Prenatal methadone exposure impairs adolescent cognition and GABAergic neurodevelopment in a novel rat model of maternal methadone treatment
Introduction
Opioid use in pregnancy has risen in recent years with one in five women from high-income countries reported to use either illicit or prescribed opioids at some point during pregnancy (Patrick et al., 2015). To date, the preferred treatment for the management of opioid dependence during pregnancy is oral methadone maintenance therapy (MMT) (Jones et al., 2012). In contrast to continual heroin use or no treatment, MMT facilitates better prenatal care, decreases maternal/fetal withdrawal and reduces the frequency and severity of birth complications, including intrauterine growth restrictions, premature birth, and miscarriage (Kandall et al., 1976; Mattick et al., 2009; Monnelly et al., 2019). However, MMT was introduced into clinical practice prior to the implementation of controlled longitudinal studies, leaving the long-lasting neurodevelopmental effects of MMT on exposed offspring unclear.
Several clinical studies indicate that in utero methadone exposure can result in deleterious deficits compared to non-exposed neonates, including decreased birthweight, head circumference, and the worsening of neonatal abstinence syndrome (NAS) (Monnelly et al., 2019; Sharpe and Kuschel, 2004). Methadone crosses the placenta and blood-brain-barrier, exposing the foetus to methadone at critical periods of brain development. Imaging studies report that infants exposed to methadone in utero display altered maturation of cerebral connective tracts (Walhovd et al., 2012) and reduced neuroanatomical volumes (Yuan et al., 2014). In line with these neurodevelopmental findings, a recent meta-analysis found that methadone-exposed infants exhibited reduced Mental Developmental Indices, including cognitive, behavioural and language deficits, compared to non-exposed peers (Monnelly et al., 2019). Furthermore, these cognitive deficits appear to extend into adolescence with a large cohort study reporting that infants exposed to opioids in utero, and subsequently diagnosed with NAS, scored lower on standardised school tests from school years 3 to 7 (approximately 10 to 13 years old) compared to non-exposed peers matched for gestational age, socioeconomic status and gender (Oei et al., 2017). While there is increasing evidence indicating in utero methadone exposure may place individuals at risk of neurodevelopmental deficits, it is difficult to determine a causal relationship due to confounding variables, such as polysubstance drug abuse or the quality of the child's postnatal environment.
Based on this growing clinical evidence, there has been an increased demand for animal studies to eliminate confounding influences and to establish the potential extent of these neurodevelopmental complications. Several animal models investigating the effects of in utero opioid exposure exist but are beset with issues that weaken their clinical and scientific relevance. An ideal animal model of prenatal opioid exposure should reflect human conditions as closely as possible, with careful consideration of the methodological parameters employed, including the dose, route, and timing of administration. Previous experimental paradigms commence opioid treatment at a fixed dose mid-gestation using subcutaneous, intraperitoneal or intravenous injections (Chen et al., 2015; Chiang et al., 2015; Zagon et al., 1979). However, pregnant women are typically opioid-dependent for months or years prior to conception and are administered ascending opioid doses as tolerance develops. While osmotic pumps deliver a stable opioid dose without frequent handling of the animal, delivery of increasing doses to parallel the increasing weight gain during pregnancy proves difficult. Moreover, neither of these routes of administration mimic the oral drug delivery typically observed in the use of MMT in the clinic. More clinically relevant animal models of prenatal methadone exposure could be achieved using an ascending dose of methadone, orally self-administered prior to conception and aimed at a stable dose/weight throughout mating, gestation and lactation. Such a treatment paradigm would better emulate the clinical drug delivery profile of methadone and alleviate any additional stress or injury that may result from toxic doses, unstable methadone plasma levels or repeated injections. Currently, no single animal model encapsulates all of these clinically relevant facets of MMT which are required to accurately investigate the potential biological avenues that may underpin the neurodevelopmental deficits observed in longitudinal population studies.
