Biology subjects

Bolton, J. L.

Publications and source records attributed to Bolton, J. L..

13 recordsLinked to original sources

bioRxiv2026

Early-life adversity alters the adult blood proteome primarily in female mice

Background: Early-life adversity (ELA) is one of the strongest predictors of poor mental and physical health later in life, increasing an individual's risk for disorders ranging from depression to metabolic syndrome. However, whether and how ELA shapes immune memory through adulthood, subsequently affecting susceptibility to immune-associated diseases such as autoimmunity, remains elusive. Methods: To address this question, we employed a translationally and ethologically relevant mouse model of resource deprivation, the limited bedding and nesting model, to study the long-term effects of ELA on the immune system. ELA was induced by limited bedding and nesting material in the cage during postnatal days (P)2-9, which provokes fragmented and unpredictable maternal behavior in the dams, and results in chronic stress in the pups. Nucleic acid Linked ImmunoSandwich Assay (NULISA) was used to survey the proteome of adult (~P90) mice of both sexes that experienced ELA vs. control conditions. Results: Using a novel proteomic method, we detected significant upregulation in the levels of IL-17A in the adult blood, primarily in females. Within females, ELA also upregulated IL-13 and CCL3. Three proteins, HGF, PDGFA, and WNT16, were significantly downregulated in ELA mice of both sexes. Conclusions: Being a hallmark proinflammatory cytokine, IL-17A is found to increase in patients with systemic lupus erythematosus (SLE), as well as in animals with lupus-like diseases in virtually all reports. Importantly, autoimmune diseases like SLE have a higher prevalence in women than men, and our data support this sex bias. Together, our data suggest that ELA shapes long-term immune function in a way that may increase risk for autoimmune diseases, with promise for the development of more targeted and personalized therapies for autoimmunity.

immunology↗

bioRxiv2025

Mismatch negativity develops in adolescence and independently of microglia

Higher brain functions and cognition undergo a critical period of development during adolescence, when psychiatric disorders such as schizophrenia typically onset. Understanding how developmental processes during adolescence interact with schizophrenia pathophysiology and risk remains a central goal in psychiatry. Here we show that a well-established biomarker of schizophrenia, mismatch negativity, matures during adolescence in mouse primary visual cortex, along with a strengthening of fronto-visual functional connectivity. Because microglia are implicated in schizophrenia risk and disease states, we further investigated what role microglia may play in the development of mismatch responses under physiological conditions. We found that microglial depletion with PLX5622 in adolescence arrests the development of resting oscillations in frontal areas, but does not affect the development of deviance detection, other signatures of visual context processing, or prefrontal-visual functional connectivity. Our findings suggest (a) a key component of mismatch negativity develops in adolescence, a period of vulnerability to schizophrenia, and (b) the development underlying this component does not require robust microglia activity, clarifying the developmental role of microglia in higher order visual processing.

neuroscience↗

bioRxiv2025

A common Iba1 antibody labels vasopressin neurons in mice

There are a wide variety of commercially available antibodies for labeling microglial cells based on different protein targets, as well as antibodies for the same protein target made in different species. While this array of targets and hosts allows for flexibility in immunohistochemical experiments, it is important to validate that different antibodies provide comparable and accurate immunodetection prior to experimental data collection. We found that a commercially available anti-Iba1 antibody, made in goat, produces irregular staining patterns in specific regions of the mouse brain, prompting a further investigation into the phenomenon. This Iba1-goat antibody displayed increased numbers of labeled cells when compared to expression of a CX3CR1-GFP reporter and IHC detection of P2RY12, two common microglial markers. Furthermore, immunodetection by other common anti-Iba1 antibodies made in rabbit and chicken did not display the excessive cell labeling when compared to the CX3CR1-GFP reporter. Upon further investigation, this Iba1-goat antibody was observed to highly colocalize with vasopressin neurons in the paraventricular nucleus of the hypothalamus (PVN) and the supraoptic nucleus of the hypothalamus (SON), the two main sites of vasopressin production in the brain. Other anti-Iba1 antibodies made in other species did not show this same colocalization with vasopressin. Finally, this effect was species-specific, as Wistar rats did not display erroneous cell labeling by the Iba1-goat antibody. In sum, the present study employs both qualitative and quantitative data to highlight the importance of validating antibody efficacy and specificity in a region- and species-specific manner. Significance StatementMicroglia are the primary immune cells of the brain and are involved in many neurodevelopmental, as well as neurodegenerative, processes, thus making the study of microglia an important area of neuroscience research. There is a wide array of antibodies available to label microglia. Specific detection of microglia using immunohistochemistry is crucial for understanding differences in cell density, morphology, and interactions with other cells in various contexts. In the present study, a common anti-Iba1 antibody made in goat was found to display erroneous labeling of vasopressin neurons in specific regions of the mouse brain, inconsistent with other microglial markers, which emphasizes the importance of validating antibody efficacy and specificity in a region- and species-specific manner prior to beginning experimental data collection.

