Edited by: Shuji Mizumoto, Meijo University, Japan
Reviewed by: Shinji Miyata, Tokyo University of Agriculture and Technology, Japan; Alexandra Yurievna Tsidulko, Federal Research Center of Fundamental and Translational Medicine, Russia
†These authors have contributed equally to this work and share first authorship
This article was submitted to Signaling, a section of the journal Frontiers in Cell and Developmental Biology
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The calyx of Held is a giant nerve terminal mediating high-frequency excitatory input to principal cells of the medial nucleus of the trapezoid body (MNTB). MNTB principal neurons are enwrapped by densely organized extracellular matrix structures, known as perineuronal nets (PNNs). Emerging evidence indicates the importance of PNNs in synaptic transmission at the calyx of Held. Previously, a unique differential expression of aggrecan and brevican has been reported at this calyceal synapse. However, the role of hyaluronan and proteoglycan binding link proteins (HAPLNs) in PNN formation and synaptic transmission at this synapse remains elusive. This study aimed to assess immunohistochemical evidence for the effect of HAPLN4 on differential PNN formation at the calyx of Held. Genetic deletion of
Perineuronal nets (PNNs) are pericellular coats of condensed matrix that enwrap the cell bodies and dendrites of certain neurons in the adult central nervous system. In the brain, PNNs are completed at the end of developmental critical periods for experience-dependent plasticity. They contribute to the stabilization of specific connection patterns, thus limiting plasticity. More recently, PNNs have been revealed to have myriad actions in many central nervous system functions, including memory, psychiatric disease, and neurodegeneration (
Perineuronal nets are typically found around fast-spiking GABAergic interneurons expressing parvalbumin. However, of note is that they also exist surrounding other neurons, such as the medial nucleus of the trapezoid body (MNTB). Each principal cell within the MNTB is contacted by a single giant terminal called the calyx of Held, which is characterized by fast and highly reliable synaptic transmission (
The hyaluronan and proteoglycan binding link proteins (HAPLNs) are key molecules in the formation and control of hyaluronan-based condensed perineuronal matrix in the adult brain (
Previous electron microscopic investigations delineated the precise distribution pattern of aggrecan and brevican at the calyx of Held (
These findings led us to conclude that different HAPLN–lectican molecular sets may orchestrate distinct PNNs with functional relevance. To verify this hypothesis, we evaluated the effect of the genetic deletion of
Homozygous
Immunohistochemistry was performed as previously described (
The sections were permeabilized with 0.2% Triton X-100 in PBS and then blocked in 10% donkey serum (Sigma, St. Louis, MO, United States; D9663) in PBS or a DuolinkTM blocking solution. Regarding aggrecan immunolabeling, pretreatment with chondroitinase ABC (ChABC, 0.1 U/ml; Sigma, C2905) for 40 min at 37°C was required. The specimens were incubated overnight at 4°C with specific primary antibodies diluted in PBS with 0.2% Triton X-100 and 1.5% donkey serum. Then, the slices were incubated for 4 h at room temperature with secondary antibodies. The following primary antibodies were used: goat anti-HAPLN4 polyclonal antibody (R&D Systems, Minneapolis, MN, United States; AF4085,
A biotinylated HA-binding protein (b-HABP: Hokudo, Sapporo, Japan, BC41) derived from versican using recombinant human G1 domain was used for hyaluronan staining, as described previously (
After tissue processing, the sections were mounted on microscope slides with a fluorescence mounting medium (Dako, S3023) or Duolink®
In a previous report, two distinct PNN-type proteoglycan expressions have been reported in the MNTB (
In the MNTB of WT, linear immunolabeling of HAPLN4, which colocalized with that of brevican, was observed (
Distribution of HAPLNs and lecticans in the medial nucleus of the trapezoid body (MNTB) of adult wild-type (WT) mice. Immunohistochemical detection of HAPLNs (green) and lecticans (magenta) in the MNTB of 4-month-old adult WT mice using HAPLN4
In our previous reports, the diffuse expression of brevican in the MNTB of the young adult
Ectopic expression of brevican in the medial nucleus of the trapezoid body (MNTB) of
To better understand the molecular mechanism for the ectopic expression of brevican in the MNTB of
Detection of specific HAPLN–lectican molecular interactions by an
A pair of primary antibodies of HAPLN1 and aggrecan generated abundant signals at the surrounding extracellular space with neuropil-like morphology around VGLUT1 staining in both WT and KO mice (
This study provides direct evidence for differential interactions between HAPLNs and lecticans. In WT form, a specific combination of HAPLN and lectican is located at the specific extracellular milieu, in which HAPLN4 and brevican are located in the perisynaptic space near the synaptic cleft, HAPLN1, and aggrecan surrounding the whole calyx of Held terminals (
Schematic representation of HAPLN-dependent perineuronal net (PNN) micro-organization at the calyx of Held synapses.
In this study, we hypothesized that distinct HAPLNs would regulate the micro-organization of PNN
A query could be raised about how each HAPLN-dependent microorganization would be distinctively formed. The authors assumed that some of the following requirements were necessary to achieve this: (i) differences in binding affinity between HAPLNs and G1 domain of lecticans, (ii) tenascin-R crosslinking of aggrecan
Each principal neuron in the MNTB receives a single input from a giant axosomatic terminal: the calyx of Held. High transmission reliability and consistency in timing and amplitude have been confirmed in the mature calyx of Held synapses (
Perineuronal nets have been implicated in a number of psychiatric disorders (
Hyaluronan and proteoglycan binding link proteins play an important role as organizers of PNNs. Moreover, the current study indicated that each HAPLN may contribute to the formation of distinct PNNs with different functional relevance. The main limitation to this study is the lack of functional studies on aberrant PNN at the calyx of Held in
Our results demonstrated a clear ectopic shift of brevican localization from the perisynaptic space between the calyx of Held terminals and principal neurons to the surrounding neuropil. In contrast, aggrecan expression showed a consistent localization at the surrounding neuropil together with HAPLN1 and tenascin-R in both KO and WT mice.
The original contributions presented in the study are included in the article/
The animal study was reviewed and approved by the Animal Care and Use Committee of Okayama University.
TO, TH, and LV contributed to the study concept and design. KN and HM contributed to the performance of the experiments. KN, HM, TH, LV, and TO contributed to the analysis and interpretation of data. TO, HM, and KN prepared the manuscript initial draft. All authors participated in the critical correction of the manuscript and approved the final version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
This study was funded by the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grant Numbers 26110713 and 19H04754 to TO), and the Operational Programme Research, Development, and Education in the framework of the project “Center of Reconstructive Neuroscience,” registration number CZ.02.1.01/0.0./0.0/15_003/0000419, to LV.
We acknowledge Midori Edamatsu, Koichi Nosaka, and Takahiro Ishii for their excellent technical support and Mitsuaki Ono, Tomoko Yonezawa, and other members of our laboratory who provided discussion and comments on this manuscript. We also acknowledge Takako Sasaki (Oita University) for providing antibodies against brevican and Reinhard Fässler (Max Planck Institute of Biochemistry) for their support in generating the knockout mice, and Editage (
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