Consequences of Parental Opioid Exposure on Neurophysiology, Behavior, and Health in the Next Generations

Abstract

Substance abuse and the ongoing opioid epidemic represents a large societal burden. This review will consider the long-term impact of opioid exposure on future generations. Prenatal, perinatal, and preconception exposure are reviewed with discussion of both maternal and paternal influences. Opioid exposure can have long-lasting effects on reproductive function, gametogenesis, and germline epigenetic programming, which can influence embryogenesis and alter the developmental trajectory of progeny. The potential mechanisms by which preconception maternal and paternal opioid exposure produce deleterious consequences on the health, behavior, and physiology of offspring that have been identified by clinical and animal studies will be discussed. The timing, nature, dosing, and duration of prenatal opioid exposure combined with other important environmental considerations influence the extent to which these manipulations affect parents and their progeny. Epigenetic inheritance refers to the transmission of environmental insults across generations via mechanisms independent of the DNA sequence. This topic will be further explored in the context of prenatal, perinatal, and preconception opioid exposure for both the maternal and paternal lineage.

The beginning of the 21st century saw an unprecedented rise in opioid use and abuse in North America and around the world. Indeed, in 2017, the United States declared the opioid epidemic a national public health emergency. The severity of this crisis has been made evident by the dramatic increase in opioid overdoses reported, with opioid-related overdose deaths tripling between 2000 and 2015 (Rudd et al. 2016). For young adults, opioid overdose surpassed car accidents as the leading cause of death (National Center for Health Statistics). Every population of people is impacted by this widespread opioid use including future generations. Here, we describe a growing body of evidence that the effects of the opioid epidemic are reaching future generations through two distinct paths: (1) prenatal (in utero) exposure, and (2) gamete exposure in either males or females leading to persistent changes that occur prior to conception, which impact subsequent offspring.

PRENATAL EXPOSURE TO OPIOIDS IN CLINICAL POPULATIONS

With the overall number of people using and/or abusing opioids increasing, so too has the number of women of reproductive age using and/or abusing opioids. The prevalence of opioid use disorder or dependency during pregnancy more than doubled between 1998 and 2011 (Maeda et al. 2014). Indeed, 22% of women in the obstetric population are prescribed opioids during their pregnancy (Bateman et al. 2014; Cho et al. 2014; Desai et al. 2014). There are two primary indications for opioid prescription during pregnancy: management of pain and opioid use disorder. The current recommendation by the American Academy of Pediatrics and American College of Obstetricians and Gynecologists is opioid maintenance therapy as the first line of treatment for women suffering with opioid use disorder during pregnancy (ACOG Committee on Health Care for Underserved Women; American Society of Addiction Medicine 2012). This is primarily because opioid maintenance therapy decreases the rate of relapse and increases the quality of prenatal care, nutrition, and fetal health as well as preventing cyclical bouts of maternal/fetal withdrawal (Kandall et al. 1976; Maas et al. 1990; Binder and Vavrinková 2008; ACOG Committee on Health Care for Underserved Women; American Society of Addiction Medicine 2012; Committee on Obstetric Practice 2017). Indeed, the safety of detoxification has been debated and long-term outcomes of withdrawal for the child are unknown. Recent reports suggest an increase in birth weight and gestational age in children born to mothers that underwent detoxification during gestation compared with those with illicit drug use at delivery (Stewart et al. 2013; Haabrekke et al. 2014; Walhovd et al. 2015). Yet, the use of buprenorphine or methadone during delivery hospitalizations is associated with decreased maternal morbidity in women with opioid use disorder and, subsequently, the number of prescriptions has increased from 457 to 844 per 100,000 deliveries from 2006 to 2015 (Duffy et al. 2018). Thus, obstetric patients with opioid use disorder can be further divided between women undergoing treatment via opioid maintenance therapy, those that continue to use opioids illicitly, and those that attempt complete abstinence with or without episodes of relapse, all of which could produce different outcomes for the offspring. Regardless of the underlying reason, there is a growing population of neonates exposed to opioids in utero.

IMMEDIATE CONSEQUENCES FOR THE NEONATE

Along with consequences for the mother, such as increased risk of death during hospitalization, cardiac arrest, and cesarean delivery (Bauer et al. 2002; Goler et al. 2008; Nezvalová-Henriksen et al. 2011), there are also myriad risks for the infant. The most consistent finding with prenatal opioid exposure is poor fetal growth resulting in low birth weight, low gestational age, decreased head circumference, and small-for-gestational-age infants (Kandall et al. 1976; Binder and Vavrinková 2008; Hunt et al. 2008; Mactier et al. 2014; Maeda et al. 2014). Additionally, some reports find that in utero exposure leads to intrauterine growth restriction (Pinto et al. 2010), placental abruption (although there are conflicting reports and this may be due to other illicit drug-dependent or abusing women [Bauer et al. 2002; Goler et al. 2008; Nezvalová-Henriksen et al. 2011]), increased length of hospital stay, preterm labor (Pinto et al. 2010; Creanga et al. 2012; Mactier et al. 2014; Maeda et al. 2014), stillbirth, and premature rupture of membranes (Nezvalová-Henriksen et al. 2011; Maeda et al. 2014). Some studies have even suggested an increased prevalence of cardiac defects, autonomic dysregulation (Paul et al. 2014), and varying vision conditions such as crossed eyes (strabismus) and nystagmus (Gill et al. 2003; Gupta et al. 2012). Finally, neonatal opioid withdrawal syndrome (NOWS), or opioid withdrawal experienced postnatally by the infant, has more than tripled between 1999 and 2013 and presents with troubling symptoms (Kandall et al. 1976; Binder and Vavrinková 2008; Hunt et al. 2008). Extended hospitalizations associated with NOWS contribute to the financial burden of addiction on our society. Symptoms of NOWS include high-pitched crying, irritability, seizures, gastrointestinal issues, increased muscle tone, and autonomic dysregulation such as inability to regulate temperature (Wiles et al. 2014). Among the devastating health consequences of NOWS are poor postnatal growth, seizures, and deleterious effects on neurodevelopment (Farid et al. 2008; Baldacchino et al. 2014). Currently, it is not possible to predict which opioid-exposed newborns will require pharmacotherapy for NOWS. Specific symptoms and severity of NOWS are variable and emerge between 24 hours and 5 days following birth, depending on the opioid (Liu et al. 2010; Kocherlakota 2014; Wiles et al. 2014; Klaman et al. 2017). Opioid dose is a poor predictor of NOWS (Rees and Inder 2005). Some factors that have been associated with NOWS severity include co-occurring exposure to benzodiazepines (Hunt et al. 2008; Nygaard et al. 2015), antidepressants (Bunikowski et al. 1998; Seligman et al. 2010), marijuana use or heavy smoking (Seligman et al. 2008), as well as genetic factors (Kaltenbach et al. 2012; Patrick et al. 2015). However, none of these considerations are sufficient to reliably predict NOWS severity, and the mechanisms by which they impact NOWS are not well understood. Further, decisions to initiate pharmacotherapy rely partly on subjective observer-rated scoring (Choo et al. 2004). A substantial proportion of newborns with gestational methadone or buprenorphine exposure will require pharmacotherapy (Kraft et al. 2016). Both buprenorphine and methadone are commonly prescribed to pregnant women with opioid use disorder. Whereas the preferred replacement opioid is still actively debated in the field, both are associated with NOWS, although methadone withdrawal emerges more slowly following birth because of its long half-life. NOWS symptoms are often treated with tapering doses of an opioid, which further extends the period of opioid exposure in neonates. Thus, prenatal opioid exposure is likely to remain a significant public health issue for many years.

