Imaging circuit formation in zebrafish

Imaging Circuit Formation in Zebrafish

Nikolas Nikolaou, Martin P. Meyer

MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Hospital Campus,London SE1 1UL, United Kingdom

Received 25 August 2010; accepted 9 January 2011

ABSTRACT: The study of nervous system develop-

ment has been greatly facilitated by recent advances in

molecular biology and imaging techniques. These

approaches are perfectly suited to young transparent

zebrafish where they have allowed direct observation of

neural circuit assembly in vivo. In this review we will

highlight a number of key studies that have applied opti-

cal and genetic techniques in zebrafish to address ques-

tions relating to axonal and dendritic arbor develop-

ment, synapse assembly and neural plasticity. These

studies have revealed novel cellular phenomena and

modes of growth that may reflect general principles

governing the assembly of neural circuits. ' 2011 Wiley

Periodicals, Inc. Develop Neurobiol 72: 346–357, 2012

Keywords: zebrafish; imaging; synapse; dendrite; axon;

circuit; plasticity; molecular biology

INTRODUCTION

Perception and behavior are dependent on the estab-

lishment of precise synaptic connections within the

developing nervous system. Two fundamental goals

in developmental neuroscience are to understand how

neurons target one another during periods of synapto-

genesis, and how synapses themselves are assembled.

The structure of developing neurons is also highly

plastic. How does the structural plasticity of individ-

ual neurons relate to circuit assembly, and how is

such plasticity regulated? Recent advances in

imaging technology, molecular biology, and the GFP

revolution have made live imaging the tool of choice

for studying circuit assembly and plasticity (Niell and

Smith, 2004; Lichtman and Smith, 2008). These tech-

nological advances have enabled researches to label

and follow over time virtually any feature of the nerv-

ous system from populations of neurons (Godinho

et al., 2005; Mumm et al., 2006), to dendrites and

axons of individual neurons (O’Rourke et al., 1994;

Jontes et al., 2000; Trachtenberg et al., 2002; Bishop

et al., 2004; Haas et al., 2006), to subcellular struc-

tures within neurons such as dendritic spines (Trach-

tenberg et al., 2002; Bishop et al., 2004; Keck et al.,

2008; Yang et al., 2009) and synaptic vesicles (Meyer

and Smith, 2006; Ruthazer et al., 2006). Live imaging

of these structures during development has revealed

new cellular phenomena and modes of growth, and

has generated hypotheses on the mechanisms of cir-

cuit assembly and plasticity. In this review we

describe the features of zebrafish as a model organism

that make it ideally suited to take advantage of these

technological developments. Rather than provide a

comprehensive review of the literature we have

focused on a few key studies that illustrate how live

imaging in zebrafish has enabled researchers to

address key questions in the field. We will finish by

discussing how the latest generation of optogenetic

tools and their use in zebrafish are likely to advance

our understanding of circuit formation, function, and

plasticity.

Correspondence to:M.P. Meyer ([email protected]).Contract grant sponsor: Medical Research Council; contract

grant number: G0801242.Contract grant sponsor: Medical Research Council Career Devel-

opment; contract grant number: G0600107.

' 2011 Wiley Periodicals, Inc.Published online 19 January 2011 in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/dneu.20874

346

Arbor Growth and Synapse Formation inthe Retinotectal Projection: A Process ofTrial and Error

A crucial step in establishing synaptic connectivity is

the elaboration of axonal and dendritic arbors. Arbor

size, shape, and position determine patterns of synap-

tic connectivity, and hence the functional properties

of neural circuits. The visual system is a prominent

model system for studying how arbor morphogenesis

underlies the formation of precise neuronal circuitry.

Connections in the visual system are topographically

organized. That is, neighboring retinal ganglion cells

(RGCs) project to neighboring neurons in the optic

tectum, the main RGC target in the zebrafish brain.

Targeting of RGC axons to correct termination zones

within the tectum, and restriction of arbor size within

the termination zone are crucial for generating pre-

cise topography. Of equal importance is the develop-

ment of dendritic arbors in postsynaptic tectal cells,

since these determine how RGC inputs to the brain

are sampled, and ultimately how visual information is

processed.

