Sutcliffe, B. | Search bioRxiv

bioRxiv2018

Neurogenetic dissection of the Drosophila innate olfactory processing center

Animals exhibit innate behaviours in response to a variety of sensory stimuli such as olfactory cues. In Drosophila, a higher olfactory centre called the lateral horn (LH) is implicated in innate behaviour. However, our knowledge of the structure and function of the LH is scant, due to the lack of sparse neurogenetic tools for this brain region. Here we generate a collection of split-GAL4 driver lines providing genetic access to 82 LH cell-types. We identify the neurotransmitter and axo-dendritic polarity for each cell-type. Using these lines were create an anatomical map of the LH. We found that [~]30% of LH projections converge with outputs from the mushroom body, the site of olfactory learning and memory. Finally, using optogenetic activation of small groups of LH neurons. We identify cell-types that drive changes in either valence or specific motor programs, such as turning and locomotion. In summary we have generated a resource for manipulating and mapping LH neurons in both light and electron microscopy and generated insights into the anatomy and function of the LH.

neuroscience↗

bioRxiv2016

Second Generation Drosophila Chemical Tags: Sensitivity, Versatility and Speed

Labeling and visualizing cells and sub-cellular structures within thick tissues, whole organs and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form sub-micron synapses but have arbors that may span millimeters in length. Traditionally labeling is achieved by immunofluorescence; however diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the centre of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo and TMP for tissue labeling; this resulted in >100 fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS and LexA responsive CLIP, SNAPf and Halo chemical labeling reagents for flies. These multimerized tags with translational enhancers display up to 64 fold increase in sensitivity over first generation reagents. In addition we developed a suite of conditional reporters (4xSNAPf tag and CLIP-SNAP-Halo) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

neuroscience↗