Neurotransmitter release is stabilized by homeostatic plasticity. Presynaptic homeostatic potentiation (PHP) operates on timescales ranging from minute- to life-long adaptations and likely involves reorganization of presynaptic active zones (AZs). At Drosophila melanogaster neuromuscular junctions, earlier work ascribed AZ enlargement by incorporating more Bruchpilot (Brp) scaffold protein a role in PHP. We use localization microscopy (direct stochastic optical reconstruction microscopy [dSTORM]) and hierarchical density-based spatial clustering of applications with noise (HDBSCAN) to study AZ plasticity during PHP at the synaptic mesoscale. We find compaction of individual AZs in acute philanthotoxin-induced and chronic genetically induced PHP but unchanged copy numbers of AZ proteins. Compaction even occurs at the level of Brp subclusters, which move toward AZ centers, and in Rab3 interacting molecule (RIM)-binding protein (RBP) subclusters. Furthermore, correlative confocal and dSTORM imaging reveals how AZ compaction in PHP translates into apparent increases in AZ area and Brp protein content, as implied earlier.

Introduction: Neurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.Methods: We employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.Results and discussion: Electrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13^GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13^GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13^GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13^GFSTF protein levels.

(1) Background: Bone substitutes are essential in orthopaedic surgery to fill up large bone defects. Thus, the aim of the study was to compare diverse bone fillers biomechanically to each other in a clinical-relevant test set-up and to detect differences in stability and handling for clinical use. (2) Methods: This study combined compressive strength tests and screw pullout-tests with dynamic tests of bone substitutes in a clinical-relevant biomechanical fracture model. Beyond well-established bone fillers (ChronOS^TM Inject and Graftys^® Quickset), two newly designed bone substitutes, a magnesium phosphate cement (MPC) and a drillable hydrogel reinforced calcium phosphate cement (CPC), were investigated. (3) Results: The drillable CPC revealed a comparable displacement of the fracture and maximum load to its commercial counterpart (Graftys^® Quickset) in the clinically relevant biomechanical model, even though compressive strength and screw pullout force were higher using Graftys^®. (4) Conclusions: The in-house-prepared cement allowed unproblematic drilling after replenishment without a negative influence on the stability. A new, promising bone substitute is the MPC, which showed the best overall results of all four cement types in the pure material tests (highest compressive strength and screw pullout force) as well as in the clinically relevant fracture model (lowest displacement and highest maximum load). The low viscosity enabled a very effective interdigitation to the spongiosa and a complete filling up of the defect, resulting in this demonstrated high stability. In conclusion, the two in-house-developed bone fillers revealed overall good results and are budding new developments for clinical use.

Revealing the molecular organization of anatomically precisely defined brain regions is necessary for refined understanding of synaptic plasticity. Although three-dimensional (3D) single-molecule localization microscopy can provide the required resolution, imaging more than a few micrometers deep into tissue remains challenging. To quantify presynaptic active zones (AZ) of entire, large, conditional detonator hippocampal mossy fiber (MF) boutons with diameters as large as 10 µm, we developed a method for targeted volumetric direct stochastic optical reconstruction microscopy (dSTORM). An optimized protocol for fast repeated axial scanning and efficient sequential labeling of the AZ scaffold Bassoon and membrane bound GFP with Alexa Fluor 647 enabled 3D-dSTORM imaging of 25 µm thick mouse brain sections and assignment of AZs to specific neuronal substructures. Quantitative data analysis revealed large differences in Bassoon cluster size and density for distinct hippocampal regions with largest clusters in MF boutons.

Chemical synaptic transmission involves neurotransmitter release from presynaptic active zones (AZs). The AZ protein Rab-3-interacting molecule (RIM) is important for normal Ca²⁺-triggered release. However, its precise localization within AZs of the glutamatergic neuromuscular junctions ofDrosophila melanogasterremains elusive. We used CRISPR/Cas9-assisted genome engineering of therimlocus to incorporate small epitope tags for targeted super-resolution imaging. A V5-tag, derived from simian virus 5, and an HA-tag, derived from human influenza virus, were N-terminally fused to the RIM Zinc finger. Whereas both variants are expressed in co-localization with the core AZ scaffold Bruchpilot, electrophysiological characterization reveals that AP-evoked synaptic release is disturbed in rim^V5−Znfbut not in rim^HA−Znf. In addition, rim^HA−Znfsynapses show intact presynaptic homeostatic potentiation. Combining super-resolution localization microscopy and hierarchical clustering, we detect ∼10 RIM^HA−Znfsubclusters with ∼13 nm diameter per AZ that are compacted and increased in numbers in presynaptic homeostatic potentiation.

Traumatic brain injury (TBI) is the leading cause of death and disability in polytrauma and is often accompanied by concomitant injuries. We conducted a retrospective matched-pair analysis of data from a 10-year period from the multicenter database TraumaRegister DGU^® to analyze the impact of a concomitant femoral fracture on the outcome of TBI patients. A total of 4508 patients with moderate to critical TBI were included and matched by severity of TBI, American Society of Anesthesiologists (ASA) risk classification, initial Glasgow Coma Scale (GCS), age, and sex. Patients who suffered combined TBI and femoral fracture showed increased mortality and worse outcome at the time of discharge, a higher chance of multi-organ failure, and a rate of neurosurgical intervention. Especially those with moderate TBI showed enhanced in-hospital mortality when presenting with a concomitant femoral fracture (p = 0.037). The choice of fracture treatment (damage control orthopedics vs. early total care) did not impact mortality. In summary, patients with combined TBI and femoral fracture have higher mortality, more in-hospital complications, an increased need for neurosurgical intervention, and inferior outcome compared to patients with TBI solely. More investigations are needed to decipher the pathophysiological consequences of a long-bone fracture on the outcome after TBI.