neuroscience↗

bioRxiv2025

Inhibition of the Microglial Phagocytic Receptor MerTK Underlies ELA-induced Changes in Synapses and Behavior in Male Mice

BackgroundEarly-life adversity (ELA) is a significant risk factor for emotional disorders like depression, likely by provoking changes in stress-related circuit development. We have previously shown that ELA increases the number of excitatory synapses onto corticotropin-releasing hormone (CRH)-expressing neurons in the paraventricular nucleus (PVN) by decreasing microglial synapse engulfment. Here, we hypothesize that ELA induces microglial dysfunction via inhibition of the microglial phagocytic receptor, MerTK, thus resulting in the observed changes in synapses and stress-related behavior. MethodsTo determine whether deleting MerTK in microglia phenocopies the effects of ELA, microglia-specific (m)MerTK-KO (CX3CR1-Cre+::MerTKfl/fl) mice were crossed with wild-type (CX3CR1-Cre-::MerTKfl/fl) mice and their litters were reared in either a control or ELA (induced by limited bedding and nesting paradigm) environment, from postnatal days (P)2-10. Excitatory synapses in the PVN were assessed at P10, and adult offspring were tested in a behavioral battery to measure threat-response (known to be dependent on PVN-CRH+neurons) and anxiety-like behavior, followed by acute restraint stress to measure the neuroendocrine stress response. ResultsFollowing ELA at P10, excitatory, but not inhibitory, synapses in the PVN were increased in males, which was mimicked by mMerTK-KO in control males, but caused no further increase in ELA males. However, females already had higher numbers of excitatory synapses at baseline, and showed no further increase with ELA or mMerTK-KO. Remarkably, the pattern of threat-response behavior in males closely matched the excitatory synapses, with mMerTK-KO control males escaping more from the simulated predator threat in the looming-shadow threat task, similar to ELA males. Again, females did not show any significant changes due to ELA or mMerTK-KO in the threat-response, although they did show ELA-induced changes in anxiety-like behavior. ELA provoked a greater corticosterone response to acute stress in males, but not females, although females were again higher at baseline. ConclusionsOur results demonstrate that ELA provokes increased excitatory synapses in the PVN, leading to an increased active response to threat in the looming-shadow test in males only. Deleting MerTK specifically from microglia recapitulates both the synaptic and behavioral effects in control males, but does not have an effect in ELA males or control females, suggesting that the MerTK pathway is already inhibited by ELA in males and less active in females at baseline. Our work is the first to elucidate the mechanisms underlying the male-biased microglial dysfunction caused by ELA, with promise for the development of better preventative and therapeutic strategies for at-risk children.

neuroscience↗

bioRxiv2024

Constitutive expression of CX3CR1-BAC-Cre introduces minimal off-target effects in microglia

