Characterization of neuronal subsets surrounded by perineuronal nets in the rhesus auditory brainstem

Abstract

The distribution of perineuronal nets and the potassium channel subunit Kv3.1b was studied in the subdivisions of the cochlear nucleus, the medial nucleus of the trapezoid body, the medial and lateral superior olivary nuclei, the lateral lemniscal nucleus and the inferior colliculus of the rhesus monkey. Additional sections were used for receptor autoradiography to visualize the patterns of GABAA and GABAB receptor distribution. The Kv3.1b protein and perineuronal nets [visualized as Wisteria floribunda agglutinin (WFA) binding] were revealed, showing corresponding region-specific patterns of distribution. There was a gradient of labelled perineuronal nets which corresponded to that seen for the intensity of Kv3.1b expression. In the cochlear nucleus intensely and faintly stained perineuronal nets were intermingled, whereas in the medial nucleus of the trapezoid body the pattern changed to intensely stained perineuronal nets in the medial part and weakly labelled nets in its lateral part. In the inferior colliculus, intensely labelled perineuronal nets were arranged in clusters and faintly labelled nets were arranged in sheets. Using receptor autoradiography, GABAB receptor expression in the anterior ventral cochlear nucleus was revealed. The medial part of the medial nucleus of the trapezoid body showed a high number of GABAA binding sites whereas the lateral part exhibited more binding sites for GABAB. In the inferior colliculus, we found moderate GABAB receptor expression. In conclusion, intensely WFA-labelled structures are those known to be functionally involved in high-frequency processing.

Fig. 1

Comparison of the distribution patterns of perineuronal nets as revealed by WFA binding sites and Kv3.1b protein in the relay stations of the auditory brainstem of the rhesus monkey. The distribution patterns change from non-compartmentalization in the anterior ventral cochlear nucleus (AVCN) with intermingled intensely and weakly stained WFA to clusters (see window) in the posterior ventral cochlear nucleus (PVCN), via subnucleation into a medial part with intensely stained WFA and a lateral part of the MNTB (additionally revealed by GABA_A and GABA_B receptors) to clusters in the external nucleus of the inferior colliculus (IC); LLN, lateral lemniscal nucleus. The heavily stained WFA binding was revealed by confocal laser scanning microscopy as thick, red perineuronal nets and is co-localized with green labelled Kv3.1b protein at the soma of the auditory neuron. In the overview, yellow colour indicates co-localization of Kv3.1b with dark WFA binding sites in a cluster.

Fig. 2

Fluorescence photomicrographs of an octopus cell (A) and a multipolar cell (B) surrounded by WFA-stained perineuronal nets.

Fig. 3

Comparison of the distribution patterns of GABA_A and GABA_B receptors in the relay stations of the auditory brainstem of the rhesus monkey. At the level of the dorsal cochlear nucleus (DCN) the superficial granular cell layer is labelled; VCN, ventral cochlear nucleus. For a better orientation, at the level of the SOC Nissl stainings are provided. The MSO is the needle-shaped structure indicated by arrows; MNTB, medial nucleus of the trapezoid body. For the CN and the IC, GABA_A receptor densities were below detection levels. GABA_B receptors were found especially in the DCIC (dorsal cortex of the IC) whereas the CIC (central nucleus of the IC) was weakly labelled.

Fig. 4

Overviews of WFA binding sites in the IC of the rhesus monkey revealing changes in the distribution patterns at different levels: the intensity of the WFA binding sites in the neuropil decreased rostrally (D). DCIC, dorsal cortex of the IC; LLN, lateral lemniscal nucleus; ECIC, external cortex of the IC; CIC, central nucleus of the IC; SC, superior colliculus.

Fig. 5

Distribution patterns of WFA binding sites in the IC of the rhesus monkey at Bregma −21.75 mm. DCIC, dorsal cortex of the IC; LLN, lateral lemniscal nucleus; ECIC, external cortex of the IC; CIC, central nucleus of the IC; open arrows indicate clusters of very large neurons with intensely stained WFA binding sites (B); windows in the CIC indicate examples of sheets of small neurons surrounded by faintly labelled perineuronal nets (D,E).