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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 66
| Issue : 2 | Page : 65-72 |
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Comparisons of stress-related neuronal activation induced by restraint in adult male rat offspring with prenatal exposure to buprenorphine, methadone, or morphine
Chia-Yen Wu1, Hwei-Hsien Chen2, Pao-Luh Tao2, Zung Fan Yuan3
1 Department of Physiology, Tzu Chi University, Hualien, Taiwan
2 Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan
3 Department of Physiology, Tzu Chi University; Master Program in Biomedical Sciences, School of Medicine, Tzu Chi University, Hualien, Taiwan
| Date of Submission | 01-Feb-2023 |
| Date of Decision | 25-Feb-2023 |
| Date of Acceptance | 06-Mar-2023 |
| Date of Web Publication | 20-Apr-2023 |
Correspondence Address:
Dr. Zung Fan Yuan
Department of Physiology, Tzu Chi University, 701, Section 3, Chung-Yang Road, Hualien, 970
Taiwan
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/cjop.CJOP-D-23-00015
Prenatal opioid exposure may impede the development of adaptive responses to environmental stimuli by altering the stress-sensitive brain circuitry located at the paraventricular nucleus of the hypothalamus (PVH) and locus coeruleus (LC). Corticotropin-releasing factor (CRF) released from neurons in the PVH has emerged as a key molecule to initiate and integrate the stress response. Methadone (Meth) and buprenorphine (Bu) are two major types of synthetic opioid agonists for first-line medication-assisted treatment of opioid (e.g., morphine, Mor) use disorder in pregnant women. No studies have compared the detrimental effects of prenatal exposure to Meth versus Bu on the stress response of their offspring upon reaching adulthood. In this study, we aimed to compare stress-related neuronal activation in the PVH and LC induced by restraint (RST) stress in adult male rat offspring with prenatal exposure to the vehicle (Veh), Bu, Meth, or Mor. CFos-immunoreactive cells were used as an indicator for neuronal activation. We found that RST induced less neuronal activation in the Meth or Mor exposure groups compared with that in the Bu or Veh groups; no significant difference was detected between the Bu and Veh exposure groups. RST-induced neuronal activation was completely prevented by central administration of a CRF receptor antagonist (α-helical CRF9-41, 10 μg/3 μL) in all exposure groups, suggesting the crucial role of CRF in this stress response. In offspring without RST, central administration of CRF (0.5 μg/3 μL)-induced neuronal activation in the PVH and LC. CRF-induced neuronal activation was lessened in the Meth or Mor exposure groups compared with that in the Bu or Veh groups; no significant difference was detected between the Bu and Veh exposure groups. Moreover, RST- or CRF-induced neuronal activation in the Meth exposure group was comparable with that in the Mor exposure group. Further immunohistochemical analysis revealed that the Meth and Mor exposure groups displayed less CRF neurons in the PVH of offspring with or without RST compared with the Bu or Veh groups. Thus, stress-induced neuronal activation in the PVH and LC was well preserved in adult male rat offspring with prenatal exposure to Bu, but it was substantially lessened in those with prenatal exposure to Meth or Mor. Lowered neuronal activation found in the Meth or Mor exposure groups may be, at least in part, due to the reduction in the density of CRF neurons in the PVH.
