Nicotine alters cellular activity and mRNA expression of patterns of Astrocytes

Leslie Sewell; James J Cray

doi:10.1371/journal.pone.0325529

. 2025 Jun 20;20(6):e0325529. doi: 10.1371/journal.pone.0325529

Nicotine alters cellular activity and mRNA expression of patterns of Astrocytes

Leslie Sewell ¹, James J Cray ^1,^2,^*

Editor: Henning Ulrich³

PMCID: PMC12180639 PMID: 40540470

Abstract

Nicotine exposure during neural development presents a significant public health concern. Nicotine, the primary addictive component of tobacco, influences the central nervous system by interacting with various cell types, including the glial cell termed astrocytes. Astrocytes are cells that are critical for supporting neurons, regulating neurotransmitter balance, and managing neuroinflammation. This current study explored nicotine’s effects on astrocytes, examining cellular activity and gene expression within an acute exposure period. Murine C8D1A astrocytic (garnered as a cell line from postnatal day 8 tissue) cells were treated with nicotine (0–500 ng/mL) in vitro, with assays measuring cell viability and apoptosis at 12, 18, 24, and 48 hours to establish a critical concentration gradient for nicotine. Nicotine exposure increased astrocyte viability at later time points (24 and 48 hours), while apoptosis rose initially but declined over time allowing for the establishment of pharmacologically and clinically relevant nicotine concentrations of 25,50 and 100ng/ml for subsequent experiments. Real-time quantitative PCR revealed that nicotine influenced inflammatory signaling, with pro-inflammatory (A1) markers (IL-6, IFNγ, TNFα) increasing in a dose- and time-dependent manner, while anti-inflammatory (A2) markers (ARG1, IL-10, TGFβ) displayed a more complex pattern after nicotine exposures to astrocytes. These results suggest that nicotine disrupts astrocyte function and inflammatory balance, which may contribute to neurodevelopmental disruptions and heightened neuroinflammatory risks in adults. Further research is needed to investigate the prolonged impact of nicotine on brain health, addiction, and associated neurological conditions.

Introduction

Use of nicotine containing products and affects in health continues to be a significant public health concern [1–3]. Nicotine is the primary psychoactive compound included in cigarettes, nicotine replacement therapies, and emerging electronic cigarette delivery systems or “vape” technologies [2–10]. Despite societal endeavors to reduce smoking, nicotine addiction continues to be a major public health crisis.

Although nicotine is infrequently abused in its isolated form, it is predominantly consumed as a constituent of tobacco, most commonly via inhalation of smoke from conventional or electronic cigarettes [10,11]. Tobacco smoke comprises a complex mixture of hundreds of chemical compounds, many of which may potentiate the psychoactive properties of nicotine. Nonetheless, nicotine remains the principal agent underlying tobacco dependence, primarily through its capacity to reinforce drug-seeking and drug-taking behaviors. A key factor contributing to this dependence is nicotine’s relatively short plasma half-life of approximately two hours, indicating that half of the administered dose is metabolized and cleared from the body within that timeframe [10]. This rapid pharmacokinetic profile results in a transient duration of action, often compelling individuals to engage in frequent re-administration to sustain its psychoactive effects. Chronic exposure to nicotine induces neuroadaptive changes within the central nervous system, involving alterations across cellular, tissue, and metabolic domains, thereby reinforcing its addictive potential [9,12,13].

One such cell that may be targeted by nicotine is the astrocyte. Astrocytes, resident glial cells of the central nervous system, play crucial roles in supporting neurons, facilitating synapse formation, maintaining the integrity of the blood-brain barrier, regulating neurotransmitter levels, contributing to energy metabolism, and influencing the development of neurological diseases [11–18]. Further astrocytes exert many integral functions during gray and white matter development. After the initial production of neurons, astrocytes can operate as guides for neuronal migration. Behaving as neuronal precursors, radial glia affords a scaffold for neuronal positioning in addition to offering an area in which neurons can travel [19].

Astrocytes undergo diverse morphological, structural, metabolic, and molecular signaling modifications in response to insults including disease and exposures like nicotine [16,20–22]. These adaptations have notable consequences for the typical functioning of astrocytes. These modifications can impact synaptic communication with neurons [20,22–25]. While reward signaling associated with nicotine exposure are intricately associated with the activities of neuronal circuits [21,26], it is reasonable to suggest that astrocytes can exert substantial influence on these neuronal circuitries.

Given the limited data on the specific effects of nicotine on astrocytes, we conducted targeted in vitro studies to determine whether astrocytes are responsive to nicotine and whether nicotine exposure alters their cell cycle in a dose- and time-dependent manner. Based on reported plasma nicotine concentrations following the consumption of a single cigarette (5–30 ng/mL; [1,27–29]), we selected a range of nicotine concentrations (0, 10, 25, 50, 100, 250, and 500 ng/mL) to capture a spectrum of cellular responses in initial screening experiments to establish a concentration gradient of effects. Subsequent analyses focused on concentrations (0, 25, 50, and 100 ng/mL) that elicited measurable cellular activity. We then assessed astrocytic mRNA expression profiles to test the hypothesis that nicotine exposure shifts the balance between neurotoxic/pro-inflammatory and neuroprotective/pro-reparative markers. Our hypothesis what that nicotine would act in a dose dependent manner to drive greater markers of neuroinflammation over our acute exposure time course.

Materials and methods

Cells and culture conditions

Murine astrocytic C8D1A cells [30] were obtained from the American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin (Sigma, USA). The cells were maintained at a temperature of 37°C with 5% CO² with media changes twice per week until 95% confluence was reached. At the time of confluence, cells were seeded at an optimal cell density of 2,500 cells per well for cell proliferation and apoptosis assays. Optimal cell density of 2500 cells per well was determined through preliminary experimentation (Supplementary Fig 1).

Functional assays

Cell viability was assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega), which quantifies the conversion of MTS tetrazolium to Formazan—a process directly proportional to the number of viable cells and often prescribed as a measure of cellular proliferation. C8D1A astrocyte cells were seeded in 96-well plates at a density of 2,500 cells per well and treated with nicotine (Sigma Aldrich, St. Louis, MO, USA, N3876) at concentrations ranging from 0 to 500 ng/mL, suspended in standard culture media. Assays were conducted at 12-, 18-, 24-, and 48-hour post-treatment. To evaluate apoptosis under the same treatment conditions, the Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega) was used. At each time point, 100 μL of Caspase-3/7 substrate/buffer solution (1:100 dilution) was added per well. Plates were shaken for 30 seconds at 300 rpm and incubated at room temperature for 1 hour. Fluorescence was then measured using a 96-well plate reader (BioTek) with excitation at 485 nm and emission at 530 nm. All experiments were performed in triplicate. These experiments were utilized to establish the appropriate concentration gradient of effect while maintaining focus on clinically relevant circulating nicotine values.

