Question: Hypocretin neurones induce panicprone state in rats.

Title: A KEY ROLE FOR OREXIN IN PANIC ANXIETY Content: Panic disorder is a severe anxiety disorder with recurrent, debilitating panic attacks. In individuals with panic disorder there is evidence of decreased central gamma-aminobutyric acid (GABA) activity as well as marked increases in autonomic and respiratory responses after intravenous infusions of hypertonic sodium lactate. In a rat model of panic disorder, chronic inhibition of GABA synthesis in the dorsomedial-perifornical hypothalamus of rats produces anxiety-like states and a similar vulnerability to sodium lactate-induced cardioexcitatory responses. The dorsomedial-perifornical hypothalamus is enriched in neurons containing orexin (ORX, also known as hypocretin), which have a crucial role in arousal, vigilance and central autonomic mobilization, all of which are key components of panic. Here we show that activation of ORX-synthesizing neurons is necessary for developing a panic-prone state in the rat panic model, and either silencing of the hypothalamic gene encoding ORX (Hcrt) with RNAi or systemic ORX-1 receptor antagonists blocks the panic responses. Moreover, we show that human subjects with panic anxiety have elevated levels of ORX in the cerebrospinal fluid compared to subjects without panic anxiety. Taken together, our results suggest that the ORX system may be involved in the pathophysiology of panic anxiety and that ORX antagonists constitute a potential new treatment strategy for panic disorder.

Title: Chronic hypoxia induces modification of the N-methyl-D-aspartate receptor in rat brain. Content: This study examined [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from cerebral cortex, hippocampus and corpus striatum of 3 week old rats exposed to 10 weeks of intermittent hypobaric hypoxia (4300 m; 450 Torr) and compared results with those of normoxic controls. The cortex, hippocampus and striatum of hypoxic animals had a 36, 35 and 31% reduction in binding sites (Bmax) and a 29, 32 and 17% decrease (reflecting increased affinity) in the dissociation constant (Kd) when compared to controls. In the cerebral cortex, both glutamate (100 microM) and glycine (10 microM) enhanced 3[H]MK-801 binding by two to 3-fold. Coagonist glutamate, however, had a higher EC50 (0.44 microM) in the hypoxic cortical membranes when compared to controls (0.28 microM). No significant differences were found in the EC50 of glycine. The results show that the NMDA receptor is altered in several brain regions of rats developing in a hypoxic environment.

Title: Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes Content: Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression.

Title: Potentiation of NMDA currents by pituitary adenylate cyclase activating polypeptide in neonatal rat sympathetic preganglionic neurons. Content: Whole cell patch-clamp recordings were made from sympathetic preganglionic neurons (SPNs) in the intermediolateral cell column of thoracolumbar spinal cord slices of 12- to 16-day-old rats, and the effects of pituitary adenylate cyclase activating polypeptide (PACAP)-38 on N-methyl-D-aspartate (NMDA)- and kainate (KA)-induced inward currents were examined. PACAP, in concentrations (10-30 nM) that caused no significant change of holding currents, reversibly increased NMDA-induced currents but not KA-induced currents. At higher concentrations (>30 nM), the peptide produced a sustained inward current. The potentiating effect of PACAP was nullified by prior incubation of the slices with the adenylate cyclase inhibitor MDL-12,330A (25 microM). Further, superfusing the slices with the membrane-permeable cyclic AMP analogue N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (100-300 microM) in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (700 microM) increased the NMDA currents. This result suggests that PACAP selectively increases NMDA-receptor-mediated responses in the rat SPNs, probably via a cyclic-AMP-dependent mechanism, providing evidence that the peptide may be involved in synaptic plasticity.

Title: Role of 5-HT in the regulation of the brain-pituitary-adrenal axis: effects of 5-HT on adrenocortical cells. Content: Serotonin (5-HT) plays a pivotal role in the regulation of the brain-pituitary-adrenal axis. In particular, 5-HT has been shown to control the activity of hypothalamic CRF neurons and pituitary corticotrope cells through activation of 5-HT1A and (or) 5-HT(2A/2C) receptor subtypes. 5-HT, acting through 5-HT2 receptors, can also trigger the renin-angiotensin system by stimulating renin secretion and consequently can enhance aldosterone production. At the adrenal level, 5-HT produced locally stimulates the secretory activity of adrenocortical cells through a paracrine mode of communication. The presence of 5-HT in the adrenal gland has been demonstrated immunohistochemically and biochemically in various species. In the frog, rat, and pig adrenal gland, 5-HT is synthesized by chromaffin cells, while in the mouse adrenal cortex, 5-HT is contained in nerve fibers. In man, 5-HT is present in perivascular mast cells. In vivo and in vitro studies have shown that 5-HT stimulates corticosteroid secretion in various species (including human). The type of receptor involved in the mechanism of action of 5-HT differs between the various species. In frogs and humans, the stimulatory effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype positively coupled to adenylyl cyclase and calcium influx. In the rat, the effect of 5-HT on aldosterone secretion is mediated via activation of 5-HT7 receptors. Clinical studies indicate that 5-HT4 receptor agonists stimulate aldosterone secretion in healthy volunteers and in patients with corticotropic insufficiency and primary hyperaldosteronism. Local serotonergic control of corticosteroid production may be involved in the physiological control of the activity of the adrenal cortex as well as in the pathophysiology of cortisol and aldosterone disorders.

Title: In vivo effect of chronic hypoxia on the neurochemical profile of the developing rat hippocampus. Content: The cognitive deficits observed in children with cyanotic congenital heart disease suggest involvement of the developing hippocampus. Chronic postnatal hypoxia present during infancy in these children may play a role in these impairments. To understand the biochemical mechanisms of hippocampal injury in chronic hypoxia, a neurochemical profile consisting of 15 metabolite concentrations and 2 metabolite ratios in the hippocampus was evaluated in a rat model of chronic postnatal hypoxia using in vivo 1H NMR spectroscopy at 9.4 T. Chronic hypoxia was induced by continuously exposing rats (n = 23) to 10% O2 from postnatal day (P) 3 to P28. Fifteen metabolites were quantified from a volume of 9-11 microl centered on the left hippocampus on P14, P21, and P28 and were compared with normoxic controls (n = 14). The developmental trajectory of neurochemicals in chronic hypoxia was similar to that seen in normoxia. However, chronic hypoxia had an effect on the concentrations of the following neurochemicals: aspartate, creatine, phosphocreatine, GABA, glutamate, glutamine, glutathione, myoinositol, N-acetylaspartate (NAA), phosphorylethanolamine, and phosphocreatine/creatine (PCr/Cr) and glutamate/glutamine (Glu/Gln) ratios (P < 0.001 each, except glutamate, P = 0.04). The increased PCr/Cr ratio is consistent with decreased brain energy consumption. Given the well-established link between excitatory neurotransmission and brain energy metabolism, we postulate that elevated glutamate, Glu/Gln ratio, and GABA indicate suppressed excitatory neurotransmission in an energy-limited environment. Decreased NAA and phosphorylethanolamine suggest reduced neuronal integrity and phospholipid metabolism. The altered hippocampal neurochemistry during its development may underlie some of the cognitive deficits present in human infants at risk of chronic hypoxia.

Title: P0 glycoprotein peptides 56–71 and 180–199 dose-dependently induce acute and chronic experimental autoimmune neuritis in Lewis rats associated with epitope spreading Content: Two synthetic peripheral nerve myelin P0 protein peptides, an immunodominant (amino acids 180-199) and a cryptic (amino acids 56-71) one, induced an acute or chronic course of experimental autoimmune neuritis (EAN) in Lewis rats, when given at low dose (50-100 microg/rat) or high dose (250 microg/rat), respectively. Corresponding to the different clinical course, pathological changes and immune responses were found: (1) Onset of clinical signs of P0 peptide 56-71 (P0 56-71) induced EAN was 1-3 days later than in P0 peptide 180-199 (P0 180-199) induced EAN at all immunizing doses, whereas the peak of the disease occurred at a similar time point post immunization (p.i.), i.e. at days 14-16 p.i. in P0 56-71 induced EAN and at day 16 p.i. in P0 180-199 induced EAN. (2) Intramolecular epitope spreading as assessed by delayed type hypersensitivity response occurred in P0 56-71 induced EAN at both low and high antigen doses and in P0 180-199 induced EAN at high antigen dose (250 microg/rat) only. (3) P0 180-199 stimulated higher levels of interferon-gamma production in P0 180-199 induced EAN than in P0 56-71 induced EAN and vice versa. (4) Histopathologic evaluation revealed a similar grade of mononuclear cell infiltration in the sciatic nerves of both types of EAN, but more severe demyelination was found in P0 180-199 induced EAN compared to P0 56-71 induced EAN. The results support the hypothesis that high dose autoantigen immunization induces extensive determinant spreading and chronic course of autoimmune diseases.

Title: Lateral Hypothalamic Signaling Mechanisms Underlying Feeding Stimulation: Differential Contributions of Src Family Tyrosine Kinases to Feeding Triggered Either by NMDA Injection or by Food Deprivation Content: In rats, feeding can be triggered experimentally using many approaches. Included among these are (1) food deprivation and (2) acute microinjection of the neurotransmitter l-glutamate (Glu) or its receptor agonist NMDA into the lateral hypothalamic area (LHA). Under both paradigms, the NMDA receptor (NMDA-R) within the LHA appears critically involved in transferring signals encoded by Glu to stimulate feeding. However, the intracellular mechanisms underlying this signal transfer are unknown. Because protein-tyrosine kinases (PTKs) participate in NMDA-R signaling mechanisms, we determined PTK involvement in LHA mechanisms underlying both types of feeding stimulation through food intake and biochemical measurements. LHA injections of PTK inhibitors significantly suppressed feeding elicited by LHA NMDA injection (up to 69%) but only mildly suppressed deprivation feeding (24%), suggesting that PTKs may be less critical for signals underlying this feeding behavior. Conversely, food deprivation but not NMDA injection produced marked increases in apparent activity for Src PTKs and in the expression of Pyk2, an Src-activating PTK. When considered together, the behavioral and biochemical results demonstrate that, although it is easier to suppress NMDA-elicited feeding by PTK inhibitors, food deprivation readily drives PTK activity in vivo. The latter result may reflect greater PTK recruitment by neurotransmitter receptors, distinct from the NMDA-R, that are activated during deprivation-elicited but not NMDA-elicited feeding. These results also demonstrate how the use of only one feeding stimulation paradigm may fail to reveal the true contributions of signaling molecules to pathways underlying feeding behavior in vivo.

Title: Spare respiratory capacity rather than oxidative stress regulates glutamate excitotoxicity after partial respiratory inhibition of mitochondrial complex I with rotenone. Content: Partial inhibition of mitochondrial respiratory complex I by rotenone reproduces aspects of Parkinson's disease in rodents. The hypothesis that rotenone enhancement of neuronal cell death is attributable to oxidative stress was tested in an acute glutamate excitotoxicity model using primary cultures of rat cerebellar granule neurons. As little as 5 nM rotenone increased mitochondrial superoxide (O2*-) levels and potentiated glutamate-induced cytoplasmic Ca2+ deregulation, the first irreversible stage of necrotic cell death. However, the potent cell-permeant O2*- trap manganese tetrakis (N-ethylpyridinium-2yl) porphyrin failed to prevent the effects of the inhibitor. The bioenergetic consequences of rotenone addition were quantified by monitoring cell respiration. Glutamate activation of NMDA receptors used the full respiratory capacity of the in situ mitochondria, and >80% of the glutamate-stimulated respiration was attributable to increased cellular ATP demand. Rotenone at 20 nM inhibited basal and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-stimulated cell respiration and caused respiratory failure in the presence of glutamate. ATP synthase inhibition by oligomycin was also toxic in the presence of glutamate. We conclude that the cell vulnerability in the rotenone model of partial complex I deficiency under these specific conditions is primarily determined by spare respiratory capacity rather than oxidative stress.

