Suchergebnisse

In einer Währungsunion besteht für die Mitgliedsländer keine Möglichkeit mehr, durch eine vorübergehende Störung verursachte binnenwirtschaftliche Ungleichgewichte mittels Anpassung des nominalen Wechselkurses zu korrigieren. Wenn die supranationale Geldpolitik unionsweiten, die nationale Fiskalpolitik jedoch in erster Linie einzelstaatlichen Zielen verpflichtet ist, stellt sich die Frage nach den stabilisierungspolitischen und wohlfahrtstheoretischen Konsequenzen eines aus dieser Konstellation möglicherweise erwachsenden Zielkonfliktes zwischen den politischen Akteuren.

In einer Währungsunion besteht für die Mitgliedsländer keine Möglichkeit mehr, durch eine vorübergehende Störung verursachte binnenwirtschaftliche Ungleichgewichte mittels Anpassung des nominalen Wechselkurses zu korrigieren. Wenn die supranationale Geldpolitik unionsweiten, die nationale Fiskalpolitik jedoch in erster Linie einzelstaatlichen Zielen verpflichtet ist, stellt sich die Frage nach den stabilisierungspolitischen und wohlfahrtstheoretischen Konsequenzen eines aus dieser Konstellation möglicherweise erwachsenden Zielkonfliktes zwischen den politischen Akteuren.

In einer Währungsunion besteht für die Mitgliedsländer keine Möglichkeit mehr, durch eine vorübergehende Störung verursachte binnenwirtschaftliche Ungleichgewichte mittels Anpassung des nominalen Wechselkurses zu korrigieren. Wenn die supranationale Geldpolitik unionsweiten, die nationale Fiskalpolitik jedoch in erster Linie einzelstaatlichen Zielen verpflichtet ist, stellt sich die Frage nach den stabilisierungspolitischen und wohlfahrtstheoretischen Konsequenzen eines aus dieser Konstellation möglicherweise erwachsenden Zielkonfliktes zwischen den politischen Akteuren.

In einer Währungsunion besteht für die Mitgliedsländer keine Möglichkeit mehr, durch eine vorübergehende Störung verursachte binnenwirtschaftliche Ungleichgewichte mittels Anpassung des nominalen Wechselkurses zu korrigieren. Wenn die supranationale Geldpolitik unionsweiten, die nationale Fiskalpolitik jedoch in erster Linie einzelstaatlichen Zielen verpflichtet ist, stellt sich die Frage nach den stabilisierungspolitischen und wohlfahrtstheoretischen Konsequenzen eines aus dieser Konstellation möglicherweise erwachsenden Zielkonfliktes zwischen den politischen Akteuren.

