Quantitative analysis of neuronal morphologies in the mouse retina visualized by using a genetically directed reporter

Abstract

An alkaline phosphatase (AP) reporter has been used to visualize detailed morphologies for all major classes of retinal neurons in the adult mouse. The analysis was performed on retinas in which AP expression was activated by Cre-mediated DNA recombination in a small fraction of cells. Recombination was controlled pharmacologically and, to a first approximation, appears to have occurred randomly. The morphologies of 794 inner retinal neurons have been analyzed by measuring arbor area, stratification level, and neurite branching patterns. When analyzed in this multidimensional parametric space, the cells can be clustered into subgroups by visual inspection and by using the Ward's and K-means algorithms. One application of this cell morphology data set and cluster analysis is as a standard for comparison with the retinas of genetically altered mice. This work illustrates the utility and feasibility of genetically directed marking methods for large-scale surveys of neuronal morphology.

Brain-Derived Neurotrophic Factor and TrkB Modulate Visual Experience-Dependent Refinement of Neuronal Pathways in Retina

“…Morphologically, 11-14 subtypes of mouse RGCs have been reported (Masland, 2001;Sun et al, 2002;Badea and Nathans, 2004;Coombs et al, 2006). We found that Thy-1-YFP transgenelabeled RGCs were members of most of the RGC subtypes reported previously, except that a subpopulation of large RGCs having diameters of dendritic coverage from 350 to 450 m (Sun et al, 2002) was not represented in our YFP-labeled RGC samples (data not shown).…”

“…Double immunostainings were performed on whole-mounted retinas with antibodies against YFP, conjugated to Alexa Fluor fluorophore 488 (1:500; Invitrogen, San Diego, CA), and tyrosine hydroxylase (TH) (1:100; Chemicon, Temecula, CA), which labels dopaminergic neurons at the inner nuclear layer (INL) and IPL border. For each RGC, a total of 15-24 optical sections obtained with a Zeiss (Oberkochen, Germany) Pascal confocal microscope were collected at intervals of 0.8 -1.5 m. We followed the procedure of Badea and Nathans (2004) to quantify stratification patterns of RGC dendrites in the IPL. Measurements of the inner and outer edge of dendrites in the IPL were normalized to retinal thickness.…”

The neuron classification problem

“…They are usually determined monothetically, although polythetic analyses also refer to them (Kong et al, 2005;Badea and Nathans, 2004). As discussed above, the lowest hierarchy levels ideally are determined polythetically, and at the heart of these levels is the neuron type, a distinct cluster in parametric space (T n in Fig.…”

“…Interestingly, all rat retinal ganglion cell classification schemas so far are monothetic, although polythetic classifications have been introduced recently for other species (Kong et al, 2005;Badea and Nathans, 2004;Cohen and Sterling, 1992). In rat, most are based on morphological characters, but some use axonal projections, physiological properties, or gene expression patterns-or combinations of morphological characters and expressed molecules.…”

“…Therefore, terms included in neuron type and neuron group levels may change when polythetic analyses are performed. However, polythetic retinal neuron analysis in other species (Badea and Nathans, 2004) preserves neuron group as an intermediary level between neuron type and neuron class. Upper hierarchy levels are invariant in different classification schemas because, as discussed above, they were deduced from general retinal (neuron class level [Masland, 2001a]) and vertebrate CNS (category and division levels [Swanson, 2003;Swanson, 2004]) organization principles.…”