EM connectomics reveals axonal target variation in a sequence-generating network

Abstract

The sequential activation of neurons has been observed in various areas of the brain, but in no case is the underlying network structure well understood. Here we examined the circuit anatomy of zebra finch HVC, a cortical region that generates sequences underlying the temporal progression of the song. We combined serial block-face electron microscopy with light microscopy to determine the cell types targeted by HVC(RA) neurons, which control song timing. Close to their soma, axons almost exclusively targeted inhibitory interneurons, consistent with what had been found with electrical recordings from pairs of cells. Conversely, far from the soma the targets were mostly other excitatory neurons, about half of these being other HVC(RA) cells. Both observations are consistent with the notion that the neural sequences that pace the song are generated by global synaptic chains in HVC embedded within local inhibitory networks.DOI: http://dx.doi.org/10.7554/eLife.24364.001

Modeling temperature manipulations in a circular model of birdsong production

“…For the last type of syllables, we show that the breaking is a natural consequence of the slowing of the activity in the telencephalon during thermal manipulation of HVC while keeping the brainstem activity unchanged. As each syllable type needs a different pattern of activity in our model, we support a recent view in which different parallel pathways are represented anatomically in HVC [25,26].…”

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

“…Paired recordings in vitro have found sparse evidence for HVC (RA) ‐HVC (RA) connections (Kosche et al, ; Mooney & Prather, ), motivating a number of alternative models in which disynaptic inhibition within HVC is sufficient to generate song‐related sequences (Armstrong & Abarbanel, ; Gibb et al, ). In a previous study, using an electron microscopic approach capable of revealing the identity of postsynaptic partners of HVC (RA) neurons, we found that 17% of synapses made by HVC (RA) axons form connections onto other HVC (RA) neurons (Kornfeld et al, ) and that the total density of these connections is ∼75 synapses/mm (Kornfeld et al, ). With this data set, we update our previous estimate (Kornfeld et al, ) for the average axonal length of HVC (RA) collaterals (13.31 ± 1.44 mm mean ± SEM ) by including data from an additional nine neurons.…”

“…On the day of the experiment, a region of dense retrograde labeling in HVC was defined using two‐photon microscopy (MOM, Sutter Instrument Company, Novato, CA), and a small pipette access hole was drilled in the cover glass adjacent to this region. In each animal, one to two cells were targeted under two‐photon guidance and loaded with 3% Neurobiotin (Vector Labs, Burlingame, CA) in 150 mM K‐Gluconate using a glass electrode (4–6 MΩ) in a juxtacellular configuration by applying 250 ms current pulses (2–15 nA; 2 Hz) for ∼10 min (Kornfeld et al, ; Narayanan et al, ; Pinault, ). Pipettes were visualized by adding 40 µM of Alexa 488 (Invitrogen) to the internal solution.…”

“…During singing, both HVC projection neuron types exhibit sparse bursts that form sequences of activity at the network level, with different neurons precisely active at different moments throughout the song (Hahnloser, Kozhevnikov, & Fee, ; Hamaguchi, Tschida, Yoon, Donald, & Mooney, ; Kozhevnikov & Fee, ; Long, Jin, & Fee, ; Lynch, Okubo, Hanuschkin, Hahnloser, & Fee, ; Picardo et al, ; Vallentin & Long, ). The importance of local processing within HVC for the generation of these neural sequences remains controversial, in part because the structure of circuits within HVC are only partially understood (Kornfeld et al, ; Kosche, Vallentin, & Long, ; Mooney & Prather, ; Stauffer et al, ).…”

“…A major gap in our knowledge of HVC function stems from an incomplete picture of the local wiring within that nucleus, especially with respect to the axon collaterals of excitatory projection neurons. These local processes have been shown to form interconnections within HVC (Kornfeld et al, ; Kosche et al, ; Mooney & Prather, ) and have been strongly implicated in the generation of sequences of neural activity (Galvis, Wu, Hyson, Johnson, & Bertram, ; Gibb, Gentner, & Abarbanel, ; Hanuschkin, Diesmann, & Morrison, ; Jin, ; Long et al, ). Although the basic morphological characteristics of HVC neurons have been investigated through bulk labeling (Benton, Cardin, & DeVoogd, ; Fortune & Margoliash, ; Nixdorf, Davis, & DeVoogd, ; Vates, Broome, Mello, & Nottebohm, ; Yip, Miller‐Sims, & Bottjer, ) or partially reconstructed single‐cell fills (Dutar et al, ; Katz & Gurney, ; Mooney, ), surprisingly little is known about their anatomy because these reconstructions were incomplete.…”