A history of opioid exposure in females increases the risk of metabolic disorders in their future male offspring.

Worldwide consumption of opioids remains at historic levels. Preclinical studies report intergenerational effects on the endogenous opioid system of future progeny following preconception morphine exposure. Given the role of endogenous opioids in energy homeostasis, such effects could impact metabolism in the next generation. Thus, we examined diet-induced modifications in F1 male progeny of morphine-exposed female rats (MORF1). When fed a high fat-sugar diet (FSD) for 6 weeks, MORF1 males display features of emerging metabolic syndrome; they consume more food, gain more weight, and develop fasting-induced hyperglycemia and hyperinsulinemia. In the hypothalamus, proteins involved in energy homeostasis are modified and RNA sequencing revealed down-regulation of genes associated with neuronal plasticity, coupled with up-regulation of genes associated with immune, inflammatory, and metabolic processes that are specific to FSD-maintained MORF1 males. Thus, limited preconception morphine exposure in female rats increases the risk of metabolic syndrome/type 2 diabetes in the next generation.

Modeling prenatal opioid exposure in animals: Current findings and future directions.

The past decade has seen a drastic rise in the number of infants exposed to opioids in utero. It is unclear what lasting effect this exposure may have on these children. Animal models of prenatal opioid exposure may provide insight into potential areas of vulnerability. The present review summarizes the findings across animal models of prenatal opioid exposure, including exposure to morphine, methadone, buprenorphine, and oxycodone. Details regarding the drug, doses, and duration of treatment, as well as key findings, are summarized in tables with associated references. Finally, significant gaps in the current preclinical literature and future directions are discussed.

Impaired cocaine-induced behavioral plasticity in the male offspring of cocaine-experienced sires.

Our previous work indicated that male, but not female, offspring of cocaine-experienced sires display blunted cocaine self-administration. We extended this line of investigation to examine behavioral sensitization, a commonly used model of cocaine-induced behavioral and neuronal plasticity. Results indicated that male, but not female, offspring of cocaine-taking sires showed deficits in the ability of repeated systemic cocaine injections to induce augmented locomotor activity. The reduced cocaine sensitization phenotype in male progeny was associated with changes in histone post-translational modifications, epigenetic processes that regulate gene expression by controlling the accessibility of genes to transcriptional machinery, in the nucleus accumbens of first-generation male progeny. Thus, five histone post-translational modifications were significantly altered in the male progeny of cocaine-exposed sires. In contrast, self-administration of nicotine was unaltered in male and female offspring suggesting that the intergenerational effects of paternal cocaine taking may be drug-specific. Interestingly, the reduced sensitivity to cocaine previously observed in the male offspring of cocaine-taking sires dissipated in the grand-offspring. Both male and female grand-progeny of cocaine-exposed sires showed unaltered cocaine-induced behavioral sensitization and cocaine self-administration. Taken together, these findings indicate that paternal cocaine taking produces changes in multiple cocaine addiction-related behaviors in male progeny, which do not persist beyond the first generation of offspring. Moreover, the altered sensitivity to cocaine in first-generation male progeny of cocaine-sired male offspring was associated with epigenetic modifications in the nucleus accumbens, a nucleus that plays a critical role in cocaine-associated behavioral plasticity.

Enhanced anxiety in the male offspring of sires that self-administered cocaine.

We previously showed that paternal cocaine exposure reduced the reinforcing efficacy of cocaine in male offspring. Here, we sought to determine whether paternal cocaine experience could also influence anxiety levels in offspring. Male rats were allowed to self-administer cocaine (controls received saline passively) for 60 days and then were bred with naïve females. Measures of anxiety and cocaine-induced anxiogenic effects were assessed in the adult offspring. Cocaine-sired male offspring exhibited increased anxiety-like behaviors, as measured using the novelty-induced hypophagia and defensive burying tasks, relative to saline-sired males. In contrast, sire cocaine experience had no effect on anxiety-like behaviors in female offspring. When challenged with an anxiogenic (but not anorectic) dose of cocaine (2.5 mg/kg, i.p.), anxiety-like behavior was enhanced in all animals to an equal degree regardless of sire drug experience. Since anxiety and depression are often co-morbid, we also assessed measures of depressive-like behavior. Sire cocaine experience had no effect on depression-like behaviors, as measured by the forced swim task, among male offspring. In a separate group of naïve littermates, select neuronal correlates of anxiety were measured. Male offspring of cocaine-experienced sires showed increased mRNA and protein expression of corticotropin-releasing factor receptor 2 in the hippocampus. Together, these results indicate that cocaine-experienced sires produce male progeny that have increased baseline anxiety, which is unaltered by subsequent cocaine exposure.

