Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes.

Neuronal activity causes the rapid expression of immediate early genes that are crucial for experience-driven changes to synapses, learning, and memory. Here, using both molecular and genome-wide next-generation sequencing methods, we report that neuronal activity stimulation triggers the formation of DNA double strand breaks (DSBs) in the promoters of a subset of early-response genes, including Fos, Npas4, and Egr1. Generation of targeted DNA DSBs within Fos and Npas4 promoters is sufficient to induce their expression even in the absence of an external stimulus. Activity-dependent DSB formation is likely mediated by the type II topoisomerase, Topoisomerase IIß (Topo IIß), and knockdown of Topo IIß attenuates both DSB formation and early-response gene expression following neuronal stimulation. Our results suggest that DSB formation is a physiological event that rapidly resolves topological constraints to early-response gene expression in neurons.

Induction of AP-1 by YAP/TAZ contributes to cell proliferation and organ growth.

Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway to control cell growth and organ size, of which dysregulation yields to tumorigenesis or hypertrophy. Upon activation, YAP/TAZ translocate into the nucleus and bind to TEAD transcription factors to promote transcriptional programs for proliferation or cell specification. Immediate early genes, represented by AP-1 complex, are rapidly induced and control later-phase transcriptional program to play key roles in tumorigenesis and organ maintenance. Here, we report that YAP/TAZ directly promote FOS transcription that in turn contributes to the biological function of YAP/TAZ. YAP/TAZ bind to the promoter region of FOS to stimulate its transcription. Deletion of YAP/TAZ blocks the induction of immediate early genes in response to mitogenic stimuli. FOS induction contributes to expression of YAP/TAZ downstream target genes. Genetic deletion or chemical inhibition of AP-1 suppresses growth of YAP-driven cancer cells, such as Lats1/2-deficient cancer cells as well as Gαq/11 mutated uveal melanoma. Furthermore, AP-1 inhibition almost completely abrogates the hepatomegaly induced by YAP overexpression. Our findings reveal a feed-forward interplay between immediate early transcription of AP-1 and Hippo pathway function.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus.

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Single-molecule imaging of transcriptionally coupled and uncoupled splicing.

Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA-binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, whereas the preceding and succeeding introns are removed cotranscriptionally. PAPERCLIP:

Fos regulates macrophage infiltration against surrounding tissue resistance by a cortical actin-based mechanism in Drosophila.

The infiltration of immune cells into tissues underlies the establishment of tissue-resident macrophages and responses to infections and tumors. Yet the mechanisms immune cells utilize to negotiate tissue barriers in living organisms are not well understood, and a role for cortical actin has not been examined. Here, we find that the tissue invasion of Drosophila macrophages, also known as plasmatocytes or hemocytes, utilizes enhanced cortical F-actin levels stimulated by the Drosophila member of the fos proto oncogene transcription factor family (Dfos, Kayak). RNA sequencing analysis and live imaging show that Dfos enhances F-actin levels around the entire macrophage surface by increasing mRNA levels of the membrane spanning molecular scaffold tetraspanin TM4SF, and the actin cross-linking filamin Cheerio, which are themselves required for invasion. Both the filamin and the tetraspanin enhance the cortical activity of Rho1 and the formin Diaphanous and thus the assembly of cortical actin, which is a critical function since expressing a dominant active form of Diaphanous can rescue the Dfos macrophage invasion defect. In vivo imaging shows that Dfos enhances the efficiency of the initial phases of macrophage tissue entry. Genetic evidence argues that this Dfos-induced program in macrophages counteracts the constraint produced by the tension of surrounding tissues and buffers the properties of the macrophage nucleus from affecting tissue entry. We thus identify strengthening the cortical actin cytoskeleton through Dfos as a key process allowing efficient forward movement of an immune cell into surrounding tissues.

The Role of C1473G Polymorphism in Mouse Triptophan Hydroxylase 2 Gene in the Acute Effects of Ethanol on the c-fos Gene Expression and Metabolism of Biogenic Amines in the Brain.