CX3CR1-Cre mouse lines have produced important advancements in our understanding of microglial biology. Recent studies have demonstrated the adverse effects of tamoxifen-induced CX3CR1-Cre expression during development, which include changes in microglial density, phenotype, and DNA damage, as well as anxiety-like behavior. However, the unintended effects of constitutive CX3CR1-BAC-Cre expression remain unexplored. Here, we characterized the effects of CX3CR1-BAC-Cre expression on microglia in CX3CR1-BAC-Cre+/- and CX3CR1-BAC-Cre-/-male and female littermates during early postnatal development and adulthood in multiple brain regions. Additionally, we performed anxiety-like behavior tests to assess changes caused by Cre expression. We found that CX3CR1-BAC-Cre expression causes subtle region- and sex-specific changes in microglial density, volume, and morphology during development, but these changes normalized by adulthood in all brain regions except the hippocampus. No behavioral effects were found. Our findings suggest that the constitutive-Cre model might be less detrimental than the inducible model, and highlight the need for proper controls.

neuroscience↗

bioRxiv2023

Maternal diesel exposure and maternal choline supplementation interactions in fetal and placental immune factors

Air pollution causes widespread inflammatory changes in the body and brain. When exposure to air pollution occurs early in development, children exhibit impaired working memory ability (Sunyer et al., 2015). In addition, prenatal exposure to diesel particulate matter (DEP) increases inflammatory cytokine expression in the whole brain of embryonic day 18 (E18) males and leads to adverse long-term negative outcomes (Bolton et al., 2012). In contrast, dietary choline supplementation is negatively correlated with inflammatory cytokine production in adult rats and cultured human cells (Zhang et al., 2018; Jiang et al., 2014). When administered as a supplement to pregnant rats, choline also improves working memory in adulthood (Meck et al., 2008; Meck & Williams, 1999; 1997). The current study sought to determine if prenatal dietary choline supplementation protects against the effects of air pollution in the developing brain and in the placenta and fetal liver. These data revealed region-specific microglial morphology alterations in fetal brain and in inflammatory gene expression in the placenta and fetal liver (specifically, Tnf, Tlr2, Tlr4, and Itgam) due to maternal choline supplementation and/or maternal air pollution exposure. We found that DEP led to changes in microglial morphology in the fetal dentate gyrus of E18 male, but not female, fetuses. In the placenta and fetal liver of males, inflammatory gene expression was affected by both DEP and maternal choline supplementation. However, maternal choline supplementation alone upregulated inflammatory gene expression in females, which may indicate an alteration in maturation rate. These data further contribute to the growing literature indicating region- and tissue-specificity in the developmental immune system in the context of maternal exposures.

neuroscience↗

bioRxiv2022

Inhibition of Aromatase by Hops, Licorice Species, and their bioactive compounds in Postmenopausal Breast Tissue

Breast cancer risk continues to rise post menopause. Endocrine therapies are employed to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer preventionatural products such as hops (Humulus lupulus) and pharmacopoeial licorice (Glycyrrhiza) species, which are used for managing menopausal symptoms, have demonstrated estrogenic and chemopreventive properties. Their beneficial effects on aromatase activity and expression as important factors in postmenopausal breast carcinogenesis are understudied. The presented data show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plants. Moreover, phytoestrogens such as 8-prenylnaringenin from hops as well as liquiritigenin and 8-prenylapigenin from licorice are shown to be potent bioactives, in line with computational docking studies. 8-Prenylnaringenin, GI extract, liquiritigenin, and licochalcone A all suppress aromatase expression in postmenopausal womens breast tissue. Collectively, these data suggest that these natural products may have breast cancer prevention potential for high-risk postmenopausal women.

