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. — **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.

© MPI of Neurobiology / Ammer & Mauss

**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.

© MPI of Neurobiology / Ammer & Mauss

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. — **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.

© MPIN / Alex Mauss

**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.

© MPIN / Alex Mauss

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

Akerboom J, et al. (2012) Optimization of a GCaMP calcium indicator for neural activity imaging. J Neurosci 32(40):13819–13840.

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.

Borst A (2009) Drosophila's view on insect vision. Curr Biol 19:R36-R47.

Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263–1268.

Govorunova EG, Sineshchekov OA, Janz R, Liu X, Spudich JL (2015) Natural light-gated anion channels: A family of microbial rhodopsins for advanced optogenetics. Science 349(6248):647–650.

Haikala V, Joesch M, Borst A, Mauss AS (2013) Optogenetic control of fly optomotor responses. J Neurosci 33:13927-13934.

Joesch M, Plett J, Borst A, Reiff D (2008) Response properties of motion-sensitive visual interneurons in the lobula plate of Drosophila melanogaster. Curr Biol 18:368-374.

Joesch M, Weber F, Eichner H, Borst A (2013) Functional specialization of parallel motion detection circuits in the fly. J Neurosci 33:902-905.

Joesch M, Schnell B, Raghu S, Reiff D, Borst A (2010) ON and OFF pathways in Drosophila motion vision. Nature 468:300-304.

Kitamoto T (2001) Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons. J Neurobiol 47:81-92.

Maisak MS, Haag J, Ammer G, Serbe E, Meier M, Leonhardt A, Schilling T, Bahl A, Rubin GM, Nern A, Dickson BJ, Reiff DF, Hopp E, Borst A (2013) A directional tuning map of Drosophila elementary motion detectors. Nature 500:212-216.

Mank M, Santos A, Direnberger S, Mrsic-Flogel T, Hofer S, Stein V, Hendel T, Reiff D, Levelt C, Borst A, Bonhoeffer T, Hübener M, Griesbeck O (2008) A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nature Methods 5:805-811.

Marella S, Fischler W, Kong P, Asgarian S, Rueckert E, Scott K (2006) Imaging taste responses in the fly brain reveals a functional map of taste category and behavior. Neuron 49:285-295.

Marvin JS, Borghuis BG, Tian L, Cichon J, Harnett MT, Akerboom J, Gordus A, et al. (2013) An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat Methods 10(2):162-170.

Mauss AS, Meier M, Serbe E, Borst A (2014) Optogenetic and pharmacologic dissection of feedforward inhibition in Drosophila motion vision. J Neurosci 34: 2254-2263.

Mauss AS, Pankova K, Arenz A, Nern A, Rubin GM, Borst A (2015) Neural circuit to integrate opposing motions in the visual field. Cell 162: 351-362.

Mauss AS, Busch C, Borst A (2017) Optogenetic neuronal silencing in Drosophila during visual processing. Sci Reports 7: 13823.

Pulver S, Pashkovski S, Hornstein N, Garrity P, Griffith L (2009) Temporal dynamics of neuronal activation by Channelrhodopsin-2 and TRPA1 determine behavioral output in Drosophila larvae. J Neurophysiol 101:3075-3088.

Reiff D, Plett J, Mank M, Griesbeck O, Borst A (2010) Visualizing retinotopic half-wave rectified input to the motion detection circuitry of Drosophila. Nat Neurosci 13:973-978.

Richter FG, Fendl S, Haag J, Drews MS, Borst A (2018) Glutamate signaling in the fly visual system. iScience 7:85-95.

Rister J, Pauls D, Schnell B, Ting CY, Lee CH, Sinakevitch I, Morante J, Strausfeld NJ, Ito K, Heisenberg M (2007) Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster. Neuron 56:155-170.

Schnell B, Raghu S, Nern A, Borst A (2012) Columnar cells necessary for motion responses of wide-field visual interneurons in Drosophila. J Comp Physiol A 198:389-395.

Sweeney S, Broadie K, Keane J, Niemann H, O'Kane C (1995) Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14:341-351.

Venken K, Simpson J, Bellen H (2011) Genetic manipulation of genes and cells in the nervous system of the fruit fly. Neuron 72: 202-230.