Tryptophan hydroxylase 2 is a key enzyme in the synthesis of the neurotransmitter serotonin, which plays an important role in the regulation of behavior and various physiological functions. We studied the effect of acute ethanol administration on the expression of the early response c-fos gene and metabolism of serotonin and catecholamines in the brain structures of B6-1473C and B6-1473G congenic mouse strains differing in the single-nucleotide substitution C1473G in the Tph2 gene and activity of the encoded enzyme. Acute alcoholization led to a significant upregulation of the c-fos gene expression in the frontal cortex and striatum of B6-1473G mice and in the hippocampus of B6-1473C mice and caused a decrease in the index of serotonin metabolism in the nucleus accumbens in B6-1473C mice and in the hippocampus and striatum of B6-1473G mice, as well as to the decrease in the norepinephrine level in the hypothalamus of B6-1473C mice. Therefore, the C1473G polymorphism in the Tph2 gene has a significant effect of acute ethanol administration on the c-fos expression pattern and metabolism of biogenic amines in the mouse brain.

In Vitro Imaging and Molecular Characterization of Ca2+ Flux Modulation by Nanosecond Pulsed Electric Fields.

In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

An Angiotensin-Responsive Connection from the Lamina Terminalis to the Paraventricular Nucleus of the Hypothalamus Evokes Vasopressin Secretion to Increase Blood Pressure in Mice.

Blood pressure is controlled by endocrine, autonomic, and behavioral responses that maintain blood volume and perfusion pressure at levels optimal for survival. Although it is clear that central angiotensin type 1a receptors (AT1aR; encoded by the Agtr1a gene) influence these processes, the neuronal circuits mediating these effects are incompletely understood. The present studies characterize the structure and function of AT1aR neurons in the lamina terminalis (containing the median preoptic nucleus and organum vasculosum of the lamina terminalis), thereby evaluating their roles in blood pressure control. Using male Agtr1a-Cre mice, neuroanatomical studies reveal that AT1aR neurons in the area are largely glutamatergic and send projections to the paraventricular nucleus of the hypothalamus (PVN) that appear to synapse onto vasopressin-synthesizing neurons. To evaluate the functionality of these lamina terminalis AT1aR neurons, we virally delivered light-sensitive opsins and then optogenetically excited or inhibited the neurons while evaluating cardiovascular parameters or fluid intake. Optogenetic excitation robustly elevated blood pressure, water intake, and sodium intake, while optogenetic inhibition produced the opposite effects. Intriguingly, optogenetic excitation of these AT1aR neurons of the lamina terminalis also resulted in Fos induction in vasopressin neurons within the PVN and supraoptic nucleus. Further, within the PVN, selective optogenetic stimulation of afferents that arise from these lamina terminalis AT1aR neurons induced glutamate release onto magnocellular neurons and was sufficient to increase blood pressure. These cardiovascular effects were attenuated by systemic pretreatment with a vasopressin-1a-receptor antagonist. Collectively, these data indicate that excitation of lamina terminalis AT1aR neurons induces neuroendocrine and behavioral responses that increase blood pressure.SIGNIFICANCE STATEMENT Hypertension is a widespread health problem and risk factor for cardiovascular disease. Although treatments exist, a substantial percentage of patients suffer from "drug-resistant" hypertension, a condition associated with increased activation of brain angiotensin receptors, enhanced sympathetic nervous system activity, and elevated vasopressin levels. The present study highlights a role for angiotensin Type 1a receptor expressing neurons located within the lamina terminalis in regulating endocrine and behavioral responses that are involved in maintaining cardiovascular homeostasis. More specifically, data presented here reveal functional excitatory connections between angiotensin-sensitive neurons in the lamina terminals and vasopressin neurons in the paraventricular nucleus of the hypothalamus, and further indicate that activation of this circuit raises blood pressure. These neurons may be a promising target for antihypertensive therapeutics.

Dynamic Change of Endocannabinoid Signaling in the Medial Prefrontal Cortex Controls the Development of Depression After Neuropathic Pain.