Keywords: Buprenorphine, methadone, morphine, prenatal opioid exposure, restraint
How to cite this article:
Wu CY, Chen HH, Tao PL, Yuan ZF. Comparisons of stress-related neuronal activation induced by restraint in adult male rat offspring with prenatal exposure to buprenorphine, methadone, or morphine. Chin J Physiol 2023;66:65-72 |
How to cite this URL:
Wu CY, Chen HH, Tao PL, Yuan ZF. Comparisons of stress-related neuronal activation induced by restraint in adult male rat offspring with prenatal exposure to buprenorphine, methadone, or morphine. Chin J Physiol [serial online] 2023 [cited 2023 May 29];66:65-72. Available from: https://www.cjphysiology.org/text.asp?2023/66/2/65/374411 |
| Introduction | |  |
Opioid use in pregnancy has escalated in recent years, raising great concerns regarding its negative effect on both the pregnant woman and her child.[1] Prenatal opioid exposure has been shown to negatively affect various long-term neurocognitive outcomes in children or in adult offspring,[2],[3],[4] including the stress response.[4],[5],[6] The major neural pathway conveying stress-related signals originates from the activation of certain neurons in the paraventricular nucleus of the hypothalamus (PVH) to secrete corticotropin-releasing factor (CRF), which stimulates the release of the adrenocorticotropic hormone from the pituitary and glucocorticoid from the adrenal glands.[6],[7] This hypothalamic-pituitary-adrenal (HPA) axis has been recognized as one of the main stress response pathways.[6],[7] In addition, stress activates certain neurons in the locus coeruleus (LC) to release norepinephrine (NE), one of the key neurochemical mediators of stress responses in the brain.[6],[8] Reciprocal neural connections exist between the CRF neurons of the PVH and NE neurons of the LC, suggesting that these two sites are major central coordinators of the stress system.[6],[9] Evidence suggests that stress-induced activation of neurons at these two sites is under opioidergic modulation.[10],[11] Notably, many animal studies have reported that the response to stress is dysregulated in adult rats subsequent to prenatal opioid exposure.[4],[5],[6],[12],[13],[14],[15],[16],[17],[18],[19],[20] Given that the ability to activate the stress response system is a normative response serving as an adaptive function, the disrupted stress response suggests damaged adaptive capability and vulnerability to mood or behavioral disorders in offspring.[5],[6],[9] Accordingly, prenatal opioid exposure may impede the development of adaptive responses to environmental stimuli by altering the stress-sensitive brain circuitry or HPA axis.[4],[6],[15]
Methadone (Meth) and buprenorphine (Bu) are two major types of synthetic opioid agonists for the first-line medication-assisted treatment of opioid use disorder in pregnant women.[1],[21] Meth is a full opioid agonist with high affinity at the μ receptor, whereas Bu is a partial opioid agonist at the μ receptor and an antagonist at the κ receptor.[1],[21] Meth has an over 50-year history of medical use, whereas Bu was approved by the U. S. Food and Drug Administration in 2002.[1] Previous investigations have reported that prenatal exposure to either Meth or Bu has a negative impact on cognitive, psychomotor, and behavioral outcomes in children up to 15 years old.[2],[3],[21],[22],[23],[24] Several experimental studies have also shown that in utero exposure to either Meth or Bu impairs neurocognitive, locomotive, or sensory functions in rats up to 5 months old (equivalent to a teenager in humans).[22] As these two medications have several advantages and disadvantages for pharmacotherapy in pregnancy,[1],[22] much interest has been paid to compare the benefit/risk profile of prenatal exposure to Meth versus Bu, mainly regarding short-term neonatal outcomes.[25],[26] However, findings regarding long-term neurocognitive outcomes in adults following prenatal opioid exposure are unreliable due to existing confounding factors and lack of control.[1],[2],[3],[4],[22] This knowledge gap can be closed by the observations from animal studies.[1],[4],[22] Nevertheless, no study has compared the detrimental effects of prenatal exposure to Meth versus Bu on the stress-sensitive brain circuitry.
In this study, we aimed to compare stress-related neuronal activation induced by restraint (RST) stress in adult male rat offspring with prenatal exposure to Bu, Meth, or morphine (Mor). To achieve this goal, we measured the number of cFos-positive cells in PVH and LC as an index of neuronal activation in a well-established prenatal opioid-exposed animal model.[27],[28],[29] Offspring with prenatal exposure to Mor served as the positive control group.
| Materials and Methods | | |
Animals
Before labor, pregnant Sprague Dawley rats (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) at embryonic day 2 (E2) were housed in cages (two per cage) in a colony room with controlled temperature (25°C), humidity (50% ±10%), and a 12 h day–night cycle (light on 7:00 a.m.–7:00 p.m.) at the Laboratory Animal Center of the National Health Research Institutes. After labor, the mother rats and their offspring were immediately transferred to a separate cage until weaning at postnatal day 28. The weaned offspring were then transported to Tzu Chi University. Four to five rats were housed per cage in a controlled colony room (light on 7:00 a.m. – 7:00 p.m., room temperature [20°C–25°C], and humidity [55%–60%]) in the Laboratory Animal Center of Tzu Chi University. All the animals were provided with food and water ad libitum. At 7–8 weeks old, the rats were allowed to undergo experimental procedures. The offspring from the same dam were randomly assigned to different experiments to avoid the litter effect. All drug injections and RST or sham treatment procedures were performed from 7:30 a.m. to 9:00 a.m., and all rats were sacrificed before 12:00 noon. All animal experiments were approved by the Institutional Animal Care and Use Committee of Tzu Chi University (approval number: 100014).