A number of key features make the zebrafish reti-

notectal projection particularly amenable to live

imaging studies. First, zebrafish embryos and larvae

are translucent, develop externally, and RGC axons

and tectal cell dendrites arborize just beneath the skin

overlying the optic tectum. These arbors are therefore

easily accessible to imaging without the need for sur-

gery, even with a standard confocal microscope. Sec-

ond, development of the visual system is rapid. The

first RGC axons reach the tectum between 2 and 3

days post-fertilization (dpf), and visually evoked

behaviors can be elicited soon thereafter (Easter and

Nicola, 1996). Indeed the bulk of RGC axon and tec-

tal dendritic arbor growth is complete by 6dpf (Niell

et al., 2004; Meyer and Smith, 2006). Thus, in one

over-night time-lapse imaging session a considerable

degree of arbor development can be observed. Third,

injection of plasmids into single cell stage embryos

results in transient mosaic expression of transgenes at

larval stages of development. This versatile and

straightforward technique has been exploited exten-

sively to achieve Golgi-like labeling of single neu-

rons with fluorescent proteins in vivo, and has

enabled fine details such as dendritic filopodia to be

resolved and followed over time using confocal or

two photon microscopy. Live in vivo imaging of

RGC axonal and tectal cell dendritic arbors in zebra-

fish has revealed dynamic aspects of arbor growth

that would be missed from analysis of static images.

For example, when presented with static images of a

developing axonal or dendritic arbor one would

assume that these arbors grow much like a tree, with

continuous elongation of branches, and a gradual

increase in arbor complexity as new branches are

added. Time-lapse imaging in zebrafish has shown

that the process of RGC axonal and tectal dendritic

arbor growth is much more dynamic. During develop-

mental periods growing axonal and dendritic arbors

are continually remodeled by the concurrent exten-

sion and retraction of filopodia, only a small fraction

of which are maintained as branches in the mature

arbor (Kaethner and Stuermer, 1992, 1997; Niell

et al., 2004; Meyer and Smith, 2006). Thus, time-

lapse imaging has shown that arbor development is

more a process of trial-and-error rather than simple

forward monotonic growth.

How is the structural plasticity of developing

axons and dendrites related to circuit formation? By

imaging RGC axons and tectal cell dendrites express-

ing fluorescent pre- and postsynaptic marker proteins,

respectively, it was found that the dynamic turnover

of axonal and dendritic branches is accompanied by

the turnover of many nascent synapses (Niell et al.,

2004; Meyer and Smith, 2006) (see Fig. 1). Thus, the

dynamic rearrangement of arbors serves to sample

many potential synaptic partners, and generates a

large pool of nascent synaptic connections, of which

only a small number are kept. Importantly, the pro-

cess of arbor growth and synapse formation appear to

be mechanistically linked. Maintenance of a synapse

stabilizes the axonal or dendritic filopodium on which

it is located and stabilized filopodia mature into

branches. Synapses are also the sites of new branch

addition. Imaging with high temporal resolution over

many hours has shown that axonal and dendritic

arbors grow by iterative rounds of filopodium exten-

sion from, and selective stabilization by synapses

(Niell et al., 2004; Meyer and Smith, 2006). These

studies suggest that by modulating two distinct

aspects of arbor growth—branch formation and

branch stability, the process of synapse formation

itself could guide growth of axonal and dendritic

arbors. Modeling studies have suggested that such a

\synaptotropic" mechanism can generate specific

dendritic arborization patterns independently of den-

dritic guidance molecules or genetically predeter-

mined branching programs (Niell, 2006; see Fig. 2).

Instead, formation of a correctly shaped and posi-

tioned dendrite depends solely on the distribution of

presynaptic partners, and on a local stochastic process

of branch extension and selective stabilization. In

other words, the extent and complexity of a dendritic

arbor will arise as a consequence of local interactions

with neighboring axons. Doesn’t this just offload the

Imaging Circuit Formation in Zebrafish 347

Developmental Neurobiology

task of circuit patterning onto the precise positioning

of axons? To a certain extent the answer is yes, but

since in the retinotectal projection a synaptotropic

mechanism also operates in presynaptic RGCs, the

initial targeting of axons need only be approximate.

A synaptotropic mechanism operating in both pre-

and postsynaptic neurons could facilitate self-organi-

zation of precise circuitry from only approximately

positioned axons. Indeed, the prevailing view in the

field of visual map development is that genetically

encoded axon guidance cues establish crude retino-

topic positioning of axons that are then refined by

neural activity (Ruthazer and Cline, 2004; Huberman

et al., 2008). It is therefore possible that activity at

nascent synapses modulates both axon branch stabil-

ity and branch formation resulting in a tight control

of axonal arbor structure, and hence map refinement.

In the following section we will describe how imag-

ing in zebrafish has been used to ask precisely how

neural activity modulates the structural development

of RGC axons.

