TRP channels influence the development and excitability of cerebellar Purkinje cells. This project uses a cerebellum-like circuit in the brain of the fruit fly to pinpoint the mechanisms by which TRP channels shape the growth and function of neurons. It combines genetics, in vivo electrophysiology, and two-photon microscopy.
Project Details
Background:
The Trpc3 gene is associated with hereditary forms of ataxia. Mice with a gain-of-function mutation in this gene, which encodes the transient receptor potential channel TRPC3, feature a striking form of ataxia (1). Since the first report of the moonwalker phenotype, these mice have been teetering in circles. Sixteen years later, the pathophysiology of this channelopathy remains a mystery. Microscopic evidence hints at a developmental defect affecting the dendrites of cerebellar Purkinje cells (1), but the signaling pathways downstream of TRPC3 that control dendritogenesis are unknown. We propose to investigate this problem in Drosophila melanogaster where developmental, structural, and functional studies can be carried out with ease. The Drosophila mushroom body bears remarkable similarity to the vertebrate cerebellum (2): Kenyon cell axons resemble the parallel fibers of granule cells; in place of Purkinje cells, they form synapses with mushroom body output neurons (MBONs). Like their mammalian counterparts, MBONs have elaborate dendrites and express trp, the eponymous homologue of the mouse Trpc3 gene (3). The aim of the proposed project is to pinpoint the mechanisms by which TRP channels shape the growth and function of neurons.
Hypothesis and Objectives:
1) The Drosophila trp gene influences the morphology and excitability of MBONs. Our objective is to interfere with the expression and function of trp in MBONs at distinct developmental stages and assess neuronal structure and function.
2) TRP channels localize to distinct subcellular domains. The student will label TRP channels using an endogenous tag and visualize their subcellular distribution throughout development.
3) TRP channel activity changes over developmental stages and starts intracellular signalling cascades. Transcriptional, morphological and functional changes in response to acute TRP channel perturbation will be recorded using patch-seq experiments.
Methodology:
The student will use RNA interference and photopharmacology to control TRP channels in genetically defined MBONs and quantify neuronal structure and function using confocal microscopy and patch clamp electrophysiology in vivo. The generation of transgenic Drosophila lines will allow for the recombinase-dependent labelling of endogenous TRP channels in defined cell types. Tagged channels will be visualized during dendritogenesis using super-resolution microscopy. The student is expected to combine the acquired skills with the transcriptomic profiling of single cells to identify TRP-dependent signaling pathways. This project will provide training in Drosophila genetics, confocal and super-resolution microscopy, molecular biology, and in vivo electrophysiology.
References
- Becker et al. (2009) A point mutation in TRPC3 causes abnormal Purkinje cell development and cerebellar ataxia in moonwalker mice. Proc Natl Acad Sci U S A 106(16): 6706–11.
- Groschner & Miesenböck (2019) Mechanisms of sensory discrimination: Insights from Drosophila olfaction. Annu Rev Biophys 48(1):209–29.
- Davis et al. (2020) A genetic, genomic, and computational resource for exploring neural circuit function. eLife 9:e50901.
People Involved
Primary supervisor: Lukas Groschner
Collaborators: