New Cre/lox Genetic Tools Figure 3. R26IAP is a sensitive reporter for Cre-mediated recombination in individual cells. (A–D) R26CreER;R26IAP mice either in the absence (A,B) or following intra-ocular injection (C,D) of 4HT show widespread Cre-mediated recombination in diverse cell types. A, brain; B, retina, C, lens, D, cornea. AP+ lens fiber and lens epithelial cells are seen in C. The overall frequency of AP-expressing cells in the brain and retina in the absence of 4HT is ,0.01%. (E–G) R26IAP mice that received an IP injection of ,184 mg of purified His-NLS-Cre protein show scattered Cre-mediated recombination only in epithelial cells lining the peritoneal cavity or in the muscle fibers of the abdominal wall immediately beneath the peritoneal surface. E, liver; F, abdominal wall; G, stomach. (H) R26IAP mice that received an intraocular injection of ,7 mg of purified His-NLS-Cre protein at ,P5 show scattered Cre-mediated in RGCs and in occasional astrocytes. RGC axons are converging on the optic nerve head at the top of the panel. (I,J) Retina (I) and cornea (J) following an intra-ocular injection of a replication defective adenovirus expressing Cre. Cre-mediated recombination and AP expression are confined to tissues near the injection site. In the retina, the compact appearance of the AP+ cells and the absence of AP+ axons suggests that most of the labeled cells are astrocytes and/or Muller glia. (K–O) Tissues from R26IAP mice in the absence (K) or following IV injection (L–O) of a replication defective adenovirus expressing Cre (Ad-Cre). K–N, kidney; O, liver. The boxed region of the renal pelvis in L is enlarged in M. N shows a region of renal cortex from a different mouse that received an IV injection of Ad-Cre in which Cre-mediated recombination was also observed in a subset of cortical tubules. Scale bars are: 1 mm for E and O; and 200 mm for D,F,G,H,J,M, and N. doi:10.1371/journal.pone.0007859.g003 interest, and, as a result, the anatomy of the cholinergic and catecholaminergic systems has been intensively studied in both normal and diseased brains in a wide variety of species. In most studies, cholinergic and catecholaminergic neurons have been visualized by immunostaining for the biosynthetic enzymes choline acetyl transferase (ChAT) and tyrosine hydroxylase (TH), respectively, a method the reveals the cell bodies and processes of all neurons of the particular neurotransmitter type. Due to the high density and wide coverage of immunoreactive processes, this approach generally reveals only minimal information about the morphologies of individual neurons. In the present study, we have developed a complementary method for visualizing systems of neurons defined by neurotransmitter type. In this method, cell- type specific, but relatively low frequency, Cre-mediated recom- bination randomly labels a subset of neurons within the population of interest to generate an atlas that reveals representative soma locations, projections, and individual morphologies for all of the neurons of that class. Using ChAT-IRES-CreER;R26IAP Mice to Produce an Atlas of Cholinergic Neurons The application of this sampling method to the cholinergic system is illustrated in Figure 4. In this experiment, a ChAT-IRES- PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7859