RNA isolation

Upon completion of functional assays, RNA studies were devised based on the stablished nicotine concentration gradient. Briefly cells were seeded at a density of 100,000 cells per well in 6 well culture plates and treated for 24-, 48-, and 72- hours with standard culture media supplemented with 0ng/ml, 25ng/ml, 50ng/ml, and 100ng/ml concentration of nicotine (Sigma Aldrich, St. Louis, MO, USA, N3876). RNA was isolated using the OMEGA BioTek E.Z.N.A. Total RNA Kit (Omega BioTek, Norcross, GA) according to manufacturer’s protocol. Quality and quantity of RNA was assessed using a Synergy Hi Microplate reader and a Take3 Microvolume Plate (BioTek), with purity being assessed as 260/280 values >2.0. Complimentary DNA synthesis was performed using Quanta qScript cDNA Synthesis reagents following manufacturer’s protocol (Quanta Biosciences, Beverly, MA).

real time quantitative polymerase chain reaction for Astrocyte polarization markers

To quantify the expression levels of targets associated with astrocyte reactivity in response to stress or injury (often termed “polarization”), we conducted quantitative PCR (qPCR) on the complementary DNA (cDNA). For this purpose, we utilized the Applied Biosystems TaqMan Gene Expression Master Mix along with targeted TaqMan gene expression assays. Specifically, we focused on specific astrocyte targets defined as having high expression under neurotoxic or neuroprotective conditions (Table 1). To ensure reliable data interpretation, we employed the ΔCT method for data normalization. In this approach, we used 18S (Mm03928990_g1) ribosomal RNA expression as the reference target. Quantitative data were compared for gene expression changes due to treatment with nicotine by ΔΔCT methodology. Previously published statistical analysis methodology was used to determine differences for gene expression after nicotine related to the target of interest [31,32]. Differences were considered significant if p ≤ 0.05.

Table 1. Pro and Anti-Inflammatory Targets with Astrocyte Reactivity. In the table below are targets that are associated with anti- and pro-inflammatory states after injury or insult.

Inflammatory Targets
A1	A2
IFNγ (Mm01188134_m1)	ARG1 (Mm00475988_m1)
IL-6 (Mm00446190_m1)	IL-10 (Mm01288386_m1)
NOS2 (Mm00440502_m1)	TGFβ-1 (Mm01178820_m1)
TNF (Mm00443258_m1)	VEGF (Mm00437306_m1)

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Statistical analysis

Quantitative data was compared to control for gene expression after pharmacological exposures for targets of interest. We used statistical analyses for qrt-PCR data utilizing ΔΔCT methodology to assess differences in gene expression. Differences were considered significant if p ≤ .05. Raw data is provided in supplemental file (S1 Data).

Results

Functional assays

C8D1A cell viability assessment following nicotine exposure was assessed using the MTS assay at 12, 18, 24, and 48 hours. Early time points (12 and 18 hours) revealed a modest reduction in cell viability across most nicotine concentrations, with a slight rebound observed at 100 ng/mL at 12 hours and a similar trend at 18 hours following 50 ng/mL treatment. These findings suggest an initial sensitivity of astrocytes to nicotine. By 24 and 48 hours, nicotine exposure exhibited a stimulatory effect on cell proliferation across all tested concentrations. However, this proliferative response plateaued at concentrations above 50 ng/mL, indicating that increasing nicotine concentration beyond 50 ng/mL did not result in proportionally greater increases in cell proliferation—the beneficial effect of nicotine started to level off, or even decrease, despite higher doses (Fig 1).

Cell proliferation as indicated by MTS Assay at 12-, 18-, 24- and 48-hour time points. Note the increase by time and relative consistency by nicotine concentration. This suggests a potential effect for nicotine where excessive amounts result in diminishing returns or no greater effects based on MTS cellular assay.

After Caspase 3/7 assay administration, apoptosis levels were measured for nicotine treatment at 12-, 18-, 24- and 48-hour effects. The relationships suggested an initial increase in apoptosis for all concentrations pf nicotine with return to homeostatic levels (similar to control) at 18 hours. At 24 hours little apoptosis was observed for control conditions with noted apoptosis activity in the 10–100ng/ml nicotine treated concentrations. At 48 hours of note was increase apoptosis activity evidenced with the 25 mg/ml concentrations (Fig 2).

Apoptosis of C8D1A Cells as indicated by positive Caspase-3/7 Assay at 12-, 18-, 24- and 48-hour time points. Note the greatest amount of apoptosis occurs rapidly by the 12 hour time point with decreased evidence thereafter. Note similar relationship for nicotine concentration to that observed for MTS assay.

Nicotine exposure’s effect on A1 polarization

To determine the effect of nicotine on astrocyte polarization, C8D1A cells were treated in vitro with 25ng/ml, 50ng/ml, and 100ng/ml nicotine for 24-, 48-, and 72- hours based on derived values for MTS and Caspase experimentation. Assessment of A1 markers at 24 hours indicated a slight stepwise increase in expression were seen with IL-6 (p = 0.264, 0.278, 0.003), IFNγ (p = 0.131, 0.101, 0.002), NOS2 (p = 0.299, 0.142, 0.152), where TNFα maintained low expression levels across all treatments p = 0.299, 0.142, 0.152). Further, at 48 hours, IL-6 (p = 0.299, 0.142, 0.152), IFNγ (p = 0.071, < .001, 0.025), and TNFα (p = 0.019, < .001, < .001) all showed significant stepwise expression across increasing concentrations of nicotine. However, NOS2 showed an increase of expression at 25ng/ml, then a decrease at 50ng/ml, with a slight increase with 100ng/ml p = 0.044, 0.002, 0.013). At 72 hours, IL-6 (p = 0.052, 0.014, 0.001), IFNγ (p = 0.011, 0.004, 0.005), and TNFα (p = 0.004, 0.003, 0.003) showed a slight increase in expression at 100ng/ml. NOS2 (p = 0.003, 0.008, 0.003) showed a slight increase of expression with only 50ng/ml (Fig 3).

