A subset of major depressive disorder (MDD) is characterized by immune system dysfunction, but the intracellular origin of these immune changes remains unclear. Here we tested the hypothesis that abnormalities in endoplasmic reticulum (ER) stress, inflammasome activity and mitochondrial biogenesis contribute to the development of systemic inflammation in MDD. RT-qPCR was used to measure mRNA expression of key organellar genes from peripheral blood mononuclear cells (PBMCs) isolated from 186 MDD and 67 healthy control (HC) subjects. The comparative CT (2-ΔΔCT) method was applied to quantify mRNA expression using GAPDH as the reference gene. After controlling for age, sex, BMI, and medication status using linear regression models, expression of the inflammasome (NLRC4 and NLRP3) and the ER stress (XBP1u, XBP1s, and ATF4) genes was found to be significantly increased in the MDD versus the HC group. Sensitivity analyses excluding covariates yielded similar results. After excluding outliers, expression of the inflammasome genes was no longer statistically significant but expression of the ER stress genes (XBP1u, XBP1s, and ATF4) remained significant and the mitochondrial biogenesis gene, MFN2, was significantly increased in the MDD group. NLRC4 and MFN2 were positively correlated with serum C-reactive protein concentrations, while ASC trended significant. The altered expression of inflammasome activation, ER stress, and mitochondrial biogenesis pathway components suggest that dysfunction of these organelles may play a role in the pathogenesis of MDD.

BACKGROUND:Major depressive disorder has a complex, bidirectional relationship with metabolic dysfunction, but the neural correlates of this association are not well understood. METHODS:In this cross-sectional investigation, we used a 2-step discovery and confirmatory strategy utilizing 2 independent samples (sample 1: 288 participants, sample 2: 196 participants) to examine the association between circulating indicators of metabolic health (leptin and adiponectin) and brain structures in individuals with major depressive disorder. RESULTS:We found a replicable inverse correlation between leptin levels and cortical surface area within essential brain areas responsible for emotion regulation, such as the left posterior cingulate cortex, right pars orbitalis, right superior temporal gyrus, and right insula (standardized beta coefficient range: -0.27 to -0.49, puncorrected < .05). Notably, this relationship was independent of C-reactive protein levels. We also identified a significant interaction effect of leptin levels and diagnosis on the cortical surface area of the right superior temporal gyrus (standardized beta coefficient = 0.26 in sample 1, standardized beta coefficient = 0.30 in sample 2, puncorrected < .05). We also observed a positive correlation between leptin levels and atypical depressive symptoms in both major depressive disorder groups (r = 0.14 in sample 1, r = 0.29 in sample 2, puncorrected < .05). CONCLUSIONS:The inverse association between leptin and cortical surface area in brain regions that are important for emotion processing and leptin's association with atypical depressive symptoms support the hypothesis that metabolic processes may be related to emotion regulation. However, the molecular mechanisms through which leptin may exert these effects should be explored further.

Chronic alcohol exposure leads to a neuroinflammatory response involving activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome and proinflammatory cytokine production. Acute ethanol (EtOH) exposure activates GABAergic synapses in the central and basolateral amygdala (BLA)ex vivo, but whether this rapid modulation of synaptic inhibition is because of an acute inflammatory response and alters anxiety-like behavior in male and female animals is not known. Here, we tested the hypotheses that acute EtOH facilitates inhibitory synaptic transmission in the BLA by activating the NLRP3 inflammasome-dependent acute inflammatory response, that the alcohol-induced increase in inhibition is cell type and sex dependent, and that acute EtOH in the BLA reduces anxiety-like behavior. Acute EtOH application at a binge-like concentration (22–44 mm) stimulated synaptic GABA release from putative parvalbumin (PV) interneurons onto BLA principal neurons inex vivobrain slices from male, but not female, rats. The EtOH facilitation of synaptic inhibition was blocked by antagonists of the Toll-like receptor 4 (TLR4), the NLRP3 inflammasome, and interleukin-1 receptors, suggesting it was mediated by a rapid local neuroinflammatory response in the BLA.In vivo, bilateral injection of EtOH directly into the BLA produced an acute concentration-dependent reduction in anxiety-like behavior in male but not female rats. These findings demonstrate that acute EtOH in the BLA regulates anxiety-like behavior in a sex-dependent manner and suggest that this effect is associated with presynaptic facilitation of parvalbumin-expressing interneuron inputs to BLA principal neurons via a local NLRP3 inflammasome-dependent neuroimmune response.SIGNIFICANCE STATEMENTChronic alcohol exposure produces a neuroinflammatory response, which contributes to alcohol-associated pathologies. Acute alcohol administration increases inhibitory synaptic signaling in the brain, but the mechanism for the rapid alcohol facilitation of inhibitory circuits is unknown. We found that acute ethanol at binge-like concentrations in the basolateral amygdala (BLA) facilitates GABA release from parvalbumin-expressing (PV) interneuron synapses onto principal neurons inex vivobrain slices from male rats and that intra-BLA ethanol reduces anxiety-like behaviorin vivoin male rats, but not female rats. The ethanol (EtOH) facilitation of inhibition in the BLA is mediated by Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation and proinflammatory IL-1β signaling, which suggests a rapid NLRP3 inflammasome-dependent neuroimmune cascade that plays a critical role in acute alcohol intoxication.

