Viability and Contractility of Rat Brain Pericytes in Conditions That Mimic Stroke; an in vitro Study.

Reopening of the cerebral artery after occlusion often results in "no-reflow" that has been attributed to the death and contraction (rigor mortis) of pericytes. Since this hypothesis still needs to be confirmed, we explored the effects of oxygen glucose deprivation (OGD) on viability and cell death of primary rat pericytes, in the presence or absence of neurovascular unit-derived cytokines. Two morphodynamic parameters, single cell membrane mobility (SCMM) and fractal dimension (Df), were used to analyze the cell contractions and membrane complexity before and after OGD. We found a marginal reduction in cell viability after 2-6 h OGD; 24 h OGD caused a large reduction in viability and a large increase in the number of apoptotic and dead cells. Application of erythropoietin (EPO), or a combination of EPO and endothelial growth factor (VEGFA1-165) during OGD significantly reduced cell viability; application of Angiopoietin 1 (Ang1) during OGD caused a marginal, insignificant increase in cell viability. Simultaneous application of EPO, VEGFA1-165, and Ang1 significantly increased cell viability during 24 h OGD. Twenty minutes and one hour OGD both significantly reduced SCMM compared to pre-OGD values, while no significant difference was seen in SCMM before and after 3 h OGD. There was a significant decrease in membrane complexity (Df) at 20 min during the OGD that disappeared thereafter. In conclusion, OGD transiently affected cell mobility and shape, which was followed by apoptosis in cultured pericytes. Ang1 may have a potentiality for preventing from the OGD-induced apoptosis. Further studies could clarify the relationship between cell contraction and apoptosis during OGD.

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. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce "kiss-and-run" exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential "kiss-and-run" and "full-collapse" exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release.

Quantitative Determination of Cellular-and Neurite Motility Speed in Dense Cell Cultures.

Mobility quantification of single cells and cellular processes in dense cultures is a challenge, because single cell tracking is impossible. We developed a software for cell structure segmentation and implemented 2 algorithms to measure motility speed. Complex algorithms were tested to separate cells and cellular components, an important prerequisite for the acquisition of meaningful motility data. Plasma membrane segmentation was performed to measure membrane contraction dynamics and organelle trafficking. The discriminative performance and sensitivity of the algorithms were tested on different cell types and calibrated on computer-simulated cells to obtain absolute values for cellular velocity. Both motility algorithms had advantages in different experimental setups, depending on the complexity of the cellular movement. The correlation algorithm (COPRAMove) performed best under most tested conditions and appeared less sensitive to variable cell densities, brightness and focus changes than the differentiation algorithm (DiffMove). In summary, our software can be used successfully to analyze and quantify cellular and subcellular movements in dense cell cultures.

Use-dependent inhibition of synaptic transmission by the secretion of intravesicularly accumulated antipsychotic drugs.

Antipsychotic drugs are effective for the treatment of schizophrenia. However, the functional consequences and subcellular sites of their accumulation in nervous tissue have remained elusive. Here, we investigated the role of the weak-base antipsychotics haloperidol, chlorpromazine, clozapine, and risperidone in synaptic vesicle recycling. Using multiple live-cell microscopic approaches and electron microscopy of rat hippocampal neurons as well as in vivo microdialysis experiments in chronically treated rats, we demonstrate the accumulation of the antipsychotic drugs in synaptic vesicles and their release upon neuronal activity, leading to a significant increase in extracellular drug concentrations. The secreted drugs exerted an autoinhibitory effect on vesicular exocytosis, which was promoted by the inhibition of voltage-gated sodium channels and depended on the stimulation intensity. Taken together, these results indicate that accumulated antipsychotic drugs recycle with synaptic vesicles and have a use-dependent, autoinhibitory effect on synaptic transmission.

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.

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. Microtubule severing by overexpressed wild-type spastin caused reduced vesicle velocity. S44L and ΔN mutations, which were functionally impaired, showed similar velocities as control cells. K388R-transfected cells exhibited an intermediate velocity profile. The results support the idea that spastin mutations not only alter axonal transport, but in addition regulate intracellular trafficking in the cell soma as well.

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. 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.

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. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

The role of ceramide in major depressive disorder.

Major depression is a severe mood disorder with a lifetime prevalence of more than 10%. The pharmacokinetic hypothesis claims that a slow accumulation of antidepressant drugs by acid trapping mainly into lysosomes is responsible for the therapeutic latency and that a lysosomal target mediates the antidepressant effects. The lysosomal lipid metabolizing enzyme acid sphingomyelinase (ASM) cleaves sphingomyelin into ceramide and phosphorylcholine. In a pilot study, the activity of this enzyme was increased in peripheral blood cells of patients with major depressive disorder (MDD), making the ASM an interesting molecular target of antidepressant drugs. Indeed, several antidepressant drugs functionally inhibit ASM. The ASM/ceramide pathway might be a missing link unifying independent findings in neurobiology and the treatment of MDD such as therapeutic latency, oxidative stress, immune activation and increased risk of cardiovascular disease.

