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© 2016, Biomedical Engineering Society. Nanoporous gold (np-Au) is a promising multifunctional material for neural electrodes. We have previously shown that np-Au nanotopography reduces astrocyte surface coverage (linked to undesirable gliosis) while maintaining high neuronal coverage in a cortical primary neuron-glia co-culture model as long as 2 weeks in vitro. Here, we investigate the potential influence of secreted soluble factors from cells grown on np-Au leading to the cell type-specific surface coverage on conventional tissue culture plastic. We then test the hypothesis that secretion of factors is responsible for inhibiting astrocyte coverage on np-Au. In order to assess whether factors secreted from cells grown on np-Au surfaces reduced surface coverage by astrocytes, we seeded fresh primary rat neuron-glia co-cultures on conventional polystyrene culture dishes, but maintained the cells in conditioned media from co-cultures grown on np-Au surfaces. After 1 week in vitro, a preferential reduction in astrocyte surface coverage was not observed, suggesting that soluble factors are not playing a role. In contrast, 4 h after cell seeding there were a significant number of non-adhered, yet still viable, cells for the cultures on np-Au surfaces. We hypothesize that the non-adherent cells are mainly astrocytes, because: (i) there was no difference in neuronal cell coverage between np-Au and pl-Au for long culture durations and (ii) neurons are post-mitotic and not expected to increase in number upon attaching to the surface. Overall, the results suggest that the np-Au topography leads to preferential neuronal attachment shortly after cell seeding and limits astrocyte-specific np-Au surface coverage at longer culture durations.

We previously demonstrated that the organophosphate diisopropylfluorophosphate (DFP) induced region-specific delayed neuronal injury in rat brain. The goal of this study was to examine the spatiotemporal pattern of the astroglial response after acute intoxication of rats with DFP. Rats were euthanized at varying times between 1 h to 7 days after DFP administration (9mg/kg, i.p.). The distribution of activated astrocytes in different brain regions was determined by immunostaining for glial fibrillary acidic acid (GFAP). GFAP immunoreactivity increased in the hippocampus, and piriform/entorhinal cortex at 1 h, peaked between 4-8 h then decreased from 16 to 24 h. GFAP labeling in the amygdala gradually increased over the 24 h period. A large increase in GFAP labeling was seen at 3-7 days following DFP administration in the hippocampus, piriform/entorhinal cortex and dorsolateral thalamus. At 3 days post-DFP exposure, GFAP immunoreactivity was totally absent in central areas of the hippocampus, piriform/entorhinal cortex and dorsolateral thalamus that contained numerous FluoroJade B (FJB) labeled neurons. By 7 days, GFAP expression was highly upregulated in all areas of each brain region. The mRNA expression of GFAP in hippocampus slowly increased at 24 h following DFP administration then dramatically increased at 3 days. This study suggests that activation of glial cells may contribute to the early neuropathological changes and later neuronal repair/plasticity following acute DFP intoxication.

BackgroundBMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons.ResultsSympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG) during times of initial growth and rapid expansion of the dendritic arbor.ConclusionsThese data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

We have examined the effects of collagen IV on the morphological development of embryonic rat sympathetic neurons in vitro. In short-term (less than or equal to 24 h) culture, collagen IV accelerated process outgrowth, causing increases in the number of neurites and total neuritic length. Analysis of proteolytic fragments of collagen IV indicated that the NC1 domain was nearly as active as the intact molecule in stimulating process outgrowth; in contrast, the 7S domain and triple helix-rich fragments of collagen IV were inactive. Moreover, anti-NC1 antiserum inhibited neuritic outgrowth on collagen IV by 79%. In long-term (up to 28 d) cultures, neurons chronically exposed to collagen IV maintained a single axon but failed to form dendrites. Thus, the NC1 domain of collagen IV can alter neuronal development by selectively stimulating axonal growth. Comparison of collagen IV's effects to those of laminin revealed that these molecules exert quantitatively different effects on the rate of initial axon growth and the number of axons extended by sympathetic neurons. Moreover, neuritic outgrowth on collagen IV, but not laminin, was blocked by cycloheximide. We also observed differences in the receptors mediating the neurite-promoting activity of these proteins. Two different antisera that recognize beta 1 integrins each blocked neuritic outgrowth on both collagen IV and laminin; however, an mAb (3A3) specific for the alpha 1 beta 1 integrin inhibited collagen IV but not laminin-induced process growth in cultures of both sympathetic and dorsal root neurons. These data suggest that immunologically distinct integrins mediate the response of peripheral neurons to collagen IV and laminin.

