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Background In chronic kidney disease (CKD) patients, increased levels of fibroblast growth factor 23 (FGF23) are associated with cardiovascular mortality. The relationship between FGF23 and heart hypertrophy has been documented, however, it is not known whether FGF23 has an effect on vasculature. Vascular smooth muscle cells VSMCs may exhibit different phenotypes; our hypothesis is that FGF23 favours a switch from a contractile to synthetic phenotype that may cause vascular dysfunction. Our objective was to determine whether FGF23 may directly control a change in VSMC phenotype. Methods This study includes in vitro, in vivo and ex vivo experiments and evaluation of patients with CKD stages 2–3 studying a relationship between FGF23 and vascular dysfunction. Results In vitro studies show that high levels of FGF23, by acting on its specific receptor FGFR1 and Erk1/2, causes a change in the phenotype of VSMCs from contractile to synthetic. This change is mediated by a downregulation of miR-221/222, which augments the expression of MAP3K2 and PAK1. miR-221/222 transfections recovered the contractile phenotype of VSMCs. Infusion of recombinant FGF23 to rats increased vascular wall thickness, with VSMCs showing a synthetic phenotype with a reduction of miR-221 expression. Ex-vivo studies on aortic rings demonstrate also that high FGF23 increases arterial stiffening. In CKD 2–3 patients, elevation of FGF23 was associated with increased pulse wave velocity and reduced plasma levels of miR-221/222. Conclusion In VSMCs, high levels of FGF23, through the downregulation of miR-221/222, causes a change to a synthetic phenotype. This change in VSMCs increases arterial stiffening and impairs vascular function, which might ultimately worsen cardiovascular disease.

Fibroblast growth factor 23 (FGF23) increases phosphorus excretion and decreases calcitriol (1,25(OH)2D) levels. FGF23 increases from early stages of renal failure. We evaluated whether strict control of phosphorus intake in renal failure prevents the increase in FGF23 and to what extent inflammation impairs regulation of FGF23. The study was performed in 5/6 nephrectomized (Nx) Wistar rats fed diets containing 0.2–1.2% phosphorus for 3 or 15 days. FGF23 levels significantly increased in all Nx groups in the short-term (3-day) experiment. However, at 15 days, FGF23 increased in all Nx rats except in those fed 0.2% phosphorus. In a second experiment, Nx rats fed low phosphorus diets (0.2 and 0.4%) for 15 days received daily intraperitoneal lipopolysaccharide (LPS) injections to induce inflammation. In these rats, FGF23 increased despite the low phosphorus diets. Thus, higher FGF23 levels were needed to maintain phosphaturia and normal serum phosphorus values. Renal Klotho expression was preserved in Nx rats on a 0.2% phosphorus diet, reduced on a 0.4% phosphorus diet, and markedly reduced in Nx rats receiving LPS. In ex vivo experiments, high phosphorus and LPS increased nuclear β-catenin and p65-NFκB and decreased Klotho. Inhibition of inflammation and Wnt signaling activation resulted in decreased FGF23 levels and increased renal Klotho. In conclusion, strict control of phosphorus intake prevented the increase in FGF23 in renal failure, whereas inflammation independently increased FGF23 values. Decreased Klotho may explain the renal resistance to FGF23 in inflammation. These effects are likely mediated by the activation of NFkB and Wnt/β-catenin signaling.

Samples of Nicoletiidae (order Zygentoma = Thysanura s. str.) collected in two localities from Puerto Rico belonging to the genus Anelpistina Silvestri, 1905 are studied. One of them, collected in the same cave where A. puertoricensis Espinasa and Baker Alpheis, 2003 was found, allows for the description of the female of this species, together with some additional information on the variability and postembryonic development of this troglobitic insect. The second consists of specimens collected in litter of the islet of Palominos, near the northeastern coast of the main island of Puerto Rico; these specimens are identified as a new species. This species, named Anelpistina naarae sp. nov., is described and compared with related species. A genetic analysis of its DNA and 16S rRNA compared with the available data of the remaining species points to the chronology of the lineages of Anelpistina in this island and their relationships with continental species of the genus.

Molecular studies using COI and 28S sequence data strongly identify a clade within the Heterolepismatinae distinct from the majority of species so far sequenced. The independence of the clade is supported by several morphological characters including a glabrous anterior margin to the frons, large trapezoidal thoracic sternites, tarsal trichobothria, long, conical parameres which in some species consist of two segments, and the presence of triangular or rounded subrectangular scales on the femora, tibia and clypeus. This clade is described as a new genus Visma n. gen. containing ten new species V. advenum n. sp., V. bingara n. sp., V. brayi n. sp., V. bundjalung n. sp., V. brigalowsum n. sp., V. capricornia n. sp., V. pallidum n. sp., V. powellheueri n. sp., V. tenebrosum n. sp. and V. xanthorrhoea n. sp.. Heterolepisma stilivarians Silvestri, 1908 is redescribed from the holotype and transferred to the new genus. The remaining H. stilivarians type series is found to be different to the holotype and removed from the type series. It is considered possible that H. annectens Silvestri, 1924 may also belong to this genus. Scanning electron microscopy of scale shape and rib-spacing is shown to be a useful tool to separate at least some species of the genus and considered to have greater potential if well preserved material is available.

Major depressive disorder (MDD) is a multidimensional disorder that is characterized by the presence of alterations in mood, cognitive capacity, sensorimotor, and homeostatic functions. Given that about half of the patients diagnosed with MDD do not respond to the various current treatments, new techniques are being sought to predict not only the course of the disease but also the characteristics that differentiate responders from non-responders. Using the electroencephalogram, a noninvasive and inexpensive tool, most studies have proposed that patients with MDD have some lateralization in brain electrical activity, with alterations in alpha and theta rhythms being observed, which would be related to dysfunctions in emotional capacity such as the absence or presence of responses to the different existing treatments. These alterations help in the identification of subjects at high risk of suffering from depression, in the differentiation into responders and nonresponders to various therapies (pharmacological, electroconvulsive therapy, and so on), as well as to establish in which period of the disease the treatment will be more effective. Although the data are still inconclusive and more research is needed, these alpha and theta neurophysiological markers could support future clinical practice when it comes to establishing an early diagnosis and treating state disorders more successfully and accurately of mood disorders.

