Annual Review of Neuroscience
Dense Circuit Reconstruction
Annu. Rev. Neurosci. 2021. 44:275–93
First published as a Review in Advance on
March 17, 2021
The Annual Review of Neuroscience is online at
neuro.annualreviews.org
https://doi.org/10.1146/annurev-neuro-110220-
013050
Copyright © 2021 by Annual Reviews.
All rights reserved
to Understand Neuronal
Computation: Focus on
Zebrafish
Rainer W. Friedrich1,2 and Adrian A. Wanner3
1
Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland;
email: Rainer.Friedrich@fmi.ch
2
Faculty of Natural Sciences, University of Basel, 4003 Basel, Switzerland
3
Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey 08544, USA;
email: adrian.wanner@princeton.edu
Keywords
neuronal circuit, wiring diagram, zebrafish, neuronal computation, volume
electron microscopy
Abstract
The dense reconstruction of neuronal wiring diagrams from volumetric
electron microscopy data has the potential to generate fundamentally new
insights into mechanisms of information processing and storage in neuronal
circuits. Zebrafish provide unique opportunities for dynamical connectomics
approaches that combine reconstructions of wiring diagrams with measure-
ments of neuronal population activity and behavior. Such approaches have
the power to reveal higher-order structure in wiring diagrams that cannot
be detected by sparse sampling of connectivity and that is essential for neu-
ronal computations. In the brain stem, recurrently connected neuronal mod-
ules were identified that can account for slow, low-dimensional dynamics
in an integrator circuit. In the spinal cord, connectivity specifies functional
differences between premotor interneurons. In the olfactory bulb, tuning-
dependent connectivity implements a whitening transformation that is based
on the selective suppression of responses to overrepresented stimulus fea-
tures. These findings illustrate the potential of dynamical connectomics in
zebrafish to analyze the circuit mechanisms underlying higher-order neu-
ronal computations.
275
 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

DENSE CIRCUIT RECONSTRUCTION: AIMS AND CHALLENGES

The synaptic connectivity among neurons is one of the most fundamental determinants of neu-
ronal circuit function. During learning, experience-driven modifications of synaptic connections
are thought to store information in the connectivity pattern—the wiring diagram—to optimize
responses to future inputs. Detailed analyses of neuronal connectivity at synaptic resolution are
therefore critical to understand neuronal computations and memory. The space of possible con-
nectivity patterns is astronomical even in small circuits: In networks of N neurons with binary
connections, the number of possible wiring diagrams (2N×N ) exceeds the number of atoms in
the universe when the number of neurons exceeds 17. This simple numbers game highlights not
only the potential of network connectivity as a resource for information processing and memory
but also the principal problem of simulating biologically realistic networks without knowledge of
realistic wiring diagrams. Recent technical developments in volume electron microscopy (vEM)
and automated image analysis made it possible to determine wiring diagrams experimentally by
reconstructing large neuronal populations and their synaptic connections at high density (Denk
et al. 2012, Kornfeld & Denk 2018, Swanson & Lichtman 2016). These methods can be com-
bined with activity measurements across the same set of neurons, for example, by calcium imaging
prior to vEM (Figure 1a). We refer to this approach as dynamical connectomics because it can
establish direct relationships between the dynamics of neuronal population activity and the un-
derlying connectivity (Litwin-Kumar & Turaga 2019). An attractive vertebrate model organism
for dynamical connectomics is the zebrafish because its small brain provides important advantages
both for large-scale optical activity measurements and for dense circuit reconstruction (Friedrich
et al. 2010, 2013).
Ever since the pioneering work of Golgi and Ramón y Cajal, most high-resolution neu-
roanatomical approaches have relied on sparse labeling and imaging of neurons. As a conse-
quence, these approaches have fundamental limitations for the reconstruction of neuronal wiring
diagrams. For example, single-neuron atlases generated by projecting sparsely sampled neuronal
morphologies into a common reference brain (Kunst et al. 2019, Oh et al. 2014) are highly valu-
able resources but cannot reveal the precise connectivity among neurons. Other approaches to
map neuronal connectivity such as available molecular methods to visualize synaptic connections
276 Friedrich • Wanner
 Dense reconstruction Paired recording
a b

Number of synapses
Brain
Mitral cells

Fixation IN type 1
Staining
Embedding IN type 2
Calcium imaging Volume EM

IN type 3

Monosynaptic tracing Genetic targeting

Presynaptic
Stack alignment
Neuron tracing
Synapse annotation

Wiring diagram (structure)

