Toxicology and Applied Pharmacology 239 (2009) 1–12
Contents lists available at ScienceDirect
Toxicology and Applied Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Acute nicotine exposure and modulation of a spinal motor circuit in
embryonic zebrafish
Latoya T. Thomas, Lillian Welsh, Fernando Galvez, Kurt R. Svoboda ⁎
Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana, 70803, USA
a r t i c l e
i n f o
Article history:
Received 12 May 2008
Revised 17 August 2008
Accepted 19 August 2008
Available online 14 December 2008
Keywords:
Embryo
Behavior
Desensitization
Spinal cord
Nicotinic acetylcholine receptor
a b s t r a c t
The zebrafish model system is ideal for studying nervous system development. Ultimately, one would like to
link the developmental biology to various aspects of behavior. We are studying the consequences of nicotine
exposure on nervous system development in zebrafish and have previously shown that chronic nicotine
exposure produces paralysis. We also have made observations that the embryos moved in the initial minutes
of the exposure as the bend rates of the musculature increased. This nicotine induced behavior manifests as
an increase in the rate of spinal musculature bends, which spontaneously begin at ∼ 17 h post fertilization.
The behavioral observations prompted the systematic characterization of nicotine-induced modulation of
zebrafish embryonic motor output; bends of the trunk musculature.
We first characterized embryonic motor output in zebrafish embryos with and without their chorions.
We then characterized the motor output in embryos raised at 28 °C and 25 °C. The act of dechorionation
along with temperature influenced the embryonic bend rate. We show that nicotine exposure increases
embryonic motor output. Nicotine exposure caused the musculature bends to alternate in a left–right–left
fashion. Nicotine was able to produce this phenotype in embryos lacking supraspinal input. We then
characterize the kinetics of nicotine influx and efflux and demonstrate that nicotine as low as 1 μM can
disrupt embryonic physiology. Taken together, these results indicate the presence of nicotinic acetylcholine
receptors (nAChRs) associated with a spinal motor circuit early in embryogenesis.
© 2008 Elsevier Inc. All rights reserved.
Introduction
Zebrafish are typically thought of as a model system to investigate
fundamental principles of developmental biology and genetics.
Within the past 10–15 years, the zebrafish model has been established
as a vertebrate platform for investigating sensory systems and how
they interact with the CNS to generate coordinated motor behaviors
(for review see Fetcho, 2007). More recently, the zebrafish model has
gained prominence for investigating aspects of chemical toxicity (for
review see Hill et al., 2005). When the disciplines of development,
motor control, and chemical toxicity converge, they allow for a
potentially unparalleled opportunity to assess the consequences of
chemical toxicity in an in vivo context. In this context, we have been
studying nicotine toxicity in zebrafish and have discovered that
zebrafish embryos are very responsive to acute nicotine exposure. The
exposure increases the rate of musculature bends or twitches
generally associated with early embryonic motor output. This overactivity during early embryogenesis may also be detrimental to the
organism.
Zebrafish embryos display bends of the musculature when
removed from their chorions or when left in their protective chorions
⁎ Corresponding author. Fax: +1 225 578 2597.
E-mail address: ksvobo1@lsu.edu (K.R. Svoboda).
0041-008X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2008.08.023
as early as 17–19 h post fertilization (Downes and Granato, 2006; Cui
et al., 2005; Saint-Amant and Drapeau, 1998; Sipple, 1998; Kimmel et
al., 1974). The frequency of these contractions peaks around 19 hpf
and then declines gradually (Saint-Amant and Drapeau, 1998; Sipple
1998). This motor output can be reduced by strychnine as early as
19 hpf (Downes and Granato, 2006), indicating that a neuronal circuit
comprised partially of inhibitory interneurons exists in embryonic
spinal cord which is capable of producing a motor output.
Vertebrate spinal neurons use acetylcholine and glutamate as
excitatory neurotransmitters to produce motor outputs. In lamprey
spinal cord, bath application of acetylcholine modulates a rhythmic
motor output (Quinlan et al., 2004). In the Xenopus spinal cord,
motoneuron collaterals project back to the interneurons that generate
swimming and excite them with acetylcholine. This serves to help
maintain an excitatory drive which sustains swimming (Roberts and
Perrins, 1995). In embryonic mouse and chick spinal cords, application
of nicotinic acetylcholine receptor (nAChR) antagonists dampens the
frequency of spontaneously occurring motor output (Myers et al.,
2005; Milner and Landmesser, 1999). Together, these findings
demonstrate the presence of nAChRs within vertebrate spinal circuits
that produce locomotion. This receptor distribution is conserved from
fishes to mammals.
The spinal circuitry that generates the motor output in zebrafish is
thought not to be overly complicated, resembling those circuits that
2
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
produce locomotion in other swimming vertebrates (Fetcho, 2007;
Downes and Granato, 2006). In zebrafish embryos, it is likely that
nAChRs are expressed by cells within spinal circuits that produce
movement in accordance with other vertebrates.
Nicotine and acetylcholine are both agonists of nAChRs and in this
paper, we demonstrate that they can modulate an embryonic motor
output in acute exposure paradigms. We hypothesize that the
exogenous nicotine is activating nAChRs within an embryonic spinal
rhythm generator because the resulting motor output alternates from
left to right and because the motor output can be activated at the level
of the spinal cord. We further characterize the actual amount of
nicotine that gets incorporated into the embryo from the waterborne
concentration. We demonstrate that the embryonic motor output is
activated by a fraction of the total nicotine accumulated during
waterborne exposures. Furthermore, we show that 1 μM nicotine can
act as an antagonist desensitizing nAChRs, making them unavailable
to respond to higher concentrations of nicotine during subsequent
acute exposures. The establishment of a reliable nicotine induced
endpoint, in this case a behavioral endpoint, serves as a launch-point
to investigate the distribution and function of nicotinic acetylcholine
receptors in embryonic zebrafish within developmental, behavioral,
and toxocological contexts.
Materials and methods
Zebrafish embryos and maintenance.
Fertilized eggs were obtained
from natural spawnings of adult zebrafish according to the Zebrafish
Book (Westerfield, 1995). The embryos used in this study were
obtained from a variety of different wild-type and transgenic lines of
zebrafish. The results reported were not dependent upon which line
of fish was used and collectively are referred to as embryos. The
wild-type lines used were TL, AB, WIK, and fishery reared (Ekkwill
Waterlife Resources, Gibsonton, Fla). The transgenic lines used were
Tg(isl1:gfp), Tg(fli1:gfp), and Tg(nbt:mapt-gfp).
Adult fish were kept at standard conditions of 28.2 °C on a 14 h
light: 10 h dark photoperiod (Westerfield, 1995) in a recirculating
system and fed three times daily with either the zebrafish diet
(Zeigler) or live artemia (Aquatic Habitats, Apopka Florida). Embryos
were collected from group spawns or paired spawns, and were rinsed
several times in embryo medium prior to experiments. Embryos were
raised at 28 °C until 12–13 hpf and thereafter were raised in laboratory
conditions at 25–26 °C unless otherwise noted.
