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Purpose: This study aimed at comparing clinical outcome, recanalization success and time metrics in the "drip and ship" (DS) vs. "drive the doctor" (DD) concept in a comparable setting. Methods: This is a retrospective analysis of thrombectomy registries of a comprehensive stroke center (CSC) and a thrombectomy-capable stroke center (TSC). Patients, who were transferred from the TSC to the CSC, were classified as DS. Patients treated at the TSC by an interventionalist transferred from the CSC were classified as DD. Good outcome was defined as mRS 0-2 or equivalent to premorbid mRS at discharge. Recanalization (TICI 2b-3 or equivalent) and time metrics were compared in both groups. Results: In total, 295 patients were included, of which 116 (39.3%) were treated in the DS concept and 179 (60.7%) in the DD concept. Good clinical outcome was similarly achieved in DS and DD (DS 25.0% vs. DD 31.3%, P = 0.293). mRS on discharge (DS median 4, DD median 4, P = 0.686), NIHSS improvement (DS median 4, DD median 5, P = 0.582) and NIHSS on discharge (DS median 9, DD median 7, P = 0.231) were similar in both groups. Successful reperfusion was achieved similarly in DS (75.9%) and DD as well (81.0%, P = 0.375). Time from onset to reperfusion (median DS 379 vs. DD 286 min, P = 0.076) and time from initial imaging to reperfusion were longer in DS compared to DD (median DS 246 vs. DD 162 min, P < 0.001). Conclusion: The DD concept is time saving while achieving similar clinical outcome and recanalization results.
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Erbin, Lano, Scribble, and Densin-180 belong to LAP (leucine-rich repeats and PDZ domain) adaptor proteins involved in cell signaling pathways. Previously, we identified Erbin, Lano, and Scribble, but not Densin-180, in muscle cells, where they are involved in regulating the aggregation of nicotinic acetylcholine receptors in vitro. Here, we analyzed their cellular localization at the neuromuscular junction (NMJ) in skeletal muscles of mice. Erbin, Lano, and Scribble were significantly accumulated at NMJs and localized in different synaptic cells. Moreover, we used mouse mutants to analyze the role of Erbin at the NMJ. We used two Erbin mutant mouse strains that either completely lack Erbin protein (Erbinnull/null ) or express a truncated Erbin mutant where the carboxy-terminal PDZ domain is replaced by ß-galactosidase (ErbinΔC/ΔC ) thereby abolishing its interaction with ErbB receptor tyrosine kinases. Neither the lack of the PDZ domain of Erbin, nor its complete absence interfered with the general localization of LAP proteins at NMJs, but Lano and Scribble transcript levels were up-regulated in homozygous Erbin-null muscles. Furthermore, grip strength was reduced and neural transmission impaired in homozygous aged Erbin-null but not Erbin-ΔC mice. Erbin-null skeletal muscles did not reveal any conspicuous impairment of the muscle fiber. Localization of other NMJ marker proteins was not affected either. Quantitative 3D morphometry showed that NMJs of Erbin-null muscles were significantly smaller and fragmented in the soleus. We speculate that Erbin, Lano, and Scribble act at the post-synaptic membrane of NMJs in a concerted fashion to regulate nicotinic acetylcholine receptors cluster morphology and neural transmission. Cover Image for this issue: doi: 10.1111/jnc.13340.
Assuntos
Junção Neuromuscular/fisiologia , Proteínas/genética , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Força da Mão/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Repetições Ricas em Leucina , Masculino , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/fisiologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/inervação , Mutação/genética , Proteínas do Tecido Nervoso , Junção Neuromuscular/ultraestrutura , Domínios PDZ/genéticaRESUMO
Amyloid-ß (Aß)-peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aß precursor protein (APP) by ß-site APP-cleaving enzyme (BACE). Using live-cell imaging of APP and BACE labeled with pH-sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α-secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual-color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/metabolismo , Células HeLa , Humanos , Transporte Proteico , ProteóliseRESUMO
In compensatory endocytosis, scission of vesicles from the plasma membrane to the cytoplasm is a prerequisite for intravesicular reacidification and accumulation of neurotransmitter molecules. Here, we provide time-resolved measurements of the dynamics of the alkaline vesicle population which appears upon endocytic retrieval. Using fast perfusion pH-cycling in live-cell microscopy, synapto-pHluorin expressing rat hippocampal neurons were electrically stimulated. We found that the relative size of the alkaline vesicle population depended significantly on the electrical stimulus size: With increasing number of action potentials the relative size of the alkaline vesicle population expanded. In contrast to that, increasing the stimulus frequency reduced the relative size of the population of alkaline vesicles. Measurement of the time constant for reacification and calculation of the time constant for endocytosis revealed that both time constants were variable with regard to the stimulus condition. Furthermore, we show that the dynamics of the alkaline vesicle population can be predicted by a simple mathematical model. In conclusion, here a novel methodical approach to analyze dynamic properties of alkaline vesicles is presented and validated as a convenient method for the detection of intracellular events. Using this method we show that the population of alkaline vesicles is highly dynamic and depends both on stimulus strength and frequency. Our results implicate that determination of the alkaline vesicle population size may provide new insights into the kinetics of endocytic retrieval.