This study aimed to establish an animal model of prenatal methadone exposure that better reflects the clinical situation in which methadone is utilised in the clinic. We also sort to determine the resulting cognitive outcomes of exposed offspring and examine potential neurochemical alterations, in particular, the major excitatory and inhibitory neurotransmitter systems, glutamate and gamma-aminobutyric acid (GABA), due to their critical role in cognition. This knowledge will provide better insight into the possible long-term neurobehavioural consequences of MMT and provide a treatment paradigm to investigate current and novel therapies for opioid-dependent pregnant women and their fetuses.
Section snippets
Animals and ethics statement
Six male (12 months old) and 16 virgin female (seven weeks old) Sprague-Dawley rats were obtained from the Animal Resources Centre (Perth, WA, Australia). Male rats were single-housed and female rats pair-housed (using a perforated plastic divider) in plastic Tecniplast cages. Rats were maintained with food and water ad libitum under controlled temperature (20 °C) with a 12:12 h light-dark cycle (illuminated from 07:00 to 19:00 h). Animals were given two weeks to acclimatise to their
Gestational, litter and offspring characteristics
Prenatal methadone exposure had no significant effect on pregnancy success or litter survival and size (all p > 0.05; Table 1). However, methadone-exposed offspring displayed significantly lower body and brain weights than their non-exposed counterparts at PN0 (body: −11%; p < 0.001, brain: −32%; p = 0.005; Table 1). These differences were not evident at PN9 (p > 0.05; Table 1) or at PN46 (p > 0.05). Gestational weight gain varied significantly with ‘gestation day’ (F6,20 = 31.370, p < 0.0001;
Discussion
MMT during gestation is a safer option than uncontrolled heroin or morphine use, however there is a growing body of clinical data indicating that MMT per se may place exposed infants at neurodevelopmental risk. The extent and causality of these risks remain poorly understood in part due to a lack of animal models that recapitulate the clinical treatment paradigm. The present model aimed to deliver methadone at a sufficient oral dose, prior to conception and in an incremental manner, similar to
Conclusion
Currently there is a global opioid epidemic, with the number of pregnant women undergoing MMT increasing at a substantial rate. Despite the widely appreciated magnitude of this complication and extensive efforts to overcome this problem, there is still great contention in the field as to the long-term effects of prenatal methadone exposure, with limited studies investigating the underlying neurochemical alterations of these effects. In line with clinical studies, the present study reveals
Role of funding source
This study was supported by Small Grants from the Faculty of Science, Medicine and Health University of Wollongong and the Illawarra Health and Medical Research Institute (IHMRI) awarded to IMW, in addition to a philanthropic donation awarded to JSL. KMB and SJM were supported by Australian Government Research Training Program Scholarships from the University of Wollongong.
Ethical statement
All experimental procedures were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (NHMRC, 2013) and were approved by the Animal Ethics Committee of the University of Wollongong (AE17/17). All efforts were made to minimise the number and suffering of animals.
Author contribution
JSL, JW, KAN, and IMW conceptualized the study; JSL, KMB, JW, SJM and KAN performed the experiments; JSL, KMB and SP analysed the data; JSL and KMB and wrote the first draft of the manuscript; JSL, KMB, JW, SJM, SP, KAN and IMW contributed to the interpretation of the data and final manuscript. All authors have approved the final manuscript.
Declaration of interest
The authors declare no conflict of interest.
References (61)
- et al.
Effects of environmental enrichment on behavioral deficits and alterations in hippocampal BDNF induced by prenatal exposure to morphine in juvenile rats
Neuroscience
(2015) - et al.
Subsequent anxiety-related behavior in rats exposed to low-dose methadone during gestation, lactation or both periods consecutively
Pharmacol. Biochem. Behav.
(2012) - et al.
Methadone trough levels in pregnancy
Am. J. Obstet. Gynecol.
(2002) - et al.
Oral and intravenous methadone use: some clinical and pharmacokinetic aspects
Drug Alcohol Depend.
(1999) - et al.
Prenatal administration of methadone using the osmotic minipump: effects on maternal and offspring toxicity, growth, and behavior in the rat
Neurotoxicol. Teratol.
