New Cre/lox Genetic Tools Figure 5. Survey of catecholaminergic neuronal morphologies and projections in the brain of TH-IRES-CreER;R26IAP mice. Animals were injected with 100 mg 4HT at P3. (A–E,G,I,K,L) 300 mm coronal sections at Bregma positions 1.7, 1.1, 0, 21.6, 22.2, 23.2, 24.7, 25.5, 28.2, respectively, as shown schematically in (N). (F,H,J,M) enlargements of boxed regions. A1, A1 noradrenergic cell group; A2, A2 noradrenergic cell group; AcN, accumbens nucleus; AN, arcuate nucleus; CNA, central nucleus of the amygdala; DRN, dorsal raphe nucleus; LC, locus coeruleus; ME, median eminence; PPTN, pedunculopontine tegmental nucleus; SN(PR), substantia nigra (pars reticulata); St, striatum; VP, ventral pallidum; VTA, ventral tegmental area; ZI, zona incerta. doi:10.1371/journal.pone.0007859.g005 neurons and processes observed in mouse and rat brains by TH immunostaining [16–21]. Below we summarize the distribution of AP+ neurons and processes in the TH-IRES-CreER;R26IAP brain, proceeding in a rostral to caudal direction. In the TH-IRES-CreER;R26IAP forebrain, dense AP+ processes, but very few AP+ cell bodies, were seen in the nucleus accumbens, striatum, ventral pallidum, and the central nucleus of the amygdala (Figure 5A–D), as expected for these principal targets of innervation by midbrain dopaminergic and noradrenergic neurons. At the level of the hypothalamus, scattered AP+ cell bodies with a small number of thick processes were present in the arcuate nucleus (Figure 5E and F). Intense AP staining was seen in the median eminence, and AP+ neurons with numerous finer processes were seen in the zona incerta (Figure 5E and F). The greatest number of AP+ neuronal cell bodies were found in a contiguous zone encompassing the substantia nigra pars reticulata and the ventral tegmental area and extending caudally as far as the pedunculopontine tegmental nucleus, nuclei that give rise to the principal dopaminergic projections to the forebrain (Figure 5G–J). At the junction of the midbrain and pons, large numbers of AP+ cells populate the dorsal raphe nucleus, a major source of projections to the nucleus accumbens (Figure 5I). The locus coeruleus, the site with the highest concentration of central noradrenergic neurons, was densely populated with AP+ cells (Figure 5K). Finally, AP+ neurons were observed in the caudal medulla in the locations predicted for A1 and A2 noradrenergic cell groups (Figure 5L and M). Using TH-IRES-CreER;R26IAP Mice to Visualize the Morphologies of Wide-Field Tyrosine Hydroxylase- Expressing Amacrine Cells As a further test of the utility of the R26IAP line, we have asked whether we could use it to characterize the unusually large arbors of dopaminergic amacrine (DA) cells in the mouse retina. DA cells have been found in the retinas of a wide variety of vertebrates. Both rodent and primate retinas have a distinctive type of TH+ amacrine cells (type 1 cells) characterized by large cell bodies with two groups of neurites: one group forms a simple tree composed of relatively thick dendrites, whereas the second is composed of extremely long, thin, and sparsely branching axon-like processes [22–24]. The dendritic and axon-like arbors are confined to the outermost sublamina of the inner plexiform layer. In the primate retina, the dendritic arbors of DA cells have diameters of ,400 mm near the fovea and ,600 mm in the periphery, whereas the axon-like arbors encompass a diameter of at least 6 mm, the exact value being difficult to ascertain due to uncertainty in the extent of diffusion of the injected label [24]. Type 1 DA cells are unusually sparse. In the primate retina they have a density of ,50/mm2 near the fovea and about 10/mm2 in the periphery, and in the mouse and rabbit they have an average density of ,20/ mm2, equivalent to ,450 and ,6,000 cells per retina, respectively [24–27]. Despite their relatively small numbers, dopamine released by DA cells plays a central role in modulating retinal signaling in response to changes in ambient illumination or circadian cues [28–32]. PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7859