pharmacology and toxicology↗

bioRxiv2021

Maternal diet disrupts the placenta-brain axis in a sex-specific manner

High maternal weight is associated with a number of detrimental outcomes in offspring, including increased susceptibility to neurological disorders such as anxiety, depression, and communicative disorders (e.g. autism spectrum disorders)1-8. Despite widespread acknowledgement of sex-biases in the prevalence, incidence, and age of onset of these disorders, few studies have investigated potential sex-biased mechanisms underlying disorder susceptibility. Here, we use a mouse model to demonstrate how maternal high-fat diet, one contributor to overweight, causes endotoxin accumulation in fetal tissue, and subsequent perinatal inflammation influences sex-specific behavioral outcomes in offspring. In male high-fat diet offspring, increased macrophage toll like receptor 4 signaling results in excess phagocytosis of serotonin neurons in the developing dorsal raphe nucleus, decreasing serotonin bioavailability in the fetal and adult brain. Bulk sequencing from a large cohort of matched first trimester human fetal brain, placenta, and maternal decidua samples reveals sex-specific transcriptome-wide changes in placenta and brain tissue in response to maternal triglyceride accumulation (a proxy for dietary fat content). Further, we find that fetal brain serotonin levels decrease as maternal dietary fat intake increases in males only. These findings uncover a microglia-dependent mechanism through which maternal diet may impact offspring susceptibility for neuropsychiatric disorder development in a sex-specific manner.

neuroscience↗

bioRxiv2021

Single-Cell transcriptional changes in hypothalamic CRH-expressing neurons after early-life adversity inform enduring alterations in responses to stress

Mental and cognitive health, as well as vulnerability to neuropsychiatric disorders, involve the interplay of genes with the environment, particularly during sensitive developmental periods. Early-life stress / adversity (ELA) promotes vulnerabilities to stress-related affective disorders, yet it is unknown how a transient ELA dictates life-long neuroendocrine and behavioral reactions to stress. The population of hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons that regulate stress-responses is a promising candidate to mediate the enduring influences of ELA on stress-related behavioral and hormonal responses via enduring transcriptional and epigenetic mechanisms. Capitalizing on a well-characterized model of ELA, we examined here the ELA-induced changes in gene expression profiles of stress-sensitive CRH-neurons in the hypothalamic paraventricular nucleus (PVN) of male mice. Given the known heterogeneity of these neuronal populations, we employed single-cell RNA sequencing (RNA-seq) approaches. The use of single-cell transcriptomics identified distinct CRH-expressing neuronal populations characterized by both their gene expression repertoire and their neurotransmitter profiles. Expression changes provoked by ELA clustered around genes involved in neuronal differentiation, synapse formation, altered energy metabolism and the cellular responses to stress and injury. Notably, the ELA-induced transcriptional changes took place primarily in subpopulations of glutamatergic CRH cells. Finally, ELA-induced transcriptional reprogramming of hypothalamic CRH-expressing neurons heralded significant, enduring disruptions of both hormonal and behavioral responses to stress throughout life.

neuroscience↗

bioRxiv2021

Impaired developmental microglial pruning of excitatory synapses on CRH-expressing hypothalamic neurons exacerbates stress responses throughout life

The developmental origins of stress-related mental illnesses are well-established, and early-life stress/adversity (ELA) is an important risk factor. However, it is unclear how ELA impacts the maturation of salient brain circuits, provoking enduring vulnerability to stress and stress-related disorders. Here we find that ELA increases the number and function of excitatory synapses onto stress-sensitive hypothalamic corticotropin-releasing hormone (CRH)-expressing neurons, and implicate disrupted synapse pruning by microglia as a key mechanism. Microglial process dynamics on live imaging, and engulfment of synaptic elements by microglia, were both attenuated in ELA mice, associated with deficient signaling of the microglial phagocytic receptor Mer. Accordingly, selective chemogenetic activation of ELA microglia increased microglial process dynamics and reduced excitatory synapse density to control levels. Selective early-life microglial activation also mitigated the adrenal hypertrophy and prolonged stress responses in adult ELA mice, establishing microglial actions during development as powerful contributors to experience-dependent sculpting of stress-related brain circuits.

neuroscience↗

bioRxiv2019

On the origins of a selective vulnerability to opioid addiction.

