Neurogenesis continues in the adult songbird brain. Many telencephalic song control regions incorporate new neurons into their existing circuits in adulthood. One song nucleus that receives many new neurons is Area X. Because this striatal region is crucial for song learning and song maintenance the recruitment of new neurons into Area X could influence these processes. As an entry point into addressing this possibility, we investigated the maturation and connectivity within the song circuit and behavioral activation of newly generated Area X neurons. Using BrdU birth dating and virally mediated GFP expression we followed adult-generated neurons from their place of birth in the ventricle to their place of incorporation into Area X. We show that newborn neurons receive glutamatergic input from pallial/cortical song nuclei. Additionally, backfills revealed that the new neurons connect to pallidal-like projection neurons that innervate the thalamus. Using in situ hybridization, we found that new neurons express the mRNA for D1- and D2-type dopamine receptors. Employing DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) and EGR-1 (early growth response protein 1) as markers for neural maturation and activation, we established that at 42 days after labeling approximately 80% of new neurons were mature medium spiny neurons (MSNs) and could be activated by singing behavior. Finally, we compared the MSN density in Area X of birds up to seven years of age and found a significant increase with age, indicating that new neurons are constantly added to the nucleus. In summary, we provide evidence that newborn MSNs in Area X constantly functionally integrate into the circuit and are thus likely to play a role in the maintenance and regulation of adult song.

The transcription factor FOXP2 is crucial for the formation and function of cortico-striatal circuits. FOXP2 mutations are associated with specific speech and language impairments. In songbirds, experimentally altered FoxP2 expression levels in the striatal song nucleus Area X impair vocal learning and song production. Overall FoxP2 protein levels in Area X are low in adult zebra finches and decrease further with singing. However, some Area X medium spiny neurons (MSNs) express FoxP2 at high levels (FoxP2 high MSNs) and singing does not change this. Because Area X receives many new neurons throughout adulthood, we hypothesized that the FoxP2 high MSNs are newly recruited neurons, not yet integrated into the local Area X circuitry and thus not active during singing. Contrary to our expectation, FoxP2 protein levels did not predict whether new MSNs were active during singing, assayed via immediate early gene expression. However, new FoxP2 high MSNs had more complex dendrites, higher spine density and more mushroom spines than new FoxP2 low MSNs. In addition, FoxP2 expression levels correlated positively with nucleus size of new MSNs. Together, our data suggest that dynamic FoxP2 levels in new MSNs shape their morphology during maturation and their incorporation into a neural circuit that enables the maintenance and social modulation of adult birdsong.The forkhead box P2 transcription factor (FOXP2) is linked to speech and language disorders. Heterozygous FOXP2 mutations in humans affect both the coordination of fine orofacial movements and language perception 1-3 . Because songbirds -like humans -need to learn most of their communicative vocalizations, they offer a unique model to study the role of FoxP2 (for nomenclature FOXP2/FoxP2 see Methods) for vocal learning and for the maintenance of learned vocalizations as adults 4 . Studying the relationship between FoxP2 and vocal learning in songbirds may inform the neurogenetic mechanism underlying the speech deficits in patients carrying FOXP2 mutations for the following reasons. The FoxP2 protein coding sequence is highly conserved between humans and songbirds as are the brain expression patterns, notably in the cerebellum and striatum 5-7 . Moreover, genetic manipulations of FoxP2 expression levels in the striatal song nucleus Area X during the critical phase of song learning lead to inaccurate and incomplete imitation of the tutor's song and more variable vocal production 3,8-10 . This phenotype bears similarities to the specific speech deficits called developmental verbal dyspraxia, DVD (or childhood apraxia of speech), that patients carrying FOXP2 mutations suffer from. The core-phenotype of DVD consists of altered precision, consistency and sequencing of movements underlying speech in the absence of neuromuscular deficits 11 . In addition, altered FoxP2 levels in adult Area X affect the dopaminergic modulation of corticostriatal signaling important to song variability and affect song maintenance 12,13 , stressing the fact that tight regulation of FoxP2 expressi...

Placozoans are a promising model system to study fundamental regeneration processes in a morphologically and genetically very simple animal. We here provide a brief introduction to the enigmatic Placozoa and summarize the state of the art of animal handling and experimental manipulation possibilities.

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