An Integrated Systems Approach to Decode the Impact of Adolescent Nicotine Exposure in Utero and Postnatally Oxycodone Exposed Offspring

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine’s widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found alterations of the blood-brain barrier (B.B.B.) and synaptic proteins. RT-qPCR further validated immune dysfunction in the central nervous system (CNS) consistent with compromised B.B.B. Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning PNO & IUO, a predictor of greater addiction risk. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal.


Introduction
The opioid epidemic represents the advent of signi cant public health challenges, setting the stage for substantial public health cost (Seaton et al., 2007;Pouryahya et al., 2020). The most readily prescribed and abused opioids include buprenorphine, morphine, and oxycodone (OXY), each often prescribed to women as an analgesic for postpartum treatment (Hodder et al., 2021). OXY is teratogenic and readily crosses the placental barrier leading to impairments in fetal development (Pouryahya et al., 2020).
Several of our previously published studies have provided evidence of phenotypic, behavioral, biochemical, and synaptic vulnerabilities of postnatal OXY-exposed offspring (PNO) and in utero OXY- vulnerabilities identi ed in opioid-exposed offspring is further complicated when considering routinely abused substances (nicotine and alcohol). The interaction between perinatal OXY exposure and adolescent vulnerability is a signi cant gap in our knowledge.
Youth are frequently tempted with nicotine and alcohol in adolescence, a vulnerable timeframe.
Adolescence (years [14][15][16][17][18][19] is a critical developmental window characterized by decreased inhibition, increased impulsivity, and the emergence of neuropsychiatric dis-orders (Caballero et al., 2016;Dwyer et al., 2019). At this age range development of the rodent forebrain follows a similar trajectory to human development, which undergoes considerable restructuring as it matures (Kopec et al., 2018). Nicotine use has been shown to disrupt this restructuring (Dwyer et al., 2019). Studies also provide evidence nicotine can disrupt the development of tertiary mechanisms for in uencing impulsivity such as neurotransmitter re-uptake and metabolism (van der Put et al., 2014). Due to these vulnerabilities to nicotine in adolescence we hypothesize synaptic and behavioral out-comes will be exacerbated in these offspring.
Our integrated systems approach found phenotypic, molecular, and synaptic de cits in these offspring.
De cits were modest compared to known de cits animals experience. However, this study aimed to use a novel dose escalation of nicotine to mirror natural progression of nicotine habit forming. Therefore, results shown here are clinically relevant.  7,9,15]. The IUO animals were orally gavaged with 15 mg/kg/day (chronic oxycodone dosage) oxycodone HCl (Sigma Aldrich, St. Louis, MO, U.S.A.) dissolved in saline vehicle. PNO groups were gavaged starting at P0, and IUO groups were gavaged starting one week before mating with a proven breeder. The IUO group was dosed throughout the pregnancy and both groups continued drug treatment until weening. Nicotine treatment followed a dose escalation model to re ect more natural progression of habit formation. However, these doses are considered appropriate because of the gestational vulnerabilities mentioned. Nicotine hydrogen tartrate salt dissolved in saline vehicle was administered subcutaneously (S.C.) using the following escalation paradigm, 1.) initial injections of 0.1 mg/kg/day on days P28-29, 2.) 0.2 mg/kg/day injection days P30-31, 3.) 0.4 mg/kg/day days P32-33 and 4.) 0.4 mg/kg 3 times a day for the remainder of the drug treatment (P32-43). Single day dosing was performed at 9:00 AM; dosing three times a day was performed at 9:00 AM, 12:00 PM, 3:00 PM. The nicotine regimen is represented in Fig. 1. All animals were monitored daily. At P43 tissue was harvested and stored at -80°C until subsequent analyses. Animals that underwent a 24-hour withdrawal period were sacri ced on P44.

Liver protein isolation
Liver tissue was homogenized with T-PER Lysis Buffer (Thermo Fischer Scienti c) containing protease/phosphatase inhibitors. Samples were spun supernatant was collected. Pierce B.C.A. protein assay kit (Thermo Fischer Scienti c) was used to determine protein concentration for western blot analysis.

Brain lysates and synaptosomes
Brain tissue from the frontal cortex was homogenized with 10x volume of lysis buffer containing protease/phosphatase inhibitors and spun at 0.3 x g for 5 minutes. The super-natant was collected, and 100 µl aliquot (brain lysate) was collected for western blotting of blood-brain barrier proteins. The supernatant was spun at 12,000 x g for 20 minutes, dis-carded, and the remaining pellet was resuspended in 200 µl of 1X PBS containing protease/phosphatase inhibitors. Pierce B.C.A. protein assay kit (Thermo Fischer Scienti c) was used to quantify and prepare samples for further analysis.

