Images were taken with a Zeiss LSM510 confocal microscope

Images were taken with a Zeiss LSM510 confocal microscope. overlapping clusters of PSD-95 and NMDA receptor subunit NR1. Selectively decreasing levels of Kal7 decreases the density of PSD-95-positive, bassoon-positive clusters along the dendritic shaft of hippocampal interneurons. Overexpression of Kal7 increases dendritic branching, inducing formation of spine-like structures along the dendrites and on the soma of normally aspiny hippocampal interneurons. Essentially all of the spine-like structures formed in response to Kal7 are apposed to vesicular glutamate transporter 1-positive, bassoon-positive presynaptic endings; GAD-positive, Trelagliptin Succinate (SYR-472) vesicular GABA transporter-positive inhibitory endings are unaffected. Almost every Kal7-positive dendritic cluster contains PSD-95 along with NMDA (NR1) and AMPA (GluR1 and GluR2) receptor subunits. Kal7-induced formation of spine-like structures requires its PDZ binding motif, and interruption of interactions between the PDZ binding motif and its interactors decreases Kal7-induced formation of spine-like structures. Kal7 thus joins Shank3 and GluR2 as molecules with a level of expression at excitatory synapses that titrates the number of dendritic spines. gene generates at least 10 transcripts encoding functionally distinct proteins. The largest isoform, Kal12, has multiple spectrin-like repeats, two Rho GEF domains, and a kinase domain. Kal7 has a single GEF domain followed by a PDZ binding motif. The isoforms of Kalirin are generated from a different promoter, and antibodies and probes to the unique region of Kal7 also recognize Kal7. are high-power images from boxed areas of are high-power Rabbit Polyclonal to OR4A15 images from boxed Trelagliptin Succinate (SYR-472) areas of images (DIV)} were incubated with the R7-Kal7CT (1, 5, 10, 20 m, peptides dissolved in medium) for 45 min; neurons were then washed with medium and returned to Trelagliptin Succinate (SYR-472) the incubator with half the original medium. Control neurons were treated with mutant R7-Kal7CT (1, 5, 10, and 20 m) or vehicle at the same time under the same conditions. Neurons were fixed with 4% paraformaldehyde 1 h, 12 h, 24 h, or 3 d after removing peptide and double stained with a polyclonal antibody to Kal7 (which detects both the peptide and endogenous Kal7) and a monoclonal antibody to Kal7 (which detects endogenous Kal7, but not R7-Kal7CT). Based on signal-to-noise ratio, the optimal concentration of peptide is 10 m. To determine whether R7-Kal7CT can alter Kal7-induced spine formation, dissociated hippocampal neurons were transfected with vector encoding Kal7GFP at DIV1. The R7-Kal7CT peptide (10 m) was added to the cultures at 7 DIV and 13 DIV. Control neurons received mutant R7-Kal7CT (10 m) or vehicle at the same times. Cells were fixed with 4% paraformaldehyde and triple stained at 18 DIV with antibodies to GFP, MAP2, and GAD65. Preparation of rat hippocampal organotypic cultures Hippocampal slices were prepared from P9 Sprague Dawley rats as described previously (Ma et al., 2003). Briefly, hippocampi were dissected into ice-cold, sterile Gey’s balanced salt solution (Sigma) containing 0.5% glucose. Medial hippocampi were sliced transversely (400 m) using a slice chopper. Slices were kept in ice-cold Gey’s balanced salt solution and then placed onto 30 mm Millicell CM membrane inserts in Petri dishes filled with 1.1 ml of culture medium containing 0.5 basal Eagle’s medium, 0.25 HBSS, 0.25 horse serum (defined, heat-inactivated), 25 U/ml penicillin, 25 g/ml streptomycin, and 1 mm l-glutamine (Invitrogen). Slices were kept under 5% CO2 at 37C, with media changes at 1 DIV and every 3 d thereafter. Immunohistochemistry (brain sections and slices) Coronal sections (12 m) were cut and mounted on gelatin-coated slides. Antibody specificity was evaluated by replacement of antibody with preimmune serum and preincubation of antibody with its antigen (10 g/l) as described previously (Ma et al., 2003); staining was eliminated in both controls. Sections were blocked in PBS containing 1% BSA/10% normal goat serum/0.25% Triton X-100, pH 7.4, for 1 h at room temperature. {Sections were stained subsequently with polyclonal Kal7 and GAD65/67 antibodies at 4C overnight.|Sections were stained with polyclonal Kal7 and GAD65/67 antibodies at Trelagliptin Succinate (SYR-472) 4C overnight subsequently.} {Sections were stained simultaneously with polyclonal Kal7 and monoclonal parvalbumin antibodies.|Sections were stained with polyclonal Kal7 and monoclonal parvalbumin antibodies simultaneously.} Primary antibodies were visualized with Cy3-labeled donkey anti-rabbit IgG (The Jackson Laboratory, Bar Harbor, ME) and FITC-labeled goat anti-mouse IgG (The Jackson Laboratory). Images were taken with a Zeiss LSM510 confocal microscope. Replacement of the Kal7 antibody with preimmune serum or preincubation of the antibody with its antigen (10 g/ml).