The perineuronal net and the control of CNS plasticity

Perineuronal nets (PNNs) are reticular structures that surround the cell body of many neurones, and extend along their dendrites. They are considered to be a specialized extracellular matrix in the central nervous system (CNS). PNN formation is first detected relatively late in development, as the mature synaptic circuitry of the CNS is established and stabilized. Its unique distribution in different CNS regions, the timing of its establishment, and the changes it undergoes after injury all point toward diverse and important functions that it may be performing. The involvement of PNNs in neuronal plasticity has been extensively studied over recent years, with developmental, behavioural, and functional correlations. In this review, we will first briefly detail the structure and organization of PNNs, before focusing our discussion on their unique roles in neuronal development and plasticity. The PNN is an important regulator of CNS plasticity, both during development and into adulthood. Production of critical PNN components is often triggered by appropriate sensory experiences during early postnatal development. PNN deposition around neurones helps to stabilize the established neuronal connections, and to restrict the plastic changes due to future experiences within the CNS. Disruption of PNNs can reactivate plasticity in many CNSs, allowing activity-dependent changes to once again modify neuronal connections. The mechanisms through which PNNs restrict CNS plasticity remain unclear, although recent advances promise to shed additional light on this important subject.     

Composition of perineuronal net extracellular matrix in rat brain: a different disaccharide composition for the net-associated proteoglycans

We developed a method to extract differentially chondroitin sulfate proteoglycans (CSPGs) that are diffusely present in the central nervous system (CNS) matrix and CSPGs that are present in the condensed matrix of perineuronal nets (PNNs). Adult rat brain was sequentially extracted with Tris-buffered saline (TBS), TBS-containing detergent, 1 m NaCl, and 6 m urea. Extracting tissue sections with these buffers showed that the diffuse and membrane-bound CSPGs were extracted in the first three buffers, but PNN-associated CSPGs remained and were only removed by 6 m urea. Most of the CSPGs were extracted to some degree with all the buffers, with neurocan, brevican, aggrecan, and versican particularly associated with the stable urea-extractable PNNs. The CSPGs in stable complexes only extractable in urea buffer are found from postnatal day 7-14 coinciding with PNN formation. Disaccharide composition analysis indicated a different glycosaminoglycan (GAG) composition for PGs strongly associated with extracellular matrix (ECM). For CS/dermatan sulfate (DS)-GAG the content of nonsulfated, 6-O-sulfated, 2,6-O-disulfated, and 4,6-O-disulfated disaccharides were higher and for heparan sulfate (HS)-GAG, the content of 6-O-sulfated, 2-N-, 6-O-disulfated, 2-O-, 2-N-disulfated, and 2-O-, 2-N-, 6-O-trisulfated disaccharides were higher in urea extract compared with other buffer extracts. Digestions with chondroitinase ABC and hyaluronidase indicated that aggrecan, versican, neurocan, brevican, and phosphacan are retained in PNNs through binding to hyaluronan (HA). A comparison of the brain and spinal cord ECM with respect to CSPGs indicated that the PNNs in both parts of the CNS have the same composition.     

Extracellular matrix of the central nervous system: from neglect to challenge

The basic concept, that specialized extracellular matrices rich in hyaluronan, chondroitin sulfate proteoglycans (aggrecan, versican, neurocan, brevican, phosphacan), link proteins and tenascins (Tn-R, Tn-C) can regulate cellular migration and axonal growth and thus, actively participate in the development and maturation of the nervous system, has in recent years gained rapidly expanding experimental support. The swift assembly and remodeling of these matrices have been associated with axonal guidance functions in the periphery and with the structural stabilization of myelinated fiber tracts and synaptic contacts in the maturating central nervous system. Particular interest has been focused on the putative role of chondroitin sulfate proteoglycans in suppressing central nervous system regeneration after lesions. The axon growth inhibitory properties of several of these chondroitin sulfate proteoglycans in vitro, and the partial recovery of structural plasticity in lesioned animals treated with chondroitin sulfate degrading enzymes in vivo have significantly contributed to the increased awareness of this long time neglected structure.     

Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

The dual role of the extracellular matrix in synaptic plasticity and homeostasis

Recent studies have deepened our understanding of multiple mechanisms by which extracellular matrix (ECM) molecules regulate various aspects of synaptic plasticity and have strengthened a link between the ECM and learning and memory. New findings also support the view that the ECM is important for homeostatic processes, such as scaling of synaptic responses, metaplasticity and stabilization of synaptic connectivity. Activity-dependent modification of the ECM affects the formation of dendritic filopodia and the growth of dendritic spines. Thus, the ECM has a dual role as a promoter of structural and functional plasticity and as a degradable stabilizer of neural microcircuits. Both of these aspects are likely to be important for mental health.     

Hyaluronan is an abundant constituent of the extracellular matrix of Xenopus embryos

The spatiotemporal distribution of hyaluronan (HA), a major constituent of the vertebrate extracellular matrix, was analyzed during early embryonic development of Xenopus laevis. This polysaccharide is abundantly present in ventricular structures such as the blastocoel, the archenteron as well as later on in the hepatic cavity, the brain ventricles and the developing heart. At the blastula stage, HA was detected in the extracellular matrix of the ecto- and mesodermal primordia. Shortly before gastrulation, it becomes enriched at the basal site of the superficial cell layer of the ectoderm. During gastrulation, enhanced synthesis of HA takes place in the involuting marginal zone, shortly before invagination starts, hence, resulting in a torus-like deposition in the deep layer of the equatorial mesodermal primordium. After gastrulation, HA appears to accumulate within the extracellular matrix demarcating the primary germ layers. During tailbud stages, it is found highly enriched in many mesodermal derivatives, e.g., in mesenchyme, the heart, precordal cartilage and the lung primordia. Furthermore, extracellular matrix of the ventral mesodermal cell layer in the trunk region and the immediate proximity of blood vessels contain high amounts of HA.     

Extracellular matrix alterations in brains lacking four of its components

The organization of the brain extracellular matrix appears to be based on aggregates of hyaluronan and proteoglycans, connected by oligomeric glycoproteins. Mild phenotypical consequences were reported from several mouse strains lacking components of this matrix such as neurocan, brevican, tenascin-R, and tenascin-C. To further challenge the flexibility of the extracellular matrix network of the brain, mice lacking all four brain extracellular matrix molecules were generated, which were found to be viable and fertile. Analysis of the brains of 1-month-old quadruple KO mice revealed increased protein levels of fibulin-1 and fibulin-2. Histochemical analysis showed an unusual parenchymal deposition of these fibulins. The quadruple KO mice also displayed obvious changes in the pattern of deposition of hyaluronan. Further, an almost quadruple knockout like extracellular environment was noticed in the brains of triple knockout mice lacking both tenascins and brevican, since these brains had strongly reduced levels of neurocan.     

Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan

Perineuronal nets (PNs) in the brains of tenascin-R-deficient (tn-r^−/−) mice develop in temporal concordance with those of wild-type (tn-r^+/+) mice. However, the histological appearance of PNs is abnormal in adult tn-r^−/− mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r^−/− mice and whether the structure of PNs could be normalized. In tn-r^−/− cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r^+/+ mice. Experiments to normalize the structure of PNs in tn-r^−/− organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r^+/+ and tn-r^−/− brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.     

Differential expression of several molecules of the extracellular matrix in functionally and developmentally distinct regions of rat spinal cord

