Epigenetics evaluation of the oncogenic mechanisms of two closely related bovine and human deltaretroviruses: A system biology study.

Human T-cell lymphotropic virus (HTLV-1) and bovine leukemia virus (BLV) are oncogenic deltaretroviruses, which are the cause of adult T cell leukemia/lymphoma (ATLL) and enzootic bovine leukosis (EBL), respectively. In this study, to evaluate the virus-host interactions in the manifestation of the associated malignancy, four pooled RNA samples of each host (three RNAs in each sample) were applied to RNA-seq. Differential expression analyses were conducted separately between ATLL and EBL groups, in comparison with the healthy group, to identify functional Gene Ontology (GO) terms and hub genes, using DAVID database and MCODE plugin in Cytoscape software, respectively. A broad range of effective genes, involved in the ATLL and EBL, was up- and downregulated. In the virus side, in both malignancy, Tax was expressed very low, but the HTLV-1-HBZ and BVL-As2 transcripts were highly expressed. Some upregulated hub genes, IL2, TOP2A, MKI67, TP73, MYC, and downregulated FOS gene family (FOS, FOSB, and FOSL2), are similarly activated in both human and bovine hosts, in related cell cycle and growth factors. Taken together, it seems that in preventing the infections and cell transformations, Tax must be targeted as a viral factor, and shared peptide in virological and immunological synapses as host factors. Therefore, in the malignant stages, HBZ and As2 transcripts along with growth factors, particularly IL-2R-γ and T-bet or TOP2A, and MKI67 should be targeted in both hosts. Additional studies at the protein level are necessary to elucidate the more useful targets for the therapy of these life-threatening diseases.

More than just inflammation: Ureaplasma species induce apoptosis in human brain microvascular endothelial cells.

BACKGROUND: Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death. METHODS: Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties. RESULTS: Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05). CONCLUSIONS: By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp.

Integrative In Silico and In Vitro Transcriptomics Analysis Revealed Gene Expression Changes and Oncogenic Features of Normal Cholangiocytes after Chronic Alcohol Exposure.

Cholangiocarcinoma (CCA) is a malignant tumor originating from cholangiocyte. Prolonged alcohol consumption has been suggested as a possible risk factor for CCA, but there is no information about alcohol's mechanisms in cholangiocyte. This study was designed to investigate global transcriptional alterations through RNA-sequencing by using chronic alcohol exposure (20 mM for 2 months) in normal human cholangiocyte MMNK-1 cells. To observe the association of alcohol induced CCA pathogenesis, we combined differentially expressed genes (DEGs) with computational bioinformatics of CCA by using publicly gene expression omnibus (GEO) datasets. For biological function analysis, Gene ontology (GO) analysis showed biological process and molecular function related to regulation of transcription from RNA polymerase II promoter, while cellular component linked to the nucleoplasm. KEGG pathway presented pathways in cancer that were significantly enriched. From KEGG result, we further examined the oncogenic features resulting in chronic alcohol exposure, enhanced proliferation, and migration through CCND-1 and MMP-2 up-regulation, respectively. Finally, combined DEGs were validated in clinical data including TCGA and immunohistochemistry from HPA database, demonstrating that FOS up-regulation was related to CCA pathogenesis. This study is the first providing more information and molecular mechanisms about global transcriptome alterations and oncogenic enhancement of chronic alcohol exposure in normal cholangiocytes.

Brain-wide maps of Fos expression during fear learning and recall.

Fos induction during learning labels neuronal ensembles in the hippocampus that encode a specific physical environment, revealing a memory trace. In the cortex and other regions, the extent to which Fos induction during learning reveals specific sensory representations is unknown. Here we generate high-quality brain-wide maps of Fos mRNA expression during auditory fear conditioning and recall in the setting of the home cage. These maps reveal a brain-wide pattern of Fos induction that is remarkably similar among fear conditioning, shock-only, tone-only, and fear recall conditions, casting doubt on the idea that Fos reveals auditory-specific sensory representations. Indeed, novel auditory tones lead to as much gene induction in visual as in auditory cortex, while familiar (nonconditioned) tones do not appreciably induce Fos anywhere in the brain. Fos expression levels do not correlate with physical activity, suggesting that they are not determined by behavioral activity-driven alterations in sensory experience. In the thalamus, Fos is induced more prominently in limbic than in sensory relay nuclei, suggesting that Fos may be most sensitive to emotional state. Thus, our data suggest that Fos expression during simple associative learning labels ensembles activated generally by arousal rather than specifically by a particular sensory cue.