(1992) - et al.
Prenatal opioid exposure: the next neonatal neuroinflammatory disease
Brain Behav. Immun.
(2020) - et al.
24-month neurobehavioral follow-up of children of methadone-maintained mothers
Infant Behav. Dev.
(1984) - et al.
Prenatal exposure to methadone or buprenorphine: early childhood developmental outcomes
Drug Alcohol Depend.
(2018) - et al.
Prenatal exposure to methadone or buprenorphine impairs cognitive performance in young adult rats
Drug Alcohol Depend.
(2020) - et al.
Early inhibitory control and working memory abilities of children prenatally exposed to methadone
Early Hum. Dev.
(2018)
High-dose methadone maintenance in pregnancy: maternal and neonatal outcomes
Am. J. Obstet. Gynecol.
Prenatal methadone exposure is associated with altered neonatal brain development
NeuroImage: Clinical
Prenatal opiate exposure impairs radial arm maze performance and reduces levels of BDNF precursor following training
Brain Res.
Neural tract development of infants born to methadone-maintained mothers
Pediatr. Neurol.
The effects of prenatal exposure to buprenorphine or methadone on infant visual evoked potentials
Neurotoxicol. Teratol.
Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation
Neuron
Sex differences in the adolescent developmental trajectory of parvalbumin interneurons in the hippocampus: a role for estradiol
Psychoneuroendocrinology
Learning ability in adult female rats perinatally exposed to methadone
Pharmacol. Biochem. Behav.
Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study ’recognition memory
Nat. Protoc.
Mechanism of mu-opioid receptor-mediated presynaptic inhibition in the rat hippocampus in vitro
J. Physiol.
Buprenorphine, methadone, and morphine treatment during pregnancy: behavioral effects on the offspring in rats
Neuropsychiatr Dis Treat
Beneficial effects of co-treatment with dextromethorphan on prenatally methadone-exposed offspring
J. Biomed. Sci.
T-maze alternation in the rodent
Nat. Protoc.
μ-Opioid receptors are in discrete hippocampal interneuron subpopulations
Hippocampus
Prenatal exposure to methadone HCL in relationship to body and brain growth in the rat
Acta Neurol. Scand.
Complementary modulation of somatic inhibition by opioids and cannabinoids
J. Neurosci.
The d- and l-isomers of methadone bind to the non-competitive site on the N-methyl-D-aspartate (NMDA) receptor in rat forebrain and spinal cord
Neurosci. Lett.
Parvalbumin-containing fast-spiking basket cells generate the field potential oscillations induced by cholinergic receptor activation in the hippocampus
J. Neurosci.
Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways
Nat. Neurosci.
Methadone during pregnancy in the rat: dose level effects on maternal and perinatal mortality and growth in the offspring’
Journal of Pharmacology and Experimental Therapeutics
Cited by (7)
Effects of prenatal opioid exposure on synaptic adaptations and behaviors across development
2023, NeuropharmacologyCitation Excerpt :In the following sections, we will first discuss what we know about the effects of POE on hippocampus-dependent plasticity and learning, and then discuss the synaptic and behavioral effects of POE on some of the cortical and mesocorticolimbic reward circuits across development. POE is known to impair hippocampal-dependent behaviors in juveniles (Niu et al., 2009; Yang et al., 2003; Nasiraei-Moghadam et al., 2013) which persists through adolescence (Lum et al., 2021; Chen et al., 2015; Wang and Han, 2009) and even into adulthood (Nasiraei-Moghadam et al., 2013; Slamberova et al., 2005; Ahmadalipour et al., 2018; Ahmadalipour and Rashidy-Pour, 2015) (we refer the readers to Table 1 for the remainder of citations). The hippocampus is particularly susceptible to perturbation from opioid exposure due to opioid-mediated regulation of hippocampal plasticity (Drake et al., 2007; Stumm et al., 2004; Simmons and Chavkin, 1996).
- 1
Both authors contributed equally to this work.