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date: 26 May 2019

Perineuronal Nets in the Superior Olivary Complex: Development, Function, and Plasticity

Abstract and Keywords

This chapter addresses perineuronal nets in the superior olivary complex, a collection of nuclei in the auditory brainstem that are involved in the processing of sound source location. Perineuronal nets, a specific form of extracellular matrix, are believed to control synaptic plasticity. They surround neuronal somata and dendrites of specific types of neurons, among which are many neurons of the superior olivary complex. The chapter describes the distribution of perineuronal nets in the superior olivary complex, focusing on controversial results and discussing underlying reasons. In addition, it considers the development of perineuronal nets and highlights differences between the main components of perineuronal nets, including the proteoglycans aggrecan, brevican, and neurocan. Finally, it introduces current concepts on the function of perineuronal nets that are specifically based on experimental data collected in the superior olivary complex and point to a contribution of perineuronal nets to synaptic transmission and neuronal excitability.

Keywords: superior olivary complex, auditory brainstem, perineuronal nets, extracellular matrix, development, aggrecan, brevican, neurocan, synaptic transmission

Introduction

The central nervous system (CNS) has nowadays to be considered as a functional network of neurons, glia, and extracellular matrix (ECM) molecules. Compared to neurons and glia, the ECM has received less attention despite the fact that the ECM constitutes up to 20% of brain volume (Bignami, Hosley, & Dahl, 1993; Lau, Cua, Keough, Haylock-Jacobs, & Yong, 2013). Perhaps the most interesting and complex functions of the CNS are the ability to encode new information (learning) and to store this information (memory). During learning a high degree of plasticity is required, while the storage of memory requires a high degree of stability (Alberini, 2011). The identification of molecules and mechanisms that modulate these processes are therefore key to the understanding of CNS function. Recent studies suggest that the neural ECM and especially the perineuronal nets (PNs) are important regulators of this plasticity (for review see Takesian & Hensch, 2013; Wang & Fawcett, 2012).

PNs are composed of different ECM molecules that form a lattice-like sheath that surrounds the somata and proximal dendrites of particular neurons. Different chondroitin sulfate proteoglycans (CSPGs) of the lectican family (e.g., aggrecan, brevican, and neurocan) bind via their G1 domains to hyaluronan, which is constantly secreted by the neuronal membrane- anchored hyaluronan synthase. This hyaluronan–CSPG bond is stabilized via hyaluronan and proteoglycan link proteins (HAPLN 1–4). Finally tenascin-R is bound to the CSPGs, cross-linking the complexes of hyaluronan, HAPLN, and CSPGs and stabilizing PNs into a quaternary complex (Chiquet-Ehrismann & Tucker, 2011; Morawski, Bruckner, Arendt, & Matthews, 2012; Yamaguchi, 2000). A special feature of the CSPGs is the numerous attached glycosaminoglycan chains (GAGs), creating the chondroitin sulfate (CS) rich region which is responsible for the specific hydrodynamic properties of strongly hydrated polyanionic ECM (Morawski et al., 2015). Each glycosaminoglycan chain consists of repeated pairs of glucuronic acid and N-acetylgalactosamine. The latter can be sulfated in position 6, 4, or even be unsulfated leading to hundreds of potential glycan combinations (Cummings, 2009); for review see (Caterson, 2012).

The systematic investigation of PNs in the CNS of different species under physiological and pathological conditions has revealed possible functions of PNs in brain development, neuronal signal processing, synaptic plasticity and neuroprotection (Bruckner et al., 2000; Matthews et al., 2002; Morawski, Bruckner, Jager et al., 2012; Morawski, Bruckner, Riederer, Bruckner, & Arendt, 2004; for reviews see Takesian & Hensch, 2013; Wang & Fawcett, 2012; Zimmermann & Dours-Zimmermann, 2008). It was even hypothesized that PNs are part of the cortical long-term memory storage system (Tsien, 2013). Still, to date a specific function of the PN remains elusive. However, there is increasing evidence that the different CSPGs may serve distinct functions in the PN as a structural entity (Avram, Shaposhnikov, Buiu, & Mernea, 2014; Balmer, 2016; Blosa et al., 2015; Favuzzi et al., 2017; Suttkus et al., 2014).

PNs are neither evenly distributed in the CNS nor generally present on most types of neurons. They only appear on specialized subsets of neurons. In the cortex, around 60–80% of these neurons belong to the group of parvalbumin-positive GABAergic interneurons, which are characterized by high-frequency action potential activity (i.e., fast-spiking neurons”) and the expression of the high-voltage gated potassium channel subunit Kv3.1 (Hartig et al., 1999; Suttkus et al., 2014). While in most cortical areas only a minor proportion of neurons are surrounded by PNs (Bruckner et al., 2000; Morawski, Pavlica et al., 2010; Seeger, Brauer, Hartig, & Bruckner, 1994; Suttkus et al., 2014), the nuclei of the central auditory system, especially those of the superior olivary complex (SOC), have exceptionally high proportions of PN-bearing neurons. As the functional role of PNs might best be studied in brain areas rich in these ECM components, the SOC has gained increasing attention in the last years and has become a model system to study the cellular and systemic function of PNs and their subcomponents in the brain (Sonntag, Blosa, Schmidt, Rubsamen, & Morawski, 2015).

