Early-life adversity increases anxiety-like behavior and modifies synaptic protein expression in a region-specific manner.

Early-life adversity (ELA) can induce persistent neurological changes and increase the risk for developing affective or substance use disorders. Disruptions to the reward circuitry of the brain and pathways serving motivation and emotion have been implicated in the link between ELA and altered adult behavior. The molecular mechanisms that mediate the long-term effects of ELA, however, are not fully understood. We examined whether ELA in the form of neonatal maternal separation (MatSep) modifies behavior and synaptic protein expression in adults. We hypothesized that MatSep would affect dopaminergic and glutamatergic signaling and enhance sensitivity to methamphetamine (Meth) reward or increase anxiety. Male Wistar rats were subjected to MatSep for 180 min/d on postnatal days (PND) 2-14 and allowed to grow to adulthood (PND 60) with no further manipulation. The hippocampus (Hipp), medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and caudate putamen (CPu) were isolated from one subgroup of animals and subjected to Western blot and protein quantitation for tyrosine hydroxylase (TH), α-synuclein (ALPHA), NMDA receptor (NMDAR), dopamine receptor-1 (D1) and -2 (D2), dopamine transporter (DAT), and postsynaptic density 95 (PSD95). Separate group of animals were tested for anxiety-like behavior and conditioned place preference (CPP) to Meth at 0.0, 0.1, and 1.0 mg/kg doses. MatSep rats displayed an increase in basal levels of anxiety-like behavior compared to control animals. MatSep rats also demonstrated CPP to Meth, but their responses did not differ significantly from controls at any drug dose. Increased NMDAR, D2, and ALPHA expression was observed in the NAc and CPu following MatSep; D2 and ALPHA levels were also elevated in the mPFC, along with DAT. MatSep rats had reduced D1 expression in the mPFC and Hipp, with the Hipp also showing a reduction in D2. Only the CPu showed elevated TH and decreased DAT expression levels. No significant changes were found in PSD95 expression in MatSep rats. In conclusion, ELA is associated with long-lasting and region-specific changes in synaptic protein expression that diminish dopamine neurotransmission and increase anxiety-like behavior in adults. These findings illustrate potential mechanisms through which ELA may increase vulnerability to stress-related illness.

Neonatal Maternal Separation Modifies Proteostasis Marker Expression in the Adult Hippocampus.

Exposure to early-life stress (ELS) can persistently modify neuronal circuits and functions, and contribute to the expression of misfolded and aggregated proteins that are hallmarks of several neurodegenerative diseases. The healthy brain is able to clear dysfunctional proteins through the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP). Accumulating evidence indicates that impairment of these pathways contributes to enhanced protein aggregation and neurodegeneration. While stress is a known precipitant of neurological decline, few specific mechanistic links underlying this relationship have been identified. We hypothesized that neonatal maternal separation (MatSep), a well-established model of ELS, has the ability to alter the levels of UPS and ALP components in the brain, and thus has the potential to disrupt proteostasis. The expression of proteostasis-associated protein markers was evaluated by immunoblotting in the hippocampus and cortex of adult Wistar rats that were previously subjected to MatSep. We observed multiple sex- and MatSep-specific changes in the expression of proteins in the ALP, mitophagy, and UPS pathways, particularly in the hippocampus of adult animals. In contrast, MatSep had limited influence on proteostasis marker expression in the cortex of adult animals. Our results indicate that MatSep can selectively modify the intracellular protein degradation machinery in ways that may impact the development and progression of neurodegenerative disease.

Maternal separation induces retinal and peripheral blood mononuclear cell alterations across the lifespan of female rats.