Western blot
Western blot was adopted from studies performed by Odegaard and colleagues (Odegaard et al., 2020a).
Brain and liver lysates, and synaptosomes from each animal were loaded into SDS PAGE 10 well gels (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen) and immunodetection. Concentrated Superblock was used to block nonspeci c binding (Thermo Fisher Scienti c, Waltham, MA, USA). After blocking, membranes were incubated overnight at 4°C with a primary antibody. Primary and secondary antibody dilutions were done according to the manufacturer's suggestion and are shown inTable 1.. Blots were developed using Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA) with Super-Signal West Pico Chemiluminescent Substrate (Thermo Fischer Scienti c).

Cotinine ELISA
Serum cotinine level was measured using Cotinine ELISA Kit (cat. no. KA0930; Abnova; Taipei, Taiwan) according to the manufacturer's guidelines and as previously described (Gupta et al., 2019). ELISA was then analyzed using Epoch System. Values were determined using the interpolated best t (P4 curve).

Histology
Protocols used were adopted from Yang et al (Yang et al., 2016). For immunohistochemistry, sections (10 µm thick) from para n-embedded whole brains were depara nized and re-hydrated. Immunostaining with GFAP and IBA-1 antibodies involved boiling the tissue sections in citrate buffer (pH 7) for about 8 minutes for e cient antigen retrieval. Sections were blocked with 10% normal goat serum (NGS) for 2 h at RT and then incubated in primary anti-GFAP (1:500), or anti-IBA1 (1:250), antibody at 4°C overnight. The sections were then incubated with immuno uorescent secondary antibody at a 1:1000 dilution and DAPI (1:333) for 2 h. GFAP is shown in red. IBA-1 shown in green. Histological counting was performed in the ImageJ software. Figures represent hand counting.

Marble burying
Marble burying was performed as described in our previous studies (Odegaard et al., 2020b). Testing was performed at P43. Animals that underwent a 24-hour withdrawal period were analyzed at P44. A rat cage (929 cm2, 43.18 × 21.59 × 20.32 cm) contained a leveled 5 cm layer of ¼-inch corncob bedding (Envigo #7097), and 20 standard glass marbles (15 mm diameter, 5.2 g) were lightly placed in a 5 × 4 arrangement along the bedding. The subject was placed into the cage, and the cage was covered for 30 min. The animal was removed, and the marbles were imaged and scored by a scorer blinded to the conditions. A marble was considered buried if more than 2/3 of a marble was under the bedding.

Hot plate
Testing was performed P43-44. Animals that underwent a 24-hour withdrawal peri-od were analyzed at P44. Each animal was placed on an Incremental Hot Plate set at 25°C (IITC Life Science, Irving, CA, U.S.A.). Temperature increased at a rate of 5°C per minute with maximum temperature of 50°C. During testing animals were constantly monitored. When the animal licked its back paw, a standardized behavior to avoid heat, the test was stopped. Duration and intensity of heat was recorded using software provided by IITC Life Science and recorded by hand. The testing plate was sanitized between each animal to prevent litter mate scent from interfering with performance.

Data and statistical analysis
All data presented reported mean as ± S.E.M. All data in each analysis was normally distributed.
Signi cance was determined using two-way ANOVA approach followed by Tukey's test or Dunnett's correction when appropriate with a signi cance criterion of p ≤ .05. Graph Pad.

Phenotypic and metabolic vulnerabilities
PNO and IUO weights trended lower than controls as shown in Fig. 2. Weight re-ductions at P28 show a signi cant in uence of perinatal OXY exposure (Fig. 2b.). P43 overall weight reductions demonstrate the impact of nicotine to exacerbate trends observed from OXY exposure. When observing the sex-based breakdown, PNO-groups over-all demonstrated the most di culty gaining weight under nicotine treatment remaining lower in weight throughout the entire treatment regimen. Signi cantly impaired ability to gain weight was most notably displayed in males with lower weight observed in all conditions Fig. 2d. These ndings suggest a weight-based vulnerability with opioid use and an increased susceptibility for males also using nicotine. The latter nding also suggests a potential hormonal element, as males tend to have slower and longer growth patterns.
We next assessed the levels of cotinine, a principal metabolite of nicotine in the liver samples from the different treatments that showed unique regulation of nicotine metabolism (Fig. 3). PNO offspring trended higher in enzyme level. While IUO-offspring showed signi cant increases in enzyme level. Next, we used ELISA to assess the level cotinine in the serum. We found PNO offspring had signi cantly lowered cotinine in their serum than saline offspring at the end of nicotine treatment.

