Lack of Laminar Shear Stress Facilitates the Endothelial Uptake of Very Small Superparamagnetic Iron Oxide Nanoparticles by Modulating the Endothelial Surface Layer.

Purpose: To study whether the absence of laminar shear stress (LSS) enables the uptake of very small superparamagnetic iron oxide nanoparticles (VSOP) in endothelial cells by altering the composition, size, and barrier function of the endothelial surface layer (ESL). Methods and Results: A quantitative particle exclusion assay with living human umbilical endothelial cells using spinning disc confocal microscopy revealed that the dimension of the ESL was reduced in cells cultivated in the absence of LSS. By combining gene expression analysis, flow cytometry, high pressure freezing/freeze substitution immuno-transmission electron microscopy, and confocal laser scanning microscopy, we investigated changes in ESL composition. We found that increased expression of the hyaluronan receptor CD44 by absence of shear stress did not affect the uptake rate of VSOPs. We identified collagen as a previously neglected component of ESL that contributes to its barrier function. Experiments with inhibitor halofuginone and small interfering RNA (siRNA) demonstrated that suppression of collagen expression facilitates VSOP uptake in endothelial cells grown under LSS. Conclusion: The absence of laminar shear stress disturbs the barrier function of the ESL, facilitating membrane accessibility and endocytic uptake of VSOP. Collagen, a previously neglected component of ESL, contributes to its barrier function.

Immuno-Electron and Confocal Laser Scanning Microscopy of the Glycocalyx.

The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.

BDNF Expression in Cortical GABAergic Interneurons.

Brain-derived neurotrophic factor (BDNF) is a major neuronal growth factor that is widely expressed in the central nervous system. It is synthesized as a glycosylated precursor protein, (pro)BDNF and post-translationally converted to the mature form, (m)BDNF. BDNF is known to be produced and secreted by cortical glutamatergic principal cells (PCs); however, it remains a question whether it can also be synthesized by other neuron types, in particular, GABAergic interneurons (INs). Therefore, we utilized immunocytochemical labeling and reverse transcription quantitative PCR (RT-qPCR) to investigate the cellular distribution of proBDNF and its RNA in glutamatergic and GABAergic neurons of the mouse cortex. Immunofluorescence labeling revealed that mBDNF, as well as proBDNF, localized to both the neuronal populations in the hippocampus. The precursor proBDNF protein showed a perinuclear distribution pattern, overlapping with the rough endoplasmic reticulum (ER), the site of protein synthesis. RT-qPCR of samples obtained using laser capture microdissection (LCM) or fluorescence-activated cell sorting (FACS) of hippocampal and cortical neurons further demonstrated the abundance of BDNF transcripts in both glutamatergic and GABAergic cells. Thus, our data provide compelling evidence that BDNF can be synthesized by both principal cells and INs of the cortex.

Distinct Localization of SNAP47 Protein in GABAergic and Glutamatergic Neurons in the Mouse and the Rat Hippocampus.

