Sex differences in behavioral and brainstem transcriptomic neuroadaptations following neonatal opioid exposure in outbred mice

Kristyn N. Borrelli, Emily J. Yao, Will W. Yen, Qiu T. Ruan, Melanie M. Chen, Julia C. Kelliher, Carly R. Langan, Julia L. Scotellaro, Richard K. Babbs, Jacob C. Beierle, Ryan W. Logan, William Evan Johnson, Elisha M. Wachman, Alberto Cruz-Martín, Camron D. Bryant

Posted on: 28 April 2021 , updated on: 4 November 2021

Preprint posted on 4 April 2021

Article now published in eneuro at

Neonatal Opioid Withdrawal Syndrome: sex differences in outbred mice.

Selected by Nándor Lipták


Opioid addiction is increasing in prevalence, particularly in the United States (Kertesz & Gordon 2019). As a result, there has been an increase in Neonatal Opioid Withdrawal Syndrome (NOWS) in infants born to women who were dependent on opioids. (Armbruster et al. 2021). The main symptoms of NOWS are reduced weight gain, severe irritability, sleep fragmentation, restlessness, hypertension and hyperalgesia. (Sutter et al. 2014). Several mouse models were developed for NOWS, most of which were inbred mouse strains. Mice are good models for NOWS, because in newborn mice, synaptogenesis and myelination in brain reach their peak in early postnatal days (P1-14), these phenomena are similar to the neurodevelopment of infants during the third trimester of human pregnancy (Barr et al. 2011).
The aim of this preprint was to develop and characterize a novel mouse model for NOWS using an outbred Carworth Farms White mouse strain.

Key findings

Similarly to a previous study (Robinson et al. 2020), morphine was administrated subcutaneously (s.c.) to newborn mice of both sexes form P1 to P14. Behavioral tests (self-righting, ultrasonic vocalization, thermal nociception) were performed on P7 and P14, 16 hours after the saline or morphine injection. On P15, mice were sacrificed, and their brain removed for further RNA isolation (pons, medulla) and transcriptome analysis. Open-field test was applied to assess anxiety-like behavior in a subgroup of mice on P21. Conditions or drugs that decrease time spent in the center are associated with anxiety-like behavior, whereas increased time spent in the center is associated with an anxiolytic effect.

Reduced body weight after chronic morphine treatment

Morphine-injected mice had significantly lower body weight from P2 to P14 compared with the control group; this trend was also observed on P21 and P51.

Behavioral characterization

In the self-righting test (turning from back to abdomen), female morphine-injected mice had an increase in self-righting latency, both on P4 and P7, suggesting that neurodevelopmental delay was more expressed in female mice after morphine administration.
Morphine administration from P1-P14 led to an increase in ultrasonic vocalizations compared with the saline-treated mice.
Thermal hyperalgesia was detected both in male and female mice on P7 and P14 using the hot-plate and tail withdrawal methods. Morphine treated females had shorter tail withdrawal latencies on P7 compared with morphine treated males.
Anxiety-like behavior (less time spent in center of the open field box) was observed in morphine treated mice compared with saline controls on P21.

Brain transcriptome on P15 (pons and medulla)

In female mice, 320 developmental genes were identified at log2FC > ±0.26 and p<0.05, including the downregulation of Nup35 nucleoporin gene, and Serpind1, part of the serpin gene superfamily.
In male mice, 328 developmental genes were identified at log2FC > ±0.26 and p<0.05, including Meg3, encoding a long noncoding RNA involved in brainstem development. Moreover, the upregulation of voltage-dependent calcium channels, Cacna1c, Cacna1d, and Cacna1e was also found.
In case of several genes, correlations were found between the changes in gene expression and a behavioral test, 63% of the selected genes were correlated at least one behavioral symptom, for example, hot-plate latency was correlated with the changes of Prokr1, Prickle3, Cdca5, Prdm6 expressions.