Blood-brain-barrier integrity
We next looked at the BBB, which tightly regulates the peripheral and central nervous (CNS) interactions and is often altered in neurodegenerative disorders. As seen in Fig. 4, these proteins were subtly downregulated in western blot analysis. Non-disruptive alterations such as those depicted suggest a subtle change in barrier permeability. Subtle changes in permeability can give rise to in ammatory phenotypes in the brain and changes in function at the synapse level that are di cult to evaluate.

Synaptic alterations
To further understand the molecular impact of BBB dysregulation we analyzed crude synaptosomes isolated from the cortex for essential synaptic proteins associated with synaptic transmission and synaptogenesis (Fig. 5). Of the proteins analyzed, all were expressed with distinct patterns in response to treatment. Most remarkably, synaptophysin was downregulated in IUO-withdrawal and nicotine groups when compared to IUO-sham, indicating potential transmission challenges because of synaptophysin's role in vesicle formation and over reactivity in withdrawal. Interestingly, saline animals experience an upward trend of excitatory amino acid transporter (EAAT) across nicotine and withdrawal while IUOgroups observed an inverse response. However, PNO demonstrates the opposite trend beginning high expression in control (PNO-sham) and low ex-pression in nicotine with even lower expression during withdrawal. IUO displayed di-minished downward trends suggesting the regulation of excess of excitatory neurotransmitters is diminished. Moreover, the up-and down-regulation of proteins associated with synaptogenesis, vesicular transport (SNARE complex), and excitatory signaling indicate vulnerabilities in adolescence and could persist into adulthood with unknown sequela.

Neuroimmune evaluation
RT-qPCR was employed to monitor the in ammatory milieu and immune response found in our other studies. Interleukin-6 (IL-6) was signi cantly downregulated between nicotine use and sham Saline-and PNO-animals, as shown in Fig. 6. Next, IBA-1 was used to characterize microglia. IBA-1 transcription was lower in saline nicotine animals when compared to saline sham animals. IUO-withdrawal animals corroborated the trend toward lower IBA-1 transcription. PNO-sham animals showed di-minished IBA-1 in opposition to IUO and Saline shams.
IUO sham displayed diminished IBA-1. However, IHC staining shown in summarized in Fig. 6. did not corroborate trends in microglia or astrocyte. These ndings provide some evidence that PNO and IUO react with nonclassical immune responses suggesting nicotine and withdrawal elicit an altered microglia activation and in ammatory response.

Behavioral de cits
PCR demonstrated that nicotine treatment in Saline-and PNO-offspring had lower in ammatory potential that interestingly did not occur in IUO-offspring. Analgesic and anxiogenic properties are de ning features of opioids and nicotine use. These distinct in ammatory signatures and general knowledge of nicotine and OXY exposure lead us to hypothesize that under stress these PNO-and IUO-offspring exposed to nicotine in early adolescence will have distinct reactions. To address this hypothesis, we used the marble bury assay and hot plate assay.
Nicotine regulation and diminished cotinine levels in OXY-exposed groups suggest enhanced anxietyliked behavior (compulsive-like) and lowered pain sensitivity. Accordingly, we used marbles buried to show anxiety level and delay to nocifensive response as an indicator pain threshold. Modest anxiety-like behaviors were observed in all animals, as shown in Fig. 7, inconsistent with the literature. This trend was not found in IUO groups. Hot plate testing revealed no signi cant difference between groups, as shown in Fig. 7. These ndings suggest distinct nociceptive regulation of PNO and IUO groups during withdrawal and nicotine use. Furthermore, hot plate tested nociception in the dorsal root ganglion and demonstrated hot stimulus does not produce a signi cant difference in animal reaction.

