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| Status |
Public on Nov 27, 2019 |
| Title |
Sex differences in behavioral and brainstem transcriptomic neuroadaptations following neonatal opioid exposure in outbred mice |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
RNA isolation and RNA-seq. RNA was isolated using previously described methods involving Trizol (Qiagen), ethanol precipitation, filtering columns for clean-up (Qiagen), and elution with sterile, double deionized water(Yazdani et al. 2015). Samples were diluted to 50 ng/ul. RNA library preparation (poly-A selection) and RNA-seq was conducted at the University of Chicago Genomics Facility on an Illumina NovaSEQ6000 using a NovaSEQ SP-100 bp flowcell/reagent cassette. The 12 multiplexed, pooled samples [from six saline (SAL)-treated and six morphine (MOR)-treated mice] were sequenced (100 bp single-end) on a single lane of the two-lane flowcell. Reads were trimmed for quality using Trimmomatic (Bolger et al. 2014). Trimmed reads were then aligned to the mm10 mouse reference genome (Ensembl) to generate BAM files for alignment using STAR (Dobin et al. 2013). The featureCounts read summarization program was used to count reads mapping to the “exon” feature in a GTF file obtained from Ensembl. Differential gene expression analysis of normalized read counts from Rsubread (Liao et al. 2019) was performed in R using edgeR (Robinson et al. 2010). In the sex-combined analysis, we included both “Sex” and “Dam” as covariates account for variance between sexes and across families. In the female-only and male-only analyses, we included “Dam” as a covariate to account for variance across families.
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| Web link |
https://pubmed.ncbi.nlm.nih.gov/34479978/
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| Contributor(s) |
Bryant C, Borrelli K, Ruan Q |
| Citation(s) |
34479978 |
| Submission date |
Nov 26, 2019 |
| Last update date |
Sep 17, 2021 |
| Contact name |
Camron D Bryant |
| E-mail(s) |
camron@bu.edu
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| Phone |
6173589577
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| Organization name |
Boston University School of Medicine
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| Department |
Pharmacology
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| Lab |
Laboratory of Addiction Genetics
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| Street address |
72 East Concord St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (12)
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| Relations |
| BioProject |
PRJNA591918 |
| SRA |
SRP233366 |
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