At 24 hours, slight stepwise increases in expression were seen with IL-6 (p = 0.264, 0.278, 0.003), IFNγ (p = 0.131, 0.101, 0.002), NOS2 (p = 0.299, 0.142, 0.152), where TNFα maintained low expression levels across all treatments p = 0.299, 0.142, 0.152). At 48 hours, IL-6 (p = 0.299, 0.142, 0.152), IFNγ (p = 0.071, < .001, 0.025), and TNFα (p = 0.019, < .001, < .001) all showed significant stepwise expression across increasing concentrations of nicotine. However, NOS2 showed an increase of expression at 25ng/ml, then a decrease at 50ng/ml, with a slight increase with 100ng/ml p = 0.044, 0.002, 0.013). At 72 hours, IL-6 (p = 0.052, 0.014, 0.001), IFNγ (p = 0.011, 0.004, 0.005), and TNFα (p = 0.004, 0.003, 0.003) showed a slight increase in expression at 100ng/ml. NOS2 (p = 0.003, 0.008, 0.003) showed a slight increase of expression with only 50ng/ml. These data suggest nicotine can drive A1 polarization of astrocytes.

Nicotine exposure’s effect on A2 polarization

To determine the effect of nicotine on Astrocyte polarization and inflammation, C8D1A cells were treated with nicotine in vitro with 25ng/ml, 50ng/ml, and 100ng/ml nicotine for 24, 48, and 72 hours. Assessment of A2 markers at 24 hours indicated ARG expression increased with 50ng/ml exposure (p = 0.027, 0.002,0.078), IL-10 expression increased with 100ng/ml exposure (p = 0.185, 0.023, 0.009), TGFβ showed a stepwise increase in expression from 25ng/ml to 100ng/ml (p = 0.016, 0.009, 0.014), and VEGF showed slight stepwise increase in expression from 25ng/ml to 100ng/ml (p = 0.087, 0.069, 0.111). At 48 hours, ARG expression appeared to double at 100ng/ml when compared to 25ng/ml (p = 0.01, 0.014, 0.002). IL-10 expression increased significantly at 100ng/ml exposure (p = 0.002, 0.005, < .001), TGFβ decreased with 50ng/ml and showed a slight increase in expression with 100ng/ml exposure (p = 0.035, 0.049, 0.036), and VEGF showed a similar decrease in expression from 25ng/ml to 50ng/ml with a slight increase with 100ng/ml (p = 0.576, 0.035, 0.153). At 72 hours, all cytokines showed a slight increase at 50ng/ml, IL-10 (p = 0.024, 0.011, 0.011) and TGFβ (p = 0.013, 0.065, 0.006) decreasing in expression at 100ngml and ARG (p = 0.018, 0.081, 0.006) and VEGF (p = 0.129, 0.011, 0.004) maintaining expression levels from 50ng/ml (Fig 4).

At 24 hours, ARG expression increased with 50ng/ml exposure (p = 0.027, 0.002,0.078), IL-10 expression increased with 100ng/ml exposure (p = 0.185, 0.023, 0.009), TGFβ showed a stepwise increase in expression from 25ng/ml to 100ng/ml (p = 0.016, 0.009, 0.014), and VEGF showed slight stepwise increase in expression from 25ng/ml to 100ng/ml (p = 0.087, 0.069, 0.111). At 48 hours, ARG expression appeared to double at 100ng/ml when compared to 25ng/ml (p = 0.01, 0.014, 0.002). IL-10 expression increased significantly at 100ng/ml exposure (p = 0.002, 0.005, < .001), TGFβ decreased with 50ng/ml and showed a slight increase in expression with 100ng/ml exposure (p = 0.035, 0.049, 0.036), and VEGF showed a similar decrease in expression from 25ng/ml to 50ng/ml with a slight increase with 100ng/ml (p = 0.576, 0.035, 0.153). At 72 hours, all cytokines showed a slight increase at 50ng/ml, IL-10 (p = 0.024, 0.011, 0.011) and TGFβ (p = 0.013, 0.065, 0.006) decreasing in expression at 100ngml and ARG (p = 0.018, 0.081, 0.006) and VEGF (p = 0.129, 0.011, 0.004) maintaining expression levels from 50ng/ml to 100ng/ml. These data suggest nicotine that although not as consistent as A1 markers, nicotine can drive some A2 polarization of astrocytes.

Discussion

Inclusive data establishes that astrocytes are sensitive to nicotine exposure, with nicotine eliciting a stimulatory effect on the cells at the 24- and 48-hour time points. Across all nicotine concentrations, this effect manifested as increased cell viability, which is interpreted as likely evidence of cellular proliferation.

Further analysis reveals an initial increase in apoptosis across all nicotine concentrations, with a return to homeostatic levels (comparable to control) at 18 hours. By 24 hours, minimal apoptosis was observed under control conditions, suggesting the cells had stabilized in culture. However, apoptosis activity persisted in nicotine-treated samples, particularly at concentrations ranging from 10 to 100 ng/ml. By 48 hours, apoptosis was significantly elevated, especially at a concentration of 25 mg/ml, indicating dose-dependent effects. This observation aligns with expectations, as increased cell density due to proliferation may trigger programmed cell death to maintain balance within the culture system [33–35].

Further, we aimed to assess the hypothesis that nicotine exposure affects the normal functioning of astrocytes, as measured by changes in mRNA expression. Previous research suggests that in astrocytes, as well as in other cell types like macrophages, markers often lack specificity for distinguishing between pro-inflammatory and pro-reparative activities [14,36,37]. Our findings are consistent with this, as nicotine exposure led to altered mRNA expression, but no clear distinction between A1 (pro-inflammatory) and A2 (anti-inflammatory) phenotypes was observed. This ambiguity may also be related to the selection of markers, although these markers (Table 2) are typically abundant during tissue insult and subsequent resolution to homeostasis [14,38–41].

Table 2. Reference table for molecular targets studied.

