Wisteria Floribunda Research Articles

Light and confocal laser-scanning microscopical evidences for complementary patterns of glial fibrillary acidic protein and Wisteria floribunda agglutinin

We investigated the pattern of glial fibrillary acidic protein (GFAP) and Wisteria floribunda agglutinin (WFA) labeled structures in the superior colliculus and in the somatosensory cortex of humans and rats of different age groups using immunohistochemical methods, light and confocal laser-scanning microscopy. We never found a double labeling of WFA and GFAP positive structures neither in the superior colliculus nor in the cortex of both man and rat. The complementary pattern of WFA and GFAP labeling was present both at the macroscopic and microscopic level. We found a clear prevalence of either WFA or GFAP expression in the arborization of the astrocytes as well as in the pattern of lamination.

The perineuronal proteoglycan surface coat in the adult rat brain, with special reference to its reactions to Gömöri's ammoniacal silver.

The present study showed that many neurons in the adult rat brain possessed a perineuronal sulfated proteoglycan surface coat which reacted to cationic iron colloid and aldehyde fuchsin. This surface coat was stained supravitally with Ehrlich's methylene blue and doubly stained with Ehrlich's methylene blue and aldehyde fuchsin. The surface coat was also stained with Gömöri's ammoniacal silver and doubly stained with Gömöri's ammoniacal silver and cationic iron colloid. The surface coat was usually expressed together with a nerve cell surface glycoprotein net detectable with lectin Wisteria floribunda agglutinin. These findings indicate that the perineuronal proteoglycan surface coat is identical to Cajal's superficial reticulum and contains some collagenous elements. It was further demonstrated that collagenase digestion erased Gömöri's ammoniacal silver impregnation within the perineuronal proteoglycan surface coat.

Morphology of perineuronal nets in tenascin-R and parvalbumin single and double knockout mice

Recently identified chondroitin sulphate proteoglycans in perineuronal nets include neurocan and phosphacan. However, the function and assembly of these components has yet to be resolved. In this study we show morphological alteration in Wisteria floribunda labelled nets around cortical interneurones both in tenascin-R knockout and tenascin-R/parvalbumin double knockout mice. This alteration reflects the loss of phosphacan and neurocan from cortical nets in mice deficient in tenascin-R. No effect on the membrane related cytoskeleton, as revealed by ankyrin R, was observed in any of the mice. These results on mice lacking tenascin-R substantiate previously reported in vitro interactions between tenascin-R and phosphacan and neurocan.

Perineuronal nets ensheath fast spiking, parvalbumin-immunoreactive neurons in the medial septum/diagonal band complex.

Perineuronal nets, composed of extracellular matrix material, have previously been associated with parvalbumin-immunoreactive neurons in the medial septum/diagonal band (MS/DB) complex of the rat. The aim of this study was to correlate the presence of perineuronal nets with electrophysiological properties and parvalbumin immunoreactivity in MS/DB neurons. Intracellular recordings were made from cells in a brain slice preparation maintained in vitro, and neurons were characterized into four populations: (i) slow-firing neurons, (ii) burst-firing neurons, (iii) fast spiking neurons with narrow action potentials and a small degree of spike frequency adaptation, and (iv) regular spiking neurons with broader action potentials and a high degree of spike frequency adaptation. Following electrophysiological characterization, neurons were filled with biocytin, processed for parvalbumin immunoreactivity and stained for perineuronal nets using Wisteria floribunda lectin. The three substances were viewed with triple fluorescence. Fast spiking, nonadapting neurons, shown previously to contain parvalbumin immunoreactivity, were nearly all ensheathed by perineuronal nets. There was a population of small parvalbumin-immunoreactive neurons which did not possess perineuronal nets, and which were not encountered with the intracellular electrodes. The other three neuron types in the MS/DB did not contain parvalbumin immunoreactivity or perineuronal nets. In keeping with this neurochemical profile for electrophysiologically identified neurons, burst-firing neurons had action potential parameters more similar to those of regular spiking than of fast spiking neurons. We conclude that fast spiking neurons, presumed to be GABAergic septohippocampal projection neurons, are surrounded by supportive structures to enable the high level of neuronal discharge required for producing disinhibition of hippocampal pyramidal neurons.

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.

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.

Cell surface composition of promastigote and opisthomorph forms of Herpetomonas roitmani (Kinetoplastida : Trypanosomatidae).

