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Published in final edited form as: Neurosci Lett. 2022 Jan 24;773:136479. doi: 10.1016/j.neulet.2022.136479

HPA Axis Dysfunction during Morphine Withdrawal in Offspring of Female Rats Exposed to Opioids Preconception

Fair M Vassoler 1,*, Sara B Isgate 1, Kerri E Budge 1, Elizabeth M Byrnes 1
PMCID: PMC8908356  NIHMSID: NIHMS1777952  PMID: 35085692

Abstract

Opioid use and abuse remain a significant public health problem, particularly in the United States. Indeed, it is estimated that up to 10% of youths (age 12–18) have taken opioids illicitly. A growing body of evidence suggests that this level of widespread opioid exposure can have effects that extend to subsequent generations. Utilizing a well-established rodent model of preconception adolescent opioid exposure in females, we found decreased opioid self-administration coupled with increased cocaine self-administration in adult offspring. This bidirectional effect may be related to negative affect associated with opioid withdrawal, including enhanced stress reactivity. In this study, we tested the hypothesis that the adult offspring of females exposed to morphine during adolescence will demonstrate increased signs of opioid withdrawal when compared to offspring of saline controls. Females were administered increasing doses of morphine (5–25 mg/kg s.c.) or saline (1 ml/kg) from postnatal day 30 (PND30)-PND39. They were then maintained drug free for a minimum of 4 weeks and mated with drug-naïve males on or after PND70. As adults, their male and female offspring (referred to as Mor-F1 or Sal-F1) were administered morphine (10 mg/kg s.c.) twice a day for 5 days. They were then tested for spontaneous withdrawal behaviors for the next 4 days (~PND70). Levels of corticotropin releasing hormone (Crh) and urocortin 3 (Ucn3) were examined in the amygdala at 48 hours and 96 hours of withdrawal. Circulating corticosterone was measured at 48 hours. Results indicate that Mor-F1 males are heavier than Sal-F1 males with no baseline differences in females. However, Mor-F1 females did not gain weight at the same rate as Sal-F1 females during withdrawal. While there were no differences in somatic withdrawal signs, gene expression data revealed a sex-specific and time-dependent effect on Crh as well as increased Ucn3 and corticosterone in females at 48hrs withdrawal. Overall, these data point to differences in withdrawal and stress reactivity in Mor-F1 animals that may contribute to observed differences in addiction-like behaviors.

Keywords: Intergenerational, opioid, morphine, withdrawal, crh, ucn3, corticosterone

1. Introduction

A growing body of evidence indicates that opioid exposure in one generation can have effects that extend to subsequent generations even in the absence of in utero exposure [1–18]. Such observations are particularly concerning when one considers the increased exposure to opioids experienced in many populations during the current opioid epidemic [19]. While the mechanism of transmission of such effects is still unclear, there is evidence that in addition to effects on some aspects of parental behavior, epigenetic modifications to germ cells and miRNA regulation may play a role [14, 20, 21]. Many phenotypes have been examined in offspring and grandoffspring of exposed animals, including phenotypes related to substance use disorder, anxiety, depression, learning and memory, attention, as well as endocrine and molecular modifications [14, 20].

Recently, we observed alterations in intravenous self-administration of both opioids and cocaine [11, 13] in offspring of rat dams with a history of opioid exposure during adolescence.

Intriguingly, these effects were bi-directional, with a decrease in opioid self-administration [11] and an increase in cocaine self-administration [13] in Mor-F1 animals (i.e. offspring of adolescent opioid exposed females) when compared to their Sal-F1 counterparts. These bidirectional changes were also reflected in circulating corticosterone in response to an acute injection of morphine or cocaine [12, 13]. Thus, there were increased levels of corticosterone following acute cocaine and decreased corticosterone following morphine in Mor-F1 compared to Sal-F1. Taken together, these findings suggest that activation of the hypothalamic pituitary adrenal (HPA) axis in response to both opioids and stimulants is altered in Mor-F1 animals which could impact subsequent self-administration behavior. It is also possible that the experience of opioid withdrawal in Mor-F1 animals differs from that of Sal-F1 animals, resulting in decreased opioid self-administration [22]. While reducing the symptoms of withdrawal from opioids is a key motivator for drug seeking following prolonged opioid exposure, a greater sensitivity to the aversive effects of withdrawal is hypothesized to be protective against the development of opioid use disorder [23–25]. Indeed, a recent analysis found that sensitivity to withdrawal from opioid agonists and stress reactivity were the only reliable predictors of individual differences in self-administration of drugs of abuse [26].

