Neuron glia biology最新文献

{"title":"Is astrocyte calcium signaling relevant for synaptic plasticity?","authors":"Sarrah Ben Achour,&nbsp;Lorena Pont-Lezica,&nbsp;Catherine Béchade,&nbsp;Olivier Pascual","doi":"10.1017/S1740925X10000207","DOIUrl":"https://doi.org/10.1017/S1740925X10000207","url":null,"abstract":"<p><p>Astrocytes constitute a major group of glial cells which were long regarded as passive elements, fulfilling nutritive and structural functions for neurons. Calcium rise in astrocytes propagating to neurons was the first demonstration of direct interaction between the two cell types. Since then, calcium has been widely used, not only as an indicator of astrocytic activity but also as a stimulator switch to control astrocyte physiology. As a result, astrocytes have been elevated from auxiliaries to neurons, to cells involved in processing synaptic information. Curiously, while there is evidence that astrocytes play an important role in synaptic plasticity, the data relating to calcium's pivotal role are inconsistent. In this review, we will detail the various mechanisms of calcium flux in astrocytes, then briefly present the calcium-dependent mechanisms of gliotransmitter release. Finally, we will discuss the role of calcium in plasticity and present alternative explanations that could reconcile the conflicting results published recently.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000207","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29505672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prostaglandin E2 induces glutamate release from subventricular zone astrocytes.","authors":"Kathleen A Dave,&nbsp;Jean-Claude Platel,&nbsp;Frank Huang,&nbsp;David Tian,&nbsp;Severine Stamboulian-Platel,&nbsp;Angélique Bordey","doi":"10.1017/S1740925X10000244","DOIUrl":"https://doi.org/10.1017/S1740925X10000244","url":null,"abstract":"<p><p>It was recently reported that in one of the adult neurogenetic zones, the subventricular zone (SVZ), astrocyte-like cells release glutamate upon intracellular Ca2+ increases. However, the signals that control Ca2+ activity and glutamate release from SVZ astrocytes are not known. Here, we examined whether prostaglandin E2 (PGE2), which induces glutamate release from mature astrocytes, is such a signal. Using the gramicidin-perforated patch-clamp technique, we show that the activity of N-Methyl-D-Aspartate receptor (NMDAR) channel in neuroblasts is a high fidelity sensor of ambient glutamate levels. Using such sensors, we found that application of PGE2 led to increased ambient glutamate levels in the SVZ. In parallel experiments, PGE2 induced an increase in intracellular Ca2+ levels in SVZ cells, in particular astrocyte-like cells, as shown using Ca2+ imaging. Finally, a PGE2 enzyme immunoassay showed that the choroid plexus of the lateral ventricle and to a lesser extent the SVZ (ten-fold less) released PGE2. These findings suggest that PGE2 is a physiological signal for inducing glutamate release from SVZ astrocytes that is important for controlling neuroblast survival and proliferation. This signal may be accentuated following ischemia or injury-induced PGE2 release and may contribute to the injury-associated increased neurogenesis.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000244","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29580727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Astrocyte proximity modulates the myelination gene fabric of oligodendrocytes.","authors":"Sanda Iacobas,&nbsp;Dumitru A Iacobas","doi":"10.1017/S1740925X10000220","DOIUrl":"https://doi.org/10.1017/S1740925X10000220","url":null,"abstract":"<p><p>Extensive literature documented that astrocytes release neurotransmitters, cytokines and other signaling molecules to modulate migration, maturation and myelin synthesis of oligodendrocytes through mechanisms primarily converging on cytosolic [Ca2+] transients. Considering the long-term effects, it is expected that astrocyte-conditioned medium is a major regulator of gene expression in oligodendrocytes even in the absence of cytosol-to-cytosol communication via astrocyte-oligodendrocyte gap junction channels. Indeed, by comparing the transcriptomes of immortalized precursor oligodendrocyte (Oli-neu) cells when cultured alone and co-cultured with non-touching astrocytes we found profound changes in the gene expression level, control and networking. Remarkably, the astrocyte proximity was more effective in remodeling the myelination (MYE) gene fabric and its control by cytokine receptor (CYR)-modulated intercellular Ca2+-signaling (ICS) transcriptomic network than the dibutyryl-cAMP (db-cAMP) treatment-induced transformation into myelin-associated glycoprotein-positive oligodendrocyte-like cells. Moreover, astrocyte proximity up-regulated 37 MYE genes and switched on another 14 MYE, 23 ICS and 4 CYR genes, enhancing the roles of the leukemia inhibitory factor receptor and connexins Cx29 and Cx47. The novel prominent gene analysis identified the enhancer of zeste homolog 2 as the most relevant MYE gene in the astrocyte proximity, notch gene homolog 1 in control and B-cell leukemia/lymphoma 2 in differentiated Oli-neu cells.