Title: Control of glutamate clearance and synaptic efficacy by glial coverage of neurons. Content: Analysis of excitatory synaptic transmission in the rat hypothalamic supraoptic nucleus revealed that glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons. Reduction in glutamate clearance, whether induced pharmacologically or associated with a relative decrease of glial coverage in the vicinity of synapses, affected transmitter release through modulation of presynaptic metabotropic glutamate receptors. Astrocytic wrapping of neurons, therefore, contributes to the regulation of synaptic efficacy in the central nervous system.

Title: Excitotoxic brain injury in adult zebrafish stimulates neurogenesis and long-distance neuronal integration. Content: Zebrafish maintain a greater capacity than mammals for central nervous system repair after injury. Understanding differences in regenerative responses between different vertebrate species may shed light on mechanisms to improve repair in humans. Quinolinic acid is an excitotoxin that has been used to induce brain injury in rodents for modeling Huntington's disease and stroke. When injected into the adult rodent striatum, this toxin stimulates subventricular zone neurogenesis and neuroblast migration to injury. However, most new neurons fail to survive and lesion repair is minimal. We used quinolinic acid to lesion the adult zebrafish telencephalon to study reparative processes. We also used conditional transgenic lineage mapping of adult radial glial stem cells to explore survival and integration of neurons generated after injury. Telencephalic lesioning with quinolinic acid, and to a lesser extent vehicle injection, produced cell death, microglial infiltration, increased cell proliferation, and enhanced neurogenesis in the injured hemisphere. Lesion repair was more complete with quinolinic acid injection than after vehicle injection. Fate mapping of her4-expressing radial glia showed injury-induced expansion of radial glial stem cells that gave rise to neurons which migrated to injury, survived at least 8 weeks and formed long-distance projections that crossed the anterior commissure and synapsed in the contralateral hemisphere. These findings suggest that quinolinic acid lesioning of the zebrafish brain stimulates adult neural stem cells to produce robust regeneration with long-distance integration of new neurons. This model should prove useful for elucidating reparative mechanisms that can be applied to restorative therapies for mammalian brain injury.

Title: brain organization Content: Transient activation of estrogen receptors (ER) in the developing brain during a limited perinatal "window of time" is recognized as a key mechanism of defeminization of neural control of reproductive function and sexual behavior. Two major ER isoforms, alpha and beta, are present in neural circuits that govern ovarian cycle and sexual behavior. Using highly selective ER agonists, this study provides the first evidence for distinct contribution of individual ER isoforms to the process of estrogen dependent defeminization. Neonatal activation of the ERalpha in female rats resulted in abrogation of cyclic ovarian activity and female sexual behavior in adulthood. These effects are associated with male-like alterations in the morphology of the anteroventral periventricular (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA), as well as refractoriness to estrogen-mediated induction of sexual receptivity. Exposure to an ERbeta-selective agonist induced persistent estrus and had a strong defeminizing effect on the hypothalamic gonadotropin "surge generator" AVPV. However, neonatal ERbeta activation failed to alter female sexual behavior, responsiveness to estrogens and morphometric features of the behaviorally relevant SDN-POA. Thus, although co-present in several brain regions involved in the control of female reproductive function, ER isoforms convey different, and probably not synergistic, chemical signals in the course of neonatal sex-specific brain organization.

Title: CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK Content: Ciliary neurotrophic factor (CNTF) induces weight loss and improves glucose tolerance in humans and rodents. CNTF is thought to act centrally by inducing hypothalamic neurogenesis to modulate food intake and peripherally by altering hepatic gene expression, in a manner similar to that of leptin. Here, we show that CNTF signals through the CNTFRα–IL-6R–gp130β receptor complex to increase fatty-acid oxidation and reduce insulin resistance in skeletal muscle by activating AMP-activated protein kinase (AMPK), independent of signaling through the brain. Thus, our findings further show that the antiobesogenic effects of CNTF in the periphery result from direct effects on skeletal muscle, and that these peripheral effects are not suppressed by diet-induced or genetic models of obesity, an essential requirement for the therapeutic treatment of obesity-related diseases.

Title: Nicotinic receptor-mediated neuroprotection in neurodegenerative disease models. Content: Multiple lines of evidence, from molecular and cellular to epidemiological, have implicated nicotinic transmission in the pathology of Alzheimer's disease (AD) and Parkinson's disease (PD). This review article presents evidence for nicotinic acetylcholine receptor (nAChR)-mediated protection and the signal transduction involved in this mechanism. The data is based mainly on our studies using rat-cultured primary neurons. Nicotine-induced protection was blocked by an alpha7 nAChR antagonist, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and an Src inhibitor. Levels of phosphorylated Akt, an effector of PI3K, Bcl-2 and Bcl-x were increased by nicotine administration. From these experimental data, our hypothesis for the mechanism of nAChR-mediated survival signal transduction is that the alpha7 nAChR stimulates the Src family, which activates PI3K to phosphorylate Akt, which subsequently transmits the signal to up-regulate Bcl-2 and Bcl-x. Up-regulation of Bcl-2 and Bcl-x could prevent cells from neuronal death induced by beta-amyloid (Abeta), glutamate and rotenone. These findings suggest that protective therapy with nAChR stimulation could delay the progress of neurodegenerative diseases such as AD and PD.

Title: The lateral hypothalamus: a primary site mediating excitatory amino acid-elicited eating. Content: Lateral hypothalamic (LH) injections of the excitatory neurotransmitter glutamate, or its excitatory amino acid (EAA) agonists, kainic acid (KA), D,L-alpha-amino-3-hydroxy-5-methyl-isoxazole propionic acid (AMPA), or N-methyl-D-aspartic acid (NMDA), can rapidly elicit an intense feeding response in satiated rats. To determine whether the LH is the actual locus of this effect, we compared these compounds' ability to stimulate feeding when injected into the LH, versus when injected into sites bracketing this region. Food intake in groups of adult male rats was measured 1 h after injection of glutamate (30-900 nmol), KA (0.1-1.0 nmol), AMPA (0.33-3.3 nmol), NMDA (0.33-33.3 nmol) or vehicle, through chronically implanted guide cannulas, into one of seven brain sites. These sites were: the LH, the anterior and posterior tips of the LH, the thalamus immediately dorsal to the LH, the amygdala just lateral to the LH, or the paraventricular and perifornical areas medial to the LH. The results show that across doses and agonists the eating-stimulatory effects were largest with injections into the LH. In the LH, glutamate between 300 and 900 nmol elicited a dose-dependent eating response of up to 5 g within 1 h (P < 0.01). Each of the other agonists at doses of 3.3 nmol or less elicited eating responses of at least 10 g with injections into this site. Injections into the other brain sites produced either no eating, or occasionally smaller and less consistent eating responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Title: Effect of interleukin-1 and tumor necrosis factor/cachectin on glucose turnover in the rat. Content: We studied the effect of recombinant human interleukin-1 beta (IL-1) and recombinant human tumor necrosis factor alpha/cachectin (TNF) on glucose kinetics in healthy rats by means of a primed constant infusion of D-(6-3H)glucose and D-[U-14C]glucose. During the isotope (6-hour) and monokine (4-hour) infusion, plasma levels of glucagon and insulin were determined and correlated with changes in glucose metabolism. The rates of glucose appearance (Ra) and disappearance (Rd) were elevated only with IL-1 and were associated with an increase in glucagon and a concomitant decrease in the ratio of insulin to glucagon. Plasma glucose concentration was increased early after IL-1 administration and coincided with the peak in the Ra. The augmentation of the metabolic clearance rate (MCR) and percent of flux oxidized by IL-1 suggest that this monokine induces the utilization of glucose as a substrate. TNF administration failed to modify the Ra or Rd, percent of flux oxidized, or MCR. TNF-treated rats increased the percent of glucose recycling, but not the total rate of glucose production. The results of this experiment suggest that endogenous macrophage products participate in the diverse alterations of carbohydrate metabolism seen during injury and/or infection.

Title: Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Content: The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

Title: Differential Effects of N-Acetylcysteine, Theophylline or Bicarbonate on Contrast-Induced Rat Renal Vasoconstriction Content: Background: Vasoconstriction and reactive oxygen species (ROS) accumulation following contrast media (CM) injection are the key factors triggering CM-induced nephropathy. We compared the effects of N-acetylcysteine (NAC), theophylline or sodium bicarbonate on intrarenal vasoconstriction and ROS generation in a rat model of CM-induced nephropathy. Methods: Following a 3-day dehydration, Sprague-Dawley rats received CM (Telebrix) or sham ‘CM’ injection of 0.9% saline. Part of them received NAC, theophylline or bicarbonate prior to CM. Medullar renal blood flow was estimated by laser Doppler. The animals were sacrificed 1, 15 or 30 min after the respective treatments, their kidneys allocated and intrarenal STAT-8 isoprostane, PGE2 and NO assessed. Results: Vasoconstriction was significantly attenuated by NAC. Theophylline only mildly attenuated the perfusion drop at 15 min, and was ineffective following 30 min. Unlike theophylline or bicarbonate, NAC significantly augmented intrarenal PGE2. NAC, theophylline but not bicarbonate, gradually increased intrarenal NO. In all experimental variables, CM-induced ROS accumulation, represented by STAT-8 isoprostane estimation, progressed undisturbed. Conclusions: (1) CM-induced intrarenal vasoconstriction was efficiently prohibited by NAC but not bicarbonate or theophylline; (2) the vasodilatory effect of NAC was mediated via increased PGE2 synthesis, and (3) ROS accumulation was a primary renal response to CM-induced injury, not affected by any pharmacologic manipulations.

Title: Ghrelin secretion stimulated by {beta}1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice. Content: Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding beta(1)-adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective beta(1)-adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on beta(1) receptors on the ghrelin-secreting cells of the stomach.

Title: Febrile seizures in the developing brain result in persistent modification of neuronal excitability in limbic circuits Content: Febrile (fever-induced) seizures affect 3–5% of infants and young children. Despite the high incidence of febrile seizures, their contribution to the development of epilepsy later in life has remained controversial. Combining a new rat model of complex febrile seizures and patch clamp techniques, we determined that hyperthermia-induced seizures in the immature rat cause a selective presynaptic increase in inhibitory synaptic transmission in the hippocampus that lasts into adulthood. The long-lasting nature of these potent alterations in synaptic communication after febrile seizures does not support the prevalent view of the 'benign' nature of early-life febrile convulsions.

Title: Arachidonic Acid in the Diabetic Rat Kidney Content: In the rat isolated perfused kidney, arachidonic acid elicits cyclooxygenase-dependent vasoconstriction through activation of PGH2/TxA2 receptors; responses are enhanced in kidneys from diabetic rats. This study examined the roles of cyclooxygenase-1/cyclooxygenase-2 in the enhanced renal vasoconstrictor effect of arachidonic acid in streptozotocin-diabetic rats. Release of 20-HETE was also determined, as this eicosanoid has been reported to elicit cyclooxygenase-dependent vasoconstriction. We confirmed that vasoconstrictor responses to arachidonic acid were enhanced in the diabetic rat kidney associated with a 2-fold-greater increase in the release of 6-ketoPGF1alpha, which was used as an index of cyclooxygenase activity. One and three micrograms of arachidonic acid increased perfusion pressure by 85+/-37 and 186+/-6 mm Hg, respectively, in diabetic rat kidneys compared with 3+/-1 and 17+/-8 mm Hg, respectively, in control rat kidneys. Inhibition of both cyclooxygenase isoforms with indomethacin (10 micromol/L) abolished the vasoconstrictor response to arachidonic acid in both diabetic and control rat kidneys, whereas inhibition of cyclooxygenase-2 with nimesulide (5 micromol/L) reduced perfusion pressure responses to 1 and 3 microg arachidonic acid only in the diabetic rat kidney to 15+/-8 and 108+/-26 mm Hg, respectively, consistent with a 3-fold increase in the renal cortical expression of cyclooxygenase-2. 20-HETE release from the diabetic rat kidney was reduced almost 6-fold and was not increased in response to arachidonic acid. These results demonstrate that the renal vasoconstrictor effect of arachidonic acid is solely dependent on cyclooxygenase activity, with no evidence for a contribution from 20-HETE; in the diabetic rat, cyclooxygenase-2 activity contributes to the renal vasoconstrictor effect of arachidonic acid.