LONG-TERM CONSEQUENCES OF PRENATAL OPIOID EXPOSURE

Unfortunately, there remains a paucity of data describing long-term consequences of prenatal opioid exposure for the offspring; thus, the long-term consequences of prenatal opioid exposure are less clear. There are a limited number of longitudinal studies that describe potential effects of prenatal opioid exposure on development, although few extend past childhood (Derauf et al. 2009; Walhovd et al. 2009). Findings to date have described several neurodevelopmental abnormalities. Decreased neuroanatomical volumes, particularly in the basal ganglia, have been found as well as more subtle reductions in the thalamus and cerebellar white matter (Yuan et al. 2014; Sirnes et al. 2017). White matter microstructural maturation has also been documented in school-aged children as well as abnormal neural tract development (Walhovd et al. 2007, 2010, 2012; Yuan et al. 2014). These abnormal anatomical changes may, at least partially, underlie observed neurocognitive effects including deficits in attention, cognition, and other executive functions (Ornoy et al. 1996, 2001; Moe 2002; Slinning 2004; Hunt et al. 2008; Konijnenberg and Melinder 2015; Nygaard et al. 2015, 2017; Konijnenberg et al. 2016). For example, at the preschool and elementary school ages, studies have found motor and cognitive impairments (Guo et al. 1994; Bunikowski et al. 1998; Hunt et al. 2008), deficits in attention, and increased risk for attention deficit hyperactivity disorder (ADHD) (Hickey et al. 1995; Ornoy et al. 1996, 2001; Suess et al. 1997), and hyperactivity (Ornoy et al. 1996). Some of these impairments, such as neurocognitive function, have been shown to persist in children raised in adoptive settings with strong positive environments (Walhovd et al. 2007, 2010, 2012). A direct effect of opioids on the prenatal developing central nervous system likely also contributes to long-term consequences for these children (Hu et al. 2002; Bhat et al. 2006; Walhovd et al. 2007; García-Fuster et al. 2008; Wang and Han 2009). However, these data must be interpreted with caution because of the potential impact of numerous additional risk factors in this clinical population.

The current understanding of neurodevelopmental changes in children with prenatal exposure to opioids is based on a limited number of studies, many with low sample sizes and myriad confounding variables. For example, some of the increased neurodevelopmental risks associated with prenatal opioid exposure can also be explained by environmental risk factors and low birth weight (Hans and Jeremy 2001; Konijnenberg and Melinder 2015). Additional risk factors for the child that confound these studies include maternal polysubstance use (particularly tobacco), altered mother–child bonding and parental interactions, environmental instability, parental education, socioeconomic class, nutrition, foster care, and unknown genetic vulnerabilities. Indeed, a recent systematic review and meta-analysis of studies examining prenatal opioid exposure found that infants and preschool children had minimal significant neurobehavioral deficits, although there was a tendency for overall poorer performance in exposed groups (Baldacchino et al. 2014). This suggests that the effects may be subtle or highly variable; therefore, it is difficult to draw strong conclusions from clinical studies.

PRENATAL EXPOSURE TO OPIOIDS IN PRECLINICAL STUDIES

Preclinical studies of in utero opioid exposure have the benefit of a reductionist approach with the capacity to control many more variables and examine more end points. Over the past four decades, animal models have been utilized to examine behavioral, physiological, and neuronal impacts of prenatal opioid exposure (for a comprehensive review, see Byrnes and Vassoler 2018). Varying animal models have been utilized including rodents, chicks, and sheep, with the majority conducted in rats. However, a number of physiological differences between rodent models and humans must be kept in mind when interpreting and translating preclinical prenatal opioid studies to clinical populations. For example, the timing of neurodevelopment across different species can vary widely. Rodents enjoy a 21- to 22-day gestation period and are born altricial on many neurodevelopmental measures. Therefore, at birth, they would be comparable to a developing human fetus in the late second trimester or early third trimester (Semple et al. 2013). Postnatal day 7 (PND7) in rodents is typically considered to be comparable to a newborn infant, particularly in terms of neural development. However, it must be kept in mind that this comparison is based primarily on the ratio of brain weight to body size as a determinant of rapid periods of brain growth (Dobbing and Sands 1979; Hofman 1983). Nevertheless, even models that expose dams to opioids throughout gestation are mostly comparable to use in the first and second trimester in humans. Unfortunately, it is challenging to administer additional drug during the immediate postpartum period in rodents to model exposure throughout the third trimester. Direct injections add a significant stress to both the pups and dam; maintenance of the dam on opioids passes drugs to pups via the dam's breast milk, but this results in highly variable and often lower levels of drug exposure in the pups. Many preclinical studies have examined the effect of opioid exposure during discreet time periods based on endogenous opioid system development. For example, as opioid receptor expression begins around gestation day 8, many prenatal studies also begin injections at that time. This produces the confounding effect of a surge in corticosterone circulation in the dam from first-time opioid exposure. Exposure to corticoids or other stressors during the prenatal window has been shown to induce significant outcomes for the pup, many of which overlap with prenatal opioid exposure (Kaffman and Meaney 2007; Juul et al. 2011; Hays et al. 2012; Raineki et al. 2014). Therefore, preclinical prenatal opioid studies have their own set of confounding variables and challenges that should be considered when interpreting results.

Early Developmental Changes

Even considering these caveats, preclinical studies examining in utero opioid exposure have found many of the same effects described in humans. Similar to human clinical studies, early developmental abnormalities have been noted. Most consistently across preclinical studies, and consonant with human observations, prenatal opioid exposure in rodents leads to a lower birth weight (Zagon and McLaughlin 1977a,b; Eriksson and Rönnbäck 1989; Hutchings et al. 1993; Lu et al. 2012; Hung et al. 2013; Wu et al. 2014; Devarapalli et al. 2016), decreased brain mass (Hung et al. 2013; Wu et al. 2014), and decreased somatic growth (Kunko et al. 1996). Yet, these effects tend to be drug specific, with oxycodone, buprenorphine, and methadone demonstrating the largest effects. Additionally, developmental delays and abnormalities specific to rodents have been noted such as increased righting latency (Kunko et al. 1996; Slamberová et al. 2005a) and poorer negative geotaxic response (Kunko et al. 1996). There are even some reports of craniofacial malformations (Devarapalli et al. 2016) and increased pup mortality with high doses of specific opioids (Hutchings et al. 1996).