This chapter addresses the cell-specific distribution of PNs in the nuclei of the SOC, considering species-specific differences and discussing controversial results based on the application of distinct markers and antibodies used for the detection of PNs. It further describes the development of PNs in the SOC, pointing out differences in the developmental profile and expression pattern of the PN subcomponents. The chapter concludes with current concepts on the function of PNs within the SOC, focusing on the impact of PNs on synaptic transmission, synaptic plasticity, and neuronal excitability.

Distribution of Perineuronal Nets in the Superior Olivary Complex

The SOC is composed of several nuclei that contribute to the processing of sound source location. Among those are the medial, lateral, and ventral nucleus of the trapezoid body (MNTB, LNTB, and VNTB), the lateral and medial superior olive (LSO and MSO), and the superior paraolivary nucleus (SPN). The distribution of PNs within these nuclei is well described across many mammalian species.

Medial Nucleus of the Trapezoid Body

Within the SOC, the MNTB stands out because virtually all of its principal neurons are surrounded by a PN. The high prevalence of PNs in the MNTB is consistent across many different species (gerbil: Lurie, Pasic, Hockfield, & Rubel, 1997; Sonntag, Blosa, Schmidt, Rubsamen, & Morawski, 2015; rat: Friauf, 2000; Hartig et al., 2001; Myers, Ray, & Kulesza, 2012; Seeger, Brauer, Hartig, & Bruckner, 1994; mouse: Balmer, 2016; Blosa et al., 2013; Blosa et al., 2015; Kolson et al., 2016; tenrec: Morawski, Bruckner, Jager, Seeger, Kunzle et al., 2010; dog: Fech, Calderon-Garciduenas, & Kulesza, 2017; rhesus monkey: Hilbig, Nowack, Boeckler, Bidmon, & Zilles, 2007). PNs in the MNTB densely cover the principal neurons’ cell bodies but rarely extend into proximal dendrites (Blosa et al., 2013; Hartig et al., 2001; Lurie, Pasic, Hockfield, & Rubel, 1997). They form cotton wool-like structures (Blosa et al., 2013; Hartig et al., 2001; Kolson et al., 2016; Lurie, Pasic, Hockfield, & Rubel, 1997) that deviate from the typical lattice-like appearance of PNs around cortical neurons (Blosa et al., 2013; Celio & Blumcke, 1994; Celio, Spreafico, Biasi, & Vitellaro-Zuccarello, 1998; Deepa et al., 2006; Hockfield & McKay, 1983; Zaremba, Guimaraes, Kalb, & Hockfield, 1989). The shape of PNs around MNTB principal cells is most likely an adaptation to its specialized synaptic input (Blosa et al., 2013), a single giant synapse, the calyx of Held, which is formed by the axons of the globular bushy cells in the ventral cochlear nucleus and covers ~50% of the MNTB neuron’s cell body (Morest, 1968; Satzler et al., 2002; see also chapter by Spirou in this book).

Various markers have been applied to identify PNs in the MNTB. While labeling of aggrecan and brevican yielded the highest number of PN positive MNTB neurons in all investigated species (gerbil: Lurie et al., 1997; rats: Myers et al., 2012; mice: Blosa et al., 2013; Kolson et al., 2016), results obtained using Wisteria floribunda agglutinin (WFA), which binds to N-acetylgalactosamine residues on the CS-GAG-chains of aggrecan (Giamanco, Morawski, & Matthews, 2010; Morawski, Bruckner, Arendt, & Matthews, 2012), were controversial, perhaps reflecting species differences. In dogs, the majority of MNTB principal cells (80%) showed WFA-labeled PNs (Fech et al., 2017). Similar observations were made in rats, where ~90% of neurons in the MNTB revealed WFA binding sites (Myers et al., 2012). However, in mice >90% of MNTB neurons were found to reveal aggrecan- and brevican positive PNs, while only ~25% of the neurons in the same nucleus were additionally reactive to WFA (Blosa et al., 2013). In the MNTB of rhesus monkeys, WFA labeling was found to follow a medio-lateral gradient with the number of WFA-reactive neurons in the medial MNTB being higher compared to the lateral MNTB (Hilbig et al., 2007). Further, MNTB neurons with WFA binding sites were shown to express the potassium channel subunit Kv3.1β which is supposed to facilitate high-frequency activity (Rudy & McBain, 2001) and which also exhibits a medio-lateral gradient in the MNTB (Gazula et al., 2010; Hilbig, Nowack, Boeckler, Bidmon, & Zilles, 2007; Leao et al., 2006).

Lateral Superior Olive

In contrast to the MNTB, PNs in the LSO enclose neuronal somata as well as proximal dendrites of mainly fusiform cells (Friauf, 2000), thereby exhibiting the characteristic net-like shape (Friauf, 2000; Lurie, Pasic, Hockfield, & Rubel, 1997). PNs in the LSO were identified in various species, including gerbil (Lurie et al., 1997), rat (Friauf, 2000; Myers, Ray, & Kulesza, 2012; Seeger, Brauer, Hartig, & Bruckner, 1994), dog (Atoji & Suzuki, 1992; Atoji, Yamamoto, Suzuki, Matsui, & Oohira, 1997; Fech, Calderon-Garciduenas, & Kulesza, 2017), tenrec (Morawski, Bruckner, Jager, Seeger, Kunzle et al., 2010) and rhesus monkey (Hilbig et al., 2007), though species-specific differences have been documented. While in dogs, medial LSO neurons were less frequently enclosed by WFA positive PNs (68%) than lateral LSO neurons (83%; Fech et al., 2017), the opposite was found in the rhesus monkey, where especially the medial LSO neurons exhibited strongly labeled WFA -positive PNs (Hilbig et al., 2007).