Title: G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons Content: The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Title: Phagocytosis and deposition of vascular beta-amyloid in rat brains injected with Alzheimer beta-amyloid. Content: The presence of extracellular deposits of beta-amyloid protein in the brain is a hallmark of Alzheimer's disease (AD). In an effort to determine the effect of amyloid in an animal model, the authors injected amyloid cores isolated from AD brains into the cortex and hippocampus of rats. Lipofuscin, a major contaminant of the plaque core preparation, was injected on the contralateral side and used as a control to induce an analogous phagocytic cell response. Rats were sacrificed 2 days, 7 days, and 1 month after injection and amyloid located by four histochemical techniques. Amyloid and lipofuscin move from the site of injection into otherwise undamaged neuropil, persist for at least 1 month and are both associated with increases in glial fibrillary acidic protein and microglia (OX-42) staining. By 1 week, many of the amyloid cores are ingested by phagocytes. Some of the beta-amyloid-containing phagocytes migrate to the vessels and to the ventricles, and by 1 month, a significant amount of the amyloid is directly associated with the vessels. This suggests that phagocytic cells can internalize exogenous amyloid and attempt to clear it from the central nervous system (CNS). Therefore, the observed distribution of amyloid is not necessarily the initial site of deposition.

Title: Neuronal replacement from endogenous precursors in the adult brain after stroke Content: In the adult brain, new neurons are continuously generated in the subventricular zone and dentate gyrus, but it is unknown whether these neurons can replace those lost following damage or disease. Here we show that stroke, caused by transient middle cerebral artery occlusion in adult rats, leads to a marked increase of cell proliferation in the subventricular zone. Stroke-generated new neurons, as well as neuroblasts probably already formed before the insult, migrate into the severely damaged area of the striatum, where they express markers of developing and mature, striatal medium-sized spiny neurons. Thus, stroke induces differentiation of new neurons into the phenotype of most of the neurons destroyed by the ischemic lesion. Here we show that the adult brain has the capacity for self-repair after insults causing extensive neuronal death. If the new neurons are functional and their formation can be stimulated, a novel therapeutic strategy might be developed for stroke in humans.

Title: Intrahippocampal administration of an androgen receptor antagonist, flutamide, can increase anxiety-like behavior in intact and DHT-replaced male rats. Content: Testosterone (T) and its 5alpha-reduced metabolite, dihydrotestosterone (DHT), can decrease anxiety-like behavior; however, the mechanisms underlying these effects have not been established. First, we hypothesized that if T reduces anxiety-like behavior through actions of its 5alpha-reduced metabolite, DHT, then gonadectomy (GDX) would increase anxiety-like behavior, an effect which would be reversed by systemic administration of DHT. Second, we hypothesized that if T and DHT reduce anxiety-like behavior in part through actions at intracellular androgen receptors in the hippocampus, then administration of an androgen receptor antagonist, flutamide, directly to the hippocampus should increase anxiety-like behavior of intact and DHT-replaced, but not GDX, male rats. Inserts that were empty or contained flutamide were applied directly to the dorsal hippocampus of intact, GDX, or GDX and DHT-replaced rats 2 h prior to testing in the open field, elevated plus maze, or defensive freezing tasks. GDX rats exhibited significantly more anxiety-like behaviors than intact or DHT-replaced rats. Intact and DHT-replaced rats administered flutamide to the hippocampus showed significantly more anxiety-like behavior than did intact and DHT-replaced controls. However, flutamide alone did not increase anxiety-like behavior of GDX rats. Together, these findings suggest that androgens can decrease anxiety-like behavior of male rats in part through DHT's actions at androgen receptors in the hippocampus.

Title: Activation of Serotonin 2C Receptors in Dopamine Neurons Inhibits Binge-like Eating in Mice Content: BACKGROUND Neural networks that regulate binge eating remain to be identified, and effective treatments for binge eating are limited. METHODS We combined neuroanatomic, pharmacologic, electrophysiological, Cre-lox, and chemogenetic approaches to investigate the functions of 5-hydroxytryptamine (5-HT) 2C receptor (5-HT2CR) expressed by dopamine (DA) neurons in the regulation of binge-like eating behavior in mice. RESULTS We showed that 5-HT stimulates DA neural activity through a 5-HT2CR-mediated mechanism, and activation of this midbrain 5-HT→DA neural circuit effectively inhibits binge-like eating behavior in mice. Notably, 5-HT medications, including fluoxetine, d-fenfluramine, and lorcaserin (a selective 5-HT2CR agonist), act on 5-HT2CRs expressed by DA neurons to inhibit binge-like eating in mice. CONCLUSIONS We identified the 5-HT2CR population in DA neurons as one potential target for antibinge therapies, and provided preclinical evidence that 5-HT2CR agonists could be used to treat binge eating.

Title: Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy. Content: Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABA(A)-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice.

Title: The pathophysiology of protein-overload proteinuria. Content: Alterations in glomerular function and structure were studied in protein-overload nephrosis in the rat induced by intraperitoneal administration of bovine serum albumin (BSA). Fractional clearance (C/GFR) studies using inulin and tracer proteins of different molecular size and charge revealed in proteinuric rats 1) unchanged glomerular filtration rate and renal plasma flow; 2) a 34-fold increase in C/GFR of rat serum albumin, reaching values similar to BSA; 3) a 2-fold increase in C/GFR for anionic horse radish peroxidase (HRP), but normal values for neutral and cationic HRP, and 4) an 11- and 3-fold increase for heterologous IgG and IgM, respectively. Glomerular epithelial cells showed degenerative changes, but the distribution of anionic sites in the glomerular basement membrane was found to be unaltered, as determined by polyethyleneimine binding studies. In summary, an elevation of serum albumin concentration resulted in an increased transcapillary albumin transport. This was found to lead to degenerative changes of glomerular epithelial cells with development of large pore defects, which were completely reversible.

Title: Chronic variable stress activates hematopoietic stem cells Content: Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis. Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk between the brain and immune system includes the hypothalamic-pituitary-adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic-adrenal-medullary axis, which controls stress-induced catecholamine release in support of the fight-or-flight reflex. It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic progenitors, giving rise to higher levels of disease-promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe(-/-) mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

Title: Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes. Content: Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity.

Title: Agouti-related peptide–expressing neurons are mandatory for feeding Content: Multiple hormones controlling energy homeostasis regulate the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Nevertheless, inactivation of the genes encoding NPY and/or AgRP has no impact on food intake in mice. Here we demonstrate that induced selective ablation of AgRP-expressing neurons in adult mice results in acute reduction of feeding, demonstrating direct evidence for a critical role of these neurons in the regulation of energy homeostasis.

Title: Activation of PAR(2) receptors sensitizes primary afferents and causes leukocyte rolling and adherence in the rat knee joint. Content: BACKGROUND AND PURPOSE The PAR(2) receptors are involved in chronic arthritis by mechanisms that are as yet unclear. Here, we examined PAR(2) activation in the rat knee joint. EXPERIMENTAL APPROACH PAR(2) in rat knee joint dorsal root ganglia (DRG) cells at L3-L5, retrogradely labelled with Fluoro-gold (FG) were demonstrated immunohistochemically. Electrophysiological recordings from knee joint nerve fibres in urethane anaesthetized Wistar rats assessed the effects of stimulating joint PAR(2) with its activating peptide, 2-furoyl-LIGRLO-NH(2) (1-100 nmol·100 μL(-1) , via close intra-arterial injection). Fibre firing rate was recorded during joint rotations before and 15 min after administration of PAR(2) activating peptide or control peptide. Leukocyte kinetics in the synovial vasculature upon PAR(2) activation were followed by intravital microscopy for 60 min after perfusion of 2-furoyl-LIGRLO-NH(2) or control peptide. Roles for transient receptor potential vanilloid-1 (TRPV1) or neurokinin-1 (NK(1) ) receptors in the PAR(2) responses were assessed using the selective antagonists, SB366791 and RP67580 respectively. KEY RESULTS PAR(2) were expressed in 59 ± 5% of FG-positive DRG cells; 100 nmol 2-furoyl-LIGRLO-NH(2) increased joint fibre firing rate during normal and noxious rotation, maximal at 3 min (normal; 110 ± 43%, noxious; 90 ± 31%). 2-Furoyl-LIGRLO-NH(2) also significantly increased leukocyte rolling and adhesion over 60 min. All these effects were blocked by pre-treatment with SB366791 and RP67580 (P < 0.05 compared with 2-furoyl-LIGRLO-NH(2) alone). CONCLUSIONS AND IMPLICATIONS PAR(2) receptors play an acute inflammatory role in the knee joint via TRPV1- and NK(1) -dependent mechanisms involving both PAR(2) -mediated neuronal sensitization and leukocyte trafficking.

Title: Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia. Content: The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.

Title: Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception. Content: Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH(2) into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1beta concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH(2) evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH(2), injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1beta production are independent of this channel.

Title: E-cadherin engagement stimulates proliferation via Rac1 Content: E-cadherin has been linked to the suppression of tumor growth and the inhibition of cell proliferation in culture. We observed that progressively decreasing the seeding density of normal rat kidney-52E (NRK-52E) or MCF-10A epithelial cells from confluence, indeed, released cells from growth arrest. Unexpectedly, a further decrease in seeding density so that cells were isolated from neighboring cells decreased proliferation. Experiments using microengineered substrates showed that E-cadherin engagement stimulated the peak in proliferation at intermediate seeding densities, and that the proliferation arrest at high densities did not involve E-cadherin, but rather resulted from a crowding-dependent decrease in cell spreading against the underlying substrate. Rac1 activity, which was induced by E-cadherin engagement specifically at intermediate seeding densities, was required for the cadherin-stimulated proliferation, and the control of Rac1 activation by E-cadherin was mediated by p120-catenin. Together, these findings demonstrate a stimulatory role for E-cadherin in proliferative regulation, and identify a simple mechanism by which cell–cell contact may trigger or inhibit epithelial cell proliferation in different settings.

Title: Involvement of sympathetic nervous system and brown fat in endotoxin-induced fever in rats. Content: The object of this study was to assess the role of brown adipose tissue (BAT) and the sympathetic nervous system in the rise in heat production associated with endotoxin-induced fever. Oxygen consumption (VO2) was found to be significantly increased (28%) over a 4-h period after two doses of endotoxin (Escherichia coli lipopolysaccharide, 0.3 mg/100 g body wt) given 24 h apart. Injection of a mixed beta-adrenoceptor antagonist (propranolol) reduced VO2 by 14% in endotoxin-treated rats, whereas the selective beta 1- (atenolol) or beta 2- (ICI 118551) antagonists suppressed VO2 by 10%. These drugs did not affect VO2 in control animals. BAT thermogenic activity assessed from measurements of in vitro mitochondrial guanosine 5'-diphosphate (GDP) binding was elevated by 54% in interscapular BAT and by 171% in other BAT depots. Surgical denervation of one lobe of the interscapular depot prevented these responses. Endotoxin failed to stimulate GDP binding in rats fed protein-deficient diets. This may have been because BAT thermogenic activity was already elevated in control rats fed these diets or because endotoxin caused a marked suppression of food intake in the protein-deficient animals. The results indicate that sympathetic activation of BAT is involved in the thermogenic responses to endotoxin and that these can be modified by dietary manipulation.