The purpose of the current study was to measure spontaneous withdrawal from opioids in Mor-F1 and Sal-F1 males and females to determine if differences in withdrawal play a role in decreased levels of opioid self-administration observed in Mor-F1 animals. Withdrawal from opioids can be measured in both humans and animals and while it can be induced after a single opioid exposure [27], the signs/symptoms are more severe with repeated drug exposures [28]. During withdrawal from repeated morphine exposure, animals and humans experience myriad negative physiological effects including homeostatic dysregulation, pain and discomfort. In rats, this manifests as diarrhea, excessive urination, salivation, jumping, wet dog shakes, decreased pain threshold, and writhing [29]. In addition to these behavioral and physiological signs, stress-related brain circuits are also activated during withdrawal [30]. Indeed, the amygdala is known to play a role in producing negative emotional states associated with opioid withdrawal [31] and has been shown to have increased activity during withdrawal as measured via manganese-enhanced magnetic resonance imaging (ME-MRI) [32]. In addition, the amygdala receives projections from the periaqueductal gray, which regulates pain and discomfort and is associated with anxiety, fear, and nociception linked with chronic exposure to drugs of abuse and withdrawal [33]. The amygdala is also associated with severity of opioid withdrawal symptoms in humans [34]. Therefore, the amygdala represents a key target to examine molecular changes that may underlie the negative state of opioid withdrawal.

During opioid withdrawal, there is an upregulation of corticotropin-releasing hormone (CRH) within the paraventricular nucleus of the hypothalamus as well as activation of neurons within the amygdala [35]. The CRH family of proteins also includes urocortin III (UCN3), which binds to CRH receptors and is involved in the stress response. Specifically, UCN3 is involved in the regulation of aspects of appetite changes within the stress response, changes that are also known physiological symptoms of withdrawal [36, 37]. Therefore, in addition to corticosterone released from the adrenal medulla, CRH and UCN3 are key peptides involved in the stress associated with opioid withdrawal. Therefore, we measured gene transcription of Crh and Ucn3 within the amygdala in F1 animals during spontaneous withdrawal from opioids as well as levels of circulating corticosterone as measures of withdrawal-induced stress [38–41]. Our working hypothesis was that Mor-F1 animals would demonstrate more severe signs of withdrawal, increased levels of Crh and Ucn3 within the amygdala and higher levels of circulating corticosterone during withdrawal when compared to their Sal-F1 counterparts.

2. Methods

2.1. Animals and housing

All animals were housed in standard acrylic laboratory cages (40 cm x 20 cm x 18 cm) at Cummings School for Veterinary Medicine at Tufts University. Animals were maintained on a 12-hour light/dark cycle with lights on at 7:00 am and all procedures were performed during the light phase. Food and water were available ad libitum. Animals were acclimated to the housing conditions for at least seven days prior to experimentation. All procedures were approved by the Institutional Animal Care and Use Committee of Tufts University and were carried out in accordance with the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal distress and reduce the number of animals used.

2.2. Adolescent morphine exposure

Post-natal day 23 (PND23) female Sprague-Dawley rats [Crl:CD(SD)BR] were purchased from Charles River Breeding Laboratories. All animals were housed 2–3 per cage. As previously described [10, 11, 13], beginning at PND30 females (n=8) were injected (s.c.) once daily with morphine sulfate (MS) for a total of 10 days using an increasing dosing regimen with doses increased every other day (5, 5, 10, 10, 15, 15, 20, 20, 25, 25 mg/kg) (Shown in Schematic 1). These animals are termed Mor-F0. Age-matched control animals (n=8) received saline injections (s.c) with volumes adjusted to match those of drug-treated females (Sal-F0). On PND 70–80 (4 to 6 weeks after their final injection), females were mated with drug-naïve colony males. Females remained group housed until the week prior to parturition. On PND1 (day of birth = PND0) all litters were culled to ten pups (5 male, 5 female). The weight of the pups and the dam was recorded. All litters were weighed and weaned on PND21 and housed with same-sex littermates. No differences in bodyweights were observed at either time point (data not shown). All testing was conducted once F1 animals were at least 60 days of age.

