Unique features of extracellular matrix in the mouse medial nucleus of trapezoid body – Implications for physiological functions

Abstract

The medial nucleus of the trapezoid body (MNTB) is a vital structure of sound localization circuits in the auditory brainstem. Each principal cell of MNTB is contacted by a very large presynaptic glutamatergic terminal, the calyx of Held. The MNTB principal cells themselves are surrounded by extracellular matrix components forming prominent perineuronal nets (PNs). Throughout the CNS, PNs, which form lattice-like structures around the somata and proximal dendrites, are associated with distinct types of neurons. PNs are highly enriched in hyaluronan and chondroitin sulfate proteoglycans therefore providing a charged surface structure surrounding the cell body and proximal neurites of these neurons. The localization and composition of PNs have lead investigators to a number of hypotheses about their functions including: creating a specific extracellular ionic milieu around these neurons, stabilizing synapses, and influencing the outgrowth of axons. However, presently the precise functions of PNs are still quite unclear primarily due to the lack of an ideal experimental model system that is highly enriched in PNs and in which the synaptic transmission properties can be precisely measured. The MNTB principal cells could offer such a model, since they have been extensively characterized electrophysiologically. However, extracellular matrix (ECM) in these neurons has not yet been precisely detailed. The present study gives a detailed examination of the ECM organization and structural differences in PNs of the mouse MNTB. The different PN components and their distribution pattern are scrutinized throughout the MNTB. The data are complemented by electron microscopic investigations of the unique ultrastructural localization of PN-components and their interrelation with distinct pre- and postsynaptic MNTB cell structures. Therefore, we believe this work identifies the MNTB as an ideal system for studying PN function.

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.