Title: Decreased neurogenesis after cholinergic forebrain lesion in the adult rat. Content: Adult neurogenesis has been shown to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic system of the basal forebrain is one of the key transmitter systems for learning and memory. Because adult neurogenesis has been implicated in cognitive performance, the present work aims at defining the role of cholinergic input for adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG-saporin was infused into the lateral ventricle of adult rats to selectively lesion cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the two main regions of adult neurogenesis: the dentate gyrus and the olfactory bulb. Five weeks after lesioning, neurogenesis, defined by the number of cells colocalized for bromodeoxyuridine (BrdU) and the neuronal nuclei marker NeuN, declined significantly in the granule cell layers of the dentate gyrus and olfactory bulb. Furthermore, immunotoxic lesions to the CBF led to increased numbers of apoptotic cells specifically in the subgranular zone, the progenitor region of the dentate gyrus, and within the periglomerular layer of the olfactory bulb. We propose that the cholinergic system plays a survival-promoting role for neuronal progenitors and immature neurons within regions of adult neurogenesis, similar to effects observed previously during brain development. As a working hypothesis, neuronal loss within the CBF system leads not only to cognitive deficits but may also alter on a cellular level the functionality of the dentate gyrus, which in turn may aggravate cognitive deficits.

Title: MicroRNA-29 induces cellular senescence in aging muscle through multiple signaling pathways Content: The mechanisms underlying the development of aging-induced muscle atrophy are unclear. By microRNA array and individual qPCR analyses, we found significant up-regulation of miR-29 in muscles of aged rodents vs. results in young. With aging, p85α, IGF-1 and B-myb muscle levels were lower while the expression of certain cell arrest proteins (p53, p16 and pRB) increased. When miR-29 was expressed in muscle progenitor cells (MPC), their proliferation was impaired while SA-βgal expression increased signifying the development of senescence. Impaired MPC proliferation resulted from interactions between miR-29 and the 3'-UTR of p85a, IGF-1 and B-myb, suppressing the translation of these mediators of myoblast proliferation. In vivo, electroporation of miR-29 into muscles of young mice suppressed the proliferation and increased levels of cellular arrest proteins, recapitulating aging-induced responses in muscle. A potential stimulus of miR-29 expression is Wnt-3a since we found that exogenous Wnt-3a stimulated miR-29 expression 2.7-fold in primary cultures of MPCs. Thus, aging-induced muscle senescence results from activation of miR-29 by Wnt-3a leading to suppressed expression of several signaling proteins (p85α, IGF-1 and B-myb) that act coordinately to impair the proliferation of MPCs contributing to muscle atrophy. The increase in miR-29 provides a potential mechanism for aging-induced sarcopenia.

Title: Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species. Content: Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

Title: Regulation of autophagy by cytoplasmic p53 Content: Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53−/− cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Title: The atypical antipsychotic olanzapine causes weight gain by targeting serotonin receptor 2C. Content: Atypical antipsychotics such as olanzapine often induce excessive weight gain and type 2 diabetes. However, the mechanisms underlying these drug-induced metabolic perturbations remain poorly understood. Here, we used an experimental model that reproduces olanzapine-induced hyperphagia and obesity in female C57BL/6 mice. We found that olanzapine treatment acutely increased food intake, impaired glucose tolerance, and altered physical activity and energy expenditure in mice. Furthermore, olanzapine-induced hyperphagia and weight gain were blunted in mice lacking the serotonin 2C receptor (HTR2C). Finally, we showed that treatment with the HTR2C-specific agonist lorcaserin suppressed olanzapine-induced hyperphagia and weight gain. Lorcaserin treatment also improved glucose tolerance in olanzapine-fed mice. Collectively, our studies suggest that olanzapine exerts some of its untoward metabolic effects via antagonism of HTR2C.

Title: Lateral Hypothalamic Area Glutamatergic Neurons and Their Projections to the Lateral Habenula Regulate Feeding and Reward. Content: UNLABELLED The overconsumption of calorically dense, highly palatable foods is thought to be a major contributor to the worldwide obesity epidemic; however, the precise neural circuits that directly regulate hedonic feeding remain elusive. Here, we show that lateral hypothalamic area (LHA) glutamatergic neurons, and their projections to the lateral habenula (LHb), negatively regulate the consumption of palatable food. Genetic ablation of LHA glutamatergic neurons increased daily caloric intake and produced weight gain in mice that had access to a high-fat diet, while not altering general locomotor activity. Anterior LHA glutamatergic neurons send a functional glutamatergic projection to the LHb, a brain region involved in processing aversive stimuli and negative reward prediction outcomes. Pathway-specific, optogenetic stimulation of glutamatergic LHA-LHb circuit resulted in detectable glutamate-mediated EPSCs as well as GABA-mediated IPSCs, although the net effect of neurotransmitter release was to increase the firing of most LHb neurons. In vivo optogenetic inhibition of LHA-LHb glutamatergic fibers produced a real-time place preference, whereas optogenetic stimulation of LHA-LHb glutamatergic fibers had the opposite effect. Furthermore, optogenetic inhibition of LHA-LHb glutamatergic fibers acutely increased the consumption of a palatable liquid caloric reward. Collectively, these results demonstrate that LHA glutamatergic neurons are well situated to bidirectionally regulate feeding and potentially other behavioral states via their functional circuit connectivity with the LHb and potentially other brain regions. SIGNIFICANCE STATEMENT In this study, we show that the genetic ablation of LHA glutamatergic neurons enhances caloric intake. Some of these LHA glutamatergic neurons project to the lateral habenula, a brain area important for generating behavioral avoidance. Optogenetic stimulation of this circuit has net excitatory effects on postsynaptic LHb neurons. This is the first study to characterize the functional connectivity and behavioral relevance of this circuit within the context of feeding and reward-related behavior.

Title: Interleukin-33 prevents apoptosis and improves survival after experimental myocardial infarction through ST2 signaling. Content: BACKGROUND ST2 is an interleukin (IL)-1 receptor family member with membrane-bound (ST2L) and soluble (sST2) isoforms, and sST2 is a biomarker for poor outcome in patients with myocardial infarction (MI). IL-33, the recently discovered ligand for ST2, activates nuclear factor kappaB and thus may regulate apoptotic cell death. We tested the hypothesis that IL-33 is cardioprotective after MI through ST2 signaling. METHODS AND RESULTS IL-33 protected cultured cardiomyocytes from hypoxia-induced apoptosis, and this cardioprotection was partially inhibited by sST2. IL-33 induced expression of the antiapoptotic factors XIAP, cIAP1, and survivin. To define the cardioprotective role of IL-33 in vivo, we performed a blinded and randomized study of ischemia/reperfusion in rats. IL-33 reduced cardiomyocyte apoptosis, suppressed caspase-3 activity, and increased expression of IAP family member proteins. IL-33 decreased both infarct and fibrosis volumes at 15 days; furthermore, both echocardiographic and hemodynamic studies revealed that IL-33 improved ventricular function. To determine whether cardioprotection by IL-33 is mediated through ST2 signaling, a randomized and blinded study of ST2(-/-) versus wild-type littermate mice was performed in 98 mice subjected to MI. At 4 weeks after MI, IL-33 reduced ventricular dilation and improved contractile function in wild-type mice but not in ST2(-/-) mice. Finally, IL-33 improved survival after MI in wild-type but not in ST2(-/-) mice. CONCLUSIONS IL-33 prevents cardiomyocyte apoptosis and improves cardiac function and survival after MI through ST2 signaling.

Title: Chronic Exercise Increases Both Inducible and Endothelial Nitric Oxide Synthase Gene Expression in Endothelial Cells of Rat Aorta Content: Chronic exercise upregulates endothelial nitric oxide synthase (eNOS) gene expression. Whether the expression of inducible nitric oxide synthase (iNOS) is affected by exercise is unknown. We therefore investigated the effects of chronic exercise on iNOS and eNOS expression in isolated rat aortic endothelial and smooth muscle cells separately. Five-week-old male Wistar rats were randomly divided into control and exercise groups. After 10 weeks of running training, animals were sacrificed under ether anesthesia. The standard curve quantitative competitive reverse transcriptase-polymerase chain reaction method was used to quantify NOS mRNA expression in isolated endothelial/smooth muscle cells. To evaluate the functional role of iNOS, we examined phenylephrine-induced vascular responses with or without pretreatment with aminoguanidine. We found that (1) expression of iNOS and eNOS mRNA in endothelial cells was increased by chronic exercise and (2) chronic exercise blunted phenylephrine-induced vascular responses, probably by increasing NO release via iNOS. Our results show that chronic exercise increases both iNOS and eNOS gene expression in endothelium. These alterations may be partially responsible for the change in vascular response after exercise.

Title: TGF-beta inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Content: Transforming growth factor-beta (TGF-beta) inhibits the growth of endothelial cells derived from various sources, including human umbilical vein, bovine aorta, and rat heart. Long-term exposure of rat heart endothelial cells to TGF-beta also induces dramatic changes in morphology that are characteristic of senescent cells. These changes are accompanied by a decrease in the number of high-affinity receptors for epidermal growth factor (EGF), with almost no change in total receptor number. Additionally, the EGF-induced expression of specific competence genes (c-myc, JE, KC) is decreased, whereas the induction of c-fos gene expression by EGF is unaltered by TGF-beta treatment. These data suggest that growth inhibitors such as TGF-beta may act by altering the cell's response to growth-stimulatory factors.

Title: A role for interleukin-1β in determining the lineage fate of embryonic rat hippocampal neural precursor cells. Content: Neurogenesis occurs in the hippocampus of the developing and adult brain due to the presence of multipotent stem cells and restricted precursor cells at different stages of differentiation. It has been proposed that they may be of potential benefit for use in cell transplantation approaches for neurodegenerative disorders and trauma. Prolonged release of interleukin-1β (IL-1β) from activated microglia has a deleterious effect on hippocampal neurons and is implicated in the impaired neurogenesis and cognitive dysfunction associated with aging, Alzheimer's disease and depression. This study assessed the effect of IL-1β on the proliferation and differentiation of embryonic rat hippocampal NPCs in vitro. We show that IL-1R1 is expressed on proliferating NPCs and that IL-1β treatment decreases cell proliferation and neurosphere growth. When NPCs were differentiated in the presence of IL-1β, a significant reduction in the percentages of newly-born neurons and post-mitotic neurons and a significant increase in the percentage of astrocytes was observed in these cultures. These effects were attenuated by IL-1 receptor antagonist. These data reveal that IL-1β exerts an anti-proliferative, anti-neurogenic and pro-gliogenic effect on embryonic hippocampal NPCs, which is mediated by IL-1R1. The present results emphasise the consequences of an inflammatory environment during NPC development, and indicate that strategies to inhibit IL-1β signalling may be necessary to facilitate effective cell transplantation approaches or in conditions where endogenous hippocampal neurogenesis is impaired.

Title: A temporal shift in the circuits mediating retrieval of fear memory Content: Fear memories allow animals to avoid danger, thereby increasing their chances of survival. Fear memories can be retrieved long after learning, but little is known about how retrieval circuits change with time. Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning. Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval. Accordingly, the conditioned tone responses of PVT neurons increased with time after training. The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval, sends dense projections to the PVT. Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points. In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits. Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear. Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalar networks for retrieval and maintenance of long-term fear memories.

Title: Topographic mapping of VMH → arcuate nucleus microcircuits and their reorganization by fasting Content: In the hypothalamic arcuate nucleus (ARC), pro-opiomelanocortin (POMC) neurons inhibit feeding and neuropeptide-Y (NPY) neurons stimulate feeding. We tested whether neurons in the ventromedial hypothalamic nucleus (VMH), a known satiety center, activate anorexigenic neuronal pathways in the ARC by projecting either excitatory synaptic inputs to POMC neurons and/or inhibitory inputs to NPY neurons. Using laser scanning photostimulation in brain slices from transgenic mice, we found that POMC and NPY neurons, which are interspersed in the ARC, are nevertheless regulated by anatomically distinct synaptic inputs. POMC neurons received strong excitatory input from the medial VMH (mVMH), whereas NPY neurons did not and, instead, received weak inhibitory input only from within the ARC. The strength of the excitatory input from the mVMH to POMC neurons was diminished by fasting. These data identify a new molecularly defined circuit that is dynamically regulated by nutritional state in a manner consistent with the known role of the VMH as a satiety center.