Discrepancies in the estimated number of PN-bearing LSO neurons have also been emerged with respect to the utilized PN marker. While more than 85% of LSO neurons in rats are surrounded by PNs when using WFA, only about 53% of the LSO neurons exhibit PNs when using the glycosylation-dependent anti-CSPG antibody Cat-315 (Myers et al., 2012). These results point to chemical variations in the subcomponents of PNs, such as glycosylation state (Dino, Harroch, Hockfield, & Matthews, 2006; Matthews et al., 2002) which might affect the suitability of PN markers and further lead to repeated misinterpretations in the literature (Morawski, Bruckner, Jager et al., 2012).

Medial Superior Olive

PNs have been found around principal neurons of the MSO in rats (Friauf, 2000; Myers, Ray, & Kulesza, 2012; Seeger, Brauer, Hartig, & Bruckner, 1994), gerbils (Lurie et al., 1997), and dogs (Atoji & Suzuki, 1992; Atoji, Yamamoto, Suzuki, Matsui, & Oohira, 1997; Fech, Calderon-Garciduenas, & Kulesza, 2017). PNs in the MSO are associated with bipolar shaped cell bodies and with medially and laterally oriented dendritic processes, remarkably at larger extent than dendrites of other nuclei in the SOC (Lurie et al., 1997).

Gradients in PN expression along the tonotopic axis have been reported for dogs. In the dorsal, high-frequency part of the MSO only 35% of the neurons are enclosed by WFA-positive PNs, while in the ventral, low-frequency portion of the MSO 78% of the neurons reveal WFA-positive PNs (Fech et al., 2017).

Other Nuclei of the Superior Olivary Complex

The remaining nuclei of the SOC, including the LNTB, VNTB, and SPN, are not well examined regarding the distribution of PNs. The little information that is available was collected in rats and dogs. In adult rats, about 70–80% of SPN neurons were found to be surrounded by PNs labeled with either WFA or anti-Cat-315 (Myers et al., 2012). In dogs, more than half of the LNTB (59%) and VNTB neurons (65%) revealed WFA-positive PNs.

Perineuronal Nets in the Superior Olivary Complex of Humans

Perineuronal nets have been detected in SOC nuclei of many species, ranging from rodents, such as gerbils, mice and rats, to dogs and rhesus monkey. However, PNs have also been found in the human SOC, but the investigations are mainly restricted to older individuals (>70 years old).

Although the neuroanatomy of the human SOC was described to be largely comparable to what has been reported for other species (Kulesza, 2007, 2008; Schmidt, Wolski, & Kulesza, 2010), the distribution of PNs was found to differ in some respects. In the MNTB, only about 25% of the neurons were associated with PNs, using either WFA or an anti-CSPG antibody. PNs were primarily associated with round-shaped neurons (59%) but were also found around stellate (39%) and fusiform cells (2%) within this nucleus. Similar results were found in the LNTB, VNTB, and SPN, where the number of neurons surrounded by PNs varied between 10 and 32% and were also distributed among fusiform, stellate, and round or oval-shaped neurons (Schmidt et al., 2010). Thus, the number of neurons surrounded by PNs is remarkably reduced compared to other mammalian species. Surprisingly, the labeling of PNs in the MSO and LSO by WFA or an anti-CSPG antibody did only rarely reveal net-like structure in the LSO (<1% of the neurons) and virtually never in the MSO (Schmidt et al., 2010). These data are to some extent conflicting with results documented in most of the other species tested so far where labeling of the LSO and MSO presented a clear, net-like structure surrounding the cell bodies and dendrites.

The interpretation of these results needs to be done with caution. The absence of WFA binding sites and the negative immunosignal in response to the used anti-CSPG antibody does not necessarily mean that MSO and LSO neurons in the human SOC do not express PNs. The suitability of some PN markers, including WFA, as well as PN-specific antibodies, has been increasingly doubted in the last years (Sonntag et al., 2015). WFA detects N-acetylgalactosamine residues on CS-GAG chains of aggrecan (Table 1, Figure 1; Giamanco et al., 2010). Still, studies conducted in the hippocampus and neocortex clearly demonstrated that many neurons that are surrounded by aggrecan do not reveal WFA binding sites (Ajmo, Eakin, Hamel, & Gottschall, 2008; Morawski, Bruckner, Jager, Seeger, & Arendt, 2010; Suttkus et al., 2014). Further, the glycosylation state of aggrecan may vary among neurons, which affects the binding properties of some glycosylation-dependent anti-CSPG antibodies, such as Cat-315 and Cat-316 (Table 1, Figure 1; Dino, Harroch, Hockfield, & Matthews, 2006; Matthews et al., 2002). It might thus be possible that the biochemical properties of aggrecan around LSO and MSO neurons in the human SOC deviate from that of other species, where WFA and various glycosylation-independent, anti-aggrecan, and anti-CSPG antibodies (Table 1, Figure 1) yielded clear PNs around MSO and LSO neurons (Fech, Calderon-Garciduenas, & Kulesza, 2017; Friauf, 2000; Lurie, Pasic, Hockfield, & Rubel, 1997; Morawski, Bruckner, Jager, Seeger, Kunzle et al., 2010; Myers, Ray, & Kulesza, 2012). Surprisingly, most of the currently recommended PN-specific antibodies that mainly detect the core proteins of various PN subcomponents (such as aggrecan, brevican, neurocan, and HAPLN 1 and 4), and which were demonstrated to specifically label PNs around neurons in the auditory brainstem of mice and gerbils (Blosa et al., 2013; Blosa et al., 2015; Kolson et al., 2016; Sonntag, Blosa, Schmidt, Rubsamen, & Morawski, 2015), have not been applied to the human auditory brainstem yet. It most likely can be assumed that these antibodies will prove to be the better choice to clarify whether neurons in the human MSO and LSO are surrounded by or devoid of PNs (Morawski, Bruckner, Jager et al., 2012).

