A synapse was defined by juxtaposition of the pre- and postsynaptic markers

A synapse was defined by juxtaposition of the pre- and postsynaptic markers. Immunohistochemistry Fresh frozen brains were processed as described [53]. 10(3):187C93, 2000): Dp71110m is derived from Cefpodoxime proxetil Dp71 transcripts deleted for exons 71C74 and contains the C-terminal sequence of muscle mass Rabbit Polyclonal to SLC25A11 dystrophin, while Dp71110a contains the option 31-amino acid C-terminal sequence due to the splicing of exon 78 (Austin et al., J Biol Chem 277:47106C47113, 2002). The location of the epitopes recognized by antibodies H4, DYS2 and 5F3 are indicated.(1.01 MB TIF) pone.0006574.s001.tif (985K) GUID:?6B361E9C-67ED-45EB-8914-3618B80B3FB5 Figure S2: Immunofluorescence detection of Dp71 in brain sections. Hippocampal expression of Dp71 isoforms revealed by H4 on rat brain sections. Representative images show Dp71 expression in (A) walls of blood vessels and perivascular astrocytes (arrowhead), and (B) in the granule-cell layer (gcl) of dentate gyrus as incomplete rings of immunoreactivity circling granule cell body and small to large dots around or within cell body. C. Immunolabeling of Dp71 with H4 antibody in the gcl and walls of blood vessels (arrowheads) in WT (C1), mdx (C2) and Dp71-null mice (C3).(2.61 MB TIF) pone.0006574.s002.tif (2.4M) GUID:?1ABADCDA-6A0F-4D98-A943-54656E320DFD Physique S3: Fractionation study. A. Schematic representation of the fractionation protocol (detailed description in Materials and Methods S1). B. Protein extracts from subcellular fractions obtained from control mouse brains probed with the anti-Dp71 (H4) antibody (top panel). Bottom panels show expression Cefpodoxime proxetil of the presynaptic and postsynaptic markers synaptophysin and PSD-95, respectively. Fractions as follows: H, total homogenate; S1, cell soma; P1, dense nuclei-associated membrane; S2, supernatant, P2: crude synaptosomal membrane, S3, cytosolic; P3, light membrane compartment; LS1, soluble; LP1, synaptosomal membrane; LP2, synaptic-vesicle enriched; SPM, synaptic plasma membrane; PSD1/PSD2, postsynaptic density Cefpodoxime proxetil fractions.(1.84 MB TIF) pone.0006574.s003.tif (1.7M) GUID:?30C997BC-B31F-43DC-BB92-27A3E4CA2635 Figure S4: Expression of Dp71 in cultured neurons of control (WT) and Dp71-null (KO) mice. Immunofluorescence assays were performed using the H4 antibody (reddish) that binds all dystrophin-gene products and the 5F3 antibody (green) directed against Dp71 isoforms lacking exon 78. Representative images of cultured neurons from WT (A, B) and KO mice (C, D). Both antibodies labeled neurites and perinuclear regions of control neurons. H4 immunolabeling was present in both control and Dp71-deficient cells, reflecting its binding to several dystrophin-gene products. In contrast, 5F3 antibody labeled neurites of control neurons, but not that of Dp71-deficient neurons. This staining pattern confirms that 5F3 detects specifically Dp71 isoforms lacking exon 78 and expressed in neurites. 5F3 also revealed a basal discontinuous granular labeling in the Golgi complex in both control and Dp71-deficient neurons, which may reflect binding to another dystrophin-like protein in the perinuclear region (Chvez et al., Biochem Biophys Res Commun 274:275C280, 2000). Nuclei stained with DAPI (blue).(0.66 MB TIF) pone.0006574.s004.tif (641K) GUID:?6496435E-D67D-4B11-8142-F6DEA93A608B Physique S5: Gross hippocampal anatomy is normal in Dp71-null mice. Formalin-fixed brain sections of WT and KO mice were immunostained with numerous cell markers: NeuN antibody was used to stain neuronal nuclei in CA1-4 and DG regions. Tuj-1 antibody immunostained pyramidal neurons and basal dentrites in CA1. The anti-parvalbumin antibody was used to reveal parvalbumin-containing interneurons.(3.70 MB TIF) pone.0006574.s005.tif (3.5M) GUID:?8872FC3C-BB0C-46C4-9BF8-69D718B66412 Figure S6: Basal neurotransmission and PTP in Dp71-null and WT mice. (ACB) Basal glutamatergic transmission mediated through AMPAr: (A) presynaptic fibre volley (PFV) slopes plotted against intensity; (B) AMPAr-mediated fEPSP slopes plotted against intensity. (CCD). NMDAr component of glutamatergic transmission: (C) PFV and (D) NMDAr-mediated fEPSP against intensity. WT(open symbols) and KO mice (black symbols). (E) Time-course of the post-tetanic potentiation (PTP) induced by a 2100 Hz HFS delivered in the presence of the NMDAr antagonist APV (80 M). Genotype effect: p 0.37, NS.(0.62 MB TIF) pone.0006574.s006.tif (609K) GUID:?AC140055-FA34-4945-8A31-BA3CC1043628 Figure S7: Spatial learning and reversal in the water maze. In this experiment, the pool diameter was 1.30 m and mice were submitted to 5 daily trials (Dark symbols, 10 KO, Open symbols, 16 WT). Acquisition was followed by a reversal task (arrow) on day 5. Escape latencies show delayed learning in KO mice during the first two days (p 0.05), with improved overall performance by day 3C4 and intact reversal.(0.43 MB TIF) pone.0006574.s007.tif (422K) GUID:?FDE6A419-E408-439A-A125-57864FEA3FCA Table S1: Main antibodies used in this study. *It could be the immunogen.(0.05 MB DOC) pone.0006574.s008.doc (46K) GUID:?BD10180F-8BA0-4988-994B-0093EA6A6896 Materials and Methods S1: Supplemental Materials and Methods.(0.04 MB DOC) pone.0006574.s009.doc.