Unique features of extracellular matrix in the mouse medial nucleus of trapezoid body – Implications for physiological functions
Highlights
► We studied the ECM of PNs in the MNTB of the auditory system in detail. ► Every MNTB neuron is surrounded by a remarkably prominent PN. ► We discovered a new distinct structural phenotype of PNs in MNTB. ► The Proteoglycans aggrecan and brevican show unique/differing spatial positions. ► Our data support the hypothesis that PNs affect physiological functions of neurons.
Introduction
Throughout the CNS specific subpopulations of both projection- and of interneurons are surrounded by extracellular matrix (ECM) components classified as perineuronal nets (PNs). Such PNs are associated with neural somata, dendrites and proximal axonal segments forming distinctly organized and for the respective neuron-type characteristic lattice-like structures. Despite their prominence and their specific manifestation in the CNS, the precise functional roles of PNs have remained elusive. PNs are hypothesized to stabilize synapses (Hockfield et al., 1990) and to modulate synaptic plasticity (Pizzorusso et al., 2002, Frischknecht et al., 2009, Carulli et al., 2010). While other studies have theorized that PNs influence the outgrowth of axons and/or establish the extracellular ionic milieu to maintain fast discharge properties of GABAergic interneurons (Brückner et al., 1993, Brückner et al., 1996, Hobohm et al., 1998, Härtig et al., 1999).
One of the key challenges to understanding PN functions is their inherent microheterogeneity. For example in the cerebral cortex, where they have been most intensively studied, they are expressed on only a small subset of neurons and are highly molecularly complex and heterogeneous structures (Matthews et al., 2002, Zimmermann and Dours-Zimmermann, 2008, Kind et al., 2012). Furthermore the synaptic connectivity of the net-bearing neurons in the cortex is equally complex. A more ideal model to study PN function would be a system in which all neurons are ensheathed with relatively homogenous PNs, the neurons are accessible to a detailed pre- and postsynaptic electrophysiological characterization, and a system that is experimentally manipulatable.
In this manuscript we identify the principal neurons of the medial nucleus of the trapezoid body (MNTB) as such a model system. The MNTB is the vital structure for sound localization in the auditory brainstem (Guinan and Li, 1990, Middlebrooks and Green, 1991, Oertel, 1999, Grothe et al., 2010). Every principal cell of MNTB is contacted by a very large presynaptic terminal, the calyx of Held (von Gersdorff and Borst, 2002, Hoffpauir et al., 2006). The terminals, which enwrap most of the MNTB cell somata forming multiple active transmission sites, derived from axons of globular bushy cells ascending from the cochlear nucleus (Spirou et al., 1990). The calyx of Held is a huge glutamatergic synapse (Grandes and Streit, 1989, Cant, 1991) that conveys fast and reliable signal transmission (von Gersdorff and Borst, 2002, Schneggenburger and Forsythe, 2006). One striking feature of this synapse – that makes it accessible for detailed physiological investigations – is the fact that each afferent fiber innervates only one postsynaptic principal cell (rat: Sätzler et al., 2002, Taschenberger et al., 2002, cat: Morest, 1968, Smith et al., 1991). Studies in a multitude of different species showed prominent expression of PNs as a typical feature of MNTB principal cells (mouse: Bekku et al., 2003, rat: Bertolotto et al., 1996, Friauf, 2000, Härtig et al., 2001, gerbil: Lurie et al., 1997, dog: Atoji et al., 1989, rhesus: Hilbig et al., 2007).
Throughout the brain, one of the major ECM constituents of PNs is chondroitin sulfate proteoglycans (CSPGs), which belong to the lectican-family of aggregating proteoglycans (Zaremba et al., 1989, Brückner et al., 1994, Brückner et al., 1998, Asher et al., 1995, Bertolotto et al., 1996, Lander et al., 1997, Matsui et al., 1998, Hagihara et al., 1999, Yamaguchi, 2000). The proteoglycans (aggrecan, brevican, neurocan and versican, Ruoslahti, 1996) bind hyaluronan, which is stabilized by link-proteins to form a quaternary complex with tenascin-R (Asher et al., 1995, Brückner et al., 2000, Yamaguchi, 2000, Carulli et al., 2007, Galtrey and Fawcett, 2007). The biological activity of CSPGs might be dependent on chondroitin sulfate glycosaminoglycans (GAGs) (Yamaguchi, 2000, Pizzorusso et al., 2002). The GAGs which are covalently linked to central domain of the core protein, but the number of attachment sites strongly differs between the lecticans (Yamaguchi, 2000).
