Morphological and Anatomical Classification of Neurobiotin-Filled Premotor Neurons in the Avian Motor Cortex

Abstract

Understanding neuronal morphologies is essential for unraveling the complexity of neural networks and the intricate mechanisms underlying brain function. That’s because the unique morphological signature of a neuron and its architecture dictate its function through orchestrating the neuron’s intrinsic properties and synaptic connections that paves the way to learning and behavior. The songbird’s brain circuitry serves as an excellent model for investigating the neural basis of vocal learning and production, a complex behavior that requires precise control over the vocal production system. The High Vocal Center (HVC), a premotor nucleus in the avian forebrain analogous to layer V in the motor cortex, is a crucial brain region involved in this process, as it is responsible for the production of learned song. Among the several neuronal populations in HVC, the premotor HVCRA neurons, which project to the robust nucleus of the arcopallium, play a critical role in controlling the precise timing and sequence of the vocal output. These neurons had been under long-sought investigations due to their significance in our understanding of rhythmic pattern generators in the brain, as they are known to be part of one of the most temporally precise neural sequences known in nature to date. Recent electrophysiological studies have identified four subclasses of HVCRA neurons (Daou and Margoliash, under review), yet their anatomical and morphological properties are still unclear. To address this gap, we analyzed a large dataset of 3D confocal images of HVCRA neurons collected by Daou et al. using intracellular neurobiotin injections that were processed histologically post-hoc and imaged on a confocal microscope. Our results show four unique subclasses of HVCRA neurons that show differences in soma sizes, spine densities, number of primary dendrites, branching, Sholl analysis, and other dendritic patterns and complexities. This study shed light on the morphological properties of HVCRA neurons in zebra finches, and their possible implications for the neural mechanisms underlying vocal learning and production. By identifying these differences between the subclasses of HVCRA neurons, we can gain a better understanding of how these neurons contribute to the precise control of vocal output, and ultimately, how the various morphological variations orchestrate the underlying behavior.