Consequences for neuronal structure and function observed during early developmental time periods have also been noted in rodents. For example, an imbalance in neurotransmitter levels have been observed in striatal tissue, particularly acetylcholine, dopamine, and serotonin (De Vries et al. 1991; Robinson et al. 1996, 1997; Robinson 2000, 2002). There have also been alterations in neuropeptide levels such as decreased brain-derived neurotrophic factor (BDNF) in the plasma in conjunction with decreased BDNF/TrkB-mediated signaling in cortical tissue (Hung et al. 2013; Wu et al. 2014). Indeed, the cortex is another region that seems to be particularly vulnerable to prenatal opioid exposure, with structural changes also frequently observed, including decreases in dendritic length, dendritic branching, and dendritic spine density (Ricalde and Hammer 1990; Mei et al. 2009), as well as alterations in myelin, myelin sheaths, and myelin-associated proteins (Vestal-Laborde et al. 2014). This is not surprising as oligodendrocytes, the glial cells that produce myelin, express opioid receptors on their surface, and seem to be involved in their maturation. Thus, preoligodendrocytes demonstrated accelerated maturation following prenatal opioid exposure (Vestal-Laborde et al. 2014) and more rapid myelination of the corpus callosum. This accelerated myelination could lead to disrupted neuronal communication and altered connectivity. Indeed, functional changes such as alterations in second messenger signaling are also evident at these early time points, which may also underlie long-term neurobehavioral outcomes (De Vries et al. 1991; Basheer et al. 1992; Wu et al. 2001; Bhat et al. 2006; Mithbaokar et al. 2016).

Impact on the Endogenous Opioid System

The impact of exogenous opioid exposure on the developing endogenous opioid system has also been extensively studied. The endogenous opioid system is critical for regulation of pain, reward, and stress responsivity. This line of experimentation has generated an abundance of data that demonstrate the wide-ranging impact that exogenous prenatal opioid exposure exerts on the developing opioid system. Increases and decreases in opioid receptor binding have been observed in the central nervous system, including the spinal cord and brain (hippocampus, striatum, thalamus, amygdala, and hypothalamus), which are both sex-, and developmental timing-specific (Kirby 1983; Vathy et al. 2000, 2003; Rimanóczy et al. 2001; Chiou et al. 2003; Slamberová et al. 2003a,b,c, 2005b; Hou et al. 2004; Bhat et al. 2006). Whereas there are exceptions, the pattern of receptor expression and ligand affinity appears to primarily be down-regulated immediately following exposure and up-regulated at later time points. This bimodal response likely underlies the observation that pain perception in these animals also demonstrates a diametric shift across development, with a tendency for increased basal pain perception (hyperalgesia) concurrent with a diminished analgesic response to opioids early in development that shifts toward increased sensitivity to analgesic properties of opioids in adulthood (O'Callaghan and Holtzman 1976, 1977; Castellano and Ammassari-Teule 1984; Gagin et al. 1996; Vathy and Komisaruk 2002; Chiang et al. 2010; Tao et al. 2011; Biglarnia et al. 2013). This effect persists even in the presence of cross-fostering, suggesting prenatal exogenous opioid exposure influencing the development of the endogenous opioid system (Gagin et al. 1996). Perinatal morphine exposure also induces morphine tolerance (Hovious and Peters 1984; Eriksson and Ronnback 1989; Chiang et al. 2010).

Pubertal maturation also tends to play a role in opioid receptor expression/ligand affinity and analgesic response, as sex differences are consistently observed. This is likely due to the influence of hormones on the opioids and the role of endogenous opioids in the maturation of the reproductive axis. For example, there have been numerous examples of impacts of prenatal opioid exposure on the endocrine system. Abnormalities begin with development, such as changes in anogenital distance (Devarapalli et al. 2016), through sexual maturation, with changes in sexual behavior (Vathy et al. 1985; Vathy and Katay 1992) and including changes in neuroendocrine functioning (Litto et al. 1983). Evidence also exists that supports the clinical observation that prenatally exposed children may be at great risk for substance abuse in adulthood. For example, enhanced conditioned place preference to opioids as well as methamphetamine have been observed in rats (Chiang et al. 2014, 2015; Wong et al. 2014) in addition to augmented methamphetamine reinstatement (Shen et al. 2016). This enhanced response may be due to increased reward from altered dopamine receptor expression and signaling observed within the nucleus accumbens (Chiang et al. 2014) or changes in the endogenous opioid receptors as described above. Whereas the majority of these preclinical observations are in males, females likely experience similar and unique vulnerabilities, which will require a significant amount of further investigation to fully understand.

Long-Term Consequences

A growing body of literature documents significant impacts of prenatal opioid exposure on the hypothalamic pituitary adrenal (HPA) axis. This includes an impact on adrenocorticotropin hormone (ACTH) and norepinephrine circulation at baseline and following a stressor (Dutriez-Casteloot et al. 1999; Rimanóczy et al. 2003; Slamberová et al. 2004; Laborie et al. 2005; Sithisarn et al. 2008). Whereas there is a mixed literature regarding the impact of prenatal opioid exposure on plasma corticosterone levels, it is clear that the HPA axis, functioning, and stress response are consistently impacted in a sex- and age-dependent manner. This endocrine stress response is connected to immune reactivity with a blunted stress response following an immune stressor (Hamilton et al. 2005). The immune system is also dysregulated in response to cytotoxic activity and an lipopolysaccharide (LPS) challenge (Shavit et al. 1998; Schrott and Sparber 2004; Hamilton et al. 2007). Because both dysregulation of the HPA axis and function of the immune system are associated with increased risk of disease, these findings support the notion that there will be long-term consequences for health and well-being in individuals exposed to opioids prenatally.

The hippocampal-based learning and memory system is a primary target of study because of its role in psychiatric illnesses and neurocognitive deficits. A growing body of evidence suggests that there are long-term alterations in the structural, physiological, and functional capacity of the hippocampus in both adolescents and adults following prenatal opioid exposure. Yet, it should be noted that the severity of these changes are typically moderate and suggest mild deficits rather than extensive neurocognitive impairments. For example, in adolescence, there are mild impairments in spatial memory as measured with a Y-maze (Niu et al. 2009) as well as impairments in passive avoidance memory (Nasiraei-Moghadam et al. 2013; Ahmadalipour et al. 2015), fear memory (Tan et al. 2015), and reference memory (Steingart et al. 2000a; Wang and Han 2009; Davis et al. 2010). Many of these changes are accompanied by physiological changes within the hippocampus including a decreased capacity for long-term potentiation and long-term depression, and molecular changes that point toward decreased synaptic plasticity (Yang et al. 2003, 2006; Villarreal et al. 2008; Tan et al. 2015). These changes provide a potential physiological/molecular mechanism to explain some of the observed neurocognitive deficits. Another observation has been increased neural apoptosis, which has been linked to deficits in learning and memory after prenatal heroin exposure in mice (Wang and Han 2009). Evidence also suggests that deficits in spatial learning and memory may be tied to hippocampal cholinergic alterations (Steingart et al. 2000a,b; Yaniv et al. 2004). Additionally, other neural changes have been reported following prenatal opioid exposure, including decreased dendrite length (Ricalde and Hammer 1990) and branch number in cortical neurons, decreased cortical thickness, altered myelin formation, and decreased neurogenesis (Sadraie et al. 2008; Lu et al. 2012; Vestal-Laborde et al. 2014; Wu et al. 2014). Long-term changes are also observed in other neurocognitive and psychiatric metrics such as an increase in depressive-like behavior (Hung et al. 2013; Wu et al. 2014) and increased acoustic startle response (Hutchings et al. 1993; Zmitrovich et al. 1994), which is associated with sensory-processing deficits often seen in autism and schizophrenia. This conglomeration of neurocognitive deficits observed in preclinical studies provides ample evidence that subtle deficits observed in human populations are at least in part the direct result of prenatal opioid exposure, and not other confounding variables, although these likely contribute as well.

