The calcium channel subunit α2δ-3 organizes synapses via an activity-dependent and autocrine BMP signaling pathway

Synapses are highly specialized for neurotransmitter signaling, yet activity-dependent growth factor release also plays critical roles at synapses. While efficient neurotransmitter signaling relies on precise apposition of release sites and neurotransmitter receptors, molecular mechanisms enabling high-fidelity growth factor signaling within the synaptic microenvironment remain obscure. Here we show that the auxiliary calcium channel subunit α2δ-3 promotes the function of an activity-dependent autocrine Bone Morphogenetic Protein (BMP) signaling pathway at the Drosophila neuromuscular junction (NMJ). α2δ proteins have conserved synaptogenic activity, although how they execute this function has remained elusive. We find that α2δ-3 provides an extracellular scaffold for an autocrine BMP signal, suggesting a mechanistic framework for understanding α2δ’s conserved role in synapse organization. We further establish a transcriptional requirement for activity-dependent, autocrine BMP signaling in determining synapse density, structure, and function. We propose that activity-dependent, autocrine signals provide neurons with continuous feedback on their activity state for modulating both synapse structure and function.


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Software and code
Policy information about availability of computer code Data collection Microscopy images were collected on either the FEI Tecnai G2 Spirit BioTWIN transmission electron microscope or the Zeiss LSM 800 confocal microscope. After imaging samples for Brp rings, Zeiss ZEN deconvolution software was used on images. Locomotion movies were collected on an iPhone 6s. Data collection from images was conducted using ImageJ (version 1.50c4).

Data analysis
All data was statistically analyzed using Prism (version 6.0h) software by GraphPad.
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Life sciences study design
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Sample size
No statistical methods were used to determine sample size. Previously published studies using the Drosophila neuromuscular junction provided insights into the current standard of sample sizes needed for experiments. Sample size was determined to be adequate based on the magnitude and consistency of measurable differences between groups.
Data exclusions For the Brp ring experiments, only planar rings were analyzed, as any rings imaged at a vertical orientation could not be visualized or analyzed.
Thus, not all Brp rings within a terminal bouton were analyzed, solely the planar-oriented rings.
In the pMad experiment, one larval tissue sample was excluded, as the signal to noise ratio was abnormally high. The investigator performing the pMad analysis had never seen such a high signal to noise ratio in any genotype analyzed, which was over hundreds of samples. Thus, this single tissue sample was excluded from the analysis.

Replication
Each experiment presented in the paper was repeated in multiple animals. For electron microscopy and nrx-1/syd-1 genetic interaction experiments, at least two larvae per genotype were analyzed. At least three animals were analyzed for all other experiments. The n for each genotype within an experiment is present in all graphs, and n is defined in the Materials and Methods section or the corresponding figure legend. Replicate experiments were successful and gave similar results. More than one technical replicate was performed for most of the experiments in this study.
Randomization Drosophila larvae or embryos of the appropriate genotypes were selected at random from a mating cross between male and female adult flies.

Blinding
The investigators were blinded during data collection and analysis for synapse density, Brp ring, and DVGLUT experiments involving wild-type and BMP mutant larvae. Blinding was not performed for additional control larvae in these experiments. Additionally, investigators were blinded for analysis of active zones containing T-bars in electron micrographs, larval locomotion, and Gbb-HA release.

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