Perineuronal Nets in the Superior Olivary ComplexDevelopment, Function, and PlasticityClick to view larger

Figure 1. Antibodies detecting the core protein and chondroitin sulfate glycosaminoglycan side chains of aggrecan.

Schematic illustration of the aggrecan protein (including the three globular domains G1–G3 of the core protein and attached CS side chains) and respective antibodies direct against different components of aggrecan.

CS—chondroitin sulfate; G—globular domain; GalNAc—N-acetylgalactosamine; GlcA—glucuronic acid; LP—link protein

Table 1. Commonly used antibodies/markers for the detection of aggrecan in perineuronal nets.

Marker

Detected Region

Detected Component

Specific for Aggrecan

Reference

Source

Core protein

MAB anti-human aggrecan

(clone HAG 7D4)

Aggrecan core protein at G1-G2 domain

Yes

(Bruckner, Morawski, & Arendt, 2008); (Virgintino et al., 2009)

Bio-Rad (AbD Serotec)

MAB 5284

anti-Cat-301

Aggrecan core protein at CS-region

Yes

Matthews et al. 2002

Merck

(Millipore)

AB1031

anti-aggrecan

Aggrecan core protein at CS-region

Yes

Blosa et al. 2013

Merck

(Millipore)

CS-Chain (glycoform)

AB1918

anti-CSPG

Unspecified CS-chain potentially on aggrecan

Unclear

Bruckner et al. 2000; Friauf 2000

Chemicon dis-continued

ab11570

anti-pan CS56

Unspecified CS-chain on PGs

No

(Balmer, Carels, Frisch, & Nick, 2009; Miyata, Komatsu, Yoshimura, Taya, & Kitagawa, 2012)

Merck, Abcam, LSBio, Bio-Rad

MAB 15812

anti-Cat-316

Unspecified CS-chain potentially on aggrecan

Unclear

Matthews et al. 2002

Merck

(Millipore)

CS-Chain (digested)

Anti-CS Delta Di-OS (clone 1B5)

0S CS-stub after ChABC digestion

Unclear

Caterson 2012

AMSBio

anti-CS Delta Di-4S (clone 2B6)

4S CS-stub after ChABC digestion

Unclear

Caterson 2012

AMSBio

anti-CS Delta Di-6S (clone 3B3)

6S CS-stub after ChABC digestion

Unclear

Caterson 2012

AMSBio

Oligosaccharide on PGs

MAB 1581

anti-Cat-315

In development:

RPTPζ/phosphacan

In adult: oligo-epitope on aggrecan

Unclear

Matthews et al. 2002; Dino et al. 2006

Merck

(Millipore)

N-acetyl-galactosamine

Wisteria Floribunda agglutinin (WFA)

N-acetyl-Galactosamine on CS-chain of aggrecan

Yes

(Bruckner et al., 1993); Giamanco et al. 2010

Merck (Sigma-Aldrich), Vector Labs

Antibodies and markers commonly used for the detection of aggrecan can be directed against the core protein, chondroitin sulfate (CS) chains, oligosaccharides, or N-acetylgalactosamines.

Development of Perineuronal Nets in the Superior Olivary Complex

PNs gradually mature during late stages of postnatal development, being established first in brainstem areas and last in cortical areas (Bruckner et al., 2000; Friauf, 2000). The formation of PNs coincides with the end of critical periods, which indicate developmental epochs with heightened plasticity (Kalb & Hockfield, 1988; Pizzorusso et al., 2002; for review see Zimmermann & Dours-Zimmermann, 2008).

The development of PNs in the superior olivary complex is well described in rodents (gerbil, mice and rats) and is characterized by profound changes between the end of the first postnatal week and the end of the first postnatal month (Bruckner et al., 2000; Friauf, 2000; Kolson et al., 2016; Lurie, Pasic, Hockfield, & Rubel, 1997; Myers, Ray, & Kulesza, 2012). These changes include an increase in the number of PN-positive neurons in the MNTB, LSO, and MSO (Myers et al., 2012), an increase in gene expression of PN components, demonstrated in the MNTB (Kolson et al., 2016), and an increase in staining intensity of PN components, indicating an increase in protein expression levels in the MNTB, LSO, and MSO (Bruckner et al., 2000; Friauf, 2000; Kolson et al., 2016; Lurie, Pasic, Hockfield, & Rubel, 1997).