Cell surface saccharide composition and surface charge of promastigote (PRO) and opisthomorph (OPM) forms of Herpetomonas roitmani were analyzed using labeled lectins and flow cytometry and cell electrophoresis. The FITC signals for concanavalin A, Helix pomatia agglutinin and wheat germ agglutinin were stronger in PRO forms, whereas for Limulus polyphemus agglutinin (LPA) and Wisteria floribunda agglutinin they were stronger in OPM forms. Prior treatment of the cells with neuraminidase decreased the FITC signal for LPA in OPM but not in PRO forms. Furthermore OPMs displayed a high negative charge (-15.45+/-1.10 mV) than PROs (-9.47+/-1.01 mV). Neuraminidase and phospholipase C treatment of the parasites significantly reduced the surface charge, especially in OPM forms. TLC analysis of the acidic components of H. roitmani showed the presence of N-acetyl-neuraminic acid. The results presented in this work indicate that changes in exposed cell surface components occur between PRO and OPM forms of H. roitmani obtained by growing the cells under different conditions.

Neuronal and glial structures of the superficial layers of the human superior colliculus.

We studied neuronal and glial elements in the superficial layers of the human superior colliculus by means of Nissl stains, Golgi impregnations, histochemical demonstration of NADPH-d activity and immunohistochemistry for glial fibrillary acidic protein (GFAP) in astrocytes. The glia-neuron interface was visualized with Wisteria floribunda agglutinin (WFA), which is a marker for perineuronal nets. The laminar pattern and the morphology of the major cell types closely resembled that found in other species although the thickness of the stratum zonale varied and the diversity of interneurons was greater than in other mammals. Furthermore, the stratum griseum superficiale showed a characteristic clustering of cells, the surfaces of which were intensely labeled by WFA. The clusters disappeared when GFAP expression increased.

Perineuronal nets of proteoglycans in the adult mouse brain, with special reference to their reactions to Gömöri's ammoniacal silver and Ehrlich's methylene blue.

As our previous studies have indicated, many subsets of neurons in the vertebrate brain possess a sulfated proteoglycan surface coat which reacts to cationic iron colloid and aldehyde fuchsin. The present study demonstrated that this surface coat is supravitally stained with Ehrlich's methylene blue, and doubly with this blue and aldehyde fuchsin, a finding suggesting its being identical to Cajal's superficial reticulum (red superficial) and to Golgi's reticular coating (revetement reticulare). The perineuronal surface coat was further stained with Gömöri's ammoniacal silver, and doubly with this silver and cationic iron colloid. These neurons with such a proteoglycan surface coat usually expressed cell surface glycoproteins which were labeled with lectin Wisteria floribunda agglutinin. Hyaluronidase digestion did not interfere with this lectin labeling of the glycoproteins, methylene blue and Gömöri's ammoniacal silver staining of the surface coat, while it erased the cationic iron colloid and aldehyde fuchsin staining of the surface coat. These findings suggest that the perineuronal proteoglycan surface coat is associated with some additional molecules which are resistant to hyaluronidase digestion and stainable with methylene blue and Gömöri's ammoniacal silver. The possibility is suggested that these molecules might represent "ligand proteoglycans" connecting the perineuronal proteoglycans and cell surface glycoproteins.

The extracellular matrix in the mouse brain: its reactions to endo-alpha-N-acetylgalactosaminidase and certain other enzymes.

As our previous studies have indicated, the cingulate cortex of the adult mouse brain contains many neurons with rich cell surface glycoproteins which are linked by collagenous ligands to perineuronal proteoglycans. The present study demonstrated that exclusive incubation with endo-alpha-N-acetylgalactosaminidase abolished the lectin Vicia villosa or Wisteria floribunda agglutinin (VVA or WFA) labeling of the nerve cell surface glycoproteins, while it neither interfered with the cationic iron colloid or aldehyde fuchsin stainings of the perineuronal proteoglycans nor abolished the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Double incubations with endo-alpha-N-acetylgalactosaminidase and collagenase did not eliminate the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, though they did eliminate the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans as well as the Gömöri's ammoniacal silver impregnation of the collagenous ligands. Triple incubations with endo-alpha-N-acetylgalactosaminidase, collagenase, and endo-alpha-N-acetylgalactosaminidase abolished the lectin VVA or WFA labeling of the nerve cell surface glycoproteins, and also eliminated the cationic iron colloid and aldehyde fuchsin stainings of the perineuronal proteoglycans and the Gömöri's ammoniacal silver impregnation of the collagenous ligands. These findings indicate that: the nerve cell surface glycoproteins or their terminal N-acetylgalactosamines are digested by endo-alpha-N-acetylgalactosaminidase; these galactosamines associated with the collagenous ligands or perineuronal proteoglycans are not digested by endo-alpha-N-acetylgalactosaminidase; and the terminal N-acetylgalactosamines newly exposed by collagenase incubation are digested by this galactosaminidase. It was further demonstrated that hyaluronidase incubation neither digests the collagenous ligands nor revives the lectin VVA or WFA labeling of the nerve cell surface proteoglycans.

