Biomedical subjects

M Lindau

Publications and source records attributed to M Lindau.

At least 19 recordsLinked to original sources

PubMed2006

Imaging and nanomedicine for diagnosis and therapy in the central nervous system: report of the eleventh annual Blood-Brain Barrier Disruption Consortium meeting.

The blood-brain barrier (BBB) presents a major obstacle to the treatment of malignant brain tumors and other central nervous system (CNS) diseases. The Eleventh Annual Blood-Brain Barrier Disruption Consortium Meeting was convened to discuss recent advances and future directions in imaging and nanomedicine. Two sessions, one on Cell and Molecular Imaging in the CNS and another on Nanotechnology, Nanobiology, and Nanomedicine, were held March 17-18, 2005, in Portland, Ore. CNS imaging presentations targeted differentiating tumor, neural lesions, and necrosis from healthy brain tissue; methods of delivery of imaging agents across the BBB; and new iron oxide-based nanoparticle contrast agents for MR imaging. Nanobiology presentations covered the development of new nanotechnology and its use in imaging, diagnosis, and therapy in the CNS. Discussions at this meeting stressed the role of biotechnology in the convergence of CNS imaging and nanomedicine and are summarized in this article.

Blood-Brain Barrier↗

PubMed2003

Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry.

We have studied exocytosis in rat peritoneal mast cells by cell-attached patch amperometry. Step increases in capacitance were accompanied by typical amperometric spikes due to the release of 5-hydroxytryptamine (serotonin), indicating exocytosis of typical mast cell granules. We have measured the time course of fusion pore expansion, and correlated it with release from the granule matrix. The fusion pore of mast cell granules grows in three stages. The initial expansion of the pore occurred at a rate of 5 nS/s, and in many cases an observable amperometric foot was detected. A second, rapid expansion phase occurred with a rate as high as 1000 nS/s, coinciding with the upstroke of the amperometric spike. A third, slower phase, with a rate of 5 nS/s, completed the final expansion of the fusion pore. These data reveal the very late stages in the exocytotic process, and demonstrate that the size of the fusion pore does not limit release during the upstroke of the amperometric spike or during the final, slow expansion that occurs during for the decay of the amperometric spike.

Animals↗

PubMed2003

Quantitative EEG abnormalities and cognitive dysfunctions in frontotemporal dementia and Alzheimer's disease.

OBJECTIVE: To investigate the relationship between quantitative EEG (qEEG) measurements in frontotemporal dementia (FTD), Alzheimer's disease (AD) and healthy controls and to study to what extent qEEG in FTD and AD or neuropsychological test results of FTD and AD patients or a combination of both contribute to classification accuracy. METHOD: The FTD sample consisted of 19 patients, the AD sample of 16 patients, and the control group of 19 subjects. Groups were matched on the group level with respect to demographic variables. For qEEG the global field power was calculated for six frequency bands: delta (1.0-3.5 Hz), theta (4.0-7.5 Hz), alpha (8.0-11.0 Hz), beta1 (12.0-15.5 Hz), beta2 (16.0-19.5 Hz), beta3 (20.0-23.5 Hz), and spectral ratio as the ratio of the sum of fast frequency bands alpha + beta1 + beta2 + beta3 and slow frequency bands delta + theta. RESULTS: In comparison to controls FTD patients were marked by an absence of an increase in slow qEEG activities and a decrease in fast activities, whereas AD patients were marked by an increase in slow activities and a smaller decrease in fast activities. According to the Mann-Whitney U test the cognitive functions of attention, visuospatial thinking and episodic memory were significantly better in FTD than in AD. Using logistic regression analysis the best predictors of FTD and AD were in a model using the delta and theta activities, and high levels of visuospatial ability and episodic memory. Classification accuracy of the model was 93.3%. CONCLUSION: FTD patients reveal a different pattern of qEEG changes than AD patients. This result demonstrates the importance of qEEG for FTD diagnosis. Cognition is selectively better in FTD than in AD. A combination of qEEG and neuropsychology is recommended for differential diagnoses of FTD and AD.

Alzheimer Disease↗

PubMed2001

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells.

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.

Animals↗

PubMed2001

Robust, high-resolution, whole cell patch-clamp capacitance measurements using square wave stimulation.