Many patients with chronic pain conditions suffer from depression. The mechanisms underlying pain-induced depression are still unclear. There are critical links of medial prefrontal cortex (mPFC) synaptic function to depression, with signaling through the endocannabinoid (eCB) system as an important contributor. We hypothesized that afferent noxious inputs after injury compromise activity-dependent eCB signaling in the mPFC, resulting in depression. Depression-like behaviors were tested in male and female rats with traumatic neuropathy [spared nerve injury (SNI)], and neuronal activity in the mPFC was monitored using the immediate early gene c-fos and in vivo electrophysiological recordings. mPFC eCB Concentrations were determined using mass spectrometry, and behavioral and electrophysiological experiments were used to evaluate the role of alterations in eCB signaling in depression after pain. SNI-induced pain induced the development of depression phenotypes in both male and female rats. Pyramidal neurons in mPFC showed increased excitability followed by reduced excitability in the onset and prolonged phases of pain, respectively. Concentrations of the eCBs, 2-arachidonoylglycerol (2-AG) in the mPFC, were elevated initially after SNI, and our results indicate that this resulted in a loss of CB1R function on GABAergic interneurons in the mPFC. These data suggest that excessive release of 2-AG as a result of noxious stimuli triggers use-dependent loss of function of eCB signaling leading to excessive GABA release in the mPFC, with the final result being behavioral depression.SIGNIFICANCE STATEMENT Pain has both somatosensory and affective components, so the complexity of mechanisms underlying chronic pain is best represented by a biopsychosocial model that includes widespread CNS dysfunction. Many patients with chronic pain conditions develop depression. The mechanism by which pain causes depression is unclear. Although manipulation of the eCB signaling system as an avenue for providing analgesia per se has not shown much promise in previous studies. An important limitation of past research has been inadequate consideration of the dynamic nature of the connection between pain and depression as they develop. Here, we show that activity-dependent synthesis of eCBs during the initial onset of persistent pain is the critical link leading to depression when pain is persistent.

MiR-744 Functions as an Oncogene Through Direct Binding to c-Fos Promoter and Facilitates Non-small Cell Lung Cancer Progression.

Metastasis is the leading cause of death in non-small cell lung cancer (NSCLC) patients. Previously, we reported that miR-744 exerted proto-oncogenic function in nasopharyngeal carcinoma, but the role of miR-744 during NSCLC development has not been established. We focused on the function and molecular mechanism of miR-744 in NSCLC. The clinical cohort data from TCGA were analyzed for the correlation of miR-744 and outcomes in NSCLC patients. Gain- and loss-of-function experiment was performed by transfection with miR-744 agomir or antagomir in NSCLC cell lines. The expression of mRNA and protein were analyzed by qPCR assays and Western blotting respectively. Cellular proliferation, migration, and invasion were analyzed by CCK8 assays, wound healing, and transwell assays, respectively. Promoter activities and gene transcription were analyzed by luciferase reporter assays. Xenograft model was applied for in vivo study. High miR-744 expression correlated with lymph node metastasis and poor prognosis in NSCLC patient. MiR-744 aggravated the growth, invasion, and metastasis of NSCLC cells eventually induced the malignant phenotype and promotes radio/chemoresistance in vitro. The -1195 to -1227 and -298 to -323 bp upstream of c-FOS gene was observed to bind with miR-744. Lastly, miR-744 acted as a tumor promoter in lung cancer growth and metastasis in vivo. Taken together, our results indicated that miR-744 up-regulated c-Fos by binding with its promoter contributed to development of NSCLC cells malignant phenotype. Our findings highlight the potential value of miR-744, which may serve as a possible therapeutic target for NSCLC.

2E-Decene-4,6-diyn-1-ol-acetate inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signalling pathways.