Regulation of Axonal Arbor Growth byNeural Activity

Perturbations in neural activity followed by studies of

fixed tissue can show the effect of the perturbation on

final arbor form. Live imaging can go one step further

by revealing precisely how activity in a growing neu-

ron affects the process of arbor growth. For example,

time-lapse imaging can reveal whether an increase in

the number of branches arises through an increased

rate of branch formation or branch stabilization. Two

recent studies have used molecular genetic

approaches to suppress synaptic transmission in

RGCs in zebrafish and then used time-lapse imaging

to examine the effect on axon arbor development. In

the first study by Hua et al., synaptic transmission in

single RGCs was suppressed by expression of a mu-

tant form of the synaptic vesicle protein, VAMP2/

Synaptobrevin 2 (Hua et al., 2005). Time-lapse imag-

ing revealed that singly silenced axons formed fewer

branches than active ones, and as a consequence

Figure 1 Series of images from time-lapse imaging of axonal and dendritic arbor growth and

synapse formation in the zebrafish tectum. (A) The growth of an RGC axon arbor occurs by an iter-

ative sequence of presynaptic punctum (green) formation and filopodial (red) stabilization. A filo-

podium (arrow) is seen to extend and nascent puncta (arrowheads) are formed soon after. The

selective maintenance of these synapses lead to stabilization of the filopodium which matures into

a stable branch. Two other filopodia are formed and eliminated in close proximity. Scale bar ¼2 lm. (B) Similarly, the dendritic arbor growth of tectal cells is characterized by the presence of

many transient filopodia (red) and postsynaptic puncta (green), indicated by arrows and arrow-

heads, respectively. Scale bar ¼ 5 lm. Image in (A) reproduced from Meyer and Smith (2006)

with permission (Copyright 2006 by the Society for Neuroscience), and image in (B) reproduced

from Niell et al. (2004) with permission (Copyright 2004 by Nature Publishing Group).

348 Nikolaou and Meyer


developed smaller arbors. Using similar techniques

Ben Fredj et al., suppressed synaptic transmission in

single RGCs using targeted expression of tetanus

toxin light-chain fused to EGFP (TeNT-Lc-EGFP)

(Ben Fredj et al., 2010). In contrast to the findings of

Hua et al., single RGC axons silenced by TeNT-Lc-

EGFP expression were larger than wild-type axons,

and time-lapse analysis revealed a failure of these

axons to arrest branch formation events. However,

both studies found that phenotypes were only appa-

rent when single RGCs were silenced in a field of

active ones. Widespread suppression of activity in

RGCs using either the mutant form of VAMP2 or

TeNT-Lc-EGFP resulted in arbors that were of nor-

mal size. Thus, relative activity levels or activity-de-

pendent competition between RGCs appear to regu-

late the size of territory occupied by axonal arbors. A

plausible explanation that could account for the dif-

ferent outcomes of single cell silencing is that the

degree of presynaptic suppression achieved with

the two approaches is different. Over-expression of

the VAMP2 mutant has been shown to suppress

stimulus-evoked vesicular release but does not

abolish it entirely (Sorensen et al., 2002), whereas

TeNT-Lc-EGFP is highly effective at blocking both

stimulus evoked and spontaneous release of synap-

tic vesicles. The weak residual activity in RGCs

expressing the VAMP2 mutant could therefore

induce plasticity mechanisms analogous to long-

term depression (LTD), which in the developing

retinotectal projection may lead to synapse elimina-

tion. Since synapses appear to be correlated with

both stabilization and formation of axonal branches

(Alsina et al., 2001; Meyer and Smith, 2006; Ruth-

azer et al., 2006), synapse elimination would lead

to an increased rate of branch elimination, a

reduced rate of branch formation and hence smaller

arbors. LTD, by definition, only occurs at synapses

with at least some activity. Therefore, in axons that

express TeNT-Lc-EGFP, where synaptic activity is

almost entirely abolished, LTD and synapse elimi-

nation are not induced, permitting branches to

extend beyond their normal territory. In this view,

competition punishes the weakly active axon more

than the completely silent one. While this possibil-

ity has not been explored directly, the contrasting

findings of these two studies suggest that different

manipulations of synaptic function could have very

different consequences for the growth of RGC

axons. To complicate matters further, other manipu-

lations that alter synaptic function have resulted in

non-competitive structural phenotypes. For exam-

ple, the blumenkohl (blu) mutant, initially identified

in a screen for zebrafish retinotectal projection

defects, is characterized by enlarged termination

zones and defasciculation of RGC axons (Baier et

al., 1996). The blu mutation maps to vglut2a, a

member of the vesicular glutamate transporter fam-

Figure 2 Synaptogenesis guides the growth and branching of dendritic arbors. Modeling the

sequence of events during synaptotropic dendritic growth in a situation where there is a non-homo-

geneous distribution of axons that are, receptive to synapse formation (dashed green boxes). Many

filopodia are extended (red), but only those which form synapses (green) are maintained as stable

branches (black). As a result, dendritic arbor growth is restricted to regions containing appropriate

presynaptic partners (dashed boxes). Image reproduced from Niell (2006) with permission (Copy-

right 2006 by Elsevier Ltd).