In detail, aggrecan and brevican first appear around P6-P7 in the MNTB and are mainly diffusely distributed in the neuropil (Figure 2), although occasional punctate accumulations of aggrecan were observed at the surface of principal neurons at this age (Friauf, 2000; Kolson et al., 2016; Lurie, Pasic, Hockfield, & Rubel, 1997). In contrast to this, HAPLN 1 already shows ring-like patterns around the principal neurons at this age and thus seems to develop much faster than brevican and aggrecan (Kolson et al., 2016). Similar observations were made for neurocan, which also clearly encircles MNTB principal cells already at P4–7 in mice (Figure 2; Bruckner et al., 2000). The early presence of HAPLN 1 might be directly linked to the development of neurocan, since HAPLN 1 interacts with neurocan and stabilizes the connection between this proteoglycan and hyaluronan (Zimmermann & Dours-Zimmermann, 2008). Around hearing onset (P12–P14), labeling of aggrecan, brevican, and HAPLN 1 is significantly increased in intensity and appears as a ring clearly delineating neuronal cell bodies (Bruckner et al., 2000; Friauf, 2000; Kolson et al., 2016; Lurie, Pasic, Hockfield, & Rubel, 1997). Between P21 and P28 intensity of PN labeling further increases and adult-like shape is reached (Figure 2; Friauf, 2000; Lurie, Pasic, Hockfield, & Rubel, 1997).

The developmental profiles of PNs in the LSO and MSO were largely comparable to what has been described in the MNTB. At the end of the first postnatal week, aggrecan exhibits a faint and diffuse labeling. Neuron-specific labeling of aggrecan is first detected around P11–P12, surrounding dendrites and cell bodies of the principal neurons in the LSO and MSO. During the next two postnatal weeks, intensity of aggrecan labeling heavily increases, exhibiting an adult-like appearance at P28 (Friauf, 2000; Lurie, Pasic, Hockfield, & Rubel, 1997).

In rodents, the expression and maturation of PNs in the SOC coincides with critical morphological and physiological developmental processes and the onset of response to airborne sound (Sonntag et al., 2009). In the MNTB, these changes encompass the (1) transition from multiple inputs to mono-innervation by the large calyx of Held during the first postnatal week (Bergsman, Camilli, & McCormick, 2004; Holcomb et al., 2013; Rodriguez-Contreras, van Hoeve, John Silvio Soria, Habets, Locher, & Borst, 2008; for review see Yu & Goodrich, 2014); (2) the growth and fenestration of the calyx of Held up to the age of P14 (Ford, Grothe, & Klug, 2009; Hoffpauir, Grimes, Mathers, & Spirou, 2006; Kandler & Friauf, 1993; Morest, 1968; for review see Yu & Goodrich, 2014); and (3) the acceleration of synaptic transmission speed, shortening of action potential duration, and increase in synaptic fidelity along with changes in the composition and expression of receptors and channels during the second postnatal week (Sonntag, Englitz, Typlt, & Rubsamen, 2011; Taschenberger & Gersdorff, 2000; for review see Gersdorff & Borst, 2002; Schneggenburger & Forsythe, 2006). Likewise, developmental changes with respect to refinement, pruning, and strengthening of synaptic contacts occur in the MSO and LSO during the second postnatal week, resulting in functional networks around hearing onset (Kandler, Clause, & Noh, 2009).

The strong temporal correlation between PN development and maturation of morphological and physiological features of SOC neurons suggests that PNs are involved in processes involved in closing the critical period in hearing system development. This was also suggested for the visual system where the appearance of PNs is likewise linked to the onset of sensory information processing (Hockfield & Sur, 1990; Kalb & Hockfield, 1988; Pizzorusso et al., 2002). The underlying mechanisms are not clarified up to now, but previous reports indicate that PNs stabilize synaptic contacts and functional networks by restricting synaptic plasticity. This was experimentally demonstrated in the visual cortex, where synaptic plasticity remained at juvenile levels after enzymatic digestion of the CS-GAG side chains of proteoglycans of PNs (Carulli et al., 2010; Pizzorusso et al., 2002; Wang & Fawcett, 2012).

In addition, it was shown that the expression and maturation of PNs does not only correlate with the onset of sensory information processing but might also directly be dependent on sensory information. Visual deprivation was found to be linked to reduced levels of aggrecan in the visual cortex (Hockfield & Sur, 1990). The effects of auditory deprivation on PN expression has also been investigated in neurons of the SOC in rats, yielding reduced numbers of PN bearing neurons in the MNTB and MSO compared to normal hearing controls (Myers et al., 2012).