Title: Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells Content: Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload-induced angiogenesis, indicating that AT1-independent signals maintain VEGF production in losartan-treated muscle.

Title: Astrocyte-Mediated Distributed Plasticity at Hypothalamic Glutamate Synapses Content: Afferent activity can induce fast, feed-forward changes in synaptic efficacy that are synapse specific. Using combined electrophysiology, caged molecule photolysis, and Ca(2+) imaging, we describe a plasticity in which the recruitment of astrocytes in response to afferent activity causes a fast and feed-forward, yet distributed increase in the amplitude of quantal synaptic currents at multiple glutamate synapses on magnocellular neurosecretory cells in the hypothalamic paraventricular nucleus. The plasticity is largely multiplicative, consistent with a proportional increase or "scaling" in the strength of all synapses on the neuron. This effect requires a metabotropic glutamate receptor-mediated rise in Ca(2+) in the astrocyte processes surrounding the neuron and the release of the gliotransmitter ATP, which acts on postsynaptic purinergic receptors. These data provide evidence for a form of distributed synaptic plasticity that is feed-forward, expressed quickly, and mediated by the synaptic activation of neighboring astrocytes.

Title: Development of a novel mouse glioma model using lentiviral vectors Content: We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP–controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein–positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme–like tumors, which contained CD133+ cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP–controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice.

Title: Signal transduction proteins that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized cells. Content: Permeabilized rat kidney cells rapidly released glucose 6-phosphate dehydrogenase (G6PD) following stimulation with peptide growth factors (Stanton, R.C., Seifter, J.L., Boxer, D.C., Zimmerman, E., and Cantley, L. C. (1991) J. Biol. Chem. 266, 12442-12448). To evaluate the signal transduction pathways mediating release of G6PD, two cell lines transfected with wild type or mutant platelet-derived growth factor (PDGF) receptors (PDGFR) were studied using two permeabilization protocols. G6PD release was evaluated by enzyme activity and Western blot analysis. PDGF caused a significant increase in G6PD release in 1 min in cells transfected with wild type PDGFR. PDGF did not stimulate G6PD release in cells transfected with tyrosine kinase-deficient PDGFR. PDGF did not stimulate G6PD release in cells transfected with partially autophosphorylation-deficient PDGFR in which four known signaling proteins do not associate with the PDGFR. The PDGF-stimulated release of G6PD was partially restored in PDGFR mutants in which either phosphatidylinositol-3-kinase or phospholipase C gamma 1 could associate with the PDGFR. Lastly, there was no basal or PDGF-stimulated phosphorylation of G6PD. We conclude that release of G6PD: 1) requires intrinsic PDGFR tyrosine kinase activity; 2) requires PDGFR autophosphorylation; 3) is mediated by signaling proteins that associate with the PDGFR; 4) is not mediated by direct phosphorylation of G6PD.

Title: Ionizing radiation-induced long-term expression of senescence markers in mice is independent of p53 and immune status. Content: Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16(INK4a) expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2(-/-) gammaC(-/-) mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.

Title: Identification of a Brainstem Circuit Controlling Feeding Content: Hunger, driven by negative energy balance, elicits the search for and consumption of food. While this response is in part mediated by neurons in the hypothalamus, the role of specific cell types in other brain regions is less well defined. Here, we show that neurons in the dorsal raphe nucleus, expressing vesicular transporters for GABA or glutamate (hereafter, DRNVgat and DRNVGLUT3 neurons), are reciprocally activated by changes in energy balance and that modulating their activity has opposite effects on feeding-DRNVgat neurons increase, whereas DRNVGLUT3 neurons suppress, food intake. Furthermore, modulation of these neurons in obese (ob/ob) mice suppresses food intake and body weight and normalizes locomotor activity. Finally, using molecular profiling, we identify druggable targets in these neurons and show that local infusion of agonists for specific receptors on these neurons has potent effects on feeding. These data establish the DRN as an important node controlling energy balance. PAPERCLIP.

Title: Age-related changes in kynurenic acid production in rat brain. Content: Two separate in vitro assays were used to examine the biosynthesis of the broad spectrum excitatory amino acid receptor antagonist kynurenic acid (KYNA) during the life span of the adult rat. Assessment of KYNA's anabolic enzyme kynurenine aminotransferase revealed steady increases between 3 and 24 months of age in all five brain regions examined. No changes were observed in the liver. The changes were particularly pronounced in the cortex and in the striatum where enzyme activity increased three-fold during the period studied. KYNA production from its bioprecursor L-kynurenine was also investigated in tissue slices and was found to be significantly enhanced in the cortex and hippocampus of old animals. The effect of depolarizing agents or sodium replacement was virtually identical in tissues from young and old rats. These data, which are in excellent agreement with reports on an age-dependent increase of KYNA concentration in brain tissue, suggest an enhanced KYNA tone in the aged brain. Together with the reported decline in cerebral excitatory amino acid receptor densities with age, increased production of KYNA may play a role in cognitive and memory dysfunction in old animals.

Title: A neural basis for melanocortin-4 receptor regulated appetite Content: Pro-opiomelanocortin (POMC)- and agouti-related peptide (AgRP)-expressing neurons of the arcuate nucleus of the hypothalamus (ARC) are oppositely regulated by caloric depletion and coordinately stimulate and inhibit homeostatic satiety, respectively. This bimodality is principally underscored by the antagonistic actions of these ligands at downstream melanocortin-4 receptors (MC4R) in the paraventricular nucleus of the hypothalamus (PVH). Although this population is critical to energy balance, the underlying neural circuitry remains unknown. Using mice expressing Cre recombinase in MC4R neurons, we demonstrate bidirectional control of feeding following real-time activation and inhibition of PVH(MC4R) neurons and further identify these cells as a functional exponent of ARC(AgRP) neuron-driven hunger. Moreover, we reveal this function to be mediated by a PVH(MC4R)→lateral parabrachial nucleus (LPBN) pathway. Activation of this circuit encodes positive valence, but only in calorically depleted mice. Thus, the satiating and appetitive nature of PVH(MC4R)→LPBN neurons supports the principles of drive reduction and highlights this circuit as a promising target for antiobesity drug development.

Title: Metformin Normalizes Type 2 Diabetes-Induced Decrease in Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus Content: In this study, we observed the effects of metformin, one of the most widely prescribed drugs for the treatment of type 2 diabetes, on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG) in Zucker diabetic fatty (ZDF) rats, which are a model for type 2 diabetes. For this, metformin was administered orally once a day to 14-week-old ZDF rats for 2 weeks and the animals were sacrificed at 16 weeks of age. During this period, blood glucose levels were higher in the vehicle-treated ZDF rats than in the Zucker lean control (ZLC) rats. Metformin treatment significantly decreased the blood glucose levels from 15.5 weeks of age. In the SZDG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for differentiated neuroblasts)-immunoreactive cells were much lower in the vehicle-treated ZDF rats than in the ZLC rats. In the metformin-treated ZDF group, Ki67- and DCX-immunoreactive cells were significantly increased in the SZDG compared to those in the vehicle-treated ZDF group. These results suggest that diabetes significantly reduces cell proliferation and neuroblast differentiation in the SZDG and that metformin treatment normalizes the reduction of cell proliferation and neuroblast differentiation in the SZDG in diabetic rats.

Title: Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis Content: All homeotherms use thermogenesis to maintain their core body temperature, ensuring that cellular functions and physiological processes can continue in cold environments. In the prevailing model of thermogenesis, when the hypothalamus senses cold temperatures it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue and white adipose tissue. Acting via the β(3)-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes, whereas it stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Exposure to cold temperature rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL-4 increased thermogenic gene expression, fatty acid mobilization and energy expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.

Title: Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons Content: Energy deficiency and dysfunction of the Na+, K+-ATPase are common consequences of many pathological insults. The nature and mechanism of cell injury induced by impaired Na+, K+-ATPase, however, are not well defined. We used cultured cortical neurons to examine the hypothesis that blocking the Na+, K+-ATPase induces apoptosis by depleting cellular K+ and, concurrently, induces necrotic injury in the same cells by increasing intracellular Ca2+ and Na+. The Na+, K+-ATPase inhibitor ouabain induced concentration-dependent neuronal death. Ouabain triggered transient neuronal cell swelling followed by cell shrinkage, accompanied by intracellular Ca2+ and Na+ increase, K+ decrease, cytochrome c release, caspase-3 activation, and DNA laddering. Electron microscopy revealed the coexistence of ultrastructural features of both apoptosis and necrosis in individual cells. The caspase inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-FMK) blocked >50% of ouabain-induced neuronal death. Potassium channel blockers or high K+ medium, but not Ca2+ channel blockade, prevented cytochrome c release, caspase activation, and DNA damage. Blocking of K+, Ca2+, or Na+ channels or high K+ medium each attenuated the ouabain-induced cell death; combined inhibition of K+ channels and Ca2+ or Na+ channels resulted in additional protection. Moreover, coapplication of Z-VAD-FMK and nifedipine produced virtually complete neuroprotection. These results suggest that the neuronal death associated with Na+, K+-pump failure consists of concurrent apoptotic and necrotic components, mediated by intracellular depletion of K+ and accumulation of Ca2+ and Na+, respectively. The ouabain-induced hybrid death may represent a distinct form of cell death related to the brain injury of inadequate energy supply and disrupted ion homeostasis.

Title: Nitroxergic nerves mediate vagally induced relaxation in the isolated stomach of the guinea pig. Content: Here we show that the relaxation induced by stimulation of the vagus nerve in the presence of cholinergic (muscarinic) and adrenergic blockade in the isolated stomach of the guinea pig is mediated by nitric oxide (NO). This is substantiated by inhibition of vagal relaxation by NG-monomethyl-L-arginine, an inhibitor of NO synthesis. The effect of NG-monomethyl-L-arginine was partially reversed by coincubation with L-arginine but not with D-arginine. NO activates soluble guanylate cyclase, and relaxation of the stomach induced by vagal stimulation was prevented by an inhibitor of soluble guanylate cyclase, methylene blue, further supporting our conclusions. The relaxant effect of vagal stimulation was also ablated by hexamethonium, an inhibitor of ganglionic nicotinic receptors, thereby showing that ganglionic transmission did not rely on NO, through its release from preganglionic neurons. However, hexamethonium did not inhibit the gastric relaxation brought about by increasing the intragastric pressure, which is also mediated by NO as previously described by us. The selective inhibition by hexamethonium of only the vagally mediated relaxation but not of the pressure-induced relaxation of the stomach indicates the existence of at least two separate neuronal pathways able to generate NO and bring about gastric accommodation of food or fluid.

Title: PP2A regulates tau phosphorylation directly and also indirectly via activating GSK-3beta. Content: Abnormal hyperphosphorylation of tau appears to be crucial in neurofibrillary degeneration in Alzheimer's disease (AD). Previous studies suggest that a down-regulation of protein phosphatase 2A (PP2A), the major tau phosphatase in human brain, contributes to tau hyperphosphorylation in AD. However, the effects of PP2A down-regulation on site-specific tau hyperphosphorylation is not well understood. In the present study, we showed that PP2A dephosphorylated tau at several phosphorylation sites with different efficiencies. Among the sites studied, Thr205, Thr212, Ser214, and Ser262 were the most favorable sites, and Ser199 and Ser404 were the least favorable sites for PP2A in vitro. Inhibition of PP2A with okadaic acid in metabolically active rat brain slices caused inhibition of glycogen synthase kinase-3beta (GSK-3beta) via an increase in its phosphorylation at Ser9. GSK-3beta phosphorylated tau at many sites, with Ser199, Thr205, and Ser396 being the most favorable sites in cells. The overall alterations in tau phosphorylation induced by PP2A inhibition were the result of the combined effects of both reduced tau dephosphorylation due to PP2A inhibition directly and reduced phosphorylation by GSK-3beta due to its inhibition. Because the impacts of tau phosphorylation on its biological activity and on neurofibrillary degeneration are site-specific, this study provides a new insight into the role of PP2A down-regulation in neurofibrillary degeneration in AD.

