Johannes Seeger

Extracellular matrix organization in various regions of rat brain grey matter.

SummaryPrevious studies revealed the concentration of extracellular matrix proteoglycans in the so-called perineuronal nets on the one hand and in certain zones of the neuropil on the other. This nonhomogeneous distribution suggested a non-random chemical and spatial heterogeneity of the extracellular space. In the present investigation, regions dominated by one of both distribution patterns, i.e. piriform and partietal cortex, reticular thalamic nucleus, medial septum/diagonal band complex and cerebellar nuclei, were selected for correlative light and electron microscopic analysis. The labelling was performed by the use of the N-acetylgalactosamine-binding plant lectinWisteria floribunda agglutinin visualized by peroxidase staining and additionally by photoconversion of red carbocyanine fluorescence labelling for electron microscopy. The intense labelling of the neuropil of a superificial piriform region, presumably identical with sublayer Ia, was confined to a fine meshwork spreading over the extracellular space between non-myelinated axons, dendrites and glial profiles. In the reticular thalamic nucleus the neuronal cell bodies were embedded in zones of labelled neuropil. In contrast to these patterns, the labelled extracellular matrix in different cortical layers and in the other subcortical regions was concentrated in perineuronal nets as large accumulations at surface areas of the neuronal perikarya and dendrites and the attached presynaptic boutons. Astrocytic processes usually were separated from the neuronal surface by the interposed extracellular material. Despite a great variability, the width of the extracellular space containing the labelled matrix components in all perineuronal nets appeared to be considerably larger than that in the labelled zones of neuropil and the non-labelled microenvironment of other neurons. Our results support the view that differences expressed in topographical and spatial peculiarities of the extracellular matrix constituents are related to neuron-type- and system-specific functional properties.

Old dyes for new scopes: the phagocytosis-dependent long-term fluorescence labelling of microglial cells in vivo

The nature of the interactions between dying neurons and microglial cells within the developing and injured CNS remains controversial. A new technique for labelling microglial cells is available, which enables further studies of such interactions in a direct way. The value of the method relies on retrograde filling of neurons with vital fluorescent dye, subsequent degeneration of the neurons due to either naturally occurring cell death or as the result of axotomy, and phagocytotic removal of the fluorescent cell debris by microglial cells, which thus become identifiable. The fluorescent dye can be visualized in whole-mounted tissue or after sectioning. Photoconversion of the dye into electron-dense material permits examination of the microglial and dying ganglion-cell interactions at the ultrastructural level. This new principle of the function-dependent, selective fluorescent labelling of phagocytosing microglial cells, which might now be extended to other dyes and to other neurodegenerative models, promises to shed light onto the function of microglial cells within the brain.

Selective in vivo fluorescence labelling of cholinergic neurons containing p75NTR in the rat basal forebrain

The cholinergic system of the rat basal forebrain is used as a model for the homologous region in humans which is highly susceptible to neuropathological alterations as in Alzheimers disease. Cholinergic cells in the basal forebrain express the low-affinity neurotrophin receptor p75NTR. This has been utilized for selective immunolesioning of cholinergic neurons after internalization of an immunotoxin composed of anti-p75NTR and the ribosome-inactivating toxin saporin. However, the goal of many studies may be not the lesion, but the identification of cholinergic cells after other experimentally induced alterations in the basal forebrain. Therefore, a novel cholinergic marker was prepared by conjugating the monoclonal antibody 192IgG directed against p75NTR with the bright red fluorochrome carbocyanine 3 (Cy3). Three days after intraventricular injection of Cy3-192IgG the fluorescence microscopic analysis revealed a pattern of Cy3-labelled cells matching the distribution of cholinergic neurons. Apparently the marker was internalized within complexes of p75NTR and Cy3-192IgG which were then retrogradely transported to the cholinergic perikarya of the basal forebrain. In addition to the even labelling of somata, a strong punctate-like Cy3-immunofluorescence was seen in structures resembling lysosomes. The specificity of the in vivo staining was proven by subsequent immunolabelling of choline acetyltransferase (ChAT) with green fluorescent Cy2-tagged secondary antibodies. In the medial septum, the diagonal band and the nucleus basalis only cholinergic neurons were marked by Cy3-192IgG. In parallel experiments, digoxigenylated 192IgG was not detectable within cholinergic basal forebrain neurons after intraventricular injection. Presumably, this modified antibody could not be internalized. On the other hand, digoxigenylated 192IgG was found to be an excellent immunocytochemical marker for p75NTR as shown by double labelling including highly sensitive mouse antibodies directed against ChAT. Based on the present findings, future applications of the apparently non-toxic Cy3-192IgG and other antibodies for fluorescent in vivo and in vitro labelling are discussed.