Activity (function)
Postsynaptic

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

www.annualreviews.org • Dense Circuit Reconstruction 277

 connectomics is therefore unique in its ability to address a variety of fundamental questions. Six
examples are briefly discussed below.
First, the first-order statistics of connectivity between neuron types can, in principle, be deter-
mined using pairwise genetic approaches (Feinberg et al. 2008). In practice, however, it is unreal-
istic to scale this strategy to large numbers of neuron types. In a densely reconstructed circuit, in
contrast, all pairwise connection probabilities can be determined simultaneously.
Second, a central question in neuroscience is how the connectivity between neurons depends
on activity. Relations between activity and connectivity may be inferred by sparse sampling when
neurons are organized in topographical maps or tuned to systematic, low-dimensional inputs. In
primary visual cortex (V1), for example, paired recordings showed that the connection probabil-
ity is enhanced between pyramidal neurons with similar tuning to orientation (Ko et al. 2011,
Lee et al. 2016), a continuous stimulus variable that is relevant to V1. In many other brain areas,
however, relationships between the activity of neurons are difficult to assess because their activ-
ity patterns cannot easily be characterized by low-dimensional tuning curves. Moreover, if these
relationships vary as a function of experience, pooling of data across individuals becomes prob-
lematic. Dynamical connectomics alleviates these problems because each data set contains many
neuron-neuron pairs from the same individual.
Third, anatomical and physiological results are often summarized in descriptive models that
take the form of flow diagrams (e.g., Figure 2a). As discussed below, wiring diagrams provide
important information to develop more naturalistic circuit models, which are an important step
toward mechanistic analyses of neuronal computations.
Fourth, a neuronal computation may be achieved by multiple permissive circuit configurations,
but the average over all permissive configurations may not be permissive itself (e.g., when solutions
to a computational problem lie on a curved manifold in the configuration space). In such cases,
the functional connectivity underlying a specific computation cannot be determined by pooling
data from different individuals and wiring diagrams have to be reconstructed individually.
Fifth, recordings from pairs or small sets of neurons are extremely inefficient in detecting com-
plex patterns in wiring diagrams. Dense circuit reconstruction is therefore important to reveal
higher-order structural features such as network motifs (Milo et al. 2002, Schwarz & Porter 2007),
connected ensembles, or correlated connectivity between excitatory and inhibitory neurons.
Sixth, it is widely assumed that information is stored in memory networks by experience-
dependent changes in functional connectivity between specific subsets of neurons of the same
types. Hence, the functional identity of individual neurons in such memory networks is deter-
mined primarily by their connectivity. Direct tests of this hypothesis will ultimately require the
detailed analysis of connectivity among functionally defined neurons.
The dense reconstruction of neuronal circuits from image data presents technical challenges
because it requires ultrastructural resolution throughout large volumes. This has been achieved by
vEM techniques with automated sectioning that can acquire large stacks of images either by imag-
ing serial sections or by repeated imaging of the sample block-face between sections (Briggman
& Bock 2012, Denk & Horstmann 2004, Denk et al. 2012, Hayworth et al. 2014, Kornfeld &
Denk 2018, Titze & Genoud 2016). Other important technical developments include automated
segmentation and further processing of volumetric image data ( Januszewski et al. 2018, Kornfeld
& Denk 2018, Turaga et al. 2010, Wanner et al. 2020). It is usually assumed that the voxel size
of vEM data should be ≤15 nm, preferably ≤10 nm, in two dimensions and ≤50 nm, prefer-
ably <40 nm, in the third dimension to reconstruct thin neurites, which can be less than 100 nm
in diameter. In recent years, high-resolution vEM data sets satisfying these requirements have
been acquired from various species and brain areas. In multiple studies, volumes of approximately