MECHANISMS BY WHICH OPIOIDS MAY IMPACT FETAL DEVELOPMENT

It is not surprising that opioid exposure can impact fetal development. Opioids easily cross the placenta to access the fetal bloodstream and can directly act on molecular targets in the fetus (Gerdin et al. 1990; Nekhayeva et al. 2005). Opioids often have active metabolites that can also contribute to primary and secondary effects of exposure. For example, Norbuprenorphine, a major metabolite of buprenorphine, induces fetal opioid dependence and leads to NOWS (Griffin et al. 2019). Opioid receptors are found throughout the central nervous system as well as numerous peripheral tissues including both the uterus and placenta (Wittert et al. 1996). In the developing neonate opioid, receptors and endogenous opioid peptides differ from adults and therefore likely have distinctive effects compared with adult exposure (Lenoir et al. 1984; Barg and Simantov 1989). A growing body of evidence shows that the endogenous opioid system plays a critical role in many aspects of development including neurogenesis (with opioids exerting an inhibitory effect) (Sargeant et al. 2008; Wu et al. 2014; Sasaki et al. 2015) and control of oligodendrocyte myelination and function, so interference with this system by exogenous opioids could alter the normal maturation process of the developing brain (Vestal-Laborde et al. 2014). The dopaminergic system in the fetal brain also plays an important role in normal brain development (Money and Stanwood 2013). Opioids increase levels of dopamine in the brain, and dopamine-rich areas such as the nucleus accumbens, basal ganglia, and frontal cortex show evidence of vulnerability (Derauf et al. 2009). Thus, this could underlie observed neurocognitive deficits (Rivkin et al. 2008). Opioids can also damage neural tissue through increased oxidative stress and apoptosis (Hung et al. 2013). There is decreased proliferation in the developing striatum (Harlan and Song 1994) and increased apoptosis in dopaminergic cell cultures and hippocampal tissues (Oliveira et al. 2002, 2003; Svensson et al. 2008; Wang and Han 2009). Finally, opioids exert a profound impact on the mother's physiology, which may secondarily influence the fetus via altered secretion of stress hormones and altered maternal health behaviors. Because endogenous opioids play a significant role in regulating the neuroendocrine axis (Morley 1981; Bicknell 1985), it becomes a challenge to discern those effects that are a result of opioid exposure on the fetus from indirect pathways resulting from changes in maternal endocrine pathophysiology. The unique role that endogenous opioids play in mediating hypothalamic and pituitary output during pregnancy and the postpartum period (Douglas et al. 1998; Wigger et al. 1999; Douglas and Russell 2001; Neumann 2001; Wigger and Neumann 2002; Grattan et al. 2008; Gustafsson et al. 2008; Russell et al. 2008; Brunton et al. 2009; Brunton and Russell 2011) likely contribute to the diverse range of outcomes experienced by the offspring. A new line of investigation examining the impact of preconception opioid exposure on future offspring highlights the conflated relationship between maternal physiology, epigenetic impacts, and opioidergic roles in development.

PRECLINICAL PRECONCEPTION OPIOID EXPOSURE (MATERNAL LINEAGE)

A growing body of evidence suggests that preconception exposure to opioids by either parent can also impact subsequent generations. As no clinical studies have examined preconception opioid exposure, the evidence currently exists solely within preclinical models. However, there have been other clinical studies that show correlations between ancestor behaviors or exposures and health consequences for future offspring, lending credence to the idea that environmental conditions can impact inheritance. In terms of opioids, preconception opioid exposure in females has primarily been examined with morphine as the prototypical opioid. In the predominant model, morphine is administered at increasing doses for 10 days during adolescence (PND30–39) in female rats. These adolescents then remain drug free until adulthood and for at least 3 weeks at which point they are mated with drug-naive males. Offspring from this model have been characterized across development. There have been no differences noted in litter size, weight, or sex ratio. However, it was shown that differences do emerge prior to puberty including activity levels, gene expression, and HPA axis (Vassoler et al. 2014). There were also decreases in ultrasonic vocalizations in Mor-F1 pups during separation from their dams (Bodi et al. 2016). Additionally, increases in juvenile play behavior in female Mor-F1 animals was also noted (Johnson et al. 2011).

The majority of the work, however, has examined the offspring (F1 animals) in adulthood. For example, changes in emotionality-related behaviors have been observed with decreases in anxiety-like behavior in adult Mor-F1 female rats during diestrus (Byrnes et al. 2011), yet they increased anxiety-like behavior during proestrus (Byrnes 2005) as measured with the elevated plus maze. This is accompanied by decreased levels of circulating corticosterone in response to opioids (Vassoler et al. 2018). Increased anxiety-like behavior is also observed following adult preconception opioid exposure, which may be caused by dendritic retraction in the dentate gyrus and down-regulation of insulin-like growth factor 2 signaling in the granular zone (Li et al. 2014). Additionally, in a slightly different paradigm, it was shown that preconception morphine oral self-administration decreases hippocampal long-term potentiation (LTP) in their future offspring (Sarkaki et al. 2008), a region critical for emotionality, learning, and memory.

Response to opioids have also been examined both in terms of pain management and reward. In the hot plate test, Mor-F1 male offspring demonstrated significantly increased sensitivity to the analgesic effects of acute morphine and developed tolerance more rapidly than Sal-F1 males (Byrnes et al. 2011). Mor-F1 animals also showed increased sensitivity to the rewarding effects of opioids measured with conditioned place preference (Vassoler et al. 2016). Female Mor-F1 offspring demonstrated a more rapid induction of morphine behavioral sensitization while Mor-F1 male offspring demonstrated enhanced expression of morphine sensitization (Byrnes 2005). However, F1 animals demonstrated attenuated locomotor sensitization to repeated quinpirole injections (D2 receptor agonist), indicating that these effects are drug-specific (Byrnes et al. 2013). Attenuated or blunted behavior was also observed during morphine self-administration. In adult F1 progeny trained to self-administer morphine, there was a consistent decrease during the acquisition phase as well as extinction and reinstatement in both sexes (Vassoler et al. 2017). Even at doses where acquisition and extinction were the same between Mor-F1 and Sal-F1 animals, the blunted reinstatement behavior was still observed in Mor-F1 animals (Vassoler et al. 2017). Both of these effects extend into the subsequent generation as well (F2). Thus, F2 progeny have attenuated locomotor sensitization to quinpirole as well as blunted morphine priming-induced reinstatement (Byrnes et al. 2013; Vassoler et al. 2017). Along with prenatal opioid exposure, preconception opioid exposure may induce epigenetic changes that confer immediate survival advantage, with potential costs to long-term health. This “programming hypothesis,” first proposed by Lester and Padbury in reference to children with prenatal cocaine exposure (Lester and Padbury 2009), is in line with the Barker hypothesis and the thrifty hypothesis and suits the data described above (Barker 1990). However, this seems to be a drug-specific phenomenon as Mor-F1 animals demonstrated increased responding for cocaine (Vassoler et al. 2019).