Electron microscopic analysis of nanoparticles delivering thioflavin-T after intrahippocampal injection in mouse: implications for targeting β-amyloid in alzheimer's disease

Abstract Prevention of β-amyloid (Aβ) production, aggregation and formation of Aβ deposits is a key pharmacological target in Alzheimers disease. The passage of Aβ-binding compounds through the blood–brain barrier is often hampered for free ligands, whereas it is enhanced by drug encapsulation in nanoparticles. Here, we describe the preparation and characterization of polymeric carriers containing thioflavin-T as a marker for fibrillar Aβ. This study is then focused on electron microscopic analyses of thioflavin-T after injection of thioflavin-T-containing nanoparticles into the mouse hippocampus. Therefore, the photoconversion of fluorescent thioflavin-T as model drug was performed in tissues fixed 3 days post-injection. Thioflavin-T delivered from nanospheres was predominantly found in neurons and microglia. Our data suggest that drugs delivered by nanoparticles might target Aβ in the brain.

Electrophysiological alterations and upregulation of ATP receptors in retinal glial Müller cells from rats infected with the Borna disease virus.

Infection with the neurotropic Borna disease virus (BDV) causes an immune‐mediated neurological disease in a broad range of species. In addition to encephalitis, BDV‐infected Lewis rats develop a retinitis histologically characterized by the loss of most retinal neurons. By contrast, the dominating retinal macroglia, the Müller cells, do not degenerate. It is known from several models of neurodegeneration that glial cells may survive but undergo significant alterations of their physiological parameters. This prompted us to study the electrophysiology and ATP‐induced changes of intracellular Ca2+‐concentration ([Ca2+]i) in Müller cells from BDV‐infected rat retinae. Freshly isolated cells were used for whole‐cell patch‐clamp recordings. Whereas neither zero current potentials nor membrane resistances showed significant alterations, the membrane capacitance increased in cells from BDV‐infected rats during survival times of up to 8 months. This process was accompanied by a decrease in K+ current densities. Müller cells from BDV‐infected rats were characterized by expression of a prominent fast‐inactivating A‐type K+ current which was rarely found in control cells. Moreover, the number of cells displaying Na+ currents was slightly increased after BDV‐infection. ATP evoked increases in [Ca2+]i in Müller cells within retinal wholemounts of both control and BDV‐infected animals. However, the number of ATP‐responding isolated cells increased from 24% (age‐matched controls) to 78% (cells from animals ≥18 weeks after infection). We conclude that in BDV‐induced retinopathy, reactive rat Müller cells change their physiological parameters but these changes are different from those in Müller cells during proliferative vitreoretinopathy in man and rabbit. GLIA 35:213–223, 2001.

Oxygen-enriched photoconversion of fluorescent dyes by means of a closed conversion chamber

The goal of the present study was an improvement of the widely used photoconversion technique, which still represents the major approach to the ultrastructural analysis of tissue labelled with fluorescent dyes. Since free access of oxygen to the tissue is essential for the dye-dependent photooxidation of diaminobenzidine (DAB), we attempted to facilitate the process using a closed conversion chamber (CCC), which allows photoconversion in an atmosphere of pure oxygen. Fixed rat tissue samples, containing 4Di-10ASP labelled retinal ganglion cells and Cy3 stained cortical perineuronal nets, were choosen to test the applicability and efficiency of the proposed system. The results are compared to corresponding structures photoconverted without pure oxygen. As a result, the employment of the CCC helps saving up to 50% of time required to achieve a comparable degree of photoconversion. Electron microscopical inspection showed no differences between both approaches regarding the distribution of DAB reaction product. However, probably due to the reduced time of irradiation, the ultrastructural integrity of tissue sometimes appeared considerably less affected after photoconversion in the CCC. Additionally, the chamber allowed for safety measures in handling DAB, as the unintentional emission of the presumable carcinogenic substance was completely avoided.

Immunohistochemical characterization and quantitative analysis of neurons in the myenteric plexus of the equine intestine

The present study was performed on whole-mount preparations to investigate the chemical neuroanatomy of the equine myenteric plexus throughout its distribution in the intestinal wall. The objective was to quantify neurons of the myenteric plexus, especially the predominant cholinergic and nitrergic subpopulations. Furthermore, we investigated the distribution of vasoactive intestinal polypeptide and the calcium-binding protein calretinin. Samples from different defined areas of the small intestine and the flexura pelvina were taken from 15 adult horses. After fixation and preparation of the tissue, immunofluorescence labeling was performed on free floating whole-mounts. Additionally, samples used for neuropeptide staining were incubated with colchicine to reveal the neuropeptide distribution within the neuronal soma. The evaluation was routinely accomplished using confocal laser-scanning microscopy. For quantitative and qualitative analysis, the pan-neuronal marker anti-HuC/D was applied in combination with the detection of the marker enzymes for cholinergic neurons and nitrergic nerve cells. Quantitative data revealed that the cholinergic subpopulation is larger than the nitrergic one in several different locations of the small intestine. On the contrary, the nitrergic neurons outnumber the cholinergic neurons in the flexura pelvina of the large colon. Furthermore, ganglia are more numerous in the small intestine compared with the large colon, but ganglion sizes are bigger in the large colon. However, comparison of the entire population of neurons in the different locations of the gut showed no difference. The present study adds further data on the chemoarchitecture of the myenteric plexus which might facilitate the understanding of several gastrointestinal disorders in the horse.