Assuntos
Hipocampo/metabolismo , Concentração de Íons de Hidrogênio , Sinapses/metabolismo , Potenciais de Ação , Animais , Endocitose , Hipocampo/fisiopatologia , Ratos , Ratos WistarRESUMO
Effects of the antidepressant fluoxetine in therapeutic concentration on stimulation-dependent synaptic vesicle recycling were examined in cultured rat hippocampal neurons using fluorescence microscopy. Short-term administration of fluoxetine neither inhibited exocytosis nor endocytosis of RRP vesicular membranes. On the contrary, acute application of the drug markedly increased the size of the recycling pool of hippocampal synapses. This increase in recycling pool size was corroborated using the styryl dye FM 1-43, antibody staining with αSyt1-CypHer™5E and overexpression of synapto-pHluorin, and was accompanied by an increase in the frequency of miniature postsynaptic currents. Analysis of axonal transport and fluorescence recovery after photobleaching excluded vesicles originating from the synapse-spanning superpool as a source, indicating that these new release-competent vesicles derived from the resting pool. Super resolution microscopy and ultrastructural analysis by electron microscopy revealed that short-term incubation with fluoxetine had no influence on the number of active synapses and synaptic morphology compared to controls. These observations support the idea that therapeutic concentrations of fluoxetine enhance the recycling vesicle pool size and thus the recovery of neurotransmission from exhausting stimuli. The change in the recycling pool size is consistent with the plasticity hypothesis of the pathogenesis of major depressive disorder as stabilization of the vesicle recycling might be responsible for neural outgrowth and plasticity.
Assuntos
Antidepressivos/farmacologia , Fluoxetina/farmacologia , Hipocampo/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Células Cultivadas , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Hipocampo/citologia , Hipocampo/metabolismo , Ratos , Ratos Wistar , Sinapses/metabolismo , Vesículas Sinápticas/metabolismoRESUMO
The plethora of available scientific cameras of different types challenges the biologically oriented experimenter when picking the appropriate camera for his experiment. In this study, we chose to investigate camera performances in a typical nonsingle molecule situation in life sciences, that is, quantitative measurements of fluorescence intensity changes from video data with typically skewed intensity distributions. Here, intensity profile dynamics of pH-sensors upon triggered changes of pH-environments in living cells served as a model system. The following camera types were tested: sCMOS, CCD (scientific and nonscientific) and EM-CCD (back- and front-illuminated). We found that although the EM-CCD cameras achieved the best absolute spatial SNR (signal-to-noise ratio) values, the sCMOS was at least of equal performance when the spatial SNR was related to the effective dynamic range, and it was superior in terms of temporal SNR. In the measurements of triggered intensity changes, the sCMOS camera had the advantage that it used the smallest fraction of its dynamic range when depicting intensity changes, and thus featured the best SNR at full usage of its dynamic range.
Assuntos
Proteínas de Fluorescência Verde/análise , Microscopia de Fluorescência/instrumentação , Animais , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Microscopia de Fluorescência/métodos , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
Hippocampal neurons in tissue culture develop functional synapses that exhibit considerable variation in synaptic vesicle content (20-350 vesicles). We examined absolute and fractional parameters of synaptic vesicle exocytosis of individual synapses. Their correlation to vesicle content was determined by activity-dependent discharge of FM-styryl dyes. At high frequency stimulation (30 Hz), synapses with large recycling pools released higher amounts of dye, but showed a lower fractional release compared to synapses that contained fewer vesicles. This effect gradually vanished at lower frequencies when stimulation was triggered at 20 Hz and 10 Hz, respectively. Live-cell antibody staining with anti-synaptotagmin-1-cypHer 5, and overexpression of synaptopHluorin as well as photoconversion of FM 1-43 followed by electron microscopy, consolidated the findings obtained with FM-styryl dyes. We found that the readily releasable pool grew with a power function with a coefficient of 2/3, possibly indicating a synaptic volume/surface dependency. This observation could be explained by assigning the rate-limiting factor for vesicle exocytosis at high frequency stimulation to the available active zone surface that is proportionally smaller in synapses with larger volumes.
Assuntos
Hipocampo/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Artefatos , Endocitose , Exocitose , Corantes Fluorescentes/metabolismo , Hipocampo/citologia , Hipocampo/ultraestrutura , Cinética , Microscopia de Fluorescência , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Ratos , Ratos Wistar , Propriedades de Superfície , Vesículas Sinápticas/ultraestrutura , Fatores de TempoRESUMO
Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1-43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.