In contrast to the early development, the effect of aging on the expression of PNs in the auditory brainstem is not well understood. Comparative analysis of PN expression in the SOC in 4-week old and 1-year old gerbils did not yield any differences (Lurie et al., 1997). Investigations in other brain regions yielded similar results. In the auditory cortex of CBA mice, no differences in the intensity of PN staining were found between young (1–3 months) and old (14–24 months) animals (Brewton, Kokash, Jimenez, Pena, & Razak, 2016). Likewise, the number of PN-bearing neurons in the human amygdala was not related to the age of the individuals (Pantazopoulos, Murray, & Berretta, 2008). Interestingly, the sulfation pattern of GAG side chains of PNs in the auditory, visual, and motor cortex of rats was demonstrated to change with increasing age (Foscarin, Raha-Chowdhury, Fawcett, & Kwok, 2017), indicating functional modifications in the aging brain.

Perineuronal Nets in the Superior Olivary ComplexDevelopment, Function, and PlasticityClick to view larger

Figure 2. Development of chondroitin-sulfate proteoglycans in the medial nucleus of the trapezoid body of the mouse.

Immunohistochemical labeling of the chondroitin-sulfated proteoglycans aggrecan (ACAN, top row), brevican (BCAN, middle row) and neurocan (NCAN, bottom row) at the ages P7, P14, and P28 indicates differences in the developmental expression patterns between ACAN and BCAN versus NCAN. ACAN and BCAN show weak, mainly intracellular immunolabeling at P7, consistent with incomplete transportation into the extracellular space. ACAN and BCAN immunoreactivity strongly increases during further development, finally forming the typical ring-like structures at P28. NCAN is characterized by early high-expression levels and is accumulated extracellularly around neurons already at P7, with only minor changes up to P28.

Structure and Molecular Composition of Perineuronal Nets in the Medial Nucleus of the Trapezoid Body

The characterization of the PNs in the SOC is currently mainly constrained to a qualitative and (less frequently) quantitative estimation of the distribution of PNs in the individual SOC nuclei. Structural details, such as molecular composition of PNs as well as interaction of PNs with neurons and synapses, have only been investigated in the MNTB so far (Blosa et al., 2013).

Elaborate immunohistochemical analyses confirmed the presence of all major PN constituents around MNTB principal cells, including hyaluronan, the CSPGs aggrecan, brevican, and neurocan, different oligosaccharide epitopes, HAPLN 1 and tenascin-R (Blosa et al., 2013; Blosa et al., 2015; Kolson et al., 2016). The typical, strong interaction of PNs and synapses terminating on the neuronal surface and on dendrites was also demonstrated in the MNTB, since both glutamatergic and GABAergic synaptic boutons, as well as the huge glutamatergic calyx of Held, were tightly enwrapped by aggrecan immunoreactivity. These data indicate that PNs in the MNTB enclose synapses irrespective of the prevalent neurotransmitter (Blosa et al., 2013).

Interestingly, the CSPGs seemed to differ in their extension and distribution around the neuronal surface. While aggrecan revealed a prominent, cotton wool-like appearance surrounding the entire neuron as well as the outer surface of the calyx of Held, brevican, and neurocan were found to be more delicate forming thinner rings around the neuronal cell bodies (Blosa et al., 2013; Kolson et al., 2016). In addition, electron microscopical analyses yielded that brevican is especially located in the perisynaptic space, around unmyelinated parts of presynaptic axons, and even in the calyceal, subsynaptic space, but it is sparsely present in the soma-free neuropil within the MNTB (Blosa et al., 2013; Blosa et al., 2015). Thus, PNs around MNTB principal neurons appear to be composed of an inner brevican/neurocan ring and an outer aggrecan ring (Blosa et al., 2013; Kolson et al., 2016). Remarkably, the extracellular space filled with perineuronal extracellular matrix revealed an extension of up to 2 µm in the MNTB (Blosa et al., 2013), which matches the bulky appearance of PNs around MNTB principal neurons and is significantly larger than what has been generally found around other neurons (20–70 nm (Sykova & Nicholson, 2008; van Harreveld, 1965). This 100-times bigger ECM coat around neurons in the SOC, especially in the MNTB, might point to an outstanding role of PNs for the physiology of these neurons (Morawski et al., 2015).

Function of Perineuronal Nets in the Superior Olivary Complex

The heterogeneous distribution and the different developmental periods of appearance of aggrecan, brevican and neurocan in the PN gives rise to the assumption that these CSPGS may occupy different functions within the PN. Depending on the brain region of interest and the considered PN subcomponent, PNs contribute to synaptic stabilization and plasticity, to synaptic transmission and to neuroprotection. However, the specific function of PNs or their subcomponents in the SOC remains largely elusive. Given the high density of PNs in the SOC, it is surprising that so far only two very recent studies considered the MNTB as a model system to estimate the PNs’ role in synaptic transmission and neuronal activity (Balmer, 2016; Blosa et al., 2015).