The Purkinje cell (PC) degeneration (pcd) mouse harbors a mutation in Agtpbp1 gene that encodes for the cytosolic carboxypeptidase, CCP1. The mutation causes degeneration and death of PCs during the postnatal life, resulting in clinical and pathological manifestation of cerebellar ataxia. Monogenic biallelic damaging variants in the Agtpbp1 gene cause infantile-onset neurodegeneration and cerebellar atrophy, linking loss of functional CCP1 with human neurodegeneration. Although CCP1 plays a key role in the regulation of tubulin stabilization, its loss of function in PCs leads to a severe nuclear phenotype with heterochromatinization and accumulation of DNA damage. Therefore, the pcd mice provides a useful neuronal model to investigate nuclear mechanisms involved in neurodegeneration, particularly the nucleolar stress. In this study, we demonstrated that the Agtpbp1 gene mutation induces a p53-dependent nucleolar stress response in PCs, which is characterized by nucleolar fragmentation, nucleoplasmic and cytoplasmic mislocalization of nucleolin, and dysfunction of both pre-rRNA processing and mRNA translation. RT-qPCR analysis revealed reduction of mature 18S rRNA, with a parallel increase of its intermediate 18S-5’-ETS precursor, that correlates with a reduced expression of Fbl mRNA, which encodes an essential factor for rRNA processing. Moreover, nucleolar alterations were accompanied by a reduction of PTEN mRNA and protein levels, which appears to be related to the chromosome instability and accumulation of DNA damage in degenerating PCs. Our results highlight the essential contribution of nucleolar stress to PC degeneration and also underscore the nucleoplasmic mislocalization of nucleolin as a potential indicator of neurodegenerative processes., This work was supported by the following grants: “Instituto de Salud Carlos III” (CIBERNED, CB06/05/0037) and CIBERONC (CB16/12/00352), “Instituto de Investigación Valdecilla” (IDIVAL, Santander, Spain), FIS PI16/02137 from ISCIII and SAF2016-79668-R (MINECO, Spain), SA043U16 (UIC076) and SA030P17 (UIC217) from JCyL (Spain).

In chronic kidney disease (CKD) patients, it would be desirable to reduce the intake of inorganic phosphate (P) rather than limit the intake of P contained in proteins. Urinary excretion of P should reflect intestinal absorption of P(inorganic plus protein-derived). The aim of the present study is to determine whether the ratio of urinary P to urinary urea nitrogen (P/UUN ratio) helps identify patients with a high intake of inorganic P.A cross-sectional study was performed in 71 patients affected by metabolic syndrome with CKD (stages 2&ndash, 3) with normal serum P concentration. A 3-day dietary survey was performed to estimate the average daily amount and the source of P ingested. The daily intake of P was 1086.5 &plusmn, 361.3 mg/day, 64% contained in animal proteins, 22% in vegetable proteins, and 14% as inorganic P. The total amount of P ingested did not correlate with daily phosphaturia, but it did correlate with the P/UUN ratio (p &lt, 0.018). Patients with the highest tertile of the P/UUN ratio &gt, 71.1 mg/g presented more abundant inorganic P intake (p &lt, 0.038).The P/UUN ratio is suggested to be a marker of inorganic P intake. This finding might be useful in clinical practices to identify the source of dietary P and to make personalized dietary recommendations directed to reduce inorganic P intake.

Background Inflammation is a common feature in chronic kidney disease (CKD) that appears specifically associated with cardiovascular derangements in CKD patients. Observational studies have revealed a link between low Mg levels and inflammation. In this study, we hypothesize that Mg might have a modulatory effect on the inflammation induced under the uraemic milieu. Methods In vivo studies were performed in a 5/6 nephrectomized rat model of CKD. Furthermore, a possible direct effect of Mg was addressed through in vitro studies with vascular smooth muscle cells (VSMCs). Results Uraemic rats fed a normal (0.1%) Mg diet showed a systemic inflammatory response evidenced by the elevation in plasma of the pro‐inflammatory cytokines TNF‐α, IL‐1β and IL‐6, and GPx activity, a marker of oxidative stress. Importantly, an increased expression of these cytokines in the aortic tissue was also observed. In contrast, a dietary Mg supplementation (0.6%) greatly prevented the oxidative stress and the pro‐inflammatory response. In vitro, in VSMCs cultured in a pro‐inflammatory high phosphate medium, incubation with Mg 1.6 mM inhibited the increase in the production of ROS, the rise in the expression of TNF‐α, IL‐1β, IL‐6 and IL‐8 and the activation of NF‐κB signalling that was observed in cells incubated with a normal (0.8 mM) Mg. Conclusion Mg supplementation reduced inflammation associated with CKD, exerting a direct effect on vascular cells. These findings support a possible beneficial effect of Mg supplementation along the clinical management of CKD patients.

