Download Development of functional hindbrain oculomotor circuitry

Curr Neurobiol 2016; 7 (2): 62-73
ISSN 0975-9042
Development of functional hindbrain oculomotor circuitry independent of both
vascularization and neuronal activity in larval zebrafish.
Florian Ulrich1#, Charlotte Grove2#, Jesús Torres-Vázquez1 and Robert Baker2
Department of Developmental Genetics, Skirball Institute of Molecular Medicine, 540 1st Avenue, New York City, New York
10016, USA.
2
Department of Neuroscience and Physiology, New York University Medical Center, 550 1st Avenue, New York City, New
York 10016, USA.
#
Florian Ulrich and Charlotte Grove are co-first authors.
1
Abstract
We investigated the contribution of blood vessel formation and neuronal excitability to the
development of functional neural circuitry in larval zebrafish by analyzing oculomotor
performance in response to visual and vestibular stimuli. To address the dependence of
neuronal function on the presence of blood vessels, we compared wild type embryos to
reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal
excitability impacts neuronal development and intracerebral vascularization, we blocked
neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both
slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude
and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial
induced eye rotation were also present. All of these behaviors except for visual tracking were
observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor
neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending
in motor innervations of the eye muscles, were correctly formed and generated appropriate
oculomotor behaviors without blood vessels. However, our observations indicate that
beginning at approximately six days, circulation was required for sustained behavioral
performance. We further found that blocking neuronal excitability with either TTX or
Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral
blood vessel formation in wild type larvae. After removal of drug treatments, the
oculomotor behaviors returned within hours. Thus, development of neuronal circuits that
drive oculomotor performance does not require neuronal spiking or activity. Together these
findings demonstrate that neither vascularization nor neuronal excitability are essential for
the formation of numerous oculomotor nuclei with intricately designed connectivity and
signal processing. We conclude that a genetic blueprint specifies early larval structural
and physiological features, and this developmental strategy may be viewed as a unique
adaptation required for early survival.
Keywords: Larval zebrafish, Neuronal activity, Vascularization, Oculomotor, Excitability, Neuronal development, TTX.
Accepted July 25, 2016
Introduction
In nearly all adult vertebrates, the vasculature is of key
importance for supplying nutrients and oxygen to the brain.
Throughout the nervous system, neurons and blood vessels
align closely to form the neurovascular unit [1]. Little is
known, however, about the causal role of the vasculature
during the embryonic establishment of neuronal pathways
and functional activity. In addition, little information exists
62
about the interplay between neuronal excitability and
embryonic development of the neurovascular unit. Several
studies have shown that endothelial cells can stimulate the
formation of neurons [2-4] and that neurons can instruct
blood vessel development [5,6]. Moreover, neuronal
activity can regulate vascular tonus in mouse brain slices,
suggesting that the supply of nutrients and oxygen to the
brain is tightly controlled by local energy demands in the
brain [7].
Curr Neurobiol 2016 Volume 7 Issue 2
Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish.
Shortly after the end of gastrulation in zebrafish
endothelial precursors from the anterior lateral plate
coalesce into rostral and midbrain organizing centers
[8,9]. The latter centers gives rise to the angiogenic
sprouts forming primordial hindbrain channels which then
provide the endothelial cells migrating into the center of
each rhombomere to form the hindbrain central arteries
as illustrated in Figures 1A and 1C. Interestingly, most
of the neuronal subgroups and circuits that are essential
for oculomotor performance (as shown schematically in
Supplementary Figure 1) lie close to blood vessels in the
fore-, mid- and hindbrain compartments. Nevertheless, we
recently found that the small central arteries growing into
rhombomere centers were not required for either neuronal
development or maintenance in the developing zebrafish
hindbrain (Figures 1B and 1D) [9]. Hence, we hypothesized
that endothelial sprouting and neuronal activity might also
be independent. Accordingly, we tested whether neuronal
activity depended on the presence of endothelial cells in
reck and cloche mutants that are devoid of intracerebral
blood vessels by utilizing the well established neuronal
circuitry and signal processing underlying oculomotor
behavior [10,11]. Conversely, to explore whether neuronal
activity was required for endothelial sprouting or the
emergence of oculomotor behavior, we blocked neuronal
excitability and synaptic transmission for several days
with either tetraodotoxin (TTX) or Tricaine.
Larval zebrafish exhibit many robust individual oculomotor
behaviors employing clearly defined sets of neurons that
A
WT
can be used as a read out for neuronal development and
function [12] (Figure 2A, Supplementary Figures 1A-1D).
Optokinetic tracking performance begins in the forebrain
pretectum with a visual slip velocity signal that directly
reaches the motoneurons (Supplementary Figures 1A and
1B, Direct). A pretectal pathway through the vestibular
nucleus converts the signal to eye velocity before reaching
the ocular motoneurons (Supplementary Figures 1A and 1B,
Indirect). The vestibular neurons also project to a hindbrain
neural integrator (VPNI) located in the caudal most
hindbrain rhombomere (r8) that converts the signal to eye
position (Supplementary Figures 1A and 1B). Importantly,
this hindbrain neural integrator is separately recruited
during horizontal fast phases as well as during saccades
for eye position holding (Supplementary Figures 1C and
1D). In addition, larval zebrafish have a novel vestibular
gravitoinertial system generating compensatory torsional
eye rotations in response to either upward or downward
head motion [13]. Utricular signaling alone, independent
of the semicircular canals, connects with specific subsets
of tangential neurons in r5 projecting to inferior rectus/
superior oblique, inferior oblique/superior rectus motor
nuclei and extraocular muscles (Supplementary Figures
1E and 1F). These straightforward, but highly detailed sets
of central oculomotor connections and signal processing
units therefore provides a rich repertoire of neuronal
subgroups and circuits to analyze any perturbation in
embryonic development.