Acute and long-lasting changes in extracellular-matrix chondroitin-sulphate proteoglycans induced by injection of chondroitinase ABC in the adult rat brain.

Lattice-like perineuronal accumulations of extracellular-matrix proteoglycans have been shown to develop during postnatal maturation and to persist throughout life as perineuronal nets (PNs) in many brain regions. However, the dynamics of their reorganization in adults are as yet unknown. The aim of the present study was to examine the capability of PNs for reconstitution after experimental destruction and to search for possible consequences of extracellular-matrix degradation for neurons and glial cells. The changes were induced by single intracortical injections of Proteus vulgaris chondroitinase ABC and studied after postinjection periods of 1 day to 5 months. The N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA), an antibody against chondroitin-sulphate proteoglycans, three antibodies recognizing initial chondroitin or chondroitin-sulphate moieties ('stubs') of proteoglycan core proteins, an antibody against the hyaluronan-binding protein component of versican, and biotinylated hyaluronectin, which binds to hyaluronan, were used as cytochemical markers. One day postinjection, the WFA-binding sites and hyaluronan were shown to be almost completely removed within a circumscribed digestion zone. The staining of different core-protein components revealed only fragments of PNs. These changes were found to be partly compensated 4 weeks after injection of chondroitinase ABC. After 8 and 12 weeks postinjection, the cytochemical and structural characteristics as well as the area-specific distribution patterns of PNs were progressively reconstituted. At 5 months postinjection, they could not be distinguished from those in untreated tissue. In contrast to such transient changes, a diffuse chondroitin-sulphate proteoglycan immunoreactivity persisted in the neuropil. Loss of neurons or alterations of their structure as well as reactions of glial cells were not observed. We conclude from this study that PNs, enzymatically destroyed in the adult rat brain, can be completely reconstituted, but the restoration of their extracellular-matrix components needs several months.

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes.

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H-type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.

Subdivisions of the motor and somatosensory thalamus of primates revealed with Wisteria floribunda agglutinin histochemistry

We obtained well-differentiated staining of thalamic subdivisions in rhesus macaques and squirrel monkeys using a lectin, Wisteria floribunda agglutinin (WFA), that labels extracellular matrix proteoglycans. Regional variations in staining were observed within the motor and somatosensory thalamic regions that bear on current interpretations of the organization of these regions. The pattern of WFA staining was generally similar to that obtained with Cat-301 antibody, which also stains proteoglycans. However, WFA reliably produced selective staining in both squirrel monkeys and macaques, whereas Cat-301 stained macaques more consistently than squirrel monkeys.

The ruminant parasite Haemonchus contortus expresses an alpha1,3-fucosyltransferase capable of synthesizing the Lewis x and sialyl Lewis x antigens.

Glycoproteins from the ruminant helminthic parasite Haemonchus contortus react with Lotus tetragonolobus agglutinin and Wisteria floribunda agglutinin, which are plant lectins that recognize alpha1,3-fucosylated GlcNAc and terminal beta-GalNAc residues, respectively. However, parasite glycoconjugates are not reactive with Ricinus communis agglutinin, which binds to terminal beta-Gal, and the glycoconjugates lack the Lewis x (Le(x)) antigen or other related fucose-containing antigens, such as sialylated Le(x), Le(a), Le(b) Le(y), or H-type 1. Direct assays of parasite extracts demonstrate the presence of an alpha1,3-fucosyltransferase (alpha1,3FT) and beta1,4-N-acetylgalactosaminyltransferase (beta1,4GalNAcT), but not beta1,4-galactosyltransferase. The H. contortus alpha1,3FT can fucosylate GlcNAc residues in both lacto-N-neotetraose (LNnT) Galalpha1-->4GlcNAcbeta1-->3Galbeta1-->4Glc to form lacto-N-fucopentaose III Galbeta1-->4[Fuca1-->3]GlcNAc beta1-->3Galbeta1-4GIc, which contains the Le(x) antigen, and the acceptor lacdiNAc (LDN) GalNAcbeta1-->4GlcNAc to form GalNAc beta1-->4[Fualpha1-->3]GlcNAc. The alpha1,3FT activity towards LNnT is dependent on time, protein, and GDP-Fuc concentration with a Km 50 microM and a Vmax of 10.8 nmol-mg(-1) h(-1). The enzyme is unusually resistant to inhibition by the sulfhydryl-modifying reagent N-ethylmaleimide. The alpha1,3FT acts best with type-2 glycan acceptors (Galbeta1-->4GlcNAcbeta1-R) and can use both sialylated and non-sialylated acceptors. Thus, although in vitro the H. contortus alpha1,3FT can synthesize the Le(x) antigen, in vivo the enzyme may instead participate in synthesis of fucosylated LDN or related structures, as found in other helminths.

