Styryl Dye FM1-43 Research Articles

Chloroquine Affects Frequency-Dependent Endocytosis in Diaphragm Muscle of Old Mice

Chloroquine is an antimalarial agent commonly used as an autophagy inhibitor by preventing autolysosomal fusion. Autophagy is an important cellular process through which defective cytoplasmic structures are recycled and degraded that has been implicated in aging neuromuscular effects. We recently showed that acute chloroquine treatment blunts the maximal forces generated by the diaphragm muscle, likely by impairing neuromuscular transmission generally consistent with aging effects in the diaphragm muscle. In addition, old mice show increased accumulation of autophagy proteins in phrenic motor neurons, consistent with impaired autophagy flux. In the present study, we hypothesized that chloroquine treatment results in accumulation of bulk endosomes pre-synaptically. Each hemidiaphragm with attached phrenic nerve was excised from 24-month-old male and female C57BL/6 mice to evaluate frequency dependent (0 vs 80 Hz stimulation) uptake of the styryl dye FM4-64, given that 80-Hz stimulation frequency induces bulk endocytosis after synaptic vesicle retrieval. Diaphragm-phrenic nerve preparations were incubated in 5 μM FM4-64 for 1 h prior to 6-min nerve stimulation (0.5 ms supramaximal pulses in 80 Hz, 200 ms trains delivered at 40% duty cycle) followed by a 5-min post-stimulation incubation. FM4-64 uptake at diaphragm neuromuscular junctions (NMJs) was measured using confocal microscopy. Preparations were randomly assigned to co-incubation with chloroquine (50 μM) or vehicle (Rees-Simpson solution). There was minimal FM4-64 uptake in the absence of phrenic nerve stimulation, consistent with the activity-dependent uptake of the styryl dye. With 80 Hz stimulation, high levels of FM4-64 uptake were evident in both the chloroquine and vehicle-treated groups without significant differences across groups. FM4-64 fluorescence was evident throughout the presynpatic terminal with few areas of dense accumulation, consistent with retention of endosomes. There was no evidence of greater endosome accumulation with chloroquine treatment. Of note, a slight increase in FM4-64 uptake was evident in the chloroquine treated groups compared to vehicle in the non-stimulated NMJs. These results are consistent with FM4-64 uptake reflecting bulk endocytosis that mainly occurs during high frequency stimulation. The impairment in autophagy flux following chloroquine treatment is expected to result in endosome accumulation but it is possible that the stimulation protocol was insuffcient to elucidate this possibility. Collectively, the findings of this study are consistent with impaired autophagy at diaphragm presynaptic terminals which may underlie neuromuscular deficits observed in old age. Supported by NIH grant R01 AG057052 and the Mayo Clinic. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A convenient fluorimetry-based degranulation assay using RBL-2H3 cells.

Type I hypersensitivity is triggered by mast cell degranulation, a stimulus-induced exocytosis of preformed secretory granules (SGs) containing various inflammatory mediators. The degree of degranulation is generally expressed as a percentage of secretory granule markers (such as β-hexosaminidase and histamine) released into the external solution, and considerable time and labor are required for the quantification of markers in both the supernatants and cell lysates. In this study, we developed a simple fluorimetry-based degranulation assay using rat basophilic leukemia (RBL-2H3) mast cells. During degranulation, the styryl dye FM1-43 in the external solution fluorescently labeled the newly exocytosed SGs, whose increase in intensity was successively measured using a fluorescence microplate reader. In addition to the rate of β-hexosaminidase secretion, the cellular FM1-43 intensity successfully represented the degree and kinetics of degranulation under various conditions, suggesting that this method facilitates multi-sample and/or multi-time-point analyses required for screening substances regulating mast cell degranulation.

Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives.

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5µM), at a 10-fold lower concentration (0.5µM), two of the derivatives protect OHCs from cell death caused by 48h-exposure to 5µM gentamicin.

