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Ethanol modifies zebrafish responses to abrupt changes in light intensity
Journal of Clinical Neuroscience 20 (2013) 476–477
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Short communication
Ethanol modifies zebrafish responses to abrupt changes in light intensity John Ramcharitar a,⇑, Ronnie M. Ibrahim b a b
St. Mary’s College of Maryland, 18952 East Fishers Road, St. Mary’s City, MD 20686, USA University of Maryland, College Park, MD, USA
a r t i c l e
i n f o
Article history: Received 24 August 2012 Accepted 8 September 2012
Keywords: Zebrafish behavior Ethanol Light intensity
a b s t r a c t Zebrafish exhibit a preference for dark areas and this behavior has been used to characterize anxiety. Their responses to light may also be modified by ethanol. Using high-speed video recordings, we demonstrated that untreated animals were relatively more active immediately after a bright-dim transition compared to animals exposed to low dose ethanol (2%). Additionally, ethanol-treated larvae were more prone to initiating behavioral responses following abrupt changes of light intensity. In conclusion, the larval zebrafish is an excellent model for investigating locomotory kinetics as well as drugs with anxiolytic properties. High-speed video recordings of behavioral responses in this species are indeed very promising for high-throughput screening. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction High-speed image analysis has been successfully used for studying larval zebrafish locomotory kinetics in various biologically-relevant contexts.1–6 This approach allows for the rapid acquisition of behavioral data in multi-well systems.7 By 5 day post-fertilization (dpf), zebrafish demonstrate robust responses to a host of stimuli e.g. abrupt changes to light level.6 Zebrafish exhibit a preference for dark areas and this behavior has been used to characterize anxiety.8 For example, when fish are confined to a white background, freezing behavior is observed.9 In addition, low-level light mediates robust positive phototaxis in zebrafish larvae, while high-intensity light induces a reverse effect.10–12 In a recent study, zebrafish larvae exposed to a dark-light-dark sequence exhibited higher motor activity within 10 minutes of the light-dark switch.7 Interestingly, the upswing in activity was accentuated in animals treated with acute low doses of ethanol (0.5–2%), while it was inhibited at 4% exposure. Here we investigated swimming behavior in larval zebrafish exposed to brightdim-bright changes in light intensity in control versus ethanol-treated (2%) animals. It was hypothesized that untreated fish would show higher levels of activity after the bright-dim interface (as opposed to dim-bright), and that ethanol-treated animals would be more active at both light transitions. 2. Methods In this study, a total of 27 wild-type (AB strain) zebrafish larvae (9–10 dpf) were used for experimentation. Embryos were sourced ⇑ Corresponding author. Tel.: +1 240 895 2098; fax: +1 240 895 4996. E-mail address: [email protected] (J. Ramcharitar). 0967-5868/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2012.09.010
from the Zebrafish International Resource Center (ZIRC, Oregon, USA) and were fed on a diet of Paramecium from 5 dpf until testing. Fish were first acclimated to bright light (163.8–164.0 Wmm 2) for 5 minutes and then abruptly switched to dim light (12.9– 13.4 Wmm 2) for 15 minutes, followed by a transition to bright light for 5 minutes. Fish were placed in a standard 24-well plate (one fish per well) for testing. A HiSpec 2 high-speed camera (Tech Imaging Inc., Salem, MA, USA) was used capture swimming behavior following changes in light intensity, at a frame rate of 250 fps. Frames were analyzed in 4 ms increments to detect motor activity following stimulation, and for computation of response latency. Treated fish were exposed to 2% ethanol solution for 30 minutes prior to experimentation, while controls fish were kept in regular tank water. 3. Results Fish exhibited three distinct locomotory behaviors in response to abrupt changes in light intensity–slow swimming, burst swimming and escape-type (startle). Slow swimming was characterized by relatively small body undulations while bursts were more rapid and mediated by larger head-tail displacements. Startle responses were identified by stereotyped C-start maneuvers (Fig. 1). When light intensity was abruptly switched from bright to dim, 60% of control animals (total n = 15) responded within 5000 milliseconds of the change, while 11 of 12 ethanol-treated larvae initiated swimming in the same window of time. For the dim-bright transition, six of 15 control animals demonstrated responses, and all but one ethanol-treated fish (total n = 12) initiated movements with latencies of less than five seconds. Response latencies were significantly smaller for larvae exposed to 2% ethanol after the bright-dim transition (Fig. 2, p < 0.05, t-test). No significant
J. Ramcharitar, R.M. Ibrahim / Journal of Clinical Neuroscience 20 (2013) 476–477
477
Fig. 1. Escape turn captured at 250 frames per second for a zebrafish larva exposed to 2% ethanol for 30 minutes. Frames are presented in 4 ms intervals with 0 ms representing the frame immediately before body undulation is detected.