Drugs. The (−)-nicotine used in this study was purchased from
Sigma (St. Louis, Missouri, USA, catalog # N3876—5 ml). Nicotine stock
solutions were made in distilled water and then diluted in embryo
medium to obtain final concentrations ranging between 1 and 30 μM.
Fresh nicotine was made daily as needed for all experiments.
-(−)-[N-methyl-3H] nicotine was purchased from Perkin Elmer Life
and Analytical Sciences (Wellesley, MA, USA), with a specific activity of
60.0 Ci/mmol in ethanol and stored at −20 °C. The activity of [3H]nicotine was measured with high efficiency via liquid scintillation
Fig. 1. The embryonic motor output in zebrafish. (A) The spontaneous activity of 6
embryos (27 hpf) was monitored while in their chorions. At the arrow, all embryos were
quickly dechorionated. The dechorionation caused a small magnitude increase in the
bend rate. (B) Bend rates are shown for embryos dechorionated at 22 hpf (n = 6) and
24 hpf (n = 6). Both sets of curves are plotted with a start point 5 min after the
dechorionation (black arrow). Dechorionation at 22 hpf caused an increase in the bend
rate that exceeded the bend rates of embryos dechorionated at 24 hpf. (C) The motor
output is quantified as the number of bends per minute that occurred in 5 min epochs
between 18 hpf and 25 hpf while the embryos were in their protective chorions. The
experiment was performed for embryos raised continuously at 28 °C (n = 16 embryos;
behavior analyzed at 28 °C) and embryos raised at 25 °C after 13 hpf (n = 15 embryos;
behavior analyzed at 25 °C). The black dashed circles in B and C highlight the 22 hpf
time point in each experiment for purposes of comparison. Asterisks denote
significance with p value b 0.05, Student T-test. In C, comparisons of bend rates were
made between the two groups of embryos at the individual developmental time-points.
counting (Tri Carb 2900TR, Perkin Elmer). Quench correction with the
tSIE/AEC indicator was used for radioactivity determinations. In all
experiments, radioisotope (0.2 μCi/L-(−)-[N-methyl-3H]) was added to
a known concentration of a “cold” nicotine (Sigma) stock solution. In the
manuscript, this final cocktail is referred to as [3H]-nicotine.
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
Behavior.
Embryos were either dechorionated or left within their
protective chorions. To obtain dechorionated embryos, the chorions
were removed via enzymatic digestion with 1.5 mg/ml protease
(Sigma) in 50 mm Petri dishes for 5 min at room temperature. After
this 5 min period, the embryos were swirled vigorously in the dish
causing the chorions to separate from the embryos. The dechorionated
embryos were washed for at least 15 min in fresh embryo medium.
Embryos (22–29 hpf) were placed in a 50 mm diameter Petri dish
containing embryo medium and videotaped with a Kohu video
camera connected to a Zeiss SV6 dissecting microscope. The embryos'
motor output (bends of the spinal musculature) were imaged in
control embryo medium for 3–20 min. Embryos were then transferred
to a second Petri dish containing nicotine (5–30 μM) in embryo
medium and the motor output was recorded again for all embryos for
a minimum of 3 min. They were then transferred back into a third
Petri dish containing embryo medium for an extended wash period
and their motor behavior was recorded to videotape. Dechorionated
embryos tended to move out of the field of view and sometimes had to
3
be repositioned. Because of this, sample sizes were typically less than
those experiments which analyzed embryos while still in the chorion.
All behavioral recordings were performed at 25–26 °C.
Immunohistochemistry.
Whole mount immunohistochemistry was
carried out using a modified version of our previous published
protocol (Svoboda et al., 2001, 2002; Pineda et al., 2006). Briefly,
embryos were first fixed in 4% paraformaldehyde overnight at 2–4 °C
and then stored in PBST (PBS containing 0.1% Tween 20). After
permeabilization, they were incubated in a primary antibody
overnight at 2–4 °C. The primary antibodies were prepared in PBST.
Anti-acetylated tubulin (aat 1:1000 dilution, Sigma Aldrich) was used
to label spinal sensory Rohon–Beard neurons and spinal interneurons.
The antibody zn5 (1:1000 dilution, currently available as zn8 from the
Developmental Hybridoma Bank, University of Iowa) was used to label
secondary motoneuron somata and axons. The following day, the
embryos were washed for 60 min and then incubated in a fluorescent
secondary anti-mouse antibody conjugated to Alexa 546 (1:1000
Fig. 2. Avoiding the motor output flurry, dechorionation at 12–15 hpf. (A) Photomicrographs of 30 hpf embryos that were dechorionated either at 12 hpf (n = 8) or 30 hpf (n = 7). The
embryos were labeled with the antibody aat. Filled arrows point to aat positive Rohon–Beard neurons and open arrows point to aat positive spinal interneurons. (B) Quantification
indicates an equal distribution of aat positive interneurons between both groups of embryos. (C) Quantification of bend rates for 25 hpf embryos that were dechorionated at 12 hpf
(n = 17, open circles). These embryos are being compared to 25 hpf embryos siblings that were left in their chorions (n = 19). (D) Photomicrographs of 72 hpf embryos that were
dechorionated either at 12 hpf (n = 7) or 30 hpf (n = 6). The embryos were labeled with zn5 to label secondary motoneurons. The filled arrows are pointing to dorsal projecting
secondary motoneuron axons.
4
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
dilution in PBST; Molecular Probes, Eugene Oregon) for 90 min to
reveal primary antibody labeling. They were then rinsed in PBST for
another 60 min and prepared for image analysis. Single focal plane
images of the fluorescent signals were acquired with an ORCA-ER
digital camera (Hamamatsu) mounted to a Zeiss Axiovert 200 M
inverted microscope equipped with a rhodamine filter cube using a
20× objective.
[3H]-nicotine uptake.
The accumulation of [3H]-nicotine was
quantified in embryos ranging in age from 22 to 24 h post fertilization
(hpf). Embryos were exposed to varying waterborne concentrations
(1, 5, 10, 15 and 30 μM) of [3H]-nicotine. After fluxing, the embryos
were transferred from Petri dishes to 100 μm nylon filters (BD Falcon,
Franklin Lakes, NJ, USA) and washed using a filtration apparatus. By
placing embryos in these filters, they were easily removed from the
fluxing solution quickly with minimal handling stress.
Influx methodology. Embryos were placed in Petri dishes containing
radioisotope and fluxed for 5–10 min depending on the experiment. In
some experiments, the behavior of the embryo was simultaneously
monitored during the flux period using a Kohu video camera mounted
to a Zeiss Stemi 2000C dissecting microscope and captured
simultaneously onto VCR tapes. Following the flux period, embryos
were transferred to nylon filters within a Petri dish and rinsed up to 5
times with embryo medium where all medium in the dish was
exchanged with fresh embryo medium. They were then immediately
transferred to 1.5 ml centrifuge tubes for further processing (2–3
embryos per tube).