We have examined the regional distribution of several chondroitin sulfate proteoglycans (neurocan, brevican, versican, aggrecan, phosphacan), of their glycosaminoglycan moieties, and of tenascin-R in the spinal cord of adult rat. The relationships of these molecules with glial and neuronal populations, identified with appropriate markers, were investigated by using multiple fluorescence labeling combined with confocal microscopy. The results showed that the distribution of the examined molecules was similar at all spinal cord levels but displayed area-specific differences along the dorso-ventral axis, delimiting functionally and developmentally distinct areas. In the gray matter, laminae I and II lacked perineuronal nets (PNNs) of extracellular matrix and contained low levels of chondroitin sulfate glycosaminoglycans (CS-GAGs), brevican, and tenascin-R, possibly favoring the maintenance of local neuroplastic properties. Conversely, CS-GAGs, brevican, and phosphacan were abundant, with numerous thick PNNs, in laminae III-VIII and X. Motor neurons (lamina IX) were surrounded by PNNs that contained all molecules investigated but displayed various amounts of CS-GAGs. Double-labeling experiments showed that the presence of PNNs could not be unequivocally related to specific classes of neurons, such as motor neurons or interneurons identified by their expression of calcium-binding proteins (parvalbumin, calbindin, calretinin). However, a good correlation was found between PNNs rich in CS-GAGs and the neuronal expression of the Kv3.1b subunit of the potassium channel, a marker of fast-firing neurons. This observation confirms the correlation between the electrophysiological properties of these neurons and the specific composition of their microenvironment.     

Cartilage link protein 1 (Crtl1), an extracellular matrix component playing an important role in heart development

To expand our insight into cardiac development, a comparative DNA microarray analysis was performed using tissues from the atrioventricular junction (AVJ) and ventricular chambers of mouse hearts at embryonic day (ED) 10.5-11.0. This comparison revealed differential expression of approximately 200 genes, including cartilage link protein 1 (Crtl1). Crtl1 stabilizes the interaction between hyaluronan (HA) and versican, two extracellular matrix components essential for cardiac development. Immunohistochemical studies showed that, initially, Crtl1, versican, and HA are co-expressed in the endocardial lining of the heart, and in the endocardially derived mesenchyme of the AVJ and outflow tract (OFT). At later stages, this co-expression becomes restricted to discrete populations of endocardially derived mesenchyme. Histological analysis of the Crtl1-deficient mouse revealed a spectrum of cardiac malformations, including AV septal and myocardial defects, while expression studies showed a significant reduction in versican levels. Subsequent analysis of the hdf mouse, which carries an insertional mutation in the versican gene (CSPG2), demonstrated that haploinsufficient versican mice display septal defects resembling those seen in Crtl1(-/-) embryos, suggesting that reduced versican expression may contribute to a subset of the cardiac abnormalities observed in the Crtl1(-/-) mouse. Combined, these findings establish an important role for Crtl1 in heart development.     

Chondroitin sulfate,a major component of the perineuronal net,elicits inward currents,cell depolarization,and calcium transients by acting on AMPA and kainate receptors of hippocampal neurons

Chondroitin sulfate (CS) proteoglycans (CSPGs) are the most abundant PGs of the brain extracellular matrix (ECM). Free CS could be released during ECM degradation and exert physiological functions; thus, we aimed to investigate the effects of CS on voltage‐ and current‐clamped rat embryo hippocampal neurons in primary cultures. We found that CS elicited a whole‐cell Na⁺‐dependent inward current (I_CS) that produced drastic cell depolarization, and a cytosolic calcium transient ([Ca²⁺]_c). Those effects were similar to those elicited by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) and kainate, were completely blocked by NBQX and CNQX, were partially blocked by GYKI, and were unaffected by MK801 and D‐APV. Furthermore, I_CS and AMPA currents were similarly potentiated by cyclothiazide, a positive allosteric modulator of AMPA receptors. Because CSPGs have been attributed Ca²⁺ ‐dependent roles, such as neural network development, axon pathfinding, plasticity and regeneration after CNS injury, CS action after ECM degradation could be contributing to the mediation of these effects through its interaction with AMPA and kainate receptors.     

