In most mammalian retinas, including the mouse retina, TH immunostaining can been used to identify DA cells, but the extensive intermingling of TH+ processes from different cells precludes the tracing of individual immunolabeled arbors. A similar challenge exists for transgenic approaches in which the TH promoter drives expression of a histochemical or fluorescent reporter [27,33,34]. In the present study, we have used TH-IRES-CreER;R26IAP mice injected with 100–300 mg 4HT between P3 and P8 to produce, on average, fewer than one AP+ DA cell per retina. This window of injection times was chosen to roughly match the initial appearance of TH+ amacrine cells in the rodent retina [35–37]. Among 120 adult retinas histochemically stained for AP and examined as flat mounts, a total of 147 AP+ cells were observed, and all were amacrine cells. Twenty-five of the AP+ cells exhibited morphologies indicative of type 1 DA cells: a large soma, a relatively simple dendritic arbor, and an extremely large and sparsely branching axon-like arbor, with both arbors confined to the outermost lamina of the inner plexiform layer (Figure 6A–C). This narrow lamination is in contrast to the diverse lamination within the inner plexiform layer that is seen with other large polyaxonal amacrine cells in primate and rodent retinas [5,38,39] The distribution of AP+ type 1 DA cells among the 120 retinas was consistent with a Poisson process: the number of retinas with zero, one, two, or more than two type 1 DA cells was 97, 21, two, and zero, respectively. Fourteen of the type 1 DA cells were traced (Figure 6A and B): 12 from retinas with a single type 1 DA cell, and two from one retina that had a pair of type 1 DA cells. In the retina with two type 1 DA cells (Figure 6B and C), the extensive overlap between the two axon-like arbors precludes an accurate assignment of connectivity for some of the axon-like segments. Among the collection of AP+ type 1 DA cells, the relative uniformity of the AP staining intensity along each axon-like process and the sharp termination of AP staining at the presumptive ends of these processes implies that their full extent has been visualized (Figure 6C). In general, the axon-like arbors encompassed regions of the retina both near to and far from the cell body. There were multiple examples of axon-like processes that projected close to the edge of the retina and then continued circumferentially along the retinal margin, suggesting that in the context of a larger retina this axon-like trajectory would have continued radially to greater distances from the cell body (Figure 6A). Despite the apparent absence of a fixed branching pattern, the length distributions for both the dendritic and axon-like arbors were remarkably narrow, with a mean+/2S.D. of 46,908+/24,175 mm for axon-like processes and 2,532+/2253 for dendrites [Figure 7; these values are uncorrected for the ,30% tissue shrinkage caused by clearing in benzylbenzoate:benzyl alcohol (BBBA)]. These observations imply the existence of a biochemical control system that precisely calibrates the total length (or mass) of these arbors. In addition to the 25 type 1 DA cells described above, a variety of other AP+ amacrine cell types were also found in this set of 120 retinas: 110 AII amacrine cells, four starburst amacrine cells, four large field amacrine cells, two ‘‘waterfall’’ amacrine cells, and two narrow field amacrine cells [see ref. 5 for examples of these morphologies]. Among the large field amacrine cells, at least one corresponds to the morphology described for type 2 DA cells (Figure 6D). We ascribe the occasional labeling of non-DA cell types to low level mis-expression of TH-IRES-CreER in these other neurons or in neuronal progenitors. Discussion The two new mouse lines described here add to the growing list of genetically engineered mice available for controlled in vivo New Cre/lox Genetic Tools recombination and cell labeling using the Cre-lox system. The R26rtTACreER line extends the dynamic range of Cre-mediated recombination efficiencies relative to that of other loci with a constitutively expressed CreER, and the R26IAP line complements the widely used Z/AP line by providing substantially higher efficiency Cre-mediated activation of AP. We note that Que and colleagues [40] have recently described the construction of a Rosa26 knock-in mouse line in which a hPLAP coding region is transcribed from a CMV enhancer/beta-actin promoter after Cre- mediated excision of a loxP-stop-loxP cassette. It seems likely that for applications requiring low efficiency recombination, the dynamic range and temporal control offered by R26rtTACreER represents an improvement over pre-existing lines in which the Tet system controls a constitutively active Cre recombinase [41–44]. It will be interesting to extend the rtTACreER strategy by targeting this cassette to loci that confer cell-type specific expression. Based on our observations with the R26rtTACreER locus, we would predict that this two-stage pharmacologic strategy will also confer tighter control of Cre activity relative to the standard CreER cassette at other sites in the genome. Factors Affecting the Efficiency of Cre-Mediated Recombination The roughly one thousand-fold difference between Z/AP and R26IAP recombination frequencies emphasizes the large effect of chromosomal location and/or target gene sequence on the efficiency of Cre-mediated recombination. This effect is substan- tially larger than the variation typically observed among target loci [45]. A second variable that affects the efficiency of Cre-mediated recombination is developmental age. With multiple target loci for which the efficiency of Cre-medated recombination can be easily quantified with AP histochemistry - including Brn3a, Brn3b, Z/AP, and Fz5 - we have consistently observed a decline in efficiency with increasing postnatal age when tested with a variety of CreER lines, including R26CreER, NFL-IRES-CreER, and ChAT-IRES- CreER (our unpublished observations). As the expression of the Rosa26, NFL, and ChAT genes persists in adulthood, the observed decline in recombination efficiency with age is unlikely to arise from the loss of CreER expression. With the R26rtTACreER system we observe an age dependent silencing that can be inhibited by initiating doxycycline exposure – and presumably rtTA activated transcription – in early embryonic development. These age-dependent phenomena likely represent progressive epigenetic changes in gene structure. A recent comparison of recombination efficiencies among the Z/AP, Z/EG, and R26R- EYFP loci in hematopoietic cells found a similar decline in recombination efficiency with age, as well as a strong correlation between lower recombination efficiency in adulthood and increased cytosine methylation, suggesting that age-dependent changes in chromatin structure can render loxP targets less accessible to Cre recombinase [46]. Consistent with our experience with the R26rtTACreER locus, studies of rtTA activation of target genes reveals a progressive silencing in neurons that can be overcome either by maintaining a basal level of target gene expression throughout development or by providing very high levels of rtTA [47]. In studies in which highly efficient Cre-mediated recombination is desirable, the use of a constitutively active Cre-recombinase - even in combination with an inefficient loxP target such as Z/AP - usually suffices to produce complete or nearly complete recom- bination [8]. When temporal control of recombination is also required and the CreER system is used, obtaining highly efficient PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7859