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000220","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29577091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters.","authors":"Eiji Shigetomi,&nbsp;Sebastian Kracun,&nbsp;Baljit S Khakh","doi":"10.1017/S1740925X10000219","DOIUrl":"https://doi.org/10.1017/S1740925X10000219","url":null,"abstract":"<p><p>Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and 'spotty' in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000219","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29576055","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bergmann glial ensheathment of dendritic spines regulates synapse number without affecting spine motility.","authors":"Jocelyn J Lippman Bell,&nbsp;Tamar Lordkipanidze,&nbsp;Natalie Cobb,&nbsp;Anna Dunaevsky","doi":"10.1017/S1740925X10000165","DOIUrl":"https://doi.org/10.1017/S1740925X10000165","url":null,"abstract":"<p><p>In the cerebellum, lamellar Bergmann glial (BG) appendages wrap tightly around almost every Purkinje cell dendritic spine. The function of this glial ensheathment of spines is not entirely understood. The development of ensheathment begins near the onset of synaptogenesis, when motility of both BG processes and dendritic spines are high. By the end of the synaptogenic period, ensheathment is complete and motility of the BG processes decreases, correlating with the decreased motility of dendritic spines. We therefore have hypothesized that ensheathment is intimately involved in capping synaptogenesis, possibly by stabilizing synapses. To test this hypothesis, we misexpressed GluR2 in an adenoviral vector in BG towards the end of the synaptogenic period, rendering the BG α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) Ca2+-impermeable and causing glial sheath retraction. We then measured the resulting spine motility, spine density and synapse number. Although we found that decreasing ensheathment at this time does not alter spine motility, we did find a significant increase in both synaptic pucta and dendritic spine density. These results indicate that consistent spine coverage by BG in the cerebellum is not necessary for stabilization of spine dynamics, but is very important in the regulation of synapse number.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000165","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29441147","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of functional units within trigeminal ganglia correlates with increased expression of proteins involved in neuron-glia interactions.","authors":"Paul L Durham,&nbsp;F G Garrett","doi":"10.1017/S1740925X10000232","DOIUrl":"https://doi.org/10.1017/S1740925X10000232","url":null,"abstract":"<p><p>Cell bodies of trigeminal nerves, which are located in the trigeminal ganglion, are completely surrounded by satellite glial cells and together form a functional unit that regulates neuronal excitability. The goals of this study were to investigate the cellular organization of the rat trigeminal ganglia during postnatal development and correlate those findings with expression of proteins implicated in neuron-glia interactions. During postnatal development there was an increase in the volume of the neuronal cell body, which correlated with a steady increase in the number of glial cells associated with an individual neuron from an average of 2.16 at birth to 7.35 on day 56 in young adults. Interestingly, while the levels of the inwardly rectifying K+ channel Kir4.1 were barely detectable during the first week, its expression in satellite glial cells increased by day 9 and correlated with initial formation of functional units. Similarly, expression of the vesicle docking protein SNAP-25 and neuropeptide calcitonin gene-related peptide was readily detected beginning on day 9 and remained elevated throughout postnatal development. Based on our findings, we propose that the expression of proteins involved in facilitating neuron-glia interactions temporally correlates with the formation of mature functional units during postnatal development of trigeminal ganglion.