Hyaluronan-based extracellular matrix under conditions of homeostatic plasticity

Neuronal networks are balanced by mechanisms of homeostatic plasticity, which adjusts synaptic strength via molecular and morphological changes in the pre- and post-synapse. Here, we wondered whether the hyaluronic acid-based extracellular matrix (ECM) of the brain is involved in mechanisms of homeostatic plasticity. We hypothesized that the ECM, being rich in chondroitin sulfate proteoglycans such as brevican, which are suggested to stabilize synapses by their inhibitory effect on structural plasticity, must be remodelled to allow for structural and molecular changes during conditions of homeostatic plasticity. We found a high abundance of cleaved brevican fragments throughout the hippocampus and cortex and in neuronal cultures, with the strongest labelling in perineuronal nets on parvalbumin-positive interneurons. Using an antibody specific for a brevican fragment cleaved by the matrix metalloprotease ADAMTS4, we identified the enzyme as the main brevican-processing protease. Interestingly, we found ADAMTS4 largely associated with synapses. After inducing homeostatic plasticity in neuronal cell cultures by prolonged network inactivation, we found increased brevican processing at inhibitory as well as excitatory synapses, which is in line with the ADAMTS4 subcellular localization. Thus, the ECM is remodelled in conditions of homeostatic plasticity, which may liberate synapses to allow for a higher degree of structural plasticity.     

Expression of versican in relation to chondrogenesis-related extracellular matrix components in canine mammary tumors

Versican plays a role in tumor cell proliferation and adhesion and may also regulate cell phenotype. Furthermore, it is one of the pivotal proteoglycans in mesenchymal condensation during prechondrogenesis. We have previously demonstrated accumulation of versican protein in myoepithelial-like spindle cell proliferations and myxoid tissues of complex and mixed mammary tumors of dogs. The objective of this study was to investigate whether the high expression of versican relates to prechondrogenesis in these tissues. Therefore, we aimed to identify cartilage markers, such as collagen type II and aggrecan both at mRNA and protein level in relation to versican. The neopitope of chondoitin-6-sulphate (3B3) known to be generated in developing cartilage has been investigated by immunohistochemisty and a panel of antibodies were used to characterize the phenotype of cells that are involved in cartilage formation. In addition, co-localization of versican with hyaluronan and link protein was studied. RT-PCR revealed upregulation of genes of versican, collagen type II and aggrecan in neoplastic tissues, especially in complex and mixed tumors. Immunohistochemistry showed the expression of cartilage biomarkers not only in the cartilagenous tissues of mixed tumors, but also in myoepitheliomas and in the myoepithelial-like cell proliferations and myxoid areas of complex and mixed tumors. The results show the cartilagenous differentiation of complex tumors and myoepitheliomas and indicate that the myxoid tissues and myoepithelial-like cell proliferations are the precursor tissues of the ectopic cartilage in mixed tumors. Furthermore, we suggest that cartilage formation in canine mammary tumors is a result of (myo)epithelial to mesenchymal transition.     

Sonic hedgehog is a regulator of extracellular glutamate levels and epilepsy

Sonic hedgehog (Shh), both as a mitogen and as a morphogen, plays an important role in cell proliferation and differentiation during early development. Here, we show that Shh inhibits glutamate transporter activities in neurons, rapidly enhances extracellular glutamate levels, and affects the development of epilepsy. Shh is quickly released in response to epileptic, but not physiological, stimuli. Inhibition of neuronal glutamate transporters by Shh depends on heterotrimeric G protein subunit Gα_i and enhances extracellular glutamate levels. Inhibiting Shh signaling greatly reduces epileptiform activities in both cell cultures and hippocampal slices. Moreover, pharmacological or genetic inhibition of Shh signaling markedly suppresses epileptic phenotypes in kindling or pilocarpine models. Our results suggest that Shh contributes to the development of epilepsy and suppression of its signaling prevents the development of the disease. Thus, Shh can act as a modulator of neuronal activity, rapidly regulating glutamate levels and promoting epilepsy.     

Casting a net on dendritic spines: the extracellular matrix and its receptors

Dendritic spines are dynamic structures that accommodate the majority of excitatory synapses in the brain and are influenced by extracellular signals from presynaptic neurons, glial cells, and the extracellular matrix (ECM). The ECM surrounds dendritic spines and extends into the synaptic cleft, maintaining synapse integrity as well as mediating trans-synaptic communications between neurons. Several scaffolding proteins and glycans that compose the ECM form a lattice-like network, which serves as an attractive ground for various secreted glycoproteins, lectins, growth factors, and enzymes. ECM components can control dendritic spines through the interactions with their specific receptors or by influencing the functions of other synaptic proteins. In this review, we focus on ECM components and their receptors that regulate dendritic spine development and plasticity in the normal and diseased brain.     

