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. 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.

Comparative studies on osteogenic potential of micro- and nanofibre scaffolds prepared by electrospinning of poly(ε-caprolactone).

The biocompatibility and osteogenic potential of four fibrous scaffolds prepared by electrospinning of poly(ε-caprolactone) (PCL) was studied with MG-63 osteoblast cells. Two different kinds of scaffolds were obtained by adjustment of spinning conditions, which were characterized as nano- or microfibrous. In addition of one nanofibrous, scaffold was made more hydrophilic by blending PCL with Pluronics F 68. Scaffolds were characterized by scanning electron microscopy and water contact angle measurements. Morphology and growth of MG63 cells seeded on the different scaffolds were investigated by confocal laser scanning microscopy after vital staining with fluorescein diacetate and by colorimetric assays. It was found that scaffolds composed of microfibres stipulated better growth conditions for osteoblasts probably by providing a real three-dimensional culture substratum, while nanofibre scaffolds restricted cell growth predominantly to surface regions. Osteogenic activity of cells was determined by alkaline phosphatase (ALP) and o-cresolphthalein complexone assay. It was observed that osteogenic activity of cells cultured in microfibre scaffolds was significantly higher than in nanofibre scaffolds regarding ALP activity. Overall, one can conclude that nanofibre scaffold provides better conditions for initial attachment of cells but does not provide advantages in terms of scaffold colonization and support of osteogenic activity compared to scaffolds prepared from microfibres.

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. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

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. 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.

Immobilization of Candida antarctica lipase B on Polystyrene Nanoparticles.

Polystyrene (PS) nanoparticles were prepared via a nanoprecipitation process. The influence of the pH of the buffer solution used during the immobilization process on the loading of Candida antarctica lipase B (Cal-B) and on the hydrolytic activity (hydrolysis of p-nitrophenyl acetate) of the immobilized Cal-B was studied. The pH of the buffer solution has no influence on enzyme loading, while immobilized enzyme activity is very dependent on the pH of adsorption. Cal-B immobilized on PS nanoparticles in buffer solution pH 6.8 performed higher hydrolytic activity than crude enzyme powder and Novozyme 435.

Batchwise and continuous organophilic nanofiltration of Grubbs-type olefin metathesis catalysts.

A mass-tagged N-mesityl imidazolinium salt with four additional -CH(2)NCy(2) substituents was synthesized, leading to a molecular mass of nearly 1100 g mol(-1) in the corresponding carbene ligand. This mass-tagged ligand was used to generate the respective Grubbs II and Grubbs-Hoveyda type complexes. The catalytic activity of the latter complex was tested in several olefin metathesis reactions and found to be slightly superior to that of the related N-mesityl based complex. In batchwise solvent resistant nanofiltration experiments the ruthenium complex dissolved in toluene and following a metathesis reactions was efficiently retained (>99.8 %) by a single nanofiltration; the permeate contained less than 4 ppm of Ru. Equally efficient catalyst retention was observed in a membrane reactor utilized for the continuous synthesis of a RCM product.