Fig. 3. Cholinergic agonists activate an embryonic motor output. (A) 28 hpf dechorionated embryos were exposed to 1 or 10 mM acetylcholine (n = 6 and n = 7, respectively) at
minute 3 of the recording (black bar). (B) 28 hpf embryos were exposed to 15 μM nicotine at minute 3 of the recording (n = 6). Video stills are from an episode of nicotine induced
motor output in a 27 hpf embryo exposed to 30 μM nicotine. Note the left–right–left nature of the bending. This nicotine induced behavior output was observed at all time-points
analyzed (22 hpf-29 hpf). (C) Quantification of behavioral data obtained from a 23 hpf embryo exposed to nicotine. The number of bends occurring per minute is shown as filled
triangles (▴). The number of doublet bends that occurred as a fraction of total bends per minute is shown by open triangles (△). The nicotine was added at minute 20 and was applied
for only 3 min. As the bend rate (▴) increased, the percentage of doublet bends that occurred per total bends (△) approached 0%. During the wash period beginning at minute 23, the
bend rate began to decrease. As the bend rate decreased, the percentage of doublet bends that occurred per total number of bends increased. Asterisks denote a significant increase
from control (pre-exposed) bend rates; ‡ denotes significant difference in bend rates between 1 mM and 10 mM exposed embryos (p valueb 0.05, Anova, Student T-test in B).
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
5
Efflux methodology.
After the flux period (see Influx methodology
above), embryos were separated and kept in individual Petri dishes
(minimum of 6 embryos per dish) containing 3 ml of embryo medium
and then removed from these wash rinses at 30 min intervals. At the
conclusion of each wash interval, embryos were then transferred to
nylon filters on a filtration apparatus and briefly rinsed with embryo
medium. Following the rinse period, they were transferred to 1.5 ml
centrifuge tubes (2–3 embryos per tube) for further processing.
Sample processing
For all experiments, water samples were taken at the start and end
of the flux period for monitoring of waterborne [3H]-nicotine.
Following radioisotope fluxing, zebrafish embryos were transferred
to a filtration apparatus where they were washed with embryo
medium to remove external unincorporated isotope. Zebrafish were
transferred to centrifuge tubes and weighed using a micro-balance.
Afterward, zebrafish were digested overnight in 1 N trace-metal grade
HNO3 acid at 60 °C. Digests were centrifuged to collect supernatants.
Five mL of Ultima Gold scintillant (Perkin Elmer) was added to the
supernatants and radioactivity levels were then measured by liquid
scintillation counting. Specific activities were used to convert radioactivity into nmol values. These absolute uptake rates were then
expressed per mg wet weight.
Statistics. All values are reported as means ± standard error of the
mean (SEM). Analysis of Variance (one-way repeated measures
analysis of variance with Holm Sidak Correction) or Student T-test
(SigmaStat 3.5) was performed to test for significance, which was
assigned if the p value was b0.05.
Results
Exogenous cholinergic agonists activate a motor output in
zebrafish embryos
Nicotine exposure in developing zebrafish embryos produces two
behavioral phenotypes. Embryos exposed chronically to 33 μM
nicotine exhibit almost complete paralysis by 66 hpf (Svoboda et al.,
2002). We have also observed that in the early minutes of the
exposure, the embryos exhibited an increase in their embryonic motor
output. Thus, we characterized the nicotine induced modulation of
motor output during early zebrafish development.
Experiments were first performed on 27–28 hpf embryos that
were removed from their chorions. We chose this point because
embryos exhibit a robust swim mechanism and touch response at this
age (Ribera and Nusslein-Volhard, 1998; Saint-Amant and Drapeau,
1998; Grunwald et al. 1988). When embryos were dechorionated at
27 hpf, an increase in the musculature bend rate occurred. At 27 hpf,
the simple act of dechorionation did not pose a problem for us because
the increase in the bend rate was not large in magnitude (Fig. 1A).
However, dechorionation between 22 and 25 hpf produced a dramatic
increase in the embryos' bend rate, often requiring between 1 and 2 h
to recover back to baseline levels. Dechorionation of younger embryos
produced higher frequency bend rates than dechorionation in older
embryos (Fig. 1B). These results are in accordance with those results
obtained by others showing that dechorionation increases the bend
rates of embryos (Saint-Amant and Drapeau, 1998).
Systematic, minute by minute or hour by hour evaluations of
embryonic bend rates of embryos in the chorions are rare in the
literature. The work of Sipple (1998) simultaneously monitored
numbers of embryos in the chorion over time while quantifying the
bend rates with computer software. In that work, the embryonic
muscle bend rates peaked at 19–20 hpf as reported by Saint-Amant
and Drapeau, but the maximum number of bends was around 22
bends per minute which is lower than the bend rates in dechorionated
Fig. 4. Pharmacology of nicotine induced motor behavior. (A) Motor output of 23 hpf
embryos (n = 3, in chorion). At minute 5, the embryos were transferred to embryo
medium with a 7.8 pH (first black bar). At minute 10, the embryos were transferred into
embryo medium containing 30 μM nicotine (second black bar). (B) ∼ 22 hpf embryos
(n = 18; dechorionated) were videotaped for 5 min. After this initial recording, the
embryos were separated into 3 groups. They were then exposed to 5, 15 or 30 μM
nicotine at minute 5 (n = 6 for each concentration). Video stills from one of the embryos
exposed to 15 μM nicotine are shown at the bottom. (C) A single 23 hpf embryo was
exposed to 5, 10 and 30 μM nicotine. A dose response was apparent in the same embryo.
Asterisks denote significant difference from control bend rates. ‡ denotes significance
between all exposure conditions (p value b 0.001, Anova). Embryos used under panel A
were raised until experimentation at 28 °C.
6
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
Fig. 5. Nicotine activation of spinal circuits. Bend rates are plotted for two individual tails (29 hpf) exposed to 5 μM and 30 µM nicotine respectively. Nicotine exposure evoked an
increase in each tail's bend rate. At the right, the graph plotting the bend rate for the tail exposed to 5 µM nicotine is expanded; below are video stills of that tail. This result was
replicated in 11 tails exposed to 30 μM nicotine. The maximum bend rate observed was 120 bends per minute (30 μM exposure) in an individual tail.
embryos (Saint-Amant and Drapeau, 1998, our results under Fig. 1B).
Thus, we felt that we needed to characterize the embryonic motor
output before we continued with our acute nicotine exposure studies
because we felt that the dramatic increase in the bend rates caused by
dechorionation could compromise our studies.
Embryos were videotaped each hour from 17–25 h at 28 °C and
their motor behavior was analyzed. In this paradigm, the muscle bend
rates peaked at around 19–20 hpf with the average bend rate
approximating 26 bends per minute. For embryos raised at 25–
26 °C, the bend rates still peaked at 19–20 hpf, but the frequency was
decreased (Fig. 1C). In young embryos (18–21 hpf) raised at either
28 °C or 25–26 °C, we also noticed that the spontaneously occurring
bends occurred in an alternating, left–right fashion. By 23–24 hpf, a
typical pattern of embryonic activity consisted of successive bends of
the musculature to the same side of the body, and then a bend to the
opposite side. The sustained alternating pattern of activity was lost as
the embryos aged (data not shown).
