Modulation of perineuronal nets and parvalbumin with developmental song learning

Abstract

Neural circuits and behavior are shaped during developmental phases of maximal plasticity known as sensitive or critical periods. Neural correlates of sensory critical periods have been identified, but their roles remain unclear. Factors that define critical periods in sensorimotor circuits and behavior are not known. Birdsong learning in the zebra finch occurs during a sensitive period similar to that for human speech. We now show that perineuronal nets, which correlate with sensory critical periods, surround parvalbumin-positive neurons in brain areas that are dedicated to singing. The percentage of both total and parvalbumin-positive neurons with perineuronal nets increased with development. In HVC (this acronym is the proper name), a song area important for sensorimotor integration, the percentage of parvalbumin neurons with perineuronal nets correlated with song maturity. Shifting the vocal critical period with tutor song deprivation decreased the percentage of neurons that were parvalbumin positive and the relative staining intensity of both parvalbumin and a component of perineuronal nets. Developmental song learning shares key characteristics with sensory critical periods, suggesting shared underlying mechanisms.

Figure 1.

Perineuronal nets are found in the songbird brain and notably demarcate song nuclei and primary sensory areas. A parasagittal section of an adult finch brain was stained with an antibody to chondroitin sulfate, which labels PNNs. In the epifluorescence photomontage, PNNs are labeled in the brainstem, thalamus, primary pallial sensory areas, and song nuclei. PNN stain demarcates the boundaries of the song nuclei HVC, RA, LMAN, and Area X. PNN stain also highlights the primary sensory pallial areas field L (L; auditory), wulst (W; visual), and nucleus basalis (B; somatosensory). Anterior is left, and dorsal is up. Scale bar, 2 mm.

Figure 2.

As in mammals, PNNs in the adult song system surround neurons that contain the calcium-binding protein parvalbumin. A–L, Each row contains representative data from a single song nucleus at low (column 1; scale bar, 250 μm) and higher (columns 2, 3; scale bar, 20 μm) magnification. Small white squares in column 1 indicate the enlarged frame in columns 2 and 3. Columns 1 and 2 show PNNs as revealed by α-CS label, whereas column 3 shows the overlay of PNN and parvalbumin stains. Note the double-labeled cells in all song nuclei in column 3.

Figure 3.

Song system perineuronal nets are developmentally regulated. Data are shown from two of the four song nuclei examined, but the same trends in PNN expression were seen in all (Table 1) (supplemental Figs. 7, 8, available at www.jneurosci.org as supplemental material). Representative confocal data from the HVCs of a 33-d-old juvenile (A–C) and an adult (D–F) show the same section stained and imaged for Nissl (A, D), α-PV (B, E), and α-CS (C, F). Note the developmental increase in PNNs (C, F). RA PNNs were also developmentally regulated (G–L). Representative confocal data from the RAs of a 33-d-old juvenile (G–I) and adult (J–L) show the same section stained and imaged for Nissl (G, J), α-PV (H, K), and α-CS (I, L). Note the developmental increase in PNNs (I, L). Scale bar, 250 μm.

Figure 4.

HVC perineuronal nets predict the maturity of song, as measured by temporal variance. A, C, The percentage of parvalbumin-positive HVC neurons (red) with PNNs (green) varied in 65-d-old zebra finches. Representative micrographs are shown for a juvenile HVC that had either a low (A; 7.2%) or high (C; 66.7%) percentage of PNNs around PV⁺ neurons. The orange and cyan dots in the top left corners correspond to the colors in B and F–I. Scale bar, 50 μm. B, In 65 d olds, the percentage of PV⁺ neurons with PNNs was highly variable and significantly different from 33 d olds but not adults. The data from the finches in A and C are shown in orange and cyan, respectively. D, E, Representative sonograms of the same juveniles with the corresponding histology data directly above suggest that fewer PNNs correlate with simpler songs. Note the simple, repetitive syllable in D produced by the finch with few PNNs in A. Correspondingly, note the more complex song in E from the finch with more PNNs in C. F, G, Each panel quantifies the relationship of the percentage of PV⁺ HVC neurons with PNNs to different spectral features of song syllables (left, medians for each finch for each syllable feature). The colored data correspond to the specific finches shown in A–E. On the right, the feature data for each syllable produced by these specific finches are shown relative to duration. The following song features are shown: entropy variance (F), mean frequency variance (G), mean entropy (H), and mean frequency (I). Note that the temporal variance of both entropy (F) and mean frequency (G) were significantly correlated with PNNs.

Figure 5.

Isolation from a song tutor alters critical period indicators in HVC. Representative data from an age-matched control (A–C) and 90-d-old isolate (D–F) are shown. The same section was triple stained and imaged for the following: Nissl (blue stain in A, D), PV (red stain in B, E), and PNNs (green stain in C, F). Note the lack of punctate PV-positive neurons in E and the less striking PNNs in F. Scale bar, 250 μm.