BACKGROUND: The 'developmental origins of adult disease' hypothesis was originally derived from evidence linking low birth weight to cardiovascular diseases including stroke. Subsequently, it has been expanded to include developmental exposures to environmental contaminants as risk factors for adult onset disease. OBJECTIVE: Our goal in this study was to test the hypothesis that developmental exposure to polychlorinated biphenyls (PCBs) alters stroke outcome in adults. METHODS: We exposed rats to the PCB mixture Aroclor 1254 (A1254) at 0.1 or 1 mg/kg/day in the maternal diet throughout gestation and lactation. Focal cerebral ischemia was induced at 6-8 weeks of age via middle cerebral artery occlusion, and infarct size was measured in the cerebral cortex and striatum at 22 hr of reperfusion. PCB congeners were quantified in brain tissue by gas chromatography with microelectron capture detection, and cortical and striatal expression of Bcl2 and Cyp2C11 were quantified by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Developmental exposure to A1254 significantly decreased striatal infarct in females and males at 0.1 and 1 mg/kg/day, respectively. Predominantly ortho-substituted PCB congeners were detected above background levels in brains of adult females and males exposed to A1254 at 1 but not 0.1 mg/kg/day. Effects of developmental A1254 exposure on Bcl2 and Cyp2C11 expression did not correlate with effects on infarct volume. CONCLUSION: Our data provide proof of principle that developmental exposures to environmental contaminants influence the response of the adult brain to ischemic injury and thus represent potentially important determinants of stroke susceptibility. [ABSTRACT FROM AUTHOR]

Polychlorinated biphenyls (PCBs) and their metabolites are linked to developmental neurotoxicity, but their levels in the gestational and lactational environment remain unexplored. This study investigated the effects of dietary exposure to the Fox River Mixture (FRM) on serum levels of PCBs and their metabolites in female C57BL/6 J mice. Mice were exposed to 0.1, 1.0, or 6.0 mg/kg body weight/day of FRM beginning two weeks before mating and throughout gestation and lactation. Serum samples collected from the dams at weaning were analyzed using gas chromatograph-tandem mass spectrometry and nontarget liquid chromatography-high resolution mass spectrometry. Results showed complex and dose-dependent differences in PCB and metabolite profiles. Untargeted metabolomics revealed alterations in metabolites involved in glucuronidation. Network analysis suggested disturbances in heme and amino acid metabolism associated with higher chlorinated PCBs. These findings suggested that PCBs and metabolites present in the gestational and lactation environment of mice may contribute to developmental neurotoxicity in rodents., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pamela J. Lein reports a relationship with Friedman Rubin (Seattle, WA) that includes: paid expert testimony. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Neurodevelopmental disorders have a strong male bias that is poorly understood. Placenta is a rich source of molecular information about environmental interactions with genetics (including biological sex), that affect the developing brain. We investigated placental-brain transcriptional responses in an established mouse model of prenatal exposure to a human-relevant mixture of polychlorinated biphenyls (PCBs)., Results: To understand sex, tissue, and dosage effects in embryonic (E18) brain and placenta by RNAseq, we used weighted gene correlation network analysis (WGCNA) to create correlated gene networks that could be compared across sex or tissue. WGCNA revealed that expression within most correlated gene networks was significantly and strongly associated with PCB exposures, but frequently in opposite directions between male-female and placenta-brain comparisons. In both WGCNA and differentially expressed gene analyses, male brain showed more PCB-induced transcriptional changes than male placenta, but the reverse pattern was seen in females. Furthermore, non-monotonic dose responses to PCBs were observed in most gene networks but were most prominent in male brain. The transcriptomic effects of low dose PCB exposure were significantly reversed by dietary folic acid supplementation across both sexes, but these effects were strongest in female placenta. PCB-dysregulated and folic acid-reversed gene networks were commonly enriched in functions in metabolic pathways involved in energy usage and translation, with female-specific protective effects enriched in PPAR, thermogenesis, glycerolipids, and O-glycan biosynthesis, as opposed to toxicant responses in male brain., Conclusions: The female protective effect in prenatal PCB exposures appears to be mediated by dose-dependent sex differences in transcriptional modulation of metabolism in placenta.