With this baseline characterization at hand, we realized that
dechorionation of early age embryos nearing 24 hpf just prior to the
nicotine exposure was not an option because the dechorionation
caused an artificial increase in the bend rate. If we waited for the bend
rate to return to baseline, an embryo dechorionated at 22 hpf would
now be 23–24 hpf. To get around this problem, we decided to
dechorionate embryos between 12 and 15 hpf and then perform the
nicotine exposures at the desired developmental time-points. This
early dechorionation had no obvious consequences on the anatomy
(Figs. 2A, B, D) or behavior (Fig. 2C) of the embryos. Consequently, for
all dechorionated embryos that were exposed to cholinergic agonists
in this study, the dechorionation was performed at 12–15 hpf, prior to
the onset of embryonic motor output.
In 27–28 hpf dechorionated embryos, exposure to 1–10 mM
acetylcholine produced an increase in the musculature bend rate of
the embryos (Fig. 3A). Nicotine exposures (5–30 μM) also produced
increases in the musculature bend rates of embryos (Fig. 3B, 15 μM
example is shown). Further analysis of the nicotine induced motor
behaviors revealed that the bends alternated in a left–right–left
fashion (Fig. 3C). The alternating pattern of bends caused by nicotine
exposure was observed at all time points analyzed (22–29 hpf). As
the bend rate increased, the percentage of consecutive bends
(referred to as doublets) that occurred to a particular side of the
body decreased. When quantified, the percentage of doublet bends
that occurred per total number of bends approached zero when the
embryo was in nicotine (quantified for 8 individual embryos). During
the wash period, the doublet bends occurred on a more regular basis
(Fig. 3C).
Since the pH of the nicotine stock solution was 7.8, and that of the
embryo medium was only 7.0, we wondered if the nicotine induced
behavior was associated with this pH difference. In experiments to
test this, embryos transferred from embryo medium at pH 7.0 to pH
7.8 did not exhibit a change in musculature bend rate (Fig. 4A). Those
same embryos were then exposed to 30 μM nicotine and an increase in
the bend rate occurred (Fig. 4A).
A dose response was apparent with higher waterborne concentrations of nicotine producing a more robust motor output (Fig. 4B). This
dose response could be demonstrated in the same embryo (Fig. 4C).
For the embryo shown in Fig. 4C, the nicotine induced increase in
muscular bend rate was reversible and when the nicotine was
removed. The bend activity again alternated in a left–right–left bend
fashion as the nicotine waterborne concentration was raised from 5 to
30 μM (not shown).
Cholinergic activation of spinal neurons and activation of an early
motor output
Some vertebrate brainstem neurons projecting to spinal cord can
be excited by endogenous acetylcholine (Le Ray et al., 2003). Thus,
application of nicotine in these brainstem regions may actually
activate spinal rhythm generators for swimming. To test if nicotine
was activating brainstem mechanisms to increase the motor output
in embryonic zebrafish, studies were performed in 27–30 hpf
embryos that were decapitated between spinal segment number 1–
3. The 27–30 hpf time window was chosen primarily because at
27 hpf, zebrafish embryos reliably respond to touch and can move.
We used this behavioral criterion to demonstrate that the tails alone
could move in response to an exogenous stimulus (Downes and
Granato, 2006). When placed in 5–30 μM nicotine, the musculature
bend rate increased and it appeared as if the tail was trying to move
(Fig. 5) as was the case for intact embryos exposed to nicotine. The
response to nicotine is not a reflex as it is a sustained bending of the
musculature in the presence of the drug. These results indicate that
nicotine is capable of activating a motor output in zebrafish embryos
without the brain and strongly suggest that the spinal neurons
responsible for generating the motor behavior are candidate cells to
express nAChR(s).
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
Desensitization of the nicotine induced motor output
When the dose response experiments were performed, the
concentration of nicotine was typically increased from low to high.
However, if the embryos were exposed to the high concentration first,
7
and then exposed to lower nicotine concentrations after a wash-out
period, they did not exhibit an increase in musculature bend rates.
This prompted a more thorough analysis of a potential desensitization
phenomenon occurring in the acute exposure paradigms.
Desensitization experiments were performed in 22–27 hpf
embryos. To facilitate video recording as well to increase sample
sizes per experiment, some experiments were performed on embryos
while in their chorions. The dechorionated embryos often moved out
of the field of view and would need repositioning throughout the
experiment. Exposure to 30 μM nicotine caused approximately a 4-fold
increase in the musculature bend rate of 25 hpf embryos in the chorion
(Fig. 6A). However, after a two hour wash period, 30 μM nicotine failed
to elicit the same magnitude response in 27 hpf embryos. This
attenuated response to nicotine was not associated with the developmental stage, since stage matched, 27 hpf control embryos had a robust
response to nicotine (Fig. 6A, open circles). We also observed this
“desensitized” motor output phenotype in embryos as young as 24 hpf;
when they were exposed to nicotine at 22 hpf (data not shown).
The same experiment was then performed on individual 26 hpf
embryos that had been dechorionated. Again, an apparent desensitization of the response occurred (Fig. 6B) as the embryos did not
respond to the second 30 μM nicotine application at 28 hpf. However,
they did respond to tactile simulation with a vigorous response to
touch indicating that the muscle-specific nAChRs had not been
desensitized by the nicotine exposure (Fig. 6B, inset). Thus, we felt
confident that the mechanism underlying this desensitized response
was upstream of muscle nAChRs. There was some variability in the
magnitude of the effect in these experiments, but the response to the
second application of 30 μM nicotine was invariably reduced or
abolished as shown in Fig. 6B.
Nicotine influx and efflux in zebrafish embryos
Radioisotopic flux experiments were performed to determine how
much nicotine was accumulating in embryos during exposures, and to
assess clearance rates during the washout phase of the experiments.
Similar types of experiments have been performed analyzing dioxin
uptake in embryonic zebrafish (Henry et al., 1997). Zebrafish embryos
were bathed in [3H]-nicotine and the amount of nicotine incorporation
was determined (Figs. 7A–C). [3H]-nicotine was quickly accumulated
in embryos at 30 μM, reaching a steady state burden with a 10 min
exposure (Fig. 7A). These results corroborate results obtained from
behavioral assays, where the nicotine induced behavioral response
peaked within 5 min of exposure onset. Subsequent influx exposures
demonstrated that [3H]-nicotine accumulation increased with increasing external nicotine concentrations up to 30 μM. However, in every
case, the amount of [3H]-nicotine uptake was less than the nominal
waterborne concentrations in the external medium (Fig. 7B).
The radioactive nicotine incorporation studies were then coupled
with the behavioral studies. After embryos were fluxed in the [3H]-
Fig. 6. Desensitization of the nicotine induced motor output. (A) (Top) Behavior
quantification from 25–27 hpf embryos (n = 6) in the chorion. 30 μM nicotine
application occurred at the first black bar resulting in an increased bend rate. After
120 min, the bend rate almost returned to baseline. The second application of 30 μM
nicotine did not produce an increase in the bend rate. Stage matched controls not
previously exposed to nicotine (open circles) respond to the nicotine exposure with an
increased bend rate. (Middle) Minutes 1 through 10 are shown as expanded view. The
nicotine exposure occurred between minute 5 and minute 10. (Bottom) Minutes 131
through 140 are shown as expanded view. The nicotine exposure occurred between
minute 135 and minute 140. (B) A 26 hpf embryo (chorion removed) was exposed to
30 μM nicotine (black bars). After an extended wash period, the second application of
nicotine did not produce an increase in the bend rate. However, the embryo still
exhibited a response to touch (inset), thus muscle nAChRs were not desensitized. In A,
asterisks denote significant difference from bend rates obtained prior to minute 5 or
135. ‡ denotes significant difference in bend rates when comparing the experimental
embryos (exposed to nicotine at minute 5) and controls (those not previously exposed
to nicotine) at the second application of nicotine (p valueb 0.001, Anova).