An imbalance of osteoclasts and osteoblasts can result in a variety of bone-related diseases, including osteoporosis. Thus, decreasing the activity of osteoclastic bone resorption is the main therapeutic method for treating osteoporosis. 2E-Decene-4, 6-diyn-1-ol-acetate (DDA) is a natural bioactive compound with anti-inflammatory and anti-cancer properties. However, its effects on osteoclastogenesis are unknown. Murine bone marrow-derived macrophages (BMMs) or RAW264.7 cells were treated with DDA, followed by evaluation of cell viability, RANKL-induced osteoclast differentiation, and pit formation assay. Effects of DDA on RANKL-induced phosphorylation of MAPKs were assayed by western blot analysis. Expression of osteoclast-specific genes was examined with reverse transcription-PCR (RT-PCR) and western blot analysis. In this study, DDA significantly inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells as well as in BMMs without cytotoxicity. DDA also strongly blocked the resorbing capacity of BMM on calcium phosphate-coated plates. DDA inhibited RANKL-induced phosphorylation of ERK, JNK and p38 MAPKs, as well as expression of c-Fos and NFATc1, which are essential transcription factors for osteoclastogenesis. In addition, DDA decreased expression levels of osteoclastogenesis-specific genes, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB (RANK) in RANKL-induced RAW264.7 cells. Collectively, these findings indicated that DDA attenuates RANKL-induced osteoclast formation by suppressing the MAPKs-c-Fos-NFATc1 signalling pathway and osteoclast-specific genes. These results indicate that DDA may be a potential candidate for bone diseases associated with abnormal osteoclast formation and function.

Soluble SORLA Enhances Neurite Outgrowth and Regeneration through Activation of the EGF Receptor/ERK Signaling Axis.

SORLA is a transmembrane trafficking protein associated with Alzheimer's disease risk. Although SORLA is abundantly expressed in neurons, physiological roles for SORLA remain unclear. Here, we show that cultured transgenic neurons overexpressing SORLA feature longer neurites, and accelerated neurite regeneration with wounding. Enhanced release of a soluble form of SORLA (sSORLA) is observed in transgenic mouse neurons overexpressing human SORLA, while purified sSORLA promotes neurite extension and regeneration. Phosphoproteomic analyses demonstrate enrichment of phosphoproteins related to the epidermal growth factor (EGFR)/ERK pathway in SORLA transgenic mouse hippocampus from both genders. sSORLA coprecipitates with EGFR in vitro, and sSORLA treatment increases EGFR Y1173 phosphorylation, which is involved in ERK activation in cultured neurons. Furthermore, sSORLA triggers ERK activation, whereas pharmacological EGFR or ERK inhibition reverses sSORLA-dependent enhancement of neurite outgrowth. In search for downstream ERK effectors activated by sSORLA, we identified upregulation of Fos expression in hippocampus from male mice overexpressing SORLA by RNAseq analysis. We also found that Fos is upregulated and translocates to the nucleus in an ERK-dependent manner in neurons treated with sSORLA. Together, these results demonstrate that sSORLA is an EGFR-interacting protein that activates EGFR/ERK/Fos signaling to enhance neurite outgrowth and regeneration.SIGNIFICANCE STATEMENT SORLA is a transmembrane trafficking protein previously known to reduce the levels of amyloid-ß, which is critical in the pathogenesis of Alzheimer's disease. In addition, SORLA mutations are a risk factor for Alzheimer's disease. Interestingly, the SORLA ectodomain is cleaved into a soluble form, sSORLA, which has been shown to regulate cytoskeletal signaling pathways and cell motility in cells outside the nervous system. We show here that sSORLA binds and activates the EGF receptor to induce downstream signaling through the ERK serine/threonine kinase and the Fos transcription factor, thereby enhancing neurite outgrowth. These findings reveal a novel role for sSORLA in promoting neurite regeneration through the EGF receptor/ERK/Fos pathway, thereby demonstrating a potential neuroprotective mechanism involving SORLA.

Chronic Stress Induces Maladaptive Behaviors by Activating Corticotropin-Releasing Hormone Signaling in the Mouse Oval Bed Nucleus of the Stria Terminalis.