Adolescent experience affects postnatal ultrasonic vocalizations and gene expression in future offspring.

The present study measured postnatal ultrasonic vocalization (USV) and gene expression to examine potential changes in communication and/or attachment in the offspring of mothers exposed to morphine during adolescence. Offspring of morphine-exposed (Mor-F1), saline-exposed (Sal-F1), or non-handled control (Con-F1) female Sprague-Dawley rats were tested for separation-induced distress calls and maternal potentiation of distress calls during early postnatal development. We also examined relative expression of dopamine D2 receptor and mu opioid receptor (oprm1) mRNA in the nucleus accumbens and hypothalamus in these offspring, as their activity has been implicated in the regulation of postnatal USV in response to maternal separation. The findings indicate that adolescent experiences of future mothers, including their 10 daily saline or morphine injections, can result in significant region-specific differences in gene expression. In addition, these experiences resulted in fewer numbers of separation-induced distress calls produced by offspring. In contrast, augmented maternal potentiation was only observed in Mor-F1 offspring. © 2016 Wiley Periodicals, Inc. Dev Psychobiol 58:714-723, 2016.

Raphe GABAergic neurons mediate the acquisition of avoidance after social defeat.

Serotonin (5-HT) modulates neural responses to socioaffective cues and can bias approach or avoidance behavioral decisions, yet the cellular mechanisms underlying its contribution to the regulation of social experiences remain poorly understood. We hypothesized that GABAergic neurons in the dorsal raphe nucleus (DRN) may participate in socioaffective regulation by controlling serotonergic tone during social interaction. We tested this hypothesis using whole-cell recording techniques in genetically identified DRN GABA and 5-HT neurons in mice exposed to social defeat, a model that induces long-lasting avoidance behaviors in a subset of mice responsive to serotonergic antidepressants. Our results revealed that social defeat engaged DRN GABA neurons and drove GABAergic sensitization that strengthened inhibition of 5-HT neurons in mice that were susceptible, but not resilient to social defeat. Furthermore, optogenetic silencing of DRN GABA neurons disinhibited neighboring 5-HT neurons and prevented the acquisition of social avoidance in mice exposed to a social threat, but did not affect a previously acquired avoidance phenotype. We provide the first characterization of GABA neurons in the DRN that monosynaptically inhibit 5-HT neurons and reveal their key role in neuroplastic processes underlying the development of social avoidance.

Deep brain stimulation of the nucleus accumbens shell attenuates cocaine reinstatement through local and antidromic activation.

Accumbal deep brain stimulation (DBS) is a promising therapeutic modality for the treatment of addiction. Here, we demonstrate that DBS in the nucleus accumbens shell, but not the core, attenuates cocaine priming-induced reinstatement of drug seeking, an animal model of relapse, in male Sprague Dawley rats. Next, we compared DBS of the shell with pharmacological inactivation. Results indicated that inactivation using reagents that influenced (lidocaine) or spared (GABA receptor agonists) fibers of passage blocked cocaine reinstatement when administered into the core but not the shell. It seems unlikely, therefore, that intrashell DBS influences cocaine reinstatement by inactivating this nucleus or the fibers coursing through it. To examine potential circuit-wide changes, c-Fos immunohistochemistry was used to examine neuronal activation following DBS of the nucleus accumbens shell. Intrashell DBS increased c-Fos induction at the site of stimulation as well as in the infralimbic cortex, but had no effect on the dorsal striatum, prelimbic cortex, or ventral pallidum. Recent evidence indicates that accumbens DBS antidromically stimulates axon terminals, which ultimately activates GABAergic interneurons in cortical areas that send afferents to the shell. To test this hypothesis, GABA receptor agonists (baclofen/muscimol) were microinjected into the anterior cingulate, and prelimbic or infralimbic cortices before cocaine reinstatement. Pharmacological inactivation of all three medial prefrontal cortical subregions attenuated the reinstatement of cocaine seeking. These results are consistent with DBS of the accumbens shell attenuating cocaine reinstatement via local activation and/or activation of GABAergic interneurons in the medial prefrontal cortex via antidromic stimulation of cortico-accumbal afferents.