Developmental exposures to PCBs are implicated in the etiology of neurodevelopmental disorders (NDDs). This observation is concerning given the continued presence of PCBs in the human environment and the increasing incidence of NDDs. Previous studies reported that developmental exposure to legacy commercial PCB mixtures (Aroclors) or single PCB congeners found in Aroclors caused NDD-relevant behavioral phenotypes in animal models. However, the PCB congener profile in contemporary human samples is dissimilar to that of the legacy Aroclors, raising the question of whether human-relevant PCB mixtures similarly interfere with normal brain development. To address this question, we assessed the developmental neurotoxicity of the Fox River Mixture (FRM), which was designed to mimic the congener profile identified in fish from the PCB-contaminated Fox River that constitute a primary protein source in the diet of surrounding communities. Adult female C57BL/6 J mouse dams (8-10 weeks old) were exposed to vehicle (peanut oil) or FRM at 0.1, 1.0, or 6.0 mg/kg/d in their diet throughout gestation and lactation, and neurodevelopmental outcomes were assessed in their pups. Ultrasonic vocalizations (USVs) and measures of general development were quantified at postnatal day (P) 7, while performance in the spontaneous alternation task and the 3-chambered social approach/social novelty task was assessed on P35. Triiodothyronine (T3) and thyroxine (T4) were quantified in serum collected from the dams when pups were weaned and from pups on P28 and P35. Developmental exposure to FRM did not alter pup weight or body temperature on P7, but USVs were significantly decreased in litters exposed to FRM at 0.1 or 6.0 mg/kg/d in the maternal diet. FRM also impaired male and female pups' performance in the social novelty task. Compared to sex-matched vehicles, significantly decreased social novelty was observed in male and female pups in the 0.1 and 6.0 mg/kg/d dose groups. FRM did not alter performance in the spontaneous alternation or social approach tasks. FRM increased serum T3 levels but decreased serum T4 levels in P28 male pups in the 1.0 and 6.0 mg/kg/d dose groups. In P35 female pups and dams, serum T3 levels decreased in the 6.0 mg/kg/d dose group while T4 levels were not altered. Collectively, these findings suggest that FRM interferes with the development of social communication and social novelty, but not memory, supporting the hypothesis that contemporary PCB exposures pose a risk to the developing brain. FRM had sex, age, and dose-dependent effects on serum thyroid hormone levels that overlapped but did not perfectly align with the FRM effects on behavioral outcomes. These observations suggest that changes in thyroid hormone levels are not likely the major factor underlying the behavioral deficits observed in FRM-exposed animals., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pamela J. Lein was hired as an expert witness by lawyers representing a group of plaintiffs alleging they were harmed by exposure to PCBs in school air. In that capacity, she testified as an expert witness on PCB neurotoxicity. The defendant was Pharmacia, a successor company to Monsanto., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Acute poisoning with organophosphorus cholinesterase inhibitors (OPs), such as OP nerve agents and pesticides, can cause life threatening cholinergic crisis and status epilepticus (SE). Survivors often experience significant morbidity, including brain injury, acquired epilepsy, and cognitive deficits. Current medical countermeasures for acute OP poisoning include a benzodiazepine to mitigate seizures. Diazepam was long the benzodiazepine included in autoinjectors used to treat OP-induced seizures, but it is now being replaced in many guidelines by midazolam, which terminates seizures more quickly, particularly when administered intramuscularly. While a direct correlation between seizure duration and the extent of brain injury has been widely reported, there are limited data comparing the neuroprotective efficacy of diazepam versus midazolam following acute OP intoxication. To address this data gap, we used non-invasive imaging techniques to longitudinally quantify neuropathology in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP) with and without post-exposure intervention with diazepam or midazolam. Magnetic resonance imaging (MRI) was used to monitor neuropathology and brain atrophy, while positron emission tomography (PET) with a radiotracer targeting translocator protein (TSPO) was utilized to assess neuroinflammation. Animals were scanned at 3, 7, 28, 65, 91, and 168 days post-DFP and imaging metrics were quantitated for the hippocampus, amygdala, piriform cortex, thalamus, cerebral cortex and lateral ventricles. In the DFP-intoxicated rat, neuroinflammation persisted for the duration of the study coincident with progressive atrophy and ongoing tissue remodeling. Benzodiazepines attenuated neuropathology in a region-dependent manner, but neither benzodiazepine was effective in attenuating long-term neuroinflammation as detected by TSPO PET. Diffusion MRI and TSPO PET metrics were highly correlated with seizure severity, and early MRI and PET metrics were positively correlated with long-term brain atrophy. Collectively, these results suggest that anti-seizure therapy alone is insufficient to prevent long-lasting neuroinflammation and tissue remodeling., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pamela J Lein reports financial support was provided by the National Institute of Neurological Disorders and Stroke CounterACT program. Brad A. Hobson reports financial support was provided by a T32 training program awarded to the University of California, Davis by the National Institute of General Medical Sciences and by a scholarship from the ARCS Foundation of Northern California., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acute intoxication with organophosphate (OP) cholinesterase inhibitors poses a significant public health risk. While currently approved medical countermeasures can improve survival