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000232","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29575445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The myelin proteolipid DMα in fishes.","authors":"Christian Brösamle","doi":"10.1017/S1740925X09000131","DOIUrl":"https://doi.org/10.1017/S1740925X09000131","url":null,"abstract":"<p><p>Vertebrate myelin membranes are compacted and held in close apposition by three structural proteins of myelin, myelin basic protein, myelin protein zero (MPZ) and myelin proteolipid protein (PLP1/DMalpha). PLP1/DMalpha is considered to function as a scaffolding protein and play a role in intracellular trafficking in oligodendrocytes. In humans, point mutations, duplications or deletions of PLP1 are associated with Pelizaeus-Merzbacher disease and spastic paraplegia Type 2. PLP1 is highly conserved between mammals, but less so in lower vertebrates. This has led some researchers to question whether certain fish species express PLP1 orthologues at all, and to suggest that the function of PLP1/DMalpha in the central nervous system (CNS) may have been taken over by MPZ. Here, we review the evidence for the conservation of orthologues of PLP1/DMalpha in actinopterygian fishes and provide a comparison of currently available sequence data across 17 fish species. Our analysis demonstrates that orthologues of PLP1/DMalpha have been retained and are functionally expressed in many, if not all, extant species of bony fish. Many of the amino acids that, when mutated, are associated with severe CNS pathology are conserved in teleosts, demonstrating conservation of essential functions and justifying the development of novel disease models in species such as the zebrafish.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X09000131","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28231572","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"5-HT2B receptors are expressed on astrocytes from brain and in culture and are a chronic target for all five conventional 'serotonin-specific reuptake inhibitors'.","authors":"Shiquen Zhang,&nbsp;Baoman Li,&nbsp;Ditte Lovatt,&nbsp;Junnan Xu,&nbsp;Dan Song,&nbsp;Steven A Goldman,&nbsp;Maiken Nedergaard,&nbsp;Leif Hertz,&nbsp;Liang Peng","doi":"10.1017/S1740925X10000141","DOIUrl":"https://doi.org/10.1017/S1740925X10000141","url":null,"abstract":"<p><p>In well-differentiated primary cultures of mouse astrocytes, which express no serotonin transporter (SERT), the 'serotonin-specific reuptake inhibitor' (SSRI) fluoxetine leads acutely to 5-HT2B receptor-mediated, transactivation-dependent phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) with an EC50 of ~5 μM, and chronically to ERK1/2 phosphorylation-dependent upregulation of mRNA and protein expression of calcium-dependent phospholipase A2 (cPLA2) with ten-fold higher affinity. This affinity is high enough that fluoxetine given therapeutically may activate astrocytic 5-HT2B receptors (Li et al., 2008, 2009). We now confirm the expression of 5-HT2B receptors in astrocytes freshly dissociated from mouse brain and isolated by fluorescence-activated cell sorting (FACS) and investigate in cultured cells if the effects of fluoxetine are shared by all five conventional SSRIs with sufficiently high affinity to be relevant for mechanism(s) of action of SSRIs. Phosphorylated and total ERK1/2 and mRNA and protein expression of cPLA2a were determined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Paroxetine, which differs widely from fluoxetine in affinity for SERT and for another 5-HT2 receptor, the 5-HT2C receptor, acted acutely and chronically like fluoxetine. One micromolar of paroxetine, fluvoxamine or sertraline increased cPLA2a expression during chronic treatment; citalopram had a similar effect at 0.1-0.5 μM; these are therapeutically relevant concentrations.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000141","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40073736","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ouabain binding kinetics and FXYD7 expression in astrocytes and neurons in primary cultures: implications for cellular contributions to extracellular K+ homeostasis?","authors":"Liang Peng,&nbsp;Rong Huang,&nbsp;Shiquen Zhang,&nbsp;Leif Hertz","doi":"10.1017/S1740925X10000013","DOIUrl":"https://doi.org/10.1017/S1740925X10000013","url":null,"abstract":"<p><p>Although Na+,K+-ATPase-mediated K+ uptake into astrocytes plays a major role in re-establishing resting extracellular K+ following neuronal excitation little information is available about astrocytic Na+,K+-ATPase function, let alone mechanisms returning K+ to neurons. The catalytic units of the Na+,K+-ATPase are the astrocyte-specific α2, the neuron-specific α3 and the ubiquitously