Machine learning-based morphological quantification of replicative senescence in human fibroblasts.

Although aging has been investigated extensively at the organismal and cellular level, the morphological changes that individual cells undergo along their replicative lifespan have not been precisely quantified. Here, we present the results of a readily accessible machine learning-based pipeline that uses standard fluorescence microscope and open access software to quantify the minute morphological changes that human fibroblasts undergo during their replicative lifespan in culture. Applying this pipeline in a widely used fibroblast cell line (IMR-90), we find that advanced replicative age robustly increases (+28-79%) cell surface area, perimeter, number and total length of pseudopodia, and nuclear surface area, while decreasing cell circularity, with phenotypic changes largely occurring as replicative senescence is reached. These senescence-related morphological changes are recapitulated, albeit to a variable extent, in primary dermal fibroblasts derived from human donors of different ancestry, age, and sex groups. By performing integrative analysis of single-cell morphology, our pipeline further classifies senescent-like cells and quantifies how their numbers increase with replicative senescence in IMR-90 cells and in dermal fibroblasts across all tested donors. These findings provide quantitative insights into replicative senescence, while demonstrating applicability of a readily accessible computational pipeline for high-throughput cell phenotyping in aging research.

Chronic early life stress alters the neuroimmune profile and functioning of the developing zebrafish gut.

Chronic early life stress (ELS) potently impacts the developing central nervous and immune systems and is associated with the onset of gastrointestinal disease in humans. Though the gut-brain axis is appreciated to be a major target of the stress response, the underlying mechanisms linking ELS to gut dysfunction later in life is incompletely understood. Zebrafish are a powerful model validated for stress research and have emerged as an important tool in delineating neuroimmune mechanisms in the developing gut. Here, we developed a novel model of ELS and utilized a comparative transcriptomics approach to assess how chronic ELS modulated expression of neuroimmune genes in the developing gut and brain. Zebrafish exposed to ELS throughout larval development exhibited anxiety-like behavior and altered expression of neuroimmune genes in a time- and tissue-dependent manner. Further, the altered gut neuroimmune profile, which included increased expression of genes associated with neuronal modulation, correlated with a reduction in enteric neuronal density and delayed gut transit. Together, these findings provide insights into the mechanisms linking ELS with gastrointestinal dysfunction and highlight the zebrafish model organism as a valuable tool in uncovering how "the body keeps the score."

An open access, machine learning pipeline for high-throughput quantification of cell morphology.

Cell morphology is influenced by many factors and can be used as a phenotypic marker. Here we describe a machine-learning-based protocol for high-throughput morphological measurement of human fibroblasts using a standard fluorescence microscope and the pre-existing, open access software ilastik for cell body identification, ImageJ/Fiji for image overlay, and CellProfiler for morphological quantification. Because this protocol overlays nuclei with their cell bodies and relies on coloration differences, it can be broadly applied to other cell types beyond fibroblasts. For details on the use and execution of this protocol, please also refer to Leung et al. (2022).1.

Chronic stress-driven glucocorticoid receptor activation programs key cell phenotypes and functional epigenomic patterns in human fibroblasts.

Chronic environmental stress can profoundly impact cell and body function. Although the underlying mechanisms are poorly understood, epigenetics has emerged as a key link between environment and health. The genomic effects of stress are thought to be mediated by the action of glucocorticoid stress hormones, primarily cortisol in humans, which act via the glucocorticoid receptor (GR). To dissect how chronic stress-driven GR activation influences epigenetic and cell states, human fibroblasts underwent prolonged exposure to physiological stress levels of cortisol and/or a selective GR antagonist. Cortisol was found to drive robust changes in cell proliferation, migration, and morphology, which were abrogated by concomitant GR blockade. The GR-driven cell phenotypes were accompanied by widespread, yet genomic context-dependent, changes in DNA methylation and mRNA expression, including gene loci with known roles in cell proliferation and migration. These findings provide insights into how chronic stress-driven functional epigenomic patterns become established to shape key cell phenotypes.

Diminished Hepatocarcinogenesis by a Potent, High-Affinity Human PPARα Agonist in PPARA-Humanized Mice.

