Johannes Kacza

Perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy

The deposition of amyloid-β (Aβ) peptides in the walls of leptomeningeal and cortical blood vessels as cerebral amyloid angiopathy (CAA) is present in normal ageing and the majority of Alzheimer’s disease (AD) brains. The failure of clearance mechanisms to eliminate Aβ from the brain contributes to the development of sporadic CAA and AD. Here, we investigated the effects of CAA and ageing on the pattern of perivascular drainage of solutes in the brains of naïve mice and in the Tg2576 mouse model of AD. We report that drainage of small molecular weight dextran along cerebrovascular basement membranes is impaired in the hippocampal capillaries and arteries of 22-month-old wild-type mice compared to 3- and 7-month-old animals, which was associated with age-dependent changes in capillary density. Age-related alterations in the levels of laminin, fibronectin and perlecan in vascular basement membranes were also noted in wild-type mice. Furthermore, dextran was observed in the walls of veins of Tg2576 mice in the presence of CAA, suggesting that deposition of Aβ in vessel walls disrupts the normal route of elimination of solutes from the brain parenchyma. These data support the hypothesis that perivascular solute drainage from the brain is altered both in the ageing brain and as a consequence of CAA. These findings have implications for the success of therapeutic strategies for the treatment of AD that rely upon the health of the ageing cerebral vasculature.

Cortical areas are revealed by distribution patterns of proteoglycan components and parvalbumin in the Mongolian gerbil and rat

Cortical areas in rodents have been basically characterized by its cytoarchitecture, connectivity or by physiological parameters. In this study we show that they are revealed by distribution patterns of proteoglycans and parvalbumin-immunoreactivity. Brains of young adult Mongolian gerbils (Meriones unguiculatus) and Wistar rats were cut into series of transversal sections. Proteoglycan components were detected using the N-acetylgalactosamine binding Wisteria floribunda agglutinin (WFA) and antibodies against chondroitin sulphate proteoglycan (CSPG). Differences between cortical areas were found to exist with regard to the occurrence and the density of perineuronal nets, but were also expressed in varying staining intensities for WFA and CSPG of the neuropil. Primary neocortical areas (somatosensory, auditory, visual cortex) were characterized by an intense neuropil staining in layer IV and the upper part of layer VI. Using the same methods strong labelling was also typical of the neuropil in the retrosplenial cortex, of layer Ia in the prepiriform cortex and the hippocampal CA3 field. In tangential sections cut from gerbil cortical hemispheres, some of the heavily lectin-stained cortical areas were sharply delineated from adjacent faintly labelled regions, others showed more diffuse borders. In the rat, the area-specific staining for WFA was less clearly expressed than in the gerbil. Immunocytochemistry of the calcium-binding protein parvalbumin in alternate sections showed labelling patterns of neuropil which resembled those of WFA-binding and CSPG-immunoreactivity in the entire neocortex and hippocampus. From these results it can be concluded that functional peculiarities of cortical fields may not only be determined by neuronal network parameters but also by the spatial arrangement of extracellular matrix proteoglycans.

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.

Axon initial segment ensheathed by extracellular matrix in perineuronal nets.

Perineuronal nets of extracellular matrix are associated with distinct types of neurons in the cerebral cortex and many subcortical regions. Large complexes of aggregating proteoglycans form a chemically specified microenvironment around the somata, proximal dendrites and the axon initial segment, including the presynaptic boutons attached to these domains. The subcellular distribution and the temporal course of postnatal formation suggest that perineuronal nets may be involved in the regulation of synaptic plasticity. Here we investigate structural and cytochemical characteristics of the extracellular matrix around axon initial segments virtually devoid of synaptic contacts. Wisteria floribunda agglutinin staining, the immunocytochemical detection of aggrecan and tenascin-R, as well as affinity-labeling of hyaluronan were used to analyze perineuronal nets associated with large motoneurons in the mouse superior colliculus. The molecular composition of perineuronal nets was divergent between neurons but was identical around the different cellular domains of the individual neurons. The axon initial segments largely devoid of synapses were covered by a continuous matrix sheath infiltrating the adjacent neuropil. The periaxonal zone penetrated by matrix components often increased in diameter along the initial segment from the axon hillock toward the myelinated part of the axon. The axonal and somatodendritic domains of perineuronal nets were concomitantly formed during the first three weeks of postnatal development. The common molecular properties and major structural features of subcellular perineuronal net domains were retained in organotypic midbrain slice cultures. The results support the hypothesis that the aggrecan-related extracellular matrix of perineuronal nets provides a continuous micromilieu for different subcellular domains performing integration and generation of the electrical activity of neurons.

Unique Luminal Localization of VGAT-C Terminus Allows for Selective Labeling of Active Cortical GABAergic Synapses

Neurotransmitter uptake into synaptic vesicles is mediated by vesicular neurotransmitter transporters. Although these transporters belong to different families, they all are thought to share a common overall topology with an even number of transmembrane domains. Using epitope-specific antibodies and mass spectrometry we show that the vesicular GABA transporter (VGAT) possesses an uneven number of transmembrane domains, with the N terminus facing the cytoplasm and the C terminus residing in the synaptic vesicle lumen. Antibodies recognizing the C terminus of VGAT (anti-VGAT-C) selectively label GABAergic nerve terminals of live cultured hippocampal and striatal neurons as confirmed by immunocytochemistry and patch-clamp electrophysiology. Injection of fluorochromated anti-VGAT-C into the hippocampus of mice results in specific labeling of GABAergic synapses in vivo. Overall, our data open the possibility of studying novel GABA release sites, characterizing inhibitory vesicle trafficking, and establishing their contribution to inhibitory neurotransmission at identified GABAergic synapses.