Repeated social defeat stress (RSDS) is a model of chronic stress in rodents. There are several variants of social defeat procedures that exert robust effects in mice, but few published detailed protocols to produce a robust stress and altered immunological profile in rats. In this article, we describe the protocol for the induction of RSDS in adult male Sprague-Dawley rats. Using a resident-intruder paradigm, a physical component of stress is induced by direct attack from the resident aggressive retired breeder Long-Evans rats on the intruder experimental rats. A subsequent threat component is induced by the presence of the aggressor in the vicinity of the intruder, but with physical separation between them. The RSDS induced by this protocol produces robust immunological and behavioral changes in the experimental rats, as evidenced by development of anxiety-like behaviors in open field, social interaction, and elevated plus maze tests, as well as by changes in immune parameters (Munshi et al., 2020). This approach has been used as an ethologically relevant model of stressors that are potent enough to impact neural circuits that are similar to the neural circuits impacted in patients with depression and anxiety.

Many temperate zone animals exhibit seasonal rhythms in physiology and behavior, including seasonal cycles of reproduction, energetics, stress responsiveness, and immune function, among many others. These rhythms are driven by seasonal changes in the duration of pineal melatonin secretion. The neural melatonin target tissues that mediate several of these rhythms have been identified, though the target(s) mediating melatonin's regulation of glucocorticoid secretion, immune cell numbers, and bacterial killing capacity remain unspecified. The present results indicate that one melatonin target tissue, the paraventricular nucleus of the thalamus (PVT), is necessary for the expression of these seasonal rhythms. Thus, while radiofrequency ablations of the PVT failed to alter testicular and body mass response to short photoperiod exposure, they did block the effect of short day lengths on cortisol secretion and bacterial killing efficacy. These results are consistent with the independent regulation by separate neural circuits of several physiological traits that vary seasonally in mammals.

Pro-inflammatory cytokines produce manifestations of sickness during inflammation, such as malaise and lethargy. They also contribute to effects of inflammation on mood. Anti-inflammatory cytokines counteract damage caused by inflammatory processes and can limit the severity of inflammation. However, very little is known about the role of anti-inflammatory cytokines in sickness and mood changes during immune activation. The purpose of this study was to determine if a prototypical anti-inflammatory cytokine, interleukin 10 (IL-10), can offset sickness behavior and anxiety caused by a pro-inflammatory cytokine, and whether IL-10 itself modifies anxiety. Rodent models of sickness display suppression of behavioral activity that may reflect lethargy or malaise, while models of anxiety display reduced exploration in several tasks. The effects of peripheral single dose of cytokines on open field exploration, social interaction and elevated plus maze (EPM) tests in adult male Sprague-Dawley rats were measured at 30-50 min post-treatment. The prototypical pro-inflammatory cytokine IL-1 beta (1 mu g, i.p.) caused a decrease in locomotor activity indicative of sickness behavior, but disproportionately reduced central area exploration in the open field, open arm exploration in the EPM and lowered social interaction. IL-10 (1 mu g, i.p.) had no effect on locomotor activity, but itself produced anxiety-like behavior in the open field and EPM. However, rats co-treated with both IL-10 and IL-1 beta showed locomotor activity, open field, social interaction and EPM behaviors very similar to control groups. This data demonstrate that IL-10 is capable of mitigating the sickness and anxiogenic effects caused by IL-1 beta, but that immune imbalance toward either a pro inflammatory or an anti-inflammatory state can produce anxiety. This has importance for understanding the scope of immune changes that produce psychiatric symptoms, and provides preliminary indication that anti-inflammatory cytokines may be potentially useful in treatment of anxiety induced by inflammatory conditions.

