Cross-species conservation in the regulation of parvalbumin by perineuronal nets.

Parvalbumin (PV) neurons play an integral role in regulating neural dynamics and plasticity. Therefore, understanding the factors that regulate PV expression is important for revealing modulators of brain function. While the contribution of PV neurons to neural processes has been studied in mammals, relatively little is known about PV function in non-mammalian species, and discerning similarities in the regulation of PV across species can provide insight into evolutionary conservation in the role of PV neurons. Here we investigated factors that affect the abundance of PV in PV neurons in sensory and motor circuits of songbirds and rodents. In particular, we examined the degree to which perineuronal nets (PNNs), extracellular matrices that preferentially surround PV neurons, modulate PV abundance as well as how the relationship between PV and PNN expression differs across brain areas and species and changes over development. We generally found that cortical PV neurons that are surrounded by PNNs (PV+PNN neurons) are more enriched with PV than PV neurons without PNNs (PV-PNN neurons) across both rodents and songbirds. Interestingly, the relationship between PV and PNN expression in the vocal portion of the basal ganglia of songbirds (Area X) differed from that in other areas, with PV+PNN neurons having lower PV expression compared to PV-PNN neurons. These relationships remained consistent across development in vocal motor circuits of the songbird brain. Finally, we discovered a causal contribution of PNNs to PV expression in songbirds because degradation of PNNs led to a diminution of PV expression in PV neurons. These findings in reveal a conserved relationship between PV and PNN expression in sensory and motor cortices and across songbirds and rodents and suggest that PV neurons could modulate plasticity and neural dynamics in similar ways across songbirds and rodents.

Cross-species conservation in the regulation of parvalbumin by perineuronal nets.

Parvalbumin (PV) neurons play an integral role in regulating neural dynamics and plasticity. Therefore, understanding the factors that regulate PV expression is important for revealing modulators of brain function. While the contribution of PV neurons to neural processes has been studied in mammals, relatively little is known about PV function in non-mammalian species, and discerning similarities in the regulation of PV across species can provide insight into evolutionary conservation in the role of PV neurons. Here we investigated factors that affect the abundance of PV in PV neurons in sensory and motor circuits of songbirds and rodents. In particular, we examined the degree to which perineuronal nets (PNNs), extracellular matrices that preferentially surround PV neurons, modulate PV abundance as well as how the relationship between PV and PNN expression differs across brain areas and species and changes over development. We generally found that cortical PV neurons that are surrounded by PNNs (PV+PNN neurons) are more enriched with PV than PV neurons without PNNs (PV-PNN neurons) across both rodents and songbirds. Interestingly, the relationship between PV and PNN expression in the vocal portion of the basal ganglia of songbirds (Area X) differed from that in other areas, with PV+PNN neurons having lower PV expression compared to PV-PNN neurons. These relationships remained consistent across development in vocal motor circuits of the songbird brain. Finally, we discovered a causal contribution of PNNs to PV expression in songbirds because degradation of PNNs led to a diminution of PV expression in PV neurons. These findings reveal a conserved relationship between PV and PNN expression in sensory and motor cortices and across songbirds and rodents and suggest that PV neurons could modulate plasticity and neural dynamics in similar ways across songbirds and rodents.

Perineuronal nets in HVC and plasticity in male canary song.

Songbirds learn their vocalizations during developmental sensitive periods of song memorization and sensorimotor learning. Some seasonal songbirds, called open-ended learners, recapitulate transitions from sensorimotor learning and song crystallization on a seasonal basis during adulthood. In adult male canaries, sensorimotor learning occurs each year in autumn and leads to modifications of the syllable repertoire during successive breeding seasons. We previously showed that perineuronal nets (PNN) expression in song control nuclei decreases during this sensorimotor learning period. Here we explored the causal link between PNN expression in adult canaries and song modification by enzymatically degrading PNN in HVC, a key song control system nucleus. Three independent experiments identified limited effects of the PNN degradation in HVC on the song structure of male canaries. They clearly establish that presence of PNN in HVC is not required to maintain general features of crystallized song. Some suggestion was collected that PNN are implicated in the stability of song repertoires but this evidence is too preliminary to draw firm conclusions and additional investigations should consider producing PNN degradations at specified time points of the seasonal cycle. It also remains possible that once song has been crystallized at the beginning of the first breeding season, PNN no longer play a key role in determining song structure; this could be tested by treatments with chondroitinase ABC at key steps in ontogeny. It would in this context be important to develop multiple stereotaxic procedures allowing the simultaneous bilateral degradation of PNN in several song control nuclei for extended periods.

Development of Perineuronal Nets during Ontogeny Correlates with Sensorimotor Vocal Learning in Canaries.

