Jens Grosche

Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R

The extracellular matrix glycoprotein tenascin‐R (TN‐R), colocalizing with hyaluronan, phosphacan, and aggregating chondroitin sulphate proteoglycans in the white and grey matter, is accumulated in perineuronal nets that surround different types of neurons in many brain regions. To characterize the role of TN‐R in the formation of perineuronal nets, we studied their postnatal development in wild‐type mice and in a TN‐R knock‐out mutant by using the lectin Wisteria floribunda agglutinin and an antibody to nonspecified chondroitin sulphate proteoglycans as established cytochemical markers. We detected the matrix components TN‐R, hyaluronan, phosphacan, neurocan, and brevican in the perineuronal nets of cortical and subcortical regions. In wild‐type mice, lectin‐stained, immature perineuronal nets were first seen on postnatal day 4 in the brainstem and on day 14 in the cerebral cortex. The staining intensity of these nets for TN‐R, hyaluronan, phosphacan, neurocan, and brevican was extremely weak or not distinguishable from that of the surrounding neuropil. However, all markers showed an increase in staining intensity of perineuronal nets reaching maximal levels between postnatal days 21 and 40. In TN‐R‐deficient animals, the perineuronal nets tended to show a granular component within their lattice‐like structure at early stages of development. Additionally, the staining intensity in perineuronal nets was reduced for brevican, extremely low for hyaluronan and neurocan, and virtually no immunoreactivity was detectable for phosphacan. The granular configuration of perineuronal nets became more predominant with advancing age of the mutant animals, indicating the continued abnormal aggregation of chondroitin sulphate proteoglycans complexed with hyaluronan. As shown by electron microscopy in the cerebral cortex, the disruption of perineuronal nets was not accompanied by apparent changes in the synaptic structure on net‐bearing neurons. The regional distribution patterns and the temporal course of development of perineuronal nets were not obviously changed in the mutant. We conclude that the lack of TN‐R initially and continuously disturbs the molecular scaffolding of extracellular matrix components in perineuronal nets. This may interfere with the development of the specific micromilieu of the ensheathed neurons and adjacent glial cells and may also permanently change their functional properties. J. Comp. Neurol. 428:616–629, 2000.

Müller cells are living optical fibers in the vertebrate retina

Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.

Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

Role of retinal glial cells in neurotransmitter uptake and metabolism

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, gamma-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate-glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.

P2 receptor‐types involved in astrogliosis in vivo

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (α,β‐methylene adenosine 5′‐triphosphate; α,β‐meATP), P2Y1,12 (adenosine 5′‐O‐(2‐thiodiphosphate; ADP‐β‐S) and P2Y2,4,6 receptors (uridine 5′‐O‐(3‐thiotriphosphate; UTP‐γ‐S). Pyridoxalphosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) was used as a non‐selective antagonist. The astroglial reaction was studied by means of immunocytochemical double‐labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5‐bromo‐2′‐deoxyuridine (BrdU). The agonist‐induced changes in comparison to the artificial cerebrospinal fluid (aCSF)‐treated control side reveal a strong mitogenic potency of ADP‐β‐S and α,β‐meATP, whereas UTP‐γ‐S was ineffective. The P2 receptor antagonist PPADS decreased the injury‐induced proliferation when given alone and in addition inhibited all agonist effects. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up‐regulation of GFAP. A significant increase of both GFAP‐immunoreactivity (IR) and GFA‐protein content (by using Western blotting) was found after microinfusion of α,β‐meATP or ADP‐β‐S. In contrast, UTP‐γ‐S failed to increase the GFAP‐IR. The morphogenic effects were also inhibited by pre‐treatment with PPADS. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP‐labelled astrocytes. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

Expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and Bcl-2 protooncogene protein by Müller (glial) cells in retinal light damage of rats

In retinal light damage, degeneration of photoreceptors may cause alterations of glial (Müller) cells. We performed immunocytochemical studies on Müller cells isolated from retinae of rats exposed to enhanced illumination for 24 months, a procedure which leads to complete loss of photoreceptor cells. One group of rats was fed daily with Ginkgo biloba extract (EGb 761, an established free radical-scavenger) during the last 8 months of life when the remaining photoreceptors (about 50%) die. We found that (1) Müller cells respond to photoreceptor damage by increased expression of glial fibrillary acidic protein, (2) Müller cells reduce expression of glutamine synthetase when the major glutamate-releasing neurons are lost, and (3) the application of exogenous free radical scavengers prevents the expression by Müller cells of the protooncogene protein Bcl-2, a molecule assumed to activate endogenous free radical-scavenging activities.

