10/26/2011 The Journal of Experimental Biology Distinct startle responses are associated with neuroanatomical differences in pufferfishes � “The leading journal in comparative animal A. K. Greenwood, C. L. Peichel, and S. J. Zottoli physiology and is published by The Company of Journal of Experimental Biology 213, 613-620. Biologists” Biologists Published in 2010 � Launched as The British Journal of Experimental Presented by: Biology in 1923 David Tomasek and Lucy Liu October 26, 2011 � Impact factor of 3.040 for February 2010 Issue 2010 The Authors The Authors � Anna K. Greenwood � Catherine (Katie) L. Peichel � Post-Doc in the Peichel Lab at the Fred � Associate Member, FHCRC Division of Hutchinson Cancer Research Center Human Biology � B.S. Psychology (1996) Rutgers University � B.A. Molecular & Cell Biology (1991) � Ph.D. Neuroscience (2004) ( ) University of California, Berkeley y y Stanford University � Ph.D. Molecular Biology (1998) � Studies the “anatomical, developmental, Princeton University and genetic basis for evolution of anti- � Studies the “genetic and neural predator morphology and behavior” mechanisms that underlie the evolution � Spent the summer of 2007 as a Grass of behaviors” in sticklebacks Fellow at the Marine Biological Laboratory Overview The Authors � Steven J. Zottoli � Escape behavior – Mauthner Cells � Professor of Biology at Williams College � M-Cell diversity and pufferfish since 1980 � Behavioral and neuroanatomical methods and � A.B. (1969) Bowdoin College results results � Ph D (1976) University of � Ph.D. (1976) University of Massachusetts, Amherst � Key points and areas for further study � Studies focus on goldfish with spinal cord � Questions? injuries to determine the neuronal basis of startle response recovery 1
10/26/2011 Whole Genome Duplication in Teleosts C-start Behavioral Response Allow for Diversification � Mauthner cells (M-cells) initiate fast-start escape response � C-type fast-start (C-start) � Stage 1: contraction of muscles on one side of the body to form rclass WGD = C-shape C shape Super whole- � Stage 2: tail stroke for forward propulsion genome duplication � Stage 3: gliding or a burst swim Stage 1 Stage 2 Stage 3 Stimulus Sequencing of Tetraodon nigroviridis (green spotted puffer) genome. Eaton, R. C., Lee, R. K. K., & Foreman, M. B. (2001). The Mauthner cell and other identified neurons in Mulley, J. & Holland, P. (2004). Comparative genomics: Small genome, big insights. Nature 431, 916-917. the brainstem escape network of fish. Progress in Neurobiology 63, 467 ‐ 485. Video: Escape Behavior in Zebrafish The Mauthner Cell (Dorsal View) � Mauthner somas receive auditory and other sensory input C ‐ start video from Fetcho Lab � The Mauthner axon synapses on contralateral motoneurons in the spinal cord For other videos, check out the websites of Jimmy Liao and George Lauder Anterior Posterior Eaton, R. C., Lee, R. K. K., & Foreman, M. B. (2001). The Mauthner cell and other identified http://evolution.berkeley.edu/evolibrary/news/060201_zebrafish neurons in the brainstem escape network of fish. Progress in Neurobiology, 63, 467-485. M-cell Activity is Unnecessary for C-start M-cell Activity is Unnecessary for C-start � M-cell activity precedes C-start � Homologous reticulospinal neurons � Electrical stimulation of M-cells can elicit response in the fifth and sixth � HOWEVER, M-cells are not necessary; a delayed hindbrain segments are response is elicited after ablation. Why? M-cell involved with startle response in goldfish i ldfi h X and zebrafish MiD2 cm Stimuli M-cell spike C-start response � These reticulospinal neurons have cell MiD3 cm bodies and axons that are smaller than those of M-cells 100 µm Eaton, R. C., Lee, R. K. K., & Foreman, M. B. (2001). The Mauthner cell and other identified neurons in the brainstem escape network of fish. Progress in Neurobiology, 63, 467-485. 2