278 Friedrich • Wanner

 a BEL b
Visual flow m
(CCW) MoTL 0μ 700
BSP BER FER 45 μm

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

www.annualreviews.org • Dense Circuit Reconstruction 279

 connectivity rules in the thalamus (Morgan et al. 2016) and cortex (Motta et al. 2019, Schmidt
et al. 2017). Structural features of neuronal circuits for sequence generation and learning were
discovered in songbirds (Kornfeld et al. 2017, 2020). In the annelid Platynereis dumerilii, a vEM
volume containing an entire animal has been mapped onto an atlas of gene expression at cellular
resolution (Randel et al. 2015, Vergara et al. 2020). In Drosophila, multiple large vEM data sets have
been acquired (Scheffer et al. 2020, Takemura et al. 2017a, Zheng et al. 2018) and used to address
various questions, including the organization of mushroom body output networks (Eichler et al.
2017, Takemura et al. 2017a), the organization of hierarchical circuits for the selection of behaviors
(Ohyama et al. 2015), and the implementation of motion detection in the visual system (Takemura
et al. 2017b). In the olfactory system, dense reconstructions revealed nonrandom convergence of
projection neurons onto Kenyon cells in the mushroom body (Zheng et al. 2020), which was not
detected previously by sparse sampling (Sosulski et al. 2011).
This review focuses on dynamical connectomics approaches in zebrafish. We specifically dis-
cuss insights into neuronal computations that could not have been obtained without dense recon-
structions of neuronal connectivity (Dow et al. 2018; Svara et al. 2018; Vishwanathan et al. 2017,
2020; Wanner & Friedrich 2020; Wanner et al. 2016a,b). A volume containing the entire larval
zebrafish brain at 5 days postfertilization has been acquired at a resolution of 14 × 14 × 25 nm3
(F. Svara, W. Denk, H. Baier & F. Kubo, manuscript in preparation) (Figure 2b). Image data sets
that have not been used for dense circuit reconstruction such as vEM data sets acquired at low
ultrastructural resolution (Hildebrand et al. 2017) are not discussed explicitly.

CHALLENGES IN MECHANISTIC SYSTEMS NEUROSCIENCE

AND THE POTENTIAL OF ZEBRAFISH
A main advantage of zebrafish for dynamical connectomics is their small size, which greatly fa-
cilitates dense measurements of neuronal population activity and connectivity (Friedrich et al.
2010, 2013). The number of neurons in the larval zebrafish brain (∼105 ) falls in between the lar-
val and adult brains of Drosophila (∼1.5 × 104 and ∼2.5 × 105 neurons, respectively). In the adult
zebrafish brain, the number of neurons (107 ) is only approximately sevenfold lower than in the
mouse brain, despite a greater than 100-fold difference in volume. Optical measurements of neu-
ronal activity through the intact skull are feasible up to 3–4 weeks postfertilization and even in
some brain areas of adult fish, including the telencephalon (Aoki et al. 2013, Chow et al. 2020,
Huang et al. 2020, Torigoe et al. 2019). Measurements and manipulations of activity during be-
havior can be performed in head-embedded and freely swimming larvae (Cong et al. 2017, Kim
et al. 2017, O’Malley et al. 1996, Vanwalleghem et al. 2018) and in head-fixed adult fish in a virtual
reality (Huang et al. 2020, Torigoe et al. 2019). While larval zebrafish are used as model organisms
to study a broad spectrum of innate behaviors, social interactions and many forms of associative
learning emerge only later in development (Buske & Gerlai 2011, Dreosti et al. 2015, Stednitz
et al. 2018, Valente et al. 2012). Adult zebrafish show a rich set of complex and plastic behav-
iors (Orger & de Polavieja 2017, Spence et al. 2008), but their full behavioral repertoire is still
underexplored.
The ability to measure activity across brain areas is exploited to examine how sensory inputs
modulate behavior through multiple layers of computation. Two examples of visuomotor transfor-
mations that have been studied extensively in zebrafish larvae are the optokinetic reflex (OKR), an
eye movement that follows rotational visual flow and stabilizes gaze, and the optomotor response
(OMR), a swimming behavior that follows translational visual flow and compensates for physical
drift. Both reflexes are based on the detection of visual flow by direction-selective ganglion cells
in the retina. Ganglion cells are the output neurons of the retina and project to 10 different ar-
borization fields, named AF1–AF10, in the contralateral hemisphere, including the optic tectum.