Early life stress alters the function and feedback regulation of the hypothalamic-pituitaryadrenal (HPA) axis, and can contribute to neuroinflammation and neurodegeneration by modifying peripheral blood mononuclear cell (PBMC) activity. The retina, as part of the nervous system, is sensitive to immune changes induced by stress. However, the consequences of stress experienced at an early age on retinal development have not yet been elucidated. Here we aimed to evaluate the impact of maternal separation (MatSep) across three stages of the lifespan (adolescent, adult, and aged) on the retina, as well as on progression through the cell cycle and mitochondrial activity in PBMCs from female Wistar rats. Newborn pups were separated from their mother from postnatal day (PND) 2 until PND 14 for 3 h/day. Retinal analysis from the MatSep groups showed architectural alterations such as a diminished thickness of retinal layers, as well as increased expression of proinflammatory markers DJ-1, Iba-1, and CD45 and the gliotic marker GFAP. Additionally, MatSep disrupted the cell cycle and caused long-term increases in mitochondrial activity in PBMCs from adolescent and adult rats. Changes in the cell cycle profile of the PBMCs from aged MatSep rats were undetected. However, these PBMCs exhibited increased sensitivity to H2O2-induced oxidative stress in vitro. Therefore, these results suggest that early life stress can have long-term effects on retinal structure and function, possibly elicited by neonatal immune preconditioning.

Bioactive growth hormone in humans: Controversies, complexities and concepts.

OBJECTIVE: To revisit a finding, first described in 1978, which documented existence of a pituitary growth factor that escaped detection by immunoassay, but which was active in the established rat tibia GH bioassay. METHODS: We present a narrative review of the evolution of growth hormone complexity, and its bio-detectability, from a historical perspective. RESULTS: In humans under the age of 60, physical training (i.e. aerobic endurance and resistance training) are stressors which preferentially stimulate release of bioactive GH (bGH) into the blood. Neuroanatomical studies indicate a) that nerve fibers directly innervate the human anterior pituitary and b) that hind limb muscle afferents, in both humans and rats, also modulate plasma bGH. In the pituitary gland itself, molecular variants of GH, somatotroph heterogeneity and cell plasticity all appear to play a role in regulation of this growth factor. CONCLUSION: This review considers more recent findings on this often forgotten/neglected subject. Comparison testing of a) human plasma samples, b) sub-populations of separated rat pituitary somatotrophs or c) purified human pituitary peptides by GH bioassay vs immunoassay consistently yield conflicting results.

Tauopathy and neurodegeneration: A role for stress.

Neurodegenerative diseases are characterized by an irreversible and progressive loss of neuronal structure and function. While many alterations to normal cellular processes occur during neurodegeneration, a pathological accumulation of aggregated proteins constitutes a hallmark of several neurodegenerative disorders. Alzheimer's disease, specifically, is pathologically defined by the formation of amyloid plaques and tangles of hyperphosphorylated tau protein. Stress has emerged as an important factor in the development and progression of neurodegenerative diseases, including Alzheimer's. Very little is known, however, regarding the effects of stress on the mechanisms controlling abnormal protein aggregation and clearance. Chronic stress activates the hypothalamic-pituitary-adrenal (HPA) axis, causing an excessive secretion of glucocorticoids that are capable of impacting diverse physiological and cellular processes. The present review focuses on the influence of stress on a key feature of Alzheimer's disease pathology, emphasizing the relationship between tau phosphorylation and accumulation and its connection to HPA axis dysfunction.

Obesity Exacerbates the Cytokine Storm Elicited by Francisella tularensis Infection of Females and Is Associated with Increased Mortality.

Infection with Francisella tularensis, the causative agent of the human disease tularemia, results in the overproduction of inflammatory cytokines, termed the cytokine storm. Excess metabolic byproducts of obesity accumulate in obese individuals and activate the same inflammatory signaling pathways as F. tularensis infection. In addition, elevated levels of leptin in obese individuals also increase inflammation. Since leptin is produced by adipocytes, we hypothesized that increased fat of obese females may make them more susceptible to F. tularensis infection compared with lean individuals. Lean and obese female mice were infected with F. tularensis and the immunopathology and susceptibility monitored. Plasma and tissue cytokines were analyzed by multiplex ELISA and real-time RT-PCR, respectively. Obese mice were more sensitive to infection, developing a more intense cytokine storm, which was associated with increased death of obese mice compared with lean mice. This enhanced inflammatory response correlated with in vitro bacteria-infected macrophage cultures where addition of leptin led to increased production of inflammatory cytokines. We conclude that increased basal leptin expression in obese individuals causes a persistent low-level inflammatory response making them more susceptible to F. tularensis infection and heightening the generation of the immunopathological cytokine storm.