Antidepressant drugs modulate growth factors in cultured cells.

BACKGROUND: Different classes of antidepressant drugs are used as a treatment for depression by activating the catecholinergic system. In addition, depression has been associated with decrease of growth factors, which causes insufficient axonal sprouting and reduced neuronal damage repair. In this study, antidepressant treatments are analyzed in a cell culture system, to study the modulation of growth factors. RESULTS: We quantified the transcription of several growth factors in three cell lines after application of antidepressant drugs by real time polymerase chain reaction. Antidepressant drugs counteracted against phorbolester-induced deregulation of growth factors in PMA-differentiated neuronal SY5Y cells. We also found indications in a pilot experiment that magnetic stimulation could possibly modify BDNF in the cell culture system. CONCLUSION: The antidepressant effects antidepressant drugs might be explained by selective modulation of growth factors, which subsequently affects neuronal plasticity.

Light-induced exocytosis in cell development and differentiation.

Calcium-dependent exocytosis of fluorescently labeled single secretory vesicles in PC12 cells and primary embryonic telencephalon cells can be triggered by illumination with visible light and imaged by TIRF or epifluorescence microscopy. Opsin 3 was identified by quantitative PCR expression analysis as the putative light receptor molecule for light-induced exocytosis. In primary chicken telencephalon cells, light-induced exocytosis is restricted to a specific period during embryonic development, and involves fusion of rather large vesicles. Strictly calcium-dependent exocytosis starts after a delay of a few seconds of illumination and lasts for up to 2 min. We analyzed the frequency, time course and spatial distribution of exocytotic events. Exocytosis in PC12 cells and telencephalon cells occurs at the periphery or the interface between dividing cells, and the duration of single secretion events varies considerably. Our observation strongly supports the idea that light induced exocytosis is most likely a mechanism for building plasma membrane during differentiation, development and proliferation rather than for calcium-dependent neurotransmitter release.

Are obese adolescents and young adults at higher risk for mental disorders? A community survey.

OBJECTIVE: Associations between body mass index (BMI) and mental disorders meeting Axis-I diagnoses according to the Diagnostic and Statistical Manual for Mental Disorders IV (DSM-IV) were investigated in The Early Developmental Stages of Psychopathology Study in a large population-based sample, which included adolescents and young adults of both genders for the first time. RESEARCH METHODS AND PROCEDURES: A total of 3021 German subjects ranging from 14 to 24 years of age were assessed for specific DSM-IV diagnoses derived from a modified version of the standardized Composite International Diagnostic Interview, and general psychological disturbances, using the Symptom Checklist-90-Revised. BMI percentiles for age and gender were calculated to avoid systematic bias in the BMI distribution resulting from the young age range represented in the sample. Additionally, subjects with a lifetime diagnosis of any eating disorder were excluded from statistical analysis to control the confounding effect of body weight-related eating disorders on associations between BMI and psychopathology. RESULTS: The results based on logistic regression analyses and MANOVAs demonstrate that the BMI is not associated with mental disorders or general psychopathologies. There were no significant associations between BMI and mood, anxiety, substance, and somatoform disorders, a result that contrasts with almost all previous clinical studies. Additionally, in contrast to clinical investigations and most epidemiological studies, neither obesity nor underweight was significantly associated with any kind of general psychopathology. DISCUSSION: The overall finding that obesity is not significantly related to marked psychopathology in the general German population of adolescents and young adults has important clinical implications.

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells.

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.

Patch-clamp capacitance measurements: new insights into the endocytic uptake of transferrin.

Since its introduction by Neher and Sakmann in 1976 the patch-clamp technique has been extensively used to study processes such as signalling and synaptic transmission, but also for monitoring endo- and exocytosis. Since biological membranes behave like electrical capacitors high-resolution measurements of membrane capacitance allow detection of small changes in membrane surface area that accompany exocytosis and endocytosis. We here describe our recent work on patch-clamp capacitance measurements in stably transfected HeLa cells expressing HFE, the hereditary hemochromatosis gene product, under the control of a tetracycline-sensitive promotor. By means of whole-cell and cell-attached techniques we were able to reveal transferrin-induced decreases in membrane capacitance reflecting increased endocytosis at the single cell level. Moreover, cell-attached recordings revealed significant alterations in the formation of single endocytic vesicles. Time-resolved measurements of cell membrane capacitance provide a new methodological approach to study the endocytic uptake of transferrin and its regulation by HFE, the hereditary hemochromatosis protein.