The bed nucleus of the stria terminalis (BNST) is a forebrain region highly responsive to stress that expresses corticotropin-releasing hormone (CRH) and is implicated in mood disorders, such as anxiety. However, the exact mechanism by which chronic stress induces CRH-mediated dysfunction in BNST and maladaptive behaviors remains unclear. Here, we first confirmed that selective acute optogenetic activation of the oval nucleus BNST (ovBNST) increases maladaptive avoidance behaviors in male mice. Next, we found that a 6 week chronic variable mild stress (CVMS) paradigm resulted in maladaptive behaviors and increased cellular excitability of ovBNST CRH neurons by potentiating mEPSC amplitude, altering the resting membrane potential, and diminishing M-currents (a voltage-gated K+ current that stabilizes membrane potential) in ex vivo slices. CVMS also increased c-fos+ cells in ovBNST following handling. We next investigated potential molecular mechanism underlying the electrophysiological effects and observed that CVMS increased CRH+ and pituitary adenylate cyclase-activating polypeptide+ (PACAP; a CRH upstream regulator) cells but decreased striatal-enriched protein tyrosine phosphatase+ (a STEP CRH inhibitor) cells in ovBNST. Interestingly, the electrophysiological effects of CVMS were reversed by CRHR1-selective antagonist R121919 application. CVMS also activated protein kinase A (PKA) in BNST, and chronic infusion of the PKA-selective antagonist H89 into ovBNST reversed the effects of CVMS. Coadministration of the PKA agonist forskolin prevented the beneficial effects of R121919. Finally, CVMS induced an increase in surface expression of phosphorylated GluR1 (S845) in BNST. Collectively, these findings highlight a novel and indispensable stress-induced role for PKA-dependent CRHR1 signaling in activating BNST CRH neurons and mediating maladaptive behaviors.SIGNIFICANCE STATEMENT Chronic stress and acute activation of oval bed nucleus of the stria terminalis (ovBNST) induces maladaptive behaviors in rodents. However, the precise molecular and electrophysiological mechanisms underlying these effects remain unclear. Here, we demonstrate that chronic variable mild stress activates corticotropin-releasing hormone (CRH)-associated stress signaling and CRH neurons in ovBNST by potentiating mEPSC amplitude and decreasing M-current in male mice. These electrophysiological alterations and maladaptive behaviors were mediated by BNST protein kinase A-dependent CRHR1 signaling. Our results thus highlight the importance of BNST CRH dysfunction in chronic stress-induced disorders.

Role of Projections between Piriform Cortex and Orbitofrontal Cortex in Relapse to Fentanyl Seeking after Palatable Food Choice-Induced Voluntary Abstinence.

We recently developed a rat model of relapse to drug seeking after food choice-induced voluntary abstinence. Here, we used this model to study the role of the orbitofrontal cortex (OFC) and its afferent projections in relapse to fentanyl seeking. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/d) and intravenous fentanyl (2.5 µg/kg/infusion) for 12 d (6 h/d). We assessed relapse to fentanyl seeking after 13-14 voluntary abstinence days, achieved through a discrete choice procedure between fentanyl infusions and palatable food (20 trials/d). In both sexes, relapse after food choice-induced abstinence was associated with increased expression of the activity marker Fos in the OFC. Pharmacological inactivation of the OFC with muscimol plus baclofen (50 + 50 ng/side) decreased relapse to fentanyl seeking. We then determined projection-specific activation of OFC afferents during the relapse test by using Fos plus the retrograde tracer cholera toxin B (injected into the OFC). Relapse to fentanyl seeking was associated with increased Fos expression in the piriform cortex (Pir) neurons projecting to the OFC, but not in projections from the basolateral amygdala and thalamus. Pharmacological inactivation of the Pir with muscimol plus baclofen decreased relapse to fentanyl seeking after voluntary abstinence. Next, we used an anatomical disconnection procedure to determine whether projections between the Pir and OFC are critical for relapse to fentanyl seeking. Unilateral muscimol plus baclofen injections into the Pir in one hemisphere plus unilateral muscimol plus baclofen injections into the OFC in the contralateral, but not ipsilateral, hemisphere decreased relapse. Our results identify Pir-OFC projections as a new motivation-related pathway critical to relapse to opioid seeking after voluntary abstinence.SIGNIFICANCE STATEMENT There are few preclinical studies of fentanyl relapse, and these studies have used experimenter-imposed extinction or forced abstinence procedures. In humans, however, abstinence is often voluntary, with drug available in the drug environment but forgone in favor of nondrug alternative reinforcers. We recently developed a rat model of drug relapse after palatable food choice-induced voluntary abstinence. Here, we used classical pharmacology, immunohistochemistry, and retrograde tracing to demonstrate a critical role of the piriform and orbitofrontal cortices in relapse to opioid seeking after voluntary abstinence.

