Figure 2. Pcdh-γs are expressed strongly in interneurons in mature cultures. A–D, Mature (30 DIV) cultures of hippocampal neurons were double labeled with anti-Pcdh-γ-cyto and PSD-95. Many spiny, pyramidal-like cells (A, B, arrowhead) did not exhibit significant staining for Pcdh-γ. In contrast, staining of Pcdh-γ in smooth, interneuron-like cells (A, B, arrow) invariably was intense. An overlay of the Pcdh-γ and PSD-95 staining patterns in interneuron-like cells (C, D) reveals that Pcdh-γ (D, arrowhead) and PSD-95 (D, arrow) do not colocalize, indicating that Pcdh-γ is not synaptic in these cells. E, F, Strong Pcdh-γ expressors synthesize inhibitory neurotransmitters. Mature cultures were double labeled with anti-Pcdh-γ-cyto and GAD. Cells that expressed strong intracellular staining for Pcdh-γs also exhibited strong staining for GAD in their cell body (arrowhead). In contrast, cells that did not express Pcdh-γs also did not express GAD in their cell body (arrow). Overlay of the Pcdh-γ/GAD double-label reaction (F, inset) shows that Pcdh-γs are not expressed within inhibitory synapses on these cells. G, H, Mature cultures were double labeled with anti-Pcdh-γ-cyto and GAD. Purified Pcdh-γ cytoplasmic domain was added to the staining reaction in H and I. Pcdh-γ completely blocked the immunoreaction in interneurons of Pcdh-γ (H) but did not affect that of GAD (I). The field in H and I is the same. Scale bar: (in I) A–C, E–I, 50 μm; D, 10 μm.