rates, they often fail to prevent chronic neurological damage. Therefore, there is need to develop effective therapies and quantitative metrics for assessing OP-induced brain injury and its rescue by these therapies. In this study we used a rat model of acute intoxication with the OP, diisopropylfluorophosphate (DFP), to test the hypothesis that T 2 measures obtained from brain magnetic resonance imaging (MRI) scans provide quantitative metrics of brain injury and therapeutic efficacy. Adult male Sprague Dawley rats were imaged on a 7T MRI scanner at 3, 7 and 28 days post-exposure to DFP or vehicle (VEH) with or without treatment with the standard of care antiseizure drug, midazolam (MDZ); a novel antiseizure medication, allopregnanolone (ALLO); or combination therapy with MDZ and ALLO (DUO). Our results show that mean T 2 values in DFP-exposed animals were: (1) higher than VEH in all volumes of interest (VOIs) at day 3; (2) decreased with time; and (3) decreased in the thalamus at day 28. Treatment with ALLO or DUO, but not MDZ alone, significantly decreased mean T 2 values relative to untreated DFP animals in the piriform cortex at day 3. On day 28, the DUO group showed the most favorable T 2 characteristics. This study supports the utility of T 2 mapping for longitudinally monitoring brain injury and highlights the therapeutic potential of ALLO as an adjunct therapy to mitigate chronic morbidity associated with acute OP intoxication., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pamela J. Lein has patent Mitigation of epileptic seizures by combination therapy using benzodiazepines and neurosteroids issued to The Regents of the University of California., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Whole brain delineation (WBD) is utilized in neuroimaging analysis for data preprocessing and deriving whole brain image metrics. Current automated WBD techniques for analysis of preclinical brain MRI data show limited accuracy when images present with significant neuropathology and anatomical deformations, such as that resulting from organophosphate intoxication (OPI) and Alzheimer's Disease (AD), and inadequate generalizability., Methods: A modified 2D U-Net framework was employed for WBD of MRI rodent brains, consisting of 27 convolutional layers, batch normalization, two dropout layers and data augmentation, after training parameter optimization. A total of 265 T 2 -weighted 7.0 T MRI scans were utilized for the study, including 125 scans of an OPI rat model for neural network training. For testing and validation, 20 OPI rat scans and 120 scans of an AD rat model were utilized. U-Net performance was evaluated using Dice coefficients (DC) and Hausdorff distances (HD) between the U-Net-generated and manually segmented WBDs., Results: The U-Net achieved a DC (median[range]) of 0.984[0.936-0.990] and HD of 1.69[1.01-6.78] mm for OPI rat model scans, and a DC (mean[range]) of 0.975[0.898-0.991] and HD of 1.49[0.86-3.89] for the AD rat model scans., Comparison With Existing Methods: The proposed approach is fully automated and robust across two rat strains and longitudinal brain changes with a computational speed of 8 seconds/scan, overcoming limitations of manual segmentation., Conclusions: The modified 2D U-Net provided a fully automated, efficient, and generalizable segmentation approach that achieved high accuracy across two disparate rat models of neurological diseases., Competing Interests: Declaration of Competing Interest None of the authors have a conflict of interest with the work presented., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Acute intoxication with high levels of organophosphate (OP) cholinesterase inhibitors can cause cholinergic crisis, which is associated with acute, life-threatening parasympathomimetic symptoms, respiratory depression and seizures that can rapidly progress to status epilepticus (SE). Clinical and experimental data demonstrate that individuals who survive these acute neurotoxic effects often develop significant chronic morbidity, including behavioral deficits. The pathogenic mechanism(s) that link acute OP intoxication to chronic neurological deficits remain speculative. Cellular senescence has been linked to behavioral deficits associated with aging and neurodegenerative disease, but whether acute OP intoxication triggers cellular senescence in the brain has not been investigated. Here, we test this hypothesis in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats were administered DFP (4 mg/kg, s.c.). Control animals were administered an equal volume (300 µL) of sterile phosphate-buffered saline (s.c.). Both groups were subsequently injected with atropine sulfate (2 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.). DFP triggered seizure activity within minutes that rapidly progressed to SE, as determined using behavioral seizure criteria. Brains were collected from animals at 1, 3, and 6 months post-exposure for immunohistochemical analyses of p16, a biomarker of cellular senescence. While there was no immunohistochemical evidence of cellular senescence at 1-month post-exposure, at 3- and 6-months post-exposure, p16 immunoreactivity was significantly increased in the CA3 and dentate gyrus of the hippocampus, amygdala, piriform cortex and thalamus, but not the CA1 region of the hippocampus or the somatosensory cortex. Co-localization of p16 immunoreactivity with cell-specific biomarkers, specifically, NeuN, GFAP, S100β, IBA1 and CD31, revealed that p16 expression in the brain of DFP animals is neuron-specific. The spatial distribution of p16-immunopositive cells overlapped with expression of senescence associated β-galactosidase and with degenerating neurons identified by FluoroJade-C (FJC) staining. The co-occurrence of p16 and FJC was positively correlated. This study implicates cellular senescence as a novel pathogenic mechanism underlying the chronic neurological deficits observed in individuals who survive OP-induced cholinergic crisis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tsai, González, Grodzki, Bruun, Saito, Harvey and Lein.)