Perineuronal sulfated proteoglycans and cell surface glycoproteins in the visual cortex of adult and newborn cats.

Sections of the visual cortex of newborn (1-4 weeks after birth) and adult cats were stained with cationic iron colloid, aldehyde fuchsin or lectins (lectin Vicia villosa, soybean and Wisteria floribunda agglutinins). Many neurons in the adult cat visual cortex contained perineuronal sulfated proteoglycans detectable with cationic iron colloid and aldehyde fuchsin, or cell surface glycoproteins reactive to lectins. Double staining indicated that some of the lectin-labeled neurons were not stained with cationic iron colloid, and also that some of the cationic iron colloid-stained neurons were not labeled with lectins. The perineuronal sulfated proteoglycans and cell surface glycoproteins developed 3 weeks after birth. In the newborn cats 1-2 weeks after birth, no neurons were reactive to cationic iron colloid, aldehyde fuchsin or lectins. In the newborn cats 3-4 weeks after birth, it was clearly observed that the cytoplasm of the glial cells closely associated with the neurons containing the perineuronal sulfated proteoglycans showed an intense reaction to cationic iron colloid and aldehyde fuchsin, and that the Golgi complexes of the neurons with cell surface glycoproteins were intensely labeled with lectins. These findings suggest that the perineuronal sulfated proteoglycans are derived from the associated glial cells, and that the cell surface glycoproteins are produced by the associated nerve cells.

Cell-surface carbohydrates of Entamoeba invadens.

Cell-surface carbohydrates of Entamoeba invadens trophozoites were analyzed using (a) a panel of highly purified lectins specific for molecules containing N-acetylglucosamine or sialic acid, N-acetylgalactosamine, galactose, mannose-like residues, and fucose; (b) Escherichia coli K-12 with mannose-sensitive fimbria; (c) enzymatic digestion; and (d) scanning electron microscopy. The presence of galactose (D-Gal) and N-acetylgalactosamine (D-GalNAc) was detected in the amoeba. Previous trypsinization induced the appearance of Glycine max (SBA, specific for D-GalNAc residues)-binding sites, whereas such treatment completely abolished the ability of Ricinus communis (RCAI) and Axinalla polypoides (APP, specific for D-Gal) lectins and partially abolished that of Euonymus europaeus (EEL, specific for D-Gal) lectins to agglutinate the trophozoites. The agglutinating activity of E. coli K-12 adheans with the amoeba was markedly increased after trypsin digestion, indicating that mannose units become exposed after enzyme treatment. These findings were essentially confirmed by scanning electron microscopy. After neuraminidase treatment the parasites became strongly agglutinated with SBA and Arachis hypogaea (PNA, specific for D-Gal) and the cell interaction with Wisteria floribunda (WFH, specific for D-GalNAc) was markedly increased. These results suggest that in E. invadens trophozoites, sialic acid residues are linked to D-Gal and D-GalNAc.

Developmental patterns of proteoglycan-containing extracellular matrix in perineuronal nets and neuropil of the postnatal rat brain.

The extracellular matrix is involved in various morphogenetic processes which are accompanied by changes in its physicochemical properties and spatial organization. In the adult brain it contributes to cellular communication and the regulation of neuronal activity. The present study deals with the postnatal appearance and transformation into adult distribution patterns of extracellular matrix components related to chondroitin-sulphate proteoglycans (CSPGs) in the rat brain. The differential accumulation of these components in neuropil and in perineuronal nets (PNs) enriched in certain regions was examined in 0-, 7-, 14-, 21- and 35-day-old rats and adult animals using the N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA) and immunocytochemical detection of CSPGs. The lectin stained the olfactory-bulb glomerular layer and layer Ia of piriform and entorhinal cortex already in newborn animals. On postnatal day 7 diffuse neuropil staining was additionally found in certain subcortical nuclei and in deep neocortical layers. The first sharply contoured PNs were detected at this age in the brain stem, indicating the more advanced maturation of matrix components in subcortical regions. CSPG immunoreactivity yielded staining patterns largely identical to WFA-binding patterns but appeared only between postnatal day 14 and 21. The adult-like stage was revealed with both methods between 21 and 35 days after birth. The results provide further evidence that the accumulation of certain CSPGs in the extracellular space is spatiotemporally related to distinct patterns of neuronal activity at the regional and cellular level.