Accordingly, we measured components of the slower
B
Neurons
reck-/Neurons
Vessels
r8
Vessels
r7
r6 r5
Vessels r8
r4 r3
r2 r1
5 dpf
P
C
A
r5 r4
r3 r2 r1
P
5 dpf
D
WT
RSN
r7 r6
clo-/RSN
Vessels
MN
MN
r8
r8
r7
3 dpf
r6
A
r5 r4 r3 r2 r1
P
A
3 dpf
r7 r6
r5
r4 r3 r2 r1
Vessels Absent
P
A
Figure 1. Vasculature in wild type (WT), cloche and reck mutants. (A-B) Maximum intensity confocal projections of a 5 dpf
WT (A) embryo and a reck (B) mutant sibling lacking central arteries in the hindbrain. Green, pan-neuronal ngn:GFP and
red, pan-endothelial kdrl:cherry. (C-D) Maximum intensity confocal projections of a WT embryo (C) and a cloche mutant (D)
sibling lacking complete hindbrain vasculature, at 3 dpf. Red, pan-endothelial kdrl:cherry and green, retrograde labeling of
reticulospinal neurons (RSNs). Lateral orientation is shown in A-D with anterior to the right and dorsal to the top
Abbreviations: r, rhombomere; WT, Wild Type; RSN, Reticulospinal Neuron; MN, Mauthner Neuron; P and A, Posterior and Anterior
Curr Neurobiol 2016 Volume 7 Issue 2
63
Ulrich/Grove/Torres-Vázquez/Baker
A
Midbrain
Hindbrain
SC
H
SR SO
T Vestibular
LR
MR
Trochlear Oculomotor
Integrator
r8
B
r7
Neurons
r6
r5
LR
IR IO
MR
r1-3
r4
Temporal
WT
Vessels
nX
Pretectal
Burst
Internuclear
Abducens
C
reck-/-
D
clo-/-
Nasal
nIV
nVII
nV
r8
Vessels
4 dpf
r7
r6 r5
r4 r3 r2
r1
P
5 dpf
nft-/-
A
4 dpf
Figure 2. Schematic overlay of neuronal groups with vasculature. (A) Rhombomeric location of the major oculomotor neurons from
the pretectum to hindbrain that mediate horizontal, vertical and torsional eye movements. (B) Lateral confocal view of a 4 dpf wild
type embryo labeled with the pan-endothelial transgene kdrl:cherry and hindbrain motoneurons identified by the marker Islet:GFP.
(C-D) Photographs of 5 dpf reck and 4 dpf cloche larvae with eye movements illustrated in Figure 3
Abbreviations: H, Horizontal; T, Tangential; LR, Lateral Rectus; SR, Superior Rectus; MR, Medial Rectus, IR, Inferior Rectus; IO, Inferior
Oblique; SO, Superior Oblique; r, rhombomere; SC, Spinal Cord; nIV, trochlear nucleus; nV, trigeminal nucleus; nVII, facial nucleus
(visual tracking) and fast (resetting) phases of horizontal
optokinetic reflexes (OKR) as well as spontaneous
scanning saccades and eye position holding in the presence
and absence of intracerebral vessels in wild type vs. reck or
cloche mutant larvae within a 4-6 dpf window. We found both
directional OKR tracking and fast phase resets including
vestibular gravitoinertial compensatory eye rotations in
all reck mutants tested. All of these behaviors except for
visual tracking were observed in cloche mutants. While
response robustness was variable and less than that in wild
type, our findings indicate that neuronal circuits mediating
oculomotor performance were still correctly formed and
exhibited activity without blood vessels. Interestingly,
sustained blocking of neuronal excitability and synaptic
activity from embryonic onset using TTX or Tricaine did
not interfere with neuronal development, since oculomotor
behavioral patterns could be found with a near normal
profile after a short recovery from the drug treatments.
Moreover, blocking neuronal activity did not noticeably
interfere with intracerebral blood vessel formation. Our
findings, therefore, emphasize the remarkable extent to
which neurons differentiate, form precise connections and
acquire functional oculomotor activity independent of the
presence of either vasculature or excitability.
64
Methods
Zebrafish Lines and Maintenance
We handled and maintained adult, larval and embryonic
zebrafish under standard laboratory conditions following
NYU’s IACUC/IBC-approved protocols. For wildtype
specimens, we used EK [14] TL, Wik, AB and mixed
genetic backgrounds [15]. For vasculature mutant lines
we used recky72 [8] and cloches5 [16]. Recky72 is a recessive
null allele of a reversion-inducing cysteine-rich protein
with Kazal motifs, a gene encoding a membrane-anchored
tumor suppressor glycoprotein required for promoting
canonical Wnt signaling that enables cerebrovascular
development. Reck is both expressed and required in
endothelial cells for intracerebral vascularization and
proper expression of molecular markers associated
with blood-brain barrier. Reck mutants display a brainspecific vascularization deficit (Figure 1B). Cloche is
an early acting zebrafish gene that is required by both
the endothelial and hematopoietic lineages; however,
the gene affected has not been identified. Cloche (clo)
mutants display a severe deficit in the specification of
both the endothelial and hematopoietic head lineages
(Figure 1D).
Curr Neurobiol 2016 Volume 7 Issue 2
Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish.
To enhance the vasculature in vivo, we took advantage of the
following transgenic endothelial reporters: Tg(kdrl:RFP)
s896
[17] and Tg(kdrl:EGFP)la116 [18]. To enhance neurons,
we used Tg(neurog1:eGFP)w61 and Tg(isl1:GFP)rw0 lines
[19]. These transgenic reporters were used in wild type
lines (see above) and mutants: recky72 [8] and cloches5
[16]. For visualizing reticulospinal neurons, we inserted a
Rhodamine-Dextran crystal into the hindbrain/spinal cord
at the level of myotome 5 as described in [9].
Drug Treatments
For Tetraodotoxin (TTX) applications, we anesthetized
embryos at 24–28 hpf in 0.016% Tricaine in embryo
medium and mounted them vertically by carefully
inserting their tails into individual holes in an agarose
plate. Thus, the heads were sticking out with the hindbrain
facing in dorsal direction. We then injected 1 nL of 1 mM
TTX in Danieau’s solution into the hindbrain ventricle,
washed the Tricaine out and kept the specimen in embryo
medium at 28.5ºC until measuring neuronal function or
confocal imaging. For sustained Tricaine treatments, we
kept embryos in 0.016% Tricaine @ 28.5ºC from 18 hpf
to 96 hpf. We refreshed the medium once a day to avoid
bacterial growth. Paralysis induced by either TTX or
Tricaine was assessed by lack of response to dish taps and
tail touches with an eyelash tool. In addition, paralyzed
embryos did not show any vestibular righting reflex and
often remained upside-down with yolk facing upwards.
