Fig 2: Schematic representation of HAPLN-dependent perineuronal net (PNN) micro-organization at the calyx of Held synapses. (A,C,D) In wild-type mice, HAPLN4-brevican-type PNN is enriched in the perisynaptic space between the calyx of Held terminals and the principal neuron, and HAPLN1–aggrecan–tenascin-R-type PNN surrounds the whole calyx of Held terminals. (B,E,F) In Hapln4-KO, the ectopic expression of brevican is detectable with HAPLN1 association at the surrounding extracellular space in the neuropil in addition to the HAPLN1–aggrecan–tenascin-R complex. Acan, aggrecan; Bcan, brevican; TN-R, tenascin-R; HA, hyaluronan; PSD, postsynaptic density.

Fig 3: Ectopic expression of brevican in the medial nucleus of the trapezoid body (MNTB) of Hapln4-KO mice. To clarify the spatial location of perineuronal net components, the immunoreactivity of HAPLNs (A,B,E,F), brevican or aggrecan (C,D,G,H), and tenascin-R (I,J) (green) is examined in the MNTB of 4-month-old adult mice using VGLUT1 as a marker of the calyces of Held (magenta). The intensity profiles of the fluorescence signals along the white lines are shown in the lower panels. The fluorescence intensities are normalized by the highest intensities of individual proteins within the lines except in (A,B). Since these have a relatively low signal-to-noise ratio of the anti-HAPLN4 antibodies, the background is high when the intensities are normalized as previously described. Of note is the observed ectopic shift of brevican immunolocalization from the perisynaptic space in the wild type (arrows) to the surrounding extracellular space in the neuropil in Hapln4-KO (arrowheads). Acan, aggrecan; Bcan, brevican; TN-R, tenascin-R. The scale bar represents 10 µm.