Ppara-null and PPARA-humanized mice are refractory to hepatocarcinogenesis caused by the peroxisome proliferator-activated receptor-α (PPARα) agonist Wy-14,643. However, the duration of these earlier studies was limited to approximately 1 year of treatment, and the ligand used has a higher affinity for the mouse PPARα compared to the human PPARα. Thus, the present study examined the effect of long-term administration of a potent, high-affinity human PPARα agonist (GW7647) on hepatocarcinogenesis in wild-type, Ppara-null, or PPARA-humanized mice. In wild-type mice, GW7647 caused hepatic expression of known PPARα target genes, hepatomegaly, hepatic MYC expression, hepatic cytotoxicity, and a high incidence of hepatocarcinogenesis. By contrast, these effects were essentially absent in Ppara-null mice or diminished in PPARA-humanized mice, although hepatocarcinogenesis was observed in both genotypes. Enhanced fatty change (steatosis) was also observed in both Ppara-null and PPARA-humanized mice independent of GW7647. PPARA-humanized mice administered GW7647 also exhibited increased necrosis after 5 weeks of treatment. Results from these studies demonstrate that the mouse PPARα is required for hepatocarcinogenesis induced by GW7647 administered throughout adulthood. Results also indicate that a species difference exists between rodents and human PPARα in the response to ligand activation of PPARα. The hepatocarcinogenesis observed in control and treated Ppara-null mice is likely mediated in part by increased hepatic fatty change, whereas the hepatocarcinogenesis observed in PPARA-humanized mice may also be due to enhanced fatty change and cytotoxicity that could be influenced by the minimal activity of the human PPARα in this mouse line on downstream mouse PPARα target genes. The Ppara-null and PPARA-humanized mouse models are valuable tools for examining the mechanisms of PPARα-induced hepatocarcinogenesis, but the background level of liver cancer must be controlled for in the design and interpretation of studies that use these mice.

Species Differences between Mouse and Human PPARα in Modulating the Hepatocarcinogenic Effects of Perinatal Exposure to a High-Affinity Human PPARα Agonist in Mice.

Evidence suggests that species differences exist between rodents and humans in their biological responses to ligand activation of PPARα. Moreover, neonatal/postnatal rodents may be more sensitive to the effects of activating PPARα. Thus, the present studies examined the effects of chronic ligand activation of PPARα initiated during early neonatal development and continued into adulthood on hepatocarcinogenesis in mice. Wild-type, Ppara-null, or PPARA-humanized mice were administered a potent, high-affinity human PPARα agonist GW7647, and cohorts of mice were examined over time. Activation of PPARα with GW7647 increased expression of known PPARα target genes in liver and was associated with hepatomegaly, increased hepatic cytotoxicity and necrosis, increased expression of hepatic MYC, and a high incidence of hepatocarcinogenesis in wild-type mice. These effects did not occur or were largely diminished in Ppara-null and PPARA-humanized mice, although background levels of hepatocarcinogenesis were also noted in both Ppara-null and PPARA-humanized mice. More fatty change (steatosis) was also observed in both Ppara-null and PPARA-humanized mice independent of GW7647 administration. Results from these studies indicate that the mouse PPARα is required to mediate hepatocarcinogenesis induced by GW7647 in mice and that activation of the human PPARα with GW7647 in PPARA-humanized mice are diminished compared with wild-type mice. Ppara-null and PPARA-humanized mice are valuable tools for examining species differences in the mechanisms of PPARα-induced hepatocarcinogenesis, but background levels of liver cancer observed in aged Ppara-null and PPARA-humanized mice must be considered when interpreting results from studies that use these models. These results also demonstrate that early life exposure to a potent human PPARα agonist does not enhance sensitivity to hepatocarcinogenesis.

Prolonged Glucocorticoid Exposure Does Not Accelerate Telomere Shortening in Cultured Human Fibroblasts.