Schematic 1. Timeline of experimental procedures.

Schematic 1.

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The manipulations of the dams (F0 generation) and the offspring (F1 generation) are depicted on the top and bottom lines, respectively. Morphine injections are depicted in blue and withdrawal timepoints are depicted in red.

2.3. F1 Morphine Regimen and Withdrawal Behavior

As shown in schematic 1, a total of 64 male and female Sal-F1 and Mor-F1 rats (>PND60) were weighed daily and administered subcutaneous injections of morphine (10mg/kg) at 8am and 4pm daily for 5 days. The withdrawal behavior was monitored and recorded at 24h, 48h, 72h, and 96h after their last injection in a clear Plexiglas observation chamber (40 cm x 20 cm x 18 cm). Withdrawal monitoring included both counted signs and checked signs modified from the Gellert and Holtzman rating scale. The counted signs of interest were teeth chattering, wet dog shakes, writhing, jumping, climbing, and digging; the checked signs were porphyrin staining, diarrhea, and ptosis [29, 42]. These symptoms were observed for a total of 10min. The withdrawal monitoring signs and behaviors were added together to make a single withdrawal score. All withdrawal signs and behaviors were weighted equally. Forty-eight hours into withdrawal (after the final injection of morphine), half of the animals were sacrificed by exposure to CO2 for less than 1min and decapitation; their brains extracted, and flash frozen in 2-methylbutane (−20C). The remaining animals continued withdrawal observations at 72, and 96hr after their last morphine injection. These animals were then be sacrificed according to the previously mentioned procedure.

2.4. Quantitative Real Time Polymerase Chain Reaction (qRT-PCR)

Animals sacrificed at 48 and 96h withdrawal timepoints were utilized for qRT-PCR analysis. To collect samples for analysis of gene expression, frozen brains were mounted on a cryostat and bilateral micropunches were taken from the amygdala (2 mm; starting at: −1.7 mm A/P, +/−3.5 mm M/L, −8 mm D/V, a region encompassing basal lateral amygdala, central amygdala and basal medial amygdala) according to Paxinos and Watson [43]. Tissue punches were homogenized in lysis buffer and total RNA extracted using the RNeasy kit from Qiagen (Valencia, CA, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using the RETROscript kit from Applied Biosystems (Carlsbad, CA, USA).

PCR was performed using an AB 7500 (Applied Biosystems) under standard amplification conditions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C. All PCR primers were TaqMan® Gene Expression Assays purchased from Applied Biosystems. The amplification efficiency of each of these assays has been validated by Applied Biosystems and averages 100% (± 10). Assay ID and accession numbers were as follows: HPRT: Mm03024075_m1; Rn01Crh: Rn01462137_m1; Ucn3:Rn02091611_s1. Final quantification of mRNA was obtained using the comparative cycle threshold (CT) method (User Bulletin #2, Applied Biosystems). Data are reported as relative transcription relative to a calibrator cDNA. In brief, the housekeeping gene HPRT was used as an internal control against which each target signal was normalized (ΔCT). Validation studies confirmed that the raw CT values of HPRT did not vary by treatment group. The ΔCT was then normalized against a calibrator (i.e. the mean of the control group for the target gene in each brain region) to provide the ΔΔCT relative to the control group. Finally, data are transformed 2^ ΔΔCT and expressed as fold change.

2.5. Corticosterone Enzyme Linked Immunosorbent Assay (ELISA)

Plasma corticosterone was determined using a standard ELISA kit according to manufacturer’s directions (Enzo Life Sciences #ADI-900–097, Farmingdale, NY). All samples were run in duplicate and an internal control was utilized across plates. Some samples were diluted 1:1. The coefficient of variance among the duplicates was less than 16%. The intraassay variability was less than 10%. Plates were read and quantified (4-parametric logistics, non-linear regression model) using GenTech plate reader and Gen5.0 software for analysis and standard curve interpolation.

2.6. Statistics

Bodyweight during morphine injections, during withdrawal, and the overall withdrawal scores were analyzed with two-way ANOVAs with maternal preconception adolescent exposure and day as factors. Total change in bodyweight data as well corticosterone data were analyzed using a two-tailed students’s t-test. Bodyweight change during withdrawal as well as qPCR data were analyzed using two-way ANOVAs with maternal preconception adolescent exposure and time as factors. Sidak or Bonferroni’s post hocs were used when appropriate. Significance was defined as p<0.05. Both sexes were analyzed in order to analyze sex as a biological variable. However, sexes were analyzed separately as we were not specifically interested in sex differences. Power analysis performed with G*Power (version 3.1.9.4) using effect sizes calculated with our data, alpha error probability set to 0.05 and power set to 0.80 revealed appropriately powered experiments.

