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Dense Circuit Reconstruction To Understand Neuronal Computation: Focus On Zebrafish
Dense Circuit Reconstruction To Understand Neuronal Computation: Focus On Zebrafish
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Contents
DENSE CIRCUIT RECONSTRUCTION: AIMS AND CHALLENGES . . . . . . . . . 276
CHALLENGES IN MECHANISTIC SYSTEMS NEUROSCIENCE
AND THE POTENTIAL OF ZEBRAFISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
INSIGHTS INTO NEURONAL COMPUTATIONS FROM DYNAMICAL
CONNECTOMICS IN ZEBRAFISH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
The Combinatorial Logic of Motion Selectivity in the Pretectum . . . . . . . . . . . . . . . . . 282
Direction-Selective Connectivity in Mechanosensory Organs
of the Lateral Line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Wiring Specificity of Speed-Related Motor Circuits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
A Brain Stem Oculomotor Module Underlying Temporal Integration
Controlling Eye Position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Whitening of Odor Representations in the Olfactory Bulb
by Tuning-Dependent Reciprocal Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
CONCLUSIONS AND OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Number of synapses
Brain
Mitral cells
Fixation IN type 1
Staining
Embedding IN type 2
Calcium imaging Volume EM
IN type 3
Presynaptic
Stack alignment
Neuron tracing
Synapse annotation
10 μm 1.5 μm 500 nm
Figure 1
Dynamical connectomics. (a) Dynamical connectomics workflow. (b) Dense and sparse sampling of connectivity. Connectivity is shown
between different types of neurons in the olfactory bulb (OB) of a zebrafish larva [mitral cells and three types of interneurons (INs)] (top
left). For simplicity, 10 neurons of each class were randomly drawn from the full wiring diagram of more than 1,000 neurons (Wanner
& Friedrich 2020). The other charts illustrate hypothetical fragments of the wiring diagram that may be obtained with sparse sampling
methods. Genetic targeting refers to methods that collectively detect all synaptic connections between two neuron types in one
direction. (c) Volumetric reconstructions of four neurons in the adult zebrafish OB showing anatomical structure beyond the diffraction
limit of light microscopy, including an overview (left), an enlargement of a subregion (center), and raw electron microscopy (EM) image
data (right). Overlays show cross sections of neurites from neurons with corresponding colors. Images illustrate that neuronal
reconstructions from volume EM data contain a wealth of ultrastructural information in addition to information about connectivity.
Reconstructions were performed by N. Moenig and M. Januszewski.
between defined neuron types (Feinberg et al. 2008) and methods to trace monosynaptically con-
nected neurons (Talay et al. 2017, Wickersham et al. 2007) also lack the power to resolve higher-
order network structure (Figure 1b). For example, monosynaptic viral tracing may reveal that
two neurons A and B are presynaptic to the same target neuron C, but it cannot be determined
whether the three neurons form a loop because the connectivity between A and B is unknown.
A comprehensive understanding of network structure therefore requires dense reconstructions of
wiring diagrams.
Fundamental concepts in neuroscience assume, often implicitly, that higher brain functions
and memory depend on connections among functionally defined ensembles of neurons. However,
many of these concepts are difficult to test without dense circuit reconstructions. Dynamical
MoNL MoNR
Right
eye FELR FEL FSP
320 μm
Midline
Left FELR FER FSP
eye MoNR MoNL 26,000 sections
@ 25 nm
BSP BEL FEL
MoTR ca. 80,000 neurons
BER
Excitatory interactions
Inhibitory interactions c
250 μm
Figure 2
Reconstruction of neurons in the larval zebrafish brain. (a) Flow diagram illustrating the propagation of
activity in a proposed network of pretectal neurons in response to counterclockwise rotational visual flow.
Boxes and ovals represent different functional classes of pretectal neurons; color highlights represent neuron
classes receiving input. Proposed interactions involving neuron types that do not receive input in response to
this stimulus (gray) are not shown. For details and proposed flow diagrams for other inputs, see Kubo et al.