Region and lamina-specific distribution of extracellular matrix proteoglycans, hyaluronan and tenascin-R in the mouse hippocampal formation

The extracellular matrix is known to show region-specific characteristics in the adult brain. Our comparative cytochemical study is focused on the laminar organisation of major extracellular matrix constituents in the murine hippocampal formation, including the regions CA1, CA2 and CA3 of the hippocampus proper, the dentate gyrus, the subiculum and the presubiculum. Components related to chondroitin sulphate proteoglycans were detected by N-acetylgalactosamine-binding Wisteria floribunda agglutinin, colloidal iron staining, and antibodies to different proteoglycan domains, including the Cat-301 and Cat-315 epitopes of aggrecan, as well as neurocan, brevican and phosphacan. The distribution patterns of these components were correlated with the patterns revealed for hyaluronan and the brain-specific extracellular matrix glycoprotein, tenascin-R, known to be ligands of extracellular matrix proteoglycans. Lectin binding clearly labelled perineuronal nets of the extracellular matrix around interneurons, which were preferentially located within or near the principal cell layers in all regions. In the hippocampus proper, the CA2 subfield showed an intense labelling of the neuropil around pyramidal cell bodies and the neuropil zones in the strata oriens and radiatum. These patterns were also seen after immunoreaction for chondroitin proteoglycan domains, brevican and phosphacan, as well as after detection of hyaluronan and tenascin-R. Characteristic laminar and intralaminar patterns were additionally expressed in the neuropil in all regions. In the dentate gyrus, the staining intensity for brevican, phosphacan and tenascin-R was predominant in the middle molecular layer, and for Cat-315 in the inner molecular layer, whereas immunoreactivity for neurocan increased within the outer molecular layer towards the hippocampal fissure. Our findings indicate that proteoglycans, hyaluronan and tenascin-R show differential patterns of co-expression in the individual regions and laminae of the hippocampal formation. The inhomogeneous composition of these major components suggests that the extracellular matrix is specifically adapted to the functional domains of intrahippocampal connections and afferent fibre systems.

Nmda-activated currents in Bergmann glial cells

NMDA receptors play a crucial role in synaptic plasticity of the central nervous system and were thought to be exclusive to neurones. In this study we provide evidence that Bergmann glial cells from mouse cerebellar slices show intrinsic responses to NMDA. As in neurones, NMDA increased membrane conductance and the responses were blocked by the NMDA antagonist ketamine, but not by the non-NMDA glutamate receptor antagonist CNQX. In contrast to responses in neurones, the current voltage relation of the glial NMDA-induced current was linear, reversed at -40 mV, currents were not blocked by Mg2+ or enhanced by glycine and NMDA did not induce an increase in cytosolic Ca2+ as recorded with a fura-2 imaging system. These data imply the presence of distinct NMDA receptors on Bergmann glial cells; these glial receptors could be the substitute for complex neurone-glia interactions in the cerebellum.

Human intravenous immunoglobulin provides protection against Aβ toxicity by multiple mechanisms in a mouse model of Alzheimer's disease

BackgroundPurified intravenous immunoglobulin (IVIG) obtained from the plasma of healthy humans is indicated for the treatment of primary immunodeficiency disorders associated with defects in humoral immunity. IVIG contains naturally occurring auto-antibodies, including antibodies (Abs) against β-amyloid (Aβ) peptides accumulating in the brains of Alzheimers disease (AD) patients. IVIG has been shown to alleviate AD pathology when studied with mildly affected AD patients. Although its mechanisms-of-action have been broadly studied, it remains unresolved how IVIG affects the removal of natively formed brain Aβ deposits by primary astrocytes and microglia, two major cell types involved in the neuroinflammatory responses.MethodsWe first determined the effect of IVIG on Aβ toxicity in primary neuronal cell culture. The mechanisms-of-action of IVIG in reduction of Aβ burden was analyzed with ex vivo assay. We studied whether IVIG solubilizes natively formed Aβ deposits from brain sections of APP/PS1 mice or promotes Aβ removal by primary glial cells. We determined the role of lysosomal degradation pathway and Aβ Abs in the IVIG-promoted reduction of Aβ. Finally, we studied the penetration of IVIG into the brain parenchyma and interaction with brain deposits of human Aβ in a mouse model of AD in vivo.ResultsIVIG was protective against Aβ toxicity in a primary mouse hippocampal neuron culture. IVIG modestly inhibited the fibrillization of synthetic Aβ1-42 but did not solubilize natively formed brain Aβ deposits ex vivo. IVIG enhanced microglia-mediated Aβ clearance ex vivo, with a mechanism linked to Aβ Abs and lysosomal degradation. The IVIG-enhanced Aβ clearance appears specific for microglia since IVIG did not affect Aβ clearance by astrocytes. The cellular mechanisms of Aβ clearance we observed have potential relevance in vivo since after peripheral administration IVIG penetrated to mouse brain tissue reaching highest concentrations in the hippocampus and bound selectively to Aβ deposits in co-localization with microglia.ConclusionsOur results demonstrate that IVIG promotes recognition and removal of natively formed brain Aβ deposits by primary microglia involving natural Aβ Abs in IVIG. These findings may have therapeutic relevance in vivo as IVIG penetrates through the blood-brain barrier and specifically binds to Aβ deposits in brain parenchyma.

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.