Active zone compaction in presynaptic homeostatic potentiation

Abstract

19 20 * Correspondence should be adressed to M.H. (heckmann@uni-wuerzburg.de) and 21 M.M.P. (mila.paul@uni-wuerzburg.de) 22molecular environment for fast and precise neurotransmitter release (Kittel et al., 2006; 75 Held and Kaeser, 2018). Whereas the Brp N-term was mapped in membrane-76 proximity, its C-term covers an area of about 0.1 µm 2 , localizes about 155 nm above 77 the postsynaptic receptors (Fouquet et al., 2009; Liu et al., 2011) and is important for 78 tethering of synaptic vesicles (Hallermann et al., 2010). Remarkably, Brp is distributed 79 heterogeneously within the AZ forming about 15 subclusters of 1.6 x 10 -3 µm 2 size 80 (Ehmann et al., 2014). 81 82Alterations in protein number contribute to synaptic differentiation and plasticity. This 83 is well established for receptor channels in postsynaptic densities (Tang et al., 2016). 84Less clear is to what extend presynaptic protein number changes contribute to 85 plasticity. Imaging Brp and other AZ components using confocal and STED microscopy 86 provided evidence for an increase in AZ area and cluster numbers in acute and chronic 87 homeostatic plasticity (Weyhersmüller et al., 2011;Goel et al., 2017; Böhme et al., 88 2019). We suggest that beside an increase in absolute protein numbers per AZ, local 89 protein rearrangements leading to alterations in synaptic strength are conceivable. To 90 address this hypothesis, we performed localization microscopy of AZs in phasic and 91 tonic presynapses at the NMJ and induced presynaptic homeostatic plasticity as a 92 model for acute changes in release (Davis and Müller, 2015). Hierarchical density-93 based spatial clustering (HDBSCAN) enabled unbiased analysis of our datasets. We 94 identified striking differences in AZ scaffold organization with more compact Brp 95 arrangement, increased molecular density and narrower positioning of AZ subclusters 96 in high release probability, phasic type Is boutons compared to low probability, tonic 97 type Ib boutons. Furthermore, we observed similar changes following induction of 98 acute presynaptic plasticity only in tonic type Ib boutons. In summary, our data suggest 99 AZ compaction to be a structural correlate of enhanced synaptic transmission in 100 synaptic differentiation and homeostatic plasticity. 101 5 MATERIAL AND METHODS 102 Fly stocks 103 Flies were raised on standard cornmeal and molasses medium at 25 °C. Drosophila 104 melanogaster wildtype strain w 1118 (Bloomington Drosophila Stock Center) male 3 rd 105 instar larvae were used for experiments. 106 107 Philanthotoxin treatment and and larval preparation 108 Philanthotoxin 433 tris (trifluoroacetate) salt (PhTx, P207 Sigma) was dissolved in 109 dimethyl sulfoxide (DMSO) to obtain a stock solution of 4 mM and stored at -20 °C. For 110 each experiment, the respective volume was further diluted with freshly prepared 111 haemolymph-like solution (HL-3, Stewart et al., 1994) to a final PhTx concentration of 112 20 µM in 0.5 % DMSO. Control experiments were performed with the same DMSO 113 (Dianova)...

Distinct Target-Specific Mechanisms Homeostatically Stabilize Transmission at Pre- and Post-synaptic Compartments

“…Finally, given these reductions in the density and intensity of active zone components, the total intensity of both BRP and Cac sfGFP per hyper-innervated NMJ was not significantly different from wild type despite the increase in their total number ( Figure 3G ). These results parallel recent studies that have shown that while the number and intensity of individual active zones can vary at NMJs, the total abundance of active zone protein remains constant (Graf et al, 2009 ; Goel et al, 2019a , b ) or can reflect nanoscale remodeling of active zone components (Böhme et al, 2019 ; Mrestani et al, 2020 ). Together, hyper-innervated NMJs express a target-specific reduction in both the number and intensity of release sites per bouton and a parallel reduction in presynaptic release probability that stabilizes synaptic strength, while no reciprocal changes are observed at hypo-innervated counterparts.…”

“…Although a similar reduction in the abundance of active zone proteins at individual release sites has recently been found in mutations that cause synaptic overgrowth at the NMJ (Goel et al, 2019a , b ), the adaptations observed in the case of hyper-innervation are distinct in that they are: (1) target-specific; and (2) involve a reduction in active zone density in addition to their apparent intensity. Although increased fluorescence intensity is typically interpreted to reflect enhanced protein abundance, a recent study using Localization Microscopy showed that increased active zone intensity may instead reflect a more compact nanoscopic arrangement (Mrestani et al, 2020 ). Nonetheless, it is remarkable that both the number and intensity of active zone components can be selectively reduced and calibrated at hyper-innervated terminals without any apparent changes at adjacent terminals shared by the same neuron on the hypo-innervated target.…”

Distinct molecular pathways govern presynaptic homeostatic plasticity

“…PhTx application at the Drosophila NMJ results in an increase in immunofluorescence intensity of several active-zone proteins, including the scaffolding protein Bruchpilot (Brp), suggesting increased Brp abundance and/or reorganization ( Böhme et al, 2019 ; Mrestani et al, 2021 ; Gratz et al., 2019 ; Weyhersmüller et al., 2011 ). We therefore examined whether Gyki-induced PHP also involves Brp modulation.…”

A Picture Worth a Thousand Molecules—Integrative Technologies for Mapping Subcellular Molecular Organization and Plasticity in Developing Circuits

“…Developmental studies of the NMJ using ExM imaging show a progressive increase in BRP area and complexity as the synapse matures (Jiang et al, 2018; Figure 5D). STED and STORM have further characterized the rapid remodeling of BRP and other active zone components during synaptic plasticity, showing that the presynaptic proteins regulating transmission are modulated to maintain synaptic efficacy during homeostatic scaling (Weyhersmüller et al, 2011;Böhme et al, 2019;Mrestani et al, 2019). STORM imaging of the NMJ has further resolved individual BRP clusters within rings at the active zone (Ehmann et al, 2014) and was later used to discover species differences in subsynaptic NMJ active zone organization in mouse vs. humans (Jones et al, 2017).…”