(2014). This example is characteristic for the description of neuronal circuits by flow diagrams. Panel a
adapted from Kubo et al. (2014). (b) Three-dimensional outline of a larval zebrafish brain five days
postfertilization that was imaged completely by serial block-face scanning electron microscopy at a
resolution of 14 × 14 × 25 nm3 . (c) Visualization of 10 pretectal neurons out of a larger sample of
reconstructed neurons in this volume. Note long-range projections across the midline. Abbreviations: BEL,
excited by backward translation, left; BER, excited by backward translation, right; BSP, specific for backward
translation; CCW, counterclockwise; FEL, excited by forward translation, left; FELR, excited by forward
translation, left or right; FER, excited by forward translation, right; FSP, specific for forward translation;
MoNL, monocular neuron sensitive to nasalward motion, left; MoNR, monocular neuron sensitive to
nasalward motion, right; MoTL, monocular neuron sensitive to temporalward motion, left; MoTR,
monocular neuron sensitive to temporalward motion, right. Data for panels b and c from F. Kubo, F. Svara,
H. Baier, and W. Denk.
100 × 200 × 300 μm3 have been achieved, and novel technological developments are pushing
toward a cubic millimeter (https://microns-explorer.org/) (Kornfeld & Denk 2018).
Pioneering studies used vEM and neuron reconstruction to determine the structural basis of
direction selectivity in retinal ganglion cells (Briggman et al. 2011). Additional studies provided
insights into mechanisms of sensory processing in the retina (Ding et al. 2016, Kim et al. 2014,
Marc et al. 2013) and V1 (Bock et al. 2011, Lee et al. 2016) and uncovered previously unknown
Feature suppression
Mitral cells
(output)
Mitral cells:
Excitatory interactions output r low
Inhibitory interactions (steady state)
c
Connectivity
Disynaptic connectivity
Activity (odor tuning)
Interneurons
1.5
× Odors
(normalized)
Mitral cells ×
1.0 Mitral cells
0.5
Mitral cells Mitral cells
Disynaptic × Correlation
×
connectivity 0 Odors
Interneurons –1 0 1
Tuning correlation
d e
Feature suppression: Feature-responsive WMC ← IN
mechanism Mitral cells Retrograde
tracing
Interneurons
Interneurons
(monosynaptic
input)
Retrograde
Mitral cells WMC → IN tracing
Mitral cells
(Disynaptic input)
Figure 3
Whitening of odor representations in the zebrafish olfactory bulb (OB) by specific wiring. (a) Interactions
between main neuron types in the larval OB. (b) Schematic illustration of pattern decorrelation by feature
suppression. (c) Relation between tuning curve similarity (x axis, Pearson correlation, binned) and disynaptic
connectivity (y axis, means ± standard error of the mean). This analysis required dense measurements of
connections and activity. The box on the left illustrates how disynaptic connectivity between mitral cells was
computed by multiplication of monosynaptic connectivity matrices from mitral cells to interneurons and
from interneurons to mitral cells. The box on the right illustrates how correlations between tuning curves
were calculated from responses of mitral cells to eight different odors. Panel c adapted from Wanner &
Friedrich (2020). (d) Mechanism of feature suppression by selective reciprocal connectivity within
feature-responsive cohorts. (e) Schematic illustration of in silico disynaptic retrograde tracing through the
wiring diagram. Images show three mitral cells that responded to a common molecular feature (blue),
presynaptic interneurons (green), and mitral cells presynaptic to these interneurons (orange). Transparency
indicates number of connections; only the most strongly connected neurons are shown. Panel e adapted from
Wanner & Friedrich (2020). Note that strongly connected disynaptic mitral cell inputs to the
feature-selective cohort include the cohort itself (black arrowheads).
ACKNOWLEDGMENTS
We thank F. Kubo, F. Svara, H. Baier, W. Denk, A. Vishwanathan, A. Ramirez, A. Sood, M.
Goldman, E. Aksay, H. S. Seung, N. Moenig, and M. Januszewski for highly valuable input. We
are also grateful to G. Keller for comments on the manuscript. The authors are supported by the
Novartis Research Foundation, the Swiss National Science Foundation (grants 31003A_135196,
310030B_1528331, and 310030A_172925), the European Research Council under the European
Union’s Horizon 2020 Research and Innovation Program (grant 742576), and a C.V. Starr Fel-
lowship in Neuroscience from Princeton University (to A.A.W.).
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