ily, which are responsible for filling synaptic

vesicles with the excitatory neurotransmitter gluta-

mate (Smear et al., 2007). Using patch-clamp

experiments in vivo it was found that synaptic

vesicles at blu retinotectal synapses contain less

glutamate, and that these synapses fatigue prema-

turely under high-frequency stimulation. Mutant

synapses appeared to function appropriately under

normal conditions however, suggesting that other

glutamate transporters can partially compensate for

the loss of vglut2a. Labeling of individual RGC

axons in the blu mutant reveals an increase in

RGC axon branch number and coverage area in the

tectum and an increase in the frequency of minia-

ture excitatory postsynaptic currents (mEPSCs) in

postsynaptic tectal cells. The increase in mEPSC

frequency may reflect an increase in the number of

presynaptic release sites in the enlarged axonal

arbors. The findings of the blu mutant study are

similar to those in the study that used TeNT-Lc-

EGFP to suppress synaptic transmission in that

expansion of RGC axonal arbor area was observed.

However, the two studies differ in one important as-

pect. In the study that used TeNT-Lc-EGFP (and also

the study that used the VAMP mutant), structural

changes in axons were only observed under competi-

tive conditions, i.e., when only a single cell was

silenced in a field of active ones. In the blu mutant,

where all RGCs are affected, alterations in single arbor

morphology were apparent. How can these differences

be reconciled? The authors of the blu study interpret

the expansion of RGC arbors, and the increased

mEPSC frequency as a homeostatic response to the

reduced glutamate content of vesicles. This contrasts

with the competitive forms of plasticity observed in

the Hua and Ben Fredj studies. Together the studies

outlined here may therefore represent graded suppres-

sion of synaptic function from mild (the blu mutant),

to severe (TeNT-Lc-EGFP). The type of plasticity

induced in RGC axons could change accordingly; mild

suppression could induce homeostatic changes

whereas more severe manipulations could induce com-

petitive forms of plasticity.

The increase in single axon arbor size in the blumutant is also likely to lead to greater convergence in

the blu tectum; an individual tectal cell is likely to be

postsynaptic partner to a greater number of RGCs,

and therefore respond to a larger part of the visual

scene. Indeed, using electrophysiological approaches,

tectal cell receptive fields were shown to be expanded

in the blu mutant relative to those in wild-type zebra-

fish. As would be predicted from the degradation of

the retinotopic map, blu mutants show impairment in

visually driven behaviors such as prey capture (Smear

et al., 2007). The study of the blu mutant therefore

nicely illustrates the feasibility of using multiple

parallel approaches in zebrafish to ask questions

relating to circuit formation. This study started with a

phenotype identified in a forward genetic screen and

used behavioral, electrophysiological, and imaging

techniques, to map out a path from genetic lesion, to

molecular and physiological deficits at an identified

synapse, to structural and functional alterations in

circuitry. However, this study also represents one of

the few examples that use electrophysiological tech-

niques in the visual system of zebrafish [see also

(Ramdya et al., 2006)]. The small size of tectal cells

make application of these techniques a considerable

challenge in zebrafish [although electrophysiological

approaches have been used extensively in studies of

circuitry elsewhere in the zebrafish nervous system

(McDearmid et al., 2006; McLean et al., 2007; Knogler

et al., 2010)]. In Xenopus tadpoles for example, there is

a long history of using both live imaging and electro-

physiology to study development of retinotectal cir-

cuitry (Alsina et al., 2001; Engert et al., 2002; Aizen-

man et al., 2003; Ruthazer et al., 2006).

Molecular Control of Axon Arbor Growthand Laminar Targeting

In addition to neural activity, transcription factors, re-

ceptor-ligand interactions, various signaling path-

ways, and local translational machinery, have all

been identified as contributors to the organization of

axonal and dendritic arbors and the placement of

these arbors in developing neuronal circuitry

(Huberman et al., 2010; Jan and Jan, 2010). In vivoimaging of RGC axons in two zebrafish mutants,

astray (ast) and dragnet (drg) have given us insight

into the molecular mechanisms regulating axon arbor

growth and laminar targeting of axons respectively.