Discussion
We employed a novel comparative analysis of vulnerabilities in PNO & IUO exposed off-spring to nicotine use in early adolescence using an integrated systems approach to evaluate molecular, enzymatic, synaptic, and behavioral de cits. We also monitored the im-mediate withdrawal of animals as this is an understudied and dynamic stress.  Fig. 4., could indicate novel regulatory mechanisms or impaired regulation of B.B.B. permeability, potentially promoting a "leaky" brain environment in the case of PNO or an ischemic brain in IUO groups. Gap junctions and tight junctions contribute to the function of several other GPCR's and leukocyte migration. Dysregulation potentially sets the stage for further impacted neuroimmune environments in PNO-and IUO-offspring exposed to nicotine. Non-disruptive B.B.B. alterations such as those observed can also set the stage for altered in ammatory responses.
While RT-qPCR showed signi cant change concerning IL-6 production, an in-depth analysis of leukocyte activity, using ow cytometry on the spleen to investigate cell speci c changes, could enhance our understanding of the observed trends. While IUO-sham animals showed lower nonsigni cant endogenous production of IL-1b and IL-6 collectively, these results suggest IUO animals have lower capacity to mount an immune response. The typical neuroprotective effects of nicotine downregulate TNF-α and IL-10 and upregulate anti-in ammatory signaling. While TNF-α was not shown to be downregulated in these studies, evaluation of IL-10 and anti-in ammatory molecules (ar-ginase-1 or arginase-2) would help characterize the immune environment of OXY-exposed offspring. Proin ammatory cytokines were mostly downregulated in saline and PNO, which recapitulates the neuroprotective potential of nicotine. However, IL-6 can be pro-and anti-in ammatory, depending on the activity of other biomolecules such as a disintegrin and metalloproteinase domain 10 (ADAM10), indicating potential differences in the down-stream in ammatory response and neuroimmune changes. IUO's lack of IL-6 induction may indicate that the neuroprotective effects of nicotine are ameliorated because of mostly diminished innate immune function, possibly due to chronic perinatal exposure to opioids. In more typical nicotine models the anti-in ammatory effects extend to other cytokines and chemokines. Mechanistic evaluation of nicotine in the context of IUO and PNO would help clarify changes in immune responses and how they contribute to the progression of neuropsychiatric disorders. This change also suggests that the pharmacokinetics of nAChR activity is signi cantly altered in IUO, in line with the altered glutamate activity observed. As such, IUO is potentially vulnerable to numerous neurological diseases and could necessitate using novel therapeutic strategies to account for unique nAChR behavior.
While OXY and nicotine use distinct mechanisms to provide desired effects, both can be used to ease various types of pain (Bruijnzeel, 2017;Pinho-Ribeiro et al., 2017;Gao et al., 2020). These studies observed minor differences associated with nociception (hot plate) and anxiety-like behavior (Fig. 7). studies used higher nicotine concentrations in their treatment plans, 2.0 mg/kg/day via S.C., intraperitoneal, or osmotic pump. Prospective studies could increase nicotine use in treatment as PNO, and IUO only experienced subtle behavioral changes during this low dose study. Using greater nicotine dosage in adolescence could pose more robust behavioral de cits and highlight molecular and synaptic alterations discussed. However, further escalating the nicotine dose in adolescence would put these vulnerable populations at risk for more severe health effects. Doing so would also lose the clinical relevance of our low dose ramp. While the behaviors observed provide the basis for investigating a broader impact, sex-based observations would provide robust results given the rich behavioral literature of opioid-exposed animals and nicotine-exposed animals.
De cits corroborate our previous research elucidating baseline de cits of PNO-and IUO-offspring

Conclusions
Collectively, our study using an integrated systems approach shows a comparative analysis of alterations in enzymatic, synaptic, molecular, and behavioral changes in OXY off-spring exposed to nicotine in adolescence. Findings establish that acute and chronic OXY promote unique neurological compensatory mechanisms regarding nicotine exposure in early adolescence. The full impact of these changes could persist into adulthood and compromise the longevity of dual exposed offspring.
Data Availability Statement: The data that support the ndings of this study are contained within the article.   Figure 1 Nicotine and oxycodone administration paradigm. This paradigm details the nicotine administration timing, dose escalation, withdrawal condition used and termination. Generated using Biorender.com   Synaptic protein and neurotransmission analysis. Western blot analysis on synaptosomal samples isolated from PNO, IUO, and saline demonstrating unique regulation. Synaptophysin is upregulated in IUOwithdrawal group. Two-way ANOVA followed by Tukey's test when appropriate: p-value: ≤.05. Error bars represent SEM: *≤.05. N=6/group/treatment.
Marble burying assay revealed no signi cant difference in anxiety-like behavior between groups. Twoway ANOVA followed by Tukey's test: p-value: ≤.05 Error bars represent S.E.M. n-value displayed in graphs.

Supplementary Files
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