Retinal pigment epithelium melanin granules are phagocytozed by Müller glial cells in experimental retinal detachment

The ability of retinal Müller glial cells to perform phagocytosis in vivo is studied in a rabbit model of experimental retinal detachment where pigment epithelial cells are occasionally detached together with the neural retina. While macrophages and/or microglial cells phagocytoze most of the cellular debris at the sclerad surface of the detached retinae, some Müller cells accumulate melanin granules. The granules are virtually intact at the ultrastructural level, and are surrounded by a membrane. They are often located close to the sclerad end of the cells, but some are distributed throughout the outer stem process up to the soma. It is concluded that rabbit Müller cells in vivo are capable of phagocytosis and of transporting the phagocytozed material within their cytoplasm.

CORTICAL PERINEURONAL NETS IN THE GRAY SHORT-TAILED OPOSSUM (MONODELPHIS DOMESTICA) : A DISTRIBUTION PATTERN CONTRASTING WITH THAT SHOWN IN PLACENTAL MAMMALS

Abstract Extracellular matrix proteoglycans accumulated in perineuronal nets and in certain neuropil zones have been shown to influence the immediate neuronal microenvironment, and to contribute to the chemoarchitectonic characteristics of neuronal networks. Studies in different placental mammals, including the human, have suggested that the major principles of extracellular matrix distribution remained constant during phylogenesis of the different mammalian strains. However, the comparison of matrix distribution between various species also indicates that striking deviations from the basic pattern may occur, although their functional significance appears unknown as yet. This study examines the extracellular matrix in the forebrain of a basic American marsupial, which has evolved independently of placental mammals for more than 100 million years. Brain sections obtained from adult gray short-tailed opossums (Monodelphis domestica) were stained for extracellular matrix components using the N-acetylgalactosamine-binding lectin Wisteria floribunda agglutinin (WFA), a polyclonal antibody against chondroitin sulfate proteoglycans (CSPG), and biotinylated hyaluronectin for the detection of hyaluronan. In subcortical regions, the distribution patterns of WFA-stained and CSPG-immunoreactive perineuronal nets were similar to those reported previously in placental mammals. In contrast, a unique distribution was found in the neocortex. This distribution was characterized by the presence of perineuronal nets around pyramidal cells and matrix components within the adjacent neuropil that together form a continuously labeled zone in layer V. Weakly stained nets ensheathed less numerous pyramidal cells in the upper layers II/III and a few multipolar cortical neurons. Dual staining experiments showed that cortical net-associated neurons were rarely immunopositive for parvalbumin. This fact, in addition to the predominant association of extracellular matrix components with layer V pyramidal cells, differentiates the neocortex in Monodelphis from that of all placental mammals studied to date. Regarding the basic phylogenetic position of this marsupial species it remains to be shown if these distribution characteristics of extracellular matrix may represent also a basic feature of cortical organization.

Surface properties of the skin of the pilot whale Globicephala melas.

On the skin surface of delphinids small biofoulers are challenged to high shear water flow and liquid-vapor interfaces of air-bubbles during jumping. This state of self-cleaning is supported by the even, nano-rough gel-coated epidermal surface of the skin. The present study focussed on the intercellular evolution of gel formation and the chemical composition of the gel smoothing the skin surface of the pilot whale, Globicephala melas , using X-ray photoelectron spectroscopy (XPS) in combination with cryo-scanning electron microscopy (CSM), and transmission electron microscopy (TEM). In the superficial layer of the epidermis, the stratum corneum, intercellular material was shown by electron optical methods to assemble from smaller into larger covalently cross-linked aggregates during the transit of the corneocytes towards the skin surface. XPS measurements showed that the surface of the skin and the intercellular gel included approximately the same amounts of polar groups (especially, free amines and amides) and non-polar groups, corresponding to the presence of lipid droplets dispersed within the jelly material. It was concluded from the results that the gel-coat of the skin surface is a chemically heterogeneous skin product. The advantages of chemically heterogeneous patches contributing to the ablation of traces of the biofouling process are discussed.