In both vertebrates and invertebrates Roundabout

(Robo) and Slit proteins serve as repulsive axon guid-

ance cues. In zebrafish ast mutants that lack func-

tional robo2, RGC axons show pathfinding defects

consistent with the established role for these receptors

(Fricke et al., 2001). However, robo2 and one of its

ligands, slit1a are also expressed in the optic tectum,

suggesting additional roles for these signaling mole-

cules once RGC axons have reached their target

(Campbell et al., 2007). Analysis of single RGC axo-

nal arbors in ast mutants, and in zebrafish injected

with slit1a antisense morpholino oligonucleotides

revealed that both morphant and mutant arbors had

more branch tips, greater arbor area, and more

presynaptic release sites. Furthermore, time-lapse



imaging revealed that ast and slit1a morphant arbors

stabilized earlier in development than wild-type ones

(Campbell et al., 2007). Together these findings

demonstrate a novel and important role for Robo-Slit

signaling in regulating morphogenesis of RGC axonal

arbors and development of the retinotopic map.

In addition to making retinotopic decisions, RGC

axons must make layer-specific targeting decisions.

RGC axons terminate in six retinorecipient layers

within the zebrafish tectum; the most superficial is

the stratum opticum (SO), three sublayers of the stra-

tum fibrosum et griseum superficiale (SFGS), the

stratum griseum centrale (SGC) and a deep layer bor-

dering the stratum album centrale (SAC), and the

stratum periventriculare (SPV). Imaging of single

RGC axons as they innervate the tectum shows that

in zebrafish, RGCs are confined to a single lamina,

and that laminar specificity is precise from the ear-

liest developmental stages (Xiao et al., 2005; Xiao

and Baier, 2007). Unlike retinotopic organization,

formation of synaptic laminae in the zebrafish tectum

appears to be an activity-independent process (Nevin

et al., 2008). A subset of RGC axons that project into

the SO and the two deep sublayers of the SFGS are

labeled in the Pou4f3:membraneGFP-expressingtransgenic line of zebrafish. This transgenic line has

been used in a forward genetic screen to identify mo-

lecular determinants of laminar choice (Xiao et al.,

2005). In one of these mutants, drg, the orderly lami-

nar distribution of RGC axons in the Pou4f3:mGFPtransgenic is disrupted (Xiao and Baier, 2007). Imag-

ing of single axons in the mutant showed that axons

often strayed from the SFGS to the SO, a phenotype

that was never observed in wild-type larvae. The

dragnet gene encodes collagen IVa5, a component of

the basement membrane that lines the surface of the

tectum. The distribution of heparan sulfate proteogly-

cans (HSPGs, a class of extracellular matrix mole-

cules involved in RGC axon guidance) which are nor-

mally associated with this basement membrane, are

disrupted in the mutant tectum. Data from the dragnetmutant suggests that collagen IVa5 is required to

anchor HSPGs in the basement membrane, which in

turn serves as a lamina targeting cue for RGC axons

(Xiao and Baier, 2007).

Zebrafish have been the focus of many mutant

screens and the studies outlined above illustrate how

mutagenesis allows the role of candidate molecules

to be tested in imaging experiments. However, for-

ward genetic screens are long-term, labor intensive

endeavors. Until recently techniques for reverse

genetic approaches in zebrafish have been limited to

mRNA knockdown strategies using modified anti-

sense morpholinos and TILLING for point mutations

among a library of chemically mutagenized gametes.

Both strategies have shortcomings: morpholinos for

example are active for only the first few days of

development and have many off target effects (Eisen

and Smith, 2008). However, the resistance of the

zebrafish genome to targeted sequence alteration has

recently been overcome through the use of zinc-finger

nucleases (ZFNs). Although still in its infancy, this

new technology promises to overcome what has been

a major gap in the repertoire of techniques available

to zebrafish researchers (Doyon et al., 2008).