8
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
nicotine and the behavior documented, [3H]-nicotine incorporation
was quantified (Fig. 7C). In this example, only a fraction of the
waterborne concentration of nicotine got into the embryo. However,
this was still enough nicotine to elicit a robust motor output.
In the earlier portion of this study, we observed what appeared to
be a “desensitization” of the nicotine induced behavior. One way that
this could be explained would be if nicotine got into the embryo and
then was not cleared in a timely fashion. If residual nicotine was still in
the embryo, that nicotine would potentially be able to bind to nAChRs,
causing the observed dampening of the behavioral response. To
address this issue, nicotine efflux experiments were performed to
determine the clearance rates of nicotine from embryos during
washout. Following the [3H]-nicotine influx period, embryos were
found to lose nicotine quickly over the first 180 min of depuration,
after which internal levels reached an asymptote. Even up to 5 h
following the exposures, an equivalent of 1 μM nicotine was still left in
the embryo (Fig. 8, top). In comparison, the accumulated [3H]-nicotine
following exposure to 10 μM nicotine was totally cleared from the
embryo by 30 min after the exposure (Fig. 8, bottom). There was no
residual nicotine left in the embryo.
We also performed experiments where embryos were exposed to
30 μM of [3H]-nicotine for 5 min while simultaneously monitoring the
behavior (not shown). After a 1–2 h wash period, the efflux rate for
half of those embryos was determined. The other half of the embryos
was exposed to [3H]-nicotine for a second time. Again, the nicotineevoked response was dampened when compared to stage matched
controls now exposed with [3H]-nicotine for the first time. Moreover,
an equivalent of 1 μM nicotine was still left in the embryos 2 h
following the exposure (data not shown).
Residual nicotine and desensitization
We wondered if the residual 1 μM nicotine was enough to inhibit
the ability of the 30 μM waterborne nicotine to evoke a motor output.
Using the flux assays described earlier, we empirically determined
that when embryos were exposed to 1 μM waterborne nicotine, the
nicotine would be completely incorporated and equilibrated into the
embryo within 2 h of exposure onset (Fig. 9A). This was the level of
incorporation we desired to mimic the level of residual nicotine that
was not cleared from embryos during the wash periods in the acute
exposure paradigms (refer to fig 8, top). Embryos were pre-incubated
in 1 μM nicotine for 2.5 h. When challenged with 30 μM nicotine, they
exhibited a reduced motor output, but it was not completely abolished
(Fig. 9B). This same experiment was performed again where embryos
Fig. 7. Quantification of nicotine uptake in zebrafish embryos. (A) 23 hpf embryos (n = 9, for each concentration) were exposed to 30 μM of [3H]-nicotine for 10, 30, 60 and 120 min,
respectively. Nicotine incorporation was quantified via scintillation counting. In this example after a 2-hour exposure, nicotine equivalent to 10 μM was incorporated into the embryos just as it
was for the ten minute time point. (B) 23 hpf embryos were exposed to varying concentrations of [3H]-nicotine for 10 min. Nicotine incorporation into the embryos was less than the
waterborne concentration of nicotine at all waterborne concentrations analyzed. The dashed lines in A and B correspond to a nicotine concentration of 30 μM. (C) 24 hpf embryos (n =6) were
exposed to 30 μM [3H]-nicotine (black bar) and an increase in the bend rate occurred. At minute 9, the embryos were then processed for scintillation counting. The inset corresponds to the
amount of nicotine in the embryos; roughly 4 μM. The amount of nicotine incorporation is much less than the waterborne concentration. Moreover, in this example 4 μM nicotine is producing
the behavioral phenotype. The dashed line corresponds to a nicotine concentration of 6 µM. Asterisk denotes significance from control bend rate with p value b 0.05, Student T-test.
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
9
embryos. We hypothesize that this increased activity alone could
alter aspects of cell biology in the developing embryo. It has been
shown in vertebrates that the rate of spontaneous motor activity can
influence development (Hanson et al., 2008; Hanson and Landmesser,
2006; Borodinsky et al., 2004; Spitzer et al., 2004; Hanson and
Landmesser, 2004). Thus, we took advantage of the fact that
dechorionated embryos exhibited a dramatic increase in musculature
bend rates (refer to Fig. 1B). Embryos were dechorionated and allowed
to “recover” as evidenced by their bend rates approaching baseline
values. When challenged with nicotine several hours later, the
response to nicotine was diminished when compared to stage
matched controls that were dechorionated just prior to the exposure
(Fig. 10). The embryos initially responded to the nicotine but were not
able to sustain the motor output when compared to the control
embryos. This phenotype was evident in embryos that were
dechorionated between 22 and 25 hpf and then challenged with
nicotine as late as 31 hpf (not shown).
These results may seem incongruous with the results from the dose
response experiment presented under Fig. 4C where the dechorionated embryo responded to 30 μM nicotine with an increase in bend
rate. However, that response may in fact be dampened as the bend rate
peaked at ∼50 bends per minute. In the dechorionated embryos
exposed to 30 μM nicotine shown in Figs. 4B and 6B, the bend rates
were higher. Thus, the increase in the motor activity produced by the
nicotine exposure on its own may also contribute to nicotine's
dampened ability to induce a motor output with repeated applications.
Discussion
Fig. 8. Nicotine efflux rates in zebrafish embryos. (Top) Efflux was quantified for
embryos exposed to 30 μM [3H]-nicotine. 22–23 hpf embryos were exposed to nicotine
for 5 min and the incorporation was quantified (point highlighted by circle). The efflux
rate was measured for a subset of the embryos hour by hour. There was 1 μM nicotine
present in the embryos 5 h after the exposure. (Bottom) Efflux was quantified for
embryos exposed to 10 μM nicotine. 22–23 hpf embryos were exposed to the nicotine
for 5 min and the incorporation was quantified (point highlighted by circle). The efflux
rate was measured for a subset of the embryos every 30 min. The nicotine was
completely cleared from the embryos 30 min following the exposure.
were now pre-incubated in 1 μM nicotine for 4 h. Those embryos had a
significantly reduced motor output when challenged with 30 μM
nicotine (Fig. 9C). In these experiments, the embryos pre-incubated in
nicotine had a robust response when placed in embryo medium
containing high KCl. These results demonstrate that the presence of
the residual nicotine was likely contributing to the desensitization
phenomenon observed earlier in this study and is likely antagonizing
nAChRs on neurons that are involved with activating the embryonic
motor behavior. There was some variability in these responses as
demonstrated in Fig. 9C where a 4 h pre-incubation with 1 μM nicotine
did not completely abolish the 30 μM nicotine induced motor output
(Fig. 9C, right). However, the pre-incubation was still able to
substantially reduce the nicotine induced motor output.