Unique distribution of the extracellular matrix component thrombospondin in the developing mouse embryo

Immunocytochemical localization of thrombospondin (TSP), a trimeric glycoprotein constituent of extracellular matrices, produced striking regional and temporal patterns of distribution in the developing mouse embryo. TSP was present in many basement membranes, surrounded epithelial cells, and was associated with peripheral nerve outgrowth. During organogenesis, TSP was also found on the surface of myoblasts and chondroblasts, and TSP was differentially deposited in cortical layers. With differentiation of chondrocytes and myotubes immunoreactivity was decreased, and differential cortical staining was lost. Presence of TSP was associated with morphogenetic processes of proliferation, migration, and intercellular adhesion.     

Identification of a specialized extracellular matrix component in Drosophila imaginal discs

We have generated a monoclonal antibody that recognizes a major component of a specialized extracellular matrix in Drosophila imaginal discs. In mature larvae, antibody binding is observed almost exclusively on imaginal discs. On the basal surface of the thoracic discs, the antigen is localized to particular regions of the epithelium, and ultrastructural studies indicate that the antigen is found in a fibrous network secreted between the cells and the basal lamina. The localized expression indicates that the matrix is not simply related to disc differentiation, as all regions of the columnar disc epithelium are determined to secrete adult cuticle. A correlation of the antigen distribution with known developmental events leads us to propose that the antigen-containing network provides an extensible matrix for the rapid elongation of the disc epithelium during evagination; consistent with this, the antigen is a component of the matrix between the dorsal and ventral surfaces of the evaginated wing pouch. The antigen is very large (greater than 5 X 10(5) Da), can be labeled metabolically with methionine and sulfate, and is digested by chondroitinase ABC; these biochemical characteristics indicate that the antigen is a proteoglycan.     

Calcium homeostasis and modulation of synaptic plasticity in the aged brain

The level of intracellular Ca2+ plays a central role in normal and pathological signaling within and between neurons. These processes involve a cascade of events for locally raising and lowering cytosolic Ca2+. As the mechanisms for age-related alteration in Ca2+ dysregulation have been illuminated, hypotheses concerning Ca2+ homeostasis and brain aging have been modified. The idea that senescence is due to pervasive cell loss associated with elevated resting Ca2+ has been replaced by concepts concerning changes in local Ca2+ levels associated with neural activity. This article reviews evidence for a shift in the sources of intracellular Ca2+ characterized by a diminished role for N-methyl-D-aspartate receptors and an increased role for intracellular stores and voltage-dependent Ca2+ channels. Physiological and biological models are outlined, which relate a shift in Ca2+ regulation with changes in cell excitability and synaptic plasticity, resulting in a functional lesion of the hippocampus.     

Tenascin is a cytoadhesive extracellular matrix component of the human hematopoietic microenvironment

Tenascin is a large extracellular matrix (ECM) glycoprotein found in restricted tissue locations in the adult organism. It is copiously synthesized in regenerative organs or regenerating tissues and by certain tumors. We have analyzed the expression of tenascin in human long term bone marrow cultures as well as in cryostat sections of native bone marrow and found it strongly expressed by the stromal cells of the microenvironment. Two different protein subunits of 280 and 220 kD were detected by immunoblotting. These two forms are derived most likely from two different mRNA splice variants of 6 and 8 kb detected by Northern blotting. The in vivo analysis of cryostat sections showed a codistribution with other ECM molecules such as fibronectin and collagen type III in the microenvironment surrounding the maturing hematopoietic cells. Using two independent cell adhesion assays tenascin could be shown to function as a cytoadhesive molecule for hematopoietic cells. These data suggest a direct involvement of tenascin in the retention of hematopoietic progenitor cells in the stroma.     

Z-form extracellular DNA is a structural component of the bacterial biofilm matrix

1. Download : Download high-res image (254KB)
2. Download : Download full-size image