Chondroitin Sulfate Side Chains and Their Impact on Neuronal Excitability and Synaptic Plasticity

Injection of chondroitinase ABC in the visual cortex and amygdala yielded reactivation of juvenile states of synaptic plasticity in these systems (Gogolla, Caroni, Luthi, & Herry, 2009; Pizzorusso et al., 2002), thus indicating that PNs restrict synaptic plasticity after the closure of the critical window (Dityatev & Fellin, 2008; Gundelfinger, Frischknecht, Choquet, & Heine, 2010; Wang & Fawcett, 2012). This is supposed to be based on the PNs’ contribution to the stabilization of neuron-synapse and synapse-dendrite interactions (Hockfield & Sur, 1990; Vivo et al., 2013; Wang & Fawcett, 2012) and potentially also on the PNs’ control of neuronal activity. The latter theory mainly relies on the fact that PNs provide a strong polyanionic charge determined by the high number of negatively charged chondroitin sulfate side chains bound to aggrecan (Giamanco, Morawski, & Matthews, 2010; Morawski et al., 2015; Morawski, Bruckner, Arendt, & Matthews, 2012). Thus, PNs, especially aggrecan, are believed to act as a diffusion barrier or spatial buffer for positively charged ions and proteins, a function thatwas found to be important for the protection of neurons against toxic accumulations of cations or other positively charged molecules (Morawski, Bruckner, Arendt, & Matthews, 2012; Morawski, Bruckner, Jager, Seeger, & Arendt, 2010; Morawski, Bruckner, Riederer, Bruckner, & Arendt, 2004; Suttkus et al., 2014; Suttkus, Morawski, & Arendt, 2016; Suttkus, Rohn, Jager, Arendt, & Morawski, 2012) but might further be essential for high-frequency neuronal activity by controlling ion availability and homeostasis (Hartig et al., 1999; Hrabetova, Masri, Tao, Xiao, & Nicholson, 2009; Morawski et al., 2015). This idea is supported by an elaborate electrophysiological work, recently published by (Balmer, 2016), who demonstrated the impact of PNs on neuronal activity in the MNTB. After enzymatic digestion of chondroitin sulfate side chains by chondroitinase ABC, MNTB principal cells revealed greatly reduced excitability in response to white noise currents, and thus needed more current for spiking compared to untreated MNTB neurons (Figure 3). This change was not based on alterations in passive membrane properties, since resting membrane potential and input resistance were comparable between enzyme-treated and control cells. Instead, the spike thresholds of the enzyme-treated neurons were elevated. The underlying mechanisms are still unclear, but given the fact that chondroitinase ABC cuts off negatively charged side chains, loss of the polyanionic character is a likely reason for the observed physiological changes. In cortical and hippocampal slices, the diffusion of calcium is increased after chondroitinase ABC- digestion of PNs (Hrabetova et al., 2009), supporting the hypothesis that PNs bind cations (Morawski et al., 2015). Sodium and potassium ions might likewise be controlled by PNs, though not yet verified experimentally (Hartig et al., 1999; Morawski et al., 2015). It is also assumed that the voltage-dependency of ion channels is regulated by the PNs anionic side chains. By increasing the negative charge of the membrane and shifting the activation of voltage-dependent currents toward more hyperpolarized potentials, the excitability of neurons might be increased (Balmer, 2016; Morawski et al., 2015).

The elevated neuronal excitability was suggested to be one principle underlying the PN-mediated restriction of synaptic plasticity (Balmer, 2016). However, only little is known about the influence of PNs on synaptic plasticity in the auditory system. A recent study published by (Happel et al., 2014) demonstrated that the enzymatic digestion of PNs in the auditory cortex of gerbils considerably affects the reversal learning behavior which is known to be based on plastic modifications of neuronal interactions. Whether PNs also control synaptic plasticity in the SOC is virtually elusive and needs to be investigated in future studies. Previous findings demonstrate that MNTB principal cells reveal heterogeneous properties with respect to short-term plasticity (Grande & Wang, 2011). Likewise, it was shown that not all PN-bearing MNTB principal cells reveal binding sites for the lectin WFA which detects the negatively charged side chains bound to proteoglycans (Blosa et al., 2013). In this regard, it might be of importance to figure out whether PNs are heterogeneous with respect to their negative charge (Morawski et al., 2015) and whether they differ in their potential to control synaptic plasticity in the MNTB.

Brevican and Its Role in Synaptic Transmission and Organization of Subsynaptic Space

The primarily perisynaptic location of brevican was assumed to be connected to the contribution to physiological processes at synapses. Electrophysiological in vivo investigations of MNTB principal neurons of brevican-deficient mice supported this theory by showing that the duration of synaptic transmission as well as of pre- and postsynaptic action potentials were significantly prolonged in comparison to wildtype mice (Figure 3; Blosa et al., 2015). These data point to an essential role of brevican for high-speed synaptic transmission, which in turn is a precondition for high-frequency activity. These physiological changes were accompanied by a reduction of the amount of the presynaptic vesicular glutamate transporter 1, which controls the amount of glutamate in each vesicle as well as the amount of glutamate released by each vesicle. In addition, striking ultrastructural alterations at the calyx of Held were found in brevican-deficient mice. Brevican is expressed in the extracellular space underneath the calyx of Held forming subsynaptic cavities which were found to separate adjacent postsynaptic densities from each other (Blosa et al., 2013; Blosa et al., 2015). These cavities were significantly reduced in size in brevican-deficient mice (Blosa et al., 2015), indicating that brevican might contribute to the compartmentalization of this huge synapse. The calyx of Held is a morphologically specialized synapse exhibiting several hundreds of active zones (Gersdorff & Borst, 2002; Taschenberger, Leao, Rowland, Spirou, & Gersdorff, 2002). The brevican-filled subsynaptic cavities are believed to be important for the spatial and functional organization of synaptic release sites which is indispensable for fast and high-frequency transmission. In this regard, brevican is assumed to restrict transmitter spillover and to control the reuptake of glutamate into the presynaptic terminal (Blosa et al., 2013; Yamada et al., 1997). Altogether, these findings indicate that brevican acts presynaptically by controlling transmitter recycling and vesicular release (Blosa et al., 2013). However, postsynaptic actions of brevican were also suggested since in hippocampal neurons brevican physically interacts with AMPA receptor subunits GluR1, GluR2/3 and GluR4 (Favuzzi et al., 2017; Saroja et al., 2014), perhaps controlling the mobility of AMPA receptors (Frischknecht et al., 2009). Further investigations are needed to elucidate the molecular mechanisms behind the action of brevican underlying its impact on glutamatergic signaling in the MNTB.