Simultaneous Imaging ofPre and Postsynaptic Partners

The studies of axonal and dendritic arbor growth out-

lined above, while informative, have only observed

circuit assembly from either the pre- or postsynaptic

perspective. How interaction with other neurons mod-

ulates the growth process has only been inferred from

the expression of synaptic marker proteins within the

cells being imaged. Imaging pre- and postsynaptic

partners simultaneously as they contact one another

would provide a direct measure of how pre-post inter-

actions modulate axonal or dendritic arbor growth. A

time lapse imaging study in zebrafish has described

the behavior of pre- and postsynaptic neurons as they

contact one another in the spinal cord (Jontes et al.,

2000). The authors focused on Mauthner cells, which

are large, paired, hindbrain neurons that mediate the

escape response in zebrafish. Each Mauthner cell

axon crosses the ventral midline to extend down the

contralateral spinal cord to synapse on serially repeat-

ing sets of three primary motor neurons. The Mauth-

ner axon and motor neuron were visualized by label-

ing each cell type with lipophilic carbocyanide dyes

and time-lapse imaging was used to describe the

dynamic behavior of each cell during the course of

the interaction. The Mauthner growth cone was found

to move relatively smoothly down the spinal cord but

appeared to pause at, and interact with successive tar-

get cells (Fig. 3). Axonal varicosities, presumably

nascent synapses, later appeared at the points where

the axon stalled. Both the primary motor neurons and

the Mauthner axon had a large number of exploratory

filopodia, consistent with an active involvement of

both pre- and postsynaptic elements in establishing

synaptic contacts.

The above study took advantage of the relative

simplicity of the spinal cord wiring diagram, and of

the fact that the Mauthner axon forms synapses with

every motor neuron it passes. Together, these sim-

plify the task of labeling pre and postsynaptic part-



ners and of catching the initial interaction between

them. The complexity of other regions of the CNS

makes the job of labeling cells that are destined to

contact one another, and imaging the interaction,

much more challenging. However, in many regions

of the CNS, synaptic connections are arranged into

discrete layers. Such organization provides an assess-

able system for studying how axons and dendrites

target each other during circuit assembly. The inner

plexiform layer (IPL) of the vertebrate retina is a

particularly attractive model in this regard since the

cellular and synaptic organization of this structure is

highly stereotyped and almost crystalline in nature.

RGCs, amacrine cells, and biopolar cells form synap-

ses with one another in the IPL. These connections

are broadly divided into two layers: connections of

cells depolarized by light (ON cells) and cells hyper-

polarized by light (OFF cells) are localized to the

inner and outer halves of the IPL, respectively.

Within the ON and OFF layers are multiple, function-

ally distinct sublaminae. Time-lapse imaging of fluo-

rescently labeled RGCs, amacrine cells and bipolar

cells in zebrafish has revealed the sequence of events

that establish the synaptic laminae in the IPL (God-

inho et al., 2005; Mumm et al., 2006; Schroeter et al.,

2006). In the pax6-DF4::M-CFP transgenic line of

zebrafish cyan fluorescent protein is targeted to the

membranes of amacrine cells, allowing the individual

synaptic laminae within the IPL to be resolved (God-

inho et al., 2005). Time-lapse imaging of these fish

reveals that amacrine cells are the first to extend neu-

rites into the future IPL, and that laminar organization

emerges almost as soon amacrine cell neurites make

contact with one another (Godinho et al., 2005).

Bipolar cells are the last to arrive in the IPL, after the

connections between RGCs and amacrine cells have

formed (Schroeter et al., 2006). Although RGCs are

born before amacrine cells they don’t extend den-

drites into the IPL until after the amacrine cell plexu-

sus develop. Indeed, studies of the lakritz/atoh7mutant zebrafish that lacks RGCs shows that RGCs

are not absolutely required for amacrine sublamina-

tion (Kay et al., 2004). These findings suggest that

the early plexusus formed by amacrine cell neurites

may prepattern the IPL and provide lamination cues

for RGC dendrites. By time-lapse imaging single

RGCs labeled with yellow fluorescent protein within

the pax6-DF4::M-CFP background, the timing of

RGC dendrite versus amacrine cell stratification

could be observed directly (Mumm et al., 2006).

While RGCs dendrites were found to exhibit diverse

growth and stratification patterns, most were found to

precisely target and costratify with pre-existing lami-

nar plexusus of amacrine cells (Fig. 4). Collectively

these observations suggest that amacrine cells first

target one another, perhaps by homotypic interac-

tions, to give rise to sublaminae, and that this process

is largely independent of RGCs. RGC dendrites then

use cues provided by these sublaminae to achieve

their final stratification pattern. These studies reveal

unexpected specificity in the targeting of dendrites

and also elegantly demonstrate the advantages of

using zebrafish and time-lapse imaging to study

mechanisms of circuit assembly.

Figure 3 Simultaneous imaging of the Mauthner growth

cone and primary motor neuron. The time-lapse sequence

of events show that the Mauthner growth cone is highly

active and transiently interacts with the motor neuron as it

migrates down the spinal cord. Although the growth cone

moved past its postsynaptic target without collapsing or sig-

nificantly altering its morphology, this interaction had a

subtle but detectable effect on growth cone dynamics. Scale

bar 10 lm. Image reproduced from Jontes et al. (2000) with

permission (Copyright 2000 by Nature Publishing Group).