280 Friedrich • Wanner

Direction-selective ganglion cells target the optic tectum (AF10) and another arborization field
in the pretectum, AF5 (Kramer et al. 2019, Robles et al. 2014). Calcium imaging and light mi-
croscopic neuron reconstructions identified two classes of direction-selective pretectal neurons:
(a) neurons that project to the vicinity of AF5 and inherit responses of direction-selective ganglion
cells, and (b) neurons that appear to integrate inputs from different types of direction-selective pre-
tectal cells. The second class of neurons therefore extracts information about the global pattern
of visual flow that is thought to be critical for the OKR and OMR (Kramer et al. 2019, Kubo et al.
2014, Naumann et al. 2016).
To explore how visual flow signals are transformed into directed behaviors downstream of the
pretectum, the temporal structure of neuronal activity throughout much of the brain was corre-
lated with the temporal structure of stimulus features and motor outputs. This approach identi-
fied candidate neurons in multiple brain areas that may be involved in different components of
the sensory-motor transformation. Further studies using physiological gain- or loss-of-function
approaches, morphological studies, and genomic analyses then focused on brain areas where se-
lected candidate neurons are enriched. These approaches have culminated in models that propose
how information is propagated and processed between brain areas during the OKR and OMR
(Kubo et al. 2014, Naumann et al. 2016, Portugues et al. 2014, Wu et al. 2020). However, these
models cannot represent circuits as biologically realistic networks of individual neurons because
the underlying data, despite their richness, do not provide sufficient constraints (Litwin-Kumar
& Turaga 2019). Circuit function is thus described by flow diagrams with abstracted descriptions
of circuit elements (e.g., brain areas, cell types, or abstract entities representing a computation)
and their interactions (e.g., excitation or inhibition) (Figure 2a). However, more detailed circuit
models are needed to analyze mechanisms of neuronal computation in depth, a situation that is
representative for many neuronal circuits across species. Ideally, network models should consist
of individual neurons corresponding to individual neurons in the brain, and they should recapitu-
late the essence of the biological computation from first principles of biophysics. Such naturalistic
network models can then serve as starting points to extract mechanisms of biological computa-
tions by continuous simplification and abstraction. Moreover, naturalistic network models enable
manipulations beyond the reach of biological methods. The reconstruction of wiring diagrams is
a key step toward such naturalistic network models, as highlighted in recent studies that incorpo-
rated wiring diagrams into computational models of neuronal circuits in Drosophila and zebrafish
(Tschopp et al. 2018, Wanner & Friedrich 2020, Vishwanathan et al. 2020).
At the current level of granularity, experimental data on the OMR, OKR, and many other
sensory-motor transformations are consistent with multiple alternative network models. A noto-
rious problem in the field that is often ignored is that the plausibility of a given model cannot be
determined accurately because the set of alternative models is poorly understood. For example,
many direct connections in a flow diagram could also be indirect, and it is difficult to assess the
full set of possible connectivity patterns and their likelihoods. The selection of end-to-end models
for sensory-motor transformations is therefore associated with high uncertainty, which is a direct
consequence of the complexity of the underlying circuits. Dense circuit reconstruction is an ef-
fective approach to reduce this uncertainty because connectivity provides strong constraints for
network models. In zebrafish, for example, tracing of neurons in a vEM data set from the whole
larval brain can directly test key assumptions about connectivity in working models of the OMR
and OKR (Figure 2). More generally, uncertainty in model selection can be efficiently reduced by
the transition from flow diagrams to naturalistic network models. Zebrafish provide rare opportu-
nities to achieve this goal by reconstructing biologically realistic wiring diagrams and integrating
them into network models.