Recovery from these drugs was established by upright
posture, tail motion and escape-like responses soon
followed by swim-like movements.
Fixing and Staining of Embryos
To allow for an easier handling during confocal imaging,
we fixed the embryos overnight in 4% PFA in PBS at
4ºC. After washing several times with PBT (PBS+0.1%
Triton X-100), we blocked the embryos for 1h at room
temperature in blocking solution (1% BSA in PBT) and
stained the embryos overnight at 4ºC with anti-RFP (1:500,
Becton Dickinson 632496) or anti-GFP antibodies (1:200,
Invitrogen, A-11122) in blocking solution. We washed the
embryos 4x for 15 minutes in Blocking Solution and then
stained them with secondary antibodies: donkey-rabbit
Alexa 488 (A-21206) or donkey-rabbit 546 (A-10040),
each at 1:2,000 in blocking buffer overnight at 4ºC. We
then washed the embryos 4x for 15 minutes in PBT at room
temperature and kept them at 4ºC in PBT until imaging. If
embryos were labeled with both RFP and GFP, we only
stained against RFP, since GFP gave a strong signal on its
own even after fixation.
Confocal Imaging
We mounted the embryos in 1% agarose/PBS and acquired
images of the vasculature and neuroepithelium using a
Leica SP5 confocal microscope with a 40x dipping/water
immersion objective (NA=0.8). Bidirectional scans were
performed at 200 lines/s in 1024 × 1024 pixel windows;
Curr Neurobiol 2016 Volume 7 Issue 2
z-stacks were taken at 1 µm z intervals. Subsequently,
the image stacks were turned into Maximum Intensity
Projections and further edited using ImageJ and Adobe
Photoshop.
Experimental set-up and eye movement behaviors
Larval fish, 4-6 dpf, were embedded in low melting
temperature agarose (2.0%, Sigma Type VII-A). The
block of agarose was pinned to a Sylgard 182 (Dow
Corning, Midland, MI) disk and excess agarose removed
from around the eyes and head rostral to the swim bladder
enabling unhindered tracking of either a horizontal or
torsional visual stimulus (Supplementary Figures 2B
and 2C). The disk with the mounted larva was lowered
into a 19 mm diameter or 25 mm square transparent
glass specimen chamber for horizontal OKR or pitch
tilt response, respectively, containing ~ 2.5 ml of 30%
Danieau’s solution. A round cover glass slip held in
position by surface tension served as the chamber ceiling.
While the larvae remained stationary on the vestibular
turntable a rotating visual surround stimulus was produced
by a servo controlled motor (Supplementary Figures 2D
and 2E) [20]. Transparent acrylic drums of different sizes,
nested concentrically around the fish chamber provided
rotating and stationary patterned stimuli, alternating black
and white stripes at 15.5º frequency. The rotating drum
was illuminated through the table shaft by visible light
LEDs. Stimulus waveforms controlling drum motions
were produced with an Agilent (Palo Alto, CA) 33120A
phase-linked function generator. Both sinusoidal and
velocity step waveforms were available from 0.125 to 2
Hz and at up to ± 20º in amplitude.
Torsional eye rotation measurements were made in a
custom-built tilting apparatus consisting of a rotating
platform driven by a DC motor [13]. Motor command
signals were generated with a custom code written in
LabView and platform position was measured via an
attached tachometer. A high speed camera (Pike IEEE
1394b, Allied Vision Technologies) mounted to the
rotation axis recorded right eye rotation. Compensatory
vestibuloocular reflexes were produced in response to a
pitch tilt sinusoid waveform at 0.125 Hz and a maximum
amplitude of ± 20°.
Eye Movement Measurements
A three-axis micromanipulator on the mounting stage for
the specimen holder allowed the head/eyes to be centered
for focusing of the overhead CCD camera. Horizontal and
torsional eye positions in 4-6 dpf larvae were sampled at
100 Hz and 60 Hz, respectively. Infrared CCD cameras
(Supplementary Figure 2F) recorded the weakly pigmented
eyes and threshold masks (Supplementary Figure 2G)
were utilized to locate the major axes of both the body and
eyes (Supplementary Figure 2I). Video images collected
by the CCD camera were processed in real time to extract
horizontal eye position. Eye velocity was obtained by
65
Ulrich/Grove/Torres-Vázquez/Baker
digitally differentiating filtered eye position records and
OKR gain calculated as drum velocity/eye velocity [20].
Eye position changes generated by torsional stimuli were
extracted by comparing recorded images with initial 0º
reference images. Saccades, optokinetic and torsional
responses, were recorded and analyzed with a custom
code written in LabView (National Instruments, Austin,
TX) and MATLAB (MathWorks, Natick, MA) [13,21].