Antiretroviral drugs induce oxidative stress and neuronal damage in the central nervous system.

HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.

Next generation effects of female adolescent morphine exposure: sex-specific alterations in response to acute morphine emerge before puberty.

Prescription opiate use by adolescent girls has increased significantly in the past decade. Preclinical studies using rats report alterations in morphine sensitivity in the adult offspring of adolescent morphine-exposed females (MOR-F1) when compared with the offspring of adolescent saline-exposed females (SAL-F1). To begin to elucidate the development of these next generation modifications, the present study examined the effects of acute morphine administration on sedation and corticosterone secretion in prepubescent SAL-F1 and MOR-F1 male and female rats. In addition, alterations in proopiomelanocortin (POMC) gene expression in the arcuate nucleus, as well as in tyrosine hydroxylase (TH) and µ-opioid receptor (OPRM1) gene expressions in the ventral tegmental area, were analyzed using quantitative PCR, to determine whether differential regulation of these genes was correlated with the observed behavioral and/or endocrine effects. Increased morphine-induced sedation, coupled with an attenuation of morphine-induced corticosterone secretion, was observed in MOR-F1 males. Significant alterations in both POMC and OPRM1 gene expressions were also observed in MOR-F1 males, with no change in TH mRNA expression. Overall, these data suggest that the transgenerational effects of adolescent morphine exposure can be discerned before pubertal development and are more pronounced in males, and suggest dysregulation of the hypothalamic-pituitary-adrenal axis in the offspring of adolescent morphine-exposed females.

Neuroplasticity-related genes correlate with individual differences in distinct phases of oxycodone self-administration in male rats.

Opioid use disorder (OUD) is a chronic condition associated with long-lasting molecular and behavioral changes. Animals with prolonged access to opioids develop behaviors similar to human OUD. Identifying associated molecular changes can provide insight to underpinnings that lead to or maintain OUD. In pilot studies, we identified several miRNA targets that are altered by the administration of oxycodone. We selected mir182 for follow up as it was recently shown to be dysregulated in plasma of men administered oxycodone. In addition, mir182 is increased in reward-related brain regions of male rats following exposure to various addictive substances. The present study utilizes a long-access oxycodone self-administration paradigm to examine changes in mir182 and its mRNA targets associated with neuroplasticity, which may be involved in the maintenance of OUD-like phenotype in rats. Male rats were trained to self-administer oxycodone (0.1 mg/kg/infusion, i. v.) for 6 h daily sessions for 12 days. Each animal had a yoked saline control that received matched saline infusions. Animals were then tested on a progressive ratio schedule to measure motivation to obtain a single infusion of oxycodone. Drug seeking was measured following 28 days of forced abstinence using a 90-min cued/test. RTqPCR was utilized to measure mir182 and mRNA targets related to neuroplasticity (wnt3, plppr4, pou3f3, tle4, cacna2d, and bdnf) from the nucleus accumbens. Data revealed that animals responded on a continuum for oxycodone. When divided into two groups termed high- and low responders, animals diverged during self-administration acquisition and maintained differences in behavior and gene expression throughout the study. mir182 was upregulated in the nucleus accumbens of both high and low responders and negatively correlated with tle4, which showed a strong negative correlation with reinstatement behavior. mRNA target levels were correlated with behaviors associated with increased severity of OUD behavior in male rats.