Inflammatory Target	Type	Source	Function
IFNγ	Pro-inflammatory	Macrophages, T helper cells, Tc cells, B cells, NK cells	Promotes Th1 immune response – secretion of Th1 associated cytokines.
IL-6	Anti-/Pro-inflammatory	B cells, T cells, monocytes	Pro- induces acute phase response and humoral immune response. Anti- inhibition of TNFα production by macrophages.
NOS2	Pro-inflammatory; enzyme	Neurons, glia during inflammation	Induced by IFNγ; generates nitric oxide (NO)
TNFα	Pro-inflammatory	Macrophages, NK cells, B cells	Stimulates neutrophil activation, anticoagulant, tumor necrosis, stimulations adhesion molecules.
ARG	Anti-inflammatory enzyme	Macrophages	Removes excess NO; wound healing, tissue repair
IL-10	Anti-inflammatory	Macrophages, monocytes, T cells, B cells	Inhibition of macrophage/monocyte and Th1cytokine production.
TGFβ-1	Roles in both anti--/pro-inflammatory responses; growth factor	Microglia (strong polarizer)	Regulates many normal cell functions such as proliferation and cell death
VEGF	Potentially anti-/pro-inflammatory; Growth factor	Endothelial cells, macrophages, monocytes	Induces vascular permeability and macrophage activation

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For example, IL-6 is a well-established marker of inflammation but also possesses anti-inflammatory functions [10,42]. Specifically, IL-6 can inhibit TNFα production by macrophages when functioning in its anti-inflammatory role. Additionally, NOS2, which is induced by IFNγ, showed increased expression in our data. At the 24-hour time point, we observed elevated levels of both IL-6 and NOS2, with further increases at 50 ng/ml nicotine exposure at the 48- and 72-hour time points, suggesting induction of NOS2 by IFNγ.

Moreover, the observed decrease in ARG1 expression, a marker commonly associated with anti-inflammatory or pro-reparative responses [10,42] indicates a relative shift toward the A1 phenotype at 48 and 72 hours. This may be due to competition for nitric oxide (NO) between ARG1 and NOS2. Reduced ARG1 activity could reflect increased NO production by NOS2, as ARG1 normally functions to deplete NO from the cerebral environment.

Furthermore, ARG1 also plays an anti-inflammatory role by inhibiting TNFα, a pro-inflammatory cytokine that promotes glutamate release, which is critical for synaptic function and contributes to cerebral inflammation [4,9,10,43]. At 48 and 72 hours, ARG1 expression did not increase at the 50 ng/ml and 100 ng/ml nicotine concentrations, whereas TNFα expression exhibited a stepwise increase with higher nicotine concentrations. This suggests that nicotine may exacerbate pro-inflammatory signaling via TNFα, particularly under conditions of elevated exposure.

TGFβ, a pleiotropic growth factor [4,9,10,43] studied here, demonstrated decreased expression which correlates with our bioassays which showed decrease of cell proliferation. Stepwise increase in TGFβ expression at 24 hours 48 hour – increase at 100ng/ml. 72 hour – increase at 50ng/ml. This could be reflected in the stimulus influence of nicotine on the cells at the 24- and 48-hour time point with all concentrations of nicotine driving increases in proliferation, as seen in the functional assays.

Data herein suggests nicotine exposure may influence immune responses in the CNS, and that glia, specifically astrocytes, are responsive to nicotine exposure and react to this insult by morphological and signaling alterations. Previous research indicates a barrage of events follows the pro-inflammatory response (i.e., leukocyte infiltration, production of pro-inflammatory cytokines) and is associated with activation of microglia and astrocytes that have the potential to contribute further to the inflammatory cascade [4,10,42,43]. Furthermore, astrocyte reactivity and subsequent responses may be dependent on the specific stimulus. Following nicotine exposure, the increase in pro-inflammatory cytokine production appears to coincide with an upregulation of pro-reparative processes. However, these reparative efforts do not appear sufficient to induce a full shift toward an A2 phenotype.

Astrocyte-targeted modulation of nicotine dependence-associated neural circuits presents a promising avenue for the development of novel smoking cessation strategies and offers broader insight into the neuropathological underpinnings of tobacco use disorder. Investigating astrocyte reactivity following chronic drug exposure may yield valuable therapeutic targets [46–49]. Nicotine use has been consistently associated with neuroinflammation, wherein reactive astrocytes serve as key mediators. Attenuating astrocyte-driven inflammatory responses may help ameliorate withdrawal symptoms and mitigate the neuroadaptive processes that underlie relapse.

One prominent pathological hallmark of nicotine dependence is the downregulation of the astrocytic glutamate transporter GLT-1 (EAAT2) within the nucleus accumbens, contributing to impaired glutamate clearance and excitotoxic signaling. Pharmacological agents such as ceftriaxone, a β-lactam antibiotic, and N-acetylcysteine (NAC), a cysteine prodrug with antioxidant and anti-inflammatory properties, have demonstrated efficacy in targeting astrocytic dysfunction. Ceftriaxone has been shown to upregulate GLT-1 expression, thereby restoring glutamate homeostasis and significantly reducing nicotine-seeking behaviors in preclinical models. Moreover, NAC modulates neuroimmune signaling pathways, including those in the nucleus accumbens, and has been observed to attenuate drug-seeking behavior. In addition to its role in glutamatergic regulation, ceftriaxone also decreases expression of pro-inflammatory markers such as tumor necrosis factor-alpha (TNF-α), potentially contributing to its anxiolytic effects during nicotine withdrawal. Collectively, these findings underscore the therapeutic potential of targeting astrocytic mechanisms in the treatment of nicotine addiction. Drugs that modulate astrocyte function, reduce reactivity, or promote astrocyte repair mechanisms might help mitigate the long-term effects of drug exposure on the brain [1,10,44–50].

Our ongoing research is progressing along multiple complementary lines, unified by the goal of elucidating astrocyte-specific responses to nicotine exposure. One key direction involves the application of next-generation sequencing to expand the repertoire of molecular markers, enabling a deeper understanding of acute nicotine-induced changes in astrocyte cellular signaling. This high-throughput approach aims to identify novel transcriptional and regulatory networks at both single-cell and population levels. Further nicotinic receptor subunit activity and modulators of these (including available pharmaceuticals such as varenicline and buproprion) should be specifically targeted to see if these represent robust targets of manipulations for therapeutic use., Concurrently, we are completing preliminary analyses on the effects of in utero nicotine exposure on glial cellularity and activity as the prenatal time period is a sensitive one for synaptogenesis and overall development of the brain. Using histological assessments during the early postnatal period, we are characterizing the enduring impact of prenatal nicotine exposure on astrocytes and microglia. These investigations seek to clarify how prenatal insults may alter glial development and function, potentially increasing susceptibility to neuropsychiatric conditions later in life.

Building upon these findings, future studies will extend to other glial populations, including oligodendrocytes, and examine intercellular interactions among glia and neurons. Particular emphasis will be placed on co-culture systems to explore the dynamic relationship between astrocytes and neurons under nicotine exposure and how this may affect neuroprotection and synaptic regulation.