Songbirds are a powerful model to study vocal learning given that aspects of the underlying behavioral and neurobiological mechanisms are analogous in many ways to mechanisms involved in speech learning. Perineuronal nets (PNNs) represent one of the mechanisms controlling the closing of sensitive periods for vocal learning in the songbird brain. In zebra finches, PNN develop around parvalbumin (PV)-expressing interneurons in selected song control nuclei during ontogeny and their development is delayed if juveniles are deprived of a tutor. However, song learning in zebra finches takes place during a relatively short period of development, and it is difficult to determine whether PNN development correlates with the end of the sensory or the sensorimotor learning period. Canaries have a longer period of sensorimotor vocal learning, spanning over their first year of life so that it should be easier to test whether PNN development correlates with the end of sensory or sensorimotor vocal learning. Here, we quantified PNN around PV-interneurons in the brain of male canaries from hatching until the first breeding season and analyzed in parallel the development of their song. PNN development around PV-interneurons specifically took place and their number reached its maximum around the end of the sensorimotor learning stage, well after the end of sensory vocal learning, and correlated with song development. This suggests that PNN are specifically involved in the termination of the sensitive period for sensorimotor vocal learning.

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.

Seasonal changes of perineuronal nets and song learning in adult canaries (Serinus canaria).

Songbirds learn their song during a sensitive period of development associated with enhanced neural plasticity. In addition, in open-ended learners such as canaries, a sensitive period for sensorimotor vocal learning reopens each year in the fall and leads to song modifications between successive breeding seasons. The variability observed in song production across seasons in adult canaries correlates with seasonal fluctuations of testosterone concentrations and with morphological changes in nuclei of the song control system (SCS). The sensitive periods for song learning during ontogeny and then again in adulthood could be controlled by the development of perineuronal nets (PNN) around parvalbumin-expressing interneurones (PV) which limits learning-induced neuroplasticity. However, this relationship has never been investigated in the context of adult vocal learning in adult songbirds. Here we explored PNN and PV expression in the SCS of adult male Fife Fancy canaries in relation to the seasonal variations of their singing behaviour. We found a clear pattern of seasonal variation in testosterone concentrations and song production. Furthermore, PNN expression was significantly higher in two specific song control nuclei, the robust nucleus of the arcopallium (RA) and the Area X of the basal ganglia, during the breeding season and during the later stages of sensorimotor song development compared to birds in an earlier stage of sensorimotor development during the fall. These data provide the first evidence that changes in PNN expression could represent a mechanism regulating the closing-reopening of sensitive periods for vocal learning across seasons in adult songbirds.

Comparing perineuronal nets and parvalbumin development between blackbird species with differences in early developmental song exposure.

Brood parasitic songbirds are a natural system in which developing birds are isolated from species-typical song and therefore present a unique opportunity to compare neural plasticity in song learners raised with and without conspecific tutors. We compared perineuronal nets (PNN) and parvalbumin (PV) in song control nuclei in juveniles and adults of two closely related icterid species (i.e. blackbirds): brown-headed cowbirds (Molothrus ater; brood parasite) and red-winged blackbirds (Agelaius phoeniceus; non-parasite). The number of PV cells per nucleus was significantly higher in adults compared with juveniles in the nucleus HVC and the robust nucleus of the arcopallium (RA), whereas no significant species difference appeared in any region of interest. The number of PNN per nuclei was significantly higher in adults compared with juveniles in HVC, RA and Area X, but only RA exhibited a significant difference between species. PV cells surrounded by PNN (PV+PNN) also exhibited age-related differences in HVC, RA and Area X, but RA was the only region in which PV+PNN exhibited significant species differences. Furthermore, a significant interaction existed in RA between age and species with respect to PNN and PV+PNN, revealing RA as a region displaying differing plasticity patterns across age and species. Additional comparisons of PNN and PV between adult male and female cowbirds revealed that males have greater numbers of all three measures in RA compared with females. Species-, sex- and age-related differences in RA suggest that species differences in neural plasticity are related to differences in song production rather than sensitivity to song learning, despite a stark contrast in early exposure to conspecific male tutors.

Testosterone or Estradiol When Implanted in the Medial Preoptic Nucleus Trigger Short Low-Amplitude Songs in Female Canaries.