Perineuronal Nets in the Superior Olivary ComplexDevelopment, Function, and PlasticityClick to view larger

Figure 3. Impact of perineuronal nets on synaptic transmission and neuronal excitability in the medial nucleus of the trapezoid body.

Electrophysiological measurements in the medial nucleus of the trapezoid body (MNTB) after targeted manipulation of perineuronal net (PN) subcomponents indicate that brevican and chondroitin sulfate (CS) side chains, mainly bound to aggrecan, serve different functions. After genetic deletion of brevican (bcan-/-) in the mouse, a significant reduction in speed of synaptic transmission at the calyx of Held is observed (left, figures were modified from Blosa et al., 2015). After enzymatic digestion of CS side chains with chondroitinase ABC (ChABC) whole-cell patch-clamp recordings indicate a reduced excitability of MNTB principal neurons (right, figures were modified from (Balmer, 2016). *** p < 0.001

Brevican and Its Impact on Hearing

Fast, temporally precise, reliable, and high-frequency synaptic transmission are important preconditions for the proper processing of auditory information within the SOC. Disruptions of one of these critical features are supposed to result in the impairment of hearing function. Since brevican was shown to be essential for achieving the very high speed of synaptic transmission in the MNTB, it is not surprising at all that auditory signal processing in the MNTB in brevican-deficient mice was also deteriorated. In detail, in vivo single-cell recordings in brevican-deficient mice yielded reduced hearing thresholds and sound-evoked discharge rates in the MNTB (Blosa et al., 2015). Interestingly, the MNTB could be excluded as the origin of these effects since the reliability of synaptic transmission at the calyx of Held—MNTB synapse was not changed in brevican-deficient mice (Blosa et al., 2015). Since these brevican-deficient mice are a systemic knockout line, it is to be expected that brevican controls synaptic transmission throughout the entire brain, and thus the cochlear nucleus, which precedes the MNTB, might be a potential source of the demonstrated alterations in sensitivity and firing rates (Blosa et al., 2015), a possibility that remains to be examined. In addition, the questions remain whether the positive influence of brevican on synaptic transmission speed is a general phenomenon, valid for all synapses along the auditory pathway (or even in the whole CNS), and whether the absence of brevican affects the localization of sound sources, a function that strongly depends on temporally precise and fast synaptic transmission in the CN and SOC.

Conclusion

PNs are a specialized form of ECM that surround specific types of neurons in the central nervous system. In most regions of the CNS, especially in the cortex, PNs are only associated with about 1–10% of the neurons, strongly depending on the area investigated (Morawski, Pavlica et al., 2010; Suttkus et al., 2014). A remarkably high density of PNs was found in the nuclei of the auditory brainstem, especially of the SOC, which seems to be consistent across many mammalian species, including humans. Thus, the PNs might serve as a suitable marker for the identification of the SOC and its subnuclei in species where the neuroanatomy is not yet well described.

Structural details and functional aspects of PNs so far have been mainly studied in cortical and hippocampal interneurons, the preferred model system for investigation of PN function (Brakebusch et al., 2002; Favuzzi et al., 2017; Geissler et al., 2013; Pizzorusso et al., 2002). The nuclei of the SOC have only been addressed in a few studies, which is surprising given the high prevalence of PNs around the neurons within this complex. One reason might be that the neurons within the SOC form a group of very active neurons partly characterized by morphologically specialized synapses, and therefore greatly differ from most of the other PN-bearing neurons, such as interneurons in the cortex and hippocampus (Sonntag et al., 2015). This might foster doubts whether the results obtained in the SOC can be generalized for other brain regions. However, in a recent comparative study it was shown that enzymatic digestion of PNs around neurons in the MNTB and around cortical interneurons results in a similar reduction of excitability in both types of neurons, thus indicating that the function of PNs might be the same irrespective of the type of neuron or transmitter (Balmer, 2016). In addition, especially the huge calyx of Held in the MNTB, which is a unique model synapse for the investigation of glutamatergic synaptic transmission (Gersdorff & Borst, 2002; Schneggenburger & Forsythe, 2006), might greatly help to study the influence of PNs on synaptic transmission. It is currently not possible to elucidate this in the same extent in the cortex and hippocampus as the direct access to the synapses is hampered due to their small sizes.

Despite of the remaining uncertainty of whether the contribution of PNs to neuronal activity and synaptic transmission observed in the MNTB can be assumed as a general PN function, these findings strongly support the previously introduced concept of the tetrapartite synapse (Dityatev & Rusakov, 2011), where the pre- and postsynapse, glia cells, and PNs conjointly control the physiology of the synapses and neurons.

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