Title: Bacteria activate sensory neurons that modulate pain and inflammation Content: Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.

Title: Neuronal Sirt1 deficiency increases insulin sensitivity in both brain and peripheral tissues. Content: Sirt1 is a NAD(+)-dependent class III deacetylase that functions as a cellular energy sensor. In addition to its well-characterized effects in peripheral tissues, emerging evidence suggests that neuronal Sirt1 activity plays a role in the central regulation of energy balance and glucose metabolism. To assess this idea, we generated Sirt1 neuron-specific knockout (SINKO) mice. On both standard chow and HFD, SINKO mice were more insulin sensitive than Sirt1(f/f) mice. Thus, SINKO mice had lower fasting insulin levels, improved glucose tolerance and insulin tolerance, and enhanced systemic insulin sensitivity during hyperinsulinemic euglycemic clamp studies. Hypothalamic insulin sensitivity of SINKO mice was also increased over controls, as assessed by hypothalamic activation of PI3K, phosphorylation of Akt and FoxO1 following systemic insulin injection. Intracerebroventricular injection of insulin led to a greater systemic effect to improve glucose tolerance and insulin sensitivity in SINKO mice compared with controls. In line with the in vivo results, insulin-induced AKT and FoxO1 phosphorylation were potentiated by inhibition of Sirt1 in a cultured hypothalamic cell line. Mechanistically, this effect was traced to a reduced effect of Sirt1 to directly deacetylate and repress IRS-1 function. The enhanced central insulin signaling in SINKO mice was accompanied by increased insulin receptor signal transduction in liver, muscle, and adipose tissue. In summary, we conclude that neuronal Sirt1 negatively regulates hypothalamic insulin signaling, leading to systemic insulin resistance. Interventions that reduce neuronal Sirt1 activity have the potential to improve systemic insulin action and limit weight gain on an obesigenic diet.

Title: Direct conversion of fibroblasts to functional neurons by defined factors Content: Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. This raised the question of whether transcription factors could directly induce other defined somatic cell fates, and not only an undifferentiated state. We hypothesized that combinatorial expression of neural-lineage-specific transcription factors could directly convert fibroblasts into neurons. Starting from a pool of nineteen candidate genes, we identified a combination of only three factors, Ascl1, Brn2 (also called Pou3f2) and Myt1l, that suffice to rapidly and efficiently convert mouse embryonic and postnatal fibroblasts into functional neurons in vitro. These induced neuronal (iN) cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of iN cells from non-neural lineages could have important implications for studies of neural development, neurological disease modelling and regenerative medicine.

Title: β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. Content: Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

Title: Role of ghrelin in the relationship between hyperphagia and accelerated gastric emptying in diabetic mice. Content: BACKGROUND & AIMS Ghrelin is an orexigenic peptide with gastroprokinetic effects. Mice with streptozotocin (STZ)-induced diabetes exhibit hyperphagia, altered gastric emptying, and increased plasma ghrelin levels. We investigated the causative role of ghrelin herein by comparing changes in ghrelin receptor knockout (growth hormone secretagogue receptor [GHS-R](-/-)) and wild-type (GHS-R(+/+)) mice with STZ-induced diabetes. METHODS Gastric emptying was measured with the [(13)C]octanoic acid breath test. The messenger RNA (mRNA) expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin was quantified by real-time reverse-transcription polymerase chain reaction. Neural contractions were elicited by electrical field stimulation in fundic smooth muscle strips. RESULTS Diabetes increased plasma ghrelin levels to a similar extent in both genotypes. Hyperphagia was more pronounced in GHS-R(+/+) than in GHS-R(-/-) mice between days 12 and 21. Increases in NPY and AgRP mRNA expression were less pronounced in diabetic GHS-R(-/-) than in GHS-R(+/+) mice from day 15 on, whereas decreases in proopiomelanocortin mRNA levels were similar in both genotypes. Gastric emptying was accelerated to a similar extent in both genotypes, starting on day 16. In fundic smooth muscle strips of diabetic GHS-R(+/+) and GHS-R(-/-) mice, neuronal relaxations were reduced, whereas contractions were increased; this increase was related to an increased affinity of muscarinic and tachykinergic receptors. CONCLUSIONS Diabetic hyperphagia is regulated by central mechanisms in which the ghrelin-signaling pathway affects the expression of NPY and AgRP in the hypothalamus. The acceleration of gastric emptying, which is not affected by ghrelin signaling, is not the cause of diabetic hyperphagia and probably involves local contractility changes in the fundus.

Title: SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance. Content: Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

Title: The effect of N-acetylcysteine in the nucleus accumbens on neurotransmission and relapse to cocaine. Content: BACKGROUND Relapse to cocaine seeking has been linked with low glutamate in the nucleus accumbens core (NAcore) causing potentiation of synaptic glutamate transmission from prefrontal cortex (PFC) afferents. Systemic N-acetylcysteine (NAC) has been shown to restore glutamate homeostasis, reduce relapse to cocaine seeking, and depotentiate PFC-NAcore synapses. Here, we examine the effects of NAC applied directly to the NAcore on relapse and neurotransmission in PFC-NAcore synapses, as well as the involvement of the metabotropic glutamate receptors 2/3 (mGluR2/3) and 5 (mGluR5). METHODS Rats were trained to self-administer cocaine for 2 weeks and following extinction received either intra-accumbens NAC or systemic NAC 30 or 120 minutes, respectively, before inducing reinstatement with a conditioned cue or a combined cue and cocaine injection. We also recorded postsynaptic currents using in vitro whole cell recordings in acute slices and measured cystine and glutamate uptake in primary glial cultures. RESULTS NAC microinjection into the NAcore inhibited the reinstatement of cocaine seeking. In slices, a low concentration of NAC reduced the amplitude of evoked glutamatergic synaptic currents in the NAcore in an mGluR2/3-dependent manner, while high doses of NAC increased amplitude in an mGluR5-dependent manner. Both effects depended on NAC uptake through cysteine transporters and activity of the cysteine/glutamate exchanger. Finally, we showed that by blocking mGluR5 the inhibition of cocaine seeking by NAC was potentiated. CONCLUSIONS The effect of NAC on relapse to cocaine seeking depends on the balance between stimulating mGluR2/3 and mGluR5 in the NAcore, and the efficacy of NAC can be improved by simultaneously inhibiting mGluR5.

Title: Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. Content: Two studies were performed to investigate the effects of an acute bout of physical exercise on the nuclear protein kappaB (NF-kappaB) signaling pathway in rat skeletal muscle. In Study 1, a group of rats (n=6) was run on the treadmill at 25 m/min, 5% grade, for 1 h or until exhaustion (Ex), and compared with a second group (n=6) injected with two doses of pyrrolidine dithiocarbamate (PDTC, 100 mg/kg, i.p.) 24 and 1 h prior to the acute exercise bout. Three additional groups of rats (n=6) were injected with either 8 mg/kg (i.p.) of lipopolysaccharide (LPS), 1 mmol/kg (i.p.) t-butylhydroperoxide (tBHP), or saline (C) and killed at resting condition. Ex rats showed higher levels of NF-kappaB binding and P50 protein content in muscle nuclear extracts compared with C rats. Cytosolic IkappaBalpha and IkappaB kinase (IKK) contents were decreased, whereas phospho-IkappaBalpha and phospho-IKK contents were increased, comparing Ex vs. C. The exercise-induced activation of NF-kappaB signaling cascade was partially abolished by PDTC treatment. LPS, but not tBHP, treatment mimicked and exaggerated the effects observed in Ex rats. In Study 2, the time course of exercise-induced NF-kappaB activation was examined. Highest levels of NF-kappaB binding were observed at 2 h postexercise. Decreased cytosolic IkappaBalpha and increased phosphor-IkappaBalpha content were found 0-1 h postexercise whereas P65 reached peak levels at 2-4 h. These data suggest that the NF-kappaB signaling pathway can be activated in a redox-sensitive manner during muscular contraction, presumably due to increased oxidant production. The cascade of intracellular events may be the overture to elevated gene expression of manganese superoxide dismutase reported earlier (Pfluegers Arch. 442, 426-434, 2001).

Title: Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors. Content: Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Title: Inhibition of apical sodium-dependent bile acid transporter as a novel treatment for diabetes. Content: Bile acids are recognized as metabolic modulators. The present study was aimed at evaluating the effects of a potent Asbt inhibitor (264W94), which blocks intestinal absorption of bile acids, on glucose homeostasis in Zucker Diabetic Fatty (ZDF) rats. Oral administration of 264W94 for two wk increased fecal bile acid concentrations and elevated non-fasting plasma total Glp-1. Treatment of 264W94 significantly decreased HbA1c and glucose, and prevented the drop of insulin levels typical of ZDF rats in a dose-dependent manner. An oral glucose tolerance test revealed up to two-fold increase in plasma total Glp-1 and three-fold increase in insulin in 264W94 treated ZDF rats at doses sufficient to achieve glycemic control. Tissue mRNA analysis indicated a decrease in farnesoid X receptor (Fxr) activation in small intestines and the liver but co-administration of a Fxr agonist (GW4064) did not attenuate 264W94 induced glucose lowering effects. In summary, our results demonstrate that inhibition of Asbt increases bile acids in the distal intestine, promotes Glp-1 release and may offer a new therapeutic strategy for type 2 diabetes mellitus.

Title: Evaluation of oxidative status and depression-like responses in Brown Norway rats with acute myeloid leukemia Content: It has been proved that oxidative stress increases when leukemia is accompanied by depression. This fact may indicate the role of oxidative stress in the development of depression in cancer patients. The aim of this study was to determine whether the acute myeloid leukemia of Brown Norway rats, which is accompanied by oxidative stress, evoked behavioral and receptor changes resembling alterations characteristic of rat models of depression. The rats were divided into two groups: leukemic rats and healthy control. Leukemia was induced through intraperitoneal injection of 10(7) promyelocytic leukemia cells to the Brown Norway rats. Depression-like behavior was evaluated in the forced swim test at 30 or 34 days after leukemic cells injection. The rats were killed after the evaluation and the spleen, brain cortex and hippocampus were excised. The red-ox state was assessed in homogenates of tissues by measuring total glutathione (GSH) content, the ferric ion reducing ability of plasma (FRAP) level, expression of heme oxygenase-1 (HO-1), biliverdin reductase (BvR) and ferritin mRNA, superoxide dismutase (SOD) activity, as well as malondialdehyde (MDA) concentration. Radioligand binding assay was used to assess of the effect of leukemia on cortical receptors. Leukemic cells were identified using RM-124 antibody by FACS Calibur flow cytometry. Leukemia influenced locomotory activity as well as forced swim test behavior in a 34-day series of experiments. Signs of oxidative stress in leukemic rats were observed in each examined stage of leukemia development. The FRAP values and glutathione contents, were significantly lowered whereas HO-1 mRNA expression, and malonodialdehyde concentrations were significantly increased in the spleen and brain structures of leukemic rats in comparison with the healthy controls. A significant increase in the potency of glycine to displace [(3)H]L-689,560 from the strychnine-insensitive glycine site of the N-methyl-D-aspartic (NMDA) receptors receptor complex in cortical homogenates of the leukemic rats in 30- and 34-day experimental series was observed in comparison with the control. Upregulation of 5-HT(2A) receptors was observed in rat cortex after 30 days of leukemia development but not in 34-days series compared with the control. It is concluded that disturbances in antioxidant system in brain cortex were accompanied by an activation of glycine sites of the NMDA receptor complex, regardless of stage of leukemia development, which are characteristic of model of depression. Findings of our study demonstrate the link between glutamatergic activity, oxidative stress and leukemia.