Limitations include the limited markers studied for mRNA expression. Given the complexity of astrocyte phenotypes observed, which do not conform strictly to canonical A1 or A2 profiles, further resolution through flow cytometry or multi-omics array approaches is warranted. These methods will be instrumental in refining our understanding of astrocytic heterogeneity and state transitions following nicotine exposure, especially now that foundational expression data have been established.

Another limitation was simply the use of the murine in vitro model of study. However, in vivo studies represent a critical next phase, encompassing both developmental and adult models. Longitudinal investigations will be essential to determine how chronic nicotine exposure influences astrocyte reactivity over time and whether such exposure leads to persistent astrogliosis and the maintenance of a pro-inflammatory milieu. Ultimately, this work aims to delineate the cellular and molecular consequences of nicotine use, thereby informing the development of more effective therapeutic interventions to mitigate its public health burden.

Conclusions

In conclusion, astrocytes, as highly adaptable glial cells, play a crucial role in sensing and influencing damaged neurons while integrating various signals to elicit specific responses that modulate neuroinflammation. The findings from this dissertation indicate that astrocytes exhibit different phenotypes at specific time points. Notably, based on the polarization data of A1 and A2 astrocyte phenotypes, there appears to be a threshold for nicotine concentration that induces a pro-inflammatory (A1) phenotype, particularly at a concentration of 100ng/ml. At lower concentrations, astrocyte reactivity demonstrates a diverse phenotype across different brain regions. The varied expression of inflammatory targets suggests that astrocytes may adopt either a neuroprotective or neurotoxic phenotype based on the specific requirements of the affected region, considering factors such as regional stimulation or inhibition of neighboring cells and the extent of insult. Further investigations are required to elucidate the activation of neurons and glial cells within the reward circuit. Although additional studies involving induced inflammation of astrocytes and the subsequent expression of A1 and/or A2 phenotypes were planned but not expanded upon in this dissertation, they hold promise for expanding our understanding of the continuum of astrocyte reactivity.

Supporting information

Supplementary Fig 1. Preliminary data used to establish an optimal cell density and determine the nicotine concentration levels of interest. Based on these cell viability assays, an optimal cell density of 2500 cells per well was determined.

(DOCX)

pone.0325529.s001.docx^{(58.1KB, docx)}

S1 File. Data.

(XLSX)

pone.0325529.s002.xlsx^{(18KB, xlsx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The Ohio State University College of Medicine (JC).

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PLoS One. 2025 Jun 20;20(6):e0325529. doi: 10.1371/journal.pone.0325529.r001

Author response to Decision Letter 0

4 Dec 2024

PLoS One. doi: 10.1371/journal.pone.0325529.r002

Decision Letter 0

Henning Ulrich

PONE-D-24-55873Nicotine alters cellular activity and mRNA expression of patterns in vitro of murine astrocytesPLOS ONE

Dear Dr. Cray Jr.,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The conclusions need to be more precise and be in line with the here shown data. There are some issues regarding statistical significance. The introduction needs to be expanded, and the context to smoking and nicotine abuse needs to be clear.

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments:

The manuscript addresses an important topic. The study tests the effect of nicotine on murine astrocytes, focusing on cellular activity and mRNA expression. The results indicate that Nicotine impacts astrocyte activity and inflammatory signaling, suggesting its potential involvement in neuroinflammation. For better clarity the following revision is recommended:

1. The abstract as well as introduction should be modified to describe Tobacco/Nicotine consumption as a major global health concern/risk in general (in adults and in pregnancy), as nicotine teratological effect in astrocytic embryonic development was not specifically assessed in this paper. C8D1A cell line was used (Postnatal d8 astrocytic cerebellum).

2. The introduction section should be expanded to include:

a. Nicotine absorption, concentration in blood and brain, half-life (2 hours) etc. This would help understand the relevancy of selected doses and culture feeding regimen used in the study.

b. Summary of research exploring the effect of Nicotine on astrocytes should be expanded, include studies demonstrating the expression of nicotinic acetylcholine receptors (nAChRs) in astrocytes, and its downstream effects on astrocytic signaling pathways, morphological and functional changes etc.

Some recommended refs:

a. Aryal et al, Glia. 2021 Apr 14;69(8):2037–2053. doi: 10.1002/glia.24011

b. Stellwagen et al 2019, Curr Opin Neurobiol 57: 179–185. doi.org/10.1016/j.conb.2019.02.010.

c. Hernández-Morales et al, 201, Neuroscience 2014

3. Material and Methods:

a. Please include media change regimen (e.g. daily? Every other day? Etc) – might affect results interpretation (Acute exposure vs. chronic).

b. Include Nicotine source and Cat number.

4. Results:

a. MTS assay: The assay relies on cellular metabolic activity, which may not always directly correlate with cell number, if feasible please correlate with number of counted cells (e.g. neuclocounter/Hemcytometer, Brdu, ICF Dapi count), if not applicable, address this issue in discussion section.

b. Figure 1. Where applicable please add statistical significance asterisks (e.g. in comparison with non-treated-NT arm (0ng/ml) at same time point). The Y axis should be modifies to represent Cell Viability (measured by OD).

c. The reduction in cell activity/cell number or metabolic activity in un-treated arm after 24 & 48 hours compared to 12&18hr should be explained.

d. Figure 2. – Where applicable statically significance (asterisks) compared to NT should be added, Y-axis (apoptosis – measured by RFU – add abbreviation (Relative Fluorescence Units).

e. The peak in mRNA expression (in both A1/A2 markers graphs) occurs after 48 hr (higher fold change – pls note difference in scaling), should be discussed/explained.

Discussion section:

1. The effect of Nicotine was assessed only in one murine cell line (derived from Cerebellum), The potential difference in Nicotine effect between human and mouse astrocytes, as well as astrocytes heterogenous sub-populations (region/functionality/morphological) should be discussed.

2. Discuss the extrapolation between study dosing and feeding regimen and potential in-vivo consumption

3. Propose directions/strategies to overcome the resolution of A1/A2 marker expression and its cross-talk with other CNS cell population, such as including additional markers, multi-omics, in-vivo analysis.

Reviewer #2: The manuscript addresses a significant public health concern by investigating the effects of nicotine on murine astrocytes, focusing on cellular activity and mRNA expression. The study is well-designed, employing appropriate methodologies to assess astrocyte responses to nicotine exposure. However, certain areas require further refinement to enhance clarity, rigor, and relevance to the broader scientific and public health community.