Role of Ventral Subiculum in Context-Induced Relapse to Alcohol Seeking after Punishment-Imposed Abstinence.

In many human alcoholics, abstinence is self-imposed because of the negative consequences of excessive alcohol use, and relapse is often triggered by exposure to environmental contexts associated with prior alcohol drinking. We recently developed a rat model of this human condition in which we train alcohol-preferring P rats to self-administer alcohol in one context (A), punish the alcohol-reinforced responding in a different context (B), and then test for relapse to alcohol seeking in Contexts A and B without alcohol or shock. Here, we studied the role of projections to nucleus accumbens (NAc) shell from ventral subiculum (vSub), basolateral amygdala, paraventricular thalamus, and ventral medial prefrontal cortex in context-induced relapse after punishment-imposed abstinence. First, we measured double-labeling of the neuronal activity marker Fos with the retrograde tracer cholera toxin subunit B (injected in NAc shell) and demonstrated that context-induced relapse is associated with selective activation of the vSub→NAc shell projection. Next, we reversibly inactivated the vSub with GABA receptor agonists (muscimol+baclofen) before the context-induced relapse tests and provided evidence for a causal role of vSub in this relapse. Finally, we used a dual-virus approach to restrict expression of the inhibitory κ opioid-receptor based DREADD (KORD) in vSub→NAc shell projection neurons. We found that systemic injections of the KORD agonist salvinorin B, which selectively inhibits KORD-expressing neurons, decreased context-induced relapse to alcohol seeking. Our results demonstrate a critical role of vSub in context-induced relapse after punishment-imposed abstinence and further suggest a role of the vSub→NAc projection in this relapse. SIGNIFICANCE STATEMENT: In many human alcoholics, abstinence is self-imposed because of the negative consequences of excessive use, and relapse is often triggered by exposure to environmental contexts associated with prior alcohol use. Until recently, an animal model of this human condition did not exist. We developed a rat model of this human condition in which we train alcohol-preferring P rats to self-administer alcohol in one context (A), punish the alcohol-reinforced responding in a different context (B), and test for relapse to alcohol seeking in Contexts A and B. Here, we used neuroanatomical, neuropharmacological, and chemogenetic methods to demonstrate a role of ventral subiculum and potentially its projections to nucleus accumbens in context-induced relapse after punishment-imposed abstinence.

Role of Central Amygdala Neuronal Ensembles in Incubation of Nicotine Craving.

UNLABELLED: The craving response to smoking-associated cues in humans or to intravenous nicotine-associated cues in adult rats progressively increases or incubates after withdrawal. Here, we further characterized incubation of nicotine craving in the rat model by determining whether this incubation is observed after adolescent-onset nicotine self-administration. We also used the neuronal activity marker Fos and the Daun02 chemogenetic inactivation procedure to identify cue-activated neuronal ensembles that mediate incubation of nicotine craving. We trained adolescent and adult male rats to self-administer nicotine (2 h/d for 12 d) and assessed cue-induced nicotine seeking in extinction tests (1 h) after 1, 7, 14, or 28 withdrawal days. In both adult and adolescent rats, nicotine seeking in the relapse tests followed an inverted U-shaped curve, with maximal responding on withdrawal day 14. Independent of the withdrawal day, nicotine seeking in the relapse tests was higher in adult than in adolescent rats. Analysis of Fos expression in different brain areas of adolescent and adult rats on withdrawal days 1 and 14 showed time-dependent increases in the number of Fos-positive neurons in central and basolateral amygdala, orbitofrontal cortex, ventral and dorsal medial prefrontal cortex, and nucleus accumbens core and shell. In adult Fos-lacZ transgenic rats, selective inactivation of nicotine-cue-activated Fos neurons in central amygdala, but not orbitofrontal cortex, decreased "incubated" nicotine seeking on withdrawal day 14. Our results demonstrate that incubation of nicotine craving occurs after adolescent-onset nicotine self-administration and that neuronal ensembles in central amygdala play a critical role in this incubation. SIGNIFICANCE STATEMENT: The craving response to smoking-associated cues in humans or to intravenous nicotine-associated cues in adult rats progressively increases or incubates after withdrawal. It is currently unknown whether incubation of craving also occurs after adolescent-onset nicotine self-administration. The brain areas that mediate such incubation are also unknown. Here, we used a rat model of incubation of drug craving, the neuronal activity marker Fos, and the Daun02 chemogenetic inactivation method to demonstrate that incubation of nicotine craving is also observed after adolescent-onset nicotine self-administration and that neuronal ensembles in the central nucleus of the amygdala play a critical role in this incubation in adult rats.