PRECONCEPTION OPIOID EXPOSURE (PATERNAL LINEAGE)

Consequences of Paternal Opioid Exposure on Male Germline

Chronic opioid exposure has profound consequences on spermatogenesis via peripheral and central mechanisms that impact the production and maturation of spermatozoa. In healthy males, endogenous opioids and their analogs tightly regulate the levels of key reproductive hormones, including gonadotropin-releasing hormone (GnRH) as well as the sex hormones luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Delitala et al. 1983). GnRH emanates from the hypothalamus and promotes the release of FSH from the anterior pituitary, which modulates peripheral and local production of testosterone. Studies in humans and rodents have demonstrated that when males are exposed to exogenous opioids, endogenous hormonal fluctuations are disrupted peripherally and locally in the testes, which often leads to reduced fertility, hypogonadism, loss of libido, and impotency (Tokunaga et al. 1977; James et al. 1980; Singer et al. 1986; Chowdhury 1987; Daniell 2002; Ahmadnia et al. 2016). Another likely contributor to reduced fertility and poor semen quality are the low blood testosterone levels associated with chronic opiate use in males (Lafisca et al. 1985; Ragni et al. 1988; Safarinejad et al. 2013), regardless of the type of opioid consumed (Katz and Mazer 2009; Bawor et al. 2015). The role of reproductive hormones in regulating spermatogenesis rapidly changes over the course of development. Hence, the degree to which sexual function is disrupted by exogenous opioids is largely dependent upon the regimen of exposure and the developmental window during which opioids are consumed. The effects of exogenous opioids on reproduction appear to be transient and partly reversible if opioid exposure occurs during adulthood, with most reproductive functions showing recovery following cessation of opioid exposure or therapy (Cicero et al. 1989; Fabbri et al. 1989). In sharp contrast, preclinical studies suggest that opioid exposure during adolescence can have profound and long-lasting consequences, even after extended drug-free periods (Cicero et al. 1989, 1991). The changes in serum concentration of LH and testosterone caused by morphine treatment during adolescence last for weeks beyond the effects produced by adult morphine exposure (Cicero et al. 1991). The negative consequences of adolescent morphine exposure outlast the measurable impact of such treatment on peripheral levels of reproductive hormones. When males treated with morphine during adolescence are mated to drug-naive females, the resulting litters are smaller in size even when mating occurs after the morphine-related influence on reproductive hormone levels have subsided (Cicero et al. 1989). These results are consistent with the idea that insults delivered during adolescence as opposed to adulthood have more profound and longer-lasting consequences for reproductive function and fitness.

In addition to these peripheral and indirect effects on reproductive and sexual function, the opioid system plays an important role in spermatogenesis via local signaling in the testes. Reproductive hormones influence spermatogenesis via their actions in somatic Sertoli and Leydig cells of the testes (Subirán et al. 2011). Sertoli cells form the blood–testis barrier and support the germ cell progenitors by facilitating the transfer of nutrients from nearby blood supplies. Leydig interstitial cells produce testosterone locally in the testes partly in response to LH secretion from the pituitary. The concentration of testosterone in the seminiferous tubule, where spermatogenesis occurs, is 100 times higher than in peripheral blood (Morse et al. 1973). The endogenous ligands of opioid receptors, endorphins, and enkephalins are also thought to be produced locally in the male reproductive tract, evidenced by the fact that the levels of these peptides are six to 12 times higher in the seminal fluid of the testes compared to peripheral blood (Subirán et al. 2011). Moreover, the genes that encode opioid peptide precursor proteins pro-enkephalin (PENK), pro-dynorphin (PDYN), or pro-opiomelanocortin (POMC) are all expressed in the testes, suggesting that local synthesis of endogenous opioids is a critical component of testicular function and spermatogenesis (Pintar et al. 1984; Kilpatrick and Millette 1986; Kilpatrick et al. 1987). Consistent with this idea, overexpression of Penk in the testes impairs male fertility and causes morphological abnormalities as well as low sperm motility in mice (O'Hara et al. 1994). The current view based on a wealth of studies in mice and rats is that differential expression of opioid precursors in somatic and germ cells of the testes contributes to the local influence of endogenous opioids on testicular function (for a more complete review of this topic, see Subirán et al. 2011).

Impact on the Endogenous Opioid System

δ-, μ-, and κ-opioid receptors are also expressed in the testes, primarily on Sertoli cells (Tsong et al. 1982; Fabbri et al. 1985, 1988; Soverchia et al. 2006) as well as on the membranes of sperm cells (Agirregoitia et al. 2006). Evidence in humans and rodents suggest that μ-opioid receptors in the testes are functionally relevant for fertility and proper reproductive functions such as sperm motility. For example, expression of the gene Oprm1 that encodes the μ-opioid receptor in mature mouse sperm is critical for in vitro fertilization and is likely involved in early embryonic development (Olabarrieta et al. 2020). Mice lacking μ-opioid receptor function show less copulation behavior, have lower sperm count, reduced sperm motility, and smaller litter sizes (Tian et al. 1997). Moreover, incubation of human sperm with the μ receptor agonist morphine substantially reduces sperm motility, an effect that can be reversed by treatment with the μ receptor antagonist naloxone (Agirregoitia et al. 2006). Hence, direct agonism of μ-opioid receptors located on the membrane of mature sperm is likely responsible for reduced sperm motility (asthenozoospermia) reported in human patients abusing opioids (Ragni et al. 1988). Consistent with this view, studies in rodents have also demonstrated adverse outcomes of chronic morphine treatment on fertility (Smith and Joffe 1975). Male rats that were treated with morphine as adults did not show overt signs of reduced copulation or sexual behavior. However, the number of pseudo- or failed pregnancies was much higher in morphine-exposed males mated with drug-naive females than in the control condition (Cicero et al. 2002). Interestingly, and perhaps surprisingly, even an acute exposure to morphine in males produced immediate effects on litter size and survival rate of offspring produced from matings with drug-naive females 24 hours after a single morphine treatment (Smith and Joffe 1975; Cicero et al. 1995). These results posit that the impact of morphine exposure in adulthood is highly dynamic, eliciting nearly immediate and transient effects on reproductive function. In contrast to μ-mediated effects demonstrated by the aforementioned studies, stimulation of δ-opioid receptors located on mature sperm promotes sperm motility. The δ-opioid receptor antagonist naltrindole inhibits sperm motility (Agirregoitia et al. 2006; Albrizio et al. 2010). One possibility is that naltrindole displaces the endogenous ligand enkephalin from the δ-opioid receptors, which normally promotes motility. Intriguingly, high doses of enkephalin also inhibit motility, presumably via their actions at the μ-opioid receptor at higher doses (Agirregoitia et al. 2006). The κ-opioid receptors present on mature sperm do not appear to play a functional role in sperm motility. Pharmacological manipulations of mature human sperm using the κ-receptor agonist U50488 did not alter sperm motility for example (Agirregoitia et al. 2006). Overall, the three opioid receptors expressed in the testes and on mature spermatozoa reflect the fact that the endogenous opioid system is critical for proper reproductive function. These processes are substantially disrupted by both acute and chronic opioid exposure, leading to adverse outcomes on fertility and reproductive function in males.