In male songbirds, the motivation to sing is largely regulated by testosterone (T) action in the medial preoptic area, whereas T acts on song control nuclei to modulate aspects of song quality. Stereotaxic implantation of T in the medial preoptic nucleus (POM) of castrated male canaries activates a high rate of singing activity, albeit with a longer latency than after systemic T treatment. Systemic T also increases the occurrence of male-like song in female canaries. We hypothesized that this effect is also mediated by T action in the POM. Females were stereotaxically implanted with either T or with 17ß-estradiol (E2) targeted at the POM and their singing activity was recorded daily during 2 h for 28 d until brains were collected for histological analyses. Following identification of implant localizations, three groups of subjects were constituted that had either T or E2 implanted in the POM or had an implant that had missed the POM (Out). T and E2 in POM significantly increased the number of songs produced and the percentage of time spent singing as compared with the Out group. The songs produced were in general of a short duration and of poor quality. This effect was not associated with an increase in HVC volume as observed in males, but T in POM enhanced neurogenesis in HVC, as reflected by an increased density of doublecortin-immunoreactive (DCX-ir) multipolar neurons. These data indicate that, in female canaries, T acting in the POM plays a significant role in hormone-induced increases in the motivation to sing.

Timing of perineuronal net development in the zebra finch song control system correlates with developmental song learning.

The appearance of perineuronal nets (PNNs) represents one of the mechanisms that contribute to the closing of sensitive periods for neural plasticity. This relationship has mostly been studied in the ocular dominance model in rodents. Previous studies also indicated that PNN might control neural plasticity in the song control system of songbirds. To further elucidate this relationship, we quantified PNN expression and their localization around parvalbumin interneurons at key time-points during ontogeny in both male and female zebra finches, and correlated these data with the well-described development of song in this species. We also extended these analyses to the auditory system. The development of PNN during ontogeny correlated with song crystallization although the timing of PNN appearance in the four main telencephalic song control nuclei slightly varied between nuclei in agreement with the established role these nuclei play during song learning. Our data also indicate that very few PNN develop in the secondary auditory forebrain areas even in adult birds, which may allow constant adaptation to a changing acoustic environment by allowing synaptic reorganization during adulthood.

Perineuronal nets and vocal plasticity in songbirds: A proposed mechanism to explain the difference between closed-ended and open-ended learning.

Perineuronal nets (PNN) are aggregations of chondroitin sulfate proteoglycans surrounding the soma and proximal processes of neurons, mostly GABAergic interneurons expressing parvalbumin. They limit the plasticity of their afferent synaptic connections. In zebra finches PNN develop in an experience-dependent manner in the song control nuclei HVC and RA (nucleus robustus arcopallialis) when young birds crystallize their song. Because songbird species that are open-ended learners tend to recapitulate each year the different phases of song learning until their song crystallizes at the beginning of the breeding season, we tested whether seasonal changes in PNN expression would be found in the song control nuclei of a seasonally breeding species such as the European starling. Only minimal changes in PNN densities and total number of cells surrounded by PNN were detected. However, comparison of the density of PNN and of PNN surrounding parvalbumin-positive cells revealed that these structures are far less numerous in starlings that show extensive adult vocal plasticity, including learning of new songs throughout the year, than in the closed-ended learner zebra finches. Canaries that also display some vocal plasticity across season but were never formally shown to learn new songs in adulthood were intermediate in this respect. Together these data suggest that establishment of PNN around parvalbumin-positive neurons in song control nuclei has diverged during evolution to control the different learning capacities observed in songbird species. This differential expression of PNN in different songbird species could represent a key cellular mechanism mediating species variation between closed-ended and open-ended learning strategies. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 975-994, 2017.

Anatomically discrete sex differences in neuroplasticity in zebra finches as reflected by perineuronal nets.

Large morphological sex differences in the vertebrate brain were initially identified in song control nuclei of oscines. Besides gross differences between volumes of nuclei in males and females, sex differences also concern the size and dendritic arborization of neurons and various neurochemical markers, such as the calcium-binding protein parvalbumin (PV). Perineuronal nets (PNN) of the extracellular matrix are aggregates of different compounds, mainly chondroitin sulfate proteoglycans, that surround subsets of neurons, often expressing PV. PNN develop in zebra finches song control nuclei around the end of the sensitive period for song learning and tutor deprivation, known to delay the end of the song learning sensitive period, decreases the numbers of PNN in HVC. We demonstrate here the existence in zebra finches of a major sex difference (males > females) affecting the number of PNN (especially those surrounding PV-positive cells) in HVC and to a smaller extent the robust nucleus of the arcopallium, RA, the two main nuclei controlling song production. These differences were not present in Area X and LMAN, the lateral magnocellular nucleus of the anterior nidopallium. A dense expression of material immunoreactive for chondroitin sulfate was also detected in several nuclei of the auditory and visual pathways. This material was often organized in perineuronal rings but quantification of these PNN did not reveal any sex difference with the exception that the percentage of PNN surrounding PV-ir cells in the dorsal lateral mesencephalic nucleus, MLd, was larger in females than in males, a sex difference in the opposite direction compared to what is seen in HVC and RA. These data confirm and extend previous studies demonstrating the sex difference affecting PNN in HVC-RA by showing that this sex difference is anatomically specific and does not concern visual or auditory pathways.