Another variation is an intersectional technique that relies on split binary systems, pioneered by the split-GAL4 system (Luan et al., 2006b) that was recently optimized (Pfeiffer et al., 2010) (Figure 3D). The GAL4 transcription factor is split into two hemidrivers, each of

which is driven by separate regulatory elements. Obeticholic Acid Where the expression domains overlap, both halves of GAL4 are expressed, heterodimerize via leucine zippers, and reconstitute a functional activator. A similar split strategy was recently developed for LexA (Ting et al., 2011). Another intersectional strategy combines GAL4 with Flp recombinase (Golic and Lindquist, 1989), each driven by separate regulatory elements. The expression of transactivator, responder, or repressor depends on recombinase activity removing an intervening stop cassette (Struhl and Basler, 1993). Alternatively, GAL80 can be activated by Flp-In so that only cells that express GAL4

and not Flp are capable of expressing a UAS-responder element (Bohm et al., 2010) (Figure 3E). Many combinations of the orthogonal binary expression systems and Flp recombinase can be envisioned (Potter et al., 2010, Bohm et al., 2010, Yagi et al., 2010 and Potter and Luo, 2011). The development of new recombinases and alternative target sites further broadens the combinatorial palette (Nern et al., 2011 and Hadjieconomou et al., 2011). The binary SNS-032 cost systems described in the previous section can be used to overexpress reporters to label neuronal subpopulations or subcompartments of these neurons (Table 1). Numerous fluorescent reporters are available. To label the entire cytoplasmic compartment, fluorescent proteins can be overexpressed (Yeh the et al., 1995, Halfon et al., 2002 and Pfeiffer et al., 2010). Fluorescent markers fused to membrane targeted domains label the cell outline (Lee and Luo, 1999, Ritzenthaler et al., 2000, Ye et al., 2007, Yu et al., 2009a and Pfeiffer et al., 2010). Fusions with synaptic vesicle proteins predominantly label the presynaptic compartment of synaptic contacts (Estes et al., 2000, Zhang et al.,

2002 and Rolls et al., 2007). Active zones can be labeled with bruchpilot-GFP (Wagh et al., 2006) or cacophony-GFP (Kawasaki et al., 2004). While there is no generic marker for postsynaptic sites, Denmark (Nicolaï et al., 2010) or Dscam[exon 17.1] (Wang et al., 2004) preferentially labels dendrites. Fusions to neurotransmitter receptor proteins such as UAS-Rdl-HA and UAS-Dα7-GFP can also be used to identify synapses (Sánchez-Soriano et al., 2005 and Leiss et al., 2009). Markers that label subcellular organelles include fluorescent proteins fused to targeting elements specific for mitochondria, endoplasmatic reticulum, Golgi, and nucleus (LaJeunesse et al., 2004 and Yasunaga et al., 2006). A fusion with horseradish peroxidase is useful for transmission electron microscopy (Larsen et al., 2003 and Watts et al., 2004).