Results
Larval wild type 4-6 dpf zebrafish exhibit robust
oculomotor performance in response to vertical axis
visual (Supplementary Figure 3) and/or head pitch/roll
vestibular stimuli [13] with the neuronal subgroups and
connections extending from the forebrain pretectum
to the hindbrain vestibular, reticular, internuclear and
motoneurons as illustrated in the Supplementray Figures
1A-F vignettes. To address whether the establishment
of neuronal circuits and the ensuing function depends
on the presence of blood vessels, we measured the slow
(tracking) and fast (resetting) phases of the horizontal
optokinetic reflexes (OKR) in 20 reck mutant larvae from
7 separate experimental series (Figures 3 and 4). We also
recorded spontaneous scanning saccades and eye position
Spontaneous Scanning reck-/- 5dpf
Light
A
20°
Saccade
0°
Eye Position
Stable
N
B
Right Eye
Left Eye
Dark
N
T
20°
20s
T
20°
Eye Position
Leak
Saccade
0°
20°
C
Spontaneous Scanning clo-/- 4dpf
Unstable
20°
0°
Stable
Leak
20s
20°
Figure 3. Spontaneous horizontal scanning eye movements in reck and cloche mutants. (A-B) Scanning behaviors of a 5 dpf reck
embryos shown either in light (A) or dark (B). Records are from the right (red) and left (blue) eyes with nasal (N) and temporal
(T) directions as indicated. Arrows mark saccades and eye position holding that is stable in A or leaky in B. (C) Scanning eye
movements in a 4 dpf cloche mutant. Arrows illustrate stable, unstable and leaky eye position holding
A
Optokinetic StepTracking reck-/- 5dpf
Eye Position
Slow Phase
20°
0°
0.125 Hz
B
Right Eye
Eye Velocity
N
T
Left Eye
T
N
Drum Rotation
Fast Phase
Direct
20°
20°/s
0°/s
Indirect
8s
20°/s
Figure 4. Optokinetic tracking in reck mutants. (A-B) Eye movements showing eye position (A) and eye velocity (B) performance in
response to ± 20°/s drum rotation at 0.125 Hz. Arrows mark the slow and fast phases in A and the direct and indirect components
in B. Records are from the right (red) and left (blue) eyes with nasal (N) and temporal (T) directions as indicated. For both the RE
and LE the direct and indirect gains were 0.4 that were reduced by 33% from a wild type gain of 0.6 (from Supplementary Figure 3)
66
Curr Neurobiol 2016 Volume 7 Issue 2
Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish.
holding patterns in the same reck larvae and an additional
13 cloche larvae. To test vestibular performance, we
measured torsional eye rotation in another 12 reck and 13
cloche mutants in response to nose-up/nose-down pitch
(Figure 5).
Spontaneous Horizontal Scanning Eye Movements in
reck and cloche Mutants
Horizontal saccadic scanning and eye position holding
at 5 dpf was well developed in both wild type larva
(Supplementary Figure 3A) and reck mutants (Figure
3A). Conjugate saccadic movement of both the right and
left eyes occurred in their respective nasal and temporal
directions. Sustained eye position holding was observed
primarily due to the visual slip feedback provided by the
retinal-pretectal-hindbrain loop (Supplementary Figures
1A and 1B). In short, this oculomotor performance in
light demonstrates the presence and functional integrity of
nearly every neuronal group and connectivity illustrated
in Figure 2A and Supplementary Figures 1A and 1B,
respectively.
We also observed scanning saccadic behavior in the absence
A
of visual feedback (Figure 3B). In the dark, eye position
in reck mutants decayed with time constants ranging from
5-10 s similar to, but on average a few seconds less than
that of a normal wild type larva (Supplementary Figure
3A). In cloche mutants, scanning saccades were conjugate
and of reasonable peak velocity for both eyes in either the
temporal or nasal direction (Figure 3C). Since none of the
cloche mutants tested responded to any visual tracking
stimuli, eye position holding was entirely due to integrator
function which was found to be quite variable, performing
as either stable, leaky or unstable as labeled in Figure
3C. This highly variable eye position holding behavior
contrasts to the symmetrical leak in both the right and left
oculomotor integrators in reck mutants (Figure 3B) and
wild type (Supplementary Figure 3). Simultaneous stable
(LE) and leaky (RE) eye position holding in Figure 3C
could occur for the same movement direction, because
the integrator circuits are separate for the left and right
side as shown in Supplementary Figures 1C and 1D. In
conclusion, all oculomotor subnuclei and connections
for spontaneous scanning and eye position holding were
present and functional without intracerebral vasculature in
Torsion in reck-/- 4 dpf
30°
Nose-up tilt
SR-IO
RE
0°
SO-IR
Nose-down tilt
B
Torsion in clo-/- 4 dpf
20s
30°
30°
Nose-up tilt
SR-IO
RE
0°
SO-IR
Nose-down tilt
30°
Figure 5. Torsional eye movements in reck and cloche mutants. (A-B) Torsional eye rotations in response to a 30° sinusoidal
nose-up and nose-down tilt at 0.1 Hz in reck (A) and cloche (B) mutants. The compensatory direction of eye rotation and the
responding eye muscle pairs are indicated as color coded in Figure 2. Records are from the right (red) eye with VOR gains in reck
and cloche mutants of 0.36 and 0.25, respectively that were reduced by 10% and 38% from a wild type gain of 0.4 [13]
Curr Neurobiol 2016 Volume 7 Issue 2
67
Ulrich/Grove/Torres-Vázquez/Baker
reck mutants or even without any head vasculature at all
in cloche mutants.
Optokinetic Eye Movements in reck Mutants
By 5 dpf, binocular visual tracking of full field targets
is well developed in wild type zebrafish larvae with eye
velocity proportional to, but less than, stimulus velocity
(Gain=0.60, Supplementary Figure 3) [20]. Optokinetic
performance in reck mutants was comparable as shown by
the eye position records in Figure 4A (alternating slow and
fast phases) and the matching step-like eye velocity records
in Figure 4B. Each 4 second eye velocity period reflects
neuronal activity in two distinct brainstem circuits that can
be separated into an early (direct) and sustained (indirect)
component (Figure 4B) [12,22]. Notably, the indirect
component of eye velocity reflects the functional status of
horizontal vestibular neurons and their connectivity with
the abducens internuclear, motor and integrator neurons
(Supplementary Figures 1A and 1B) [12]. For both the
RE and LE in reck mutants the direct and indirect gains
were 0.4, lower by 33% from a wild type gain of 0.6 (from
Supplementary Figure 3).
The use of optokinetic sinusoidal tracking in reck mutants
(Supplementary Figure 4A) supports and extends the step
tracking findings by showing that both eye velocity phase
and gain are maintained over a wide frequency bandwidth.
For example, at 0.125 Hz both RE and LE OKR gains were
0.43 and, compared to a wild type gain of 0.6, reduced
by 28%. At two higher frequencies, 0.5 Hz and 1.0 Hz,
OKR gains were reduced by 45% and 55%, respectively
(Supplementary Figures 4B and 4C). Phases at 0.125 Hz
and 0.5 Hz were ~ 0° and at 1.0 Hz -20° that were not
significantly different than wild type [23]. In summary,
visual performance from the retina to the pretectum
through to the hindbrain oculomotor nuclei and circuitry
was similar to, but of less amplitude than that observed in
wild type larva with intact vasculature.