Germline Reprogramming Elicited by Opioid Exposure

Paternal environmental perturbations such as drug exposure can produce long-lasting consequences that are apparent in the next generation. In the case of paternally derived insults, the mode of transmission from fathers or sires to their progeny is primarily conferred via reprogramming of the germline epigenome and alterations to the developmental trajectory of the resulting progeny (Bale 2015; Yohn et al. 2015; Goldberg and Gould 2018; Pierce et al. 2018). Broadly defined, epigenetics is the study of environmental effects on the way genes are expressed. The term “epigenetics” encompasses a vast cohort of mechanisms by which DNA is regulated outside of mutations to the DNA sequence. Some of these epigenetic marks are established in the germline and survive the reprogramming events associated with spermatogenesis, facilitating a phenomenon referred to as epigenetic inheritance. These mechanisms include DNA methylation and posttranslational modification of histone proteins and the actions of small noncoding RNAs (Berger et al. 2009; Dunn et al. 2010; Jenkins and Carrell 2012; Bale 2014, 2015). There are numerous examples of environment-mediated changes in DNA and histone epigenetic modifications across the life span that alter phenotypes (Fraga et al. 2005; Poulsen et al. 2007). It is noteworthy that morphine has been shown to be a mutagen of spermatocytes in mice (Badr and Rabouh 1983), raising the possibility of transmission via genetic mutations. However, the homogeneity and consistency of some of the inherited phenotypes described below are not consistent with random mutations produced by a mutagen in the germline. The next section will address the influence of paternal exposure to opioids on the behavior and physiology of adult descendants and speculate on the mechanisms underlying the inheritance of these phenotypes. There is an important distinction between multi- and transgenerational effects: any consistent environmentally mediated changes in descendant physiology that cannot be attributed to a direct effect of the environmental event are characterized as transgenerational. In the case of paternal manipulations, transgenerational effects can emerge as early as the second (F2) generation removed from the exposure.

Several instances of transgenerational heritability in humans have been reported. For example, around the turn of the twentieth century, the remote community of Överkalix, Sweden routinely experienced periods of food abundance and famine. Retrospective studies indicated that if the paternal grandfather experienced a period of food excess during the slow growth phase of adolescence, his grandsons had increased risk of diabetes-related mortality (Kaati et al. 2002; Pembrey et al. 2006). The mechanisms underlying this phenomenon may be persistent changes to germline epigenetic marks. This is a controversial concept as, traditionally, it was thought that all epigenetic marks were erased soon after fertilization. Moreover, sperm histones are replaced by protamines, which allows for higher chromatin compaction. Following fertilization, paternal protamines are replaced by histones of maternal origin (Puri et al. 2010). However, it is now clear that some maternal DNA methylation is conserved (Hackett et al. 2013) and a limited number of paternal histones and their epigenetic marks are retained and influence development following fertilization (Hammoud et al. 2009). Findings like these provide a mechanistic basis for transgenerational epigenetic inheritance. In the context of paternal opioid exposure, the majority of the work has centered around multigenerational (also known as intergenerational) studies, where only the first generation was examined for changes in physiology or behavior. For more complete reviews of epigenetic inheritance related to drugs of abuse, see Yohn et al. (2015), Goldberg and Gould (2018), and Pierce et al. (2018). A study in male patients living with opioid use disorder found increased DNA methylation of seven CpG islands located on the OPRM1 gene-promoter region compared to controls. Interestingly, similar patterns of DNA methylation were observed in blood samples collected in this small clinical study (Chorbov et al. 2011). In rats, chronic morphine treatment during adolescence increased histone H3 acetylation of the seminiferous tubules in adults while total expression of H3 was unaffected (Vassoler et al. 2020). Given the important role of opioid signaling on regulating spermatogenesis, it is plausible that opioid-derived reprogramming of the epigenetic landscape is orchestrated by the actions of opioids in the brain, on reproductive hormone levels, and locally in the testes. A major challenge going forward is to further characterize drug-induced epigenetic changes in the germline that could influence physiology and behavior across generations. This is a significant hurdle for the entire field of transgenerational epigenetics, which will likely require the development of new methods to accurately trace the influence of a specific germline modification through embryogenesis and development.

Consequences for Future Generations

The idea that lifetime experiences in fathers can have profound influences on their progeny was once highly controversial. The “Theory of Inheritance of Acquired Characteristics” first proposed by the French zoologist Jean-Baptiste Lamarck at the turn of the 19th century was dismissed as nonsensical by most of the contemporary scientific community. However, mounting evidence in both retrospective human studies and preclinical animal manipulations have demonstrated that the nature, timing, and duration of environmental perturbations are critical in assessing the extent to which future generations will be affected by paternal insults. Moreover, the delineation of the mechanisms responsible for the transmission of environmentally derived inherited phenotypes discussed in the previous section has aided in convincing some of the most ardent skeptics that epigenetic inheritance from the paternal lineage is not only plausible but demonstrable. In the last decade, there has been a sharp rise in the number of studies examining the influence of paternal drug exposure on future generations with the majority of this work focused on stimulants and alcohol (Vassoler et al. 2013; Pierce and Vassoler 2014; Zhu et al. 2014; Finegersh et al. 2015a,b; White et al. 2016; Rompala et al. 2017; Wimmer et al. 2017, 2019; McCarthy et al. 2018). A growing proportion of studies addressing the impact of opioid consumption on future generations have probed these effects using paradigms where either one or both parents are treated with opioids prior to conception.

The previous section focused on the deleterious impact of opioid treatment on male reproductive function. Hence, the fact that these paternal exposure studies on progeny behavior and physiology are feasible at all may come as a surprise. In the majority of the articles discussed below, parental opioid exposure does not have any overt negative impact on reproductive success. There is some evidence for reduced birth weight and higher mortality rates in the progeny of sires treated with opioids (Soyka et al. 1978; Eriksson et al. 1989; Akbarabadi et al. 2018; Ashabi et al. 2018; Sadat-Shirazi et al. 2019); however, initial litter sizes at parturition and the proportion of successful pregnancies is largely intact in most of the studies discussed in this section. It is worth pointing out that the exposure regimens used in opioid studies of fertility and male reproductive function tended to rely on experimenter-delivered manipulations using high and escalating doses of opioids. A substantial proportion of articles investigating the consequences of parental opioid exposure on physiology and behavior of future generations have relied on self-administration paradigms, where animals volitionally consume opioids through drinking water or via operant responding to earn intravenous infusions of the drug. These methodological considerations may partly explain the wide range of effects observed in this line of inquiry. Keeping in mind that translation of opioid doses from rodents to humans is imperfect, the self-administration paradigms may be advantageous over experimenter-delivered approaches, adding a self-titrating volitional and translational component to this line of work.