Why I liked this preprint

Sex differences were identified in behavioral tests and in the changes of gene expression in the pons and medulla after morphine treatment. These data could be useful for further identification of novel therapeutic targets for NOWS patients.

Questions for the authors

1. The morphine treatment protocol of (Robinson et al. 2020) was selected for the experiments of the present preprint, but Robinson’s research group used 10 mg/kg dose of morphine (s.c.). Moreover, according to your data, 15% of the morphine-treated mice died before P14. This morphine-induced mortality was not observed with the 10 mg/kg protocol (Robinson et al. 2020). What was the reason of changing the morphine dosage to 15 mg/kg?
2. Both spontaneous (Buckman et al. 2009) and naloxone-precipitated morphine withdrawal (Hodgson et al. 2008; Pedron et al. 2016) evoked anxiolytic-like behavior in mice. Similar results were published using outbred male mice by our research group (Lipták et al. 2012). In contrast to the literature, in the open-field test, morphine-treated mice after 16 hours of the last injection exhibited anxiety-like behavior. What is your opinion about this contradiction?
3. Heat-induced tail withdrawal is a spinal reflex (Ramabadran et al. 1989). Would it be advantageous to perform transcriptomic analysis on spinal medulla tissue samples in your future experiments?
4. The calculation of maximum possible effect (MPE%) is an accepted way to assess the analgesic effect of any drug in mice, because baseline latencies may differ between mice, especially in outbred strains (Mogil et al. 2000). Did female or male mice display big differences in baseline latencies in the hot-plate or tail withdrawal test?


Armbruster D., Schwirian C., Mosier A., Tam W.M. & Prusakov P. (2021) Neonatal Abstinence Syndrome and Preterm Infants: A Look at Current Practice. Adv Neonatal Care 21, 107-14.
Barr G.A., McPhie-Lalmansingh A., Perez J. & Riley M. (2011) Changing mechanisms of opiate tolerance and withdrawal during early development: animal models of the human experience. ILAR J 52, 329-41.
Buckman S.G., Hodgson S.R., Hofford R.S. & Eitan S. (2009) Increased elevated plus maze open-arm time in mice during spontaneous morphine withdrawal. Behav Brain Res 197, 454-6.
Hodgson S.R., Hofford R.S., Norris C.J. & Eitan S. (2008) Increased elevated plus maze open-arm time in mice during naloxone-precipitated morphine withdrawal. Behav Pharmacol 19, 805-11.
Kertesz S.G. & Gordon A.J. (2019) A crisis of opioids and the limits of prescription control: United States. Addiction 114, 169-80.
Lipták N., Dochnal R., Babits A., Csabafi K., Szakács J., Tóth G. & Szabó G. (2012) The effect of pituitary adenylate cyclase-activating polypeptide on elevated plus maze behavior and hypothermia induced by morphine withdrawal. Neuropeptides 46, 11-7.
Mogil J.S., Chesler E.J., Wilson S.G., Juraska J.M. & Sternberg W.F. (2000) Sex differences in thermal nociception and morphine antinociception in rodents depend on genotype. Neurosci Biobehav Rev 24, 375-89.
Pedron V.T., Varani A.P. & Balerio G.N. (2016) Baclofen prevents the elevated plus maze behavior and BDNF expression during naloxone precipitated morphine withdrawal in male and female mice. Synapse 70, 187-97.
Ramabadran K., Bansinath M., Turndorf H. & Puig M.M. (1989) Tail immersion test for the evaluation of a nociceptive reaction in mice. Methodological considerations. J Pharmacol Methods 21, 21-31.
Robinson S.A., Jones A.D., Brynildsen J.K., Ehrlich M.E. & Blendy J.A. (2020) Neurobehavioral effects of neonatal opioid exposure in mice: Influence of the OPRM1 SNP. Addict Biol 25, e12806.
Sutter M.B., Leeman L. & Hsi A. (2014) Neonatal opioid withdrawal syndrome. Obstet Gynecol Clin North Am 41, 317-34.



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