3. Results

3.1. Bodyweight during morphine injections

Prior to their first morphine injection, adult Mor-F1 males weighed more than age-matched Sal-F1 males. A repeated measures two-way ANOVA was utilized to analyze bodyweights across days. As shown in Figure 1A, there was a main effect of maternal preconception adolescent exposure [F (1,29)=6.895; p=0.01] as well as a significant main effect of day [F (4, 116) = 3.219; p=0.015] but no interaction [F (4, 116) = 1.387; p=0.24]. Post hoc analysis revealed that Mor-F1 males were significantly heavier than Sal-F1 males and that all animals demonstrated a decrease in weight from days 4 compared with day 2 (p<0.05). A student’s t-test was utilized to examine differences between the Sal-F1 and Mor-F1 males’ total weight change across the 5 days of morphine injections. There was no significant difference between the bodyweight change of Mor-F1 and Sal-F1 males (p=0.16). A different pattern of effects was observed in F1 females (Figure 1B). A repeated measures two-way ANOVA revealed a significant main effect of day [F (4, 120) = 3.253; p=0.01] as well as a significant interaction [F (4, 120) = 2.82; p=0.03], but no main effect of maternal preconception adolescent exposure [F (1, 30) = 2.946; p=0.09]. Post-hoc analysis revealed that Sal-F1 but not Mor-F1 had increased bodyweight on Day 3 compared with day 1 and 5. A students’ t-test was used to examine the total body weight change across the injection days and found no significant difference between Mor-F1 and Sal-F1 females (p=0.25).

Figure 1. Bodyweight during twice daily morphine injections.

Figure 1.

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The body weight in grams (mean +/− SEM) of the Sal-F1 and Mor-F1 males (Panel A) and females (Panel B) during the 5 days of morphine injections as well as the mean +/− SEM change in bodyweight across the 5 days. *p<0.05 main effect of maternal adolescent treatment; N=16/group

3.2. Bodyweight during withdrawal

Following the 5 days of twice daily morphine injections, all animals were weighed and observed for signs of spontaneous withdrawal for 96 hours. Bodyweights during withdrawal are presented in Figure 2. Panel 2A shows mean (+/−SEM) bodyweight of F1 males on the last day of morphine injections (D5) followed by the 4 days post-withdrawal. These data were analyzed with a two-way ANOVA, which revealed a significant main effect of maternal preconception adolescent exposure [F (1, 115) = 32.11; p<0.0001] but no effect of day [F (4, 115) = 0.5151; p=0.72] and no interaction [F (4, 115) = 0.06074; p=0.99]. Consistently, the Mor-F1 males were significantly heavier than the Sal-F1 males throughout withdrawal. Because of the basal differences in bodyweight between the two groups, we examined the change in body weight from the last injection (Day 5). These data, presented in panel B, show the pattern of weight change during withdrawal in male F1 animals. These data were analyzed with a two-way ANOVA which showed a significant main effect of day [F (2, 72) = 20.89; p<0.0001] but no effect of maternal preconception adolescent exposure [F (1, 72) = 0.6338; p=0.42] and no interaction [F (2, 72) = 2.035; p=0.13]. Post hoc tests showed that animals gained weight across withdrawal time. Data from F1 females are presented in panels C and D and show a significant main effect of maternal preconception adolescent exposure [F (1, 118) = 6.149; p=0.01] on bodyweight. Post hoc analysis indicate that Mor-F1 females were heavier than Sal-F1 females (p<0.05). There was no main effect of day [ F (4, 118) = 0.7063; p=0.59] and no interaction [F (4, 118) = 0.4928; p=0.74]. When the body weight change across withdrawal timepoints was analyzed, the two-way ANOVA showed both a significant main effect of maternal preconception adolescent exposure [F (1, 74) = 7.196; p=0.009] as well as a significant main effect of day [F (2, 74) = 23.59; p<0.0001] with no interaction [F (2, 74) = 2.197; p=0.118]. Post hoc analysis revealed that only the Mor-F1 animals continued to show significant weight loss 48h post-withdrawal.