Labeling PIEZO2 activity in the peripheral nervous system

Sensory neurons detect mechanical forces from both the environment and internal organs to regulate physiology. PIEZO2 is a mechanosensory ion channel critical for touch, proprioception, and bladder stretch sensation, yet its broad expression in sensory neurons suggests it has undiscovered physiological roles. To fully understand mechanosensory physiology, we must know where and when PIEZO2-expressing neurons detect force. The fluorescent styryl dye FM 1-43 was previously shown to label sensory neurons. Surprisingly, we find that the vast majority of FM 1-43 somatosensory neuron labeling in mice invivo is dependent on PIEZO2 activity within the peripheral nerve endings. We illustrate the potential of FM 1-43 by using it to identify novel PIEZO2-expressing urethral neurons that are engaged by urination. These data reveal that FM 1-43 is a functional probe for mechanosensitivity via PIEZO2 activation invivo and will facilitate the characterization of known and novel mechanosensory processes in multiple organ systems.

Arabidopsis ADF5 Acts as a Downstream Target Gene of CBFs in Response to Low-Temperature Stress.

Low temperature is a major adverse environment that affects normal plant growth. Previous reports showed that the actin cytoskeleton plays an important role in the plant response to low-temperature stress, but the regulatory mechanism of the actin cytoskeleton in this process is not clear. C-repeat binding factors (CBFs) are the key molecular switches for plants to adapt to cold stress. However, whether CBFs are involved in the regulation of the actin cytoskeleton has not been reported. We found that Arabidopsis actin depolymerizing factor 5 (ADF5), an ADF that evolved F-actin bundling function, was up-regulated at low temperatures. We also demonstrated that CBFs bound to the ADF5 promoter directly in vivo and in vitro. The cold-induced expression of ADF5 was significantly inhibited in the cbfs triple mutant. The freezing resistance of the adf5 knockout mutant was weaker than that of wild type (WT) with or without cold acclimation. After low-temperature treatment, the actin cytoskeleton of WT was relatively stable, but the actin cytoskeletons of adf5, cbfs, and adf5 cbfs were disturbed to varying degrees. Compared to WT, the endocytosis rate of the amphiphilic styryl dye FM4-64 in adf5, cbfs, and adf5 cbfs at low temperature was significantly reduced. In conclusion, CBFs directly combine with the CRT/DRE DNA regulatory element of the ADF5 promoter after low-temperature stress to transcriptionally activate the expression of ADF5; ADF5 further regulates the actin cytoskeleton dynamics to participate in the regulation of plant adaptation to a low-temperature environment.

Molecular Tuning of Styryl Dyes Leads to Versatile and Efficient Plasma Membrane Probes for Cell and Tissue Imaging.

The plasma membrane (PM) plays a major role in many biological processes; therefore, its proper fluorescence staining is required in bioimaging. Among the commercially available PM probes, styryl dye FM1-43 is one of the most widely used. In this work, we demonstrated that fine chemical modifications of FM1-43 can dramatically improve the PM staining. The newly developed probes, SP-468 and SQ-535, were found to display enhanced photophysical properties (reduced cross-talk, higher brightness, improved photostability) and, unlike FM1-43, provided excellent and immediate PM staining in 5 different mammalian cell types including neurons (primary culture and tissue imaging). Taking advantage of these features, we successfully used SP-468 in STED super resolution neuronal imaging. Additionally, we showed that the new probes displayed differences in their internalization pathways compared to their parent FM1-43. Finally, we showed that the new probes kept the ability to stain the PM of plant cells. Overall, this work presents new useful probes for PM imaging in cells and tissues and provides insights on the molecular design of new PM targeting molecules.

Renewed proliferation in adult mouse cochlea and regeneration of hair cells

The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs.