Drugs
Bu (Sigma-Aldrich, St. Louis, MO, USA), Meth (USP, Rockville, MD, USA), and Mor (National Bureau of Controlled Drugs, Taipei, Taiwan) were dissolved in distilled water and administered subcutaneously (s.c.) at a volume of 1.0 mL/kg body weight. CRF (Sigma-Aldrich) and α-helical CRF9-41 (a CRF receptor antagonist, Sigma-Aldrich) were dissolved in saline with concentrations of 0.5 μg/3 μL and 10 μg/3 μL, respectively, and given intracerebroventricularly (i.c.v.) with a slow injection by an injector (30 G, 12 mm in length) at a volume of 3 μL within 90 s.
Prenatal opioid exposure
The doses and methods of prenatal opioid exposure have been described previously.[22],[23],[24] In brief, pregnant rats were randomly assigned to four experimental groups and received s. c. injection with one of the three opioid drugs or vehicle (Veh) during E3–E20. In Group 1 (Veh control, Veh), rats received 1 × PBS at 1 mL/kg twice per day. In Group 2 (Bu), the rats received Bu at 3 mg/kg once per day. In Group 3 (Meth), rats received Meth at 5 mg/kg twice per day during E3 and then at 7 mg/kg twice per day during E4–E20. In Group 4 (Mor), rats received Mor, 2 mg/kg (initial dose) to 4 mg/kg (final dose), twice a day with an increment of 1 mg/kg per week.
Restraint
The stress paradigm was restraining the offspring individually in a plexiglass cylinder (9 cm × 7 cm × 15 cm) for 30 min in their cages. After RST, the animals were released and stayed in their cages for 2 h before they were sacrificed. The offspring without RST (No-RST) were handled by gently picking up the animals as the sham treatment group.
Guide cannula implantation
For i.c.v. injection of CRF or CRF receptor antagonist, each rat was implanted with a stainless steel guide cannula (23G, 11 mm in length) toward the right lateral cerebral ventricle (coordinator: AP: −0.2 mm; ML: −1.2 mm; DV: −3.2 mm) under anesthesia (ketamine/xylazine/acepromazine = 50/10/2 mg/kg, s.c.) using a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). The rats were allowed to recover in their cages for 7 days before any experimental procedure, wiped with antibiotic ointment (chlortetracycline; Pfizer, Taipei, Taiwan) on the wounds, and injected subcutaneously a nonsteroidal anti-inflammatory drug (carprofen, 5 mg/kg per day; Vetnostrum, Taipei, Taiwan) for 5 days.
Process of brain tissues
The offspring rats under deep anesthesia (35% chloral hydrate in 1 mL/kg, intraperitoneal injection; Sigma-Aldrich) were perfused with saline, followed by ice-cold 4% paraformaldehyde in 0.1 M borate buffer through the ascending aorta. Their brains were removed immediately, postfixed for 3 h, and cryoprotected in 20% sucrose in 0.1 M potassium phosphate buffer (KPBS) at 4°C overnight. Five analogous series of 30 μm thick frozen coronal sections across the entire brain were collected and stored in cryoprotectant (30% ethylene glycol and 20% glycerol in 0.05 M sodium phosphate buffer) at −20°C until the immunohistochemical process. The immunoreactivity of protein was detected using an avidin-biotin-immunoperoxidase technique. Immunohistochemistry (IHC), including treatment of 0.3% hydrogen peroxide and 0.5% sodium borohydride, was performed on free-floating sections. All the antibodies were tested with several titers (including one without antibody) to obtain the lowest and most reliable titer before the IHC procedure. One series of brain sections of each rat was incubated in cFos antiserum (1:40,000; rabbit polyclonal antibody; RRID: AB_2800543; a gift from Dr. Sawchenko, LNSF, Salk Institute for Biological Studies, La Jolla, CA, USA) or CRF antibody (1:20,000, Cat#: T-4037; RRID: AB_518252; Peninsula, San Carlos, CA, USA) at 4°C for 2 days in KPBS with 0.3% Triton X-100 and 2% normal goat serum. The primary antibody was stained with VECTASTAIN Elite Reagents (Vector Laboratories, Burlingame, CA, USA), and the reaction product was developed using a nickel-enhanced glucose oxidase method. For CRF and cFos double staining, the brain sections that contained a PVH were processed as follows: cFos was revealed using 3´-diaminobenzidine (DAB, Sigma-Aldrich) intensified with nickel for staining with black, followed by CRF with DAB without nickel for staining with brown.