The act of dechorionation and its impact on nicotine induced
motor output
To this point, it appeared that the residual nicotine may be the
main cause of the dampened response to subsequent nicotine
challenges in zebrafish embryos. However, another factor may also
contribute to this dampened motor output with repeat nicotine
exposures. In acute exposure paradigms, nicotine can dramatically
increase the bend rates of the spinal musculature in zebrafish
In this study, we characterized nicotine induced modulation of
early embryonic motor behaviors in zebrafish. This study also provides
insight into the kinetics of nicotine accumulation and depuration in
zebrafish embryos during early development. We provide evidence
that nicotine, at low (μM) concentrations, leads to altered behavior
associated with nAChRs. Moreover, the nicotine induced motor
behavior can occur in the absence of brainstem input. Thus, when
we begin to define the cellular substrate of this nicotine mediated
phenotype, we will focus on spinal neurons, predicting that neurons
associated with a rhythm generator will likely express nAChRs.
Zebrafish embryonic motor behavior
Within the last 10–15 years, the zebrafish system has also emerged
as a prominent vertebrate model to study the development of motor
circuits, as well as the cellular factors and molecular factors that
influence locomotor production (Chen et al., 2008; Fetcho et al., 2008;
Fetcho, 2007; McDearmid et al., 2006; Bhatt et al., 2004; Granato et al.,
1996). Recently, Burgess and Granato (2007) have written computer
software to track behaviors of many larval zebrafish swimming
simultaneously in a Petri dish. The behavior in these “older” fish is
readily quantifiable as it is easy to keep track of the heads and tails of
swimming larva with high speed cinematography.
To date, few if any research groups have quantified the bend rates
in early embryos with computer algorithms because the embryos are
“tumbling” within their chorions making their heads and tails
sometimes hard to track. To our knowledge, the works from 3
research groups (Sipple, Saint-Amant and Drapeau, and Downes and
Granato) have been the best attempts to quantify the early embryonic
motor output in zebrafish. The work of Sipple and colleagues
quantified the early embryonic motor behaviors of embryos in their
chorions using computer software in a manner similar to the work of
Burgess and Granato (2007).
In this study, bend rates of individual embryos were quantified by
eye minute by minute in various experimental paradigms. This
analysis and quantification was somewhat tedious, but our results
are in accord with the results obtained by Sipple as we observed a
10
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
Fig. 9. One μM nicotine, equilibration and effect on embryonic physiology. (A) Embryonic equilibration of [3H]-nicotine was quantified for 1, 5 and 15 μM nicotine. (Left) thirty-one
embryos were separated into three groups, transferred to [3H]-nicotine, and videotaped for 5 min to verify that nicotine was exerting an effect. (Right) Influx was then quantified at
minutes 5, 10, 60, and 120. After 2 h, equilibration was reached for the 1 μM [3H]-nicotine. (B) 25 hpf embryos (n = 8) were pre-incubated in 1 μM nicotine for 2.5 h (filled circles).
Stage matched control embryos (n = 7) are denoted by the open circles. At minute 5, both groups of embryos were exposed to 30 μM nicotine. The pre-incubated embryos (filled
circles) have a diminished response to 30 μM nicotine compared to the controls. However, those embryos still respond with a robust output when exposed to embryo medium
containing high KCl. (C) (Left) 25 hpf embryos (n = 8) were pre-incubated in 1 μM for 4 h (filled circles). Stage matched control embryos (n = 7) are denoted by the open circles. At
minute 5, both groups of embryos were exposed to 30 μM nicotine. The embryos pre-incubated in 1 μM nicotine do not respond to 30 μM nicotine compared to the controls. However,
those embryos still respond with a robust motor output when exposed to embryo medium containing high KCL. (Right) 25 hpf embryos (n = 7) were pre-incubated in 1 μM nicotine
for 4 h (filled circles). Stage matched control embryos (n = 7) are denoted by the open circles. At minute 5, both groups of embryos were exposed to 30 μM nicotine. The preincubated embryos (filled circles) have an increase in bend rate upon 30 μM nicotine exposure compared to their bend rates prior to nicotine exposure, but it is dampened compared
to stage matched embryos that exhibit about 15 bends per minute upon nicotine exposure. Asterisks denote significant difference from pre-exposed bend rates. ‡ denotes significant
difference in bend rates between the pre-incubated and control embryos produced by exposure to nicotine (p value b 0.001, Anova).
peak in bend rate occurring at about 19 hpf. The results obtained from
dechorionated embryos were also similar to those reported by SaintAmant and Drapeau, 1998. Also, after 23–24 hpf, the spontaneously
occurring motor output rarely alternated in a sustained manner. We
also noticed that raising embryos at slightly cooler temperatures also
affected the rate of embryonic motor activity, although the peak of
activity still occurred at ∼ 19–20 hpf. So when looking at the general
biology of zebrafish embryos, the act of dechorionation as well as
temperature fluctuations (also reported by Saint-Amant and Drapeau,
1998) can easily influence early embryonic motor behaviors.
nAChRs and embryonic motor output
The findings reported here add to a list of vertebrates, either as adult
or embryos that have nAChRs distributed within spinal circuits that
modulate or produce locomotor behaviors. In adult lamprey and Xenopus embryos, exogenous ACh can modulate the motor output via an
excitatory mechanism. The fact that a motor output is modulated (speed
up or slow down) by ACh implies that nAChRs are located in the circuit
itself that produces the output, or on neuronal elements that activate the
circuits. In Xenopus, it is now known that spinal motoneurons feed back
to interneurons within the central pattern generator, release ACh onto
interneurons and this helps in providing a sustained motor output
(Roberts and Perrins, 1995; Perrins and Roberts, 1994). The direct
application of exogenous ACh to the spinal cords of Xenopus actually
activates a motor output (Panchin et al., 1991).
In embryonic chick spinal cord, administration of DHβE, an
antagonist of the α4/β2 nAChR slows down embryonic motor output,
but does not abolish it (Milner and Landmesser, 1999). This indicates
that the α4/β2 nAChR is likely distributed within the spinal circuit
that governs chick embryonic motor output. Thus, in a variety of
vertebrates including zebrafish reported in this study, nAChRs are
present early in embryonic spinal cord development.
The neurons within embryonic zebrafish spinal motor circuits,
which give rise to those circuits governing locomotion later in
adulthood, are the likely candidate cells to express nAChRs and
ultimately be innervated by local spinal cholinergic inputs. This
statement is based on the following observations. Nicotine applied to
zebrafish embryos activated an embryonic motor output. This output
appeared rhythmic, alternating in a left–right–left manner indicating
that nicotine may be activating a spinal circuit that generates rhythmic
motor output. Such a circuit is known to exist in very young embryos
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
Fig. 10. The act of dechorionation quenches the nicotine induced motor output.
Quantification of motor behavior was performed as in previous experiments. At the left,
embryos (n = 7) were dechorionated at 22 hpf (open circles) and videotaped every
hour for 5 h. Stage matched controls were left in their chorions (filled circles, n = 6)
until 27 hpf and then they were dechorionated (minute 300). After the increase in bend
rate caused by that dechorionation subsided, the embryos were exposed to 30 μM
nicotine. Those embryos dechorionated at 22 hpf (open circles) have a dampened
response to nicotine exposure, but they still do respond with a motor output (white bar
in graph at the right). Black bar corresponds to the nicotine induced bend rates of
embryos dechorionated at 27 hpf. The bend rates are shown for the first minute of the
nicotine response. Asterisk denotes significant difference in the bend rates of the two
groups of embryos upon exposure to nicotine (p value b 0.01, Anova).
