Differential Release of Exocytosis Marker Dyes Indicates Stimulation-Dependent Regulation of Synaptic Activity.

There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation.

Surface Trafficking of APP and BACE in Live Cells.

Amyloid-β (Aβ)-peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aβ precursor protein (APP) by β-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.

The Ca2+ sensor protein swiprosin-1/EFhd2 is present in neurites and involved in kinesin-mediated transport in neurons.

Swiprosin-1/EFhd2 (EFhd2) is a cytoskeletal Ca2+ sensor protein strongly expressed in the brain. It has been shown to interact with mutant tau, which can promote neurodegeneration, but nothing is known about the physiological function of EFhd2 in the nervous system. To elucidate this question, we analyzed EFhd2-/-/lacZ reporter mice and showed that lacZ was strongly expressed in the cortex, the dentate gyrus, the CA1 and CA2 regions of the hippocampus, the thalamus, and the olfactory bulb.

Dynamic properties of the alkaline vesicle population at hippocampal synapses.

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.

Common strength and localization of spontaneous and evoked synaptic vesicle release sites.

Background: Different pools and functions have recently been attributed to spontaneous and evoked vesicle release. Despite the well-established function of evoked release, the neuronal information transmission, the origin as well as the function of spontaneously fusing synaptic vesicles have remained elusive. Recently spontaneous release was found to e.

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.

Basic presynaptic functions in hippocampal neurons are not affected by acute or chronic lithium treatment.

Lithium is an effective mood-stabilizer in the treatment of bipolar affective disorder. While glycogen synthase kinase 3-mediated and inositol depletion-dependent effects of lithium have been described extensively in literature, there is very little knowledge about the consequences of lithium treatment on vesicle recycling and neurotransmitter availability. In the present study we have examined acute and chronic effects of lithium on synaptic vesicle recycling using primary hippocampal neurons.

The pH probe CypHer™5E is effectively quenched by FM dyes.

Concurrent imaging of spectrally distinct fluorescence probes has become an important method for live-cell microscopy experiments in many biological disciplines. The technique enables the identification of a multitude of causal relationships. However, interactions between fluorescent dyes beyond an obvious overlap of their fluorescent spectra are often neglected.

Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses.

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved.

Pool-independent labelling of synaptic vesicle exocytosis with single vesicle resolution in rat hippocampal neurons.

FM dyes are an established tool to analyze synaptic vesicle pools. However, quantitative measurements using FM dyes are typically based on the re-release properties of previously labelled vesicles, which might vary depending on the experimental setup. An FM dye protocol independent of the previous labelling of vesicle membrane has not been applied for quantitative measurements of individual synaptic vesicles before.

A fast and robust method for automated analysis of axonal transport.

Cargo movement along axons and dendrites is indispensable for the survival and maintenance of neuronal networks. Key parameters of this transport such as particle velocities and pausing times are often studied using kymograph construction, which converts the transport along a line of interest from a time-lapse movie into a position versus time image. Here we present a method for the automatic analysis of such kymographs based on the Hough transform, which is a robust and fast technique to extract lines from images.

Patterned anvils for high pressure measurements at low temperature.

Multiprobe high pressure measurements require electrical leads in the sample chamber. Compared to conventional wire-based techniques, metallic tracks patterned onto the anvil surface improve reliability and ease of use, and enable novel and more demanding measurements under high pressure. We have developed new anvil designs based on sputter-deposited tracks on alumina and moissanite anvils.

High pressure study of BaFe2As2--the role of hydrostaticity and uniaxial stress.

We investigate the evolution of the electrical resistivity of BaFe(2)As(2) single crystals with pressure. The samples used were from the same batch, grown using a self-flux method, and showed properties that were highly reproducible. Samples were pressurized using three different pressure media: pentane-isopentane (in a piston-cylinder cell), Daphne oil (in an alumina anvil cell) and steatite (in a Bridgman cell).

Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippocampal synapses.

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.

Background determination-based detection of scattered peaks.

In many instances of signal and image processing, it is indispensable to precisely distinguish scattered peaks from a background, e.g., camera signals in microscopy.

Synapse clusters are preferentially formed by synapses with large recycling pool sizes.

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.

Distinct intracellular vesicle transport mechanisms are selectively modified by spastin and spastin mutations.

Spastin is a microtubule severing ATPase that regulates intracellular and axonal transport of vesicles. Intracellular vesicle trafficking was analyzed in differentiated SH-SY5Y-neuroblastoma cells, transfected with spastin wild-type and three spastin mutations (ΔN, K388R, S44L) to investigate spastin-mediated effects on the velocity of vesicles, stained with LysoTracker Red®. The vesicle velocity varied considerably between mutations and detailed analysis revealed up to five distinct velocity classes.

Fluoxetine prevents stimulation-dependent fatigue of synaptic vesicle exocytosis in hippocampal neurons.

Effects of the antidepressant fluoxetine on stimulation-dependent synaptic vesicle exocytosis were examined in cultured primary hippocampal neurons. Synaptic vesicles were fluorescently labeled in vitro with FM1-43, washed and subsequently destained in two consecutive cycles. Exocytosis was triggered by electric field stimulation and imaged by fluorescence microscopy.

Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference.

Determination of axonal transport velocities via image cross- and autocorrelation.

On their way to the synapse and back, neuronal proteins are carried in cargo vesicles along axons and dendrites. Here, we demonstrate that the key parameters of axonal transport, i.e.