HVC interneurons are not renewed in adult male zebra finches.

Adult neurogenesis is a widespread phenomenon in many species, from invertebrates to humans. In songbirds, the telencephalic region, high vocal center (HVC), continuously integrates new neurons in adulthood. This nucleus consists of a heterogenous population of inhibitory interneurons (HVC(IN)) and two populations of projection neurons that send axons towards either the robust nucleus of the arcopallium (HVC(RA)) or the striatal nucleus area X (HVC(X)). New HVC neurons were initially inferred to be interneurons, because they lacked retrograde labelling from the HVC's targets. Later studies using different tracers demonstrated that HVC(RA) are replaced but HVC(X) are not. Whether interneurons are also renewed became an open question. As the HVC's neuronal populations display different physiological properties and functions, we asked whether adult HVC indeed recruits two neuronal populations or whether only the HVC(RA) undergo renewal in adult male zebra finches. We show that one month after being born in the lateral ventricle, 42% of the newborn HVC neurons were retrogradely labelled by tracer injections into the RA. However, the remaining 58% were not immunoreactive for the neurotransmitter GABA, nor for the calcium-binding proteins, parvalbumin (PA), calbindin (CB) and calretinin (CR) that characterize different classes of HVC(IN). We further established that simultaneous application of parvalbumin, calbindin and calretinin antibodies to HVC revealed approximately the same fraction of HVC neurons, i.e. 10%, as could be detected by GABA immunoreactivity. This implies that the sum of HVC(IN) expressing the different calcium-binding proteins constitute all inhibitory HVC(IN). Together these results strongly suggest that only HVC(RA) are recruited into the adult HVC.