Synapse Assembly

In the CNS chemical synapses typically form

between the axon of one neuron and the dendrites or

cell soma of other neurons. At the level of ultrastruc-

ture, synapses are composed of a presynaptic bouton,

a synaptic cleft, and a postsynaptic specialization.

Presynaptic boutons contain numerous small (*50

nm) clear-centered synaptic vesicles filled with neu-

rotransmitter. Synaptic vesicles dock, fuse and

release neurotransmitter into the synaptic cleft at a

specialized domain called the active zone which is

characterized by the presence of an electron-dense

meshwork of proteins. Directly opposed to the active

zone is the postsynaptic density (PSD), which like the

active zone, is an electron-dense matrix of proteins.

The PSD is composed of structural and scaffolding

proteins that serve to cluster postsynaptic receptors

and associated signaling molecules at the postsynap-

tic membrane (Waites et al., 2005). Both the pre- and

postsynaptic specializations are molecularly complex.

The PSD alone contains *1000 identified proteins

(Yoshimura et al., 2004; Collins et al., 2006). What is

more remarkable is that during periods of circuit

assembly a vast number of synaptic connections are

assembled in a relatively short period of time. How

are all the molecular constituents of synapses deliv-

ered to sites of contact between pre- and postsynaptic

cells? In vitro studies have proved to be useful in

answering this question since individual molecules

can be tracked (by fusing them to GFP for example)

as they are recruited to sites of axo-dendritic contact.

This reductionist approach has revealed that presyn-

aptic assembly occurs through the delivery of preas-

sembled protein complexes (Ahmari et al., 2000;

Shapira et al., 2003). These complexes contain either

Figure 4 RGC dendrites target established presynaptic amacrine strata. Time-lapse analysis of

an RGC dendrite (yellow) initially distributed toward the inner IPL. At a later time point the den-

drite is found to precisely target and costratify with pre-existing laminar plexuses of amacrine cells

(cyan). Scale bar 10 lm. Image reproduced from Mumm et al. (2006) with permission (Copyright

2006 by Elsevier Inc).



synaptic vesicle precursors or active zone structural

proteins. The mechanisms of postsynaptic assembly

are more contentious. For example, there is evidence

that the postsynsptic scaffolding molecule, PSD-95 is

trafficked to the PSD by the gradual recruitment of

individual proteins, and through the delivery of pre-

fabricated protein complexes (Prange and Murphy,

2001; Bresler et al., 2004). While in vitro assays pro-

vide a powerful means to study the cell biology of

synapse assembly they cannot provide definitive

answers about how synapses are assembled in vivo.Many factors that are likely to impact synapse assem-

bly and stability such as molecular interactions with

neighboring cells and extracellular matrix, and

normal patterns of synaptic activity are perturbed

in vitro. Live imaging in zebrafish can complement

in vitro studies of synapse formation by providing the

natural three-dimensional environment of the nervous

system, normal physiological conditions and intact

sensory and synaptic input. Time-lapse imaging of

the synaptic vesicle protein, synaptophysin, fused to

GFP (syp-GFP) in RGC axons in zebrafish revealed

highly mobile clusters of syp-GFP within developing

axons, supporting the notion that presynaptic assem-

bly occurs via the recruitment of preassembled pack-

ets (Meyer and Smith, 2006). Similarly, the dynamic

behavior of a synaptic cell adhesion molecule, N-cad-

herin, was followed in Rohon-Beard sensory neurons

in the zebrafish spinal cord (Jontes et al., 2004; Latefi

et al., 2009). N-cadherin is thought to be involved in

the initial stages of synapse assembly and also syn-

apse stabilization. Like syp-GFP, an N-cadherin-GFP

fusion protein (N-cad-GFP) was transported within

axons as highly mobile protein clusters, while more

stable accumulations of N-cad-GFP corresponded to

synaptic sites. Many of the stable accumulations of

N-cad-GFP formed rapidly in the wake of migrating

growth cones suggesting that N-cadherin is recruited

early on during development of a synapse (Jontes et

al., 2004). Structure–function analysis revealed that

the extracellular domain of N-cadherin is required for

proper trafficking and targeting, and that post-transla-

tional processing of N-cadherin may be a mechanism

for regulating synaptogenesis in vivo (Jontes et al.,

2004; Latefi et al., 2009). Imaging the postsynaptic

marker protein PSD-95-GFP in zebrafish supports the

notion that postsynaptic assembly occurs very differ-

ently from presynaptic assembly. Imaging PSD-95-

GFP in zebrafish tectal cells in vivo showed that this

scaffolding protein concentrated at PSDs by the grad-

ual accumulation from an initially diffuse distribution

in dendrites (Niell et al., 2004). The modular recruit-

ment of PSD-95 to synaptic sites that has been

observed in vitro was not apparent in tectal cells invivo.