Studies suggest that ketogenic diets (KD) may improve memory in mouse models of aging and Alzheimer's disease (AD). This study determined whether a continuous or intermittent KD (IKD) enhanced cognitive behavior in the TgF344-AD rat model of AD. At 6 months-old, TgF344-AD and wild-type (WT) littermates were placed on a control (CD), KD, or IKD (morning CD and afternoon KD) provided as two meals per day for 2 or 6 months. Cognitive and motor behavior and circulating β-hydroxybutyrate (BHB), AD biomarkers and blood lipids were assessed. Animals on a KD diet had elevated circulating BHB, with IKD levels intermediate to CD and KD. TgF344-AD rats displayed impaired spatial learning memory in the Barnes maze at 8 and 12 months of age and impaired motor coordination at 12 months of age. Neither KD nor IKD improved performance compared to CD. At 12 months of age, TgF344-AD animals had elevated blood lipids. IKD reduced lipids to WT levels with KD further reducing cholesterol below WT levels. This study shows that at 8 or 12 months of age, KD or IKD intervention did not improve measures of cognitive or motor behavior in TgF344-AD rats; however, both IKD and KD positively impacted circulating lipids.

Recent experimental evidence suggests combined treatment with midazolam and allopregnanolone is more effective than midazolam alone in terminating seizures triggered by acute organophosphate (OP) intoxication. However, there are concerns that combined midazolam and allopregnanolone increases risk of adverse cardiovascular events. To address this, we used telemetry devices to record cardiovascular responses in adult male Sprague-Dawley rats acutely intoxicated with diisopropylfluorophosphate (DFP). Animals were administered DFP (4 mg/kg, sc), followed immediately by atropine (2 mg/kg, i.m.) and 2-PAM (25 mg/kg, i.m.). At 40 min post-exposure, a subset of animals received midazolam (0.65 mg/kg, im); at 50 min, these rats received a second dose of midazolam or allopregnanolone (12 mg/kg, im). DFP significantly increased blood pressure by ~ 80 mmHg and pulse pressure by ~ 34 mmHg that peaked within 12 min. DFP also increased core temperature by ~ 3.5 °C and heart rate by ~ 250 bpm that peaked at ~ 2 h. Heart rate variability (HRV), an index of autonomic function, was reduced by ~ 80%. All acute (within 15 min of exposure) and two-thirds of delayed (hours after exposure) mortalities were associated with non-ventricular cardiac events within 10 min of cardiovascular collapse, suggesting that non-ventricular events should be closely monitored in OP-poisoned patients. Compared to rats that survived DFP intoxication without treatment, midazolam significantly improved recovery of cardiovascular parameters and HRV, an effect enhanced by allopregnanolone. These data demonstrate that midazolam improved recovery of cardiovascular and autonomic function and that the combination of midazolam and allopregnanolone may be a better therapeutic strategy than midazolam alone., (© 2024. The Author(s).)

Acute intoxication with organophosphorus (OP) cholinesterase inhibitors can produce seizures that rapidly progress to life-threatening status epilepticus. Significant research effort has been focused on investigating the involvement of muscarinic acetylcholine receptors (mAChRs) in OP-induced seizure activity. In contrast, there has been far less attention on nicotinic AChRs (nAChRs) in this context. Here, we address this data gap using a combination of in vitro and in vivo models. Pharmacological antagonism and genetic deletion of α4, but not α7, nAChR subunits prevented or significantly attenuated OP-induced electrical spike activity in acute hippocampal slices and seizure activity in mice, indicating that α4 nAChR activation is necessary for neuronal hyperexcitability triggered by acute OP exposures. These findings not only suggest that therapeutic strategies for inhibiting the α4 nAChR subunit warrant further investigation as prophylactic and immediate treatments for acute OP-induced seizures, but also provide mechanistic insight into the role of the nicotinic cholinergic system in seizure generation.

Epoxyeicosatrienoic acids (EpETrEs) are bioactive lipid mediators of arachidonic acid cytochrome P450 oxidation. In vivo, the free (unbound) form of EpETrEs regulate multiple processes including blood flow, angiogenesis and inflammation resolution. Free EpETrEs are thought to rapidly degrade via soluble epoxide hydrolase (sEH); yet, in many tissues, the majority of EpETrEs are esterified to complex lipids (e.g. phospholipids) suggesting that esterification may play a major role in regulating free, bioactive EpETrE levels. This hypothesis was tested by quantifying the metabolism of intraperitoneally injected free d11-11(12)-Epoxyeicosatrienoic acid (d11-11(12)-EpETrE) in male and female rats. Plasma and tissues (liver, adipose and brain) were obtained 3 to 4 min later and assayed for d11-11(12)-EpETrE and its sEH metabolite, d11-11,12-dihydroxyeicosatrienoic acid (d11-11,12-diHETrE) in both the free and esterified lipid fractions. In both males and females, the majority of injected tracer was recovered in liver followed by plasma and adipose. No tracer was detected in the brain, indicating that brain levels are maintained by endogenous synthesis from precursor fatty acids. In plasma, liver, and adipose, the majority (>54 %) of d11-11(12)-EpETrE was found esterified to phospholipids or neutral lipids (triglycerides and cholesteryl esters). sEH-derived d11-11,12-diHETrE was not detected in plasma or tissues, suggesting negligible conversion within the 3-4 min period post tracer injection. This study shows that esterification is the main pathway regulating free 11(12)-EpETrE levels in vivo., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Organophosphate (OP) poisoning can trigger cholinergic crisis, a life-threatening toxidrome that includes seizures and status epilepticus. These acute toxic responses are associated with persistent neuroinflammation and spontaneous recurrent seizures (SRS), also known as acquired epilepsy. Blood-brain barrier (BBB) impairment has recently been proposed as a pathogenic mechanism linking acute OP intoxication to chronic adverse neurologic outcomes. In this review, we briefly describe the cellular and molecular components of the BBB, review evidence of altered BBB integrity following acute OP intoxication, and discuss potential mechanisms by which acute OP intoxication may promote BBB dysfunction. We highlight the complex interplay between neuroinflammation and BBB dysfunction that suggests a positive feedforward interaction. Lastly, we examine research from diverse models and disease states that suggest mechanisms by which loss of BBB integrity may contribute to epileptogenic processes. Collectively, the literature identifies BBB impairment as a convergent mechanism of neurologic disease and justifies further mechanistic research into how acute OP intoxication causes BBB impairment and its role in the pathogenesis of SRS and potentially other long-term neurologic sequelae. Such research is critical for evaluating BBB stabilization as a neuroprotective strategy for mitigating OP-induced epilepsy and possibly seizure disorders of other etiologies. SIGNIFICANCE STATEMENT: Clinical and preclinical studies support a link between blood-brain barrier (BBB) dysfunction and epileptogenesis; however, a causal relationship has been difficult to prove. Mechanistic studies to delineate relationships between BBB dysfunction and epilepsy may provide novel insights into BBB stabilization as a neuroprotective strategy for mitigating epilepsy resulting from acute organophosphate (OP) intoxication and non-OP causes and potentially other adverse neurological conditions associated with acute OP intoxication, such as cognitive impairment., (Copyright © 2024 by The Author(s).)

