Decreased myelin-related gene expression in the nucleus accumbens during spontaneous neonatal opioid withdrawal in the absence of long-term behavioral effects in adult outbred CFW mice
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ABSTRACT: Prenatal opioid exposure is a major health concern in the United States, with the incidence of neonatal opioid withdrawal syndrome (NOWS) escalating in recent years. NOWS is revealed upon cessation of in utero opioid exposure and is characterized by increased irritability, disrupted sleep patterns, high-pitched crying, and erratic feeding. The main pharmacological treatment strategy for alleviating symptoms is opioid maintenance therapy. The neural mechanisms mediating NOWS and the long-term neurobehavioral effects are poorly understood. We used a third trimester-approximate model in which neonatal outbred pups (Cartworth Farms White; CFW) were administered once-daily morphine (15 mg/kg, s.c.) from postnatal day (P) day 1 through P14 and were then assessed for behavioral and transcriptomic adaptations within the nucleus accumbens (NAc) on P15. We also investigated the long-term effects of perinatal morphine exposure on adult learning and reward sensitivity. We observed significant weight deficits, spontaneous thermal hyperalgesia, and altered ultrasonic vocalization (USV) profiles following repeated morphine and during spontaneous withdrawal. Transcriptome analysis of NAc from opioid-withdrawn P15 neonates via bulk mRNA sequencing identified an enrichment profile consistent with downregulation of myelin-associated transcripts. Despite the neonatal behavioral and molecular effects, there were no significant long-term effects of perinatal morphine exposure on adult spatial memory function in the Barnes Maze, emotional learning in fear conditioning, or in baseline or methamphetamine-potentiated reward sensitivity as measured via intracranial self-stimulation. Thus, the once daily third trimester-approximate exposure regimen, while inducing NOWS model traits and significant transcriptomic effects in neonates, had no significant long-term effects on adult behaviors.
Project description:The United States is currently facing a severe opioid epidemic, therefore addressing how opioids induce rewarding behaviors could be key to a solution for this medical and societal crisis. Recently, the endogenous cannabinoid system has emerged as a hot topic in the study of opioid reward but relatively little is known about how chronic opioid exposure may affect this system. In the present study, we investigated how chronic morphine may modulate the endogenous cannabinoid system in the ventral tegmental area (VTA), a critical region in the mesolimbic reward circuitry. Our studies found that the VTA expresses 32 different proteins or genes related to the endogenous cannabinoid system; 3 of these proteins or genes were significantly affected after chronic morphine exposure. We also investigated the effects of acute and chronic morphine treatment on the production of the primary endocannabinoids, 2-Arachidonoylglycerol (2-AG) and anandamide (AEA), and identified that acute, but not chronic, morphine treatment significantly reduced AEA production in the VTA; 2-AG levels were unchanged in either condition. Lastly, our studies exhibited a systemic enhancement of 2-AG tone via inhibition of monoacylglycerol lipase (MAGL)-mediated degradation and the pharmacological activation of cannabinoid receptor 2 (CB2R) significantly suppressed chronic morphine-induced conditioned place preference. Taken together, our studies offer a broad picture of chronic morphine-induced alterations of the VTA endogenous cannabinoid system, provide several uncharacterized targets that could be used to develop novel therapies, and identify how manipulation of the endocannabinoid system can mitigate opioid reward to directly address the ongoing opioid epidemic.
Project description:Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious side effects such as addiction, immunosuppression and gastrointestinal symptoms limit long term use. In the current study using a chronic morphine-murine model a longitudinal approach was undertaken to investigate the role of morphine modulation of gut microbiome as a mechanism contributing to the negative consequences associated with opioids use. The results revealed a significant shift in the gut microbiome and metabolome within 24 hours following morphine treatment when compared to placebo. Morphine induced gut microbial dysbiosis exhibited distinct characteristic signatures profiles including significant increase in communities associated with pathogenic function, decrease in communities associated with stress tolerance. Collectively, these results reveal opioids-induced distinct alteration of gut microbiome, may contribute to opioids-induced pathogenesis. Therapeutics directed at these targets may prolong the efficacy long term opioid use with fewer side effects.
