Figure 7. Quantification of axon-like processes and dendrite lengths for type 1 DA cells. Red circles, lengths calculated from the Neuromantic tracing of BBBA clarified retinas. BBBA causes ,30% tissue shrinkage. Black circle and error bars, mean+/2S.D. doi:10.1371/journal.pone.0007859.g007 Z/EG loci reflect the high CpG content of the E. coli lacZ coding region, which is present as part of the beta-geo coding region in both loci and may promote somatic cytosine methylation and epigenetic silencing. A second strategy for reducing recombination efficiency focuses on reducing CreER expression, either by using an IRES-CreER knock-in allele, which likely decreases translation inefficiency, or, as in the R26rtTACreER locus, by overlaying a second level of transcriptional regulation. Both of these approaches for reducing CreER can eliminate background recombination in the absence of 4HT. Finally, combining both an inefficient loxP target (Z/AP) and low CreER expression reduces recombination to extremely low levels, such that fewer than one hundred neurons can be reproducibly labeled per brain depending on the IRES-CreER knock-in and the time and dose of 4HT [4]. Sparse Labeling of Defined Neuronal Types as a Tool to Survey Changes in CNS Structure A method for visualizing the morphologies of a large and representative sampling of neurons of a genetically defined type has the potential to fill a hitherto vacant niche in neuro-anatomic analyses of brain structure. Historically, the analysis of neuronal morphology has been performed either by sparse labeling methods that are not cell type specific (e.g. the Golgi stain) or by filling individual neurons with a tracer using a whole cell patch electrode, a method that is extremely laborious and not easily adaptable as a survey tool [e.g. ref. 48]. These classic methods also suffer from the problem of incomplete filling of large and/or complex neuronal processes. At the other end of the spectrum with respect to the number of labeled cells, immunostaining for markers such as ChAT or TH reveals all of the cells of a given type, but the high density of labeled cells and their interdigitating processes generally preclude an assessment of individual morphologies. Moreover, the immunostaining approach is limited to those antigens that are relatively abundant, cytosolic, and cell-type specific. Thus, many molecular markers that distinguish neuronal types, such as transcription factors, or various low abundance or transiently expressed proteins cannot be used directly to reveal the morphologies of the cells in which they are [or were] expressed. While this last challenge is being partially addressed by the creation of BAC transgenic lines that express GFP or Cre in defined subsets of cells [e.g. refs 49,50], the challenge of visualizing individual neuronal morphologies can only be met by a method that generates a relatively sparse collection of labeled neurons. The strategy of generating a sparsely labeled sampling of a single neuronal type – what we refer to here as an atlas of New Cre/lox Genetic Tools morphologies - is illustrated here for cholinergic and catechol- aminergic neurons. The method takes advantage of the flexibility afforded by 4HT dosing to noninvasively generate a collection of several dozen to several hundred labeled neurons throughout the entire CNS. Current evidence suggests that the 1–2 month delay between Cre-mediated recombination and histologic analysis provides sufficient time for AP to uniformly populate the plasma membrane of all dendritic and axonal processes even among the largest neurons [4]. This approach could be used to survey the survival and morphology of defined classes of neurons in mouse models of a variety of CNS diseases or insults, including inherited disease, stroke, infection, drug toxicity, and sensory deprivation. For unilateral perturbations - such as a surgical lesion, localized drug injection, or recombinant virus injection – labeled cells ipsilateral and contralateral to the site of the manipulation can be compared. The Morphologies of Type 1 DA Cells in the Mouse Retina DA cells, first defined as dopamine-accumulating neurons and subsequently shown to express TH, have been extensively studied in a variety of species [22–25,51]. Their wide arbors and diffuse coverage likely reflect the role of dopamine as a neuromodulator that acts on a broad and still incompletely enumerated group of target cells to modulate retinal sensitivity. The best-characterized effect of dopamine in the retina is in mediating the light-dependent uncoupling of horizontal and amacrine cell gap junctions [reviewed in ref. 28]. Recent work suggests that retinal dopamine release is regulated by both ambient light level and by the circadian clock [29–32]. To the best of our knowledge, the full morphology of individual type 1 DA cells has not been previously reported in any species owing to uncertainties regarding the completeness of tracer filling. Dacey has estimated the diameter of the type 1 primate DA cell axon-like arbor at ,8–10 mm based on a quantitative analysis of arbor density, cell density, and the morphology of HRP filled cells [24]. The cell morphologies determined here indicate that the typical type 1 DA cell in the mouse retina has axon-like processes that extend both radially and circumferentially, forming a sparse arbor that generally encompasses a substantial fraction of the retinal area and has a total length of ,6.5 cm in a retina with a flat mounted diameter of ,5 mm (Figure 7; values corrected for tissue shrinkage in BBA). If the arbors were artificially straightened and directed radially, they would extend to a distance of at least several millimeters from the cell body (Figure 6). On the assumption that signals received by the dendrites of type 1 DA cells are transmitted along the full length of their axon-like arbors, then it would appear that activation of even a small number of these cells would produce a signal with a relatively uniformly spatial output across the retina, a property consistent with the known neuromodulatory functions of retinal dopamine. In relation to the cell biological mechanisms that control the development of neuronal morphology, the large and irregularly shaped axonal arbors of the type 1 DA cells would appear to rule out any constraints on axonal trajectories based on homotypic signaling, as observed for axonal and dendritic arbors in Drosophila [52,53] and as postulated for a variety of cell types in the mammalian retina [54]. However, an interesting observa- tion regarding type 1 DA cells is the very narrow length distributions of the dendrites and axon-like processes (Figure 7). These distributions suggest that there is a stringent control mechanism that regulates the total length of each class of neuritic process, even among cell types in which the branching pattern and coverage geometry is highly variable. The hypothesized control mechanism could, for example, reflect a genetic program that PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e7859