Confocal microscopical analysis of ChAT+ neurons labelled with calretinin (CR) and different GABAergic markers. A, B, C, D, E: during postnatal development at P11 (A, B), P16 (C, D) and P22 (E), ChAT+ cells (red) sometimes also contain (white arrows) GABAergic markers (green). GABA was revealed with either a polyclonal (GABAp in A) or a monoclonal (GABAm in D) antibody. Panels C and E show labelling of GAD67; in particular the large white arrowhead in C indicates a neuron that presented triple immunoreaction for ChAT/GAD67/CR (blue). No double labelling was found using GAD65 as GABAergic marker (B). In the adult (Ad), ChAT+ cells (red) only showed occasional immunoreactivity for GABAergic markers (green), as shown in F (GAD65), G (GABAm) and H (GABAp). In G, the large white arrowhead indicates again a cell labelled also for CR (blue). I, L: in the adult transgenic (Tg) GAD67-GFP mouse, no ChAT+ neurons (red) expressed GFP (green), but two of them contained CR (blue signal in L, small white arrowheads). Small red arrowheads: single ChAT+ neurons. Small green arrowheads: single GABAergic cells. Small white arrowheads: double ChAT+/CR+ neurons. Scale bar: 23 μm in A, B and E; 20 μm in C, F and G; 33 μm in D and I; 11 μm in L.