Calcium‐binding proteins define interneurons in HVC of the zebra finch (Taeniopygia guttata)

Abstract

Nucleus HVC of the avian song system is essential to song patterning and is a prime site for auditory-vocal integration important to vocal learning. These processes require precise, high-frequency action potential activity, which, in other systems, is often correlated with the expression of calcium-binding proteins. To characterize any such functional specializations in HVC, we retrogradely labeled projection neurons innervating HVC's known targets, namely, area X or nucleus robustus arcopallialis (RA), then stained HVC sections with antibodies to the calcium-binding proteins parvalbumin, calbindin, and calretinin. Under epifluorescent illumination, neither projection neuron type exhibited detectable levels of calcium-binding protein immunoreactivity, whereas a third cell type, made up of nonprojection neurons (interneurons), was immunopositive for one, two, or all three of the calcium-binding proteins. In fact, most of these interneurons were either doubly or triply labeled. To explore the link between the electrical and calcium-binding protein properties of individual HVC neurons, we used intracellular methods in brain slices to record from identified HVC cell types based on their intrinsic electrical properties. Intracellular neurobiotin combined with immunostaining revealed that fast-spiking interneurons, but not the slower-spiking projection neurons, were positive for one or more calcium-binding proteins. Confocal microscopy confirmed these results and also revealed that RA-projecting cells might contain very low levels of parvalbumin. These results indicate that HVC interneurons are specialized in their calcium-binding proteins and suggest how it might be possible to resolve the details of HVC microcircuits underlying song selectivity and auditory-vocal learning.