Testosterone stimulates perineuronal nets development around parvalbumin cells in the adult canary brain in parallel with song crystallization

Perineuronal nets (PNN) of the extracellular matrix are dense aggregations of chondroitin-sulfate proteoglycans that usually surround fast-spiking parvalbumin-expressing inhibitory interneurons (PV). The development of PNN around PV appears specifically at the end of sensitive periods of visual learning and limits the synaptic plasticity in the visual cortex of mammals. Seasonal songbirds display a high level of adult neuroplasticity associated with vocal learning, which is regulated by fluctuations of circulating testosterone concentrations. Seasonal changes in testosterone concentrations and in neuroplasticity are associated with vocal changes between the non-breeding and breeding seasons. Increases in blood testosterone concentrations in the spring lead to the annual crystallization of song so that song becomes more stereotyped. Here we explore whether testosterone also regulates PNN expression in the song control system of male and female canaries. We show that, in both males and females, testosterone increases the number of PNN and of PV neurons in the three main telencephalic song control nuclei HVC, RA (nucleus robustus arcopallialis) and Area X and increases the PNN localization around PV interneurons. Singing activity was recorded in males and quantitative analyses demonstrated that testosterone also increased male singing rate, song duration and song energy while decreasing song entropy. Together, these data suggest that the development of PNN could provide the synaptic stability required to maintain the stability of the testosterone-induced crystallized song. This provides the new evidence for a role of PNN in the regulation of adult seasonal plasticity in seasonal songbirds.