www.annualreviews.org • Dense Circuit Reconstruction 281

 The OKR and OMR are only two examples of sensory-motor transformations that have been
examined in zebrafish larvae by population activity measurements and/or quantitative behavioral
analyses. Additional studies examined other forms of visual processing and visuomotor transfor-
mations (Bahl & Engert 2020, Barker & Baier 2015, Bollmann 2019, Chen et al. 2018, Dragomir
et al. 2020, Dunn et al. 2016, Helmbrecht et al. 2018, Romano et al. 2015, Sumbre et al. 2008,
Yildizoglu et al. 2020), responses to thermal stimuli (Haesemeyer et al. 2018, Lin et al. 2020) and
to vestibular or mechanosensory input (Favre-Bulle et al. 2018, Migault et al. 2018, Privat et al.
2019), the suppression of mechanosensory responses to self-generated movements via efference
copies (Pichler & Lagnado 2020), sleep (Chiu et al. 2016, Leung et al. 2019), startle and escape
reflexes (Lacoste et al. 2015, Marquart et al. 2019, Marsden et al. 2018, Mu et al. 2012, O’Malley
et al. 1996, Tabor et al. 2018, Yao et al. 2016), nocifensive behavior (Wee et al. 2019a), gaze control
(Aksay et al. 2007, Brysch et al. 2019, Miri et al. 2011), light preference and phototaxis (Wolf et al.
2017, Zhang et al. 2017), control of appetite (Wee et al. 2019b), neuromodulation (Lovett-Barron
et al. 2017, 2020; Reinig et al. 2017), habituation to sensory stimuli (Marquart et al. 2019, Randlett
et al. 2019, Tabor et al. 2018), specific forms of associative learning (Aizenberg & Schuman 2011,
Lin et al. 2020), and prey capture, which comprises multiple behavioral components (Bianco &
Engert 2015, Henriques et al. 2019, Kim et al. 2017, Muto et al. 2013). Moreover, large-scale
imaging was exploited to examine the hypothesis that the brain operates near a point of critical-
ity (Ponce-Alvarez et al. 2018) and provided insights into the integration of sensory experience
on timescales longer than neuronal time constants (Andalman et al. 2019, Bahl & Engert 2020,
Dragomir et al. 2020, Kawashima et al. 2016, Lin et al. 2020, Mu et al. 2019, Sumbre et al. 2008).
In addition, many other questions in systems neuroscience have been addressed in zebrafish larvae,
but a comprehensive overview of this field is beyond the scope of this review. Dense reconstruc-
tions of neuronal circuits could thus provide highly valuable information for mechanistic analyses
of a broad spectrum of neuronal computations underlying sensory and motor processing.
Juvenile or adult zebrafish show complex and plastic behaviors that have not been observed in
larvae (Buske & Gerlai 2011, Chou et al. 2016, Dreosti et al. 2015, Namekawa et al. 2018, Orger
& de Polavieja 2017, Palumbo et al. 2020, Spence et al. 2008, Stednitz et al. 2018, Tang et al.
2020, Valente et al. 2012, Yashina et al. 2019). In an operant conditioning paradigm, cue-evoked
activity in the dorsal telencephalon increased as adult zebrafish learned to avoid a visual cue (Aoki
et al. 2013). In telencephalic area Dp, the homolog of olfactory cortex, neurons receive excitatory
and inhibitory synaptic inputs during odor responses that are co-tuned and balanced (Blumhagen
et al. 2011, Rupprecht & Friedrich 2018), consistent with theoretical models of memory networks
(Hennequin et al. 2017). Odor discrimination learning (Namekawa et al. 2018) was associated
with the formation of functional neuronal assemblies, a signature of autoassociative memory, and
resulted in odor- and task-specific modifications of inhibitory subcircuits (Frank et al. 2019). Ex-
periments using a virtual reality revealed prominent responses to unexpected sensory inputs in
multiple telencephalic areas (Huang et al. 2020, Torigoe et al. 2019) consistent with predictive pro-
cessing (Keller & Mrsic-Flogel 2018). The adult zebrafish is therefore an interesting model system
to study neuronal computations underlying higher brain functions in a small vertebrate brain.

INSIGHTS INTO NEURONAL COMPUTATIONS FROM DYNAMICAL

CONNECTOMICS IN ZEBRAFISH
The Combinatorial Logic of Motion Selectivity in the Pretectum
Pretectal neurons are thought to play a pivotal role in visuomotor transformations, including the
OKR and OMR, because they extract important information about visual flow patterns. Com-
prehensive analyses identified classes of pretectal neurons that respond to distinct, nonrandom

282 Friedrich • Wanner

combinations of motion directions in the two eyes (Kubo et al. 2014, Naumann et al. 2016). A
subset of these classes are classified as monocular because their responses depend only on input
to the contralateral eye. These neurons extend neurites into AF5, suggesting that they receive
direct input from direction-selective ganglion cells in the retina (Kramer et al. 2019). Binocular
pretectal neurons, in contrast, integrate inputs from both eyes and do not project to AF5.
Following principles of parsimony, hypothetical wiring patterns have been proposed to explain
the functional properties of different classes of monocular and binocular pretectal neurons (Kubo
et al. 2014). The complexity of the proposed connectivity, together with the nontopographic
organization of the pretectum and the lack of genetic markers, makes it difficult to experimentally
test the proposed wiring schemes by sparse sampling of connectivity. A direct approach toward
a mechanistic understanding of optic flow processing in the pretectum would be to reconstruct
the connectivity between populations of functionally characterized pretectal neurons. Such an
approach requires long-range tracing of neurons across the midline to determine the integration
of information from both eyes by binocular neurons because projections of retinal ganglion cells
are 100% collateral. To address this challenge, the morphology and synaptic connectivity of
pretectal neurons are reconstructed in a volume of EM images that was acquired after measuring
responses of neuronal somata to whole-field motion in the two eyes (F. Svara, W. Denk, H. Baier
& F. Kubo, manuscript in preparation). Functionally characterized pretectal neurons and their
synapses are annotated manually, including a substantial number of neurons that cross the midline
(Figure 2). This approach can directly test current models of motion processing in the pretectum
by searching for predicted connectivity between specific types of neurons (Kubo et al. 2014).