Vestibular Behavior in reck and cloche Mutants
In order to test the status of hindbrain circuitry wiring
that exists from the vestibular sensory periphery to the
individual midbrain oculomotor subnuclei shown in Figure
2A, both reck and cloche mutants were presented with a
sinusoidal nose-up/nose-down stimulus as diagrammed in
Supplementary Figures 1E and 1F. Both mutants exhibited
eye position changes compensatory to the direction of head
tilt (e.g. for nose-up tilt clockwise rotation of the right
eye). This response pattern demonstrated that individual
tangential subgroups in rhombomere 5 established correct
connections with their respective oculomotor targets as
shown for the right eye in Figure 5 (e.g. SO-IR in the nose
down case and SR-IO in the nose-up).
For comparable head tilts compensatory eye position
amplitudes in reck mutants were nearly the same as in
normal wild type larva [13], but noticeably less in cloche
mutants (Figures 5A and 5B, n=6). Records from the right
68
(red) eye showed vestibulocular (VOR) gains in reck
and cloche mutants of 0.36 and 0.25, respectively that in
turn were lower than wild type gain by 10% and 38%. In
addition, eye velocity was step-like rather than sinusoidal
in cloche mutants (Figures 5B) suggesting somewhat
compromised circuit performance perhaps reflective
of the metabolic state. Not unexpectedly, behavioral
performance could be greatly diminished after 15-30 min
of continuous testing, and in some cases to zero, consistent
with the avascular head condition.
Together these observations provide a direct demonstration
that in either the absence of intracerebral (reck) or complete
head vasculature (cloche), all the essential neurons
and circuits are formed from the forebrain through the
hindbrain, including the pathways and function of orbital
eye muscles. Collectively these observations (Figures 3-5)
show that oculomotor function can be, and often is, nearly
equivalent to that of wild type larva, particularly with 4-5
dpf reck mutants. These findings support the hypothesis
that a hard-wired genetic blueprint gives rise to a functional
neural network in the absence of vascular patterning cues
and the accompanying metabolic resources.
Role of Neuronal Excitability in Neuronal Function and
Vascular Development
Subsequent to the previous observations demonstrating
that neuronal activity did not depend on the presence of
endothelial cells in reck or cloche mutants, we explored
the converse paradigm of whether either endothelial
sprouting or oculomotor behavior depended on neuronal
excitability and/or activity dependent behaviors. In the
first set of experiments, we injected at ~ 24 h 1 nL of a 1
mM solution of Tetraodotoxin (TTX) into the hindbrain
ventricle. Oculomotor behaviors were recorded at 4-6 dpf
between 4 to 7 h after the recovery from complete paralysis
with the appearance of spontaneous and escape-like body
movements, but not yet full righting and/or swimming
reflexes.
Comparison of wild type (Figure 6A, WT) with TTX
treated larvae (Figure 6B at 6 dpf) revealed that both the
vasculature and neuronal subgroups were not noticeably
different. Surprisingly, in all TTX larvae examined (n=12),
both spontaneous scanning and visual tracking behaviors
were present and not too different from untreated wild
type larva (Figure 7 and Supplementary Figure 3). The
frequency of spontaneous scanning saccades was often
slightly reduced, but eye position holding was stable
(Figure 7A), indicative of the presence of a visual slip
feedback from retina through the pretectum. In the
dark, decay in eye position was similar to that observed
in wild type larvae (Figure 7B and Supplementary
Figure 3B) suggesting nearly normal operation of the
oculomotor integrator network (VPNI in Supplementary
Figures 1C and 1D [12,24,25]. These observations were
complemented by records showing optokinetic tracking
and fast phase resets to be quite typical with, in this case,
Curr Neurobiol 2016 Volume 7 Issue 2
Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish.
A
B
WT
Neurons
Vessels
nVII
nV
nX
r8
r7 r6
r5
TTX
Neurons
nX
nIV
nVII
r6
r3 r2 r1
5 dpf
P
r5
r4
r3 r2 r1
5 dpf
A
nIV
nV
r8
r7
r4
Vessels
P
A
Figure 6. Vasculature in WT and TTX treated larvae. (A-B) Maximum intensity confocal projections of a WT (A) embryo and after
5 days in TTX (B) showing neurons and hindbrain central arteries. In both cases, green shows Islet-GFP and red, pan-endothelial
kdrl:cherry. Lateral orientation is shown in A-B with anterior to the right and dorsal to the top. Abbreviations are as in Figure 1
Spontaneous Scanning TTX 6dpf
A
B
Light
N
Right Eye
Left Eye
T
T
N
Stable
Saccade
Saccade
0°
Leak
C
Eye Position
20°
Dark
20s
20°
Optokinetic Step Tracking TTX 6dpf
20°
Slow Phase
0°
D
Eye Velocity
Drum Rotation
Fast Phase
8s
Direct
20°
20°/s
0°/s
Indirect
20°/s
Figure 7. Spontaneous and optokinetic behavior after TTX. (A-B) Scanning behavior of a 6 dpf embryo shown in light (A) and
dark (B). Arrows mark saccades and eye position holding (stable in A or leak in B). (C-D) Optokinetic step tracking performance
in response to ± 20°/s drum rotation at 0.125 Hz. Eye position is illustrated in C and eye velocity in D. Arrows mark the slow and
fast phases in C and the direct and indirect components in D. Records are from the right (red) and left (blue) eyes with nasal (N)
and temporal (T) directions as indicated. RE and LE direct OKR gains were 0.56 and 0.52 that were reduced by 10% and 13%
from a wild type gain of 0.6. RE and LE indirect OKR gains were 0.51 and 0.52 that were reduced by 17% and 13%, respectively
from a wild type gain of 0.6 (from Supplementary Figure 3)
the left eye direct component slightly better than the right
eye (Figures 7C and 7D). RE and LE direct OKR gains
were 0.56 and 0.52 that were lower from wild type gain
of 0.6 by 10% and 13%, respectively. However, RE and
LE indirect OKR gains were similar at 0.51 and 0.52
and lower from wild type gain of 0.6 by 17% and 13%,
respectively (from Supplementary Figure 3).
