Adult neurogenesis is a process in which new neurons are generated in neurogenic niches and become recruited into distinct regions of the mature brain. In the adult songbird brain, new neurons are incorporated into areas that facilitate learning, production and maintenance of song. The striatal song nucleus Area X constantly receives new medium spiny neurons (MSNs) throughout adulthood, but it was not known if they are functionally integrated into the preexisting circuitry. To address this question, I applied Bromodeoxyuridine (BrdU) and lentiviral vector-mediated labelling of progenitor cells and examined the maturation, connectivity and singing elicited activation and of their progeny in Area X after different survival periods. Six weeks after their birth, the majority of new neurons expressed a marker for mature MSNs, show pre- and postsynaptic connections and expressed dopamine receptors, indicative of dopaminergic innervation. The expression of the immediate early gene EGR-1 (early growth response protein 1) was used to assess if and at what age new neurons were activated by singing. Already three weeks after their labelling, a small fraction of new MSNs expressed EGR-1 after singing and this fraction increased with progressing maturation. Measuring MSN densities in zebra finches up to seven years of age provided insights into the dynamics of striatal adult neurogenesis and revealed that it is a process of constant new neuron addition. New MSNs that are recruited into Area X express the forkhead box protein P2 (FoxP2). This transcription factor has important functions in mammalian brain development and mutations in FOXP2 cause speech and language impairments in humans. In zebra finches, correct FoxP2 expression levels in Area X are crucial for successful song learning and for song modulation between different social contexts. FoxP2 levels in Area X are high during the phase of song learning but generally low in adults and are downregulated by singing. MSNs in Area X exhibit different FoxP2 expression levels. Since FoxP2 downregulation after singing only occurs in MSNs with low FoxP2 levels (FoxP2low) and not in MSNs with high FoxP2 levels (FoxP2high), I postulated that the latter were recently recruited and need to become FoxP2low MSNs before they would be activated by singing. This hypothesis was tested by measuring FoxP2 protein levels and EGR-1 expression in individual new MSNs of singing and non-singing birds at different time points after BrdU birth dating. Interestingly, FoxP2high and FoxP2low MSNs were equally activated during singing, indicating that this is a process independent of FoxP2 levels. Further, I identified that one third of new MSNs expressed FoxP2 at high levels during early stages of their maturation. However, the majority of matured MSNs expressed FoxP2 at low levels, indicating an age-related decrease of FoxP2 levels in a subset of newly recruited MSNs. Because Foxp2 was shown to enhance neuronal outgrowth and differentiation, I analyzed the dendrite morphology and the density of dendritic spines of FoxP2high and FoxP2low new MSNs that were virally labelled and expressed the green fluorescent protein. FoxP2high new MSNs had more complex dendrites and a higher density of the mature mushroom spines than FoxP2low new MSNs and thus probably received more pallial inputs during a narrow timeframe of their maturation. Comparing my results to what is known about MSNs of the direct and indirect pathway of the basal ganglia of rodents, I hypothesize that early differences in FoxP2 levels and concomitant diverging new MSNs morphology might indicate the existence of distinct MSN subtypes in Area X of zebra finches. Altogether, the presented data illustrate that new MSNs recruited into Area X of adult zebra finches are functional and might play a role for the maintenance of song. Within the first six weeks after their birth new MSNs exhibited dynamic FoxP2 expression levels which are liked to their dendritic arborization and spine density, thus broadening FoxP2 function by an implication in striatal adult neurogenesis.