A link exists between immune function and psychiatric conditions, particularly depressive and anxiety disorders. Psychological stress is a powerful trigger for these disorders and stress influences immune state. However, the nature of peripheral immune changes after stress conflicts across studies, perhaps due to the focus on few measures of pro-inflammatory or anti-inflammatory processes. The basolateral amygdala (BLA) is critical for emotion, and plays an important role in the effects of stress on anxiety. As such, it may be a primary central nervous system (CNS) mediator for the effects of peripheral immune changes on anxiety after stress. Therefore, this study aimed to delineate the influence of stress on peripheral pro-inflammatory and anti-inflammatory aspects, BLA immune activation, and its impact on BLA neuronal activity. To produce a more encompassing view of peripheral immune changes, this study used a less restrictive approach to categorize and group peripheral immune changes. We found that repeated social defeat stress in adult male Sprague-Dawley rats increased the frequencies of mature T-cells positive for intracellular type 2-like cytokine and serum pro-inflammatory cytokines. Principal component analysis and hierarchical clustering was used to guide grouping of T-cells and cytokines, producing unique profiles. Stress shifted the balance towards a specific set that included mostly type 2-like T-cells and pro-inflammatory cytokines. Within the CNS component, repeated stress caused an increase of activated microglia in the BLA, increased anxiety-like behaviors across several assays, and increased BLA neuronal firing in vivo that was prevented by blockade of microglia activation. Because repeated stress can trigger anxiety states by actions in the BLA, and altered immune function can trigger anxiety, these results suggest that repeated stress may trigger anxiety-like behaviors by inducing a pro-inflammatory state in the periphery and the BLA. These results begin to uncover how stress may recruit the immune system to alter the function of brain regions critical to emotion.

Cocaine addiction is a chronic, relapsing disorder. Stress and cues related to cocaine are two common relapse triggers. We have recently shown that exposure to repeated restraint stress during early withdrawal accelerates the time‐dependent intensification or “incubation” of cue‐induced cocaine craving that occurs during the first month of withdrawal, although craving ultimately plateaus at the same level observed in controls. These data indicate that chronic stress exposure during early withdrawal may result in increased vulnerability to cue‐induced relapse during this period. Previous studies have shown that chronic stress exposure in drug‐naïve rats increases neuronal activity in the basolateral amygdala (BLA), a region critical for behavioral responses to stress. Given that glutamatergic projections from the BLA to the nucleus accumbens are critical for the incubation of cue‐induced cocaine craving, we hypothesized that cocaine withdrawal and chronic stress exposure produce separate increases that additively increase BLA neuronal activity. To assess this, we conducted in vivo extracellular single‐unit recordings from the BLA of anesthetized adult male rats following cocaine or saline self‐administration (6 h/day for 10 days) and repeated restraint stress or control conditions on withdrawal days (WD) 6‐14. Recordings were conducted from WD15 to WD20. Interestingly, cocaine exposure alone increased the spontaneous firing rate in the BLA to levels observed following chronic stress exposure in drug‐naïve rats. Chronic stress exposure during cocaine withdrawal further increased firing rate. These studies may identify a potential mechanism by which both cocaine and chronic stress exposure drive cue‐induced relapse vulnerability during abstinence.