Direction-Selective Connectivity in Mechanosensory Organs

of the Lateral Line
The lateral line consists of discrete mechanosensory organs, the neuromasts, with hair cells simi-
lar to those in the inner ear. Individual hair cells are direction selective, responding preferentially
to water movement in either rostral or caudal direction, and make synapses onto afferent axon
terminals that transmit information toward the brain. Each afferent axon terminal receives input
only from hair cells with the same directional preference (Nagiel et al. 2008). By vEM analysis of
multiple neuromasts, Dow et al. (2018) confirmed this direction-selective connectivity and pro-
vided additional insights into the synaptic organization of neuromasts. They found that individual
hair cells innervate more than one axon terminal and that neuromasts contain one dominant axon
terminal per direction that receives most of the hair cell input. Further results demonstrated that
the development of polarity-specific connectivity is activity independent and determined by the
identity of hair cells, which is established by Notch signaling. These insights into the neuromast
microcircuit required the simultaneous reconstruction of connections between multiple hair cells
and axon terminals along with activity measurements.

Wiring Specificity of Speed-Related Motor Circuits

In fish, vigorous muscle contractions travel at high frequency from head to tail during fast swim-
ming, while muscles contract with less strength and lower frequency during slow swimming. These
rhythmic muscle contractions alternate between the left and right side of the tail and are controlled
by a neural circuit in the spinal cord known as a central pattern generator (CPG) (Goulding 2009,
Grillner 2006). With increasing contraction strength, more and larger motoneurons (MNs) of
the CPG are recruited, resulting in stronger contractions of the tail musculature (Burke et al.
1982, Ulfhake & Kellerth 1982). The rhythmic excitation of MNs is provided by circumferential

www.annualreviews.org • Dense Circuit Reconstruction 283

 descending excitatory interneurons (CiDs) (Kimura et al. 2006). During slow swimming, only ven-
tral CiDs are active while dorsal CiDs remain silent. With increasing swim speed, the activity of
the CiDs shifts dorsally and the ventral CiDs are inactivated (Ampatzis et al. 2014, McLean et al.
2008). The ordered recruitment of MNs could be a consequence of size-dependent biophysical
properties of MNs. Alternatively, or in addition, the CPG may selectively recruit subsets of MNs
through specific wiring patterns between MNs and CiDs. To probe the wiring specificity of MN
recruitment, Svara et al. (2018) acquired a complete serial block-face electron microscopy stack of
a spinal cord hemisegment of a larval zebrafish and reconstructed the wiring diagram of the MN
and CiD circuit. While the small MNs appeared to be immature and received almost no synaptic
inputs, the intermediate and large MNs were indeed differentially innervated. Both received input
by the ventral CiDs, but only the large MNs received input also from dorsal CiDs. Consequently,
the large MNs are only recruited by the dorsal CiDs during fast swimming. During slow swim-
ming, in contrast, dorsal CiDs and large MNs are both inactive, and the small and intermediate
MNs are recruited by the ventral CiDs according to their size and excitability. The mechanism
that inactivates ventral CiDs during fast swimming remains to be examined.