Alterations in Reward Processing and Pain Sensitivity

Epidemiological studies have shown that the children of parents who live with substance use disorder are at higher risk for a host of psychiatric diseases including substance abuse (Biederman et al. 2000; Peleg-Oren and Teichman 2006). Rodent studies of paternal opioid exposure have been largely consistent with these reports, with many articles showing alterations in addiction-like behaviors in the next generation. Sires treated with escalating doses of morphine during adolescence produced female offspring with increased self-administration of morphine and oxycodone (Vassoler et al. 2020). Morphine-sired female offspring also showed increased motivated responding for oxycodone on a progressive ratio reinforcement schedule, where the response criterion increases over the course of the self-administration session, suggesting an increase in the reinforcing properties of oxycodone in morphine-sired female offspring. In sharp contrast, both acquisition and motivated responding for cocaine self-administration were blunted in male and female morphine-sired offspring, demonstrating a bidirectional impact of sire morphine exposure during adolescence on reward processing for opioids and cocaine in the next generation (Vassoler et al. 2020). Consistent with these findings, opioid consumption was increased in progeny of sires and/or dams treated with morphine during adulthood. Interestingly, these effects were mitigated when sires and dams were provided environmental enrichment during the abstinence washout period prior to mating (Pooriamehr et al. 2017). This enrichment manipulation in parents also dampened the impact of parental exposure on anxiety-like and depressive-like behaviors in this model, highlighting the importance of other environmental considerations and factors prior to breeding after drug exposure. In sharp contrast to the aforementioned articles, morphine and methamphetamine self-administration were unaltered by paternal morphine self-administration that occurred during adulthood. The same study demonstrated that morphine-sired progeny were resistant to morphine conditioned place preference but more sensitive to methamphetamine conditioned place preference (Sadat-Shirazi et al. 2019). Conditioned place preference for morphine was also reduced in morphine-sired progeny in a model where parental drug exposure occurred during adolescence followed by naloxone-precipitated withdrawal prior to breeding (Azadi et al. 2019). The precipitated withdrawal component of this work could have introduced additional factors such as stress in both sires and dams, which may partly explain some of the opposite results on drug reward compared to other studies of this kind. These combined results also highlight the importance of the methods employed to assess drug reward in descendants. When the rewarding properties of opioids are measured using drug–environment associations that require intact hippocampal function, the results can diverge from those attained using operant conditioning. This was the case even when the parental interventions were exactly identical within a single study (Sadat-Shirazi et al. 2019). Moreover, the well-established decrease in spatial memory caused by paternal opioid exposure (further detailed in the following section) represents a potentially confounding factor when measuring drug–spatial cues associations in these multigenerational opioid paradigms. Overall, there are consistent reports of altered drug-taking behavior and drug reward in the offspring of sires that are exposed to opioids prior to conception. The direction, sex, and drug specificity of these effects seem to be partly tied to the exact patterns and nature of the parental exposure to opioids.

Paternal opioid exposure has a pronounced impact on pain threshold and opioid-induced antinociception in the next generation. Male rats chronically treated with morphine through a liquid diet until 5 days prior to mating with drug-naive females produced male offspring with an increased response to the analgesic action of morphine as measured by the hot plate latency pain test (Eriksson et al. 1989). The dose of morphine tested in this study was 7.5 mg/kg and female offspring were not included. In another set of experiments, morphine-derived male offspring also showed a heightened sensitivity to the antinociceptive properties of morphine at two additional doses of morphine: 10 and 12 mg/kg on a similar hot plate pain test. However, female offspring produced by sires treated with morphine during adulthood showed similar levels of morphine-derived analgesia (Cicero et al. 1995). Consistent with these findings, chronic maternal and/or paternal morphine exposure increased the analgesic properties of a low dose (1.5 mg/kg) of morphine in male progeny in a formalin-based pain assay. Interestingly, morphine-sired male progeny also showed a baseline reduction in nociception in this report (Ashabi et al. 2018). When paternal morphine exposure occurred during adolescence, the impact on nociception measured by a similar formalin pain test in male morphine-sired progeny was subtler. The initial (acute) response was intact but morphine-sired male progeny showed a less pronounced pain response during the later (chronic) part of the pain assay. Morphine (2 mg/kg) had little impact on the paternal morphine-related changes in nociception of F1 male progeny (Pachenari et al. 2018). Taken together, these results demonstrate that paternal opioid consumption or treatment produces substantial alterations in baseline nociception and in the analgesic properties of morphine in male offspring.

Deleterious Effects on Cognition

Paternal and parental opioid exposure also has negative consequences for memory formation and cognitive function in many multigenerational models of opioid abuse. In a study where sires and dams self-administered morphine orally for 21 days, followed by a 10-day washout period, the resulting male and female progeny showed deficits in passive avoidance fear memory if either one or both parents were exposed to morphine prior to conception (Akbarabadi et al. 2018). These results suggested that prenatal morphine consumption in either parent diminished hippocampal function. Consistent with this possibility, spatial memory acquisition and retrieval assessed by the Morris water maze was impaired by maternal and/or paternal morphine exposure in first-generation male progeny—female offspring were not included in this study. These deficits were accompanied by a reduction in phosphorylated CREB protein, an activity-dependent transcription factor that is known to be critical for synaptic plasticity and memory formation in the hippocampus (Ashabi et al. 2019). Expression of the calcium-binding protein S100 and the inflammatory cytokine tumor necrosis factor α (TNF-α) were decreased and increased, respectively, in the hippocampus of first-generation progeny of parents treated with morphine through their drinking water (Amri et al. 2018). Even brief parental insults spanning only 5 days of morphine treatment prior to mating with no washout period were sufficient to produce synaptic plasticity impairments in the performant path of the hippocampus in male and female F1 progeny (Sarkaki et al. 2008), a phenomenon thought to underlie memory storage within this region. In contrast, a chronic regimen of intravenous morphine self-administration spanning 60 days prior to conception in sires with no washout period produced more subtle disruptions in cognition. Female, but not male progeny of morphine-treated sires showed impairments in novel object-recognition memory. This paternal manipulation spared hippocampus-dependent fear-conditioned memory and object-location memory in both male and female offspring (Ellis et al. 2020). Taken together, these studies reveal that prenatal paternal opioid exposure negatively affects memory and cognitive function in the next generation. The degree to which these manipulations influenced F1 progeny appear to be partly dependent upon methodological considerations including the dose, length, and mode of exposure and the amount of abstinence time prior to conception.