© 2020 The Author(s)., SOS1 and SOS2 are the most universal and widely expressed family of guanine exchange factors (GEFs) capable or activating RAS or RAC1 proteins in metazoan cells. SOS proteins contain a sequence of modular domains that are responsible for different intramolecular and intermolecular interactions modulating mechanisms of self-inhibition, allosteric activation and intracellular homeostasis. Despite their homology, analyses of SOS1/2-KO mice demonstrate functional prevalence of SOS1 over SOS2 in cellular processes including proliferation, migration, inflammation or maintenance of intracellular redox homeostasis, although some functional redundancy cannot be excluded, particularly at the organismal level. Specific SOS1 gain-of-function mutations have been identified in inherited RASopathies and various sporadic human cancers. SOS1 depletion reduces tumorigenesis mediated by RAS or RAC1 in mouse models and is associated with increased intracellular oxidative stress and mitochondrial dysfunction. Since WT RAS is essential for development of RAS-mutant tumors, the SOS GEFs may be considered as relevant biomarkers or therapy targets in RAS-dependent cancers. Inhibitors blocking SOS expression, intrinsic GEF activity, or productive SOS protein-protein interactions with cellular regulators and/or RAS/RAC targets have been recently developed and shown preclinical and clinical effectiveness blocking aberrant RAS signaling in RAS-driven and RTK-driven tumors., ES and FCB were supported by grants from ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-UIC 076) and Areces Foundation (CIVP19A5942). JMRC and NZ received grant support from MINECO-FEDER (SAF2016-78852-R and Spanish Association against Cancer (AECC/CGB14142035THOM). ES and JMRC were also supported by ISCIII-CIBERONC (groups CB16/12/00352 and CB16/12/00273, respectively). Research co-financed by FEDER funds.

Monoacylglycerol lipase inhibition (MAGL) has emerged as an interesting therapeutic target for neurodegenerative disease treatment due to its ability to modulate the endocannabinoid system and to prevent the production of proinflammatory mediators. To obtain a beneficial response, it is necessary to understand how this inhibition affects the neuron&ndash, glia crosstalk and neuron viability. In this study, the effect of MAGL inhibition by KML29 was evaluated in two types of rat cortical primary cultures, mixed cultures, including neuron and glial cells, and neuron-enriched cultures. The risk of neuronal death was estimated by longitudinal survival analysis. The spontaneous neuronal risk of death in culture was higher in the absence of glial cells, a process that was enhanced by KML29 addition. In contrast, neuronal survival was not compromised by MAGL inhibition in the presence of glial cells. Blockade of cannabinoid type 2 (CB2) receptors expressed mainly by microglial cells did not affect the spontaneous neuronal death risk but decreased neuronal survival when KML29 was added. Modulation of cannabinoid type 1 (CB1) receptors did not affect neuronal survival. Our results show that neuron&ndash, glia interactions are essential for neuronal survival. CB2 receptors play a key role in these protective interactions when neurons are exposed to toxic conditions.

Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.

Phosphatidylinositol&ndash, 5 phosphate (PI5P) and other mono-phosphoinositides (mono-PIs) play second messenger roles in both physiological and pathological conditions. Despite this, their intracellular targets and mechanisms of action remain poorly characterized. Here, we show that Vav1, a protein that exhibits both Rac1 GDP/GTP exchange and adaptor activities, is positively modulated by PI5P and, possibly, other mono-PIs. Unlike other phospholipid&ndash, protein complexes, the affinity and specificity of the Vav1&ndash, lipid interaction entail a new structural solution that involves the synergistic action of the Vav1 C1 domain and an adjacent polybasic tail. This new regulatory layer, which is not conserved in the Vav family paralogs, favors the engagement of optimal Vav1 signaling outputs in lymphocytes.

Introduction Schizophrenia is a chronic illness often accompanied by metabolic disorders, diabetes, obesity and cardiovascular problems often associated with unhealthy lifestyles, as well as neuroendocrine problems caused by the disease itself. Lifestyle changes, such as regular physical exercise, have a positive effect on metabolic disorders and mental health, although the molecular changes that occur in this type of patient and how they explain the changes in their response are unknown. This study wants to analyze in a novel way the proteins and molecular pathways involved in critical plasmatic proteins in plasma to reveal the pathways involved in the implementation of physical exercise and the changes that occur among patients who participate in such programs with those who leave. Methods Twenty-one patients with chronic schizophrenia underwent a daily, 6-month aerobic training program. We divided them into a group that completed the program (12 patients) and a second group that left the training program (9 patients). The biochemical and clinical data of each patient were analyzed and the proteomic profile of the plasma was studied using ESI-LC–MS/MS. Results Proteomic analysis recognizes 21.165 proteins and peptides in each patient, of which we identified 1.812 proteins that varied between both groups linked to the metabolic and biological regulation pathways. After clinical analysis of each patient we found significant differences in weight, BMI, abdominal perimeter, diastolic blood pressure, and HDL cholesterol levels. The main change that vertebrates both groups is the Self-Assessment Anhedonia Scale, where we detected higher levels in the dropout group (no physical activity) compared to the active group. Conclusion The benefits of physical exercise are clear in chronic patients with schizophrenia, as it substantially improves their BMI, as well as their clinical and biochemical parameters. However, our study reveals the biological and molecular pathways that affect physical exercise in schizophrenia, such as important metabolic proteins such as ApoE and ApoC, proteins involved in neuronal regulation such as tenascin and neurotrophins, neuroinflammatory regulatory pathways such as lipocalin-2 and protein 14-3-3, as well as cytoskeleton proteins of cells such as spectrins and annexines. Understanding these molecular mechanisms opens the door to future therapies in the chronicity of schizophrenia.

