Prenatal methadone exposure impairs adolescent cognition and GABAergic neurodevelopment in a novel rat model of maternal methadone treatment

https://doi.org/10.1016/j.pnpbp.2021.110281Get rights and content

Highlights

  • Development of a novel rat model of maternal methadone treatment that aims to recapitulate clinical treatment scenario.

  • Prenatal methadone exposure caused recognition memory deficits in male and female adolescent offspring.

  • Prenatal methadone exposure caused learning deficits in adolescent female offspring.

  • Prenatal methadone exposure resulted in sex- and brain region-dependent reductions to GAD67 and parvalbumin protein levels.

Abstract

Methadone maintenance treatment (MMT) is the most common treatment for opioid-dependent pregnant women worldwide. Despite its widespread use, MMT is associated with a variety of adverse neurodevelopmental outcomes in exposed offspring, particularly cognitive impairments. The neurobiological abnormalities underlying these cognitive impairments are, however, poorly understood. This is, in part, due to a lack of animal models that represents the standard of care that methadone is administered in the clinic, with inconsistencies in the timing, doses and durations of treatment. Here we describe the characterisation of a clinically relevant rat model of MMT in which the long-term behavioural and neurobiological effects of prenatal methadone exposure can be assessed in adolescent offspring. Female Sprague-Dawley rats were treated orally with an ascending methadone dosage schedule (5, 10, 15, 20, 25 and 30 mg/kg/day), self-administered in drinking water prior to conception, throughout gestation and lactation. Pregnancy success, maternal gestational weight gain, litter survival and size were not significantly altered in methadone-exposed animals. Methadone-exposed offspring body and brain weights were significantly lower at birth. Novel object recognition tests performed at adolescence revealed methadone-exposed offspring had impaired recognition memory. Furthermore, the rewarded T-maze alternation task demonstrated that methadone-exposed female, but not male, offspring also exhibit working memory and learning deficits. Immunoblots of the adolescent prefrontal cortex and hippocampus showed methadone-exposed offspring displayed reduced levels of mature BDNF, in addition to the GABAergic proteins, GAD67 and parvalbumin, in a sex- and brain region-specific fashion. This rat model closely emulates the clinical scenario in which methadone is administered to opioid-dependent pregnant woman and provides evidence MMT can cause cognitive impairments in adolescent offspring that may be underlined by perturbed neurodevelopment of the GABAergic system.

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.

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