Dendritic arborization is a critical determinant of neuronal connectivity. The structure of a neuron's dendritic arbor determines the number of synaptic inputs a neuron can receive and how it processes synaptic input from other neurons. Here, we describe methods for visualizing and quantifying the dendritic arbor in primary cell cultures and in the intact rodent brain. These techniques can be used to answer significant scientific questions, such as the effects of disease processes, drugs, growth factors, and diverse environmental stressors on dendritogenesis in both in vitro and in vivo rodent models., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bile acids (BA) are among the most abundant metabolites produced by the gut microbiome. Primary BAs produced in the liver are converted by gut bacterial 7-α-dehydroxylation into secondary BAs, which can differentially regulate host health via signaling based on their varying affinity for BA receptors. Despite the importance of secondary BAs in host health, the regulation of 7-α-dehydroxylation and the role of diet in modulating this process is incompletely defined. Understanding this process could lead to dietary guidelines that beneficially shift BA metabolism. Dietary fiber regulates gut microbial composition and metabolite production. We tested the hypothesis that feeding mice a diet rich in a fermentable dietary fiber, resistant starch (RS), would alter gut bacterial BA metabolism. Male and female wild-type mice were fed a diet supplemented with RS or an isocaloric control diet (IC). Metabolic parameters were similar between groups. RS supplementation increased gut luminal deoxycholic acid (DCA) abundance. However, gut luminal cholic acid (CA) abundance, the substrate for 7-α-dehydroxylation in DCA production, was unaltered by RS. Further, RS supplementation did not change the mRNA expression of hepatic BA producing enzymes or ileal BA transporters. Metagenomic assessment of gut bacterial composition revealed no change in the relative abundance of bacteria known to perform 7-α-dehydroxylation. P. ginsenosidimutans and P. multiformis were positively correlated with gut luminal DCA abundance and increased in response to RS supplementation. These data demonstrate that RS supplementation enriches gut luminal DCA abundance without increasing the relative abundance of bacteria known to perform 7-α-dehydroxylation.

Neuropilin-1 (Nrp1), a transmembrane protein expressed on CD4 + T cells, is mostly studied in the context of regulatory T cell (Treg) function. More recently, there is increasing evidence that Nrp1 is also highly expressed on activated effector T cells and that increases in these Nrp1-expressing CD4 + T cells correspond with immunopathology across several T cell-dependent disease models. Thus, Nrp1 may be implicated in the identification and function of immunopathologic T cells. Nrp1 downregulation in CD4 + T cells is one of the strongest transcriptional changes in response to immunoregulatory compounds that act though the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. To better understand the link between AhR and Nrp1 expression on CD4 + T cells, Nrp1 expression was assessed in vivo and in vitro following AhR ligand treatment. In the current study, we identified that the percentage of Nrp1 expressing CD4 + T cells increases over the course of activation and proliferation in vivo . The actively dividing Nrp1 + Foxp3 - cells express the classic effector phenotype of CD44 hi CD45RB lo , and the increase in Nrp1 + Foxp3 - cells is prevented by AhR activation. In contrast, Nrp1 expression is not modulated by AhR activation in non-proliferating CD4 + T cells. The downregulation of Nrp1 on CD4 + T cells was recapitulated in vitro in cells isolated from C57BL/6 and NOD (non-obese diabetic) mice. CD4 + Foxp3 - cells expressing CD25, stimulated with IL-2, or differentiated into Th1 cells, were particularly sensitive to AhR-mediated inhibition of Nrp1 upregulation. IL-2 was necessary for AhR-dependent downregulation of Nrp1 expression both in vitro and in vivo . Collectively, the data demonstrate that Nrp1 is a CD4 + T cell activation marker and that regulation of Nrp1 could be a previously undescribed mechanism by which AhR ligands modulate effector CD4 + T cell responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sandoval, Malany, Thongphanh, Martinez, Goodson, Souza, Lin, Sweeney, Pennington, Lein, Kerkvliet and Ehrlich.)