Recruitment of a Neuronal Ensemble in the Central Nucleus of the Amygdala Is Required for Alcohol Dependence.

UNLABELLED: Abstinence from alcohol is associated with the recruitment of neurons in the central nucleus of the amygdala (CeA) in nondependent rats that binge drink alcohol and in alcohol-dependent rats. However, whether the recruitment of this neuronal ensemble in the CeA is causally related to excessive alcohol drinking or if it represents a consequence of excessive drinking remains unknown. We tested the hypothesis that the recruitment of a neuronal ensemble in the CeA during abstinence is required for excessive alcohol drinking in nondependent rats that binge drink alcohol and in alcohol-dependent rats. We found that inactivation of the CeA neuronal ensemble during abstinence significantly decreased alcohol drinking in both groups. In nondependent rats, the decrease in alcohol intake was transient and returned to normal the day after the injection. In dependent rats, inactivation of the neuronal ensemble with Daun02 produced a long-term decrease in alcohol drinking. Moreover, we observed a significant reduction of somatic withdrawal signs in dependent animals that were injected with Daun02 in the CeA. These results indicate that the recruitment of a neuronal ensemble in the CeA during abstinence from alcohol is causally related to excessive alcohol drinking in alcohol-dependent rats, whereas a similar neuronal ensemble only partially contributed to alcohol-binge-like drinking in nondependent rats. These results identify a critical neurobiological mechanism that may be required for the transition to alcohol dependence, suggesting that focusing on the neuronal ensemble in the CeA may lead to a better understanding of the etiology of alcohol use disorders and improve medication development. SIGNIFICANCE STATEMENT: Alcohol dependence recruits neurons in the central nucleus of the amygdala (CeA). Here, we found that inactivation of a specific dependence-induced neuronal ensemble in the CeA reversed excessive alcohol drinking and somatic signs of alcohol dependence in rats. These results identify a critical neurobiological mechanism that is required for alcohol dependence, suggesting that targeting dependence neuronal ensembles may lead to a better understanding of the etiology of alcohol use disorders, with implications for diagnosis, prevention, and treatment.

Neural correlates of food anticipatory activity in mice subjected to once- or twice-daily feeding periods.

In rodents, restricted food access to a limited period each day at a predictable time results in the appearance of food anticipatory activity (FAA). Two shorter periods of food access each day can result in two FAA bouts. In this study, we examine FAA under 12:12 and 18:6 photoperiods in mice (Mus musculus) with one or two food access periods per day and measure the activation of the suprachiasmatic, dorsomedial and arcuate nuclei by assaying Fos protein expression, while making use of tissue-type plasminogen activator knockout mice to assess the role of neural plasticity in adaptation to restricted feeding cycles. Long days were utilised to allow for temporal separation of two restricted feeding periods during the light phase. Mice fed twice per day generally divided FAA into two distinct bouts, with mice lacking tissue-type plasminogen activator showing reduced FAA. Increases in Fos expression in response to one restricted feeding period per day were seen in the dorsomedial and arcuate nuclei in both 12:12 and 18:6 conditions, with an increase seen in the SCN in only the 12:12 condition. These increases were eliminated or reduced in the two feeding time conditions (done in 18:6 only). Both activity patterns and Fos expression differed for single restricted feeding times between 18:6 and 12:12 photoperiods. Fos activation was lower during RF in 18:6 than 12:12 across all three brain regions, a pattern not reflective of changes in FAA. These data suggest that involvement of these regions in FAA may be influenced by photoperiodic context.

