High-throughput serial reconstruction of songbird brain areas

Electrophysiological and behavioral studies have begun to provide insights into the learning and production of birdsong. Complex song-related neuronal activity patterns were discovered in multiple brain areas. Efferent anatomical connections of premotor areas have been fairly well described. For example, different populations of neurons in the high vocal center (HVC) project to the robust nucleus of the archistriatum (RA) and to Area X. However, the ultra-structure of intra-area and afferent connections remains unknown.

Knowledge of the ultra-structure may be essential for understanding the functional mechanisms of neural activity pattern generation. The aim of our research is to reconstruct part of a single premotor area using microscopy. We use in-vivo injections of fluorescent tracers to label different neuron populations (retrograde tracing to label dendrites and cell bodies and anterograde tracing to label axons at the area of interest). In addition immunohistochemistry is applied to fixed brain slices to label specific cell types (such as inhibitory neurons) or parts of cells (for example postsynaptic densities). Conventional fluorescent light microscopy (FLM) provides anatomical information down to a scale of about 400 nm and therefore does not allow unambiguous imaging of finer details, such as synapses (postsynaptic densities are around 200 to 1000 nm in size). We use transmission electron microscopy (TEM) which allows sub-nanometer resolution, correlatively with FLM on ultra-thin serial sections to overcome this drawback. Our technique enables us to gather information about inter-area and afferent connectivity of neurons as well as the ultra-structure of synaptic connections.