Acute organophosphate (OP) intoxication can trigger seizures that progress to status epilepticus (SE), and survivors often develop chronic morbidities, including spontaneous recurrent seizures (SRS). The pathogenic mechanisms underlying OP-induced SRS are unknown, but increased BBB permeability is hypothesized to be involved. Previous studies reported BBB leakage following OP-induced SE, but key information regarding time and regional distribution of BBB impairment during the epileptogenic period is missing. To address this data gap, we characterized the spatiotemporal progression of BBB impairment during the first week post-exposure in a rat model of diisopropylfluorophosphate-induced SE, using MRI and albumin immunohistochemistry. Increased BBB permeability, which was detected at 6 h and persisted up to 7 d post-exposure, was most severe and persistent in the piriform cortex and amygdala, moderate but persistent in the thalamus, and less severe and transient in the hippocampus and somatosensory cortex. The extent of BBB leakage was positively correlated with behavioral seizure severity, with the strongest association identified in the piriform cortex and amygdala. These findings provide evidence of the duration, magnitude and spatial breakdown of the BBB during the epileptogenic period following OP-induced SE and support BBB regulation as a viable therapeutic target for preventing SRS following acute OP intoxication., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Developmental exposure to environmental toxicants has been linked to the onset of neurological disorders and diseases. Despite substantial advances in the field of neurotoxicology, there remain significant knowledge gaps in our understanding of cellular targets and molecular mechanisms that mediate the neurotoxicological endpoints associated with exposure to both legacy contaminants and emerging contaminants of concern. Zebrafish are a powerful neurotoxicological model given their high degree sequence conservation with humans and the similarities they share with mammals in micro- and macro-level brain structures. Many zebrafish studies have effectively utilized behavioral assays to predict the neurotoxic potential of different compounds, but behavioral phenotypes are rarely able to predict the brain structures, cell types, or mechanisms affected by chemical exposures. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI), a recently developed genetically-encoded calcium indicator, undergoes a permanent green to red switch in the presence of elevated intracellular Ca 2+ concentrations and 405-nm light, which allows for a "snapshot" of brain activity in freely-swimming larvae. To determine whether behavioral results are predictive of patterns of neuronal activity, we assessed the effects of three common neurotoxicants, ethanol, 2,2',3,5',6-pentachlorobiphenyl (PCB 95), and monoethylhexyl phthalate (MEHP), on both brain activity and behavior by combining the behavioral light/dark assay with CaMPARI imaging. We demonstrate that brain activity profiles and behavioral phenotypes are not always concordant and, therefore, behavior alone is not sufficient to understand how toxicant exposure affects neural development and network dynamics. We conclude that pairing behavioral assays with functional neuroimaging tools such as CaMPARI provides a more comprehensive understanding of the neurotoxic endpoints of compounds while still offering a relatively high throughput approach to toxicity testing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here, we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, each dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 minutes later by ALLO. Behavioral state, vital signs, arterial blood gases, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 hours after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/ml. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine., (© 2023 The Authors. Epilepsia Open published by Wiley Periodicals LLC on behalf of International League Against Epilepsy.)

Hexahydro-1,3,5-trinitro-1,3,5-triazine, or Royal Demolition Explosive (RDX), is a major component of plastic explosives such as C-4. Acute exposures from intentional or accidental ingestion are a documented clinical concern, especially among young male U.S. service members in the armed forces. When ingested in large enough quantity, RDX causes tonic-clonic seizures. Previous in silico and in vitro experiments predict that RDX causes seizures by inhibiting α1β2γ2 γ-aminobutyric acid type A (GABA A ) receptor-mediated chloride currents. To determine whether this mechanism translates in vivo, we established a larval zebrafish model of RDX-induced seizures. After a 3 h of exposure to 300 µM RDX, larval zebrafish exhibited a significant increase in motility in comparison to vehicle controls. Researchers blinded to experimental group manually scored a 20-min segment of video starting at 3.5 h post-exposure and found significant seizure behavior that correlated with automated seizure scores. Midazolam (MDZ), an nonselective GABA A R positive allosteric modulator (PAM), and a combination of Zolpidem (α1 selective PAM) and compound 2-261 (β2/3-selective PAM) were effective in mitigating RDX-triggered behavioral and electrographic seizures. These findings confirm that RDX induces seizure activity via inhibition of the α1β2γ2 GABA A R and support the use of GABA A R-targeted anti-seizure drugs for the treatment of RDX-induced seizures., (© 2023. The Author(s).)

In recent years, psychedelics have garnered significant interest as therapeutic agents for treating diverse neuropsychiatric disorders. However, the potential for these compounds to produce developmental neurotoxicity has not been rigorously assessed, and much of the available safety data is based on epidemiological studies with limited experimental testing in laboratory animal models. Moreover, the experimental safety data available thus far have focused on adult organisms, and the few studies conducted using developing organisms have tested a limited number of compounds, precluding direct comparisons between various chemical scaffolds. In the present study, 13 psychoactive compounds of different chemical or pharmacological classes were screened in a larval zebrafish model for teratological and behavioral abnormalities following acute and chronic developmental exposures. We found that the psychedelic tryptamines and ketamine were less neurotoxic to larval zebrafish than LSD and psychostimulants. Our work, which leverages the advantage of using zebrafish for higher throughput toxicity screening, provides a robust reference database for comparing the neurotoxicity profiles of novel psychedelics currently under development for therapeutic applications.