10/26/2011 Previous Studies with Pufferfish do not M-cell Diversity is Apparent in Teleosts Link Cell Diversity and Behavior � Many teleosts lack obvious M-cells, � Two previously-examined pufferfish species do not have small M-cells, M-cells with small perform the fast-start in response to a tactile stimulus axon diameter, or altered axon cap � Some pufferfish do not have M-cells, while others have structure M-cells that are small � Life history is related to M-cell variation � Life history is related to M-cell variation � Relationship?? Relationship?? � Bottom dwelling, use of crypsis or camouflage, extreme caudal fin modification � How does this diversity affect fast-start behavior? � Lumpfish larvae (lack M-cells) have delayed C-starts compared to larval zebrafish Purpose of Study Discovery Science � Description of nature through observation and analysis � Purpose : To identify a correlation between M-cell � Examples anatomy and C-start response in two pufferfish species � Cajal’s observations of neurons using Golgi’s method in sister families � Jane Goodall’s qualitative and quantitative observations of chimpanzee behavior � No hypothesis was given, why? � Can use inductive reasoning to derive generalizations from observations � “All organisms are made of cells” Campbell, N. A., and Reece, J. B. Biology (Eighth Edition). San Francisco: Benjamin Cummings, 2008. Tetraodon nigroviridis Diodon holocanthus Green spotted puffer Porcupine puffer or balloonfish � Family Tetraodontidae � Family Diodontidae � Habitat: Estuaries, freshwater streams around South � Habitat: Marine, inshore and reef areas in the tropics Asia � Predators: Sharks and large bony fish � Predators: Birds and other fish (?) Figure 1 (B) Figure 1 (A) 3
10/26/2011 Methods: Behavior Methods: Stimuli Setup � Trials of tactile and acoustic stimuli High Speed Camera � Tactile: touched body of fish with a plastic rod Mirror � Responses not analyzed quantitatively 45º Aquarium � Acoustic: hit a rubber mallet on the side of the platform holding the tank (several parameters) Tactile: Plastic rod Platform Acoustic: Rubber mallet at constant height Methods: Behavior - Acoustic Methods: Behavior - Acoustic � Latency: time between impact of mallet to axial � Angle: line extended along midline of anterior portion of movement of the head or tail fish; measured change in angle of this line Time � Probability: proportion of total trials evoking fast-start # fast start / total trials � Peak angular velocity: change in angle and time � Duration (of stage 1): stage 1 ended when fish began to � Distance moved: minimum straight-line distance that the straighten tail fish moved using a point on the midline in between the pectoral fins Stage 1 Stage 3 Stage 2 End Stimulus Response * Start Startle response + swim Methods: Neuroanatomy Methods: Neuroanatomy Retrograde Tracing � Retrograde tracing � Tracing neural connections from the point of termination (synapse) to the source (cell body) using retrograde � Silver stain and plastic sections transport of material of small molecular weight http://www.invitrogen.com/site/us/en/home/References/Molecular ‐ Probes ‐ The ‐ Handbook/Fluorescent ‐ Tracers ‐ of ‐ Cell ‐ Morphology ‐ and ‐ Fluid ‐ Flow/Polar ‐ Tracers.html 4
10/26/2011 Methods: Neuroanatomy Methods: Neuroanatomy How retrograde tracing works How retrograde tracing works � Spinal cords were cut � To visualize BDA, incubated sections in avidin- horseradish peroxidase (avidin-HRP) � Biotin dextran amine (BDA) was applied to the cut to identify all cells that project from the brain into the spinal � Avidin binds to biotin—this locates the cells that took up cord BDA � Axons take up biotin dextran amine (BDA) and transport A ons take p biotin de tran amine (BDA) and transport � The HRP bound to avidin catalyzes the breakdown of The HRP bo nd to a idin catal es the breakdo n of it back to the cell body H 2 O 2 to H 2 O and O 2 � Brains were removed, frozen, and were sectioned � Add DAB, which is oxidized (by HRP) and turns black � So the cells that had synapses in the spinal cord are now black Discussion with Prof. Hopkins and http://www.vectorlabs.com/catalog.aspx?dpID=11&locID=14193 Discussion with Prof. Hopkins and http://www.vectorlabs.com/catalog.aspx?dpID=11&locID=14193 Methods: Neuroanatomy Results: Fast-start behavior detected in Silver stain and plastic sections both Species � Silver staining � Brains removed and prepared for staining � Morse’s modification of Bodian’s silver technique � Embedding in plastic for thin sectioning � Sections were mounted on slides and stained with Toluidine Blue � Sections were mounted on slides and stained with Toluidine Blue Figure 1 (C) and (D) Silhouettes of (C) T. nigroviridis and (D ) D. holocanthus at 2 ms intervals *Recall: latency = time between impact of mallet to when fish commenced axial movement of the head or tail Results: Reduced Fast Start in Results: Neuroanatomy D. holocanthus Telencephalon Optic tectum Green-spotted Porcupinefish Dorsal puffer Cerebellum Hindbrain Anterior Posterior Diencephalon Ventral Transverse Table 1 Sagitta l Horizontal Figure 2 (A) 5
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