High-resolution, whole cell capacitance measurements are usually performed using sine wave stimulation using a single frequency or a sum of two frequencies. We present here a high-resolution technique for whole-cell capacitance measurements based on square-wave stimulation. The square wave represents a sum of sinusoidal frequencies at odd harmonics of the base frequency, the amplitude of which is highest for the base frequency and decreases as the frequency increases. The resulting currents can be analyzed by fitting the current relaxations with exponentials, or by a phase-sensitive detector technique. This method provides a resolution undistinguishable from that of single-frequency sine wave stimulation, and allows for clear separation of changes in capacitance, membrane conductance, and access resistance. In addition, it allows for the analysis of more complex equivalent circuits as associated with the presence of narrow fusion pores during degranulation, tracking many equivalent circuit parameters simultaneously. The method is insensitive to changes in the reversal potential, pipette capacitance, or widely varying cell circuit parameters. It thus provides important advantages in terms of robustness for measuring cell capacitances, and allows analysis of complicated changes of the equivalent circuits.

Animals↗

PubMed2001

Voltage-dependent membrane capacitance in rat pituitary nerve terminals due to gating currents.

We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.

Animals↗

PubMed2000

Amyloid Abeta40 CSF concentrations correlate to frontal lobe atrophy in frontotemporal dementia.

We wanted to further study amyloid Abeta protein alterations in non-AD neurodegenerative diseases. Cerebrospinal fluid concentrations of the amyloid Abeta protein with 40 (Abeta40) and 42 (Abeta42) amino acid residues were measured in eleven patients with frontotemporal dementia (FTD). Abeta40 and Abeta42 concentrations were related to the degree of frontal lobe atrophy as assessed with MRI volumetry. Abeta40 concentrations showed a statistically significant linear correlation with degree of frontal lobe atrophy (r = -0.77, p<0.02). Similar results have not been found in previous studies of CSF Abeta40 concentrations and atrophy in patients with AD which suggest that the role of Abeta40 differs between the pathological processes of FTD and AD.

Aged↗

PubMed2000

Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells.

Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.

Animals↗

PubMed2000

Resolution of patch capacitance recordings and of fusion pore conductances in small vesicles.

We investigated the noise levels in cell-attached patch capacitance recordings with a lock-in amplifier. The capacitance noise level decreases with increasing sine wave frequency up to 20-40 kHz. With a 20-mV rms sine wave the rms noise level above 8 kHz is <50 aF. With increasing sine wave amplitudes a further reduction down to 14 aF could be achieved. Capacitance measurements with a lock-in amplifier may also be used to measure the conductance of fusion pores connecting the vesicular lumen to the extracellular space. It is estimated that at noise levels of 14 aF fusion pore conductances between 20 pS and 700 pS may be resolved in vesicles with 380-aF capacitance by using a 50-kHz sine wave. This corresponds to vesicles with a approximately 110-nm diameter. It is suggested that with low-noise techniques fusion pores may be detectable in vesicles approaching the size of large synaptic vesicles.

Animals↗

PubMed1999

Membrane capacitance techniques to monitor granule exocytosis in neutrophils.

Cell membranes behave like electrical capacitors and changes in cell capacitance therefore reflect changes in the cell area. Monitoring capacitance can thus be used to study dynamic cellular phenomenon involving rapid changes in cell surface, such as exo- and/or endocytosis. In this review focus is on the use of capacitance techniques to study exocytosis in human neutrophils. We compare the whole-cell and the cell-attached capacitance techniques, and we review the complete literature dealing with capacitance measurements in human neutrophils.

Animals↗

PubMed1999

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism.

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.

Animals↗

PubMed1998

Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms.

Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor alpha-naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.

Animals↗

PubMed1998

Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.

We have studied exocytosis of single small granules from human neutrophils by capacitance recordings in the cell-attached configuration. We found that 2.2% of the exocytotic events were flickers. The flickers always ended with a downward step. This indicates closing of the fusion pore. During flickering, the fusion pore conductance remained below 1 nS, and no net membrane transfer was detectable. After fusion pore expansion beyond 1 nS the pore expanded irreversibly, leading to rapid full incorporation of the granule/vesicle into the plasma membrane. Following exocytosis of single granules, a capacitance decrease directly related to the preceding increase was observed in 7% of the exocytotic events. This decrease followed immediately after irreversible pore expansion, and is presumably triggered by full incorporation of the vesicle into the patch membrane. The capacitance decrease could be interpreted as endocytosis triggered by exocytosis. However, the gradual decrease could also reflect a decrease in the "free" patch area following incorporation of an exocytosed vesicle. We conclude that non-stepwise capacitance changes must be interpreted with caution, since a number of factors go into determining cell or patch admittance.

Biophysical Phenomena↗

PubMed1998

Mechanism of peptide-induced mast cell degranulation. Translocation and patch-clamp studies.