expressed α1. In the present work, Bmax and KD values for α1, α2 and α3 subunits were computed in cultured cerebro-cortical mouse astrocytes and cerebellar granule neurons by non-linear regression as high-affinity (α2, α3) and low-affinity (α1) [3H]ouabain binding sites, which stoichiometrically equal transporter sites. Cellular expression was also determined of the brain- and α1-β1 isoform-specific FDYX7, regulating Na+,K+-ATPase efficiency and K+-sensitivity. From ouabain-sensitive K+ uptake rates published by ourselves (Walz and Hertz, 1982) or others (Atterwill et al., 1985), Na+,K+-ATPase turnover was determined. Subunits α2 and α3 showed Bmax of 15-30 pmol/mg protein, with maximum turnover rates of 70-80/s. Bmax of the α1 subunit was low in neurons but very high in astrocytes (645 pmol/mg protein), where turnover rate was slow, reflecting expression of selectively expressed FXYD7, and binding was increased by K+. The role of these characteristics for K+ homeostasis are discussed.</p>","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000013","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"28740081","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Central role of glia in disease research.","authors":"R Douglas Fields","doi":"10.1017/S1740925X10000177","DOIUrl":"https://doi.org/10.1017/S1740925X10000177","url":null,"abstract":"Widening interest in glial involvement in neurological and psychiatric illness is evident from the increasing number and diversity of studies with a focus on glia published in leading journals. Examples of this include recent articles published in Neurology and the Proceedings of the National Academy of Science, and all of the papers in this issue of Neuron Glia Biology. \u0000 \u0000Multiple sclerosis (MS) is perhaps the prototypic glial disease, but the disorder is characterized by wide ranging variation in severity and disease progression among individuals. In large part this reflects the relapsing-remitting cycles of autoimmune damage to myelin and its repair, but increasingly, disorders once lumped together under the diagnosis of MS are being isolated as separate diseases with distinct pathophysiological mechanisms. In the process, more diverse roles for glia are being revealed. \u0000 \u0000Neuromylitis optica (NMO) is an inflammatory disease that attacks the optic nerve and spinal cord, causing white matter lesions that result in blindness and paralysis. NMO is now seen as distinct from MS, and the most recent evidence supports the hypothesis that the direct target of the disorder is not oligodendrocytes, but rather astrocytes. A Neurology paper by Takano and colleagues (2010) reports that glial fibrillary acidic protein (GFAP) in cerebral spinal fluid (CSF) is massively increased in patients with NMO compared to patients with MS. This is consistent with previous studies linking NMO with anti-aquaporin-4 (AQP4) autoantibodies (Lennon et al., 2005), and the depletion of astrocytic foot processes and loss of AQP-4 (water channel) in pathological studies of MNO patients. This astrocyte-specific biomarker in cerebral spinal fluid can distinguish NMO from MS and it can be used to track the efficacy of treatments (Giovannoni 2010). Such markers are extremely desirable in monitoring CNS disease where biopsy is rarely performed. At the same time the results illustrate the interdependence of astrocytes, olgodendrocytes, and axons for normal function. \u0000 \u0000GFAP in the CSF is an indicator of astrocyte damage, but many signals are released from glia to regulate nervous system development, response to injury, and synaptogenesis. Levels of the protein S100 in CSF, for example, showed a similar trend to AQP-4 in NMO and MS patients. S100 protein is secreted from astrocytes and it acts as a neuromodulator. A study published by Gomez-Casati et al (2010) addresses the question of how the release of signaling molecules from glia that promote synaptogenesis is regulated by signals from neurons. I these studies signaling by the neuregulin tyrosine kinase receptor (erbB) was eliminated in glial cells in the inner ear of mice by expression of a DN-erbB4 receptor under control of the GFAP promoter. The researchers report that BDNF is the synaptogenic signal produced by nonneuronal cells in the vestibular sensory epithelium in response to the growth factor neuregulin (NRG1), which is p","PeriodicalId":19153,"journal":{"name":"Neuron glia biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2010-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/S1740925X10000177","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"29365973","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}