Experiment protocols
A total of 100 rats (weight of 280–350 g) were used to conduct four series of experiments [Figure 1]. These rats were the offspring from Groups 1 to 4 with different prenatal exposures. In these experiments, after RST treatments and/or drug interventions, the rats were allowed to stay in their home cages for 2 h before being sacrificed. Their brains were removed immediately and then sliced at 30 μm thickness for five mutual series of sections. For each rat, one series of sections was used for staining cFos and/or CRF to evaluate the levels of neuronal activation or density of CRF neurons. Experiment 1 was performed to investigate the effects of prenatal opioid exposure on RST-induced neuronal activation. Twelve offspring from each prenatal exposure group were randomly divided into the RST and No-RST groups (each n = 6). Experiment 2 was performed to study the effect of a CRF receptor antagonist on RST-induced neuronal activation. At 10 min before RST, six offspring from each prenatal exposure group received i.c.v. α-helical CRF9-41 (10 μg/3 μL). Experiment 3 was performed to investigate the neuronal activation induced by CRF. Six offspring from each prenatal exposure group without any manipulation received central administration of CRF (0.5 μg/3 μL, i.c.v.). Experiment 4 was performed to investigate the effect of prenatal opioid exposure on the density of CRF neurons. One offspring from each prenatal exposure group without any manipulation was used for staining of CRF under basal conditions, whereas one other offspring from each prenatal exposure group with RST was used for double staining of cFos and CRF under stress conditions. | Figure 1: Schematic illustration showing the experimental protocols of this study. Four series of experiments were conducted. A total of 100 adult male rat offspring with prenatal exposure to vehicle, buprenorphine, methadone, or morphine were used. RST treatment: Restraint treatment, No-RST: Sham treatment, i.c.v. injection: Intracerebroventricular injection, CRF: Corticotropin-releasing factor.
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Data analysis for cFos-ir or corticotropin-releasing factor-ir profiles
The quantification of cFos-immunoreactive (cFos-ir) or CRF-ir profiles was performed under a light microscope (DM 2000, Leica, Germany). The investigator who conducted this step was blinded to the treatment received by the offspring. The cFos-positive nuclear profiles (diameter >4.5 μm) at both sides of PVH and LC were counted under a magnification of 20×. The borders of PVH and LC were defined based on Nissl-stained reference samples. Three to four consecutive sections contained the PVH, whereas four to five sections contained the LC. The total number of cFos profiles in each bilateral nucleus was estimated by multiplying by 5 and corrected for double-counting errors through Abercrombie correction.[30]
Statistical analysis
Data obtained from six rats per group are expressed as mean ± standard error of the mean. Data were evaluated by two-way (Experiment 1 and 2) or one-way ANOVA (Experiment 3), followed by Student–Newman–Keuls post hoc pairwise comparisons. In the two-way ANOVA, one factor was the exposure factor, while the other factor was the treatment factor (RST or antagonist). Statistical analyses were performed with SigmaPlot software v12.0 (Systat Software, San Jose, CA, USA). P < 0.05 was considered statistically significant.