abrupt changes in light intensity may be different to those elicited by light stimuli of constant intensity. Finally, we found that fish acutely exposed to 2% ethanol showed greater incidences of behavioral responses to both light intensity transitions, compared to untreated fish. However, the effect of ethanol has been shown to be dose-dependent and it also varies with developmental stage.7,13 In conclusion, the larval zebrafish is an excellent model for investigating locomotory kinetics as well as drugs with anxiolytic properties. High-speed video recordings of behavioral responses in this species are indeed very promising for high-throughput screening (which is not feasible in rodent models). References Fig. 2. Average response latencies following abrupt light intensity change from bright to dim. Response latency was significantly higher for control zebrafish larvae (p < 0.05; t-test). Standard error bars are shown.
difference in response latency was observed after the dim-bright interface for control versus treated fish.
4. Discussion In summary, we demonstrated that untreated animals were relatively more active immediately after the bright-dim transition, compared to when light was switched from dim to bright. Additionally, ethanol-treated larvae were more prone to initiating behavioral responses following abrupt changes of light intensity in either direction, compared to control animals. The data therefore supported our hypotheses. The observation of increased motor activity in darkened conditions is consistent with previous reports.7,8 Interestingly, 40% of control animals initiated responses following the abrupt change in light intensity from dim to bright, while freezing responses have been observed in fish placed in a bright environment.9 Our data are however consistent with those from Esch et al.,7 for 5–7 dpf larvae transitioned from dark to light. Indeed, behavioral responses to
1. Bettinger JC, Leung K, Bolling MH, et al. Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans. PLoS One 2012;7:e35192. 2. Guarnieri DJ, Heberlein U. Drosophila melanogaster: a genetic model system for alcohol research. Int Rev Neurobiol 2003;54:199–228. 3. Silberman Y, Ariwodola OJ, Weiner JL. Differential effects of GABAB autoreceptor activation on ethanol potentiation of local and lateral paracapsular GABAergic synapses in the rat basolateral amygdala. Neuropharmacology 2009;56:886–95. 4. Budick SA, O’Malley DM. Locomotor repertoire of the larval zebrafish: swimming, turning and prey capture. J Exp Biol 2000;203:2565–79. 5. Feitl KE, Ngo V, McHenry MJ. Are fish less responsive to a flow stimulus when swimming? J Exp Biol 2010;213:3131–7. 6. Colwill RM, Creton R. Imaging escape and avoidance behavior in zebrafish larvae. Rev Neurosci 2011;22:63–73. 7. de Esch C, van der Linde H, Slieker R, et al. Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neurotoxicol Teratol 2012;34:425–33. 8. Blaser RE, Chadwick L, McGinnis GC. Behavioral measures of anxiety in zebrafish (Danio rerio). Behav Brain Res 2010;208:56–62. 9. Maxinimo C, de Brito TM, Colmanetti R, et al. Parametric analyses of anxiety in zebrafish scototaxis. Behav Brain Res 2010;210:1–7. 10. Brockerhoff SE, Hurley JB, Janssen-Bienhold U, et al. A behavioral screen for isolating zebrafish mutants with visual system defects. Proc Natl Acad Sci USA 1995;92:10545–9. 11. Orger MB, Baier H. Channeling of red and green cone inputs to the zebrafish optomotor response. Vis Neurosci 2005;22:275–81. 12. Burgess HA, Schoch H, Granato M. Distinct retinal pathways drive spatial orientation behaviors in zebrafish navigation. Curr Biol 2010;20:381–6. 13. MacPhail RC, Brooks J, Hunter DL, et al. Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Neurotoxicology 2009;30:52–8.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Neuroscience journal homepage: www.elsevier.com/locate/jocn
Short communication
Ethanol modifies zebrafish responses to abrupt changes in light intensity John Ramcharitar a,⇑, Ronnie M. Ibrahim b a b
St. Mary’s College of Maryland, 18952 East Fishers Road, St. Mary’s City, MD 20686, USA University of Maryland, College Park, MD, USA
a r t i c l e
i n f o
Article history: Received 24 August 2012 Accepted 8 September 2012
Keywords: Zebrafish behavior Ethanol Light intensity