Neural circuits in the cerebral cortex consist of excitatory pyramidal cells and inhibitory interneurons. These two main classes of cortical neurons follow largely different genetic programs, yet they assemble into highly specialized circuits during development following a very precise choreography. Previous studies have shown that signals produced by pyramidal cells influence the migration of cortical interneurons, but the molecular nature of these factors has remained elusive. Here, we identified Neuregulin 3 (Nrg3) as a chemoattractive factor expressed by developing pyramidal cells that guides the allocation of cortical interneurons in the developing cortical plate. Gain- and loss-of-function approaches reveal that Nrg3 modulates the migration of interneurons into the cortical plate in a process that is dependent on the tyrosine kinase receptor ErbB4. Perturbation of Nrg3 signaling in conditional mutants leads to abnormal lamination of cortical interneurons. Nrg3 is therefore a critical mediator in the assembly of cortical inhibitory circuits. ; uterus, and 1 m g/ m L pCAG - Gfp or Nrg3 (kindly provided by C. Lai, Indiana Uni- versity, Bloomington, and subcloned into pCAGGS) plasmids were injected into the lateral ventricle of the telencephalon through the uterine wall. Square electric pulses of 45 V and 50 ms were passed through the uterus five times, spaced 950 ms, using a square pulse electroporator. The uterine horns were placed back in the abdominal cavity, which was then suture closed, and the female was allowed to recover. Explant Cultures For COS cell confrontation assays, COS7 cells were transfected with plasmids encoding Rfp alone, Rfp and Cxcl12 , Rfp and Nrg3 , Rfp and CRD-Nrg1 ,or Rfp and Ig-Nrg1 , and cell aggregates were prepared by diluting transfected cells with Matrigel in a 1:1 proportion. After jellification, COS cell aggregates were cut with a scalpel in small rectangular prisms of approximately 400 3 400 3 800 m m and confronted to MGE explants obtained from GFP-expressing trans- genic mice in 3D Matrigel pads. The cDNA used for expression of Cxcl12 was obtained from Invitrogen (clone number: 3483088; accession number: BC006640). Nrg3 was kindly provided by Cary Lai (Indiana University, Bloo- mington). The sequences used for expression of type I NRG1 ( Ig-Nrg1 ) and type III NRG1 ( CRD-Nrg1 ) correspond to the accession numbers AY648976 and AY648975, respectively. For Cxcl12 chemokine-blocking experiments, SU6656 (Sigma; 330161-87-0) was added to the medium at a final concentra- tion of 15 m M. Previous worked has shown that Src functions downstream of Cxcr4 activation ( Cabioglu et al., 2005 ). In Vitro Focal Electroporation Coronal slice cultures were obtained as described previously ( Anderson et al., 1997 ). A pCAGG-based dsRed plasmid was pressure injected focally into the MGE of coronal slice cultures by a Pneumatic PicoPump through a glass micropipette. Slices were then electroporated within a setup of two hor- izontally oriented platinum electrodes powered by a Electro-Square-Porator, as described before ( Flames et al., 2004 ). Time-Lapse Videomicroscopy Slices were transferred to the stage of an upright Leica DMLFSA or inverted Leica DMIRE2 microscope coupled to a confocal spectral scanning head (Leica; TCS SL) and viewed through 10–60 3 water immersion or 20 3 oil objec- tives. Slices were continuously superfused with warmed (32 C) artificial cere- brospinal fluid at a rate of 1 mL/min or maintained in supplemented Neurobasal medium. To block Cxcl12 function, SU6656 (Sigma; 330161-87-0) was added to the medium at a final concentration of 15 m M. Stripe Assay Purified CXCL12 protein was obtained from PeproTech (250-20A) and used at 1 ng/ m L. GST and EGF-Nrg3-GST were purified using standard protocols and used at 10 m g/mL. Alternating lanes, 50 m m wide, were laid down on a poly- lysine-coated plastic dish. Alexa 555-labeled anti-rabbit IgGs were added to the GST, EGF-Nrg3-GST, and CXCL12 protein solution for lane identification. The lanes were further coated with laminin. MGE explants were dissected out of GFP + brain slices, plated on top of the protein stripes, and incubated in methylcellulose-containing Neurobasal medium for 48 hr. FACS We dissected the sensorimotor cortex of E17.5 embryos and P4 pups following in utero electroporation at E14.5. Cortical tissue was dissociated as described previously ( Catapano et al., 2001 ). GFP + cells were purified using fluorescent activated cell sorting (FACSARIA III; BD Biosciences), and the re- sulting pellet was kept at 80 C. TaqMan Gene