Preconception opioids interact with mouse strain to alter morphine withdrawal in the next generation.

RATIONALE: Transgenerational effects of preconception morphine exposure in female rats have been reported which suggest that epigenetic modifications triggered by female opioid exposure, even when that exposure ends several weeks prior to pregnancy, has significant ramifications for their future offspring. OBJECTIVE: The current study compares two mouse strains with well-established genetic variation in their response to mu opioid receptor agonists, C57BL/6J (BL6) and 129S1/svlmJ (129) to determine whether genetic background modifies the impact of preconception opioid exposure. METHODS: Adolescent females from both strains were injected daily with morphine for a total of 10 days using an increasing dosing regimen with controls receiving saline. Several weeks after their final injection, aged-matched BL6 and 129 morphine (Mor-F0) or saline (Sal-F0) females were mated with drug naïve males to generate Mor-F1 and Sal-F1 offspring, respectively. As adults, F1 mice were made morphine dependent using thrice daily morphine injections for 4 days. On day 5, mice were administered either saline or morphine followed 3 h later by naloxone. Behavioral and physiological signs of withdrawal were then measured. RESULTS: Regardless of strain or sex, morphine-dependent Mor-F1 mice had significantly lower levels of withdrawal-induced corticosterone but significantly higher glucose levels when compared to Sal-F1 controls. In contrast, both strain- and preconception opioid exposure effects on physical signs of morphine dependence were observed.

Vascular endothelial growth factor is upregulated by L-dopa in the parkinsonian brain: implications for the development of dyskinesia.

Angiogenesis and increased permeability of the blood-brain barrier have been reported to occur in animal models of Parkinson's disease and l-dopa-induced dyskinesia, but the significance of these phenomena has remained unclear. Using a validated rat model of l-dopa-induced dyskinesia, this study demonstrates that chronic treatment with l-dopa dose dependently induces the expression of vascular endothelial growth factor in the basal ganglia nuclei. Vascular endothelial growth factor was abundantly expressed in astrocytes and astrocytic processes in the proximity of blood vessels. When co-administered with l-dopa, a small molecule inhibitor of vascular endothelial growth factor signalling significantly attenuated the development of dyskinesia and completely blocked the angiogenic response and associated increase in blood-brain barrier permeability induced by the treatment. The occurrence of angiogenesis and vascular endothelial growth factor upregulation was verified in post-mortem basal ganglia tissue from patients with Parkinson's disease with a history of dyskinesia, who exhibited increased microvascular density, microvascular nestin expression and an upregulation of vascular endothelial growth factor messenger ribonucleic acid. These congruent findings in the rat model and human patients indicate that vascular endothelial growth factor is implicated in the pathophysiology of l-dopa-induced dyskinesia and emphasize an involvement of the microvascular compartment in the adverse effects of l-dopa pharmacotherapy in Parkinson's disease.

Intergenerational effects of preconception opioids on glucose homeostasis and hepatic transcription in adult male rats.