Figure 2. Bodyweight during withdrawal.

Figure 2.

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The body weight in grams (Mean +/− SEM) of the SAL-F1 and Mor-F1 males (Panel A) and females (Panel C) on the final day of injections (D5) and first 96 hours of withdrawal. The mean +/− SEM change in bodyweight from the last injection (D5) is also shown for males (Panel B) and females (Panel D). *p<0.05 N=8–16/group

3.3. Withdrawal behaviors

Withdrawal signs over time are presented in Figure 3 utilizing a single withdrawal score. As illustrated in Panel A there was a significant main effect of day [F (3, 86) = 3.023; p=0.034] on the number of withdrawal signs in male F1 rats. However, the main effect of maternal preconception adolescent exposure did not reach statistical significance [F (1, 86) = 2.9; p=0.09] and there was no interaction [F (3, 86) = 1.243; p=0.29]. For the females, there were no significant effects of maternal preconception adolescent exposure [F (1, 88) = 1.913; p=0.17], day [F (3, 88) = 2.266; p=0.09], or an interaction [F (3, 88) = 0.7501; p=0.53].

Figure 3. Withdrawal behavior score.

Figure 3.

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The mean +/− SEM combined withdrawal score of all checked and counted signs of withdrawal in males (Panel A) and females (Panel B) across the first 96 hours of withdrawal. N=8–16/group

3.4. Gene Expression Changes during Withdrawal

Half of all animals were euthanized following the 48h of withdrawal, and the other half at the 96-hour timepoint. Corticotropin releasing hormone (Crh) and urocortin 3 (Ucn3) were analyzed via qRT-PCR from tissue punches taken from the amygdala. Males and females were analyzed separately by two-way ANOVA. For the F1 males, Crh data showed a significant main effect of maternal preconception adolescent exposure [F (1, 27) = 4.369; p=0.04], as well as withdrawal time [F (1, 27) = 6.645; p=0.01], and a significant interaction [F (1, 27) = 5.211; p=0.03]. Post hoc analyses revealed that Mor-F1 males had significantly elevated levels of Crh in the amygdala 48 hours into withdrawal (Figure 4A). For Ucn3, there were no significant effects of maternal preconception adolescent exposure [F (1, 27) = 0.1239; p=0.72] or withdrawal time [F (1, 27) = 0.0003579; p=0.98], and no interaction [F (1, 27) = 0.116; p=0.73] (Figure 4B). For the females, analysis of Crh revealed a significant main effect of maternal preconception adolescent exposure [F (1, 28) = 6.392; p=0.01], as well as a main effect of withdrawal time [F (1, 28) = 22.78; p<0.0001], and a significant interaction [F (1, 28) = 4.802; p=0.03]. Post hoc analyses showed that Mor-F1 females had significantly lower levels of Crh in the amygdala 48 hours into withdrawal (Figure 4C). For Ucn3, there was no main effect of maternal preconception adolescent exposure [F (1, 27) = 1.263; p=0.27], nor a main effect of withdrawal time [F (1, 27) = 1.568; p=0.22], however, there was a significant interaction [F (1, 27) = 6.765; p=0.01]. Post hoc analyses found that Mor-F1 females had significantly higher levels of Ucn3 in the amygdala 48 hours into withdrawal (Figure 4D).

Figure 4. Gene expression in the amygdala during withdrawal.

Figure 4.

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The mean +/− SEM fold change in gene expression of Crh in males (Panel A) and females (Panel C) as well as Ucn3 in males (Panel B) and females (Panel D) at 48h and 96h withdrawal. *p<0.05 compared with Sal-F1 control. N=8/group

3.5. Circulating Corticosterone levels at 48h withdrawal

No differences in levels of corticosterone between the Sal-F1 and Mor-F1 males were observed [t(14)=0.068; p=0.9465] (Figure 5A). However, as shown in Figure 5B, there was a significant increase in corticosterone in the Mor-F1 females compared with the Sal-F1 controls [t(14)=2.287; p=0.04]. Unfortunately, due to a freezer failure, plasma samples from the 96 hour time point were unavailable.

Figure 5. Circulating plasma corticosterone during withdrawal.

Figure 5.