Glucocorticoids attenuate interleukin-6-induced c-Fos and Egr1 expression and impair neuritogenesis in PC12 cells.

Interleukin-6 (IL-6) is a cytokine primarily known for immune regulation. There is also growing evidence that IL-6 triggers neurogenesis and impacts neural development, both life-long occurring processes that can be impaired by early-life and adult stress. Stress induces the release of glucocorticoids by activation of the hypothalamic-pituitary-adrenal (HPA) axis. On the cellular level, glucocorticoids act via the ubiquitously expressed glucocorticoid receptor. Thus, we aimed to elucidate whether glucocorticoids affect IL-6-induced neural development. Here, we show that IL-6 signalling induces neurite outgrowth in adrenal pheochromocytoma PC12 cells in a mitogen-activated protein kinase (MAPK) pathway-dependent manner, since neurite outgrowth was diminished upon Mek-inhibitor treatment. Using quantitative biochemical approaches, such as qRT-PCR analysis of Hyper-IL-6 treated PC12 cells, we show that neurite outgrowth induced by IL-6 signalling is accompanied by early and transient MAPK-dependent mRNA expression of immediate early genes coding for proteins such as early growth response protein 1 (Egr1) and c-Fos. This correlates with reduced proliferation and prolonged G0/G1 cell cycle arrest as determined by monitoring the cellular DNA content using flow cytometry. These results indicate for IL-6 signalling-induced neural differentiation. Interestingly, the glucocorticoid Dexamethasone impairs early IL-6 signalling-induced mRNA expression of c-Fos and Egr1 and restrains neurite outgrowth. Impaired Egr1 and c-Fos expression in neural development is implicated in the aetiology of neuropathologies. Thus, it appears likely that stress-induced release of glucocorticoids, as well as therapeutically administered glucocorticoids, contribute to the development of neuropathologies by reducing the expression of Egr1 and c-Fos, and by restraining IL-6-dependent neural differentiation.

Neuronal mechanisms of adenosine A2A receptors in the loss of consciousness induced by propofol general anesthesia with functional magnetic resonance imaging.

Propofol is the most common intravenous anesthetic agent for induction and maintenance of anesthesia, and has been used clinically for more than 30 years. However, the mechanism by which propofol induces loss of consciousness (LOC) remains largely unknown. The adenosine A2A receptor (A2A R) has been extensively proven to have an effect on physiological sleep. It is, therefore, important to investigate the role of A2A R in the induction of LOC using propofol. In the present study, the administration of the highly selective A2A R agonist (CGS21680) and antagonist (SCH58261) was utilized to investigate the function of A2A R under general anesthesia induced by propofol by means of animal behavior studies, resting-state magnetic resonance imaging and c-Fos immunofluorescence staining approaches. Our results show that CGS21680 significantly prolonged the duration of LOC induced by propofol, increased the c-Fos expression in nucleus accumbens (NAc) and suppressed the functional connectivity of NAc-dorsal raphe nucleus (DR) and NAc-cingulate cortex (CG). However, SCH58261 significantly shortened the duration of LOC induced by propofol, decreased the c-Fos expression in NAc, increased the c-Fos expression in DR, and elevated the functional connectivity of NAc-DR and NAc-CG. Collectively, our findings demonstrate the important roles played by A2A R in the LOC induced by propofol and suggest that the neural circuit between NAc-DR maybe controlled by A2A R in the mechanism of anesthesia induced by propofol.

Facilitation of dopamine-dependent long-term potentiation in the medial prefrontal cortex of male rats follows the behavioral effects of stress.