Project description:Opioid use during pregnancy can lead to negative infant health outcomes, including neonatal opioid withdrawal syndrome (NOWS). NOWS comprises gastrointestinal, autonomic nervous system, and neurological dysfunction that manifest during spontaneous withdrawal. Current treatments involve non-pharmacological and pharmacological interventions, however, there is no one standardized approach, in part because of variability in NOWS severity. To effectively model NOWS traits in mice, we used a third trimester-approximate opioid exposure paradigm, where neonatal inbred FVB/NJ and outbred Carworth Farms White (CFW) pups were injected twice-daily with morphine (10-15 mg/kg, s.c.) or saline (0.9%, 20 ul/g, s.c.) from postnatal day (P) one to P14. We observed reduced body weight gain, hypothermia, thermal hyperalgesia, and increased ultrasonic vocalizations (USVs). Neonatal USVs are emitted exclusively in isolation to communicate distress and thus serve as a model behavior for affective states. On P14, we observed altered USV syllable profiles during spontaneous morphine withdrawal, including an increase in Complex 3 syllables in FVB/NJ females (but not males) and in CFW mice of both sexes. Brainstem transcriptomics revealed an upregulation of the kappa opioid receptor (Oprk1), whose activation has previously been shown to contribute to withdrawal-induced dysphoria. Treatment with the kappa opioid receptor (KOR) antagonist, nor-BNI (30 mg/kg, s.c.), significantly reduced USV emission in FVB/NJ females, but not FVB/NJ males during spontaneous morphine withdrawal. Furthermore, treatment with the KOR agonist, U50,488h (0.625 mg/kg, s.c.), was sufficient to increase USV emission on P10 (both sexes) and on P14 (females only) in FVB/NJ mice. Together, these results indicate a female-specific recruitment of the dynorphin/KOR system in neonatal opioid withdrawal symptom severity.
Project description:Morphine addiction causes major medical and social problems worldwide. Chronic morphine exposure results in the development of behavioral sensitization, accompanied by the disruption of brain homeostasis. As a key brain reward region, nucleus accumbens (NAc) plays a central role in brain reward mechanisms. However, the contribution of morphine exposure to NAc is poorly understood. Here we indicated that chronic morphine exposure induced neuroinflammation, abnormal neuronal physiology, and dysregulation of glycolytic metabolism in NAc. In summary, our findings illustrate the effects of morphine in NAc, and provide a new insight for development of future morphine addiction therapeutics.
Project description:The Opioid Use Disorder epidemic led to an increase in cases of Nenonatal Opioid Withdrawal Syndrome (NOWS) in infants born to opioid-dependent mothers. Hallmark features include weight loss, irritability, inconsolability, insomnia, and increased pain sensitivity. The neurobiological basis of NOWS is largely unknown. Improved mouse models will facilitate mechanistic and treatment discovery. We treated neonatal outbred Cartworth Farms White (CFW) mice (Swiss Webster) with morphine sulfate (15 mg/kg, s.c.) twice daily on postnatal day (P)1 through P14, the approximate third trimester-equivalent of human gestation. Weight loss was monitored and behavioral symptoms were measured on P7 and P14 at 16 h post-morphine. Brainstem containing pons and medulla was collected on P14 and processed for transcriptome analysis via mRNA sequencing (RNA-seq). Morphine induced weight loss from P2 to P14 that remained at P21 and P50. Repeated morphine also induced a delayed self-righting latency at P4 and a persistent, female-specific delay at P14. Morphine-treated females also showed an earlier increase in ultrasonic vocalizations (USVs) on P7. Both morphine-treated sexes showed a large increase in USVs on P14. Furthermore, thermal nociception via hot plate and tail withdrawal assays indicated that mice exhibited thermal hyperalgesia on P7 and P14, with females showing greater hyperalgesia (tail withdrawal) on P7. Morphine-treated mice also exhibited anxiety-like behavior at P21 (open field). Finally, brainstem transcriptome analysis identified a canonical gene set relevant to opioid signaling in males and a distinct gene set in females that was enriched for ribosomal proteins, mitochondrial function and neurodegenerative disorders. Sex-specific transcriptomic neuroadaptations implicate sex-specific treatments.
Project description:All drugs of abuse induce long-lasting changes in synaptic transmission and neural circuit function that underlie substance use disorders. Another recently appreciated mechanism of neural circuit plasticity is mediated through activity-regulated changes in myelin that can tune circuit function and influence cognitive behavior1. Here, we explored the role of myelin plasticity in dopaminergic circuity and reward learning. We demonstrate that dopaminergic neuronal activity-regulated myelin plasticity is a key modulator of dopaminergic circuit function and opioid reward. Oligodendroglial lineage cells respond to dopaminergic neuronal activity evoked by either optogenetic stimulation of dopaminergic neurons, optogenetic inhibition of GABAergic neurons, or administration of morphine or cocaine. These oligodendroglial changes are evident selectively within the ventral tegmental area (VTA), but not along the axonal projections in the medial forebrain bundle nor within the target nucleus accumbens (NAc). Genetic blockade of oligodendrogenesis dampens dopamine release dynamics in nucleus accumbens and impairs behavioral conditioning to morphine. Taken together, these findings underscore a critical role for oligodendrogenesis in reward learning and identify dopaminergic neuronal activity-regulated myelin plasticity as an important circuit modification that is required for opioid reward.