A Brain Stem Oculomotor Module Underlying Temporal Integration

Controlling Eye Position
The control of eye position by the oculomotor system is a key component of vision. Eye position
signals originate in the premotor velocity-to-position neural integrator (VPNI) circuit that tem-
porally integrates angular velocity command signals into angular position commands. The popu-
lation activity across integrator neurons lies on a low-dimensional manifold that is parametrized
by the angular position of the eyes (Seung 1996), similar to the dynamics of other circuits for
motor control, memory, and decision-making (Gallego et al. 2017). Such systems are of broad
interest because they are suitable to support short-term memory functions, but the underlying
mechanisms are not well understood.
Vishwanathan et al. (2017, 2020) acquired a large serial-section electron microscopy stack from
the brain stem of a zebrafish larva, a region containing both a key extraocular MN population and
many oculomotor interneurons. Using advanced automated segmentation methods, they deter-
mined the synaptic connectivity between approximately 3,000 nodes, each representing a full or
partial reconstruction of a neuron. From the connectivity matrix they computed the eigencen-
trality, which measures the relative influence of a node in a graph, and defined nodes with high
eigencentrality as the center of the graph. These nodes are recurrently connected and expected to
have a strong influence on the dynamics of neuronal population activity. Other nodes providing
input to, or receiving output from, the center are referred to as the periphery. Further analysis us-
ing graph clustering methods revealed that the center was organized into two modules, each with
denser intramodule than intermodule connectivity. In one group (the axial module), cells had weak
recurrent connectivity and made synapses onto spinally projecting neurons in the periphery. In
another group (the oculomotor module), cells had denser recurrent connectivity and projected
to extraocular MNs in the periphery. Comparison with calcium imaging data from age-matched
conspecifics showed that neurons with oculomotor functional signatures were twice as likely to be
associated with the oculomotor module than with the axial module.
The recurrent connectivity and peripheral connections of the oculomotor module are con-
sistent with proposed computational models of the VPNI. To further test the hypothesis that
the oculomotor module corresponds to the VPNI, a network model was created that directly
incorporated the synaptic connections from the reconstructed connectome. This model success-
fully predicted characteristic tuning properties of integrator neurons. The dense reconstruction of

284 Friedrich • Wanner

neuronal connectivity in the brain stem, together with network analyses and functional imaging,
therefore revealed a complex web of interneuronal connections that is consistent with the function
of a neural integrator. It will now be interesting to examine whether the structural signatures of the
oculomotor module in the zebrafish brain stem generalize to other circuits performing temporal
integration and short-term memory.

Whitening of Odor Representations in the Olfactory Bulb

by Tuning-Dependent Reciprocal Connectivity
In the olfactory bulb (OB), odors evoke distributed patterns of activity across an array of discrete
input channels, the olfactory glomeruli, where axons of receptor neurons expressing the same
odorant receptor converge. OB output is transmitted by mitral cells to multiple targets, includ-
ing higher brain areas that are thought to function as autoassociative memory networks (Haberly
2001). While activity patterns across glomeruli overlap in response to similar odors and strongly
depend on odor concentration (Friedrich & Korsching 1997), activity patterns across mitral cells
are rapidly decorrelated after stimulus onset and are more concentration invariant (Friedrich &
Laurent 2001, Niessing & Friedrich 2010, Zhu et al. 2013). Odor representations therefore un-
dergo a transformation akin to whitening, a fundamental operation that supports pattern classifi-
cation and memory formation (Cohen et al. 2020, Yamins & DiCarlo 2016). This transformation
cannot be explained by global operations such as normalization or contrast enhancement, imply-
ing that it requires specific interactions between subsets of mitral cells (Friedrich & Wiechert
2014). The structure of these interactions needs to be adapted to the structure of input patterns
that are transmitted to the OB via the array of glomeruli. Hence, whitening is thought to involve
an evolutionary memory of olfactory stimulus space that is embedded in the wiring diagram.
The matrix of functional interactions between mitral cells cannot be uncovered by sparse,
monosynaptic sampling of connections because mitral cells communicate polysynaptically via in-
hibitory interneurons (Figure 3a). Wanner and colleagues (Wanner & Friedrich 2020; Wanner
et al. 2016a,b) therefore used a dynamical connectomics approach to analyze mechanisms of
whitening in the OB. They measured odor-evoked activity across approximately 30% of all neu-
rons in the OB of a zebrafish larva, subsequently acquired a vEM image stack containing the
entire OB, and reconstructed almost all of the 1,047 neurons and their synaptic connections.
As observed previously in the adult OB (Friedrich & Wiechert 2014), mitral cell activity pat-
terns evoked by similar odors were initially highly correlated because small cohorts of mitral cells
showed strong responses to common molecular features. Subsequently, correlations between these
activity patterns decreased because cohorts of strongly responding mitral cells were selectively in-
hibited (Figure 3b). Hence, pattern decorrelation is the consequence of a feature suppression
mechanism that attenuates the representation of redundant features relative to the representation
of stimulus-specific pattern components.
A comprehensive analysis of disynaptic connectivity between mitral cells revealed that disyn-
aptic interactions are overrepresented between mitral cells with similar odor tuning (Figure 3c).
Moreover, reciprocal connections were highly overrepresented in disynaptically connected mitral
cell–interneuron–mitral cell triplets. As a consequence, cohorts of mitral cells tuned to common
molecular features strongly downscale their own activity when and only when the molecular
feature is present (Figure 3d). A naturalistic network model of the OB precisely reproduced
this mechanism of feature suppression when neurons were connected according to the measured
wiring diagram but not when connectivity was randomized. More specific manipulations of
the wiring diagram further confirmed that this feature suppression mechanism can account for
whitening in the OB. Whitening is therefore mediated by higher-order structure in the wiring

