Automated synapse-level reconstruction of neural circuits in the larval zebrafish brain.

Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm3. We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system.

Local Axonal Conduction Shapes the Spatiotemporal Properties of Neural Sequences.

Sequential activation of neurons has been observed during various behavioral and cognitive processes, but the underlying circuit mechanisms remain poorly understood. Here, we investigate premotor sequences in HVC (proper name) of the adult zebra finch forebrain that are central to the performance of the temporally precise courtship song. We use high-density silicon probes to measure song-related population activity, and we compare these observations with predictions from a range of network models. Our results support a circuit architecture in which heterogeneous delays between sequentially active neurons shape the spatiotemporal patterns of HVC premotor neuron activity. We gauge the impact of several delay sources, and we find the primary contributor to be slow conduction through axonal collaterals within HVC, which typically adds between 1 and 7.5 ms for each link within the sequence. Thus, local axonal "delay lines" can play an important role in determining the dynamical repertoire of neural circuits.

High-precision automated reconstruction of neurons with flood-filling networks.

Reconstruction of neural circuits from volume electron microscopy data requires the tracing of cells in their entirety, including all their neurites. Automated approaches have been developed for tracing, but their error rates are too high to generate reliable circuit diagrams without extensive human proofreading. We present flood-filling networks, a method for automated segmentation that, similar to most previous efforts, uses convolutional neural networks, but contains in addition a recurrent pathway that allows the iterative optimization and extension of individual neuronal processes. We used flood-filling networks to trace neurons in a dataset obtained by serial block-face electron microscopy of a zebra finch brain. Using our method, we achieved a mean error-free neurite path length of 1.1 mm, and we observed only four mergers in a test set with a path length of 97 mm. The performance of flood-filling networks was an order of magnitude better than that of previous approaches applied to this dataset, although with substantially increased computational costs.

Volume EM Reconstruction of Spinal Cord Reveals Wiring Specificity in Speed-Related Motor Circuits.

Spinal interneurons coordinate the activity of motoneurons to generate the spatiotemporal patterns of muscle contractions required for vertebrate locomotion. It is controversial to what degree the orderly, gradual recruitment of motoneurons is determined by biophysical differences among them rather than by specific connections from presynaptic interneurons to subsets of motoneurons. To answer this question, we mapped all connections from two types of interneurons onto all motoneurons in a larval zebrafish spinal cord hemisegment, using serial block-face electron microscopy (SBEM). We found specific synaptic connectivity from dorsal but not from ventral excitatory ipsilateral interneurons, with large motoneurons, active only when strong force is required, receiving specific inputs from dorsally located interneurons, active only during fast swims. By contrast, the connectivity between inhibitory commissural interneurons and motoneurons lacks any discernible pattern. The wiring pattern is consistent with a recruitment mechanism that depends to a considerable extent on specific connectivity.

Progress and remaining challenges in high-throughput volume electron microscopy.

Recent advances in the effectiveness of the automatic extraction of neural circuits from volume electron microscopy data have made us more optimistic that the goal of reconstructing the nervous system of an entire adult mammal (or bird) brain can be achieved in the next decade. The progress on the data analysis side-based mostly on variants of convolutional neural networks-has been particularly impressive, but improvements in the quality and spatial extent of published VEM datasets are substantial. Methodologically, the combination of hot-knife sample partitioning and ion milling stands out as a conceptual advance while the multi-beam scanning electron microscope promises to remove the data-acquisition bottleneck.

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

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.

Prediction of folding equilibria of differently substituted peptides using one-step perturbation.

Computer simulation using long molecular dynamics (MD) can be used to simulate the folding equilibria of peptides and small proteins. However, a systematic investigation of the influence of the side-chain composition and position at the backbone on the folding equilibrium is computationally as well as experimentally too expensive because of the exponentially growing number of possible side-chain compositions and combinations along the peptide chain. Here, we show that application of the one-step perturbation technique may solve this problem, at least computationally; that is, one can predict many folding equilibria of a polypeptide with different side-chain substitutions from just one single MD simulation using an unphysical reference state. The methodology reduces the number of required separate simulations by an order of magnitude.