Title: Induction of human neuronal cells by defined transcription factors Content: Somatic cell nuclear transfer, cell fusion, or expression of lineage-specific factors have been shown to induce cell-fate changes in diverse somatic cell types. We recently observed that forced expression of a combination of three transcription factors, Brn2 (also known as Pou3f2), Ascl1 and Myt1l, can efficiently convert mouse fibroblasts into functional induced neuronal (iN) cells. Here we show that the same three factors can generate functional neurons from human pluripotent stem cells as early as 6 days after transgene activation. When combined with the basic helix-loop-helix transcription factor NeuroD1, these factors could also convert fetal and postnatal human fibroblasts into iN cells showing typical neuronal morphologies and expressing multiple neuronal markers, even after downregulation of the exogenous transcription factors. Importantly, the vast majority of human iN cells were able to generate action potentials and many matured to receive synaptic contacts when co-cultured with primary mouse cortical neurons. Our data demonstrate that non-neural human somatic cells, as well as pluripotent stem cells, can be converted directly into neurons by lineage-determining transcription factors. These methods may facilitate robust generation of patient-specific human neurons for in vitro disease modelling or future applications in regenerative medicine.

Title: Amyloid accumulation is a late event in sporadic Alzheimer's disease-like pathology in nontransgenic rats Content: The amyloid cascade hypothesis posits that deposition of the amyloid β (Aβ) peptide in the brain is a key event in the initiation of Alzheimer's disease (AD). Nonetheless, it now seems increasingly unlikely that amyloid toxicity is the cause of sporadic AD, which leads to cognitive decline. Here, using accelerated-senescence nontransgenic OXYS rats, we confirmed that aggregation of Aβ is a later event in AD-like pathology. We showed that an age-dependent increase in the levels of Aβ₁₋₄₂ and extracellular Aβ deposits in the brain of OXYS rats occur later than do synaptic losses, neuronal cell death, mitochondrial structural abnormalities, and hyperphosphorylation of the tau protein. We identified the variants of the genes that are strongly associated with the risk of either late-onset or early-onset AD, including App, Apoe4, Bace1, Psen1, Psen2, and Picalm. We found that in OXYS rats nonsynonymous SNPs were located only in the genes Casp3 and Sorl1. Thus, we present proof that OXYS rats may be a model of sporadic AD. It is possible that multiple age-associated pathological processes may precede the toxic amyloid accumulation, which in turn triggers the final stage of the sporadic form of AD and becomes a hallmark event of the disease.

Title: Exercise-linked FNDC5/irisin rescues synaptic plasticity and memory defects in Alzheimer’s models Content: Defective brain hormonal signaling has been associated with Alzheimer's disease (AD), a disorder characterized by synapse and memory failure. Irisin is an exercise-induced myokine released on cleavage of the membrane-bound precursor protein fibronectin type III domain-containing protein 5 (FNDC5), also expressed in the hippocampus. Here we show that FNDC5/irisin levels are reduced in AD hippocampi and cerebrospinal fluid, and in experimental AD models. Knockdown of brain FNDC5/irisin impairs long-term potentiation and novel object recognition memory in mice. Conversely, boosting brain levels of FNDC5/irisin rescues synaptic plasticity and memory in AD mouse models. Peripheral overexpression of FNDC5/irisin rescues memory impairment, whereas blockade of either peripheral or brain FNDC5/irisin attenuates the neuroprotective actions of physical exercise on synaptic plasticity and memory in AD mice. By showing that FNDC5/irisin is an important mediator of the beneficial effects of exercise in AD models, our findings place FNDC5/irisin as a novel agent capable of opposing synapse failure and memory impairment in AD.

Title: Interactive effects of oligofructose and obesity predisposition on gut hormones and microbiota in diet-induced obese rats. Content: OBJECTIVE Oligofructose (OFS) is a prebiotic that reduces energy intake and fat mass via changes in gut satiety hormones and microbiota. The effects of OFS may vary depending on predisposition to obesity. The aim of this study was to examine the effect of OFS in diet-induced obese (DIO) and diet-resistant (DR) rats. METHODS Adult, male DIO, and DR rats were randomized to: high-fat/high-sucrose (HFS) diet or HFS diet + 10% OFS for 6 weeks. Body composition, food intake, gut microbiota, plasma gut hormones, and cannabinoid CB(1) receptor expression in the nodose ganglia were measured. RESULTS OFS reduced body weight, energy intake, and fat mass in both phenotypes (P < 0.05). Select gut microbiota differed in DIO versus DR rats (P < 0.05), the differences being eliminated by OFS. OFS did not modify plasma ghrelin or CB(1) expression in nodose ganglia, but plasma levels of GIP were reduced and PYY were elevated (P < 0.05) by OFS. CONCLUSIONS OFS was able to reduce body weight and adiposity in both prone and resistant obese phenotypes. OFS-induced changes in gut microbiota profiles in DIO and DR rats, along with changes in gut hormone levels, likely contribute to the sustained lower body weights.

Title: Olanzapine depot exposure in male rats: Dose-dependent lipogenic effects without concomitant weight gain. Content: Treatment with second-generation antipsychotic agents such as olanzapine frequently results in metabolic adverse effects, e.g. hyperphagia, weight gain and dyslipidaemia in patients of both genders. The molecular mechanisms underlying metabolic adverse effects are still largely unknown, and studies in rodents represent an important approach in their exploration. However, the validity of the rodent model is hampered by the fact that antipsychotics induce weight gain in female, but not male, rats. When administered orally, the short half-life of olanzapine in rats prevents stable plasma concentrations of the drug. We recently showed that a single intramuscular injection of long-acting olanzapine formulation yields clinically relevant plasma concentrations accompanied by several dysmetabolic features in the female rat. In the current study, we show that depot injections of 100-250 mg/kg olanzapine yielded clinically relevant plasma olanzapine concentrations also in male rats. In spite of transient hyperphagia, however, olanzapine resulted in weight loss rather than weight gain. The resultant negative feed efficiency was accompanied by a slight elevation of thermogenesis markers in brown adipose tissue for the highest olanzapine dose, but the olanzapine-related reduction in weight gain remains to be explained. In spite of the absence of weight gain, an olanzapine dose of 200mg/kg or above induced significantly elevated plasma cholesterol levels and pronounced activation of lipogenic gene expression in the liver. These results confirm that olanzapine stimulates lipogenic effects, independent of weight gain, and raise the possibility that endocrine factors may influence gender specificity of metabolic effects of antipsychotics in the rat.

Title: Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion Content: BACKGROUND Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms. METHODS Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways. RESULTS Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect. CONCLUSIONS Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

Title: Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone Content: AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.

Title: Hypoxia-induced energy stress regulates mRNA translation and cell growth. Content: Oxygen (O2) deprivation, or hypoxia, has profound effects on cell metabolism and growth. Cells can adapt to low O2 in part through activation of hypoxia-inducible factor (HIF). We report here that hypoxia inhibits mRNA translation by suppressing multiple key regulators, including eIF2alpha, eEF2, and the mammalian target of rapamycin (mTOR) effectors 4EBP1, p70S6K, and rpS6, independent of HIF. Hypoxia results in energy starvation and activation of the AMPK/TSC2/Rheb/mTOR pathway. Hypoxic AMP-activated protein kinase (AMPK) activation also leads to eEF2 inhibition. Moreover, hypoxic effects on cellular bioenergetics and mTOR inhibition increase over time. Mutation of the TSC2 tumor suppressor gene confers a growth advantage to cells by repressing hypoxic mTOR inhibition and hypoxia-induced G1 arrest. Together, eIF2alpha, eEF2, and mTOR inhibition represent important HIF-independent mechanisms of energy conservation that promote survival under low O2 conditions.

Title: The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy. Content: Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started approximately 1 h after seizure onset, peaked at 3-6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5-10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Title: Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments. Content: Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17 beta-estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17 beta-Estradiol up-regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up-regulated. Treatment of estrogen receptor (ER) alpha knockout mice with 17 beta-estradiol or treatment of C57BL/6 mice with 17 alpha-estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17 beta-estradiol effects on apoE are either directly or indirectly mediated by ER alpha in the diencephalon, while the effects in the cortex may be mediated by a non-classical mechanism or by ER beta. Measurement of mRNA levels in estrogen versus placebo-treated wild-type mice indicated that the effect of 17 beta-estradiol on apoE was not associated with changes in apoE mRNA levels.

Title: Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Content: Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

Title: 15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer. Content: Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.

Title: The death receptor CD95 activates adult neural stem cells for working memory formation and brain repair. Content: Adult neurogenesis persists in the subventricular zone and the dentate gyrus and can be induced upon central nervous system injury. However, the final contribution of newborn neurons to neuronal networks is limited. Here we show that in neural stem cells, stimulation of the "death receptor" CD95 does not trigger apoptosis but unexpectedly leads to increased stem cell survival and neuronal specification. These effects are mediated via activation of the Src/PI3K/AKT/mTOR signaling pathway, ultimately leading to a global increase in protein translation. Induction of neurogenesis by CD95 was further confirmed in the ischemic CA1 region, in the naive dentate gyrus, and after forced expression of CD95L in the adult subventricular zone. Lack of hippocampal CD95 resulted in a reduction in neurogenesis and working memory deficits. Following global ischemia, CD95-mediated brain repair rescued behavioral impairment. Thus, we identify the CD95/CD95L system as an instructive signal for ongoing and injury-induced neurogenesis.

Title: Hydrogen sulfide interacts with nitric oxide in the heart: possible involvement of nitroxyl. Content: AIMS The present study aims to investigate the interaction between nitric oxide (NO) and hydrogen sulfide (H(2)S), the two important gaseous mediators in rat hearts. METHODS AND RESULTS Intracellular calcium in isolated cardiomyocytes was measured with a spectrofluorometric method using Fura-2. Myocyte contractility was measured with a video edge system. NaHS (50 µM, an H(2)S donor) had no significant effect on the resting calcium level, electrically induced (EI) calcium transients, and cell contractility in ventricular myocytes. Stimulating endogenous NO production with l-arginine or exogenous application of NO donors [sodium nitroprusside (SNP) and 2-(N,N-diethylamino)-diazenolate-2-oxide] decreased myocyte twitch amplitudes accompanied by slower velocities of both cell contraction and relaxation. Surprisingly, NaHS reversed the negative inotropic and lusitropic effects of the above three NO-increasing agents. In addition, the mixture of SNP + NaHS increased, whereas SNP alone decreased, the resting calcium level and the amplitudes of EI calcium transients. Angeli's salt, a nitroxyl anion (HNO) donor, mimicked the effect of SNP + NaHS on calcium handling and myocyte contractility. Three thiols, N-acetyl-cysteine, l-cysteine, and glutathione, abolished the effects of HNO and SNP + NaHS on myocyte contraction. Neither Rp-cAMP [a protein kinase A (PKA) inhibitor] nor Rp-cGMP [a protein kinase G (PKG) inhibitor] affected the effects of SNP + NaHS, suggesting a cAMP/PKA- or cGMP/PKG-independent mechanism. CONCLUSION H(2)S may interact with NO to form a thiol sensitive molecule (probably HNO) which produces positive inotropic and lusitropic effects. Our findings may shed light on the interaction of NO and H(2)S and provide new clues to treat cardiovascular diseases.