For treatment with Tricaine, wild type zebrafish were
raised from 18 h to 4-5 days in 0.016% Tricaine solutions.
All spontaneous and/or evoked movements, save for
cardiovascular function, were absent until the larvae
were removed from the Tricaine. Righting and swimming
reflexes were often quite slow to recover (similar to the
TTX treatments), however, larval escape responses often
were regained within minutes; we thus tested oculomotor
behaviors within 1-4 h after removal from Tricaine.
Curr Neurobiol 2016 Volume 7 Issue 2
Somewhat unexpectedly, and despite severe edema, 14 of
the 17 tested Tricaine treated larvae exhibited spontaneous
scanning saccadic behavior with reasonably high velocity
saccades, but clearly with a diminished frequency (Figures
8A and 8B). A pretectal visual feedback loop appeared to
be present as shown by the stable right eye position holding
in the nasal direction (RE, Stable), but not for the other
direction (RE, temporal leak). However, on comparison
with scanning in the dark (Figure 8B), these behaviors
much more likely reflected the individual neural integrator
(VPNI) functions for the nasal and temporal directions (as
illustrated by the circuits in Supplementary Figures 1C
and 1D).
Optokinetic tracking showed that a direct retinal-pretectal
connection to motoneurons was intact as can be seen in
the step-like changes in eye position (Figure 8C) and the
69
Ulrich/Grove/Torres-Vázquez/Baker
A
Spontaneous Scanning Tricaine 5dpf
B
Light
Stable
Stable
Saccade
Dark
10°
Saccade
0°
Right Eye
C
N
Leak
Left Eye
10°
Leak
N
T
Eye Position
T
Optokinetic Step Tracking Tricaine 5dpf
20s
10°
Fast Phase
0°
D
Eye Velocity
Drum Rotation
Slow Phase
4s
10°
Direct
10°/s
0°/s
Indirect
10°/s
Figure 8. Spontaneous and optokinetic behavior after Tricaine. (A-B) Scanning behavior of a 5 dpf embryo shown in either
light (A) or in dark (B). Arrows mark saccades and eye position holding showing either stable (RE) or leak (LE) in A and the
same pattern in B. (C-D) Optokinetic step tracking performance in response to ± 20°/s drum rotation at 0.125 Hz. Eye position
is illustrated in C and eye velocity in D. Arrows mark the slow and fast phases in C and the direct and indirect components in D.
Records are from the right (red) and left (blue) eyes with nasal (N) and temporal (T) directions as indicated. Both RE and LE direct
OKR gains were 0.4 and reduced by 33% from wild type gain of 0.6 (from Supplementary Figure 3). RE and LE indirect OKR gains
were 0.15 and 0.1, respectively and were reduced by 75% and 83% from a wild type gain of 0.6 (from Supplementary Figure 4)
accompanying direct component of eye velocity (Figure
8D, Direct). Both RE and LE direct OKR gains were 0.4,
reduced by 33% from wild type gain of 0.6. Also, the
saccadic reset circuitry was functional as indicated by the
low velocity fast phases (Figure 8C, Fast Phase). However,
in all the Tricaine raised larva, the indirect component
of step velocity was severely compromised (Figure 8D,
Indirect), clearly indicating the pretectal to vestibular
connectivity was affected (Supplementary Figures 1A and
1B). RE and LE indirect OKR gains were 0.15 and 0.1 and
lower than wild type gains by 75% and 83%, respectively.
In summary, at 4-5 days, after removal of drug treatments
that prevented neuronal spiking and synaptic activity,
oculomotor behavioral performance quickly returned
within hours, but much more robustly for the TTX, rather
than Tricaine, treated larvae.
Discussion
In this study we tested the hypothesis that the complex
circuitry that gives rise to robust horizontal eye tracking,
saccadic and torsional responses at 4-6 dpf is able to
form and become functional in the absence of either
metabolic support or neuronal activity. We found that reck
and cloche mutants, each lacking intracerebral vessels
and thus metabolic support for neuronal development,
exhibited all oculomotor behaviors. Wild type larvae in
which neuronal activity was blocked from 1 to 5 days also
exhibited oculomotor responses comparable to untreated
larvae once the block was removed. Our findings clearly
indicate that neural network developmental patterning
and central processing is independent of vasculature and
neural excitability.
70
Our study provides an assessment of functional neuronal
development through the observation of horizontal and
torsional oculomotor performance, which is dependent
on identified and well documented types of neurons and
circuits residing in the forebrain, midbrain, hindbrain and
periphery [12,13]. The presence of spontaneous scanning
saccades in the dark for both reck and cloche mutants
(Figures 3B and 3C) demonstrated the presence of a
pattern generator (r3-4) interconnected with burst (r5) and
integrator (r8) neurons both targeting Abd Mns and Abd
Int neurons (r5-6) (Supplementary Figures 1C and 1D).
All reck and many of the illustrated cloche decay time
constants were similar to those of the wild type larvae.
The degree to which larvae were able to maintain eye
position and counteract opposing forces pulling the eye
back in the original position is dependent, in part, on
the maturity of the velocity to position neural network
[24,26]. Reck mutants exhibited eye position holding with
a network maturity on par with 5 dpf wild type larvae,
albeit often with a slightly faster decay of eye position
holding. Because the cloche mutants lacked visual
performance and often exhibited considerable edema in
the lateral orbital eye socket (Figure 2D), a wide variation
of horizontal eye holding behavior was not unexpected.
The eye socket edema likely compromised eye plant
performance whose effect could not be separated from
that of the integrator circuitry; however the presence of
time constants far exceeding normal age wild type larvae
suggested that the integrator circuitry was correctly
established and operational. By contrast, the reck mutants
did not experience any overt eye socket edema (Figure
2C) and thus the oculomotor integrator exhibited the
Curr Neurobiol 2016 Volume 7 Issue 2
Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish.
typical exponential eye holding position decay in dark,
Figure 3B.