| Results | | |
Effects of prenatal opioid exposure on CRF-mediated restraint-induced neuronal activation
The bright-field pictures in [Figure 2] show the profiles of the cFos-ir neurons (an indicator for neuronal activation) in offspring with four different prenatal exposures. In No-RST offspring, few cFos-ir neurons were observed. Thus, RST-induced neuronal activation in both the PVH and LC of the four study groups with different prenatal exposures, as evidenced by increases in cFos-ir neurons. Compared with the profile of the Veh exposure group, RST-induced neuronal activation was found to be lessened in offspring with prenatal exposure to Meth or Mor but not in those with prenatal exposure to Bu. Analysis of the grouped data revealed that in both the PVH and LC, the numbers of cFos-ir neurons were minimal in No-RST rats [Figure 3]a but largely increased in RST rats in all four exposure groups [[Figure 3]b; P < 0.001]. In rats with RST [Figure 3]b, the number of cFos-ir neurons in the Bu exposure group did not differ from that in the Veh group (P > 0.05), but it was significantly greater than that in the Meth or Mor exposure group (Bu vs. Meth, P < 0.001; Bu vs. Mor, P < 0.001). The number of cFos-ir neurons in the Meth exposure group was slightly smaller than that in the Mor exposure group (P < 0.03). The RST-induced increases in the numbers of cFos-ir neurons in all four exposure groups were completely prevented by treatment with a CRF receptor antagonist [Figure 3]c; no significant differences were detected among the four exposure groups (P > 0.05). | Figure 2: Stress-induced neuronal activation in the PVH (a) and LC (b) of four study groups with different prenatal exposures. Bright-field representative micrographs show cFos expressing profiles in adult male rat offspring with prenatal exposure to Veh, Bu, Meth, or Mor. cFos-ir cells were used as an indicator for neuronal activation. RST, offspring with restraint; No-RST, offspring without restraint; III, the third ventricle; IV, the fourth ventricle; Me5, mesencephalic trigeminal nucleus; scp, superior cerebellar peduncle. Scale bar = 35 m. Note that RST-induced neuronal activation was lessened in the offspring with prenatal exposure to Meth or Mor but not in those with prenatal exposure to Bu. Veh: Vehicle, Bu: Buprenorphine, Meth: Methadone, Mor: Morphine, cFos-ir: cFos-immunoreactive, LC: Locus coeruleus. PVH: Paraventricular nucleus in the hypothalamus.
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 | Figure 3: Effects of prenatal exposure to different opioids on stress-induced neuronal activation in the PVH and LC. (a) Offspring without restraint (No-RST). (b) Offspring with RST. (c) Offspring with RST plus CRF receptor antagonist (α-helical CRF9-41; 10 μg/3 μL, i.c.v.). Data were obtained from adult male rat offspring with prenatal exposure to Veh, Bu, Meth, or Mor. cFos-ir cells were used as an indicator for neuronal activation. Data in each group are the mean ± SEM from six rats. *, P < 0.05, compared with Veh; #, P < 0.05, compared with Bu; +, P < 0.05, compared with Meth. Note that RST induced less neuronal activation in offspring with prenatal exposure to Meth or Mor, compared with those with prenatal exposure to Bu or Veh. This neuronal activation was completely prevented by CRF receptor antagonist in all study groups with RST. CRF: Corticotropin-releasing factor, Veh: Vehicle, Bu: Buprenorphine, Meth: Methadone, Mor: Morphine, cFos-ir: cFos-immunoreactive, LC: Locus coeruleus, PVH: Paraventricular nucleus in the hypothalamus.
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Effects of prenatal opioid exposure on corticotropin-releasing factor-induced neuronal activation
In No-RST offspring, central administration of CRF-induced neuronal activation in both the PVH and LC. Analysis of the grouped data [Figure 4] revealed that in both the PVH and LC, the numbers of cFos-ir neurons in the Bu exposure group did not differ from those in the Veh group (P > 0.05), but they were significantly greater than those in the Meth or Mor exposure group (Bu vs. Meth, P < 0.001; Bu vs. Mor, P < 0.01). In the PVH, the number of cFos-ir neurons in the Meth exposure group was slightly smaller than that in the Mor exposure group (P < 0.03). | Figure 4: CRF-induced neuronal activation in the PVH (a) and LC (b) of No-RST offspring with different prenatal exposures. Data were obtained from adult male rat offspring with prenatal exposure to Veh, Bu, Meth, or Mor. CRF was given at a dose of 0.5 μg/3 μL i.c.v. cFos-ir cells were used as an indicator for neuronal activation. Data in each group are the mean ± SEM from six rats. *P < 0.05, compared with Veh; #P < 0.05, compared with Bu; +P < 0.05, compared with Meth. Note that CRF induced less neuronal activation in offspring with prenatal exposure to Meth or Mor, compared with those with prenatal exposure to Bu or Veh. PVH: Paraventricular nucleus in the hypothalamus, CRF: Corticotropin-releasing factor, Veh: Vehicle, Bu: Buprenorphine, Meth: Methadone, Mor: Morphine, cFos-ir: cFos-immunoreactive, LC: Locus coeruleus, i.c.v.: Intracerebroventricular.