© 2019 The Authors., Circulating neutrophils are, by necessity, quiescent and relatively unresponsive to acute stimuli. In regions of inflammation, mediators can prime neutrophils to react to acute stimuli with stronger proinflammatory, pathogen‐killing responses. In neutrophils G protein‐coupled receptor (GPCR)‐driven proinflammatory responses, such as reactive oxygen species (ROS) formation and accumulation of the key intracellular messenger phosphatidylinositol (3,4,5)‐trisphosphate (PIP3), are highly dependent on PI3K‐γ, a Ras‐GTP, and Gβγ coincidence detector. In unprimed cells, the major GPCR‐triggered activator of Ras is the Ras guanine nucleotide exchange factor (GEF), Ras guanine nucleotide releasing protein 4 (RasGRP4). Although priming is known to increase GPCR–PIP3 signaling, the mechanisms underlying this augmentation remain unclear. We used genetically modified mice to address the role of the 2 RasGEFs, RasGRP4 and son of sevenless (SOS)1/2, in neutrophil priming. We found that following GM‐CSF/TNFα priming, RasGRP4 had only a minor role in the enhanced responses. In contrast, SOS1/2 acquired a substantial role in ROS formation, PIP3 accumulation, and ERK activation in primed cells. These results suggest that SOS1/2 signaling plays a key role in determining the responsiveness of neutrophils in regions of inflammation., This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/J004456/1 and BB/P013384/1).

Using Sos1 knockout (Sos1-KO), Sos2-KO, and Sos1/2 double-knockout (Sos1/2-DKO) mice, we assessed the functional role of Sos1 and Sos2 in skin homeostasis under physiological and/or pathological conditions. Sos1 depletion resulted in significant alterations of skin homeostasis, including reduced keratinocyte proliferation, altered hair follicle and blood vessel integrity in dermis, and reduced adipose tissue in hypodermis. These defects worsened significantly when both Sos1 and Sos2 were absent. Simultaneous Sos1/2 disruption led to severe impairment of the ability to repair skin wounds, as well as to almost complete ablation of the neutrophil-mediated inflammatory response in the injury site. Furthermore, Sos1 disruption delayed the onset of tumor initiation, decreased tumor growth, and prevented malignant progression of papillomas in a DMBA (7,12-dimethylbenz[α]anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate)-induced skin carcinogenesis model. Finally, Sos1 depletion in preexisting chemically induced papillomas resulted also in decreased tumor growth, probably linked to significantly reduced underlying keratinocyte proliferation. Our data unveil novel, distinctive mechanistic roles of Sos 1 and Sos2 in physiological control of skin homeostasis and wound repair, as well as in pathological development of chemically induced skin tumors. These observations underscore the essential role of Sos proteins in cellular proliferation and migration and support the consideration of these RasGEFs as potential biomarkers/therapy targets in Ras-driven epidermal tumors., This study was supported by grants FIS PI16/02137 from ISCIII (MINECO), SA043U16 (UIC 076) from JCyL, and AECC Spain (to E.S.); by MINECO grant SAF2015-66015-R; and by MSyC grants ISCIII-RETIC RD12/0036/0009, PIE 15/00076, and CB/16/00228 (to J.M.P.). This research was cofinanced by FEDER funds

Son of Sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the small GTPases RAC and RAS. Although the molecular mechanisms of RAS GEF catalysis have been unveiled, how SOS1 acquires RAC GEF activity and what is the physio-pathological relevance of this activity is much less understood. Here we show that SOS1 is tyrosine phosphorylated on Y1196 by ABL. Phosphorylation of Y1196 controls SOS1 inter-molecular interaction, is required to promote the exchange of nucleotides on RAC in vitro and for platelet-derived growth factor (PDGF) activation of RAC- and RAC-dependent actin remodeling and cell migration. SOS1 is also phosphorylated on Y1196 by BCR-ABL in chronic myelogenous leukemic cells. Importantly, in these cells, SOS1 is required for BCR-ABL-mediated activation of RAC, cell proliferation and transformation in vitro and in a xenograft mouse model. Finally, genetic removal of Sos1 in the bone marrow-derived cells (BMDCs) from Sos1 fl/fl mice and infected with BCR-ABL causes a significant delay in the onset of leukemogenesis once BMDCs are injected into recipient, lethally irradiated mice. Thus, SOS1 is required for full transformation and critically contribute to the leukemogenic potential of BCR-ABL., This work has been supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC 10168 and 18621), the Italian Ministries of Education- University-Research (MIUR-PRIN-2009X23L78), the International Association For Cancer Research (AICR-09-0582 and 14-0335), the CARIPLO Foundation (2010-0737) and the European Research Council (Advanced-ERC268836). ALI was supported by a Grant from der German Jose Carreras Stiftung (DJCLS R14/22) and a Grant from the Government Baden-Württemberg (MWK). CM was supported form a AECC fellowship, Spain.

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability., This work was supported by grants FIS PI13/02846, RTICC RD12/0036/0001 and RD12/0036/0048 from ISCIII (MINECO) and grants SA181U13 and BIO/SA03/14 from JCyL, Spain. PL-B, RG-N and CG were supported by FEDER-JCyL, Fondo Social Europeo and AECC, respectively.

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro-inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively., Grant sponsor: ‘‘Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) Instituto de Salud Carlos III’’; Grant number: CB06/05/0037; Grant sponsor: Ministerio de Ciencia y Tecnología; Grant numbers: BFU2010-18284, BFU2011-23983; Grant sponsors: ‘‘Instituto de Formación e Investigación Marqués de Valdecilla’’ (IFIMAV), Junta de Castilla y León, Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León y Fundación Memoria D. Samuel Solórzano-Barruso, Spain.

The serotonergic centrifugal system innervating the main olfactory bulb (MOB) plays a key role in the modulation of olfactory processing. We have previously demonstrated that this system suffers adaptive changes under conditions of a lack of olfactory input. The present work examines the response of this centrifugal system after mitral cell loss in the Purkinje cell degeneration (pcd) mutant mice. The distribution and density of serotonergic centrifugal axons were studied in the MOB of control and pcd mice, both before and after the loss of mitral cells, using serotonin (5-HT) and 5-HT transporter immunohistochemistry. Studies of the amount of 5-HT and its metabolite, 5-hydroxyindole acetic acid (5-HIAA), were performed by means of high-performance liquid chromatography (HPLC), and the relative amounts of brain-derived neurotrophin factor, BDNF, and its major receptor, tropomyosin-related kinase B (TrkB), were measured by Western blot. Our study revealed that the serotonergic system develops adaptive changes after, but not before, mitral cell loss. The lack of the main bulbar projection cells causes a decrease in the serotonergic input received by the MOB, whereas the number of serotonergic cells in the raphe nuclei remains constant. In addition, one of the molecules directly involved in serotonergic sprouting, the neurotrophin BDNF and its main receptor TrkB, underwent alterations in the MOBs of the pcd animals even before the loss of mitral cells. These data indicate that serotonergic function in the MOB is closely related to olfactory activity and that mitral cell loss induces serotonergic plastic responses.