a b s t r a c t Zebrafish exhibit a preference for dark areas and this behavior has been used to characterize anxiety. Their responses to light may also be modified by ethanol. Using high-speed video recordings, we demonstrated that untreated animals were relatively more active immediately after a bright-dim transition compared to animals exposed to low dose ethanol (2%). Additionally, ethanol-treated larvae were more prone to initiating behavioral responses following abrupt changes of light intensity. In conclusion, the larval zebrafish is an excellent model for investigating locomotory kinetics as well as drugs with anxiolytic properties. High-speed video recordings of behavioral responses in this species are indeed very promising for high-throughput screening. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction High-speed image analysis has been successfully used for studying larval zebrafish locomotory kinetics in various biologically-relevant contexts.1–6 This approach allows for the rapid acquisition of behavioral data in multi-well systems.7 By 5 day post-fertilization (dpf), zebrafish demonstrate robust responses to a host of stimuli e.g. abrupt changes to light level.6 Zebrafish exhibit a preference for dark areas and this behavior has been used to characterize anxiety.8 For example, when fish are confined to a white background, freezing behavior is observed.9 In addition, low-level light mediates robust positive phototaxis in zebrafish larvae, while high-intensity light induces a reverse effect.10–12 In a recent study, zebrafish larvae exposed to a dark-light-dark sequence exhibited higher motor activity within 10 minutes of the light-dark switch.7 Interestingly, the upswing in activity was accentuated in animals treated with acute low doses of ethanol (0.5–2%), while it was inhibited at 4% exposure. Here we investigated swimming behavior in larval zebrafish exposed to brightdim-bright changes in light intensity in control versus ethanol-treated (2%) animals. It was hypothesized that untreated fish would show higher levels of activity after the bright-dim interface (as opposed to dim-bright), and that ethanol-treated animals would be more active at both light transitions. 2. Methods In this study, a total of 27 wild-type (AB strain) zebrafish larvae (9–10 dpf) were used for experimentation. Embryos were sourced ⇑ Corresponding author. Tel.: +1 240 895 2098; fax: +1 240 895 4996. E-mail address: [email protected] (J. Ramcharitar). 0967-5868/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jocn.2012.09.010
from the Zebrafish International Resource Center (ZIRC, Oregon, USA) and were fed on a diet of Paramecium from 5 dpf until testing. Fish were first acclimated to bright light (163.8–164.0 Wmm 2) for 5 minutes and then abruptly switched to dim light (12.9– 13.4 Wmm 2) for 15 minutes, followed by a transition to bright light for 5 minutes. Fish were placed in a standard 24-well plate (one fish per well) for testing. A HiSpec 2 high-speed camera (Tech Imaging Inc., Salem, MA, USA) was used capture swimming behavior following changes in light intensity, at a frame rate of 250 fps. Frames were analyzed in 4 ms increments to detect motor activity following stimulation, and for computation of response latency. Treated fish were exposed to 2% ethanol solution for 30 minutes prior to experimentation, while controls fish were kept in regular tank water. 3. Results Fish exhibited three distinct locomotory behaviors in response to abrupt changes in light intensity–slow swimming, burst swimming and escape-type (startle). Slow swimming was characterized by relatively small body undulations while bursts were more rapid and mediated by larger head-tail displacements. Startle responses were identified by stereotyped C-start maneuvers (Fig. 1). When light intensity was abruptly switched from bright to dim, 60% of control animals (total n = 15) responded within 5000 milliseconds of the change, while 11 of 12 ethanol-treated larvae initiated swimming in the same window of time. For the dim-bright transition, six of 15 control animals demonstrated responses, and all but one ethanol-treated fish (total n = 12) initiated movements with latencies of less than five seconds. Response latencies were significantly smaller for larvae exposed to 2% ethanol after the bright-dim transition (Fig. 2, p < 0.05, t-test). No significant
J. Ramcharitar, R.M. Ibrahim / Journal of Clinical Neuroscience 20 (2013) 476–477
477
Fig. 1. Escape turn captured at 250 frames per second for a zebrafish larva exposed to 2% ethanol for 30 minutes. Frames are presented in 4 ms intervals with 0 ms representing the frame immediately before body undulation is detected.
