Background

The Drosophila visual system provides an excellent model to understand the mechanisms controlling neuronal circuit development and function. Each adult Drosophila compound eye is made up of roughly 800 single eye units called ommatidia. The ommatidia reside in the most distal portion of the visual system, called the retina. Each ommatidium is composed of 8 light-sensitive photoreceptors (R cells), with the motion-sensitive outer R1-R6 cells forming a stereotypical horseshoe shape around the color-sensitive inner R7 and R8 cells. The R7 cell body lies in the distal retina, directly above the more proximal R8 cell body. The R1-R6 cells are responsible for motion detection and express rhodopsin(Rh)-1 which detects light in a broad spectrum (Montell, 2012; Behnia and Desplan, 2015). R7 and R8 photoreceptors are responsible for color vision and express Rh3 or Rh4 and Rh5 or Rh6, respectively (Rister and Heisenberg, 2006; Morante and Desplan, 2008).

R7 and R8 photoreceptors can be further classified into either short-wavelength pale (p) or long-wavelength yellow (y) subtypes depending on the rhodopsin they express. R7p and R7y express Rh-3 and Rh-4 respectively, and detect light in the UV-spectrum, while R8p and R8y express Rh-5 and Rh-6 respectively, and detect blue and green light, respectively (Morante and Desplan, 2008). This specification of the ommatidia to either a yellow or pale fate is not evenly distributed, with ~70% of the ommatidia containing the yellow (long-wave UV and green) and ~30% containing the pale (short-wave UV and blue) (Yamaguchi et al., 2010; Montell, 2012). All R cells have their cell bodies within the retina, and project the axons into the optic lobe where they form synaptic connections with their respective targets (Fischbach and Dittrich, 1989).

The difference in spectral sensitivity among R7 (UV light) and R8 (blue or green light) subtypes, and that flies demonstrate phototactic behavior (the preference to move towards a light source) (Yamaguchi et al., 2010), allow us to test the differential functionality of R7 and R8 photoreceptors by developing a behavioral choice paradigm with “UV vs. blue” or “UV vs. green” light sources. This is an example of “differential phototaxis”, where flies will choose between two light sources. Conversely, “fast phototaxis” can be observed when flies in a “light vs. dark” paradigm rush quickly towards the light source (Benzer, 1967), believing it to be an escape. The development of a rapid phototactic T-maze assay to assess the difference in color preference is very useful for understanding the differential requirements of genes for the development and function of R7 and R8 photoreceptors, which form synaptic contacts with different post-synaptic partners (Rister et al., 2015; Millard and Pecot, 2018; Shaw et al., 2019).

Electrophysiological recordings and calcium imaging have been able to elegantly demonstrate photoreceptor subtype functionality (Harris et al., 1976; Dolph et al., 2011; Weir et al., 2016; Schnaitmann et al., 2018). However, these approaches require expensive equipment and are technically challenging. By comparison, the phototactic T-maze paradigm behavioral assay is an affordable, simple, and high throughput method, which can be readily used in large-scale genetic screens for genes controlling color vision. The behavioral T-maze choice assay we describe here is modified from the phototactic choice paradigm described previously by Yamaguchi et al. (2010), whose design in turn was based off of the original odor paradigm generated by Tully and Quinn (1985). In this protocol, we describe in detail the schematics for building the T-maze apparatus and the use of this system for the analysis of color vision. In our recent study (Shaw et al., 2019), we used this T-maze system to examine the role of the transmembrane protein Borderless (Bdl) in the regulation of color vision. Our apparatus has a horizontal design, as oppose to the traditional vertical design, for increased stability and convenience. Additionally, our data analysis takes into account the animals that do not make a choice (neutral flies that remain at the choice point), which would significantly affect the results in certain experimental paradigms.