(Downes and Granato, 2006; Saint-Amant and Drapeau, 1998). This
motor output could be activated by nicotine in the absence of
brainstem inputs indicating an activation of nAChRs located on spinal
neurons. Candidate cell types to be activated by nicotine include
interneurons within the rhythm generator itself and sensory neurons
known as RB neurons which are known to make synapses with spinal
interneurons. During normal development, the nAChRs are likely
expressed on these spinal neurons, but they are just not being overly
excited by endogenous acetylcholine. Hence, the receptors are poised
to be activated, but they just typically are not activated early in
embryogenesis to produce a sustained motor output.
The nicotine induced behavioral output reported here, was
reduced with reported nicotine exposure, suggesting a possible
receptor desensitization of neuronal nAChRs. This phenotype was
evident between 22 and 28 hpf. In a variety of systems, nicotine is very
effective at desensitizing the same neuronal nAChRs it activates,
particularly the nAChR composed of all α7 subunits and the one
composed of α4/β2 subunits (for review, see Mudo et al., 2007).
Pharmacological evidence from our previous work suggested that
neurons in embryonic zebrafish spinal cord could express α4/β2
nAChRs (Svoboda et al., 2002). Importantly, the muscle nicotinic
acetylcholine receptor does not appear to desensitize as quickly as the
neuronal nAChRs because the 28 hpf embryo still responded to tactile
stimulation even when that embryo has failed to respond to repeated
nicotine exposure (Fig. 6B). Thus, it may be the case that desensitization of an α4/β2-like nAChR or other neuronal nAChRs is underlying
the desensitized behavior in zebrafish reported here.
Speeding up embryonic motor activity: implications for
abnormal development
The act of dechorionation also results in an abnormally fast motor
output. Thus, we have two means whereby we can increase the
motor output in zebrafish embryos, either via nicotine exposure or
by the act of dechorionation. Both can dramatically increase the
embryonic motor output at a developmental time-point where the
11
embryo simply does not usually move fast. This abrupt increase in
activity may have an effect on development. As the muscle output is
a reflection of CNS output, we would argue that the increase in
motor activity, which is likely caused by an inappropriate excitation
of the embryonic nervous system, will alter the cell biology of
neurons in the embryo (Hanson and Landmesser, 2006; Borodinsky
et al., 2004) as well as embryonic muscle. Over-activity caused by
dechorionation dampened the nicotine induced motor output. This
over-activity could alter biological functions including protein
expression. For example, if neuronal nAChR expression was altered
by the increase in activity caused by dechorionation, or if the nAChRs
themselves were altered, this could contribute to the dampened
effect. It also may be the case that the over-activity may be altering
muscle properties, or muscle AChRs themselves. Lastly, the cell
biology of the neurons within the spinal rhythm generator may in
fact be altered at the anatomical, physiological, or biochemical levels
by the over-activity. These possibilities all warrant further
investigation.
Embryonic motor output: a diagnostic tool for studying vertebrate
nicotine toxicity and nAChR distribution
Using a combination of behavior and pharmacological techniques,
we have demonstrated that nicotine exposure at a very low
concentration (1 μM) can disrupt zebrafish embryonic physiology.
When embryos were pre-incubated in 1 μM nicotine for 2.5–4 h and
then challenged with 30 μM nicotine, they did not exhibit the robust
increase in bend rates typical for embryos exposed to the higher
concentrations. The ability of 1 μM nicotine exposure to have an
impact on embryonic physiology is significant to us because this low
concentration was clearly interacting with nAChRS in the zebrafish
embryo without producing overt toxic effects. In mammalian studies
utilizing embryonic explants, concentrations between 0.6 μM and
6 μM nicotine can result in abnormal development. Embryonic
lethality occurred in those explants when they were exposed to
6 μM nicotine (Zhao and Reece, 2005).
We are interested in using the zebrafish as both a model system to
study the consequences of nicotine toxicity and also to understand the
normal role of nAChRs in vertebrate spinal neuron development. Our
results obtained with 1 μM coupled with our observations that 5 μM
nicotine exposure can disrupt motoneuron axonal pathfinding
(Menelaou and Svoboda, 2009), suggest that the nAChRs in
vertebrates may be behaving in a similar fashion when exposed to
nicotine. It has been shown with epibatidine binding studies that
there are two specific types of binding sites which recognize
epibatidine in the 48 hpf developing zebrafish CNS. These binding
sites may possibly correspond to α4/β2 nAChRs or other related
nAChRs (Zirger et al., 2003). Similarly, two epibatidine binding sites
also exist in 9–11-week prenatal human brain with binding constants
that are similar to those detected in the zebrafish. Thus, the zebrafish
nAChRs during embryogenesis appear to be very similar to mammalian (human) nAChRs expressed during embryogenesis. Our behavioral data including the desensitization data from embryos younger
than 28 hpf are consistent with those observations.
The nicotine mediated behavioral phenotype reported here will be
used as a diagnostic endpoint in future studies where we plan to
elucidate which specific nAChR subtypes underlie this behavior. We
ultimately will determine which individual cells express those same
nAChRs and believe that nAChRs will be found on cells located within
the spinal circuit that produces embryonic motor output or on cells
that activate the motor output. Once this distribution is established,
we will then be poised to knockdown expression of the specific nAChR
receptor subtypes and determine if that knockdown impinges on
nicotine's ability to activate the embryonic motor output as well as to
determine if reducing expression of the specific nAChR subtypes,
alters neuronal development.
12
L.T. Thomas et al. / Toxicology and Applied Pharmacology 239 (2009) 1–12
Conflicts of interests
The authors declare that there are no conflicts of interests.
Acknowledgments
This work was supported by grants from the Louisiana Board of
Regents LEQSF(2005-08)-RD-A-11 and the NIH/National Institute of
Environmental Health Sciences ES016513 (KRS). We thank Ms. Robin
Pollet for providing expert zebrafish care, helping with embryo
collection and behavioral analysis.
References
Bhatt, D.H., Otto, S.J., Depoister, B., Fetcho, J.R., 2004. Cyclic AMP-induced repair of
zebrafish spinal circuits. Science 305, 254–258.
Borodinsky, L.N., Rootm, C.M., Cronin, J.A., Sann, S.B., Gu, X., Spitzer, N.C., 2004. Activitydependent homeostatic specification of transmitter expression in embryonic
neurons. Nature 429, 523–530.
Burgess, H.A., Granato, M., 2007. Sensorimotor gating in larval zebrafish. J. Neurosci. 27,
4984–4994.
Chen, Y.H., Huang, F.L., Cheng, Y.C., Wu, C.J., Yang, C.N., Tsay, H.J., 2008. Knockdown of
zebrafish Nav1.6 sodium channel impairs embryonic locomotor activities.
J. Biomed. Sci. 15, 69–78.
Cui, W.W., Low, S.E., Hirata, H., Saint-Amant, L., Geisler, R., Hume, R.I., Kuwada, J.Y., 2005.