Both reck and cloche mutants made compensatory eye
movements in response to sinusoidal vertical pitch
stimulus (Figures 5A and 5B), indicating the presence
of a functioning three-neuron circuit spanning from the
hindbrain to the midbrain in both mutants. The gain of
the reck mutants in response to the pitch tilt stimulus
nearly matched that of wild type only reduced by 10%
from controls. In cloche mutants, after the beginning
of the stimulus paradigm VOR gain was initially lower
(Figure 5B, 38%) but diminished within 15-30 min likely
reflecting the onset of metabolic influences on behavior.
Two notable caveats exist to the conclusions that reck and
often cloche mutant performance is comparable to that of
wild type larvae. First, as seen in both the eye position
and velocity records in Figure 4 and Supplementary
Figure 3, there existed a ~ 4.0 Hz low amplitude (~ 2-4°/
s) back-ground rhythm that previously was attributed
to a bandwidth limiting performance of the visual slip
feedback circuitry [22]. In normal larvae, this oscillation
is often present at ~ 1°s (Supplementary Figure 3).
Under stressful circumstances, for example, large visual/
vestibular paradigm shifts, or as in the mutant cases
likely, oxygen/nutrient deficits, a larger oscillation is quite
evident from the onset of visuomotor testing. Predictably,
prolonged optokinetic performance testing ranging from
a few minutes to an hour eventually led to lower eye
velocity gains, and in some cases, behavioral performance
nearly disappeared (data not shown). We conclude that
while there may be no essential role for vascularization in
building the hindbrain oculomotor circuitry, there clearly
is a role in maintaining circuitry robustness as would
be required in a successful developmental transition to
juvenile and adult stages.
The Tricaine treated larvae did not exhibit the performance
levels observed with the TTX treated animals that were
almost indistinguishable from wild type larvae. In
particular, behaviors dependent on temporal integration of
input signals, i.e. eye position holding (compare Figures
7A and 7B to 8A and 8B) associated with spontaneous
saccades and the indirect component of OKR (compare
Figures 7C and 7D to 8C and 8D) were degraded and
involve the vestibular and velocity position neural
integrator (VPNI) networks (Supplementary Figures 1A1D). The susceptibility of these neuronal subtypes may
be a function of the intrinsic and/or extrinsic synaptic
conductances. TTX specifically blocks the family of
Na+ channels, mostly at nM concentrations. In contrast,
Tricaine, at the concentrations used in this study, has been
shown to block both Na+ and Ca2+ channels in zebrafish
[27-29]; furthermore, Tricaine and local anesthetics in
general [30,31] not only block sodium channels, but
potassium [32], nicotinic acetycholine receptor channels
[33] and G protein receptor activity [34]. Even though
integrative functions are compromised by Tricaine, the
Curr Neurobiol 2016 Volume 7 Issue 2
presence of vestibular and neural integrator neurons and
their associated connectivity is still evident as demonstrated
during some stable eye position holding epochs (Figures
8A and 8B). Central signal processing was also evident for
vestibular inputs to reach the burst pattern generator and
trigger burst neuron activity responsible for generating
fast phases and saccades (as illustrated in Supplementary
Figures 1C and 1D).
Velocity to position neural integrator neurons exhibit position
and saccadic sensitivity [11]. The integrative property of
the neural integrator neurons and associated network has
been demonstrated in the adult goldfish [10,35] as well as
the larval zebrafish [24,25]. TTX has minimal influence on
the functional development of integration as demonstrated
by spontaneous saccade and eye position holding behavior
on par with that observed in wild type larvae in the dark
(Figure 7B and Supplementary Figure 3B). These robust
behaviors also indicate the presence of the burst pattern
generator and associated functional connectivity with burst
and vestibular neurons connected to Abd Mns and Abd Int
neurons (Supplementary Figures 1C and 1D).
While the overall performance of the Tricaine treated
animals is degraded relative to the wild type and TTX
treated animals, these larvae still generated saccades and
exhibited eye position holding (Figures 8A and 8B). These
results demonstrate the presence of a sufficient population
of neurons exhibiting a correct connectivity to move the
eyes appropriately. Although the slow and fast phases of
OKR were also less than wild type controls (Figures 8C
and 8D), eye velocity still reflected the directional change
in drum velocity via the direct pretectal component to the
MR and Abd Mns (Supplementary Figures 1A and 1B).
However, the pretectal connection through the vestibular
nucleus was not able to sustain the constant eye velocity
tracking which quickly dropped by >80% (Figure 8D).
Zebrafish larvae normally exhibit little eye velocity storage
[20] and, therefore, are required to continuously convert slip
velocity to eye velocity throughout the stimulus period (as in
Supplementary Figure 3D). This constant velocity behavior
was severely compromised after Tricaine, yet the vestibular
slip velocity signal to the integrator network was adequate to
produce a step of eye position as well as trigger fast phase
resets. Overall the differences between the Tricaine and
TTX treated larvae may be related to the broadly disrupted
channel activity imposed by Tricaine compared to a more
specific disruption of sodium channels caused by TTX.
Overall in this study, we found that vascularization and
neuronal excitability were not essential for the formation
of numerous oculomotor nuclei displaying a substantial
interconnectivity and signal processing. We, therefore,
conclude that a genetic blueprint largely specifies early
larval structural and physiological features; however,
activity dependent feedback is clearly used to optimize
an age-related oculomotor performance. We suggest that
this developmental strategy appears to be an adaptation
required for early survival.
71
Ulrich/Grove/Torres-Vázquez/Baker
References
1. Carmeliet P, Jain RK. Molecular mechanisms and clinical
applications of angiogenesis. Nature 2011; 473: 298-307.
2. Louissaint A Jr, Rao S, Leventhal C, et al. Coordinated
interaction of neurogenesis and angiogenesis in the adult
songbird brain. Neuron 2002; 34: 945-960.
3. Makita T, Sucov HM, Gariepy CE, et al. Endothelins are
vascular-derived axonal guidance cues for developing
sympathetic neurons. Nature 2008; 452: 759-763.
4. Tsai HH, Niu J, Munji R, et al. Oligodendrocyte precursors
migrate along vasculature in the developing nervous system.
Science 2016; 351: 379-384.
5. Mukouyama YS, Gerber HP, Ferrara N, et al. Peripheral
nerve-derived VEGF promotes arterial differentiation via
neuropilin 1-mediated positive feedback. Development
2005; 132: 941-952.