Specific Comments on Sections

1. Introduction

o The introduction discusses the effects of nicotine during pregnancy, but it is unclear how this background connects to the study’s objectives. The authors should clarify:

� Why is the focus on nicotine exposure during pregnancy relevant to astrocyte activity in this experimental model?

� How does the current study design address the broader implications for pregnancy-related outcomes, if at all?

� If the focus is more general (e.g., nicotine's effects on the central nervous system), consider rephrasing or narrowing the discussion for better alignment with the study's aim.

o May need reference for “Discuss how findings from murine cells can be cautiously extrapolated to human physiology and potential limitations of this approach”.

2. Materials and Methods

o Cell Model:

� Why were murine astrocytic cells chosen instead of human astrocytic cells?

� Discuss how findings from murine cells can be cautiously extrapolated to human physiology and potential limitations of this approach.

o Dosage Selection:

� Clarify the rationale for the chosen nicotine dosages at each experimental step.

� Provide references or scientific justification for selecting the initial concentrations (0, 10, 25, 50, 100, 250, 500 ng/mL) and explain why the later experiments only used 0, 25, 50, and 100 ng/mL.

� How do these concentrations relate to physiologically relevant exposures in humans (e.g., plasma nicotine levels in smokers or vapers)?

o Experimental Details:

� Ensure that dosages and time points are explicitly mentioned for each assay. This will help readers replicate and interpret the study.

3. Results

o The description of findings is generally clear, but some terminology, such as "diminishing returns were observed," requires more explicit explanation for readers who may not be familiar with the concept.

o Provide a brief interpretation of how observed changes in apoptosis and proliferation reflect potential mechanisms of nicotine's impact on astrocytes.

4. Discussion

o Expand on the potential therapeutic implications of the findings (if possible):

� How might astrocyte modulation contribute to strategies for addressing nicotine addiction and neuroinflammatory disorders?

� Could targeting astrocytes play a role in smoking cessation interventions or reduce the neurological impact of nicotine exposure?

o Broaden the discussion to include potential applications of the study's findings beyond pregnancy, such as the relevance of astrocyte responses to nicotine in adolescent brain development or chronic nicotine users if possible.

Reviewer #3: In this work, the author used different functional assays to test nicotine effects on C8D1A cell viability, proliferation, and apoptosis at different time points. They also demonstrated the nicotine effects on gene expression of pro/anti-inflammatory markers. However, there lacks evidence to support the conclusion that nicotine alters astrocyte activity and inflammatory signaling. In the meanwhile, the statistical analysis in this research is not clear. The concerning are listed below:

Major

• Figure 1, your result suggests that excessive nicotine may lead to diminishing effects (no additional effects) based on the MTS assay. Have your considered testing higher dose to further explore this trend? Usually, 0.1-10µM (16-1620 ng/mL) dose of nicotine mimic as regular smoking or high environmental exposure.

• Figure 2, what is the reason for high apoptosis level in control group (0 ng/mL of nicotine) at 12, 18, and 48 hours? And there is no apoptosis measured in the same condition at 24 hours.

• In figure 2 legend, "Note similar relationship for nicotine concentration to that observed for MTS assay," please clarify or provide more information for this conclusion. In figure 1 MTS assay, the cell viability increased by nicotine treatment at lower dose at the 48-hour treatment group. However, the figure 2 shows that the apoptosis level significantly increased by nicotine treatment at lower dose at the 48-hour treatment group.

• In your caspase assay, there is a significant increase in apoptosis with the 10 ng/mL nicotine treatment at 12 hours. To strengthen your findings, consider including gene expression data corresponding to the 10 ng/mL treatment at 12 hours for Figures 3 and 4.

• For figure 3 and 4, clarity whether the significant differences are between different groups (e.g., dose comparisons) or between the control group and each treatment group.

Minor:

• Please specify the statistical methods used for analysis in the figure legends and in the research method. And specify whether the error bars represent standard error of the mean or standard deviation.

• For figure 1 and 2, include significance markers (e.g., stars) to indicate statistically significant changes.

• Define the p-value thresholds for the significance levels.

• Figure 3 and 4, please clarify the experiment number and independent repeat number for the representative blots as shown in the figures.

**********

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Reviewer #1: No

Reviewer #2: Yes: Li Feng

Reviewer #3: No

**********

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Attachment

Submitted filename: Comments-PONE-D-24-55873.docx

pone.0325529.s003.docx^{(18.9KB, docx)}

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Submitted filename: Comments-Li Feng.docx

pone.0325529.s004.docx^{(17KB, docx)}

PLoS One. 2025 Jun 20;20(6):e0325529. doi: 10.1371/journal.pone.0325529.r003

Author response to Decision Letter 1

23 Apr 2025

Response to Reviewers:

GENERAL COMMENTS

Reviewer I:

• The manuscript addresses a significant public health concern by investigating the effects of nicotine on murine astrocytes, focusing on cellular activity and mRNA expression. The study is well-designed, employing appropriate methodologies to assess astrocyte responses to nicotine exposure. However, certain areas require further refinement to enhance clarity, rigor, and relevance to the broader scientific and public health community.

Reviewer II:

• The manuscript addresses an important topic. The study tests the effect of nicotine on murine astrocytes, focusing on cellular activity and mRNA expression. The results indicate that Nicotine impacts astrocyte activity and inflammatory signaling, suggesting its potential involvement in neuroinflammation.

Specific Comments on Sections

ABSTRACT

Reviewer II:

• The abstract as well as introduction should be modified to describe Tobacco/Nicotine consumption as a major global health concern/risk in general (in adults and in pregnancy), as nicotine teratological effect in astrocytic embryonic development was not specifically assessed in this paper. C8D1A cell line was used (Postnatal d8 astrocytic cerebellum).

Response: We have updated the abstract and introduction to reflect this request. We note the cells were obtained as a cell line and maintained from a postnatal model. Most of our prior work on nicotine has been in the prenatal developmental sphere and we apologize for not taking a closer editing eye.

INTRODUCTION

Reviewer I:

• The introduction discusses the effects of nicotine during pregnancy, but it is unclear how this background connects to the study’s objectives. The authors should clarify:

• Why is the focus on nicotine exposure during pregnancy relevant to astrocyte activity in this experimental model? Response: As above it is not. We are interested in astrocyte reaction to any exposure including prenatal and have updated throughout to reflect this as the reviewer identified our approach was neither specific nor limited to a pregnancy exposure model.

• How does the current study design address the broader implications for pregnancy-related outcomes, if at all? Response: We have attempted to address this now in the discussion as we do have ongoing experiments assessing prenatal exposures on glial cells in vivo via a histological approach. As above, we apologize we were not more careful in our first submission.