Chemogenetic activation of the lateral hypothalamus reverses early life stress-induced deficits in motivational drive.

Altered motivated behaviour is a cardinal feature of several neuropsychiatric conditions including mood disorders. One well-characterized antecedent to the development of mood disorders is exposure to early life stress (ELS). A key brain substrate controlling motivated behaviour is the lateral hypothalamus (LH). Here, we examined the effect of ELS on LH activation and the motivation to self-administer sucrose. We tested whether chemogenetic activation of LH circuits could modify sucrose responding in ELS rats and examined the impact on LH cell populations. Male rat pups were maternally separated for 0 or 3 h on postnatal days 2-14. During adolescence, rats received bilateral injections of hM3D(Gq), the excitatory designer receptor exclusively activated by designer drugs, into LH. In adulthood, rats were trained to self-administer sucrose and tested under a progressive ratio schedule to determine their motivation for reward following injection with either vehicle or 5 mg/kg clozapine-N-oxide. Brains were processed for Fos-protein immunohistochemistry. ELS significantly suppressed lever responding for sucrose, indicating a long-lasting impact of ELS on motivation circuits. hM3D(Gq) activation of LH increased responding, normalizing deficits in ELS rats, and increased Fos-positive orexin and MCH cell numbers within LH. Our findings indicate that despite being susceptible to environmental stressors, LH circuits retain the capacity to overcome ELS-induced deficits in motivated behaviour.

Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.

Context-induced reinstatement of drug seeking is a well established animal model for assessing the neural mechanisms underlying context-induced drug relapse, a major factor in human drug addiction. Neural activity in striatum has previously been shown to contribute to context-induced reinstatement of heroin, cocaine, and alcohol seeking, but not yet for methamphetamine seeking. In this study, we found that context-induced reinstatement of methamphetamine seeking increased expression of the neural activity marker Fos in dorsal but not ventral striatum. Reversible inactivation of neural activity in dorsolateral but not dorsomedial striatum using the GABA agonists muscimol and baclofen decreased context-induced reinstatement. Based on our previous findings that Fos-expressing neurons play a critical role in conditioned drug effects, we assessed whether context-induced reinstatement was associated with molecular alterations selectively induced within context-activated Fos-expressing neurons. We used fluorescence-activated cell sorting to isolate reinstatement-activated Fos-positive neurons from Fos-negative neurons in dorsal striatum and used quantitative PCR to assess gene expression within these two populations of neurons. Context-induced reinstatement was associated with increased expression of the immediate early genes Fos and FosB and the NMDA receptor subunit gene Grin2a in only Fos-positive neurons. RNAscope in situ hybridization confirmed that Grin2a, as well as Grin2b, expression were increased in only Fos-positive neurons from dorsolateral, but not dorsomedial, striatum. Our results demonstrate an important role of dorsolateral striatum in context-induced reinstatement of methamphetamine seeking and that this reinstatement is associated with unique gene alterations in Fos-expressing neurons.

Inhibitory Interneurons That Express GFP in the PrP-GFP Mouse Spinal Cord Are Morphologically Heterogeneous, Innervated by Several Classes of Primary Afferent and Include Lamina I Projection Neurons among Their Postsynaptic Targets.

