Article Figures & Data
Figures
- Fig. 1.
Specificity of GKAP antibodies and primary structure of GKAPL N-terminal splice variant.A, Two independent GKAP antibodies recognize recombinantly expressed GKAP and identical 95 and 130 kDa bands in rat brain membranes. COS-7 cells transfected with GKAP cDNA, and rat cortical membranes (10 μg protein) were immunoblotted with GKAP antibodies N1564 and C9589, as indicated. Heterologously expressed GKAP comigrates with the 95 kDa immunoreactive brain protein. Ctx, Cortical membranes. B, Comparison of heterologously expressed GKAP, GKAPL, and rat hippocampal membranes, immunoblotted with N1564 antibodies. Heterologously expressed GKAPL comigrates with the ∼130 kDa immunoreactive brain protein. The same result was obtained with C9589 antibodies (data not shown). Hpc, Hippocampal membranes.C, Amino acid sequence alignment comparing the N-terminal GKAP splice variant GKAPL to GKAP.Splice, Presumed alternative splice site.
- Fig. 2.
Tissue distribution of GKAP mRNA in rat. PolyA mRNA multitissue Northern blot probed with 32P-labeled GKAP DNA. Positions of RNA molecular size markers are shown.
- Fig. 3.
Distribution of GKAP mRNA in adult rat brain analyzed by in situ hybridization. A, Horizontal (top) and sagittal (bottom) rat brain sections probed with 35S-labeled antisense GKAP cRNA. B–D, Dark-field microscopy of sagittal in situ hybridization sections.B, Hippocampal area CA1 and overlying cortex.Ctx, Cerebral cortex, so, st. oriens;cc, corpus callosum. C, Cerebellar cortex. The arrow points to a Purkinje cell.m, Molecular layer; g, granular layer;w, white matter. D, Hippo-campal formation. dg, Dentate gyrus; h, hilus. Note prominent expression of GKAP mRNA by interneurons in the hilar region of the dentate gyrus and the st. oriens of CA1. Scale bars:A, 5 mm; B–D, 0.3 mm.
- Fig. 4.
Immunohistochemical localization of GKAP protein in the hippocampus. A–C, Coronal sections were immunolabeled with GKAP antibody C9589, and visualized by DAB. A, Hippocampal formation. The dendritic fields of pyramidal neurons and dentate granule cells and a population of interneurons, are heavily labeled. B, Region CA1 of the hippocampus. Note the relative absence of staining in pyramidal cell bodies, and the darkly stained interneurons and their dendrites. The dendrites of pyramidal neurons in the st. radiatum and st. oriens are also labeled, st. oriens more strongly than st. radiatum.C, Hilar region of the dentate gyrus, filled with GKAP immunoreactive interneurons and dendrites. Note the relative absence of staining in the granule cell body layer.D–G, Coronal sections immunolabeled by GKAP antibody N1564 (D-E), or C9589 (F, G) followed by Cy3- or FITC-conjugated secondary antibody, and visualized by confocal microscopy. D, CA1 st. radiatum. Dendrites of pyramidal neurons are decorated by striking GKAP puncta. There is very slight labeling of pyramidal cell bodies.E, 2× magnification of the dendrites in D. F, An isolated interneuron in area CA1. G,Interneurons and their dendrites in the hilar region of the dentate gyrus, covered in bright GKAP immunoreactive puncta. sr, St. radiatum; other abbreviations as shown in Figure 3. Scale bars:A, 0.5 mm; B, C, 125 μm; D, G, 60 μm; E, F, 30 μm.
- Fig. 5.
Immunohistochemical localization of GKAP protein in cerebral cortex and cerebellum. A–C, Coronal sections labeled with GKAP antibody C9589 and visualized via DAB histochemistry. A, Cerebral cortex, showing widespread and diffuse neuropil staining. B, C,Cerebellar Purkinje and granule cell staining in cerebellum. Purkinje cell bodies and glomeruli of the granular cell layer are stained.D–G, Sections labeled with GKAP antibody N1564 (D) or C9589 (E–G) and Cy3- or FITC-conjugated secondary antibody and visualized by confocal microscopy.D, Apical dendrites of pyramidal neurons in cerebral cortex decorated by GKAP immunoreactive puncta. E, F,Bright GKAP puncta on dendrites of neocortical neurons.G, High-power view of the Purkinje cell layer in the cerebellum. p, Purkinje cell; other abbreviations as in Figure 3. Scale bars: A, B, 0.5 mm; C, 125 μm; D, E, G, 60 μm; F, 30 μm.
- Fig. 6.
Immunogold EM localization of GKAP in dendritic spines and PSD. A–C, Postembedding 18nm immunogold labeling of GKAP in sections from cerebral cortex layers II–III. *Presynaptic terminals.A, An asymmetric synapse showing gold particles associated with PSD; some particles lie against the plasma membrane, and others lie at the cytoplasmic face of the PSD. B,Three immunopositive synapses in the same field, each with gold particles overlying the PSD. One particle (just belowthe B) seems to lie within the cytoplasm, perhaps in association with a cytoskeletal element. C, An immunopositive spine cut in a longitudinal plane, permitting visualization of the spine apparatus (arrowheads) and spine neck. Scale bar, 500 nm. D-E, Quantitative analysis of the distribution of GKAP immunogold particles at synapses. Grids of neocortex (layers II–III) prepared from two animals were examined. Random electron micrographs were made of fields in which at least one gold particle seemed associated with a synaptic active zone. EM negatives were digitized, and all gold particles within 100 nm of clearly definable active zones were analyzed. The data were from 85 active zones (mean length of synaptic apposition, 338 nm; SD, 81 nm). D, Distribution of gold particles across the synapse, axis perpendicular to the synaptic cleft (“0” corresponds to the cytoplasmic leaflet of the postsynaptic membrane). GKAP-immunogold distribution peaks at 10–30 nm on the intracellular side of the postsynaptic membrane.E, Lateral distribution of gold particles. Normalized lateral distance is defined as the absolute value of [(distance from center of particle to one edge of active zone) − (distance to other edge of active zone)]/(total length of active zone). GKAP is distributed evenly along the active zone.
- Fig. 7.
GKAP colocalizes at excitatory synapses with GluR1. In hippocampal neurons at 3 weeks in culture, GKAP (A) is distributed in puncta that colocalize with clusters of GluR1 (B) at dendritic spines in pyramidal cells. Punctate GKAP immunoreactivity (C) does not colocalize with clusters of the GABA-R β2 and β3 subunits (D), markers of GABAergic inhibitory synapses. Scale bar, 10 μm; inset, 5 μm.
