Neurogenetics

Lines to genetically manipulate columnar elements. Drosophila brains expressing GFP and synaptotagmin with driver lines specific for columnar neurons as indicated. GFP labels the entire cell while synaptotagmin labels exclusively presynaptic sites. Endogenously expressed synaptic protein bruchpilot highlighting the synaptic neuropiles is shown in blue.

Brains are difficult to study because they comprise complex and densely intermingled neuronal circuits composed of vast numbers of diverse nerve cells. Transgenic approaches - highly developed in Drosophila - provide opportunities for non-invasive cell-specific manipulations and visualizations that are not limited by anatomical constraints (Borst, 2009; Venken et al., 2011). Combined with various assays, the morphology and functional roles of neurons can be determined in a highly reproducible fashion for one neuronal type at a time.

To this end, the existence of cell-specific DNA regulatory elements is exploited to generate stable transgenic fly lines in which a transcription factor derived from other organisms (usually Gal4 from yeast) is expressed specifically in a neuron of interest. For many visual interneurons, we have such lines at our disposal that can be crossed to Gal4-activatable UAS actuator lines for cell-specific visualization and manipulation. Silencing of neurons can be achieved for instance by expressing the inwardly rectifying potassium channel Kir2.1 (Baines et al., 2001), the temperature-inducible synaptic inhibitor shibirets (Kitamoto, 2001), synaptobrevin-cleaving tetanus toxin light chain (Sweeney et al., 1995), or the photo-activatable chloride channel GtACR1 (Govorunova et al., 2015; Mauss et al., 2017). As a complementary approach, neurons can be activated with heat using the TrpA1 channel (Marella et al., 2006) or with light using photo-activatable Channelrhodopsin2 (Boyden et al., 2005; Pulver et al., 2009; Haikala et al., 2013, Mauss et al., 2014).

Optogenetic and pharmacologic dissection of the lobula plate connectivity.The schematic illustrates the anatomical layout of fly visual neuropiles medulla, lobula and lobula plate. One lobula plate tangential cell (LPTC) of the vertical system (VS) is shown in green with recording electrode; the dendrites arborize in layer 4 of the lobula plate. Examples of two T4 and two T5 cells are depicted in red that receive input onto their dendrites in the medulla and lobula, respectively, and convey information to the lobula plate. To probe the synaptic connectivity functionally between T4/T5 and LPTCs we photoactivate T4/T5 cells expressing optogenetic tools and record the synaptic responses in LPTCs electrophysiologically. The micrograph shows an in vivo Drosophila preparation with T4/T5 cells expressing Channel-rhodopsin2-mCherry and an LPTC (VS cell) filled with Alexa488 via a patch electrode. This approach can be combined with pharmacologic block of certain neurotransmitter receptor types.

Combined with a suitable behavioral readout (Rister et al., 2007) or whole-cell patch-clamp recordings from LPTCs (Joesch et al., 2008), detailed circuit information and function can be inferred from such manipulations (Joesch et al., 2010; Schnell et al., 2012; Bahl et al., 2013; Joesch et al., 2013; Maisak et al., 2013, Mauss et al., 2014, Mauss et al., 2015). In addition, the cell-specific expression of fluorescence reporters of neural activity like TN-XXL (Mank et al., 2008; Reiff et al., 2010), GCaMP (Akerboom et al., 2012; Maisak et al., 2013) or iGluSnFR (Marvin et al., 2013; Richter et al., 2018) can be used establish visual response properties of neurons that are not accessible to electrodes.

References

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Bahl A, Ammer G, Schilling T, Borst A (2013) Object tracking in motion-blind flies. Nat Neurosci 16:730-738.

Baines R, Uhler J, Thompson A, Sweeney S, Bate M (2001) Altered electrical properties in Drosophila neurons developing without synaptic transmission. J Neurosci 21:1523-1531.

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