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Effects of prenatal opioid exposure on the density of CRF neurons in the PVH and CRF nerve processes in locus coeruleus
In No-RST offspring [Figure 5]a, the number of neurons expressing CRF in the Bu exposure group was comparable with that in the Veh exposure group. However, the number of neurons expressing CRF in the Meth or Mor exposure group was relatively fewer than that in the Veh or Bu exposure group. Similar profiles of neurons expressing CRF or co-expressing CRF and cFos were observed in RST offspring [Figure 5]b. Similar profiles of CRF nerve processes were observed in No-RST offspring [Figure 5]c. | Figure 5: Density of CRF neurons in the PVH of the No-RST (a) and RST offspring (b) and in the LC of No-RST offspring (c) with different prenatal exposures. Representative micrographs show CRF and/or cFos expressing profiles in adult male rat offspring with prenatal exposure to Veh, Bu, Meth, or Mor. (a and c) Staining of CRF in brain sections from the offspring without restraint (No-RST). III, the third ventricle. Scale bar = 60 μm. (b) Double staining of CRF and cFos in brain sections from offspring with restraint (RST). cFos-immunoreactive cells were used as an indicator for neuronal activation induced by RST. White arrows indicate neurons expressing CRF alone (brown). Black arrows indicate neurons expressing cFos alone (black). Hollow arrowheads indicate neurons co-expressing CRF and cFos. Scale bar = 10 μm. Note that the density of CRF neurons was reduced in offspring with prenatal exposure to Meth or Mor but not in those with prenatal exposure to Bu. CRF: Corticotropin-releasing factor, Veh: Vehicle, Bu: Buprenorphine, Meth: Methadone, Mor: Morphine, cFos-ir: cFos-immunoreactive, LC: Locus coeruleus.
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| Discussion | | |
Prenatal opioid exposure is known to dysregulate the stress response in adult offspring.[4],[5],[6],[12],[13],[14],[15],[16],[17],[18],[19],[20] Meth and Bu are two major types of synthetic opioid agonists for the medication-assisted treatment of opioid use disorder in pregnant women.[1],[21] The detrimental effects of prenatal exposure to Meth versus Bu on the stress-sensitive brain circuitry warrant investigation. The results of this study demonstrated that RST-induced neuronal activation in the PVH and LC was well preserved in adult male rat offspring with prenatal exposure to Bu, but it was substantially lessened in those with prenatal exposure to Meth or Mor. In addition, RST-induced neuronal activation was completely prevented by a CRF receptor antagonist in all exposure groups, suggesting the crucial role of CRF in this stress response. Central administration of CRF alone produced a profile of neuronal activation in the PVH and LC similar to that induced by RST. Furthermore, the lessened neuronal activation found in the Meth or Mor exposure groups may be, at least in part, due to the reduction in the density of CRF neurons in the PVH and CRF nerve processes in the LC, as evidenced by the data of our IHC analysis. Collectively, these observations suggested that, in terms of stress-induced neuronal activation in adult offspring, the detrimental effect of prenatal exposure to Bu was less than that of prenatal exposure to Meth or Mor.
