Acute nicotine exposure and modulation of a spinal motor circuit in embryonic zebrafish
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 over-activity 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 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 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.
Section snippets
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
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
Discussion
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
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.
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