Morphological study of neocortical areas in Rett syndrome.

Various neocortical areas from four females aged 16-24 years with Rett syndrome (RS) were investigated and compared with brains of therapy-resistant partial epilepsy (TRPE) patients (18-25 years), infantile autism (IA), and control brains (24 and 58 years). The cytoarchitecture of area 10 (frontal), area 21 (temporal), area 4 (primary motor cortex), and area 17 (primary visual cortex) was studied by the combined Klüver-Barrera (luxol fast blue and cresyl violet) standard procedure. Autofluorescence of lipofuscin, immunofluorescence of synaptic vesicle proteins [synaptophysin (p38)] and lectin-stained (Wisteria floribunda agglutinin) perineuronal nets (PNs) were studied in the cortices using dual-channel confocal laser scanning microscopy. The brains of RS females show various types of morphological/cytoarchitectonical abnormalities of single pyramidal neurons in layers II-III, and V-VII of different cortical areas. The abnormalities include mild losses of pyramidal neurons, more pronounced in layers II and III than in layers V and VII, and more evident in frontal and temporal areas than in the visual cortex. Microdysgenesis, including abnormalities due to neuronal migration disorders, was not found in RS, in contrast to the observations in TRPE patients, strongly indicating that RS is not a neuronal migration disorder. Lipofuscin distribution was normal but amounts were lower in RS cases than in control and TRPE brains. PNs were less expressed in cortices of the IA case but were clearly overexpressed in the motor cortex of RS. Quantitative analysis of p38 showed a decrease in the area occupied by p38 immunoreactivity by 20-40% in RS compared with controls. It is concluded that RS could best be explained by a postnatal synaptogenic developmental deficiency; the basic defect, however, is still completely unknown.

Characterization of proteoglycan-containing perineuronal nets by enzymatic treatments of rat brain sections.

Proteoglycans are among the major extracellular matrix components of the central nervous system. In the cerebral cortex and many subcortical regions, chondroitin sulphate proteoglycans, which are related to the aggrecan-versican-neurocan family, have been detected immunocytochemically in perineuronal nets that surround various types of neurons. This indicates that, in the brain, there is a nonhomogeneous but defined distribution of extracellular matrix components. The present study is a further attempt to characterize the perineuronal nets in the cerebral cortex. Sections obtained from fixed and unfixed rat brains were subjected to different enzymatic treatments prior to the visualization of perineuronal nets using N-acetylgalactosamine-binding Wisteria floribunda agglutinin, antibodies against chondroitin sulphate proteoglycans or hyaluronectin, and biotinylated hyaluronectin which detects hyaluronan. In all perineuronal nets the binding of the Wisteria floribunda agglutinin was abolished after the incubation of sections with chondroitinase ABC. The protein components of the proteoglycan complexes became easier to digest after removal of chondroitin sulphate chains or hyaluronan. Since only quantitative, and not qualitative, differences in the labelling properties and the structural appearance of cortical perineuronal nets were observed after the various treatments, it is concluded that, with regard to their proteoglycan composition, these structures have common basic properties.

Perineuronal sulfated proteoglycans and cell surface glycoproteins in adult and newborn mouse brains, with special reference to their postnatal developments.

Sections of the retrosplenial cortex from adult and newborn mouse brains were observed with a light microscope. The retrosplenial cortex of the adult animals contained many neurons (10% of the total), including some dark neurons, with perineuronal sulfated proteoglycans detectable with cationic iron colloid and aldehyde fuchsin. The retrosplenial cortex of the adult animals also contained many neurons (10% of the total) with cell surface glycoproteins reactive to lectin Vicia villosa, soybean or Wisteria floribunda agglutinin. Double staining showed that the majority (75%) of the neurons labeled with lectins were stained with cationic iron colloid, and that some (25%) of them were not stained with this colloid. Double staining also showed that some (25%) of the neurons stained with cationic iron colloid were not labeled with lectins. These findings indicate that the perineuronal sulfated proteoglycans are, at least partly, independent from the cell surface glycoproteins. Observations of the sections from the newborn animals revealed that the perineuronal sulfated proteoglycans were produced by the associated satellite astrocytes 3-4 weeks after birth, and that the cell surface glycoproteins were produced by the associated nerve cells at earlier stages, or 2-3 weeks after birth. Dark neurons began to appear 3-4 weeks after birth. These dark neurons or their Golgi complexes were also reactive to lectins, suggesting the production of cell surface glycoproteins.