Hypoculoside, a sphingoid base-like compound from Acremonium disrupts the membrane integrity of yeast cells

We have isolated Hypoculoside, a new glycosidic amino alcohol lipid from the fungus Acremonium sp. F2434 belonging to the order Hypocreales and determined its structure by 2D-NMR (Nuclear Magnetic Resonance) spectroscopy. Hypoculoside has antifungal, antibacterial and cytotoxic activities. Homozygous profiling (HOP) of hypoculoside in Saccharomyces cerevisiae (budding yeast) revealed that several mutants defective in vesicular trafficking and vacuolar protein transport are sensitive to hypoculoside. Staining of budding yeast cells with the styryl dye FM4-64 indicated that hypoculoside damaged the vacuolar structure. Furthermore, the propidium iodide (PI) uptake assay showed that hypoculoside disrupted the plasma membrane integrity of budding yeast cells. Interestingly, the glycosidic moiety of hypoculoside is required for its deleterious effect on growth, vacuoles and plasma membrane of budding yeast cells.

Alleviation of Hippocampal Endoplasmic Reticulum Stress by Allomyrina dichotoma Larvae Extract.

In the brain, endoplasmic reticulum (ER) stress results in synaptic dysfunction and eventually leads to neurodegeneration. Allomyrina dichotoma larvae are a Chinese ethnomedicine and are widely used in East Asia. In the present study, we investigated the ability of ethanol extract of A. dichotoma larvae (ADE) to improve synaptic structure and function by activating unfolded protein response (UPR) under ER stress in animal and neuron culture models. ER stress was induced in obese mice fed a high fat diet (HFD) or by treating dissociated cultures of rat embryonic (E19) hippocampal neurons with tunicamycin (TM). Western blot and real-time or conventional RT-PCR were performed to analyze the expressions of ER stress marker proteins. In dissociated hippocampal cultures, immunocytochemistry was performed for synaptic proteins, and cultures were stained with styryl dye FM1-43 to assess presynaptic activities. In HFD-fed obese mice, ADE efficiently reduced the expressions of ER stress markers, such as, xbp-1, chop, atf4, erdi4, and eIf2a, and those of the ER chaperone/foldases Bip/grp78, Ero-1l, and PDI. Unconventionally spliced xbp-1s mRNA was not detected. In primary rat hippocampal cultures under ER stress, ADE significantly lowered the nuclear expression of CHOP, inhibited the downregulations of postsynaptic proteins, such as, GluN2A, GluN2B, and PSD-95, and maintained the pool size of recycling presynaptic vesicles. The study shows that ADE potently suppressed the induction of ER stress and maintained the structure and function of hippocampal neurons, and suggests that ADE is a potentially valuable food supplement and preventive therapeutic for ER stress-related nervous disorders.

Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells

The recycling of synaptic vesicle (SV) proteins and transmitter release occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the adaptor protein 1 and 3 complex (AP-1/AP-3). As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7days invitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5min after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. Cover Image for this issue: doi. 10.1111/jnc.13801.

Erratum to: The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity.

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.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64.

The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

Live cell imaging of FM4-64, a tool for tracing the endocytic pathways in Arabidopsis root cells.

Confocal live imaging of the amphiphilic styryl dye FM4-64 is a valuable technique to monitor organelle dynamics and in particular endocytic pathways. After application in plants, FM4-64 immediately stains the plasma membrane and is then integrated on vesicles following endomembrane system-dependent internalization processes. Over time, FM4-64 becomes distributed throughout the full vesicular network from the plasma membrane to the vacuole, including the components of the secretory pathways. Here we provide succinct examples of the many important developmental processes in plants that rely on endocytosis and describe two suitable methods to trace the endocytic pathways in Arabidopsis thaliana root cells based on the uptake of FM4-64.

Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 - a comparison with annexin V-FITC.

The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophore-stimulated T-lymphocytes further increases this fluorescence [Zweifach, 2000]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca2+-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss of phospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca2+ or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca2+-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca2+-release from intracellular stores and by Ca2+ and ionophore A23187 treatment showed that the increase in FM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca2+-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.Electronic Supplementary MaterialSupplementary material is available for this article at 10.2478/s11658-014-0195-3 and is accessible for authorized users.