NADPH oxidases constitute a major source of superoxide anion (⋅O2 -) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-β 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-β 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-β 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-β 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-β 1.

14 páginas, 6 figuras, 3 tablas.-- et al., Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons., This work was supported by the Ministerio de Ciencia e Innovación (BFU2010-18284), the Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional Sobre Drogas), the Junta de Castilla y León, “MMA,” “Samuel Solórzano Barruso,” and the “Alicia Koplowitz” Foundations, and Centre for Regenerative Medicine and Cell Therapy of Castilla y León.

The periglomerular cells (PG) of the olfactory bulb (OB) are involved in the primary processing and the refinement of sensory information from the olfactory epithelium. The neurochemical composition of these neurons has been studied in depth in many species, and over the last decades such studies have focused mainly on the rat. The increasing use of genetic models for research into olfactory function demands a profound characterization of the mouse olfactory bulb, including the chemical composition of bulbar interneurons. Regarding both their connectivity with the olfactory nerve and their neurochemical fate, recently, two different types of PG have been identfied in the mouse. In the present report, we analyze both the synaptology and the chemical composition of specific PG populations in the murine olfactory bulb, in particular, those containing the neuropeptide cholecystokinin. Our results demonstrate the existence in the mouse of non-GABAergic PG and that these establish synaptic contacts with the olfactory nerve within the glomeruli. Based on previous classifications, we propose that this population would constitute a new subtype of type 1 mouse PG. In addition, we demonstrate the partial coexistence of cholecystokinin with the calcium-binding proteins neurocalcin and parvalbumin. All these findings add further data to our knowledge of the synaptology and neurochemistry of mouse PG. The differences observed from other rodents reflect the neurochemical heterogeneity of PG in the mammalian OB. © 2010 Wiley-Liss, Inc.

OBJECTIVES: Hyperleptinemia and oxidative stress play a major role in the development of cardiovascular diseases in obesity. This study aimed to investigate whether there is a relationship between plasma levels of leptin and phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and its potential relevance in the vascular remodeling in obese patients. METHODS: The study was performed in 164 obese and 94 normal-weight individuals (controls). NADPH oxidase activity was evaluated by luminescence in phagocytic cells. Levels of leptin were quantified by ELISA in plasma samples. Carotid intima-media thickness (cIMT) was measured by ultrasonography. In addition, we performed in-vitro experiments in human peripheral blood mononuclear cells and murine macrophages. RESULTS: Phagocytic NADPH oxidase activity and leptin levels were enhanced (P < 0.05) in obese patients compared with controls. NADPH oxidase activity positively correlated with leptin in obese patients. This association remained significant in a multivariate analysis. cIMT was higher (P < 0.05) in obese patients compared with controls. In addition, cIMT also correlated positively with leptin and NADPH oxidase activity in obese patients. In-vitro studies showed that leptin induced NADPH oxidase activation. Inhibition of the leptin-induced NADPH oxidase activity by wortmannin and bisindolyl maleimide suggested a direct involvement of the phosphatidylinositol 3-kinase and protein kinase C pathways, respectively. Finally, leptin-induced NADPH oxidase activation promoted macrophage proliferation. CONCLUSIONS: These findings show that phagocytic NADPH oxidase activity is increased in obesity and is related to preclinical atherosclerosis in this condition. We also suggest that hyperleptinemia may contribute to phagocytic NADPH oxidase overactivity in obesity.

The Pax6 transcription factor is a key element along brain development in both the visual and olfactory systems. The involvement of Pax6 in neural fate is well documented in the visual system, whereas in the olfactory system, and in particular in the olfactory bulb (OB), its expression during adulthood has only begun to be elucidated. In the OB, the modulation of primary sensory information is first performed by periglomerular cells (PG). A considerable body of information has unveiled the neurochemical heterogeneity of these neurons. Thus it is well known that Pax6 coexists with dopaminergic/GABAergic mouse PG. However, the presence of this transcription factor in other mouse PG subpopulations has not been studied. Here, we analyzed whether Pax6 is expressed in PG containing the calcium-binding proteins neurocalcin and parvalbumin, and the neuropeptide cholecystokinin. Our results show that Pax6 is not expressed by these PG subpopulations, suggesting that it is mainly restricted to GABAergic PG populations. These findings provide new data in the chemical characterization of mouse Pax6-positive PG.

Human serum albumin (HSA) is an effective therapeutic agent that protects neurons after cerebral ischemia or related injuries by means of its antioxidant capacity. Our aim was to test whether bovine serum albumin (BSA) might also provide protection, especially against DNA damage. Rat cortical neurons were cultured in both the presence and absence of BSA. To test the neuroprotective role of BSA against DNA damage and neuronal death, primary cultures were investigated using both gamma-H2AX and pATM immunocytochemistry, and the TUNEL assay, respectively. Quantitative analyses revealed that the cultures in the absence of BSA had a higher number of apoptotic neurons. Additionally, neurons showing DNA strand breaks were fewer when BSA was added to the medium. BSA acts as a neuroprotective molecule, reducing both the DNA damage and apoptosis rates. This effect is similar to that described for HSA, probably due to its antioxidant activity. Hence, we have demonstrated that BSA provides a neuroprotective role when DNA damage occurs. Additionally, we suggest that BSA probably shares similarities with HSA in its antioxidant activity, opening new ways in the study of stroke and related brain diseases.