Potential Neural Mechanisms

The neural mechanisms underlying behavioral changes in these multigenerational models of paternal opioid exposure remain largely unknown in many cases. However, several studies have identified some consistent electrophysiological and cellular adaptations emanating from paternal opioid history in the brain of opioid-sired offspring. The gene and protein expression of the μ-opioid receptor was increased by paternal morphine exposure in the nucleus accumbens of male progeny. In addition, gene expression of the κ- and δ-opioid receptors were also elevated in male morphine-sired offspring. In the prefrontal cortex, expression of the κ-opioid receptor transcript was increased, while δ- and μ-opioid receptors were unaffected by paternal morphine exposure (Ashabi et al. 2018). The CREB signaling pathway, which regulates activity-dependent synaptic plasticity, has also been implicated in many of these reports. However, the direction of the changes in CREB signaling diverges based on the study and on the brain region examined. In one publication, paternal and/or maternal morphine exposure increased phosphorylated CREB levels in the nucleus accumbens (Rohbani et al. 2019). However, another experiment showed the opposite effect of paternal morphine exposure in the nucleus accumbens of male progeny. Both phosphorylated CREB and the phosphorylated version of the activity-dependent kinase ERK were increased in the nucleus accumbens of male morphine-sired progeny (Ashabi et al. 2018). In the hippocampus, paternal morphine exposure decreased phospho-CREB levels in offspring (Ashabi et al. 2019). One consistent observation is that phosphorylated CREB is not changed by paternal opioid exposure in the prefrontal cortex of first-generation male progeny (Ashabi et al. 2018, 2019). The expression of the CREB target gene Bdnf was increased in the prefrontal cortex of both male and female offspring of morphine-exposed sires that were treated during adolescence (Vassoler et al. 2020). On the other hand, paternal morphine exposure decreased Bdnf expression in the nucleus accumbens of male progeny (Rohbani et al. 2019). Dopaminergic signaling is also impacted in a sex- and region-specific manner by prenatal opioid exposure. Microinjections of the D1 receptor antagonist into the hippocampus or prefrontal cortex ameliorated spatial memory deficits of male morphine-sired progeny (Ashabi et al. 2019). Protein expression of the D2-like dopamine receptors was increased in the nucleus accumbens of morphine-sired male offspring (Rohbani et al. 2019). The dopamine neurons of the ventral tegmental area (VTA) had less spontaneous burst firing in the progeny of sires exposed to morphine during adolescence (Azadi et al. 2019). The reduction in burst event frequency, burst duration, and bursting activity of the VTA is consistent with the resistance to low doses of morphine observed in F1 morphine-sired progeny observed in this publication (Azadi et al. 2019). This reduction in dopamine neuron activity could be compounded by increased degradation of dopamine in the nucleus accumbens, a prominent efferent projection of the VTA. Protein expression of the monoamine oxidase B enzyme that metabolizes dopamine was increased by paternal and/or maternal morphine exposure in the nucleus accumbens of male offspring (Sadat-Shirazi et al. 2019). Moreover, the baseline-firing rate of the nucleus accumbens and prefrontal cortex was reduced in the male progeny of morphine-treated sires (Ashabi et al. 2018). The locus coeruleus (LC) plays a pivotal role in mediating the development of tolerance associated with chronic opioid consumption. Changes in LC function in response to opioid exposure also mediate withdrawal signs produced by abstinence in opioid-dependent subjects (Rasmussen et al. 1990; Christie et al. 1997; Mazei-Robison and Nestler 2012; Scavone et al. 2013; Van Bockstaele and Valentino 2013). Interestingly, paternal exposure to morphine during adolescence changed the baseline properties of the LC in drug-naive F1 progeny. Noradrenergic neurons of morphine-sired male offspring showing decreased decay in action potential slopes and higher after hyperpolarization amplitude (Pachenari et al. 2019). Taken together, these findings demonstrate that paternal opioid exposure alters neurodevelopment in a region-specific manner that results in long-lasting adaptations in neuronal activity and cellular signaling pathways within brain regions critical for cognition, reward, and affective behaviors.

Limitations

Studying the impact of paternal opioid exposure on behavior in future generations is inherently fraught with several potentially confounding factors. Performance on the behavioral tasks discussed above can be altered by stress responsivity, motor function, as well as anxiety-like and depressive-like behaviors. These important caveats are not always considered, in part because this kind of research is extremely time-demanding and financially burdensome. There are several reports of increased anxiety-like behavior in progeny of opioid-exposed sires (Pooriamehr et al. 2017; Rohbani et al. 2019; Sabzevari et al. 2019), with some exceptions (Ellis et al. 2020). Neuroendocrine systems can also be disrupted by paternal adolescent opioid exposure. Serum testosterone and serum LH levels were deceased in male offspring of sires exposed to opioids during adolescence. The weight of adrenal glands of morphine-sired male offspring was higher compared to controls while serum corticosterone was unaltered. In contrast, serum corticosterone levels were higher in female morphine-sired progeny. Additionally, β-endorphin secretion in the hypothalamus was increased in female morphine-sired progeny (Cicero et al. 1989). These results emphasize the potential impact of paternal opioid exposure on neuroendocrine function in male and female progeny, which represent a substantial caveat that should be rigorously examined and addressed in these kinds of multigenerational behavioral assessments. It is not clear whether neuroendocrine disruptions also contribute to the sex-specific nature of many inherited phenotypes in the first-generation progeny. More research is needed to determine whether the bias toward male-specific phenotypes that are the currently prevailing trend are driven by biology or a dearth of examination of female offspring. Another important consideration that is rarely addressed in this space has to do with potential alterations in maternal behavior. Male rodents do not actively participate in raising developing pups in a laboratory setting and are often separated from the females shortly after mating. However, there is evidence that dams can sense the fitness of their mate and adjust their level of maternal care toward their pups accordingly (Drickamer et al. 2000; Sheldon and Smith 2000; Gowaty et al. 2007). The differential allocation hypothesis (Burley 1988) posits that maternal behavior could be impacted by paternal insults such as opioid exposure, a possibility that has been largely ignored in the field so far. There are many important developmental events between the preconception paternal opioid insult and behavioral assessment in adult progeny, which complicates the interpretation of results especially when the aforementioned caveats are not thoroughly and systematically addressed.

CONCLUSIONS

Opioid exposure has profound and long-lasting consequences on many biological functions in parental and future generations. The recurring theme of this rapidly growing body of literature is that the exact timing, nature, dosing, and duration of opioid exposure combined with other important environmental considerations that are not directly related to the exposure itself influence the extent to which these manipulations affect parents and their progeny. The phenotypes produced range from subtle to extremely overt. Human studies are constrained by the difficulty, complexity, and cost associated with the kinds of multigenerational and longitudinal studies. It remains difficult to determine whether and how the wealth of data coming from the preclinical literature applies to children of parents that were exposed to opioids prior to conception. As the number of individuals prescribed opioids for pain management has grown and the opioid addiction epidemic continues to ravage the world, the long-term impact on future generations remains an important area of research.