abrupt changes in light intensity may be different to those elicited by light stimuli of constant intensity. Finally, we found that fish acutely exposed to 2% ethanol showed greater incidences of behavioral responses to both light intensity transitions, compared to untreated fish. However, the effect of ethanol has been shown to be dose-dependent and it also varies with developmental stage.7,13 In conclusion, the larval zebrafish is an excellent model for investigating locomotory kinetics as well as drugs with anxiolytic properties. High-speed video recordings of behavioral responses in this species are indeed very promising for high-throughput screening (which is not feasible in rodent models). References Fig. 2. Average response latencies following abrupt light intensity change from bright to dim. Response latency was significantly higher for control zebrafish larvae (p < 0.05; t-test). Standard error bars are shown.
difference in response latency was observed after the dim-bright interface for control versus treated fish.
4. Discussion In summary, we demonstrated that untreated animals were relatively more active immediately after the bright-dim transition, compared to when light was switched from dim to bright. Additionally, ethanol-treated larvae were more prone to initiating behavioral responses following abrupt changes of light intensity in either direction, compared to control animals. The data therefore supported our hypotheses. The observation of increased motor activity in darkened conditions is consistent with previous reports.7,8 Interestingly, 40% of control animals initiated responses following the abrupt change in light intensity from dim to bright, while freezing responses have been observed in fish placed in a bright environment.9 Our data are however consistent with those from Esch et al.,7 for 5–7 dpf larvae transitioned from dark to light. Indeed, behavioral responses to
1. Bettinger JC, Leung K, Bolling MH, et al. Lipid environment modulates the development of acute tolerance to ethanol in Caenorhabditis elegans. PLoS One 2012;7:e35192. 2. Guarnieri DJ, Heberlein U. Drosophila melanogaster: a genetic model system for alcohol research. Int Rev Neurobiol 2003;54:199–228. 3. Silberman Y, Ariwodola OJ, Weiner JL. Differential effects of GABAB autoreceptor activation on ethanol potentiation of local and lateral paracapsular GABAergic synapses in the rat basolateral amygdala. Neuropharmacology 2009;56:886–95. 4. Budick SA, O’Malley DM. Locomotor repertoire of the larval zebrafish: swimming, turning and prey capture. J Exp Biol 2000;203:2565–79. 5. Feitl KE, Ngo V, McHenry MJ. Are fish less responsive to a flow stimulus when swimming? J Exp Biol 2010;213:3131–7. 6. Colwill RM, Creton R. Imaging escape and avoidance behavior in zebrafish larvae. Rev Neurosci 2011;22:63–73. 7. de Esch C, van der Linde H, Slieker R, et al. Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neurotoxicol Teratol 2012;34:425–33. 8. Blaser RE, Chadwick L, McGinnis GC. Behavioral measures of anxiety in zebrafish (Danio rerio). Behav Brain Res 2010;208:56–62. 9. Maxinimo C, de Brito TM, Colmanetti R, et al. Parametric analyses of anxiety in zebrafish scototaxis. Behav Brain Res 2010;210:1–7. 10. Brockerhoff SE, Hurley JB, Janssen-Bienhold U, et al. A behavioral screen for isolating zebrafish mutants with visual system defects. Proc Natl Acad Sci USA 1995;92:10545–9. 11. Orger MB, Baier H. Channeling of red and green cone inputs to the zebrafish optomotor response. Vis Neurosci 2005;22:275–81. 12. Burgess HA, Schoch H, Granato M. Distinct retinal pathways drive spatial orientation behaviors in zebrafish navigation. Curr Biol 2010;20:381–6. 13. MacPhail RC, Brooks J, Hunter DL, et al. Locomotion in larval zebrafish: influence of time of day, lighting and ethanol. Neurotoxicology 2009;30:52–8.