Motor circuit function is stabilized during postembryonic growth by anterograde trans-synaptic Jelly Belly - Anaplastic Lymphoma Kinase signaling

Abstract

SummaryThe brain adapts to a changing environment or growing body size by structural growth and synaptic plasticity. Mechanisms studied to date promote synaptic growth between partner neurons, while negative counterparts that inhibit such interactions have so far remained elusive. Here, we investigate the role of Jeb-Alk signaling in coordinating motor circuit growth during larval stages of Drosophila. We quantify neuronal growth dynamics by intra-vital imaging, and synaptogenesis at nanometer resolution using endogenously labeled synaptic proteins, conditionally tagged with a fluorophore, and link changes in circuit anatomy with altered synaptic physiology and behavior. We find that loss of Jeb-Alk signaling leads to increased strengthening of synaptic excitation by developmental addition of additional postsynaptic but not pre-synaptic specializations. These changes ultimately lead to an epilepsy-like seizure behavior. We thus demonstrate that trans-synaptic anterograde Jeb-Alk signaling acts to stabilize developmental plasticity and circuit function, and that it does so specifically during postembryonic growth.

New cell biological explanations for kinesin-linked axon degeneration

“…Cloning of UAS-Nox-YPet 10xUAS-IVS-Nox::YPet was generated by using the pJFRC12-10XUAS-IVS-myr-GFP vector (Addgene 26222; Pfeiffer et al, 2010) as a backbone which was modified by substituting GFP with YPet (Nguyen and Daugherty, 2005) plus an N-terminal flexible linker, amplified from dFlex_YPet_phase0 (Gärtig et al, 2019) using primer ML1 and ML2, and inserted by the Klenow Assembly Method (tinyurl.com/4r99uv8m) into the XbaI/BamHI sites producing Vector 1: pJFRC12-10xUAS-IVS-myr-linker-YPet. Nox cDNA was amplified from a DGRC (Drosophila Genomics Resource Center) cDNA library clone using primers ML5 and ML6, located in a pOTB7 vector backbone, and inserted into BamHI/XhoI sites of Vector 1.…”

The neuropeptide pigment‐dispersing factor signals independently of Bruchpilot‐labelled active zones in daily remodelled terminals of Drosophila clock neurons

“…The unchanged number of BRP FonYPet ‐labelled puncta between ZT2 and ZT14 in the sLNv is not in direct agreement with a previous study (Gorostiza et al, 2014). As the size of central synapses typically is below the resolution of confocal light microscopy, we wondered whether these results may be caused by insufficient spatial resolution and repeated the analysis with expanded brains using a previously established expansion microscopy (ExM) protocol (Gärtig et al, 2019). Unfortunately, this protocol resulted in a strong loss of staining intensity compared to the standard whole‐mount protocol and would require an optimised custom‐made microscopic setup to visualise BRP and PDF staining in the sLNv terminals (Figure 2a).…”

“…The following genotypes of Drosophila melanogaster were used for synaptic labelling: UAS‐brp::GFP‐TR725 (BL36292; Fouquet et al, 2009), dFLEX w; Pdf ‐Gal4/ brp FOnYpet , UAS‐FLP; +/MKRS (Gärtig et al, 2019). The lines for the targeted RNAi screen are listed in Supplementary Table 1 originated from the shRNA, KK and GD library of the Vienna Drosophila Resource Center (VDRC) or was a kind gift of Stephan Sigrist (Freie Universität Berlin, Germany), Paul Taghert (U Washington, St. Louis, USA) or Michael Bender (U Georgia, Athens, GA, USA).…”

“…For expansion microscopy (ExM, Chozinski et al, 2016), ICC was performed as described above, with a few additional steps as described earlier (Gärtig et al, 2019). After ICC, samples were transferred from the reaction tubes into a droplet of PBS on poly‐Lysine coated glass slides (ThermoFisher) stabilised by a bean‐shaped ring drawn with a PAP‐Pen (Merck).…”

“…We therefore switched to the dFLEx system (Gärtig et al, 2019) and expressed fluorescently labelled BRP FO-nYPet at endogenous levels in the sLNvs. Using dFLEX, we observed labelling of membrane-associated puncta in a size range around 400 nm in confocal resolution (Figure 1c) which is compatible with the data from electron microscopic measurements at the NMJ (Kaufmann et al, 2002).…”