The zebrafish shocked gene encodes a glycine transporter and is essential for the
function of early neural circuits in the CNS. J. Neurosci. 25, 6610–6620.
Fetcho, J.R., 2007. The utility of zebrafish for studies of the comparative biology of motor
systems. J. Exp. Zoolog. B Mol. Dev. Evol. 308, 550–562.
Fetcho, J.R., Higashijima, S., McLean, D.L., 2008. Zebrafish and motor control over the last
decade. Brain Res. Rev. 57, 86–93.
Downes, G.B., Granato, M., 2006. Supraspinal input is dispensable to generate
glycine-mediated locomotive behaviors in the zebrafish embryo. J. Neurobiol.
66, 437–451.
Granato, M., van Eeden, F.J., Schach, U., Trowe, T., Brand, M., Furutani-Seiki, M., Haffter,
P., Hammerschmidt, M., Heisenberg, C.P., Jiang, Y.J., Kane, D.A., Kelsh, R.N., Mullins,
M.C., Odenthal, J., Nusslein-Volhard, C., 1996. Genes controlling and mediating
locomotor behavior of the zebrafish embryo and larva. Development 123,
399–413.
Grunwald, D.J., Kimmel, C.B., Westerfield, M., Walker, C., Streisinger, G., 1988. A neural
degeneration mutation that spares primary neurons in the zebrafish. Dev. Biol. 126,
115–128.
Hanson, M.G., Landmesser, L.T., 2004. Normal patterns of spontaneous activity are
required for correct motor axon guidance and the expression of specific guidance
molecules. Neuron 43, 687–701.
Hanson, M.G., Landmesser, L.T., 2006. Increasing the frequency of spontaneous
rhythmic activity disrupts pool-specific axon fasciculation and pathfinding of
embryonic spinal motoneurons. J. Neurosci. 26, 12769–12780.
Hanson, M.G., Milner, L.D., Landmesser, L.T., 2008. Spontaneous rhythmic activity in
early chick spinal cord influences distinct motor axon pathfinding decisions. Brain
Res. Rev. 57, 77–85.
Henry, T.R., Spitsbergen, J.M., Hornung, M.W., Abnet, C.C., Petersen, R.E., 1997. Early life
stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish (Danio rerio).
Toxicol. Appl. Pharmacol. 142, 56–68.
Hill, A.J., Teraoka, H., Heideman, W., Peterson, R.E., 2005. Zebrafish as a model vertebrate
for investigating chemical toxicity. Toxicol. Sci. 86, 6–19.
Kimmel, C.B., Patterson, J., Kimmel, R.O., 1974. The development and behavioral
characteristics of the startle response in zebrafish. Dev. Psychobiol. 7, 47–60.
Le Ray, D., Brocard, F., Bourcier-Lucas, C., Auclair, F., Lafaille, P., Dubuc, R., 2003. Nicotinic
activation of reticulospinal cells involved in the control of swimming in lampreys.
Eur. J. Neurosci. 17, 137–148.
McDearmid, J.R., Liao, M., Drapeau, P., 2006. Glycine receptors regulate interneuron
differentiation during spinal network development. Proc. Natl. Acad. Sci. U. S. A. 103,
9679–9684.
Menelaou, E., Svoboda, K.R., 2009. Secondary motoneurons in juvenile and adult
zebrafish: Axonal pathfinding errors caused by embryonic nicotine exposure. J.
Comp. Neurol. 512, 305–322.
Milner, L.D., Landmesser, L.T., 1999. Cholinergic and GABAergic inputs drive patterned
spontaneous motoneuron activity before target contact. J. Neurosci. 19, 3007–3022.
Mudo, G., Belluardo, N., Fuxe, K., 2007. Nicotinic receptor agonists as neuroprotective/
neurotrophic drugs. Progress in molecular mechanisms. J. Neural Transm. 114,
135–147.
Myers, C.P., Lewcock, J.W., Hanson, M.G., Gosgnach, S., Aimone, J.B., Gage, F.H., Lee, K.F.,
Landmesser, L.T., Pfaff, S.L., 2005. Cholinergic input is required during embryonic
development to mediate proper assembly of spinal locomotor circuits. Neuron 46,
37–49.
Panchin Yu, Y., Perrins, R.J., Roberts, A., 1991. The action of acetylcholine on the
locomotor central pattern generator for swimming in Xenopus embryos. J. Exp. Biol.
161, 527–531.
Perrins, R., Roberts, A., 1994. Nicotinic and muscarinic ACh receptors in rhythmically
active spinal neurones in the Xenopus laevis embryo. J. Physiol. 478 (Pt 2), 221–228.
Pineda, R.H., Svoboda, K.R., Wright, M.A., Taylor, A.D., Novak, A.E., Gamse, J.T., Eisen, J.S.,
Ribera, A.B., 2006. Knockdown of Nav 1.6a Na+ channels affects zebrafish
motoneuron development. Development 133, 3827–3836.
Quinlan, K.A., Placas, P.G., Buchanan, J.T., 2004. Cholinergic modulation of the locomotor
network in the lamprey spinal cord. J. Neurophysiol. 92, 1536–1548.
Ribera, A.B., Nusslein-Volhard, C., 1998. Zebrafish touch-insensitive mutants reveal an
essential role for developmental regulation of sodium current. J. Neurosci. 18,
9181–9191.
Roberts, A., Perrins, R., 1995. Positive feedback as a general mechanism for sustaining
rhythmic and non-rhythmic activity. J. Physiol. Paris 89 (4–6), 241–248.
Saint-Amant, L., Drapeau, P., 1998. Time course of the development of motor behaviors
in the zebrafish embryo. J. Neurobiol. 37, 622–632.
Sipple, B. A. (1998). The Rohon-Beard cell : the formation, function, and fate of a
primary sensory system in the embryonic zebrafish, Danio rerio. Ph.D. Thesis,
Temple University.
Spitzer, N.C., Root, C.M., Borodinsky, L.N., 2004. Orchestrating neuronal differentiation:
patterns of Ca2+ spikes specify transmitter choice. Trends Neurosci. 27, 415–421.
Svoboda, K.R., Linares, A.E., Ribera, A.B., 2001. Activity regulates programmed cell death
of zebrafish Rohon–Beard neurons. Development 128, 3511–3520.
Svoboda, K.R., Vijayaraghavan, S., Tanguay, R.L., 2002. Nicotinic receptors mediate
changes in spinal motoneuron development and axonal pathfinding in embryonic
zebrafish exposed to nicotine. J. Neurosci. 22, 10731–10741.
Westerfield, M., 1995. The Zebrafish Book. University of Oregon Press, Eugene, OR.
Zhao, Z., Reece, E.A., 2005. Nicotine-induced embryonic malformations mediated by
apoptosis from increasing intracellular calcium and oxidative stress. Birth Defects
Res. B Dev. Reprod. Toxicol. 74, 383–391.
Zirger, J., Beattie, C.E., McKay, D.B., Boyd, R.T., 2003. Cloning and expression of zebrafish
neuronal nicotinic acetylcholine receptors. Gene Expression Patterns 3, 747–754.