Stress alters the expression of cancer-related genes in the prostate.

BACKGROUND: Prostate cancer is a major contributor to mortality worldwide, and significant efforts are being undertaken to decipher specific cellular and molecular pathways underlying the disease. Chronic stress is known to suppress reproductive function and promote tumor progression in several cancer models, but our understanding of the mechanisms through which stress contributes to cancer development and progression is incomplete. We therefore examined the relationship between stress, modulation of the gonadotropin-releasing hormone (GnRH) system, and changes in the expression of cancer-related genes in the rat prostate. METHODS: Adult male rats were acutely or repeatedly exposed to restraint stress, and compared to unstressed controls and groups that were allowed 14 days of recovery from the stress. Prostate tissue was collected and frozen for gene expression analyses by PCR array before the rats were transcardially perfused; and brain tissues harvested and immunohistochemically stained for Fos to determine neuronal activation. RESULTS: Acute stress elevated Fos expression in the paraventricular nucleus of the hypothalamus (PVH), an effect that habituated with repeated stress exposure. Data from the PCR arrays showed that repeated stress significantly increases the transcript levels of several genes associated with cellular proliferation, including proto-oncogenes. Data from another array platform showed that both acute and repeated stress can induce significant changes in metastatic gene expression. The functional diversity of genes with altered expression, which includes transcription factors, growth factor receptors, apoptotic genes, and extracellular matrix components, suggests that stress is able to induce aberrant changes in pathways that are deregulated in prostate cancer. CONCLUSIONS: Our findings further support the notion that stress can affect cancer outcomes, perhaps by interfering with neuroendocrine mechanisms involved in the control of reproduction.

Extended access to methamphetamine self-administration up-regulates dopamine transporter levels 72 hours after withdrawal in rats.

Previous studies have demonstrated that there are persistent changes in dopamine systems following withdrawal from methamphetamine (METH). This study examined changes in striatal dopamine transporter (DAT), tyrosine hydroxylase (TH) and dopamine receptor 2 (D2) 72 h after withdrawal from METH intravenous self- administration (IVSA). Rats were given limited (1h) or extended (6h) access to METH IVSA (0.05 mg/kg/0.1 ml infusion) for 22 days. Controls did not receive METH IVSA. The rats given extended access to IVSA displayed higher METH intake during the first hour of drug access compared to rats given limited access. Extended access to METH also produced a concomitant increase in striatal DAT levels relative to drug-naïve controls. There were no changes in TH or D2 levels across groups. Previous studies have reported a decrease in striatal DAT levels during protracted periods (>7 days) of withdrawal from METH IVSA. This study extends previous work by showing an increase in striatal DAT protein expression during an earlier time point of withdrawal from this drug. These results are an important step toward understanding the dynamic changes in dopamine systems that occur during different time points of withdrawal from METH IVSA.

Female responses to acute and repeated restraint stress differ from those in males.

Chronic stress is implicated in diseases which differentially affect men and women. This study investigated how the activation of neuronal subpopulations contributes to changes in neuroendocrine regulation that predispose members of each sex to stress-related health challenges. Adult male and female rats were restrained in single (acute) or 14 consecutive daily (repeated) 30 min sessions; brain sections were immunohistochemically stained for Fos, arginine vasopressin (AVP) or glucocorticoid receptor (GR) within the paraventricular hypothalamic nucleus (PVH). Acute restraint increased the number of PVH cells expressing Fos, with greater increases in males than females. Habituated responses were seen following repeated stress in both sexes, with no sex differences between groups. No sex differences were found in the number of neurons co-expressing Fos and AVP. Absolute counts of cellular Fos and GR co-localization mirrored Fos expression. In contrast, when doubly-labeled cells were normalized to staining for Fos alone, females showed greater numbers of Fos- and GR-positive cells than males after both acute and repeated stress. These data demonstrate that sex-specific stress responses are evident at the level of neuronal activation, and may contribute to different consequences of chronic stress in females versus males. Females may be more sensitive to glucocorticoid negative feedback, suggesting that sex-dependent differences in the efficiency of initiating and terminating stress responses may exist. Understanding the neural and endocrine pathways that mediate these functions in males and females will inform targeted therapeutic strategies to alleviate stress and the sex-specific afflictions with which it is associated.