One advantage of in vitro systems is that it is

feasible to visualize contact events between pre and

postsynaptic neurons, and then see how and when

synaptic components are recruited to contact sites. As

we have already mentioned, imaging such contact

events in vivo represents a considerable challenge,

particularly in the CNS. The neuromuscular junction

(NMJ) however is more accessible to labeling and

imaging. Transgenic lines of zebrafish, that express

GFP in motor neurons, allow visualization of motor

axons as they innervate their muscle targets. This,

coupled with staining live embryos with fluorescently

tagged a-bungarotoxin [a toxin that binds to postsy-

naptic acetylcholine receptors (AChR)] enables si-

multaneous imaging of both the presynaptic and post-

synaptic specializations in vivo. Thus, the NMJ is an

attractive model for studying the sequence of events

during synapse assembly. Furthermore, because myo-

tubes and motor neurons develop in a rostral-to-cau-

dal progression, the whole sequence of events under-

lying NMJ assembly can be viewed in a single zebra-

fish embryo (Flanagan-Steet et al., 2005; Panzer et

al., 2005, 2006). Two time lapse imaging studies

have demonstrated that prepatterned AChRs exist on

muscle fibers before the arrival of motor axons (Fla-

nagan-Steet et al., 2005; Panzer et al., 2006). Follow-

ing contact with these clusters, presynaptic vesicles

rapidly clustered in motor axons (Fig. 5). These

observations suggested that at the NMJ, postsynaptic

differentiation precedes presynaptic differentiation

and that the formation of initial postsynaptic special-

izations does not require motor axons. Postsynaptic

differentiation was nonetheless found to be nerve de-

pendent, in that axons are crucial for the maturation

and maintenance of the postsynaptic apparatus.

CONCLUDING REMARKS

By imaging nervous system development in zebrafish

we have already gained valuable insight into the

mechanisms of nervous system assembly. The future

looks as, if not more promising. The recent develop-

ment of photo-switchable ion channels and pumps

such as channelrhodopsin (ChR2) and halorhodopsin

(NpHR) has already enabled non-invasive dissection

of circuit function in zebrafish (Douglass et al., 2008;

Arrenberg et al., 2009; Schoonheim et al., 2010).

These tools could also be used to address questions

relating to nervous system plasticity. For example, by

using them to control the firing pattern and rate

within a developing neuron we could ask what kinds

of activity patterns drive structural changes in neu-

rons. Genetically encoded reporters of synaptic func-



tion could also provide a means to examine the emer-

gence of circuit function, functional plasticity at the

level of single synapses, and to relate synapse func-

tion to the structural plasticity of neurons (Dreosti et al.,

2009). For example, the functional properties of an

identified synapse could be related to the dynamic

behavior of the axonal or dendritic branch on which it

is located. Furthermore enhancer and gene trapping

approaches are generating hundreds of Gal4 driver lines

in which the expression of UAS-linked effectors can be

targeted to subpopulations of neurons (Scott et al.,

2007; Asakawa and Kawakami, 2009). This approach

promises to vastly expand the repertoire of neuronal

cell types that are accessible to manipulation and imag-

ing.

The authors thank Juan Burrone, Sarah Hammond, and

Paul Hunter for helpful comments on the manuscript.

Figure 5 Postsynaptic differentiation precedes and guides axon outgrowth. Time-lapse images of

a motor axon growth cone extending toward, contacting, and extending beyond pre-patterned AChR

clusters. The motor axon was labeled with VAMP-GFP (green) and the AChR clusters with a-bun-garotoxin (red). A number of pre-existing AChR clusters are seen ahead of the growth cone, which

eventually contacts the clusters one by one. In some cases VAMP-GFP positive clusters of presynap-

tic vesicles accumulate over pre-patterned AChR clusters (asterisks), possibly forming NMJs.

Toward the end of the time-lapse imaging the growth cone is seen to turn toward pre-patterned

AChR clusters at the dorsal end of the myotome (arrowheads). Scale bar 10 lm. Images reproduced

from Panzer et al. (2006) with permission (Copyright 2006 by the Society for Neuroscience).



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Imaging circuit formation in zebrafish