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

Jörgen Kornfeld,Rajeevan T Narayanan,Robert Egger,Fabian Svara,Marcel Oberlaender,Sam E Benezra,Michael A Long,Winfried Denk

doi:10.7554/elife.24364

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.

Highlights

We used a structural approach combining anatomical reconstructions of complete cells in light microscopy (LM) with high-throughput serial block-face electron microscopy (SBEM) (Denk and Horstmann, 2004; Seung, 2009). We used both LM and EM, because anatomically, synapses can only be identified unambiguously in EM, but currently the size of the volume that can be studied by EM is limited to several hundred microns in one dimension (Helmstaedter, 2013)
When we examined three BDA-stained axons that each emerged from labeled somata in the SBEM dataset, we found that of 121 connections, 115 terminated on dendrites of inhibitory cells but only six onto excitatory cells, four of which being other HVC(RA) cells (e.g., Figure 2e)
We have shown that the synaptic architecture in HVC contains a density of connections between HVC(RA) neurons that might be sufficient to support a synaptic-chain model, whereby precisely timed sequences of action potential bursts in HVC(RA) neurons are generated by a wave of activity propagating via synaptic connections among these neurons without the need for inhibition-mediated propagation of activity (Yildiz and Kiebel, 2011) or to involve structures outside HVC (Hamaguchi and Mooney, 2012; Goldin et al, 2013; Hamaguchi et al, 2016)

Summary

Introduction

Neural sequences are central to many models of circuit function (Diesmann et al, 1999; Jin et al, 2007; Gibb et al, 2009; Fiete et al, 2010; Mostafa and Indiveri, 2014; Cannon et al, 2015a; Rajan et al, 2016), and neurons often fire sequentially during specific behaviors (Hahnloser et al, 2002; Peters et al, 2014; Mello et al, 2015) or cognitive states (Pastalkova et al, 2008; Harvey et al, 2012), but the network properties that underlie such dynamics are poorly understood. We explore the synaptic connections within the zebra finch HVC, which is central to generating the neuronal activity necessary to coordinate activation of vocal muscles during the highly reproducible courtship song (Nottebohm et al, 1976; Vu et al, 1994; Aronov et al, 2008; Long and Fee, 2008). An HVC(RA) neuron is either silent or active in the form of a burst of action potentials that occurs at a single precise and cell-specific time (Hahnloser et al, 2002; Kozhevnikov and Fee, 2007; Long et al, 2010; Vallentin and Long, 2015).

Methods

Results

Conclusion