treatment-resistant patients.

Effects of buprenorphine on behavioral tests for antidepressant and anxiolytic drugs in mice.

RATIONALE: Buprenorphine (BPN) has been shown to rapidly improve mood in treatment-resistant depressed patients in small clinical studies. However, BPN's effects in preclinical tests for mood and antidepressant efficacy are largely unexplored. OBJECTIVE: The current study examined the effects of BPN in the forced swim test (FST) and novelty-induced hypophagia (NIH) test as measures of antidepressant and anxiolytic-like effects in C57BL/6 J mice. Microdialysis was used to measure whether BPN engaged kappa-opioid receptor (KORs) in the nucleus accumbens shell (NAcSh) at a behaviorally active dose (0.25 mg/kg). METHODS: BPN was tested in the FST at both 30 min and 24 h post-administration. Also measured in the FST at 24 h post-administration were the KOR antagonist norbinaltorphimine (nor-BNI), the MOR agonist morphine and the reference antidepressant desipramine. The anxiolytic effects of BPN were examined in the NIH test 24 h after treatment. The effects of acute injection of BPN and the KOR agonist U50,488 were measured on extracellular dopamine (DA) levels in the NAcSh. RESULTS: BPN produced significant reductions in FST immobility without changing locomotor activity and reduced approach latencies in the novel environment of the NIH test when tested 24 h after treatment. Repeated daily BPN injections for 6 days did not produce tolerance to these behavioral effects. nor-BNI produced a similar antidepressant-like response in the FST 24 h post-injection but morphine and desipramine were ineffective. BPN (0.25 mg/kg) did not alter DA levels when given alone but prevented the KOR agonist U50,488 from reducing DA levels. CONCLUSIONS: Acute and subchronic treatment with BPN produced antidepressant and anxiolytic-like responses in mice at doses that engage KORs. These studies support the clinical evidence that BPN may be a novel rapid-acting antidepressant medication and provides rodent models for investigating associated neurochemical mechanisms.

Effects of gestational allopregnanolone administration in rats bred for high affective behavior.

The anxiolytic neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one or 3alpha,5alpha-THP) has been proposed to play a developmental role in emergent neural regulation of affective behavior. This experiment examined whether allopregnanolone administered during the last week of gestation in rats would alter neonatal and adult offspring behaviors in the selectively-bred High vocalizing line, who have low levels of allopregnanolone and high levels of anxious/depressive behaviors. Dams were injected twice a day with the neurosteroid or vehicle, or handled as controls, and were tested on the elevated plus maze just before parturition. Maternal behavior was assessed throughout the first week of life, and affective behavior in the offspring was tested at one week of age (ultrasonic vocalizations test) and as adults (plus maze and forced swim tests). Offspring prenatally exposed to allopregnanolone were less anxious as neonates and less depressed as adults compared to both control groups. Only male adult offspring, however, revealed less anxious behavior on the plus maze. Neither the dams' anxiety behavior measured in late gestation nor their postnatal maternal behavior was altered compared to controls, suggesting a direct, long-lasting effect of gestational allopregnanolone on the developing fetal brain independent of mediating maternal factors. These results are discussed in light of new evidence about the developmental role of the GABA-A receptor prenatally.