Title: H2S and HS− donor NaHS releases nitric oxide from nitrosothiols, metal nitrosyl complex, brain homogenate and murine L1210 leukaemia cells Content: Nitrosoglutathione [(GSNO), 500 nmol/l] relaxed the norepinephrine precontracted rat aortic rings. The relaxation effect was pronouncedly enhanced by H2S- and HS−-donor NaHS (30 μmol/l) at 7.5 pH but not at 6.3 pH. To study molecular mechanism of this effect, we investigated whether NaHS can release NO from NO donors. Using an electron paramagnetic resonance spectroscopy method of spin trap and by measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that NaHS released NO from nitrosothiols, namely from GSNO, S-nitroso-N-acetyl-dl-penicillamine (SNAP), from metal nitrosyl complex nitroprusside (SNP) and from rat brain homogenate and murine L1210 leukaemia cells. From the observation that the releasing effect was more pronounced at 8.0 pH than 6.0 pH, we suppose that HS−, rather than H2S, is responsible for the NO-releasing effect. Since in mammals, H2S and HS− are produced endogenously, we assume that their effect to release NO from nitrosothiols and from metal nitrosyl complexes are responsible for some of their biological activities and that this mechanism may be involved in S-nitrosothiol-signalling reactions.

Title: Glutamate mediates the function of melanocortin receptor 4 on Sim1 neurons in body weight regulation. Content: The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, selective disruption of glutamate release from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.

Title: Organ-resident, nonlymphoid cells suppress proliferation of autoimmune T-helper lymphocytes. Content: Local presentation of autoantigen by organ-resident cells inappropriately expressing Ia determinants has been implicated in organ-specific autoimmunity. Experimental autoimmune uveoretinitis, induced in rats by immunization with retinal soluble antigen, is used as a model of organ-specific autoimmunity. In an in vitro system derived from this model, uveitogenic rat T-helper lymphocytes specific to the retinal soluble antigen, or control T-helper lymphocytes reactive to the purified protein derivative of tuberculin, were cocultured with Ia-expressing syngeneic retinal glial cells (Müller cells) in the presence of specific antigen. Antigen presentation was not apparent under ordinary culture conditions, and the Müller cells profoundly suppressed the proliferative response of primed T-helper lymphocytes to antigen presented on conventional antigen-presenting cells, as well as their subsequent interleukin-2 (IL-2)-dependent expansion. Suppression of proliferation was accompanied by inhibition of IL-2 production in response to antigen, as well as by reduction in high-affinity IL-2 receptor expression, and proceeded via a contact-dependent mechanism. These results suggest a role for locally acting suppression mechanisms in immune regulation and maintenance of tissue homeostasis.

Title: Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome. Content: The proinflammatory cytokine IL-1beta plays an important role in antifungal immunity; however, the mechanisms by which fungal pathogens trigger IL-1beta secretion are unclear. In this study we show that infection with Candida albicans is sensed by the Nlrp3 inflammasome, resulting in the subsequent release of IL-1beta. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome, as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL- 1beta secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans, which is consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.

Title: The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase Content: Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation. To circumvent this, we used two outbred lines of rats, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.

Title: Downregulation of Superoxide Dismutase Activity and Gene Expression in Cultured Rat Brain Astrocytes after Incubation with Vitamin C Content: Reactive oxygen species have been linked with neuropathological changes in the central nervous system. Epidemiological studies supported the beneficial effect of supplementation of antioxidants. Superoxide dismutase (SOD) is an endogenous enzyme which can scavenge reactive oxygen species. This study investigated the effect of supplementation with ascorbic acid (vitamin C) on the changes of SOD in cultured neurological cells. Rat brain astrocytes (RBA-1 cells) were incubated with vitamin C and divided into four groups: a control group (without vitamin C) and three treatment groups with vitamin C at 40, 80, and 160 µmol/l. After short-term (2 days) and long-term (7 days) incubation, SOD activity, SOD mRNA level by Northern blotting, and SOD protein amounts by Western blotting were measured. After 2 days of incubation, vitamin C resulted in a decrease in the activity of SOD in a concentration-dependent manner (Mn-SOD from 14.8 ± 1.2 to 13.2 ± 0.5 U/mg protein and Cu/Zn-SOD from 64.8 ± 1.2 to 51.7 ± 0.9 U/mg protein; p < 0.05), and vitamin C also attenuated the Cu/Zn-SOD mRNA level from 100 to 86.3 ± 6.7%; p < 0.01), whereas the protein amounts of these two SODs remained unchanged. After 7 days of incubation with vitamin C, the SOD activity of RBA-1 cells decreased significantly (Mn-SOD from 14.9 ± 0.3 to 11.8 ± 0.3 U/mg protein and Cu/Zn SOD from 61.8 ± 1.8 to 54.6 ± 0.9 U/mg protein; p < 0.01), and the mRNA level was also attenuated (Mn-SOD from 100 to 86.8 ± 8.7% and Cu/Zn-SOD from 100 to 84.7 ± 4.8%; p < 0.01). These results suggest that 2 and 7 days of incubation with relatively high concentrations of vitamin C may downregulate activity and gene expression of SOD in cultured RBA-1 cells.

Title: Fibroblast Growth Factor 21 Prevents Atherosclerosis by Suppression of Hepatic Sterol Regulatory Element-Binding Protein-2 and Induction of Adiponectin in Mice Content: BACKGROUND Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF21 in the cardiovascular system remains elusive. METHODS AND RESULTS The roles of FGF21 in atherosclerosis were investigated by evaluating the impact of FGF21 deficiency and replenishment with recombinant FGF21 in apolipoprotein E(-/-) mice. FGF21 deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF21 protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription factor sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF21-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF21 are abrogated by hepatic expression of sterol regulatory element-binding protein-2. CONCLUSIONS FGF21 protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

Title: No effect of dietary fat on short-term weight gain in mice treated with atypical antipsychotic drugs Content: Rationale:Atypical antipsychotic drugs (AAD) induce significant weight gain in female C57BL/6J mice. The effect of dietary fat on weight gain and serum lipids in this model is unknown. Objectives: Test the hypothesis that the obesigenic effects of these drugs are greater in the presence of a high-fat diet. Methods:Female C57BL/6J mice were treated with atypical antipsychotics for 3 weeks and fed either a low-fat or high-fat diet (4.6 vs 15.6% fat by wt). Food intake (FI), body weight (BW), body composition, and serum lipids were measured during treatment with optimized doses of olanzapine, quetiapine, and risperidone. Energy intake (EI) and feed efficiency (FE) were calculated. Group differences in change were analyzed via repeated measures analysis of variance (ANOVA). Serum lipid concentrations, EI and FE were compared using two-way ANOVA.Results:AAD-treated mice gained significantly more weight than controls after 3 weeks (P<0.001). Treatment and diet had significant effects on FI and EI over time (P<0.001). AAD-treated mice had significantly higher FE than controls (P<0.05); however, there was no significant drug by diet interaction (P=0.65). Risperidone low-fat mice gained significantly more absolute fat mass than placebo low-fat mice (P<0.05). All treatment groups, except quetiapine low-fat and olanzapine high-fat, gained significantly more absolute lean mass than placebo controls (P<0.05). Cholesterol levels were significantly lower in quetiapine and risperidone than placebo (P<0.05). Risperidone low-fat mice had significantly higher triglyceride levels than placebo and risperidone high-fat mice (P<0.05).Conclusions:A high-fat diet does not increase AAD-induced BW gain in female mice during a 3-week treatment period.

Title: Sema3A regulates bone-mass accrual through sensory innervations Content: Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a−/− mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a−/− mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1−/− and Sema3aosx−/− mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin−/− and Sema3anestin−/− mice) had low bone mass, similar to Sema3a−/− mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin−/− mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin−/− mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a−/− mice, such as olfactory development, were identified in Sema3asynasin−/− mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a−/− mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

Title: Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy Content: Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Title: Exercise causes a tissue-specific change of NO production in the kidney and lung. Content: Nitric oxide (NO) is produced in the vascular endothelium and is a potent vasodilator substance that participates in the regulation of local vascular tone. Exercise causes peculiar changes in systemic and regional blood flow, i.e., an increase of systemic blood flow and a redistribution of local tissue blood flow, by which the blood flow is greatly increased in the working muscles, whereas it is decreased in some organs such as the kidney and intestine. Thus we hypothesized that exercise causes a tissue-specific change of NO production in some internal organs. We studied whether exercise affects expression of NO synthase (NOS) mRNA and protein, NOS activity, and tissue level of nitrite/nitrate (stable end products of NO) in the kidneys (in which blood flow during exercise is decreased) and lungs (in which blood flow during exercise is increased with the increase of cardiac output) of rat. Rats ran on a treadmill for 45 min at a speed of 25 m/min. Immediately after this exercise, kidneys and lungs were quickly removed. Control rats remained at rest during this 45-min period. Expression of endothelial NOS (eNOS) mRNA in the kidneys was markedly lower in exercise rats than in control rats, whereas that in the lungs was significantly higher in exercise rats than in control rats. Western blot analysis confirmed down- and upregulation of eNOS protein in the kidney and lung, respectively, after exercise. On the other hand, neither expression of neuronal NOS (nNOS) mRNA and nNOS protein nor inducible NOS (iNOS) mRNA and iNOS protein in the kidneys and lungs differed between exercise and control rats. NOS activity in the kidney was significantly lower in exercise rats than in control rats, whereas that in the lung was significantly higher in exercise rats than in control rats. On the other hand, the iNOS activity in the kidneys and lungs did not differ between exercise rats and control rats. Tissue nitrite/nitrate level in the kidneys was markedly lower in exercise rats, whereas that in the lungs was significantly higher in exercise rats. The present results show that production of NO is markedly and tissue-specifically changed in the kidney and lung by exercise.

Title: Behavioral characterization of neuropeptide Y knockout mice. Content: An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception.

Title: Methionine restriction decreases visceral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction. Content: Reduced dietary methionine intake (0.17% methionine, MR) and calorie restriction (CR) prolong lifespan in male Fischer 344 rats. Although the mechanisms are unclear, both regimens feature lower body weight and reductions in adiposity. Reduced fat deposition in CR is linked to preservation of insulin responsiveness in older animals. These studies examine the relationship between insulin responsiveness and visceral fat in MR and test whether, despite lower food intake observed in MR animals, decreased visceral fat accretion and preservation of insulin sensitivity is not secondary to CR. Accordingly, rats pair fed (pf) control diet (0.86% methinone, CF) to match the food intake of MR for 80 weeks exhibit insulin, glucose, and leptin levels similar to control-fed animals and comparable amounts of visceral fat. Conversely, MR rats show significantly reduced visceral fat compared to CF and PF with concomitant decreases in basal insulin, glucose, and leptin, and increased adiponectin and triiodothyronine. Daily energy expenditure in MR animals significantly exceeds that of both PF and CF. In a separate cohort, insulin responses of older MR animals as measured by oral glucose challenge are similar to young animals. Longitudinal assessments of MR and CF through 112 weeks of age reveal that MR prevents age-associated increases in serum lipids. By 16 weeks, MR animals show a 40% reduction in insulin-like growth factor-1 (IGF-1) that is sustained throughout life; CF IGF-1 levels decline much later, beginning at 112 weeks. Collectively, the results indicate that MR reduces visceral fat and preserves insulin activity in aging rats independent of energy restriction.

Title: Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors Content: PEROXISOMES are cytoplasmic organelles which are important in mammals in modulation of lipid homeostasis, including the metabolism of long-chain fatty acids and conversion of cholesterol to bile salts (reviewed in refs 1 and 2). Amphipathic carboxylates such as clofibric acid have been used in man as hypolipidaemic agents and in rodents they stimulate the proliferation of peroxisomes. These agents, termed peroxisome proliferators, and all-trans retinoic acid activate genes involved in peroxisomal-mediated β-oxidation of fatty acids1–4. Here we show that the receptor activated by peroxisome proliferators5 and the retinoid X receptor-α (ref. 6) form a heterodimer that activates acyl-CoA oxidase gene expression in response to either clofibric acid or the retinoid X receptor-α ligand, 9-cis retinoic acid, an all-trans retinoic acid metabolite7,8; simultaneous exposure to both activators results in a synergistic induction of gene expression. These data demonstrate the coupling of the peroxisome proliferator and retinoid signalling pathways and provide evidence for a physiological role for 9-cis retinoic acid in modulating lipid metabolism.