• If the focus is more general (e.g., nicotine's effects on the central nervous system), consider rephrasing or narrowing the discussion for better alignment with the study's aim. Response: We hope our edits reflect our appreciation for the reviewer bringing this to our attention.

• May need reference for “Discuss how findings from murine cells can be cautiously extrapolated to human physiology and potential limitations of this approach”. Response: That is a fantastic point and not simply limited to murine to human comparison but also cell line vs primary cell approaches. We wanted a robust high throughput system for study and chose the murine astrocyte as it is an established cell line and with the fore knowledge, we were conducting prenatal exposure in an in vivo model we planned to study histologically for glial cell effects. We have added this to discussion, limitations, and future directions.

Reviewer II:

• The introduction section should be expanded to include:

• Nicotine absorption, concentration in blood and brain, half-life (2 hours) etc. This would help understand the relevancy of selected doses and culture feeding regimen used in the study. Response: Thank you for this comment. The selected feeding program was based on previous work by our own group on mesenchymal cell sensitivity to nicotine exposure, correlative cotinine concentrations for active nicotine use (30-300ng/ml) and our inclusive bioassays (FIGURE 1 and 2). We utilized both MTS and Apoptosis assay to define dose concentration and timepoints to be used for mRNA studies. Our range after considering IC50 and EC50 values led us to the 25-100ng/ml dose range that was used.

* Tobacco smoke comprises a complex mixture of hundreds of chemical compounds, many of which may potentiate the psychoactive properties of nicotine. Nonetheless, nicotine remains the principal agent underlying tobacco dependence, primarily through its capacity to reinforce drug-seeking and drug-taking behaviors. A key factor contributing to this dependence is nicotine’s relatively short plasma half-life of approximately two hours, indicating that half of the administered dose is metabolized and cleared from the body within that timeframe [10]. This rapid pharmacokinetic profile results in a transient duration of action, often compelling individuals to engage in frequent re-administration to sustain its psychoactive effects.

• Summary of research exploring the effect of Nicotine on astrocytes should be expanded, include studies demonstrating the expression of nicotinic acetylcholine receptors (nAChRs) in astrocytes, and its downstream effects on astrocytic signaling pathways, morphological and functional changes etc. Response: Thank you. We had previously edited this down for brevity but have included this now in discussion. We are relying primarily on published literature as in our own laboratory we have only conducted experiments with the alpha7 subunit for obvious reasons as it homodimerizes and is involved in addiction. We have expanded our discussion accordingly.

MATERIALS AND METHODS

Reviewer I:

• Cell Model:

• Why were murine astrocytic cells chosen instead of human astrocytic cells? Response: We are a preclinical teratology focused. Our next step is to use a translational in utero exposure model, so our preliminary experimentations were done with murine astrocyte cells.

• Discuss how findings from murine cells can be cautiously extrapolated to human physiology and potential limitations of this approach. Response: We have updated the discussion to address these concerns.

* Rationale for Using Murine Astrocytic Cells:

* Murine astrocytic cells (C8D1A) were selected for this study due to their well-characterized, reproducible behavior in vitro and their widespread use as a model for studying astrocyte physiology and response to pharmacological agents. These cells provide a controlled and genetically stable platform for investigating mechanisms of cellular response, including proliferation, apoptosis, and gene expression. While human astrocytes offer species-specific insights, they present limitations such as donor variability, ethical constraints, and reduced scalability for high-throughput assays. Moreover, murine models remain highly relevant for translational research, as they are commonly used in in vivo studies of neuroinflammation, addiction, and neurodegeneration, facilitating continuity between in vitro and in vivo experimentation.

• Dosage Selection (ADDRESSED IN INTRO):

• Clarify the rationale for the chosen nicotine dosages at each experimental step.

* Given the limited data on the specific effects of nicotine on astrocytes, we conducted targeted in vitro studies to determine whether astrocytes are responsive to nicotine and whether nicotine exposure alters their cell cycle in a dose- and time-dependent manner. Based on reported plasma nicotine concentrations following the consumption of a single cigarette (5–30 ng/mL; Benowitz & Jacob, 1994), we selected a range of nicotine concentrations (0, 10, 25, 50, 100, 250, and 500 ng/mL) to capture a spectrum of cellular responses. Subsequent analyses focused on concentrations (0, 25, 50, and 100 ng/mL) that elicited measurable cellular activity. In parallel, we assessed astrocytic mRNA expression profiles to test the hypothesis that nicotine exposure shifts the balance between neurotoxic/pro-inflammatory and neuroprotective/pro-reparative markers.

• Provide references or scientific justification for selecting the initial concentrations (0, 10, 25, 50, 100, 250, 500 ng/mL) and explain why the later experiments only used 0, 25, 50, and 100 ng/mL. Response: These doses allowed for maximal effect without unnecessary toxicity.

* After preliminary experimentation, two concentrations of nicotine, namely 25ng/ml and 50ng/ml, were selected based on their significance as determined by cell viability assays. Cell viability, indicative of proliferation, was evaluated using the colorimetric MTS assay. Apoptosis was assessed using the Apo-ONE Caspase-3/7 fluorescence assay. The results from cell viability assays led to the determination of 25ng/ml and 50ng/ml being those concentrations of interest. The 100ng/ml nicotine concentration was added to gauge effects of an exorbitant exposure of nicotine. We also decided to explore these concentrations at 24-, 48-, and 72-hour time points.

• How do these concentrations relate to physiologically relevant exposures in humans (e.g., plasma nicotine levels in smokers or vapers)? RESPONSE: Literature suggests 30-300 ng/ml is indicative of active nicotine use.

* Active smokers typically consume between 10 and 100 mg of nicotine per day, while individuals using alternative nicotine delivery systems may be exposed to an even broader range of nicotine levels (Benowitz 1984; L Benowitz, et al 1988)

• Experimental Details:

• Ensure that dosages and time points are explicitly mentioned for each assay. This will help readers replicate and interpret the study. Response: This has been updated throughout.

Reviewer II:

• Material and Methods:

• Please include media change regimen (e.g. daily? Every other day? Etc.) – might affect results interpretation (Acute exposure vs. chronic). All regimens were acute and only necessitated one feed as indicated by the initial dose schema gating of the MTS and Caspase assays (Figures 1 and 2)

• Include Nicotine source and Cat number.

