Update on Perineuronal Net Staining With Wisteria floribunda Agglutinin (WFA)

Abstract

As chemically specialized forms of the extracellular matrix in the central nervous system, polyanionic perineuronal nets (PNs) contain diverse constituents, including chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid, and tenascins. They are detectable by various histological approaches such as colloidal iron binding and immunohistochemical staining to reveal, for instance, the CSPGs aggrecan, neurocan, phosphacan, and versican. Moreover, biotin, peroxidase, or fluorescein conjugates of the lectins Vicia villosa agglutinin and soybean agglutinin enable the visualization of PNs. At present, the N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA) is the most widely applied marker for PNs. Therefore, this article is largely focused on methodological aspects of WFA staining. Notably, fluorescent WFA labeling allows, after its conversion into electron-dense adducts, electron microscopic analyses. Furthermore, the usefulness of WFA conjugates for the oftentimes neglected in vivo and in vitro labeling of PNs is emphasized. Subsequently, we discuss impaired WFA-staining sites after long-lasting experiments in vitro, especially in autoptic brain samples with long postmortem delay and partial enzymatic degradation, while immunolabeling of aggrecan and CSPG link proteins under such conditions has proven more robust. In some hippocampal regions from perfusion-fixed mice, more PNs are aggrecan immunoreactive than WFA positive, whereas the retrosplenial cortex displays many WFA-binding PNs devoid of visible aggrecan immunoreactivity. Additional multiple fluorescence labeling exemplarily revealed in ischemic tissue diminished staining of WFA-binding sites and aquaporin 4 and concomitantly upregulated immunolabeling of neurofilament, light chains, and collagen IV. Finally, we briefly discuss possible future staining approaches based on nanobodies to facilitate novel technologies revealing details of net morphology.

A Comprehensive Atlas of Perineuronal Net Distribution and Colocalization with Parvalbumin in the Adult Mouse Brain

“…It is important to note that this analysis reflects gene expression and PNNs at the level of brain areas and not single cells. To validate our approach, we selected a few genes known to be related to PNN structure and function: Aggrecan ( Acan ), a major proteoglycan core protein present in PNNs (Dauth et al, 2016; Fawcett et al, 2019; Ueno et al, 2018; Härtig et al, 2022; Oohashi et al, 2015; Yamada et al, 2017), Hyaluronan and proteoglycan link protein 1 ( Hapln1 ), coding for a link protein essential for PNNs structure (Carulli et al, 2010); hyaluronan synthase 3 ( Has3 ), a necessary component for PNN aggregation (Kwok et al, 2010); Matrix metallopeptidase 9 ( Mmp9 ), an enzyme known to regulate PNN and PV development (Pirbhoy et al, 2020); A disintegrin and metalloproteinase with thrombospondin motifs ( Adamts5 also known as Adamts11 ), an aggrecan-degrading protease (Held-Feindt et al, 2006) that is expressed by PV interneurons with a PNN (Rossier et al, 2015), and parvalbumin ( Pvalb ). All these genes were significantly correlated with both PNN energy and WFA diffuse fluorescence ( Figure 6 A, B).…”

Neural Extracellular Matrix Remodeling Signatures in Genetic and Acquired Mouse Models of Epilepsy

“…The plant lectin WFA is a widely used indicator for a PNN-typical glycosaminoglycan subtype, which is preferentially linked to Aggrecan (Härtig et al, 2022). We found a wide-spread and evident upregulation of WFA-binding chondroitin sulfates paralleled by an overall loss of Brevican, with both effects being most pronounced in the hippocampus.…”

Extracellular Matrix Regulation in Physiology and in Brain Disease

“…PNNs within the spinal cord are found around large somas of spinoparabrachial projecting neurons in Lamina I of the dorsal horn. Three days after spared nerve injury (SNI), the intensity of WFA staining was significantly reduced around these neurons [ 116 ]. WFA signal was detected within microglial lysosomes, indicating that microglia are phagocytosing PNN.…”