Peripheral inflammation often causes changes in mood and emergence of depressive behavior, and is characterized by a group of physical manifestations including lethargy, malaise, listlessness, decreased appetite, anhedonia, and fever. These behavioral changes are induced at the molecular level by pro-inflammatory cytokines like interleukin (IL)-1 beta, IL-6 and TNF-alpha. The basolateral amygdala (BLA) is a key brain region involved in mood and may mediate some of the behavioral effects of inflammation. However, it is unknown whether peripheral inflammatory state affects the activity of BLA neurons. To test this, adult male Sprague-Dawley rats were treated with IL-1 beta (1 mu g, intraperitoneal (i.p.)), and behavioral and electrophysiological measures were obtained. IL-1 beta reduced locomotion in the open-field test and also reduced home-cage mobility, consistent with features of sickness-like behavior. Using in vivo single-unit extracellular electrophysiological recordings from anesthetized rats, we found that spontaneous BLA neuronal firing was acutely (<30 min) increased after IL-1 beta, followed by a return to baseline level, particularly in the basal nucleus of the BLA complex. To verify and expand on effects of peripheral inflammation, we tested whether another, long-lasting inflammagen also changes BLA neuronal firing. Lipopolysaccharide (250 mu g/kg, i.p.) increased BLA firing rate acutely (<30 min) and persistently. The findings demonstrate a rapid effect of peripheral inflammation on BLA activity and suggest a link between BLA neuronal firing and triggering of behavioral consequences of peripheral inflammation. These findings are a first step toward understanding the neuronal basis of depressive behavior caused by acute peripheral inflammation. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Chronic psychological stress is one of the most common triggers of clinical depression and anxiety, and many studies suggest involvement of the immune system in these effects. The basolateral amygdala (BLA), a key brain region involved in emotion, mediates many effects of stress on emotion and may be an intermediary in the link between immune function and depression. Repeated social defeat stress (RSDS) is a powerful model of social stress with known impact on immune function. The peripheral immune system includes pro‐inflammatory and anti‐inflammatory aspects that, as a whole, may exert very different influences on behavior. The aim of this study is (1) to determine the effect of RSDS on the balance between pro‐inflammatory and anti‐inflammatory aspects of peripheral immune function, and (2) to determine if a shift in this balance causes a change in BLA function. Adult male Sprague Dawley rats were exposed to the resident‐intruder model of RSDS using unfamiliar aggressor Long Evans rats for five consecutive days, or control handling. After three days, the following were evaluated: (a) peripheral circulating T‐cell counts and their intracellular cytokine profile (Th1, Th2) by flow‐cytometry, (b) pro‐ and anti‐inflammatory serum cytokine levels by ELISA, and (c) anxiety‐like behavior in the open field test (OFT). RSDS decreased the frequency of dual positive (DP, CD4+CD8+) T‐cells as well as the CD4+ T‐cells with no significant changes in that of the CD8+ T‐cells. Additionally, RSDS increased the frequency of dual negative (DN, CD4‐CD8‐) precursor T‐cells in the circulation. Frequency of CD4+ T‐cells positive for the Th2‐like (anti‐inflammatory) cytokine was increased with similar increase in those of the CD8+ T‐cells positive for the Tc2‐like (anti‐inflammatory) cytokine. RSDS also caused changes in many different serum cytokines. OFT showed that RSDS induced an increase in anxiety‐like behavior. To test whether a pro‐inflammatory state can induce a change in the BLA physiology, in vivo extracellular electrophysiological recordings were performed in a different set of rats that underwent peripheral immune challenge with interleukin‐1β. Interleukin‐1β caused a trend of a time‐dependent alteration of the evoked local field potential as well as alteration of the spontaneous neuronal firing‐rate in the BLA. Taken together, these data suggest that RSDS affects differentiation of peripheral circulatory T‐cells and shifts peripheral immune balance. In addition, a shift in immune balance impacts BLA neuronal activity. This could contribute to the effects of RSDS on anxiety behavior. The results have implications in understanding how chronic stress might alter BLA physiology by recruitment of the peripheral immune system. Support or Funding Information NIH Grant MH084970 and MH109484 to JAR.

Brain activities of the mitochondrial enzyme α-ketoglutarate dehydrogenase complex (KGDHC) are reduced in Alzheimer's disease and other age-related neurodegenerative disorders. The goal of the present study was to test the consequences of mild impairment of KGDHC on the structure, protein signaling and dynamics (mitophagy, fusion, fission, biogenesis) of the mitochondria. Inhibition of KGDHC reduced its in situ activity by 23–53% in human neuroblastoma SH-SY5Y cells, but neither altered the mitochondrial membrane potential nor the ATP levels at any tested time-points. The attenuated KGDHC activity increased translocation of dynamin-related protein-1 (Drp1) and microtubule-associated protein 1A/1B-light chain 3 (LC3) from the cytosol to the mitochondria, and promoted mitochondrial cytochrome c release. Inhibition of KGDHC also increased the negative surface charges (anionic phospholipids as assessed by Annexin V binding) on the mitochondria. Morphological assessments of the mitochondria revealed increased fission and mitophagy. Taken together, our results suggest the existence of the regulation of the mitochondrial dynamism including fission and fusion by the mitochondrial KGDHC activity via the involvement of the cytosolic and mitochondrial protein signaling molecules. A better understanding of the link among mild impairment of metabolism, induction of mitophagy/autophagy and altered protein signaling will help to identify new mechanisms of neurodegeneration and reveal potential new therapeutic approaches.