Adolescence represents a period of significant neurodevelopment during which adverse experiences can lead to prolonged effects on disease vulnerability, including effects that can impact future offspring. Adolescence is a common period for the initiation of drug use, including the use of opioids. Beyond effects on central reward, opioids also impact glucose metabolism, which can impact the risk of diabetes. Moreover, recent animal models suggest that the effects of adolescent opioids can effect glucose metabolism in future offspring. Indeed, we demonstrated that the adult male offspring of females exposed to morphine for 10 days during adolescence (referred to as MORF1 males) are predisposed to the adverse effects of an obesogenic diet. As adults, MORF1 males fed a high fat moderate sucrose diet (FSD) for just 6 weeks had increased fasting glucose and insulin levels when compared to age-matched offspring of females exposed to saline during adolescence (SALF1 males). Clinically, a similar profile of impaired fasting glucose has been associated with hepatic insulin resistance and an increased risk of non-alcoholic fatty liver disease. Thus, in the current study, we used RNA sequencing to determine whether adult MORF1 males demonstrate significant alterations in the hepatic transcriptome suggestive of alterations in metabolism. Age-matched SALF1 and MORF1 males were fed either FSD or control diet (CD) for 8 weeks. Similar to our previous observations, FSD-maintained MORF1 males gained more weight and displayed both fasting hyperglycemia and hyperinsulinemia when compared to FSD-maintained SALF1 males, with no significant effect on glucagon. No differences in bodyweight or fasting-induce glucose were observed in control diet (CD)-maintained F1 males, although there was a trend for CD MORF1 males to display elevated levels of fasting insulin. Unexpectedly, transcriptional analyses revealed profound differences in the hepatic transcriptome of CD-maintained MORF1 and SALF1 (1686 differentially expressed genes) with no significant differences between FSD-maintained MORF1 and SALF1 males. As changes in the hepatic transcriptome were not revealed under 8 weeks FSD conditions, we extended the feeding paradigm and conducted a glucose tolerance test to determine whether impaired fasting glucose observed in FSD MORF1 males was due to peripheral insulin resistance. Impaired glucose tolerance was observed in both CD and FSD MORF1 males, and to a more limited extent in FSD SALF1 males. These findings implicate intergenerational effects of adolescent morphine exposure on the risk of developing insulin resistance and associated comorbidities, even in the absence of an obesogenic diet.

HPA axis dysfunction during morphine withdrawal in offspring of female rats exposed to opioids preconception.

Opioid use and abuse remain a significant public health problem, particularly in the United States. Indeed, it is estimated that up to 10% of youths (age 12-18) have taken opioids illicitly. A growing body of evidence suggests that this level of widespread opioid exposure can have effects that extend to subsequent generations. Utilizing a well-established rodent model of preconception adolescent opioid exposure in females, we found decreased opioid self-administration coupled with increased cocaine self-administration in adult offspring. This bidirectional effect may be related to negative affect associated with opioid withdrawal, including enhanced stress reactivity. In this study, we tested the hypothesis that the adult offspring of females exposed to morphine during adolescence will demonstrate increased signs of opioid withdrawal when compared to offspring of saline controls. Females were administered increasing doses of morphine (5-25 mg/kg s.c.) or saline (1 ml/kg) from postnatal day 30 (PND30)-PND39. They were then maintained drug free for a minimum of 4 weeks and mated with drug-naïve males on or after PND70. As adults, their male and female offspring (referred to as Mor-F1 or Sal-F1) were administered morphine (10 mg/kg s.c.) twice a day for 5 days. They were then tested for spontaneous withdrawal behaviors for the next 4 days (∼PND70). Levels of corticotropin releasing hormone (Crh) and urocortin 3 (Ucn3) were examined in the amygdala at 48 h and 96 h of withdrawal. Circulating corticosterone was measured at 48 h. Results indicate that Mor-F1 males are heavier than Sal-F1 males with no baseline differences in females. However, Mor-F1 females did not gain weight at the same rate as Sal-F1 females during withdrawal. While there were no differences in somatic withdrawal signs, gene expression data revealed a sex-specific and time-dependent effect on Crh as well as increased Ucn3 and corticosterone in females at 48hrs withdrawal. Overall, these data point to differences in withdrawal and stress reactivity in Mor-F1 animals that may contribute to observed differences in addiction-like behaviors.

Ventral tegmental afferents in stress-induced reinstatement: the role of cAMP response element-binding protein.

The transcription factor cAMP response element-binding protein (CREB) is required for stress- but not drug-induced reinstatement of cocaine conditioned place preference. To reveal the neural circuitry associated with this CREB dependence, we injected a retrograde tracer into the ventral tegmental area (VTA) and identified afferents that were activated after stress or cocaine exposure in both naive and cocaine-conditioned mice. Neuronal activation, as assessed by Fos expression, was greatly reduced in the dorsal and ventral bed nucleus of the stria terminalis (BNST), lateral septum, and nucleus accumbens shell in mice lacking CREB (CREBαΔ mice) after a 6 min swim stress but not after cocaine exposure (20 mg/kg). Additionally, activation of VTA afferent neurons in the ventral BNST and the infralimbic cortex in CREBαΔ mice was blunted in response to stress. This pattern of neuronal activation persisted in mice that were conditioned to a cocaine place preference procedure before stress exposure. Furthermore, lidocaine inactivation (0.4 µl, 4%) studies demonstrated the necessity of BNST activation for swim-stress-induced reinstatement of cocaine-conditioned reward. Together, the present studies demonstrate that CREB is required for the activation of a unique circuit that converges on the dopamine reward pathway to elicit reinstatement of drug reward and points to the BNST as a key intersection between stress and reward circuits.

