(A) Structure of the R26IAP knock-in. In the absence of Cre-mediated recombination, the 3′ half of the AP coding region is inverted in the germline configuration. It assumes the correct orientation following Cre-mediated recombination between inverted loxP sites. (B) P30 retina from Chat-IRES-CreER;R26IAP mice treated with 4HT. AP histochemistry labels cholinergic (starburst) amacrine cells. Scale bar, 100 µm. (C–F) P30 brain from Chat-IRES-CreER;R26IAP mice treated with high dose 4HT at P5. AP histochemistry labels numerous axons throughout the cortex (D) and hippocampus (F), as well as cranial motor neurons (E), the axons of which are seen exiting the brain stem (red arrows). Scale bars in D–F, 200 µm. (G and H) Coronal sections of P30 forebrain from Chat-IRES-CreER;R26-LSL-nGFP mice treated with high dose 4HT at P4. Approximately 50% of cholinergic neurons in the basal forebrain, medial septal nucleus, striatum, and spinal cord (visualized with ChAT immunohistochemistry) are GFP+. Medial to the striatum, a distinctive group of GFP+ cell is ChAT−; these cells presumably expressed Chat (and, therefore, Cre) in the early postnatal period and then repress Chat expression in adulthood. In (H), arrows point to ChAT+;GFP− neurons and arrowheads point to ChAT+; GFP+ neurons. Scale bar, 50 µm.