Cellular health depends on the normal function of the endoplasmic reticulum (ER) to fold, assemble, and modify critical proteins to maintain viability. When the ER cannot process proteins effectively, a condition known as ER stress ensues. When this stress is excessive or prolonged, cell death via apoptotic pathways is triggered. Interestingly, most major diseases have been shown to be intimately linked to ER stress, including diabetes, stroke, neurodegeneration, and many cancers. Thus, controlling ER stress presents a significant strategy for drug development for these diseases. The goal of this review is to present various small molecules that alleviate ER stress with the intention that they may serve as useful starting points for therapeutic agent development.

Chronic neurodegenerative diseases are a group of devastating neurological disorders that result in significant morbidity and mortality in the elderly population worldwide. Recent researches have shown some interesting associations of the classical antiobesity hormone leptin with two most important neurodegenerative diseases—Alzheimer’s disease (AD) and Parkinson’s disease (PD). Although several clinical studies have found the procognitive and memory-enhancing role of this peptide hormone in leptin-deficient patients, surprisingly it has not been used in any clinical trials involving patients with developing or full-blown neurodegenerative conditions. This review article is an attempt to bring together the existing information about the clinical associations of leptin with AD and PD. It starts with the basic understanding of leptin action in the brain and its derangements in these diseases and eventually discusses the potential of this hormone as a neuroprotective agent in clinical scenario.

Background: Gynecological carcinomas form a significant proportion of all malignancies in women across the globe. These are associated with an increase in the serum concentrations of certain tumor markers such as cancer antigen (CA)125, CA19.9 and carcinoembryonic antigen (CEA) that correlate with the tumor burden. Methods: Pre- and post-treatment serum levels of CA125, CA19.9 and CEA were determined in 36 patients of ovarian carcinoma, 31 patients of cervical carcinoma and 20 patients of endometrial carcinoma using enzyme-linked immunosorbent assay. The pre- and the post-treatment levels of these markers have been compared and correlated. Results: With primary treatment, CA125 level was significantly reduced in ovarian, endometrial (ps < 0.001) and cervical (p = 0.001) carcinomas and that of CA19.9 was significantly decreased in cervical and endometrial carcinomas (ps < 0.001). Surprisingly, post-treatment CEA level was significantly increased in cervical carcinoma (p = 0.001) with significant increase after radiotherapy (p = 0.003), but not after surgery (p = 0.091). Treatment had no effects on CA19.9 level in ovarian carcinoma and on CEA levels in ovarian and endometrial carcinomas. Pre- and post-treatment levels of CA125, CA19.9 and CEA showed strong positive correlation in cervical carcinoma, while those of CA19.9 showed very strong positive correlation in endometrial carcinoma. Conclusions: Post-treatment serum CA125 level best reflects the treatment effect in all three types of gynecological carcinomas. CA19.9 is reliable to evaluate treatment effect in patients of cervical and endometrial carcinomas. Studies involving larger population size should be conducted to identify the changes in CEA while assessing treatment effect in cervical carcinoma patients.

a b a c Aditi Kishore Shinde, M.S. , Soumyabrata Munshi, M.D. , Subhash Chandra Biswas, M.D. , Avijit Hazra, M.D. , b Sasanka Chakrabarti, M.D. Department of Obstetrics and Gynecology, Institute of Post-Graduate Medical Education and Research, 244 Acharya J.C. Bose Road, Kolkata 700020, India. Department of Biochemistry, Institute of Post-Graduate Medical Education and Research, 244 Acharya J.C. Bose Road, Kolkata 700020, India. c Department of Pharmacology, Institute of Post-Graduate Medical Education and Research, 244 Acharya J.C. Bose Road, Kolkata 700020, India.

Introduction: The cell viability assay by alamar blue is based on the principle of reduction of the non-fluorescent reagent (resazurin) to a fluorescent compound (resarufin) by the intracellular reducing environment of living cells over time. In the present study, we have for the first time shown that even in the absence of cells, there occurs significant interaction between alamar blue and cell-culture media causing an increase in fluorescence. Methods: We have used Opti-MEM, DMEM and 1:1 DMEM: Opti-MEM as three different media and determined the changes in their relative fluorescence units (RFUs) over time after the addition of 10% (v/v) alamar blue using two-way repeated measures analysis of variance (RM-ANOVA) followed by Tukey's post-hoc test. Results: Our results show that upon the addition of alamar blue, there occurs a significant increase in RFUs in all the three media over time along with a significantly higher RFU for the Opti-MEM overall (p < 0.05). We also show that the time-dependent change in RFU of 1:1 DMEM: Opti-MEM was more gradual compared to that of the other two media. Discussion: These findings indicate that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue. In addition our results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability. (C) 2014 Elsevier Inc. All rights reserved.