The superficial dorsal horn of the spinal cord contains numerous inhibitory interneurons, which regulate the transmission of information perceived as touch, pain, or itch. Despite the importance of these cells, our understanding of their roles in the neuronal circuitry is limited by the difficulty in identifying functional populations. One group that has been identified and characterized consists of cells in the mouse that express green fluorescent protein (GFP) under control of the prion protein (PrP) promoter. Previous reports suggested that PrP-GFP cells belonged to a single morphological class (central cells), received inputs exclusively from unmyelinated primary afferents, and had axons that remained in lamina II. However, we recently reported that the PrP-GFP cells expressed neuronal nitric oxide synthase (nNOS) and/or galanin, and it has been shown that nNOS-expressing cells are more diverse in their morphology and synaptic connections. We therefore used a combined electrophysiological, pharmacological, and anatomical approach to reexamine the PrP-GFP cells. We provide evidence that they are morphologically diverse (corresponding to "unclassified" cells) and receive synaptic input from a variety of primary afferents, with convergence onto individual cells. We also show that their axons project into adjacent laminae and that they target putative projection neurons in lamina I. This indicates that the neuronal circuitry involving PrP-GFP cells is more complex than previously recognized, and suggests that they are likely to have several distinct roles in regulating the flow of somatosensory information through the dorsal horn.

Mimicking the tumour microenvironment of chronic lymphocytic leukaemia in vitro critically depends on the type of B-cell receptor stimulation.

BACKGROUND: The B-cell receptor (BCR) has a key role in the cross-talk between chronic lymphocytic leukaemia (CLL) cells and the tissue microenvironment, which favours disease progression by promoting proliferation and drug resistance. In vitro studies on downstream signalling and functional effects of CLL BCR ligation often report contradictory results, in part owing to the lack of a standardised stimulation protocol. Our aim was to define a biologically relevant and robust in vitro stimulation method with regard to cellular phenotypic and transcriptional responses. METHODS: We evaluated mRNA (FOS, MYC, LPL) and protein (CD54, CD19, CD62L, CD184) expression of genes modulated by BCR triggering in immunoglobulin heavy-chain variable region genes (IGHV)-mutated and -unmutated CLL cells, after stimulation using soluble or immobilised anti-IgM antibodies from different suppliers. RESULTS: The effect of BCR stimulation on gene and protein expression was comparable in all CLL patients, irrespective of IGHV mutation status. However, immobilised anti-IgM stimulation elicited clear and robust changes in gene and protein expression, whereas the response to soluble anti-IgM was far less obvious. CONCLUSIONS: These data indicate that the method of BCR stimulation is of major importance regarding responsiveness of CLL cells in the context of the tumour microenvironment, whereas genetic differences in the BCR pathway are less critical.

Crk and CrkL are required for cell transformation by v-fos and v-ras.

Crk and CrkL are SH2- and SH3-containing cytosolic adaptor proteins that can induce anchorage-independent growth of fibroblasts. Crk and CrkL play key roles in maintaining cytoskeletal integrity, cell motility and migration. We investigated the role of these two proteins in oncogenic transformation induced by v-fos and v-ras oncogenes using cell lines and fibroblasts carrying conditional alleles of Crk or CrkL. Transformation was assessed by cell morphology, saturation density and anchorage-independent growth in soft agar. We found that cell lines expressing v-fos or v-ras in the absence of Crk or CrkL displayed no evident morphological alterations and reduced anchorage-independent growth compared to those retaining Crk and CrkL. Similarly, overexpression of v-fos in mouse embryonic fibroblasts conferred a growth advantage and induced morphological changes, both of which were abrogated in the absence of either Crk or CrkL. In contrast, Crk, but not CrkL, contributed to v-ras-induced transformation of embryonic fibroblasts. These results suggest that both Crk and CrkL are required for the acquisition of cellular transformation by v-fos, whereas Crk plays a more prominent role than CrkL in v-ras-induced transformation.

Ncor2 is required for hematopoietic stem cell emergence by inhibiting Fos signaling in zebrafish.

Nuclear receptor corepressors (Ncors) are important for developmental and homeostatic processes in vertebrates, which exert transcriptional repression by coordinating with histone deacetylases. However, little is known about their roles in definitive hematopoiesis. In this study, we show that in zebrafish, ncor2 is required for hematopoietic stem cell (HSC) development by repressing fos-vegfd signaling. ncor2 is specifically expressed in the aorta-gonad-mesonephros (AGM) region in zebrafish embryos. ncor2 deficiency reduced the population of HSCs in both the AGM region and T cells in the thymus. Mechanistically, ncor2 knockdown upregulated fos transcription by modulating the acetylation level in the fos promoter region, which then enhanced Vegfd signaling. Consequently, the augmented Vegfd signaling induced Notch signaling to promote the arterial endothelial fate, therefore, possibly repressing the hemogenic endothelial specification, which is a prerequisite for HSC emergence. Thus, our findings identify a novel regulatory mechanism for Ncor2 through Fos-Vegfd-Notch signaling cascade during HSC development in zebrafish embryos.