The effect of stress on animal behavior and brain activity has been attracting growing attention in the last decades. Stress dramatically affects several aspects of animal behavior, including motivation and cognitive functioning, and has been used to model human pathologies such as post-traumatic stress disorder. A key question is whether stress alters the plastic potential of synaptic circuits. In this work, we evaluated if stress affects dopamine (DA)-dependent synaptic plasticity in the medial prefrontal cortex (mPFC). On male adolescent rats, we characterized anxiety- and depressive-like behaviors using behavioral testing before and after exposure to a mild stress (elevated platform, EP). After the behavioral protocols, we investigated DA-dependent long-term potentiation (DA-LTP) and depression (DA-LTD) on acute slices of mPFC and evaluated the activation of DA-producing brain regions by western and dot blot analysis. We show that exposure to the EP stress enhances DA-LTP and that desipramine (DMI) treatment abolishes this effect. We also found that DA-LTD is not affected by EP stress unless when this is followed by DMI treatment. In addition, EP stress reduces anxiety, an effect abolished by both DMI and ketamine, while motivation is promoted by previous exposure to EP stress independently of pharmacological treatments. Finally, this form of stress reduces the expression of the early gene cFOS in the ventral tegmental area. These findings support the idea that mild stressors can promote synaptic plasticity in PFC through a dopaminergic mechanism, an effect that might increase the sensitivity of mPFC to subsequent stressful experiences.

Olfactory learning and memory in the greater short-nosed fruit bat Cynopterus sphinx: the influence of conspecifics distress calls.

This study was designed to test whether Cynopterus sphinx distress calls influence olfactory learning and memory in conspecifics. Bats were exposed to distress calls/playbacks (PBs) of distress calls/modified calls and were then trained to novel odors. Bats exposed to distress calls/PBs made significantly fewer feeding attempts and bouts of PBs exposed to modified calls, which significantly induced the expression of c-Fos in the caudomedial neostriatum (NCM) and the amygdala compared to bats exposed to modified calls and trained controls. However, the expression of c-Fos in the hippocampus was not significantly different between the experimental groups. Further, protein phosphatase-1 (PP-1) expression was significantly lower, and the expression levels of E1A homologue of CREB-binding protein (CBP) (P300), brain-derived neurotrophic factor (BDNF) and its tyrosine kinase B1 (TrkB1) receptor were significantly higher in the hippocampus of control/bats exposed to modified calls compared to distress calls/PBs of distress call-exposed bats. Exposure to the call possibly alters the reciprocal interaction between the amygdala and the hippocampus, accordingly regulating the expression levels of PP1, P300 and BDNF and its receptor TrkB1 following training to the novel odor. Thus, the learning and memory consolidation processes were disrupted and showed fewer feeding attempts and bouts. This model may be helpful for understanding the contributions of stressful social communications to human disorders.

Rarity of human T helper 17 cells is due to retinoic acid orphan receptor-dependent mechanisms that limit their expansion.

The reason why CD4(+) T helper 17 (Th17) cells, despite their well-known pathogenic role in chronic inflammatory disorders, are very rare in the inflammatory sites remains unclear. We demonstrate that human Th17 cells exhibit low ability to proliferate and to produce the T cell growth factor interleukin-2 (IL-2), in response to combined CD3 and CD28 stimulation. This was due to the upregulated expression of IL-4-induced gene 1 (IL4I1) mRNA, a secreted L-phenylalanine oxidase, which associated with a decrease in CD3ζ chain expression and consequent abnormalities in the molecular pathway that allows IL-2 production and cell proliferation. High IL4I1 mRNA expression was detectable in Th17 cell precursors and was strictly dependent on Th17 cell master gene, the retinoid acid related orphan receptor (RORC). Th17 cells also exhibited RORC-dependent CD28 hyperexpression and the ability to produce IL-17A after CD28 stimulation without CD3 triggering. Our findings suggest that the rarity of human Th17 cells in inflamed tissues results from RORC-dependent mechanisms limiting their expansion.