Acute and repeated restraint differentially activate orexigenic pathways in the rat hypothalamus.

Stress and obesity are highly prevalent conditions, and the mechanisms through which stress affects food intake are complex. In the present study, stress-induced activation in neuropeptide systems controlling ingestive behavior was determined. Adult male rats were exposed to acute (30 min/d × 1 d) or repeated (30 min/d × 14 d) restraint stress, followed by transcardial perfusion 2 h after the termination of the stress exposure. Brain tissues were harvested, and 30 µm sections through the hypothalamus were immunohistochemically stained for Fos protein, which was then co-localized within neurons staining positively for the type 4 melanocortin receptor (MC4R), the glucagon-like peptide-1 receptor (GLP1R), or agouti-related peptide (AgRP). Cell counts were performed in the paraventricular (PVH), arcuate (ARC) and ventromedial (VMH) hypothalamic nuclei and in the lateral hypothalamic area (LHA). Fos was significantly increased in all regions except the VMH in acutely stressed rats, and habituated with repeated stress exposure, consistent with previous studies. In the ARC, repeated stress reduced MC4R cell activation while acute restraint decreased activation in GLP1R neurons. Both patterns of stress exposure reduced the number of AgRP-expressing cells that also expressed Fos in the ARC. Acute stress decreased Fos-GLP1R expression in the LHA, while repeated restraint increased the number of Fos-AgRP neurons in this region. The overall profile of orexigenic signaling in the brain is thus enhanced by acute and repeated restraint stress, with repeated stress leading to further increases in signaling, in a region-specific manner. Stress-induced modifications to feeding behavior appear to depend on both the duration of stress exposure and regional activation in the brain. These results suggest that food intake may be increased as a consequence of stress, and may play a role in obesity and other stress-associated metabolic disorders.

A discrete GABAergic relay mediates medial prefrontal cortical inhibition of the neuroendocrine stress response.

Complementing its roles in cognitive and affective information processing, the medial prefrontal cortex (mPFC) is a nodal point of a limbic forebrain circuit that modulates stress-related homeostatic mechanisms, including the hypothalamic-pituitary-adrenal (HPA) axis. mPFC influences on HPA output are predominantly inhibitory and emanate from the prelimbic and/or dorsal anterior cingulate cortical fields (PL and ACd, respectively). mPFC projections do not target HPA effector neurons in the paraventricular hypothalamic nucleus (PVH) directly, distributing instead to nearby forebrain regions, including some that house GABAergic neurons implicated in inhibitory PVH control. To identify pathway(s) subserving HPA-inhibitory mPFC influences, an initial screen for sources of GABAergic input to PVH whose sensitivity to an acute emotional (restraint) stress was diminished by PL/ACd lesions identified a discrete region of the anterior bed nucleus of the stria terminalis (aBST) as a candidate for fulfilling this role. Anatomical tracing experiments confirmed projections from PL (but not ACd) to implicated aBST cell groups, and from these to PVH. Finally, selective immunotoxin-mediated ablation of GABAergic aBST neurons recapitulated the effects of PL/ACd lesions on acute stress-induced activation of HPA output. The identification of a proximate mediator of HPA-inhibitory limbic influences provides a framework for clarifying how inhibitory neural and hormonal controls of HPA output are integrated, adaptations of the axis to chronic stress are effected, and how endocrine abnormalities may contribute to stress-related psychiatric illnesses in which mPFC dysfunction is implicated.