The origins and neural bases of the current opioid addiction epidemic are unclear. Genetics plays a major role in addiction vulnerability, but cannot account for the recent exponential rise in opioid abuse, so environmental factors must contribute. Individuals with history of early-life adversity (ELA) are disproportionately prone to opioid addiction, yet whether ELA interacts with factors such as increased access to opioids to directly influence brain development and function and cause opioid addiction vulnerability is unknown. We simulated ELA in female rats and this led to a striking opioid addiction-like phenotype. This was characterized by resistance to extinction, increased relapse-like behavior, and, as in addicted humans, major increases in opioid economic demand. By contrast, seeking of a less salient natural reward was unaffected by ELA, whereas demand for highly palatable treats was augmented. These discoveries provide novel insights into the origins and nature of reward circuit malfunction that may set the stage for addiction.

neuroscience↗

bioRxiv2019

Associations of ‘Relative corticosterone deficiency’ with genetic variation in CYP17A1 and metabolic syndrome features

Context and objectiveCommon genetic variants in CYP17A1 associate with higher blood pressure, putatively from impaired 17-hydroxylase activity and mineralocorticoid excess. However, the same variants protect against obesity and insulin resistance. We tested whether CYP17A1 variants that enhance 17-hydroxylase activity cause relative corticosterone deficiency. Since corticosterone is thought to contribute disproportionately to negative feedback in the hypothalamic-pituitary-adrenal axis, we also tested whether lower corticosterone associates with higher cortisol and hence with metabolic syndrome.\n\nDesignCross-sectional studies within the population-based Orkney Complex Disease Study (ORCADES; n=2018), VIKING Health Study Shetland (VIKING; n=2098), East Hertfordshire study (EHERTS; n=279), Edinburgh Type 2 Diabetes Study (ET2DS; n=903), and the Swiss Kidney Project on Genes in Hypertension (SKIPOGH; n=888).\n\nOutcome measuresCortisol and corticosterone in morning plasma samples in ORCADES, VIKING and ET2DS, and in EHERTS in plasma following overnight dexamethasone suppression (0.25mg) and 30 mins after ACTH1-24 (1{micro}g); cortisol and corticosterone metabolites in day and night urine samples in SKIPOGH. Features of the metabolic syndrome including body mass index, systolic blood pressure, lipid profile, fasting glucose, fasting insulin and HOMA-IR.\n\nResultsIn ORCADES, ET2DS and SKIPOGH, CYP17A1 variants were associated with corticosterone:cortisol ratio. In ORCADES, VIKING and ET2DS there were consistent associations of morning plasma cortisol and corticosterone with BMI, blood pressure, lipid profile, fasting glucose and HOMA-IR. In EHERTS, however, after dexamethasone suppression and ACTH1-24 stimulation, impaired glucose tolerance and insulin sensitivity were associated with higher cortisol but lower corticosterone. Similarly, in SKIPOGH, low corticosterone:cortisol metabolite ratios were associated with high BMI and dyslipidemia.\n\nConclusions Relative corticosterone deficiency, due to a primary alteration in adrenal steroidogenesis favouring cortisol over corticosterone, may mediate the associations of genetic variation in CYP17A1 with metabolic syndrome. However, additional determinants of variation in plasma corticosterone are likely to explain its generally positive associations with features of metabolic syndrome.

epidemiology↗

bioRxiv2018

Intra-individual methylomics detects the impact of early-life adversity

Genetic and environmental factors interact during sensitive periods early in life to influence mental health and disease via epigenetic processes such as DNA methylation. However, it is not known if DNA methylation changes outside the brain provide an epigenetic signature of early-life experiences. Here, we employed a novel intra-individual approach by testing DNA methylation from buccal cells of individual rats before and immediately after exposure to one week of typical or adverse life experience. We find that whereas inter-individual changes in DNA methylation reflect the effect of age, DNA methylation changes within paired DNA samples from the same individual reflect the impact of diverse neonatal experiences. Genes coding for critical cellular-metabolic enzymes, ion channels and receptors were more methylated in pups exposed to the adverse environment, predictive of their repression. In contrast, the adverse experience was associated with less methylation on genes involved in pathways of death and inflammation as well as cell-fate related transcription factors, indicating their potential upregulation. Thus, intra-individual methylome signatures indicate large-scale transcription-driven alterations of cellular fate, growth and function.

genomics↗