Perineuronal nets and song learning-related neuroplasticity in the songbird brain

Perineuronal nets (PNN) are aggregations of extracellular matrix components surrounding the soma of some neurons, mainly GABAergic interneurons expressing the calcium binding protein parvalbumin (PV+). In mammals, the development of PNN limits synaptogenesis around PV+ neurons and PNN have been validated as a marker characterizing the end of critical periods for visual learning. In songbirds, song learning is limited to critical periods during ontogeny in close-ended learners such as zebra finches and to specific phases of the annual cycle in open-ended learners such as canaries that are able to modify their song seasonally. Sensitive periods for song learning are associated with neuroplasticity including morphological changes due to neurogenesis and synaptic reorganization in the song control nuclei during development and adult seasonal song modifications. The hormonal control of developmental and seasonal neuroplasticity is well documented in songbirds but little is known about the possible regulation of sensitive periods for vocal learning by PNN. First, to explore the expression of PNN throughout the development, we used zebra finches brains collected at different key ages in the song learning process (10, 20, 30, 40, 50, 60, 90, 120 days post-hatch, dph) and we quantified PNN expression and their colocalization around PV+ interneurons. The number of PNN and the % of PNN around PV+ interneurons increased progressively during developmental song learning in 3 of the main song control nuclei (HVC, RA and Area X). Moreover, we confirmed that females that never sing have fewer PNN than males in HVC and RA, two song nuclei involved in song production, at all ages after the peak in PNN numbers seen in males between 50 and 90 dph. Secondly we used adult male and female canaries (in 2 different experiments) treated with a subcutaneous implant filled with testosterone or left empty in control subjects to mimic what happens in the spring when the seasonal modification of the song ends and the song crystallizes. Testosterone significantly increased the number of PNN in the main song control nuclei in both sexes. Together these data suggest that increased expression of PNN in the songbird brain might limit neuroplasticity at the end of developmental and seasonal vocal learnin