Psychosocial stress, especially when chronic or excessive, can increase disease risk and accelerate biological aging. Although the underlying mechanisms are unclear, in vivo studies have associated exposure to stress and glucocorticoid stress hormones with shorter telomere length. However, the extent to which prolonged glucocorticoid exposure can shorten telomeres in controlled experimental settings remains unknown. Using a well-characterized cell line of human fibroblasts that undergo gradual telomere shortening during serial passaging in culture, we show that prolonged exposure (up to 51 days) to either naturalistic levels of the human endogenous glucocorticoid cortisol or the more potent synthetic glucocorticoid dexamethasone is not sufficient to accelerate telomere shortening. While our findings await extension in other cell types and biological contexts, they indicate that the in vivo association of psychosocial stress with telomere shortening is unlikely to be mediated by a direct and universal glucocorticoid effect on telomere length.

Prepronociceptin-Expressing Neurons in the Extended Amygdala Encode and Promote Rapid Arousal Responses to Motivationally Salient Stimuli.

Motivational states consist of cognitive, emotional, and physiological components controlled by multiple brain regions. An integral component of this neural circuitry is the bed nucleus of the stria terminalis (BNST). Here, we identify that neurons within BNST that express the gene prepronociceptin (PnocBNST) modulate rapid changes in physiological arousal that occur upon exposure to motivationally salient stimuli. Using in vivo two-photon calcium imaging, we find that PnocBNST neuronal responses directly correspond with rapid increases in pupillary size when mice are exposed to aversive and rewarding odors. Furthermore, optogenetic activation of these neurons increases pupillary size and anxiety-like behaviors but does not induce approach, avoidance, or locomotion. These findings suggest that excitatory responses in PnocBNST neurons encode rapid arousal responses that modulate anxiety states. Further histological, electrophysiological, and single-cell RNA sequencing data reveal that PnocBNST neurons are composed of genetically and anatomically identifiable subpopulations that may differentially tune rapid arousal responses to motivational stimuli.

Social Stimuli Induce Activation of Oxytocin Neurons Within the Paraventricular Nucleus of the Hypothalamus to Promote Social Behavior in Male Mice.

Oxytocin (OT) is critical for the expression of social behavior across a wide array of species; however, the role of this system in the encoding of socially relevant information is not well understood. In the present study, we show that chemogenetic activation of OT neurons within the paraventricular nucleus of the hypothalamus (PVH) of male mice (OT-Ires-Cre) enhanced social investigation during a social choice test, while chemogenetic inhibition of these neurons abolished typical social preferences. These data suggest that activation of the OT system is necessary to direct behavior preferentially toward social stimuli. To determine whether the presence of a social stimulus is sufficient to induce activation of PVH-OT neurons, we performed the first definitive recording of OT neurons in awake mice using two-photon calcium imaging. These recordings demonstrate that social stimuli activate PVH-OT neurons and that these neurons differentially encode social and nonsocial stimuli, suggesting that PVH-OT neurons may act to convey social salience of environmental stimuli. Finally, an attenuation of social salience is associated with social disorders, such as autism. We therefore also examined possible OT system dysfunction in a mouse model of autism, Shank3b knock-out (KO) mice. Male Shank3b KO mice showed a marked reduction in PVH-OT neuron number and administration of an OT receptor agonist improved social deficits. Overall, these data suggest that the presence of a social stimulus induces activation of the PVH-OT neurons to promote adaptive social behavior responses.SIGNIFICANCE STATEMENT Although the oxytocin (OT) system is well known to regulate a diverse array of social behaviors, the mechanism in which OT acts to promote the appropriate social response is poorly understood. One hypothesis is that the presence of social conspecifics activates the OT system to generate an adaptive social response. Here, we selectively recorded from OT neurons in the paraventricular hypothalamic nucleus (PVH) to show that social stimulus exposure indeed induces activation of the OT system. We also show that activation of the OT system is necessary to promote social behavior and that mice with abnormal social behavior have reduced numbers of PVH-OT neurons. Finally, aberrant social behavior in these mice was rescued by administration of an OT receptor agonist.

Paraventricular Thalamus Projection Neurons Integrate Cortical and Hypothalamic Signals for Cue-Reward Processing.