Substance P and other polycationic peptides are thought to stimulate mast cell degranulation via direct activation of G proteins. We investigated the ability of extracellularly applied substance P to translocate into mast cells and the ability of intracellularly applied substance P to stimulate degranulation. In addition, we studied by reverse transcription--PCR whether substance P-specific receptors are present in the mast cell membrane. To study translocation, a biologically active and enzymatically stable fluorescent analogue of substance P was synthesized. A rapid, substance P receptor- and energy-independent uptake of this peptide into pertussis toxin-treated and -untreated mast cells was demonstrated using confocal laser scanning microscopy. The peptide was shown to localize preferentially on or inside the mast cell granules using electron microscopic autoradiography with 125I-labeled all-D substance P and 3H-labeled substance P. Cell membrane capacitance measurements using the patch-clamp technique demonstrated that intracellularly applied substance P induced calcium transients and activated mast cell exocytosis with a time delay that depended on peptide concentration (delay of 100-500 s at concentrations of substance P from 50 to 5 microM). Degranulation in response to intracellularly applied substance P was inhibited by GDPbetaS and pertussis toxin, suggesting that substance P acts via G protein activation. These results support the recently proposed model of a receptor-independent mechanism of peptide-induced mast cell degranulation, which assumes a direct interaction of peptides with G protein alpha subunits subsequent to their translocation across the plasma membrane.

Animals↗

PubMed1997

The exocytotic event in chromaffin cells revealed by patch amperometry.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at <3 nm for a variable period, which can last for several seconds, before it expands. Transmitter is released much faster through this pore than in mast cells, generating a 'foot' signals which precedes the amperometric spike. Occasionally, the narrow pore forms only transiently and does not expand, allowing complete transmitter release without full fusion of the vesicle with the plasma membrane.

Catecholamines↗

PubMed1997

Exocytotic competence and intergranular fusion in cord blood-derived eosinophils during differentiation.

We studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5'-O-(3-thiotriphosphate) (GTP) gamma S after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from approximately 700 microns2 to approximately 400 microns2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.

Cell Differentiation↗

PubMed1996

A slowly activating voltage-dependent K+ current in rat pituitary nerve terminals.

1. A novel slowly activating voltage-dependent K+ current was observed in isolated nerve terminals from rat neurohypophysis using the whole-cell configuration of the patch-clamp technique. 2. The activation kinetics of the slow current could be fitted assuming Hodgkin--Huxley-type kinetics, an exponential, n, of 1.3 and activation time constants decreasing from 4 s at -50 mV to 0.7s at +40 mV. 3. A positive shift of reversal potential was observed when [K+] was increased in the bath solution. The current is carried mainly but not exclusively by K+ ions. 4. When intracellular free [Mg2+] was low (approximately 60 microM), average current density was 74 pA pF-1 at membrane potentials around 0 mV. In 83% of nerve terminals current amplitude was > 10 pA pF-1. 5. The slow current was never observed when the pipette contained 4.6 mM free Mg2+. At a physiological level of free Mg2+ (0.5 mM) the average current density was 16 pA pF-1. 6. When nerve terminals were analysed after patch-clamp experiments for vasopressin content by immunodetection, no difference in current amplitude was found between the terminals containing vasopressin and all analysed terminals. 7. The voltage dependence of activation was fitted by a Boltzmann equation giving a half-activation potential of -37 mV and a slope factor of about 9 mV. 8. Tail current deactivation kinetics was biexponential with time constants of 0.12 and 1.5s. Kinetics was dependent on the duration of the activating pulse. 9. Noise analysis of the slow current indicated a single-channel current of 0.33 pA at +6 mV, corresponding to a single-channel conductance of 4.3 pS. 10. This is the first demonstration of a current similar to the slow K+ current, IKs, in a neurone, suggesting that a protein similar to the IKs-inducing channel protein IsK (minK) may be present in peptidergic nerve terminals. 11. The activation properties are consistent with a role of the slow current in inhibition of excitability, at least at the level of the nerve terminal.

Animals↗

PubMed1995

A novel Ca(2+)-dependent step in exocytosis subsequent to vesicle fusion.

Exocytosis begins with formation of a small fusion pore which then expands allowing rapid release of granular contents. We studied the influence of cytoplasmic free Ca2+ ([Ca2+]i) on the conductance of the initial pore and on the dynamics of subsequent expansion in horse eosinophils using the patch clamp technique. The mean initial conductance is approximately 200 pS independent of [Ca2+]i. This value is close to that previously found in beige mouse mast cells. The pore subsequently expands by 18 nS/s at [Ca2+]i < 10 nM, by 40 nS/s at [Ca2+]i = 1.5 microM and by 90 nS/s at [Ca2+]i = 10 microM. These results show that the structure of the initial fusion pore is independent of cytoplasmic Ca2+. However, the pore expansion is a Ca(2+)-dependent process modulating secretion at a step later than vesicle-plasma membrane fusion.

Animals↗