Adipocyte-derived angiopoietin-1 supports neurite outgrowth and synaptogenesis of sensory neurons

Sensory and sympathetic innervation of the white fat tissue (WAT) contributes to lipolysis. In addition, both fiber types adapt in density to weight gain and loss. Because these findings are indicative for a tight control of nerve fiber plasticity by adipokines, we tested whether adipocytes control neurite growth of sensory neurons through angiopoietin‐1 (Ang‐1). We further considered initial hints that Ang‐1‐induced neuritogenesis involves transactivation of the high‐affinity nerve growth factor (NGF) receptor trkA. Coculturing dorsal root ganglion (DRG) cells with 3T3‐L1 adipocytes supported neurite outgrowth. These neurotrophic effects were associated with the increased expression of Ang‐1 (presumably in adipocytes) as well as of trkA. The effects were abolished upon inactivating Ang‐1 in culture with selective antibodies. Likewise, neurite outgrowth was impaired in the presence of inactivating NGF antibodies as well as upon inhibition of the NGF high‐affinity trkA receptor with the antagonist K252a, indicating a tight cooperation of Ang‐1 and NGF in the control of neuritogenesis. DRG‐adipipocyte cocultures were further used to establish whether sensory neurons would form synaptic contacts with adipocytes. Electron microscopy demonstrated that cultured sensory neurons develop predominantly neuroneuronal synapses but seem to affect adipocytes by synapses en passant. Comparably to the case for neuritogenesis, expression of the presynaptic protein synaptophysin as well of the postsynaptic protein PSD‐95 correlated with Ang‐1 levels in culture. It is concluded that adipocyte‐secreted Ang‐1 supports neurite outgrowth, which is involved in synaptogenesis. The novel function of Ang‐1 appears to play a physiological role in WAT plasticity.

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.

A novel quantification of blood-brain barrier damage and histochemical typing after embolic stroke in rats.

Treatment strategies in acute ischemic stroke are still limited. Considering numerous translation failures, research is tending to a preferred use of human-like animal models, and a more-complex perspective of tissue salvaging involving endothelial, glial and neuronal components according to the neurovascular unit (NVU) concept. During ischemia, blood-brain barrier (BBB) alterations lead to brain edema and hemorrhagic transformation affecting NVU components. The present study aims on a novel quantification method of BBB damage and affected tissue following experimental cerebral ischemia, closely to the human condition. Wistar rats underwent embolic middle cerebral artery occlusion, followed by an intravenous application of fluorescein isothiocyanate (FITC)-tagged albumin (≈70kDa) and/or biotinylated rat IgG (≈150kDa) as BBB permeability markers. Both fluorescent agents revealed similar leakage and allow quantification of BBB permeability by fluorescence microscopy, and after immunohistochemical conversion into a permanent diaminobenzidine label at light-microscopical level. The following markers were identified for sufficient detection of NVU components: Rat endothelial cell antigen-1 (RECA) and laminin for vessels, Lycopersicon esculentum and Griffonia simplicifolia agglutinin for vessels and microglial subpopulations, ionized calcium binding adaptor molecule 1 (Iba1), CD68 and CD11b for macrophages, activated microglia, monocytes and neutrophils, S100β for astroglia, as well as NeuN and HuC/D for neurons. This is the first report confirming the usefulness of simultaneously applied FITC-albumin and biotinylated rat IgG as BBB permeability markers in experimental stroke, and, specifying antibodies and lectins for multiple fluorescence labeling of NVU components. Newly elaborated protocols might facilitate a more-complex outcome measurement in drug development for cerebral ischemia.

Photonic crystal light collectors in fish retina improve vision in turbid water.

Seeing in the Dark Elephantnose fish are known to use electrosensing to navigate their murky freshwater environment. However, unlike some other animals from dark environments, they have retained their eyes and some dependence on vision. While most vertebrate vision optimizes either photon catch (for increased light capture) or visual acuity, Kreysing et al. (p. 1700) show that the unique structures of the grouped retinae found in the eyes of this species matches rod and cone sensitivity, which allows for the simultaneous use of both types of photoreceptors over a large range of dim light intensities. Layering cones on top of rods allows the elephantnose fish to see low-contrast objects in a murky environment. Despite their diversity, vertebrate retinae are specialized to maximize either photon catch or visual acuity. Here, we describe a functional type that is optimized for neither purpose. In the retina of the elephantnose fish (Gnathonemus petersii), cone photoreceptors are grouped together within reflecting, photonic crystal–lined cups acting as macroreceptors, but rod photoreceptors are positioned behind these reflectors. This unusual arrangement matches rod and cone sensitivity for detecting color-mixed stimuli, whereas the photoreceptor grouping renders the fish insensitive to spatial noise; together, this enables more reliable flight reactions in the fish’s dim and turbid habitat as compared with fish lacking this retinal specialization.