The paraventricular thalamus (PVT) is an interface for brain reward circuits, with input signals arising from structures, such as prefrontal cortex and hypothalamus, that are broadcast to downstream limbic targets. However, the precise synaptic connectivity, activity, and function of PVT circuitry for reward processing are unclear. Here, using in vivo two-photon calcium imaging, we find that PVT neurons projecting to the nucleus accumbens (PVT-NAc) develop inhibitory responses to reward-predictive cues coding for both cue-reward associative information and behavior. The multiplexed activity in PVT-NAc neurons is directed by opposing activity patterns in prefrontal and lateral hypothalamic afferent axons. Further, we find that prefrontal cue encoding may maintain accurate cue-reward processing, as optogenetic disruption of this encoding induced long-lasting effects on downstream PVT-NAc cue responses and behavioral cue discrimination. Together, these data reveal that PVT-NAc neurons act as an interface for reward processing by integrating relevant inputs to accurately inform reward-seeking behavior.

Obesity remodels activity and transcriptional state of a lateral hypothalamic brake on feeding.

The current obesity epidemic is a major worldwide health concern. Despite the consensus that the brain regulates energy homeostasis, the neural adaptations governing obesity are unknown. Using a combination of high-throughput single-cell RNA sequencing and longitudinal in vivo two-photon calcium imaging, we surveyed functional alterations of the lateral hypothalamic area (LHA)-a highly conserved brain region that orchestrates feeding-in a mouse model of obesity. The transcriptional profile of LHA glutamatergic neurons was affected by obesity, exhibiting changes indicative of altered neuronal activity. Encoding properties of individual LHA glutamatergic neurons were then tracked throughout obesity, revealing greatly attenuated reward responses. These data demonstrate how diet disrupts the function of an endogenous feeding suppression system to promote overeating and obesity.

Sex-specific differences in genotoxic and epigenetic effects of 1,3-butadiene among mouse tissues.

Exposure to environmental chemicals has been shown to have an impact on the epigenome. One example is a known human carcinogen 1,3-butadiene which acts primarily by a genotoxic mechanism, but also disrupts the chromatin structure by altering patterns of cytosine DNA methylation and histone modifications. Sex-specific differences in 1,3-butadiene-induced genotoxicity and carcinogenicity are well established; however, it remains unknown whether 1,3-butadiene-associated epigenetic alterations are also sex dependent. Therefore, we tested the hypothesis that inhalational exposure to 1,3-butadiene will result in sex-specific epigenetic alterations. DNA damage and epigenetic effects of 1,3-butadiene were evaluated in liver, lung, and kidney tissues of male and female mice of two inbred strains (C57BL/6J and CAST/EiJ). Mice were exposed to 0 or 425 ppm of 1,3-butadiene by inhalation (6 h/day, 5 days/week) for 2 weeks. Strain- and tissue-specific differences in 1,3-butadiene-induced DNA adducts and crosslinks were detected in the liver, lung and kidney; however, significant sex-specific differences in DNA damage were observed in the lung of C57BL/6J mice only. In addition, we assessed expression of the DNA repair genes and observed a marked upregulation of Mgmt in the kidney in female C57BL/6J mice. Sex-specific epigenetic effects of 1,3-butadiene exposure were evident in alterations of cytosine DNA methylation and histone modifications in the liver and lung in both strains. Specifically, we observed a loss of cytosine DNA methylation in the liver and lung of male and female 1,3-butadiene-exposed C57BL/6J mice, whereas hypermethylation was found in the liver and lung in 1,3-butadiene-exposed female CAST/EiJ mice. Our findings suggest that strain- and sex-specific effects of 1,3-butadiene on the epigenome may contribute to the known differences in cancer susceptibility.

Metabolism and Toxicity of Trichloroethylene and Tetrachloroethylene in Cytochrome P450 2E1 Knockout and Humanized Transgenic Mice.