Fusion events lead to truncation of FOS in epithelioid hemangioma of bone.

Epithelioid hemangioma of bone is a locally aggressive vascular neoplasm. It can be challenging to diagnose because of the wide histological spectrum, which can make it difficult to differentiate from other vascular neoplasms such as epithelioid hemangioendothelioma or epithelioid angiosarcoma. COBRA-FISH karyotyping identified a balanced t(3;14) translocation. Transcriptome sequencing of the index case and two other epithelioid hemangiomas revealed a recurrent translocation breakpoint involving the FOS gene, which was fused to different partners in all three cases. The break was observed in exon 4 of the FOS gene and the fusion event led to the introduction of a stop codon. In all instances, the truncation of the FOS gene would result in the loss of the transactivation domain (TAD). Using FISH probes we found a break in the FOS gene in two additional cases, in none of these cases a recurrent fusion partner could be identified. In total, FOS was split in 5/7 evaluable samples. We did not observe point mutations leading to early stop codons in any of the 10 cases where RNA was available. Detection of FOS rearrangement may be a useful diagnostic tool to assist in the often difficult differential diagnosis of vascular tumors of bone. Our data suggest that the translocation causes truncation of the FOS protein, with loss of the TAD, which is thereby a novel mechanism involved in tumorigenesis.

Control over a stressor involves the posterior dorsal striatum and the act/outcome circuit.

Controllable/escapable tailshocks (ESs) do not produce the behavioral and neurochemical outcomes produced by equal yoked uncontrollable/inescapable tailshocks (ISs). The prelimbic cortex is known to play a key role in mediating the protective effects of control. The concepts of act/outcome learning and control seem similar, and act/outcome learning is mediated by a circuit involving the prelimbic cortex and posterior dorsomedial striatum (DMS). Thus, we tested the involvement of the DMS in the protective effect of ES, in rats. First, we examined Fos immunoreactivity in both the DMS and dorsolateral striatum (DLS) after ES and yoked IS. We then investigated the effect of blocking DMS or DLS N-methyl-d-aspartate receptors with the specific antagonist D-(-)-2-amino-5-phosphopentanoic acid (D-AP5) on the release of dorsal raphe nucleus serotonin (5-HT) during ES, as well as on the level of anxiety produced by the ES experience 24 h later. ES, but not yoked IS, produced a large increase of Fos activity in the DMS. Consistent with the Fos data, D-AP5 in the DMS, but not in the DLS, prevented the inhibition of dorsal raphe nucleus 5-HT release normally produced by ES. Furthermore, D-AP5 administered into the DMS before ES, but not into the DLS, increased anxiety 24 h later, leading to levels similar to those produced by IS. These results suggest that, as with appetitive act/outcome contingency learning, the protective effects of behavioral control over a stressor require the DMS.

Role of lateral hypothalamus in two aspects of attention in associative learning.

Orexin (hypocretin) and melanin-concentrating hormone (MCH) neurons are unique to the lateral hypothalamic (LH) region, but project throughout the brain. These cell groups have been implicated in a variety of functions, including reward learning, responses to stimulants, and the modulation of attention, arousal and the sleep/wakefulness cycle. Here, we examined roles for LH in two aspects of attention in associative learning shown previously to depend on intact function in major targets of orexin and MCH neurons. In experiments 1 and 2, unilateral orexin-saporin lesions of LH impaired the acquisition of conditioned orienting responses (ORs) and bilaterally suppressed FOS expression in the amygdala central nucleus (CeA) normally observed in response to food cues that provoke conditioned ORs. Those cues also induced greater FOS expression than control cues in LH orexin neurons, but not in MCH neurons. In experiment 3, unilateral orexin-saporin lesions of LH eliminated the cue associability enhancements normally produced by the surprising omission of an expected event. The magnitude of that impairment was positively correlated with the amount of LH damage and with the loss of orexin neurons in particular, but not with the loss of MCH neurons. We suggest that the effects of the LH orexin-saporin lesions were mediated by their effect on information processing in the CeA, known to be critical to both behavioral phenomena examined here. The results imply close relations between LH motivational amplification functions and attention, and may inform our understanding of disorders in which motivational and attentional impairments co-occur.