Background: Gestational exposure to polychlorinated biphenyls (PCBs) has been associated with elevated risk for neurodevelopmental disorders. Placental epigenetics may serve as a potential mechanism of risk or marker of altered placental function. Prior studies have associated differential placental DNA methylation with maternal PCB exposure or with increased risk of autism spectrum disorder (ASD). However, sequencing-based placental methylomes have not previously been tested for simultaneous associations with maternal PCB levels and child neurodevelopmental outcomes., Objectives: We aimed to identify placental DNA methylation patterns associated with maternal PCB levels and child neurodevelopmental outcomes in the high-risk ASD MARBLES cohort., Methods: We measured 209 PCB congeners in 104 maternal serum samples collected at delivery. We identified networks of DNA methylation from 147 placenta samples using the Comethyl R package, which performs weighted gene correlation network analysis for whole genome bisulfite sequencing data. We tested placental DNA methylation modules for association with maternal serum PCB levels, child neurodevelopment, and other participant traits., Results: PCBs 153 + 168, 170, 180 + 193, and 187 were detected in over 50% of maternal serum samples and were highly correlated with one another. Consistent with previous findings, maternal age was the strongest predictor of serum PCB levels, alongside year of sample collection, pre-pregnancy BMI, and polyunsaturated fatty acid levels. Twenty seven modules of placental DNA methylation were identified, including five which significantly correlated with one or more PCBs, and four which correlated with child neurodevelopment. Two modules associated with maternal PCB levels as well as child neurodevelopment, and mapped to CSMD1 and AUTS2, genes previously implicated in ASD and identified as differentially methylated regions in mouse brain and placenta following gestational PCB exposure., Conclusions: Placental DNA co-methylation modules were associated with maternal PCBs and child neurodevelopment. Methylation of CSMD1 and AUTS2 could be markers of altered placental function and/or ASD risk following maternal PCB exposure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Assessing health outcomes associated with exposure to polychlorinated biphenyls (PCBs) is important given their persistent and ubiquitous nature. PCBs are classified as a Group 1 carcinogen, but the full range of potential noncancer health effects from exposure to PCBs has not been systematically summarized and evaluated. We used systematic review methods to identify and screen the literature using combined manual review and machine learning approaches. A protocol was developed that describes the literature search strategy and Populations, Exposures, Comparators, and Outcomes (PECO) criteria used to facilitate subsequent screening and categorization of literature into a systematic evidence map of PCB exposure and noncancer health endpoints across 15 organs/systems. A comprehensive literature search yielded 62,599 records. After electronic prioritization steps, 17,037 studies were manually screened at the title and abstract level. An additional 900 studies identified by experts or supplemental searches were also included. After full-text screening of 3889 references, 1586 studies met the PECO criteria. Relevant study details such as the endpoints assessed, exposure duration, and species were extracted into literature summary tables. This review compiles and organizes the human and mammalian studies from these tables into an evidence map for noncancer health endpoints and PCB mixture exposure to identify areas of robust research as well as areas of uncertainty that would benefit from future investigation. Summary data are available online as interactive visuals with downloadable metadata. Sufficient research is available to inform PCB hazard assessments for most organs/systems, but the amount of data to inform associations with specific endpoints differs. Furthermore, despite many years of research, sparse data exist for inhalation and dermal exposures, which are highly relevant human exposure routes. This evidence map provides a foundation for future systematic reviews and noncancer hazard assessments of PCB mixtures and for strategic planning of research to inform areas of greater uncertainty., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SKS, PR, CRK, PJL, PFL, AS, MT, MSB, JT, TAJ, LR, JM, AFK, RB, RS, SS, CL, KS, and BI report financial support provided by the US Environmental Protection Agency under Contract 68HERC19D0003 to ICF. PJL reports a relationship with Friedman Rubin Associates that includes: paid expert testimony. CRK reports a relationship with Bausch Health Companies Inc US that includes: employment and equity or stocks., (Published by Elsevier Inc.)

Linoleic acid (LA, 18:2n-6) is an essential nutrient for optimal infant growth and brain development. The effects of LA in the brain are thought to be mediated by oxygenated metabolites of LA known as oxidized LA metabolites (OXLAMs), but evidence is lacking to directly support this hypothesis. This study investigated whether OXLAMs modulate key neurodevelopmental processes including axon outgrowth, dendritic arborization, cell viability and synaptic connectivity. Primary cortical neuron-glia co-cultures from postnatal day 0-1 male and female rats were exposed for 48h to the following OXLAMs: 1) 13-hydroxyoctadecadienoic acid (13-HODE); 2) 9-hydroxyoctadecadienoic acid (9-HODE); 3) 9,10-dihydroxyoctadecenoic acid (9,10-DiHOME); 4) 12(13)-epoxyoctadecenoic acid (12(13)-EpOME); 5) 9,10,13-trihydroxyoctadecenoic acid (9,10,13-TriHOME); 6) 9-oxo-octadecadienoic acid (9-OxoODE); and 7) 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Axonal outgrowth, evaluated by Tau-1 immunostaining, was increased by 9-HODE, but decreased by 12,13-DiHOME in male but not female neurons. Dendrite arborization, evaluated by MAP2B-eGFP expression, was affected by 9-HODE, 9-OxoODE, and 12(13)-EpOME in male neurons and, by 12(13)-EpOME in female neurons. Neither cell viability nor synaptic connectivity were significantly altered by OXLAMs. Overall, this study shows select OXLAMs modulate neuron morphology in a sex-dependent manner, with male neurons being more susceptible., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pamela J. Lein reports financial support was provided by National Institute of Child Health and Human Development., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)