* Sigma Life Science (−)-Nicotine ≥99% (GC), liquid N3876-100ml CAS Number 54-11-5 (IN TEXT: Sigma Aldrich, St. Louis, MO, USA, N3876)

RESULTS

Reviewer I:

• The description of findings is generally clear, but some terminology, such as "diminishing returns were observed," requires more explicit explanation for readers who may not be familiar with the concept. Response: We have updated our language for consistency.

* C8D1A cell viability following nicotine exposure was assessed using the MTS assay at 12, 18, 24, and 48 hours. Early time points (12 and 18 hours) revealed a modest reduction in cell viability across most nicotine concentrations, with a slight rebound observed at 100 ng/mL at 12 hours and a similar trend at 18 hours following 50 ng/mL treatment. These findings suggest an initial sensitivity of astrocytes to nicotine. By 24 and 48 hours, nicotine exposure exhibited a stimulatory effect on cell proliferation across all tested concentrations. However, this proliferative response plateaued at concentrations above 50 ng/mL, indicating that increasing nicotine concentration beyond 50 ng/mL did not result in proportionally greater increases in cell proliferation—the beneficial effect of nicotine started to level off, or even decrease, despite higher doses (Figure 1)

• Provide a brief interpretation of how observed changes in apoptosis and proliferation reflect potential mechanisms of nicotine's impact on astrocytes. Response: Although we did interpret the time course in this vein the initial experiments were simply conducted as a pharmacological bioassay to define critical concentrations to be tested for mRNA marker studies focused on astrocyte polarization.

Reviewer II:

• MTS Assay:

• The assay relies on cellular metabolic activity, which may not always directly correlate with cell number, if feasible, please correlate with number of counted cells (e.g., nucleocounter/Hemocytometer, ICF, DAPI Count). If not applicable, address this issue in the discussion section. Response: We have added this to the limitations, and although we agree MTS is still a standard in the field of experimental pharmacology and drug development to define IC25/50/75/90 and EC25/50/75/90 values, etc. We used these tools is a similar fashion to identify critical concentration schema for polarization studies.

* MTS assays were utilized in our cell culture studies to obtain a more accurate and functionally relevant assessment of cell viability. Unlike direct cell counting methods, which merely quantify total cell numbers, the MTS assay provides insight into cellular metabolic activity. Aside from determining initial seeding densities, cell counts were not employed, as they do not adequately reflect the metabolic status or viability of the cultured cells.

• Figure 1. Where applicable please add statistical significance asterisks (e.g. in comparison with non-treated-NT arm (0ng/ml) at same time point). The Y axis should be modified to represent Cell Viability (measured by OD). Response: We would be happy to revisit this if considered necessary given our above explanation of how we utilized our MTS and apoptosis information.

• The reduction in cell activity/cell number or metabolic activity in un-treated arm after 24 & 48 hours compared to 12&18hr should be explained. Response: This is likely an artifact of cell culture on plastic, cell loss is often observed in the first 24-48 hours after plating which is why control 0ng/ml group is so critical for interpretation.

• Figure 2. Where applicable statical significance (asterisks) compared to NT should be added, Y-axis (apoptosis – measured by RFU – add abbreviation (Relative Fluorescence Units). Response: We would be happy to revisit this if considered necessary given our above explanation of how we utilized our MTS and apoptosis information.

• Figure 3 & 4: The peak in mRNA expression (in both A1/A2 markers graphs) occurs after 48 hr. (higher fold change – pls note difference in scaling), should be discussed/explained. Response: As we are not comparing between timepoints we did not use the same y axis scale. We would be happy to revisit. However, we interpret the highest values changes as being the critical signaling axis peak after acute exposure. We have added this to the discussion.

DISCUSSION

Reviewer I:

• Expand on the potential therapeutic implications of the findings (if possible):

• How might astrocyte modulation contribute to strategies for addressing nicotine addiction and neuroinflammatory disorders? Response: We would like to thank the reviewer for prompting our expansion of the discussion. Astrocytes have been linked to drug seeking behaviors and transcriptional changes are crucial to interpreting biological responses to nicotine we do believe that the astrocyte may prove a useful target in the monitoring of inflammatory mechanistic processes related to spectrum of nicotine related health problems.

• Could targeting astrocytes play a role in smoking cessation interventions or reduce the neurological impact of nicotine exposure? Response: As astrocytes play a critical role in nicotine use and cessation relapse, we do believe that targeted therapies that alter extracellular levels of certain neurotransmitters (i.e. dopamine) associated with astrocyte activity may prove to be a useful therapeutic tool.

• Broaden the discussion to include potential applications of the study's findings beyond pregnancy, such as the relevance of astrocyte responses to nicotine in adolescent brain development or chronic nicotine users if possible. Response: We agree our focus was previously too heavily reliant on interpretations of prenatal exposures. We have updated throughout.

Reviewer II:

• The effect of Nicotine was assessed only in one murine cell line (derived from Cerebellum), The potential difference in Nicotine effect between human and mouse astrocytes, as well as astrocytes heterogenous sub-populations (region/functionality/morphological) should be discussed. Response: Agreed. This is similar to a previously addressed comment above.

• Discuss the extrapolation between study dosing and feeding regimen on potential in-vivo consumption. Response: Agreed. This is similar to a previously addressed comment above.

• Propose directions/strategies to overcome the no specificity of A1/A2 markers, such as including additional markers, multi-omics, in-vivo analysis. Respon

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Nicotine alters cellular activity and mRNA expression of patterns of astrocytes

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PERMALINK

Nicotine alters cellular activity and mRNA expression of patterns of Astrocytes

Leslie Sewell

James J Cray

Roles

Abstract

Introduction

Materials and methods

Cells and culture conditions

Functional assays

RNA isolation

real time quantitative polymerase chain reaction for Astrocyte polarization markers

Table 1. Pro and Anti-Inflammatory Targets with Astrocyte Reactivity. In the table below are targets that are associated with anti- and pro-inflammatory states after injury or insult.

Statistical analysis

Results

Functional assays

Fig 1. Cell viability and nicotine responsiveness.

Fig 2. Apoptosis and Nicotine Responsiveness.

Nicotine exposure’s effect on A1 polarization

Fig 3. Quantification of A1 polarization.

Nicotine exposure’s effect on A2 polarization

Fig 4. Quantification of A2 polarization.

Discussion

Table 2. Reference table for molecular targets studied.

Conclusions

Supporting information

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Decision Letter 0

Henning Ulrich

Roles

Author response to Decision Letter 1

Decision Letter 1

Henning Ulrich

Roles

Acceptance letter

Henning Ulrich

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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