Cocaine-induced chromatin remodeling increases brain-derived neurotrophic factor transcription in the rat medial prefrontal cortex, which alters the reinforcing efficacy of cocaine.

Cocaine self-administration alters patterns of gene expression in the brain that may underlie cocaine-induced neuronal plasticity. In the present study, male Sprague Dawley rats were allowed to self-administer cocaine (0.25 mg/infusion) 2 h/d for 14 d, followed by 7 d of forced abstinence. Compared with yoked saline control rats, cocaine self-administration resulted in increased brain-derived neurotrophic factor (BDNF) protein levels in the rat medial prefrontal cortex (mPFC). To examine the functional relevance of this finding, cocaine self-administration maintained under a progressive ratio schedule of reinforcement was assessed after short hairpin RNA-induced suppression of BDNF expression in the mPFC. Decreased BDNF expression in the mPFC increased the cocaine self-administration breakpoint. Next, the effect of cocaine self-administration on specific BDNF exons was assessed; results revealed selectively increased BDNF exon IV-containing transcripts in the mPFC. Moreover, there were significant cocaine-induced increases in acetylated histone H3 (AcH3) and phospho-cAMP response element binding protein (pCREB) association with BDNF promoter IV. In contrast, there was decreased methyl-CpG-binding protein 2 (MeCP2) association with BDNF promoter IV in the mPFC of rats that previously self-administered cocaine. Together, these results indicate that cocaine-induced increases in BDNF promoter IV transcript in the mPFC are driven by increased binding of AcH3 and pCREB as well as decreased MeCP2 binding at this BDNF promoter. Collectively, these results indicate that cocaine self-administration remodels chromatin in the mPFC, resulting in increased expression of BDNF, which appears to represent a compensatory neuroadaptation that reduces the reinforcing efficacy of cocaine.

Consequences of Parental Opioid Exposure on Neurophysiology, Behavior, and Health in the Next Generations.

Substance abuse and the ongoing opioid epidemic represents a large societal burden. This review will consider the long-term impact of opioid exposure on future generations. Prenatal, perinatal, and preconception exposure are reviewed with discussion of both maternal and paternal influences. Opioid exposure can have long-lasting effects on reproductive function, gametogenesis, and germline epigenetic programming, which can influence embryogenesis and alter the developmental trajectory of progeny. The potential mechanisms by which preconception maternal and paternal opioid exposure produce deleterious consequences on the health, behavior, and physiology of offspring that have been identified by clinical and animal studies will be discussed. The timing, nature, dosing, and duration of prenatal opioid exposure combined with other important environmental considerations influence the extent to which these manipulations affect parents and their progeny. Epigenetic inheritance refers to the transmission of environmental insults across generations via mechanisms independent of the DNA sequence. This topic will be further explored in the context of prenatal, perinatal, and preconception opioid exposure for both the maternal and paternal lineage.

Transgenerational effects on anxiety-like behavior following adolescent morphine exposure in female rats.