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The mean +/− SEM total circulating corticosterone levels (ng/ml) measured from plasma in males (Panel A) and females (Panel B) at 48h withdrawal. *p<0.05 compared with Sal-F1 control. N=8/group

4. Discussion

While there were no significant differences in somatic withdrawal behavioral signs between the Sal-F1 and Mor-F1 females, Mor-F1 females did demonstrate extended weight loss during withdrawal compared to Sal-F1 females, particularly at the 48-hour time point. This time point was also associated with alterations in stress-related peptides within the amygdala in both males and females. Mor-F1 females had decreased levels of Crh and increased levels of Ucn3 compared with Sal-F1 controls while the males demonstrated increased Crh in the amygdala at the 48-hour time point. This study also revealed significant changes in bodyweight at baseline. Mor-F1 males weighed significantly more than Sal-F1 males prior to and during morphine injections.

These findings support our recent findings demonstrating metabolic effects in Mor-F1 males when maintained on a high fat diet [8]. Increased levels of food-seeking, particularly of highly palatable foods, is known to have overlapping neural circuitry with substance use disorder [44, 45]. Therefore, it is possible that Mor-F1 animals have neurodevelopment alterations that impact both food consumption and drug self-administration. It should be noted that this phenotype is not consistently observed in Mor-F1 animals maintained on a diet of standard chow. That said, the baseline differences in bodyweight observed between Mor-F1 and Sal-F1 males likely did not contribute to the molecular differences observed during withdrawal as all doses are adjusted for weight and there is no literature to suggest that a 20–40g difference in an adult rat will impact gene expression. Yet, as this phenotype was observed in this study, it suggests that the neural circuitry underling feeding and/or metabolism may be vulnerable to the intergenerational effects of female adolescent morphine exposure. To what extent these effects relate to the bi-directional nature of opioid versus stimulant self-administration remains unknown.

In this study we did not observe significant differences in withdrawal between Mor-F1 and Sal-F1 animals. Withdrawal was measured by blinded observers and utilized a combination of counted and checked signs based on previous studies [29, 42]. While there may have been marginal trends between Mor-F1 and Sal-F1 males at 96 hours (p=0.07), generally the quantity of somatic signs of withdrawal were similar between F1 groups. Typically, physical signs of withdrawal increase and intensify between 48- and 96-hours following secession of opioid administration. This pattern was observed in all of our animals with withdrawal score increasing and peaking at 72 hours for both Mor-F1 and Sal-F1 animals of both sexes. For the male Mor-F1 animals, their overall withdrawal score was still elevated at 96 hours, while scores were decreasing for all other groups. While withdrawal can be consistently measured and observed using this strategy, such measures represent only one aspect of withdrawal.

Weight loss is another common sign of opioid withdrawal. In the current study, decreased body weight during withdrawal was observed in Mor-F1 females when compared to their Sal-F1 counterparts. This effect could suggest prolonged GI distress or a change in motivated feeding. Moreover, we cannot rule out differences in other aspects of withdrawal, including changes in emotional stress, which have been documented previously during opioid withdrawal [46]. To that end, we observed substantial differences in expression of stress-related genes in the amygdala during withdrawal as well as increased circulating corticosterone in Mor-F1 females. At the 48-hour timepoint, both Crh and Ucn3 were significantly dysregulated in Mor-F1 females compared to Sal-F1 controls, albeit in opposite directions, with Crh was downregulated and Ucn3 was upregulated. Interestingly, recent work has demonstrated that stimulation of the Crh system within the amygdala can promote reward consumption, so a decrease in Crh may be associated with a negative affective state of withdrawal [47]. In addition, Ucn3 is associated with food regulation during negative affective states such as withdrawal, so increased Ucn3 within the amygdala may partially underlie the reduced weight gain observed in females at the 48h time point [36, 37]. Taken together with increased corticosterone observed at this time, these data support the hypothesis that the stress associated with withdrawal may be exacerbated in Mor-F1 females. Future studies would benefit from examination of depression- and anxiety-like behaviors during withdrawal.