www.annualreviews.org • Dense Circuit Reconstruction 285

 Olfactory bulb Odor 1 Odor 2
a b
Glomeruli
(input) Mitral cells:
r high
input
Interneurons W

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).

286 Friedrich • Wanner

diagram that is adapted to the correlation structure of input patterns (Wanner & Friedrich 2020).
This correlation structure appears to be dominated by molecular features such as functional
groups, probably as a necessary consequence of physical interactions between odorant receptors
and their ligands. Attenuating representations of these molecular features therefore decorrelates
inputs and facilitates pattern classification. Interestingly, the disynaptic inhibitory connectivity
among mitral cells in the OB is inversely related to monosynaptic excitatory connectivity among
co-tuned principal neurons in V1 (Ko et al. 2011, Lee et al. 2016), which has been proposed to
amplify representations of visual features. However, functional interactions between pyramidal
cells with similar tuning appear to be dominated by inhibition rather than excitation (Chettih &
Harvey 2019). It will thus be interesting to further compare mechanisms of feature processing in
the OB and visual cortex and their specific adaptations to natural inputs.

CONCLUSIONS AND OUTLOOK

Methods for the automated acquisition and analysis of vEM data have enabled the transition from
sparse reconstructions of individual neurons to dense reconstructions of entire networks. This
ability provides fundamentally new opportunities to analyze neuronal computations and to test
mechanistic models of learning, abstraction, and cognition. While precise knowledge of wiring
diagrams is critical, wiring diagrams alone are insufficient to understand circuit function without
additional information. We argue that the combination of dense circuit reconstruction with pop-
ulation activity measurements—dynamical connectomics—is particularly informative because it
can provide direct links between the synaptic inputs of individual neurons and their outputs, and
between higher-order structure of a network and its dynamics.
vEM data not only are important for the reconstruction of wiring diagrams but also contain
other valuable information (Figure 1c). For example, the ultrastructural reconstruction of entire
neurons allows for comprehensive analyses of synapse distributions along neurites, and dense re-
constructions of a brain area can identify novel rare cell types (Morgan et al. 2016, Motta et al.
2019, Wanner et al. 2016b). The ultrastructure of neurites and synapses may be informative about
synaptic weights and other functional properties (Bartol et al. 2015, Kornfeld et al. 2020, Motta
et al. 2019), and it will be interesting to explore how cellular ultrastructure is related to gene
expression (Vergara et al. 2020).
A particular advantage of zebrafish for dynamical connectomics is their small size, which en-
ables the reconstruction of neuronal connectivity across brain areas (Figure 2). Small brains also
facilitate reconstructions of equivalent circuits in multiple individuals, which will be important
to explore interindividual variations of wiring diagrams and their modifications by specific ex-
periences. Even in adult zebrafish, it is feasible to reconstruct entire brain areas such as distinct
forebrain regions using available technology. Future applications of dynamical connectomics in ze-
brafish may thus address a broad spectrum of questions. In larvae, the ability to determine neuronal
connectivity across brain areas may be exploited to pinpoint the neuronal circuitry underlying
sensory-motor transformations. Generally, dynamical connectomics has unique potential to de-
termine how information is stored in the wiring diagram by experience-dependent modifications
of synaptic connections and to explore neuronal implementations of computational strategies such
as predictive processing. This challenge may be addressed in adult zebrafish, which provide the
opportunity to analyze complex behaviors, including associative learning (Aoki et al. 2013, Chou
et al. 2016, Frank et al. 2019, Huang et al. 2020, Palumbo et al. 2020). Dynamical connectomics
in zebrafish is therefore a promising approach to explore mechanisms of neuronal computations
underlying higher brain functions and cognition.

www.annualreviews.org • Dense Circuit Reconstruction 287

 DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

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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