Expression Assays We isolated GFP + pyramidal cells by FACS at E17.5 and P4 after in utero elec- troporation at E14.5. mRNA was then extracted using the RNeasy Micro Kit (QIAGEN) according to the manufacturer's instructions. RNA quality was as- sessed using a bioanalyzer (Agilent Technologies) and then retro-transcribed into single-stranded cDNA. The RNA was sent to Unidad Geno ́ mica (Funda- cio ́ n Parque Cientı ́fico de Madrid) for quality control and retro-transcription. Relative gene expression levels from three independent samples were analyzed using custom designed TaqMan low-density array (TLDA) plates (Micro Fluidic Cards; Applied Biosystems). Each plate contained duplicates for all the genes shown in Table S1 . Data were collected and analyzed using the threshold cycle (Ct) relative quantification method. The housekeeping gene 18 RNA was included in the array for assessing RNA quality and sample normalization. Western Blot Cortical lysates were prepared from P30 control and Nestin-Cre;Nrg3 F/F and Nex-Cre;Nrg3 F/F mutants as described before ( Fazzari et al., 2010; Vullhorst et al., 2009 ) and blotted using mouse anti- b -Actin (1:4,000; Sigma) and rabbit anti-Nrg3 (1:500; Abcam). Signals were detected with a luminescent image analyzer (LAS-1000PLUS; Fujifilm) and quantified with Quantity One 1D Anal- ysis Software (Bio-Rad Laboratories). Image Analysis and Quantification Images were acquired using fluorescence microscopes (DM5000B, CTR5000, and DMIRB from Leica, or Apotome.2 from Zeiss) coupled to digital cameras (DC500 or DFC350FX, Leica; OrcaR2, Hamamatsu), or in an inverted Leica TCS SP8 confocal microscope. All images were analyzed with ImageJ (Fiji). For the quantification of migration in MGE explants, the distance migrated by the30furthestcellswasmeasured.Forthequantificationofshort-rangechemo- attraction, the colocalizing area between MGE and COS cells was measured. For the analysis of the interneuron angle of migration, we draw a grid of virtual radial lines (lines perpendicular to the ventricular zone and the pial surface) and oriented each cell in relation to the most adjacent ''radial line.'' Cells that deviated less than 25 from radial lines were considered as radially oriented; those that deviate more than 25 were designated as tangentially oriented. We systematically exclude from this analysis those cells located in the more lateral or medial regions of the cortex, so that the curvature of the slice in those regions would not interfere with our analysis ( Martini et al., 2009 ). For the quan- tification of cell migration in MGE explants, we measured the distance migrated by the30furthest cellsand normalized theaverage migrateddistance tothedis- tancebetweenMGEandCOSexplants.Forthequantificationofthecolocalizing area between migrating interneurons and COS cells in the short-distance confrontation assays, we quantified the colocalizing area using ImageJ (Fiji). Stripes were quantified by counting thenumber of neuronscontained in a virtual grid containing five black and five red lines. The same area was used for all explants. Sections from control and mutant mice were imaged during the same imaging session. Data acquisition was performed using the same laser power, photomultiplier gain, pinhole, and detection filter settings (1,024 3 1,024 resolution; 12 bits). Quantifications were done using ImageJ (Fiji). Layers were drawn following nuclear staining. For in situ hybridization, the area quanti- fied was divided in ten equal bins, and the percentage of cells in each bin was calculated. The bins were then matched to the appropriate layers. Statistical Analyses Statistical analysis was carried out in SPSS (SPSS, Inc.). The p values below 0.05 were considered statistically significant. Data are presented as mean and SEM throughout the manuscript ( Table S3 We thank I. Andrew, S. Bae, M.A. Casillas, M. Ferna ́ndez, and T. Gil for excel-lent technical assistance and laboratory support; A. Caler for excellent support with FACS experiments; G. Expo ́sito for support with imaging; L. Lim for help with quantitative methods; V. Borrell, R. Hevner, C. Lai, V. Pachnis, C. Redies,B. Rico, J.L.R. Rubenstein, and M. Tessier-Lavigne for plasmids and anti-bodies; and A. Barco, M.A. Nieto, and K. Nave for mouse strains. We are grateful to members of the Flames, O.M., and Rico laboratories for stimulating dis- cussions and ideas. This work was supported by grants from European Research Council (ERC-2011-AdG 293683) and the Spanish Government (CSD2007-00023 and SAF2011-28845) to O.M. O.M. is a Wellcome Trust Investigator. ; Sí