Morphological characterization of HVC projection neurons in the zebra finch (Taeniopygia guttata)

Abstract

Singing behavior in the adult male zebra finch is dependent upon the activity of a cortical region known as HVC (proper name). The vast majority of HVC projection neurons send primary axons to either the downstream premotor nucleus RA (robust nucleus of the arcopallium, or primary motor cortex) or Area X (basal ganglia), which play important roles in song production or song learning, respectively. In addition to these long-range outputs, HVC neurons also send local axon collaterals throughout that nucleus. Despite their implications for a range of circuit models, these local processes have never been completely reconstructed. Here, we use in vivo single-neuron Neurobiotin fills to examine 40 projection neurons across 31 birds with somatic positions distributed across HVC. We show that HVC and HVC neurons have categorically distinct dendritic fields. Additionally, these cell classes send axon collaterals that are either restricted to a small portion of HVC ("local neurons") or broadly distributed throughout the entire nucleus ("broadcast neurons"). Overall, these processes within HVC offer a structural basis for significant local processing underlying behaviorally relevant population activity.

Local axonal conduction delays underlie precise timing of a neural sequence

“…To estimate these delays, we needed a measure for axonal pathlength as well as conduction velocity. Axonal pathlengths to each synapse were determined using previous observations from our laboratory; the relative position and abundance of synapses were measured using electron microscopy (Kornfeld et al, 2017) and the total axonal extent for each neuron was obtained from 22 complete light microscopic reconstructions of local axonal collaterals (Figure 3B) (Benezra et al, 2018). Our first estimate for conduction velocity was 0.3 mm/ms, a value reported for local unmyelinated axons in mammalian neocortex (Helmstaedter et al, 2008; Hirsch and Gilbert, 1991; Shu et al, 2007).…”

“…We estimate that conduction delays are significantly larger than those afforded by other biophysical parameters, such as synaptic delays (Sabatini and Regehr, 1996) (~0.1 ms) and postsynaptic integration time (Long et al, 2010) (~5 ms during singing). Notably, the dendrites of HVC premotor neurons are highly compact (i.e., a spatial extent of less than 200µm) (Benezra et al, 2018) and are unlikely to contribute significantly, although to date no direct measurements of their electric signaling properties exist (Magee and Cook, 2000; Williams and Stuart, 2002). Therefore, approximately half of the total elapsed time of the HVC sequence could be attributed to local axonal conduction, a comparatively inflexible process that may underlie the behavioral stereotypy inherent in the adult zebra finch song (Lombardino and Nottebohm, 2000) by rendering the circuit less sensitive to perturbations (Hamaguchi et al, 2016; Swadlow et al, 1981).…”

“…We estimated the possible locations of synapses between HVC premotor neurons along local axons by combining results from previously reported anatomical data sets (Benezra et al, 2018; Kornfeld et al, 2017). We used a database of 22 reconstructions of local axon collaterals of in vivo labeled HVC premotor neurons (Benezra et al, 2018) to determine possible synapse locations irrespective of the postsynaptic target along the local axons of each neuron by sampling points along the reconstructed axons at the average distance between synapses, which has previously been determined using EM measurements along HVC premotor neuron axons (Kornfeld et al, 2017). We then estimated which of these possible synapse locations could target other HVC premotor neurons.…”

“…For each neuron, we generated N Syn * L Neuron / L Avg samples from this log-normal distribution. Here, N Syn = 170 is the average number of synapses made by each HVC premotor neuron onto other premotor neurons, L Neuron is the total axonal pathlength of this specific premotor neuron and L Avg is the average axonal pathlength across all 22 premotor neurons (Benezra et al, 2018). Samples beyond the maximum pathlength distance to the soma were repeated.…”

Addition of new neurons and the emergence of a local neural circuit for precise timing

“…Only a single connection between a pair of neurons was allowed. Parameter σ RA → I was chosen to match the upper bound on the number of postsynaptic HVC INT partners for an HVC RA neuron [14, 59]. On average an HVC RA neuron contacted 11.6% of HVC INT neurons.…”