Several rodent studies have reported long-term changes in the behavioral and hormonal responses to stress-provoking stimuli following perinatal opioid exposure. For example, the adult offspring exposed to opioids prenatally exhibited diminished anxiety-like behavior in the light–dark transition test,[27] increased duration of immobility time in the forced swim test,[16] abnormally slow response to the hot-plate test,[17] difficulties in the active avoidance tests,[18] decreased ability to respond to a conditioning stimulus to avoid an electric shock,[19] less line crossing and fewer rearing in the open field,[20] and decreased time struggling during the swim tests.[5] Although these observations indicated that perinatal opioid exposure produced hyporesponsiveness to stress in adult offspring, the underlying mechanisms are not completely known. In the study of hormonal responses, offspring with in utero exposure to opioids displayed diminished changes in serum corticosterone and blood glucose after the forced swim test[5] and the reduced response of serum adrenocorticotropic hormone after RST stress.[14],[15] Thus, dysregulation of the HPA axis following perinatal opioid exposure has been proposed to be one possible mechanism.[4],[6],[14],[15] In this regard, prenatal opioid exposure may impede the development of adaptive responses to environmental stimuli by altering the stress-sensitive brain circuitry.[4],[6] The LC is one of the key components of the stress-sensitive circuit by releasing NE in the brain.[31] The LC is innervated by CRF-containing terminals.[31] In response to stress, the release of CRF from these terminals can increase LC tonic neuronal firing, which in turn promotes the release of NE in the forebrain.[31] There are reciprocal neural connections existing between the CRF neurons of the PVH and NE neurons of the LC and this suggests that these two sites are major central coordinators of the stress system.[6],[9] CRF released from neurons in the PVH has emerged as a key molecule to initiate and integrate the stress response. No study has investigated the detrimental effects of perinatal opioid exposure on the central stress system. Thus, our findings regarding the lessened stress-induced neuronal activation in the PVH and LC and reduced density of CRF neurons in the PVH may be possible mechanisms underlying the hyporesponsiveness to stress in adult offspring after perinatal opioid exposure.
Meth and Bu are two synthetic opioid agonists widely used in the substitution treatment of pregnant women who are addicted to opioids.[1],[21] Given that these two medications have several advantages and disadvantages for pharmacotherapy in pregnancy,[1],[22] much interest has been paid to compare the benefit/risk profile of prenatal exposure to Meth versus Bu. Studies comparing the long-term neurocognitive outcomes in human adults following prenatal exposure to Meth versus Bu are lacking due to existing confounding factors and lack of control.[1],[2],[3],[4],[17] To date, only three rodent studies have addressed this issue.[25],[26],[27] Compared with those exposed prenatally to Meth, the adult rat offspring exposed prenatally to Bu had a higher preference index in the novel object test, a longer total social interaction time, a longer time spent in the open arms in the elevated plus maze test, and less sensitivity to Mor-induced antinociceptive effects.[25],[26],[27] No study has compared the detrimental effects of prenatal exposure to Meth versus Bu on the central stress system. Our findings regarding the comparison of stress-induced neuronal activation in the PVH and LC and the density of CRF neurons in the PVH provide novel evidence to support the notion that Bu is superior to Meth as the substitution treatment of opioid use disorder in pregnant women.
One issue that should be addressed is that the doses of prenatal exposure of Bu, Meth, and Mor were different in this study. These doses and methods of prenatal opioid exposure were adopted from previous studies with the consideration of the differences in lethal effects among these opioid drugs.[22],[23],[24] Whether the differences in the doses of these opioid drugs may contribute to the differences in the neuronal stress response we observed remains to be investigated. The other issue is that several immediate early genes (IEGs) have been found to be molecular markers for neuronal activity. The activation of these IEGs may lead to different physiological responses to different types of stimuli. For example, Egr-1 is mainly used in the study of sensory stimuli,[32] whereas of Arc is a molecular substrate for the structural and synaptic plasticity observed following stimuli.[33] In this study, we used cFos as the molecular marker for neuronal activity due to the fact that cFos has been extensively used in the study of stress stimuli in the literature.
| Conclusion | | |
The results of this study suggested that stress-induced neuronal activation in the PVH and LC was well preserved in adult male rat offspring with prenatal exposure to Bu, but it was substantially lessened in those with prenatal exposure to Meth or Mor. The lessened neuronal activation found in the Meth or Mor exposure groups may be, at least in part, due to the reduction in the density of CRF neurons in the PVH.
Acknowledgment
The authors are grateful to KGSupport Ltd. Academic Submission Services for assisting with language editing.
Authors' contributions
H. H. C, P. L. T, and Z. F. Y were responsible for conception and design of the research; C. Y. W and Z. F. Y performed the experiments and analyzed the data; Z. F. Y prepared the images and figures; C. Y. W, H. H. C, P. L. T, and Z. F. Y drafted the manuscript; Z. F. Y revised the final manuscript. All authors contributed to the article and approved the submitted version.
Financial support and sponsorship
This study is financially supported by Tzu Chi University (grant number: TCIRP100001) and National Health Research Institutes, Taiwan (grant numbers: NHRI-PHPP31, NHRI-NPSP01).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
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