Trichloroethylene (TCE) and tetrachloroethylene (PCE) are structurally similar olefins that can cause liver and kidney toxicity. Adverse effects of these chemicals are associated with metabolism to oxidative and glutathione conjugation moieties. It is thought that CYP2E1 is crucial to the oxidative metabolism of TCE and PCE, and may also play a role in formation of nephrotoxic metabolites; however, inter-species and inter-individual differences in contribution of CYP2E1 to metabolism and toxicity are not well understood. Therefore, the role of CYP2E1 in metabolism and toxic effects of TCE and PCE was investigated using male and female wild-type [129S1/SvlmJ], Cyp2e1(-/-), and humanized Cyp2e1 [hCYP2E1] mice. To fill in existing gaps in our knowledge, we conducted a toxicokinetic study of TCE (600 mg/kg, single dose, i.g.) and a subacute study of PCE (500 mg/kg/day, 5 days, i.g.) in 3 strains. Liver and kidney tissues were subject to profiling of oxidative and glutathione conjugation metabolites of TCE and PCE, as well as toxicity endpoints. The amounts of trichloroacetic acid formed in the liver was hCYP2E1≈ 129S1/SvlmJ > Cyp2e1(-/-) for both TCE and PCE; levels in males were about 2-fold higher than in females. Interestingly, 2- to 3-fold higher levels of conjugation metabolites were observed in TCE-treated Cyp2e1(-/-) mice. PCE induced lipid accumulation only in liver of 129S1/SvlmJ mice. In the kidney, PCE exposure resulted in acute proximal tubule injury in both sexes in all strains (hCYP2E1 ≈ 129S1/SvlmJ > Cyp2e1(-/-)). In conclusion, our results demonstrate that CYP2E1 is an important, but not exclusive actor in the oxidative metabolism and toxicity of TCE and PCE.

Editor's Highlight: Collaborative Cross Mouse Population Enables Refinements to Characterization of the Variability in Toxicokinetics of Trichloroethylene and Provides Genetic Evidence for the Role of PPAR Pathway in Its Oxidative Metabolism.

Background: Trichloroethylene (TCE) is a known carcinogen in humans and rodents. Previous studies of inter-strain variability in TCE metabolism were conducted in multi-strain panels of classical inbred mice with limited genetic diversity to identify gene-environment interactions associated with chemical exposure. Objectives: To evaluate inter-strain variability in TCE metabolism and identify genetic determinants that are associated with TCE metabolism and effects using Collaborative Cross (CC), a large panel of genetically diverse strains of mice. Methods: We administered a single oral dose of 0, 24, 80, 240, or 800 mg/kg of TCE to mice from 50 CC strains, and collected organs 24 h post-dosing. Levels of trichloroacetic acid (TCA), a major oxidative metabolite of TCE were measured in multiple tissues. Protein expression and activity levels of TCE-metabolizing enzymes were evaluated in the liver. Liver transcript levels of known genes perturbed by TCE exposure were also quantified. Genetic association mapping was performed on the acquired phenotypes. Results: TCA levels varied in a dose- and strain-dependent manner in liver, kidney, and serum. The variability in TCA levels among strains did not correlate with expression or activity of a number of enzymes known to be involved in TCE oxidation. Peroxisome proliferator-activated receptor alpha (PPARα)-responsive genes were found to be associated with strain-specific differences in TCE metabolism. Conclusions: This study shows that CC mouse population is a valuable tool to quantitatively evaluate inter-individual variability in chemical metabolism and to identify genes and pathways that may underpin population differences.

Prefrontal cortex output circuits guide reward seeking through divergent cue encoding.