Presently, little is known about the precise physiological functions of PNs. In the cortex, PN-wearing interneurons are often associated with the calcium-binding protein parvalbumin (PV) and the potassium channel subunit Kv3.1b (Härtig et al., 1995, Härtig et al., 1999). In the brainstem PNs are widely expressed and especially associated with fast-spiking neurons (Arai et al., 1991, Caicedo et al., 1996, Lohmann and Friauf, 1996, Dodson et al., 2003, Elezgarai et al., 2003). In this regard the calyx of Held–MNTB principal neuron complex shows one of the fastest synaptic transmission pathways in the mammalian brain (von Gersdorff and Borst, 2002).
Interestingly previous work demonstrated the expression of PNs and of certain matrix components around principal neurons of the mouse MNTB (Bekku et al., 2003). The electrophysiology of these neurons is well described both in vitro and in vivo (Forsythe and Barnes-Davies, 1993, Borst et al., 1995, Schneggenburger et al., 2002, von Gersdorff and Borst, 2002, Kopp-Scheinpflug et al., 2008, Sonntag et al., 2009, Sonntag et al., 2011). However, a detailed molecular dissection of the PN components and structures covering MNTB neurons in mice is still lacking.
The present study provides detailed immunohistochemical and electron microscopic analysis of the ECM organization and structural characteristics of PN-comprising proteins of the mouse MNTB. Our work details the expression of PN components and their distribution pattern in different subregions of the physiologically well-characterized MNTB. Electron microscopy was used to precisely identify the ultrastructural localization of PN-components throughout the MNTB and their close association with distinct components of the calyces of Held and with glial cells ensheathing the principal cells.
Section snippets
Animals
Data were collected from C57BL/6J mice at the age of 2 months. Animals were maintained on a 12h/12 h light–dark-cycle under conditions of constant temperature (22 °C). They had free access to food and water. The mice were acquired from the Medical Experimental Center of the Medical Faculty of the University of Leipzig. All experiments were approved by the institutional animal care and use committee and the Saxonian District Government, Leipzig.
Cytochemistry
The animals were deeply anesthetized with CO2 and
Results
Immunostaining of the mouse MNTB revealed that virtually all major PN-components are detected on MNTB principal neurons. While in most areas of the CNS PN-bearing neurons are spread throughout the respective nuclei, in the MNTB every principal cell is associated with a PN, building a dense meshwork of intermingled ECM structures.
Discussion
The results of the present study indicate that most of the ECM molecules investigated are strongly expressed in the MNTB of adult mice and display distinct patterns in close association with the principal neurons. Virtually every neuron of the mice MNTB is surrounded by a PN that exhibits unique and precise localization with subcellular neuronal structures. As a whole, the PNs in MNTB present are unique and structurally distinct, possibly related to the specific morphology of the excitatory
Conclusion
Here, we describe the specialized ECM of perineuronal nets in the MNTB. The PNs in the MNTB are remarkably prominent and surround virtually every principal neuron. The specialized ECM in the MNTB represents a new structural phenotype within the brain. This is most likely caused by the huge, specialized excitatory presynaptic terminal, the calyx of Held, which endorse the assumption of a selective adaptation of the ECM to a highly specialized system.
Most strikingly, we could reveal a so far
Acknowledgements
This work was supported by the German Research Foundation MO 2249/2-1 (M.M) within the PP 1608, GRK 1097 “INTERNEURO” (T.A., M.M.), the EU-Project “Neuropro” (Grant Agreement No. 223077), the Alzheimer Forschungsinitiative e.V. (AFI #1186, M.M.) and the MD/PhD program at the University of Leipzig (M.M.).
References (118)
- et al.
Immunocytochemical evidence for inhibitory and disinhibitory circuits in the superior olive
Hear Res
(1990) - et al.
Molecular cloning of Bral2, a novel brain-specific link protein, and immunohistochemical colocalization with brevican in perineuronal nets
Mol Cell Neurosci
(2003) - et al.
Clearance of glutamate inside the synapse and beyond
Curr Opin Neurobiol
(1999) - et al.
Neurodegenerative and morphogenic changes in a mouse model of temporal lobe epilepsy do not depend on the expression of the calcium-binding proteins parvalbumin, calbindin, or calretinin
Neuroscience
(2000) - et al.
Cortical areas are revealed by distribution patterns of proteoglycan components and parvalbumin in the Mongolian gerbil and rat
Brain Res
(1994) - et al.
Region and lamina-specific distribution of extracellular matrix proteoglycans, hyaluronan and tenascin-R in the mouse hippocampal formation
J Chem Neuroanat
(2003) - et al.