Project description:Addictive drugs including opioids activate signal transduction pathways that regulate gene expression in the brain. However, changes in CNS gene expression following morphine exposure are poorly understood. We studied the effect of short- and long-term morphine treatment on gene expression in the hypothalamus and pituitary using genome-wide DNA microarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses. In the hypothalamus, we found that short-term morphine administration up-regulated (at least 2-fold) 39 genes and down-regulated six genes. Long-term morphine administration up-regulated 35 genes and down-regulated 51 hypothalamic genes. In the pituitary, we found that short-term morphine administration up-regulated (at least 2-fold) 110 genes and down-regulated 29 genes. Long-term morphine administration up-regulated 85 genes and down-regulated 37 pituitary genes. Strikingly, microarray analysis uncovered several genes involved in food intake (neuropeptide Y, agouti-related protein, and cocaine and amphetamine-regulated transcript) whose expression was strongly altered by morphine exposure in either the hypothalamus or pituitary. Subsequent RT-PCR analysis confirmed similar gene regulation of noteworthy genes in these regions. Finally, we found functional correlation between morphine-induced alterations in food intake and regulations of genes involved in this process. Changes in genes related to food intake may uncover new pathways related to some of the physiological effects of opioids. Keywords: Comparative treatment versus placebo 8 samples analyzed: 4 from hypothalamus (2 biological replicates and 2 dye swaps) and 4 from pituitary (2 biological replicates and 2 dye swaps) 8 samples analyzed: 4 from hypothalamus short term treatment (2 biological replicates and 2 dye swaps) and 4 hypothalamus long term treatment (2 biological replicates and 2 dye swaps)
Project description:Addictive drugs including opioids activate signal transduction pathways that regulate gene expression in the brain. However, changes in CNS gene expression following morphine exposure are poorly understood. We studied the effect of short- and long-term morphine treatment on gene expression in the hypothalamus and pituitary using genome-wide DNA microarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses. In the hypothalamus, we found that short-term morphine administration up-regulated (at least 2-fold) 39 genes and down-regulated six genes. Long-term morphine administration up-regulated 35 genes and down-regulated 51 hypothalamic genes. In the pituitary, we found that short-term morphine administration up-regulated (at least 2-fold) 110 genes and down-regulated 29 genes. Long-term morphine administration up-regulated 85 genes and down-regulated 37 pituitary genes. Strikingly, microarray analysis uncovered several genes involved in food intake (neuropeptide Y, agouti-related protein, and cocaine and amphetamine-regulated transcript) whose expression was strongly altered by morphine exposure in either the hypothalamus or pituitary. Subsequent RT-PCR analysis confirmed similar gene regulation of noteworthy genes in these regions. Finally, we found functional correlation between morphine-induced alterations in food intake and regulations of genes involved in this process. Changes in genes related to food intake may uncover new pathways related to some of the physiological effects of opioids. Keywords: Comparative treatment versus placebo
Project description:Substance use disorders (SUDs) induce widespread molecular dysregulation in nucleus accumbens (NAc), a brain region pivotal for coordinating motivation and reward, which is linked to neural and behavioral disturbances promoting addiction. Despite the overlapping symptomatology of SUDs, different drug classes exert partly unique influences on neural circuits, cell types, physiology, and gene expression. To better understand common and divergent molecular mechanisms governing SUD pathology, we characterized the cell-type-specific restructuring of the NAc transcriptional landscape after psychostimulant or opioid exposure. We combined fluorescence-activated nuclei sorting and RNA sequencing to profile NAc D1 and D2 medium spiny neurons (MSNs) across morphine exposure paradigms, including initial exposure, prolonged withdrawal after repeated exposure, and re-exposure post-withdrawal. Our analyses reveal that D1 MSNs display many convergent transcriptional responses, whereas D2 MSNs manifest highly divergent responses, with morphine causing far more adaptations in this cell type.
Project description:Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR gene expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine at 2 or 5 mg/kg ip bid (MS5) and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and microRNA expression profiling was performed on hippocampal tissues at P9. Overall, MS2 and MS5 morphine treatment altered expression of a total of 150 mRNAs (>1.5 fold change, P<0.05; 36 up, 114 down), and MS5 affected 63 mRNAs. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. MS5 decreased expression of miR-204-5p, miR-455-3p, miR-448-3p, and miR-574-3p.Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed microRNAs These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR gene expression in stressed neonatal mice. If permanent, morphine–mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.