Global opioid use and misuse remains high, despite efforts to decrease rates of prescribing and diversion. Chronic exposure to opioids, particularly during critical periods of development, can lead to long-lasting effects, including effects that may extend to future generations. Using a rodent model, we have demonstrated significant transgenerational effects of female adolescent morphine exposure, despite the absence of in utero drug exposure. While these effects have been observed in both sexes, effects on anxiety-like behavior were only observed in F1 females. The current study was designed to examine both inter- and transgenerational effects of adolescent morphine exposure on anxiety-like behavior. Female Sprague Dawley rats were administered increasing doses of morphine (5-25 mg/kg s.c.) or saline for 10 days during adolescence (PND30-39). Adult diestrous female offspring (MORF1 or SALF1) and grand offspring (F2) were tested for anxiety-like behavior using the elevated plus maze (EPM). F1 females cross-fostered to donor mothers were also examined. The results show that MORF1 and MORF2 females spend significantly more time on the open arms of the EPM compared to SALF1 controls, an effect that persisted in cross-fostered females. Additional studies demonstrate that this effect is estrous cycle dependent, as decreased anxiety-like behavior was observed in diestrus, while increased anxiety-like behavior was observed in estrus. These behavioral effects were not associated with any differences in circulating corticosterone either at baseline or following EPM testing. Thus, female adolescent morphine exposure alters the regulation of anxiety-like behavior in an estrous-dependent manner and this effect persists in the F2 generation.

Sexually dimorphic neuroimmune response to chronic opioid treatment and withdrawal.

Opioid use disorder is a leading cause of morbidity and mortality in the United States. Increasing pre-clinical and clinical evidence demonstrates sex differences in opioid use and dependence. However, the underlying molecular mechanisms contributing to these effects, including neuroinflammation, are still obscure. Therefore, in this study, we investigated the effect of oxycodone exposure and withdrawal on sex- and region-specific neuroimmune response. Real-time PCR and multiplex cytokine array analysis demonstrated elevated neuroinflammation with increased pro-inflammatory cytokine levels, and aberrant oligodendroglial response in reward neurocircuitry, following withdrawal from chronic oxycodone treatment. Chronic oxycodone and withdrawal treated male mice had lower mRNA expression of TMEM119 along with elevated protein levels of pro-inflammatory cytokines/chemokines and growth factors (IL-1ß, IL-2, IL-7, IL-9, IL-12, IL-15, IL17, M-CSF, VEGF) in the prefrontal cortex (PFC) as compared to their female counterparts. In contrast, reduced levels of pro-inflammatory cytokines/chemokines (IL-1ß, IL-6, IL-9, IL-12, CCL11) was observed in the nucleus accumbens (NAc) of oxycodone and withdrawal-treated males as compared to female mice. No treatment specific effects were observed on the mRNA expression of putative microglial activation markers (Iba1, CD68), but an overall sex specific decrease in the mRNA expression of Iba1 and CD68 was found in the PFC and NAc of male mice as compared to females. Moreover, a sex and region-specific increase in the mRNA levels of oligodendrocyte lineage markers (NG2, Sox10) was also observed in oxycodone and withdrawal treated animals. These findings may open a new avenue for the development of sex-specific precision therapeutics for opioid dependence by targeting region-specific neuroimmune signaling.

Soft Capsule Magnetic Millirobots for Region-Specific Drug Delivery in the Central Nervous System.

Small soft robotic systems are being explored for myriad applications in medicine. Specifically, magnetically actuated microrobots capable of remote manipulation hold significant potential for the targeted delivery of therapeutics and biologicals. Much of previous efforts on microrobotics have been dedicated to locomotion in aqueous environments and hard surfaces. However, our human bodies are made of dense biological tissues, requiring researchers to develop new microrobotics that can locomote atop tissue surfaces. Tumbling microrobots are a sub-category of these devices capable of walking on surfaces guided by rotating magnetic fields. Using microrobots to deliver payloads to specific regions of sensitive tissues is a primary goal of medical microrobots. Central nervous system (CNS) tissues are a prime candidate given their delicate structure and highly region-specific function. Here we demonstrate surface walking of soft alginate capsules capable of moving on top of a rat cortex and mouse spinal cord ex vivo, demonstrating multi-location small molecule delivery to up to six different locations on each type of tissue with high spatial specificity. The softness of alginate gel prevents injuries that may arise from friction with CNS tissues during millirobot locomotion. Development of this technology may be useful in clinical and preclinical applications such as drug delivery, neural stimulation, and diagnostic imaging.