Aggrecan-based extracellular matrix is an integral part of the human basal ganglia circuit
Neuroscience
(2008) - et al.
Changes in sialic acid expression in the lung during intrauterine development of the human fetus
Acta Histochem
(2002) - et al.
Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis
Neuron
(2000) - et al.
Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body
Neuroscience
(2003)
The role of chondroitin sulfate proteoglycans in regeneration and plasticity in the central nervous system
Brain Res Rev
Perineuronal net formation and structure in aggrecan knockout mice
Neuroscience
Signal processing in brainstem auditory neurons which receive giant endings (calyces of Held) in the medial nucleus of the trapezoid body of the cat
Hear Res
Allocation of perineuronal nets and parvalbumin-, calbindin-D28k- and glutamic acid decarboxylase-immunoreactivity in the amygdala of the rhesus monkey
Brain Res
Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations
Brain Res
Perineuronal nets in the rat medial nucleus of the trapezoid body surround neurons immunoreactive for various amino acids, calcium-binding proteins and the potassium channel subunit Kv3.1b
Brain Res
Low expression of extracellular matrix components in rat brain stem regions containing modulatory aminergic neurons
J Chem Neuroanat
Binding of calcium to glycosaminoglycans: an equilibrium dialysis study
Arch Biochem Biophys
Intracranial injury acutely induces the expression of the secreted isoform of the CNS-specific hyaluronan-binding protein BEHAB/brevican
Exp Neurol
Brevican-containing perineuronal nets of extracellular matrix in dissociated hippocampal primary cultures
Mol Cell Neurosci
The medial nucleus of the trapezoid body: comparative physiology
Neuroscience
Immunohistochemical localization of gamma-aminobutyric acid- and aspartate-containing neurons in the guinea pig superior olivary complex
Hear Res
Extracellular space parameters in the rat neocortex and subcortical white matter during postnatal development determined by diffusion analysis
Neuroscience
The cloning of a receptor-type protein tyrosine phosphatase expressed in the central nervous system
J Biol Chem
Occurrence of a N-terminal proteolytic fragment of neurocan, not a C-terminal half, in a perineuronal net in the adult rat cerebrum
Brain Res
Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member
J Biol Chem
Differential regulation of expression of hyaluronan-binding proteoglycans in developing brain: aggrecan, versican, neurocan, and brevican
Biochem Biophys Res Commun
Chondroitin sulfate proteoglycan-based extracellular matrix in chicken (Gallus domesticus) brain
Brain Res
The collateral system of the medial nucleus of the trapezoid body of the cat, its neuronal architecture and relation to the olivo-cochlear bundle
Brain Res
Vesicle pools and short-term synaptic depression: lessons from a large synapse
Trends Neurosci
Parvalbumin-immunoreactive neurons in the cortex in Pick’s disease
J Neurol
Astrocyte-induced modulation of synaptic transmission
Can J Physiol Pharmacol
On the existence of a cartilage-like proteoglycan and link proteins in the central nervous system
Glia
Extracellular matrix of the superior olivary nuclei in the dog
J Neurocytol
Proteoglycans in the developing brain: new conceptual insights for old proteins
Physiol Rev
Characterization of the rhesus monkey superior olivary complex by calcium binding proteins and synaptophysin
J Anat
Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS
J Neurochem
Bral1: its role in diffusion barrier formation and conduction velocity in the CNS
J Neurosci
Immunohistochemical mapping of perineuronal nets containing chondroitin unsulfated proteoglycan in the rat central nervous system
Cell Tissue Res
Differential expression of HNK-1 and p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro
J Comp Neurol
Pre- and postsynaptic whole-cell recordings in the medial nucleus of the trapezoid body of the rat
J Physiol (Lond)
Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain
Glia
Extracellular matrix organization in various regions of rat brain grey matter
J Neurocytol
Acute and long-lasting changes in extracellular-matrix chondroitin–sulphate proteoglycans induced by injection of chondroitinase ABC in the adult rat brain
Exp Brain Res
Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R
J Comp Neurol
Distribution of calcium-binding protein immunoreactivities in the guinea pig auditory brainstem
Anat Embryol
Projections to the lateral and medial superior olivary nuclei from the spherical and globular bushy cells of the anteroventral cochlear nucleus
Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components
J Comp Neurol
Upregulation of aggrecan, link protein 1, and hyaluronan synthases during formation of perineuronal nets in the rat cerebellum
J Comp Neurol
Animals lacking link protein have attenuated perineuronal nets and persistent plasticity
Brain
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