Synaptosomal-associated protein of 47 kDa (SNAP47) isoform is an atypical member of the SNAP family, which does not contribute directly to exocytosis and synaptic vesicle (SV) recycling. Initial characterization of SNAP47 revealed a widespread expression in nervous tissue, but little is known about its cellular and subcellular localization in hippocampal neurons. Therefore, in the present study we applied multiple-immunofluorescence labeling, immuno-electron microscopy and in situ hybridization (ISH) and analyzed the localization of SNAP47 in pre- and postsynaptic compartments of glutamatergic and GABAergic neurons in the mouse and rat hippocampus. While the immunofluorescence signal for SNAP47 showed a widespread distribution in both mouse and rat, the labeling pattern was complementary in the two species: in the mouse the immunolabeling was higher over the CA3 stratum radiatum, oriens and cell body layer. In contrast, in the rat the labeling was stronger over the CA1 neuropil and in the CA3 stratum lucidum. Furthermore, in the mouse high somatic labeling for SNAP47 was observed in GABAergic interneurons (INs). On the contrary, in the rat, while most INs were positive, they blended in with the high neuropil labeling. ISH confirmed the high expression of SNAP47 RNA in INs in the mouse. Co-staining for SNAP47 and pre- and postsynaptic markers in the rat revealed a strong co-localization postsynaptically with PSD95 in dendritic spines of pyramidal cells and, to a lesser extent, presynaptically, with ZnT3 and vesicular glutamate transporter 1 (VGLUT1) in glutamatergic terminals such as mossy fiber (MF) boutons. Ultrastructural analysis confirmed the pre- and postsynaptic localization at glutamatergic synapses. Furthermore, in the mouse hippocampus SNAP47 was found to be localized at low levels to dendritic shafts and axon terminals of putative INs forming symmetric synapses, indicating that this protein could be trafficked to both post- and presynaptic sites in both major cell types. These results reveal divergent localization of SNAP47 protein in mouse and rat hippocampus indicating species- and cell type-specific differences. SNAP47 is likely to be involved in unique fusion machinery which is distinct from the one involved in presynaptic neurotransmitter release. Nonetheless, our data suggest that SNAP47 may be involved not only postsynaptic, but also in presynaptic function.

Expression of the voltage- and Ca2+-dependent BK potassium channel subunits BKß1 and BKß4 in rodent astrocytes.

Large-conductance Ca(2+) -activated (BK) potassium channels are centrally involved in neurovascular coupling, immunity, and neural transmission. The ability to be synergistically activated by membrane depolarization, different ligands and intracellular Ca(2+) links intracellular signaling and membrane excitability. The diverse physiological functions of BK channels crucially depend on regulatory ß subunits. Although first studies characterized the neuronal distribution of BKß subunits in the rodent brain, it is largely unknown which ß subunit proteins are expressed in astrocytes and thus mediate these regulatory effects. We therefore analyzed the expression of BKß subunits in rat and mouse brain and glial cell cultures. A monospecific polyclonal antibody against the BKß4 channel subunit was raised, affinity-purified and extensively characterized. BKß4 and to a lesser degree BKß1 transcripts and protein were detected in several astrocytic populations and cultured cells. Particularly strong BKß4 immunostaining was detected in astrocytic progenitors derived from the subventricular zone. The overlapping expression of BKα and BKß4 in astrocytes implies a functional relationship and suggests that BKß4 is an important accessory ß subunit for astrocytic BK channels. In addition, BKß4 might exert effects independent of the α subunit as functional heterologous co-expression of Nav1.6 and BKß4 resulted in reduced Nav1.6 sodium currents. Thus, BKß4 expression in astrocytes likely participates in regulating astrocytic voltage gradients and maintaining K(+) homeostasis, hence enabling astrocytes to fulfill their complex regulatory influence on proper brain function.

An optimized method for simultaneous demonstration of neurons and myelinated fiber tracts for delineation of individual trunco- and palliothalamic nuclei in the mammalian brain.

The truncothalamic complex has long been considered to be a nuclear group with "non-specific" projections. More recently, it is suggested that these thalamic nuclei play an important role in regulating distinct basal ganglia circuits. To further analyze the exact biological function of individual nuclei of the truncothalamic complex a simple and reliable technique for an exact delineation of distinct nuclei is desirable. Therefore, we evaluated and optimized several potential procedures for a combined visualization of neurons and myelinated fibers. Fiber staining with gold toning or immunocytochemical visualization of myelin basic protein shows high contrast and precision but precludes sufficient demonstration of neuronal cell bodies. When the most common technique for the simultaneous visualization of both structures, the Kluver-Barrera procedure, is used, demonstration of myelinated fibers is restricted when the technique is applied to cryostat or vibratome sections. In the present report this limitation was abolished. The final protocol includes lipid extraction prior to the incubation with Luxol Fast Blue and uses carefully characterized staining conditions for Luxol Fast Blue and cresyl violet rendering microscopically controlled differentiation steps unnecessary. The optimized Kluver-Barrera technique results in high precision localization of individual axons and cell bodies and thus permits an exact and simple delineation of individual nuclei in the vertebrate thalamus.