The prefrontal cortex is a critical neuroanatomical hub for controlling motivated behaviours across mammalian species. In addition to intra-cortical connectivity, prefrontal projection neurons innervate subcortical structures that contribute to reward-seeking behaviours, such as the ventral striatum and midline thalamus. While connectivity among these structures contributes to appetitive behaviours, how projection-specific prefrontal neurons encode reward-relevant information to guide reward seeking is unknown. Here we use in vivo two-photon calcium imaging to monitor the activity of dorsomedial prefrontal neurons in mice during an appetitive Pavlovian conditioning task. At the population level, these neurons display diverse activity patterns during the presentation of reward-predictive cues. However, recordings from prefrontal neurons with resolved projection targets reveal that individual corticostriatal neurons show response tuning to reward-predictive cues, such that excitatory cue responses are amplified across learning. By contrast, corticothalamic neurons gradually develop new, primarily inhibitory responses to reward-predictive cues across learning. Furthermore, bidirectional optogenetic manipulation of these neurons reveals that stimulation of corticostriatal neurons promotes conditioned reward-seeking behaviour after learning, while activity in corticothalamic neurons suppresses both the acquisition and expression of conditioned reward seeking. These data show how prefrontal circuitry can dynamically control reward-seeking behaviour through the opposing activities of projection-specific cell populations.

Hormonal gain control of a medial preoptic area social reward circuit.

Neural networks that control reproduction must integrate social and hormonal signals, tune motivation, and coordinate social interactions. However, the neural circuit mechanisms for these processes remain unresolved. The medial preoptic area (mPOA), an essential node for social behaviors, comprises molecularly diverse neurons with widespread projections. Here we identify a steroid-responsive subset of neurotensin (Nts)-expressing mPOA neurons that interface with the ventral tegmental area (VTA) to form a socially engaged reward circuit. Using in vivo two-photon imaging in female mice, we show that mPOANts neurons preferentially encode attractive male cues compared to nonsocial appetitive stimuli. Ovarian hormone signals regulate both the physiological and cue-encoding properties of these cells. Furthermore, optogenetic stimulation of mPOANts-VTA circuitry promotes rewarding phenotypes, social approach and striatal dopamine release. Collectively, these data demonstrate that steroid-sensitive mPOA neurons encode ethologically relevant stimuli and co-opt midbrain reward circuits to promote prosocial behaviors critical for species survival.

Colloidal Nanomolybdenum Influence upon the Antioxidative Reaction of Chickpea Plants (Cicer arietinum L.).

The use of colloidal solutions of metals as micronutrients enhances plant resistance to unfavorable environmental conditions and ensures high yields of food crops. The purpose of the study was a comparative evaluation of presowing treatment with nanomolybdenum and microbiological preparation impact upon the development of adaptive responses in chickpea plants. Oxidative processes did not develop in all variants of the experiment but in variants treated with microbial preparation, and joint action of microbial and nanopreparations even declined, as evidenced by the reduction of thiobarbituric acid reactive substances in photosynthetic tissues by 15 %. The activity of superoxide dismutase increased (by 15 %) in variant "nanomolybdenum" and joint action "microbial + nanomolybdenum," but it decreased by 20 % in variants with microbial preparation treatment. The same dependence was observed in changes of catalase activity. Antioxidant status factor, which takes into account the ratio of antioxidant to pro-oxidant, was the highest in variants with joint action of microbial preparation and nanomolybdenum (0.7), the lowest in variants with microbial treatment only (0.1). Thus, the results show that the action of nanoparticles of molybdenum activated antioxidant enzymes and decreased oxidative processes, thus promoting adaptation of plants.

Visualization of cortical, subcortical and deep brain neural circuit dynamics during naturalistic mammalian behavior with head-mounted microscopes and chronically implanted lenses.

Genetically encoded calcium indicators for visualizing dynamic cellular activity have greatly expanded our understanding of the brain. However, owing to the light-scattering properties of the brain, as well as the size and rigidity of traditional imaging technology, in vivo calcium imaging has been limited to superficial brain structures during head-fixed behavioral tasks. These limitations can now be circumvented by using miniature, integrated microscopes in conjunction with an implantable microendoscopic lens to guide light into and out of the brain, thus permitting optical access to deep brain (or superficial) neural ensembles during naturalistic behaviors. Here we describe steps to conduct such imaging studies using mice. However, we anticipate that the protocol can be easily adapted for use in other small vertebrates. Successful completion of this protocol will permit cellular imaging of neuronal activity and the generation of data sets with sufficient statistical power to correlate neural activity with stimulus presentation, physiological state and other aspects of complex behavioral tasks. This protocol takes 6-11 weeks to complete.