A translocation experiment of red swamp crayfish (Procambarus clarkii) to different sites located in the River Guadiamar was performed in order to assess the ability of this species as bioindicator of heavy metal and metalloid contamination. Crayfish were placed in cages and exposed to polluted environment during either 6 or 12days in the three sites with different concentration of contaminants. Their tissues (exoskeleton+gills, hepatopancreas and abdominal muscle) were dissected and analysed by ICP-MS to assess for concentration of Cd, Cu, Zn, Pb and As. Both exposure times result in significant bioaccumulation of some metals in crayfish tissues as compared to their concentration in the environment. According to overall metal concentration, crayfish tissues rank as follows: hepatopancreas/viscera>exoskeleton/gills>abdominal muscle. Essential metals for crayfish metabolism (Cu and Zn) are always found in high concentrations independently of their quantities in the environment because of the ability of crayfish to manipulate their levels for their own metabolic profit. Metals not involved in crayfish metabolism (Cd, Pb, As) tend to increase with increasing concentration in the surrounding environment and with longer exposure times. Thus crayfish could be used as bioindicator of these pollutants because their dose- and time-dependent accumulation may be reflective of the levels of non-essential metals present in contaminated wetlands. Future guidelines in plans for monitoring contamination on polluted Mediterranean rivers and wetlands should take into account the implementation of the incubation of crayfish during 6days and their subsequent analyses of metal contents, as a routine.

The mammalian olfactory bulb (OB) has all the features of a whole mammalian brain but in a more reduced space: neuronal lamination, sensory inputs, afferences, or efferences to other centers of the central nervous system, or a contribution of new neural elements. Therefore, it is widely considered as "a brain inside the brain." Although this rostral region has the same origin and general layering as the other cerebral cortices, some distinctive features make it very profitable in experimentation in neurobiology: the sensory inputs are driven directly on its surface, the main output can be accessed anatomically, and new elements appear in it throughout adult life. These three morphological characteristics have been manipulated to analyze further the response of the whole OB. The present review offers a general outlook into the consequences of such experimentation in the anatomy, connectivity and neurochemistry of the OB after (a) sensory deprivation, mainly by naris occlusion; (b) olfactory deinnervation by means of olfactory epithelium damage, olfactory nerve interruption, or even olfactory tract disruption; (c) the removal of the principal neurons of the OB; and (d) management of the arrival of newborn interneurons from the rostral migratory stream. These experiments were performed using surgical or chemical methods, but also by means of the analysis of genetic models, some of whose olfactory components are missing, colorless or mismatching within the wild-type scenario of odor processing.

Publisher's version, Northern freshwater fish may be suitable for the genetic dissection of ecological traits because they invaded new habitats after the last ice age (∼10.000 years ago). Arctic charr (Salvelinus alpinus) colonizing streams and lakes in Iceland gave rise to multiple populations of small benthic morphotypes, often in sympatry with a pelagic morphotype. Earlier studies have revealed significant, but subtle, genetic differentiation between the three most common morphs in Lake Thingvallavatn. We conducted a population genetic screen on four immunological candidate genes Cathelicidin 2 (Cath2), Hepcidin (Hamp), Liver expressed antimicrobial peptide 2a (Leap-2a), and Major Histocompatibility Complex IIα (MHCIIα) and a mitochondrial marker (D-loop) among the three most common Lake Thingvallavatn charr morphs. Significant differences in allele frequencies were found between morphs at the Cath2 and MHCIIα loci. No such signal was detected in the D-loop nor in the other two immunological genes. In Cath2 the small benthic morph deviated from the other two (FST = 0.13), one of the substitutions detected constituting an amino acid replacement polymorphism in the antimicrobial peptide. A more striking difference was found in the MHCIIα. Two haplotypes were very common in the lake, and their frequency differed greatly between the morphotypes (from 22% to 93.5%, FST = 0.67). We then expanded our study by surveying the variation in Cath2 and MHCIIα in 9 Arctic charr populations from around Iceland. The populations varied greatly in terms of allele frequencies at Cath2, but the variation did not correlate with morphotype. At the MHCIIα locus, the variation was nearly identical to the variation in the two benthic morphs of Lake Thingvallavatn. The results are consistent with a scenario where parts of the immune systems have diverged substantially among Arctic charr populations in Iceland, after colonizing the island ∼10.000 years ago., The Palsson laboratory is supported by Icelandic Research foundation and the University of Iceland research fund. Icelandic research foundation (grant of excellence: nr 100204011) to S.S. Sigurdsson, A. Palsson, B.K. Kristjansson, Zophonias O. Jonsson and Ian A. Johnston paid for part of this work. Kalina H. Kapralova and Johannes Gudbrandsson were supported by the University of Iceland doctoral fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., Ritrýnt tímarit

Herein, we investigate whether the NADPH oxidase might be playing a key role in the degree of oxidative stress in the senescence-accelerated mouse prone-8 (SAM-P8). To this end, the activity and expression of the NADPH oxidase, the ratio of glutathione and glutathione disulfides (GSH/GSSG), and the levels of malonyl dialdehyde (MDA) and nitrotyrosine (NT) were determined in renal tissue from SAM-P8 mice at the age of 1 and 6 months. The senescence-accelerated-resistant mouse (SAM-R1) was used as control. At the age of 1 month, NADPH oxidase activity and Nox2 protein expression were higher in SAM-P8 than in SAM-R1 mice. However, we found no differences in the GSH/GSSG ratio, MDA, NT, and Nox4 levels between both groups of animals. At the age of 6 months, SAM-R1 mice in comparison to SAM-P8 mice showed an increase in NADPH oxidase activity, which is associated with higher levels of NT and increased Nox4 and Nox2 expression levels. Furthermore, we found oxidative stress hallmarks including depletion in GSH/GSSG ratio and increase in MDA levels in the kidney of SAM-P8 mice. Finally, NADPH oxidase activity positively correlated with Nox2 expression in all the animals (r = 0.382, P < 0.05). Taken together, our data allow us to suggest that an increase in NADPH oxidase activity might be an early hallmark to predict future oxidative stress in renal tissue during the aging process that takes place in SAM-P8 mice.

