Fig. 4.

Ectopic expression of Nxph1 at excitatory synapses. (A) Several Nxph1-GFP^tg/+ transgenic mouse lines express the fusion protein as evidenced by immunoblots of brain lysates. (B and C) Light microscopic images of immunohistochemistry with anti-GFP antibodies show Nxph1-GFP in brain sections of transgenic animals (B, line #10 from A). WT sections are devoid of any staining (C). CC, cerebral cortex; CA1 region, hippocampus; DG, dentate gyrus. (Scale bar, 150 µm.) (D–F) Different methods of EM immunolabeling were applied to localize Nxph1-GFP in the somatosensory cortex of transgenic brains (boxed area in B). Tokuyasu cryosectioning (D), Lowicryl postembedding (E), and Epon postembedding (F) techniques show 10-nm gold particles (circled in red) at synaptic clefts of asymmetric (presumptive excitatory) contacts. (Scale bars, 100 nm.) (G–K) In addition to the synaptic cleft, immunolabeling was found at the presynaptic plasma membrane outside active zones (G), at extrasynaptic sites of the cell surface (H), (I) intracellularly over vesicular structures in the terminal, and inside the lumen of rER (J) and Golgi cisternae (K). (Scale bars, 200 nm.)