6. Stenman JM, Rajagopal J, Carroll TJ, et al. Canonical Wnt
signaling regulates organ-specific assembly and differentiation
of CNS vasculature. Science 2008; 322: 1247-1250.
7. Gordon GR, Choi HB, Rungta RL, et al. Brain metabolism
dictates the polarity of astrocyte control over arterioles.
Nature 2008; 456: 745-749.
8. Ulrich F, Carretero-Ortega J, Menendez J, et al. Reck enables
cerebrovascular development by promoting canonical Wnt
signaling. Development 2016; 143: 147-159.
9. Ulrich F, Ma LH, Baker RG, et al. Neurovascular
development in the embryonic zebrafish hindbrain. Dev Biol
2011; 357: 134-151.
10.Aksay E, Baker R, Seung HS, et al. Anatomy and discharge
properties of pre-motor neurons in the goldfish medulla that
have eye-position signals during fixations. J Neurophysiol
2000; 84: 1035-1049.
11.Pastor AM, De la Cruz RR, Baker R. Eye position and eye
velocity integrators reside in separate brainstem nuclei. Proc
Natl Acad Sci USA 1994; 91: 807-811.
12.Ma LH, Grove CL, Baker R. Development of oculomotor
circuitry independent of hox3 genes. Nat Commun 2014; 5:
4221.
13.Bianco IH, Ma LH, Schoppik D, et al. The tangential nucleus
controls a gravito-inertial vestibulo-ocular reflex. Curr Biol
2012; 22: 1285-1295.
14.Lawson ND, Weinstein BM In vivo imaging of embryonic
vascular development using transgenic zebrafish. Dev Biol
2002; 248: 307-318.
15.Haffter P, Granato M, Brand M, et al. The identification of
genes with unique and essential functions in the development
of the zebrafish, Danio rerio. Development 1996; 123: 1-36.
16.Stainier DY, Weinstein BM, Detrich HW, et al. Cloche,
an early acting zebrafish gene, is required by both the
endothelial and hematopoietic lineages. Development 1995;
121: 3141-3150.
17.Chi NC, Shaw RM, De Val S, et al. Foxn4 directly regulates
tbx2b expression and atrioventricular canal formation. Genes
Dev 2008; 22: 734-739.
72
18.Anderson MJ, Pham VN, Vogel AM, et al. Loss of unc45a
precipitates arteriovenous shunting in the aortic arches. Dev
Biol 2008; 318: 258-267.
19.Higashijima S, Hotta Y, Okamoto H. Visualization of cranial
motor neurons in live transgenic zebrafish expressing green
fluorescent protein under the control of the islet-1 promoter/
enhancer. J Neurosci 2000; 20: 206-218.
20.Beck JC, Gilland E, Baker R, et al. Instrumentation for
measuring oculomotor performance and plasticity in larval
organisms. Methods Cell Biol 2004; 76: 385-413.
21.Mensh BD, Aksay E, Lee DD, et al. Spontaneous eye
movements in goldfish: oculomotor integrator performance,
plasticity, and dependence on visual feedback. Vision Res
2004; 44: 711-726.
22.Marsh E, Baker R. Normal and adapted visuooculomotor
reflexes in goldfish. J Neurophysiol 1997; 77: 1099-1118.
23.Beck JC, Gilland E, Tank DW, et al. Quantifying the
ontogeny of optokinetic and vestibuloocular behaviors in
zebrafish, medaka, and goldfish. J Neurophysiol 2004; 92:
3546-3561.
24.Miri A, Daie K, Arrenberg AB, et al. Spatial gradients and
multidimensional dynamics in a neural integrator circuit. Nat
Neurosci 2011; 14: 1150-1159.
25.Miri A, Daie K, Burdine RD, et al. Regression-based
identification of behavior -encoding neurons during largescale optical imaging of neural activity at cellular resolution.
J Neurophysiol 2011; 105: 964-980.
26.Daie K, Goldman MS, Aksay ER. Spatial patterns of
persistent neural activity vary with the behavioral context of
short-term memory. Neuron 2015; 85: 847-860.
27.Attili S, Hughes SM. Anaesthetic tricaine acts preferentially
on neural voltage-gated sodium channels and fails to block
directly evoked muscle contraction. PLoS One 2014; 9:
e103751.
28.Frazier DT, Narahashi T. Tricaine (MS-222): effects on ionic
conductances of squid axon membranes. Eur J Pharmacol
1975; 33: 313-317.
29.Gnuegge L, Schmid S, Neuhauss SC. Analysis of the activitydeprived zebrafish mutant macho reveals an essential
requirement of neuronal activity for the development of a
fine-grained visuotopic map. J Neurosci 2001; 21: 35423548.
30.Nau C, Wang GK. Interactions of local anesthetics with
voltage-gated Na+ channels. J Membr Bio 2004; 201: 1-8.
31.Scholz A. Mechanisms of (local) anaesthetics on voltagegated sodium and other ion channels. Br J Anaesth 2002; 89:
52-61.
32.Valenzuela C, Delpon E, Tamkun MM, et al. Stereoselective
block of a human cardiac potassium channel (Kv1.5) by
bupivacaine enantiomers. Biophys J 1995; 69: 418-427.
33.Neher E, Steinbach JH. Local anaesthetics transiently block
currents through single acetylcholine-receptor channels. J
Physiol 1978; 277: 153-176.
Curr Neurobiol 2016 Volume 7 Issue 2
Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish.
34.Hollmann MW, McIntire WE, Garrison JC, et al. Inhibition
of mammalian Gq protein function by local anesthetics.
Anesthesiology 2002; 97: 1451-1457.
35.Aksay E, Olasagasti I, Mensh BD, et al. Functional dissection
of circuitry in a neural integrator. Nat Neurosci 2007; 10:
494-504.
Correspondence to:
Robert Baker,
Department of Neuroscience and Physiology,
New York University Medical Center,
550 First Avenue New York,
NY 10016,
USA.
Tel: 914-645-2479
E-mail: [email protected]
Curr Neurobiol 2016 Volume 7 Issue 2
73