et al., Sos1 and Sos2 are ubiquitously expressed, universal Ras guanine nucleotide exchange factors (Ras-GEFs) acting in multiple signal transduction pathways activated by upstream cellular kinases. The embryonic lethality of Sos1 null mutants has hampered ascertaining the specific in vivo contributions of Sos1 and Sos2 to processes controlling adult organism survival or development of hematopoietic and nonhematopoietic organs, tissues, and cell lineages. Here, we generated a tamoxifen-inducible Sos1-null mouse strain allowing analysis of the combined disruption of Sos1 and Sos2 (Sos1/2) during adulthood. Sos1/2 double-knockout (DKO) animals died precipitously, whereas individual Sos1 and Sos2 knockout (KO) mice were perfectly viable. A reduced percentage of total bone marrow precursors occurred in single-KO animals, but a dramatic depletion of B-cell progenitors was specifically detected in Sos1/2 DKO mice. We also confirmed a dominant role of Sos1 over Sos2 in early thymocyte maturation, with almost complete thymus disappearance and dramatically higher reduction of absolute thymocyte counts in Sos1/2 DKO animals. Absolute counts of mature B and T cells in spleen and peripheral blood were unchanged in single-KO mutants, while significantly reduced in Sos1/2 DKO mice. Our data demonstrate functional redundancy between Sos1 and Sos2 for homeostasis and survival of the full organism and for development and maturation of T and B lymphocytes., Work was supported by grants FIS-PS09/01979, RTICC-RD12/0036/0001, and RD12/0036/0048 from Instituto de Salud Carlos III (Madrid, Spain) and Fundación Samuel Solórzano (Salamanca, Spain). This research was also supported by the Intramural Research Program of the CCR, NCI, NIH.

DNA repair protects neurons against spontaneous or disease-associated DNA damage. Dysfunctions of this mechanism underlie a growing list of neurodegenerative disorders. The Purkinje cell (PC) degeneration mutation causes the loss of nna1 expression and is associated with the postnatal degeneration of PCs. This PC degeneration dramatically affects nuclear architecture and provides an excellent model to elucidate the nuclear mechanisms involved in a whole array of neurodegenerative disorders. We used immunocytochemistry for histone variants and components of the DNA damage response, an in situ transcription assay, and in situ hybridization for telomeres to analyze changes in chromatin architecture and function. We demonstrate that the phosphorylation of H2AX, a DNA damage signal, and the trimethylation of the histone H4K20, a repressive mark, in extensive domains of genome are epigenetic hallmarks of chromatin in degenerating PCs. These histone modifications are associated with a large scale reorganization of chromatin, telomere clustering, and heterochromatin-induced gene silencing, all of them key factors in PC degeneration. Furthermore, ataxia telangiectasia mutated and 53BP1, two components of the DNA repair pathway, fail to be concentrated in the damaged chromatin compartments, even though the expression levels of their coding genes were slightly up-regulated. Although the mechanism by which Nna1 loss of function leads to PC neurodegeneration is undefined, the progressive accumulation of DNA damage in chromosome territories irreversibly compromises global gene transcription and seems to trigger PC degeneration and death., This work was supported by Dirección General de Investigación Grant BFU2008-00175, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas Grant CB06/05/0037 from Instituto de Salud Carlos III, Ministerio de Ciencia y Tecnología Grant BFU2010-18284, Ministerio de Sanidad, Política Social e Igualdad (Plan Nacional Sobre Drogas), Instituto de Formación e Investigación Marqués de Valdecilla Grant FMV/UC09-02, Junta de Castilla y León, and Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León.

The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.

The adult brain is considered to be a radioresistant organ since it is mainly composed of non-dividing cells. However, in adult animals there are a few neurogenic brain areas that are affected by ionizing radiation whose plasticity and capacity for recovery are still unclear. Here, mice were irradiated with a minimal lethal dose of radiation in order to determine its effects on the subventricular zone (SVZ), the rostral migratory stream (RMS), and the olfactory bulb (OB). These regions underwent a dramatic reduction in cell proliferation and ensuing morphological alterations, accompanied by a patent reactive gliosis. Bone marrow stem cell (BMSC) transplants were also performed after the radiation treatment to allow the mouse survival with a view to analyzing long-term effects. Normal proliferation rates were not recovered over time and although bone marrow-derived cells reached the brain, they were not incorporated into the SVZ-RMS-OB pathway in an attempt to rescue the damaged regions. Since neurogenesis produces new interneurones in the OB, thus feeding cell turnover, the volume and lamination of the OB were analyzed. The volume of the OB proved to be dramatically reduced at postnatal day 300 (P300), and this shrinkage affected the periependymal white matter, the granule cell layer, the external plexiform layer, and the glomerular layer. These results should be taken into account in cell therapies employing BMSC, since such cells reach the encephalon, although they cannot restore the damage produced in neurogenic areas. This study thus provides new insight into the long-term effects of ionizing radiation, widely employed in animal experimentation and even in clinical therapies for human beings.