In Mor-F1 males we observed increased Crh in the amygdala at the 48h time point. Work on this system in rodents is predominantly performed in males even though substantial sex-differences exist within these systems[48–52]. In rodents, following initial exposure to opioids (which can increase Crh and corticosterone), opioid exposure suppresses the activity of the HPA axis [53]. However, during withdrawal from chronic opioid exposure, the HPA axis can become hyper-activated [54]. Thus, the changes that we observe in Crh in the amygdala are likely a manifestation of the stress response produced form opioid withdrawal, which is specific to males. To what extent the differences in Crh in Mor-F1 males and Crh and Ucn3 play a role in the blunted opioid self-administration observed in Mor-F1 animals remains to be determined, however, the current outcomes support further investigation. Indeed, there are other brain regions and neurotransmitter systems implicated in opioid withdrawal that may be impacting the behavior of F1 animals that also warrant further study. For example, the locus coeruleus (LC) which contains the majority of brain noradrenergic neurons and the lateral paragigantocellularis (LPGi) are well established as key mediators of opioid dependence and tolerance as well as withdrawal [55]. The LC and the LPGi are also sources of the neurotransmitter peptide orexin, which represents an additional neurotransmitter system that may contribute to opioid withdrawal [55]. Intriguingly, the LPGi was shown to mediate, at least in part, a similar effect in Mor-Sired animals [56]. In this study, Azadi et al., demonstrate that with an adolescent opioid exposure model in males (as opposed to females) the Mor-Sired males have augmented naloxone precipitated opioid withdrawal somatic signs compared with Sal-Sired males and that the LPGi plays a role in the phenotype [56]. The ventral tegmental area (VTA), not only contributes to the rewarding and euphoric aspects of opioids but plasticity in the VTA including with glutamatergic, GABAergic, and dopaminergic neurotransmitter systems also likely contribute to continued drug seeking, dependence, and withdrawal [57, 58]. Thus, the complexity of brain regions and systems involved in opioid withdrawal goes far beyond the scope of this study. Future studies could examine if altered expression or plasticity within any of these systems contribute to the observed phenotypes in F1 animals.

Finally, it is important to consider the mechanisms by which such effects may be passed from one generation to the next. While this was not examined in the current study, we have previously shown that maternal behavior does not play a major role in the transmission of effects similar to these [7] [59]. Therefore, it is likely that preconception morphine exposure is either acting directly on the germ cells or producing widespread physiological changes throughout the body that eventually impact that germ cell. Opioid receptors are expressed fairly ubiquitously throughout the brain and body including brain, GI tract, immune cells, and reproductive tissues [60]. Indeed, opioids play a known role in pregnancy, parturition, and lactation [60]. Therefore, maintained epigenetic changes to these tissues may play a role in the transmission of behavioral effects from one generation to the next. The interaction between the endogenous opioid receptor system and the reproductive axis may also explain some of the sex-specificity of the observed effects. For example, androgen receptor stimulation by testosterone has been shown to transcriptionally regulate peripheral mu opioid receptor expression [61], an effect that may be altered in Mor-F1 males. Opioid receptor expression within the hypothalamus may also play a role in the observed sex-specificity. The current study did not examine potential mechanisms of transmission, but a growing body of literature suggests that DNA methylation, histone modifications, and non-coding RNAs may all play a role in the transmission of inter- and trans-generational epigenetic inheritance [9, 14, 21, 62, 63].

5. Conclusions

In the current set of studies, we demonstrated changes in physiological, and molecular response to opioid withdrawal in Mor-F1 animals compared to Sal-F1 controls. These changes may play a role in the decreased morphine self-administration previously observed with this model, though they are likely only one piece to this complex phenotype. Additionally, the increased bodyweight observed at baseline in Mor-F1 males is consonant with the notion that the neural circuitry in Mor-F1 animals is significantly altered and may play a role in the increased acquisition and consumption of cocaine in these animals. Overall, the current set of findings provide additional evidence that preconception morphine exposure in adolescent females induces intergenerational effects on neural systems regulating adaptations in response to chronic opioid. The mechanisms underlying these effects remain unknown but point to significant adaptations in the HPA axis in Mor-F1 males and females.

Highlights.

  • Maternal history of opioid exposure increases offspring bodyweight

  • No changes in somatic withdrawal in males and females

  • Alterations of stress-related peptides in the amygdala during withdrawal

  • Increased corticosterone in females during withdrawal

6. Acknowledgments

We would like to thank Cristina Wyse for technical assistance and sample collection. In addition, this work was supported by NIH NIDA R01DA025674, BBRF NARSAD YIA 26771, and the Tufts Substance Use Initiative Seed Grant.

Footnotes

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