The Antidepressant Fluoxetine Mobilizes Vesicles to the Recycling Pool of Rat Hippocampal Synapses During High Activity

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.

Studying synaptic efficiency by post-hoc immunolabelling

BackgroundIn terms of vesicular recycling, synaptic efficiency is a key determinant of the fidelity of synaptic transmission. The ability of a presynaptic terminal to reuse its vesicular content is thought to be a signature of synaptic maturity and this process depends on the activity of several proteins that govern exo/endocytosis. Upon stimulation, individual terminals in networks of cultured cerebellar granule neurons exhibit heterogeneous exocytic responses, which reflect the distinct states of maturity and plasticity intrinsic to individual synaptic terminals. This dynamic scenario serves as the substrate for processes such as scaling, plasticity and synaptic weight redistribution. Presynaptic strength has been associated with the activity of several types of proteins, including the scaffolding proteins that form the active zone cytomatrix and the proteins involved in presynaptic exocytosis.MethodsWe have combined fluorescence imaging techniques using the styryl dye FM1-43 in primary cultures of cerebellar granule cells with subsequent post-hoc immunocytochemistry in order to study synaptic efficiency in terms of vesicular release. We describe a protocol to easily quantify these results with minimal user intervention.ResultsIn this study we describe a technique that specifically correlates presynaptic activity with the levels of presynaptic markers. This method involves the use of the styryl dye FM1-43 to estimate the release capacity of a synaptic terminal, and the subsequent post-hoc immunolabelling of thousands of individual nerve terminals. We observed a strong correlation between the release capacity of the nerve terminal and the levels of the RIM1α but not the Munc13-1 protein in the active zone.ConclusionsOur findings support those of previous studies and point out to RIM1α as a crucial factor in determining synaptic efficiency. These results also demonstrate that this technique is a useful tool to analyse the molecular differences underlying the heterogeneous responses exhibited by neuronal networks.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na+ channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca2+ because its effect was abolished in Ca2+-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca2+ channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca2+ channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.

Membrane cholesterol regulates different modes of synaptic vesicle release and retrieval at the frog neuromuscular junction

We investigated the effects of cholesterol removal on spontaneous and KCl-evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl-β-cyclodextrin (MβCD) induced an increase in the frequency of miniature end-plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1-43. Treatment with MβCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MβCD were larger than those from control. In addition, treatment with MβCD reduced the fusion of synaptic vesicles that are mobilized during KCl-evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MβCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl-evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.

Two‐photon microscopy of dermal innervation in a human re‐innervated model of skin

When skin is injured, innervation can be severely disrupted. The subsequent re-innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two-photon excitation fluorescence (TPEF) microscopy of entire human skin explants re-innervated by rodent sensory neurons labelled with the styryl dye FM1-43. TPEF imaging of nerve fibres to a depth up to roughly 300μm within the dermis was demonstrated, allowing three-dimensional reconstruction of the neural tree structure. Endogenous second-harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis.

Morphological and functional abnormalities in neuromuscular junctions of Drosophila melanogaster induced by human APP gene expression

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by a loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in increased production of amyloid-β-protein (Aβ). To gain insight into the synaptic effects of APP protein in AD patients, wild-type APP, its mutant form APP-Swedish responsible for familial AD, and human beta-secretase gene were expressed in motor neurons of Drosophila melanogaster larvae. It was found that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons caused significant morphological and functional changes in neuromuscular junctions (NMJs)-a dramatic increase in the number of synaptic boutons and altered exocytosis revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed with APP (APP-Swedish) expression, as well as for double transgenes bearing APP (APP-Swedish) and human beta-secretase (BACE), which caused secretion of amyloid beta protein (Aβ). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from Aβ neurotoxic effects.