One of the sexual dimorphic differences in adult rodents is neural proliferation. Here we demonstrate that physiological hormone stages can modulate this proliferation in the adult forebrain. Female mice, both pregnant and synchronized in oestrus, exhibited higher proliferating cell percentages than males in both the rostral migratory stream (RMS) and the olfactory bulb (OB). Moreover, although the hormonal component also influenced the subventricular zone (SVZ), no differences in proliferation were observed in this region. In addition, both groups of females had higher numbers of serotonergic fibres in these regions. Serotonin may therefore be related to the mechanism of action by which hormones can affect cell proliferation of this brain region. We also evaluated cell death in the SVZ in males and females, finding that this was higher in the former. Taken together, our results support the idea that in female rodents more neuroblasts are able to reach the RMS and then proliferate, apoptosis being an additional mechanism affecting the low proliferation of cells in the RMS and OB in males. Thus, proliferation in the RMS is influenced by sexual dimorphism.

Zinc ions are selectively accumulated in certain neurons (zinc-enriched neurons). The mouse olfactory bulb is richly innervated by zinc-enriched terminals. Here, the plasticity of the zincergic system was studied in the olfactory bulb of the Purkinje Cell Degeneration mutant mouse, an animal with specific postnatal neurodegeneration of the main projection neurons of the olfactory bulb. The analysis focused particularly on the anterior olfactory nucleus since most centrifugal afferents coming to the olfactory bulb arise from this structure. Zinc-enriched terminals in the olfactory bulb and zinc-enriched somata in the anterior olfactory nucleus were visualized after selenite injections. Immunohistochemistry against the vesicular zinc transporter was also carried out to confirm the distribution pattern of zinc-enriched terminals in the olfactory bulb. The mutant mice showed a clear reorganization of zincergic centrifugal projections from the anterior olfactory nucleus to the olfactory bulb. First, all zincergic contralateral neurons projecting to the olfactory bulb were absent in the mutant mice. Second, a significant increase in the number of stained somata was detected in the ipsilateral anterior olfactory nucleus. Since no noticeable changes were observed in the zinc-enriched terminals in the olfactory bulb, it is conceivable that mitral cell loss could induce a reorganization of zinc-enriched projections coming from the anterior olfactory nucleus, probably directed at balancing the global zincergic centrifugal modulation. These results show that zincergic anterior olfactory nucleus cells projecting to the olfactory bulb undergo plastic changes to adapt to the loss of mitral cells in the olfactory bulb of Purkinje Cell Degeneration mutant mice.

A new technique, based on live Sperm Inclusion in Microgels (SIM), allows quick and easy analysis of giant spermatozoa under bright field or fluorescence microscopy. The technique has been assayed on Drosophila melanogaster (Diptera) and Eucypris virens (Ostracoda) spermatozoa and based on the inclusion of freshly obtained male gametes in low melting agarose microgels at 37 degrees C to prevent cell damage. Gametes spread onto pretreated slides are dehydrated and directly observed under phase contrast microscopy or stained with specific fluorochromes for DNA or proteins. Results show that the morphology of whole sperm is highly preserved allowing identification of sperm characteristics difficult to visualize after using standard fixation procedures. In Drosophila melanogaster, SIM allows the simultaneous visualization of the complete flagellum plus a clear delineation of the DNA. It also allows observation of morphological changes in the perforatorium as sperm elongation takes place. In E. virens, SIM slides visualized under phase contrast and fluorescence microscopy show three main morphological regions on the entire spermatozoa. Spermatozoa of this species are auto-fluorescent under wavelength excitation from 387 to 562 nm. In spite of this, clear localization of the DNA molecule at the posterior 1/3 part of the whole spermatozoa can also be achieved after single DAPI staining or in combination with Mercuri-diBrom-Fluorescein, a fluorochrome for protein targeting.

The distribution patterns of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the olfactory bulb (OB) of three species of macaque. AChE was detected by a histochemical method and ChAT immunoreactivity by immunocytochemistry. Similar results were observed in all species analyzed. With the exception of the olfactory nerve layer, all layers of the macaque monkey OB demonstrated a dense innervation of AChE- and ChAT-positive fibers. The distribution patterns of AChE- and ChAT-labeled fibers were similar for both cholinergic markers, although the number of AChE-labeled fibers was clearly higher than the number of ChAT-immunoreactive fibers. The highest density of AChE and ChAT-stained fibers was observed in the interface between the glomerular layer and the external plexiform layer and in the internal plexiform layer. Dense bundles of labeled fibers were observed in the caudal OB, coursing from the olfactory peduncle. All ChAT-immunopositive elements were identified as centrifugal fibers, derived from neurons caudal to the OB. Neither olfactory fibers nor intrinsic neurons were observed after ChAT immunocytochemistry. However, a few AChE-positive cells were observed in the glomerular layer and in both external and internal plexiform layers. These neurons were presumably identified as periglomerular cells, superficial short-axon cells, and/or external tufted cells and deep short-axon cells. Contrary to other neurotransmitters and neuroactive substances, the distribution patterns of ChAT and AChE activities in the macaque monkey OB closely resembled the patterns described in macrosmatic mammals and showed laminar differences with the distribution pattern observed in humans.

The effect of a high-frequency signal on the FitzHugh-Nagumo excitable model is analyzed. We show that the firing rate is diminished as the ratio of the high-frequency amplitude to its frequency is increased. Moreover, it is demonstrated that the excitable character of the system, and consequently the firing activity, is suppressed for ratios above a given threshold value. In addition, we show that the vibrational resonance phenomenon turns up for sufficiently large noise strength values., Comment: 4 pages, 4 figures (to appear in Physical Review E)

The focus of this paper is on the synchronous activity of a system of two intrinsically noisy Hodgkin-Huxley neurons coupled by a diffusive interaction. It is shown that conductance noise allows the relative phase of the neurons to display several different dynamical regimes ranging from phase and antiphase locking to random switching between two or more states. A synchronization diagram displaying the structure of the distribution function of the cyclic relative phase of the two neurons is presented. The addition of sinusoidal forcing terms to the equations governing the membrane voltage of both neurons gives rise to the statistical locking of those random switchings to the phase of the external signal.