Notch2 regulates the development of marginal zone B cells through Fos.

B cells are classified into several subsets depending on their functions, marker expression pattern and localization. Marginal zone B (MZB) cells are a distinct lineage from follicular B cells, and regulate host defenses against blood-borne pathogens. Notch2/RBP-J signaling regulates the development of MZB cells by interacting with delta-like 1 ligand, although the target genes for Notch2 signaling remain unclear. We identified Fos as an upregulated gene in LPS-stimulated B cells that received Notch2 signaling. Fos is expressed in CD21(high)CD23(low) MZB cells at a higher level compared to CD21(Int)CD23(high) follicular B cells. Deleting the Notch2 gene in CD19(+) B cells decreased Fos expression in B cells. Overexpression of Fos in Notch2-deficient B cells or bone marrow cells partially restored MZB development. Fos promoter activity was upregulated by Notch2 signaling, indicating that Notch2 directly controls Fos transcription associated with MZB development. These data identify Fos as one of the target genes for Notch2 signaling that is crucial for MZB development.

Chronic exposure of gestation rat to sevoflurane impairs offspring brain development.

Recently it was demonstrated that the exposure of the developing brain during the period of synaptogenesis to drugs that block NMDA glutamate receptors can trigger widespread apoptotic neurodegeneration. Sevoflurane is a new inhalation anesthetic agent commonly used in the clinic. Here we address whether sevoflurane could induce neurotoxicity in the developing brain. Sevoflurane was administered to rats before pregnancy and pregnant rats on embryonic days E6, E10, E14, and E18 1MAC for 6 h, and we employed histopathological, immunochemistry, semiquantitative RT-PCR, and Western blot to investigate the effect of the exposure of pregestation and gestation rats to sevoflurane on the offspring brain development. The results showed that the exposure of gestation but not pregestation rats to sevoflurane-induced extensive apoptotic neurodegeneration in the hippocampus of offspring at P0, P7, and P14, accompanied by altered expression of casepase-3, GAP-43, nNOS, NMDAR1, NMDAR2A, and NMDAR2B. Furthermore, upregulation of PKCα and p-JNK and downregulation of p-ERK and FOS protein levels were observed in the hippocampus of offspring at P0, P7, and P14 from rats exposed to sevoflurane at gestation, but not pregestation. In summary, our data suggest that sevoflurane induces developmental neurotoxicity in rats and this may be attributed to the upregulation of PKCα and p-JNK and downregulation of p-ERK and FOS protein in the hippocampus.

Segregation of acute leptin and insulin effects in distinct populations of arcuate proopiomelanocortin neurons.

Acute leptin administration results in a depolarization and concomitant increase in the firing rate of a subpopulation of arcuate proopiomelanocortin (POMC) cells. This rapid activation of POMC cells has been implicated as a cellular correlate of leptin effects on energy balance. In contrast to leptin, insulin inhibits the activity of some POMC neurons. Several studies have described a "cross talk" between leptin and insulin within the mediobasal hypothalamus via the intracellular enzyme, phosphoinositol-3-kinase (PI3K). Interestingly, both insulin and leptin regulate POMC cellular activity by activation of PI3K; however, it is unclear whether leptin and insulin effects are observed in similar or distinct populations of POMC cells. We therefore used dual label immunohistochemistry/in situ hybridization and whole-cell patch-clamp electrophysiology to map insulin and leptin responsive arcuate POMC neurons. Leptin-induced Fos activity within arcuate POMC neurons was localized separate from POMC neurons that express insulin receptor. Moreover, acute responses to leptin and insulin were largely segregated in distinct subpopulations of POMC cells. Collectively, these data suggest that cross talk between leptin and insulin occurs within a network of cells rather than within individual POMC neurons.