New Cre/lox Genetic Tools Figure 4. Survey of cholinergic neuronal morphologies and projections in the brain and retina of R26IAP;ChAT-IRES-CreER mice. Animals were injected with a single IP injection of 200 mg 4HT at P8. (A,C,F,I,K) 300 mm coronal sections at Bregma positions 1.2, 0, 22.5, 25.0, and 27.8, respectively, as shown schematically in (N). (B,D,E,G,H,J,L) enlargements of boxed regions. (M) In a flat mounted retina, AP-expression is present exclusively in starburst amacrine cells. In the absence of 4HT, retinas were devoid of AP+ cells. BNA, basolateral nucleus of the amygdala; DMN(VN), dorsal motor nucleus of the vagus nerve; LDTN, laterodorsal tegmental nucleus; MS, Medial septum; PPTN; pedunculopontine tegmental nucleus; SI, substantia innominata; St, striatum. doi:10.1371/journal.pone.0007859.g004 CreER;R26IAP mouse was injected IP with 200 mg 4HT at P8, and one month later a complete series of 300 mm coronal brain sections was histochemically stained for AP (Figure 4N). The distribution of AP+ cell bodies and processes was found to be in excellent agreement with the distribution of cholinergic neurons and processes observed in mouse and rat brains by ChAT immunostaining [14,15]. In the following paragraph, we summa- rize the distribution of the AP+ neurons and processes in the ChAT-IRES-CreER;R26IAP brain, proceeding in a rostral to caudal direction. AP+ neurons with well-defined processes were observed in the medial septum (Figure 4A,B) and in the basal forebrain in and near the substantia innominata (Figure 4C,D). In the striatum, AP+ neurons had both thick tortuous dendrites and a halo of much finer (presumably axonal) processes (Figure 4A,C,E). A large number of fine AP+ processes were found throughout the cortex and hippocampus, as expected for diffuse cholinergic projections originating in the basal forebrain (Figure 4F,G); an especially dense plexus of AP+ processes was present in the basolateral nucleus of the amygdala (Figure 4F-H). Near the pons/midbrain junction, AP+ neurons were localized to the laterodorsal tegmental nucleus and the pedunculopontine tegmental nucleus (Figure 4I,J). In the caudal medulla, AP+ neurons were seen in the dorsal motor nucleus of the tenth (vagus) nerve (Figure 4K,L). In retina flat mounts, AP labeling was confined to starburst amacrine cells, the only cholinergic cell type within the mammalian retina, confirm- ing the specificity of ChAT-IRES-CreER expression (Figure 4M). Using TH-IRES-CreER;R26IAP Mice to Produce an Atlas of Catecholaminergic Neurons To further extend the approach of labeling a subset of neurons within a given neurotransmitter system, we have also generated an atlas of labeled neurons for the catecholaminergic systems by targeting R26IAP recombination with TH-IRES-CreER following IP injection of 100 mg 4HT at P3. Figure 5 shows a series of 300 mm coronal sections from a TH-IRES-CreER;R26IAP brain that was histochemically stained for AP (Figure 5N). The distribution of AP+ cell bodies and processes was found to be in excellent agreement with the distribution of catecholaminergic PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7859