10/26/2011

1

Distinct startle responses are associated with neuroanatomical differences in pufferfishes

A. K. Greenwood, C. L. Peichel, and S. J. Zottoli

Journal of Experimental Biology 213, 613-620.Published in 2010

Presented by:David Tomasek and Lucy Liu

October 26, 2011

The Journal of Experimental Biology

“The leading journal in comparative animal physiology and is published by The Company of Biologists”BiologistsLaunched as The British Journal of Experimental Biology in 1923Impact factor of 3.040 for 2010

February 2010 Issue

The Authors

Anna K. GreenwoodPost-Doc in the Peichel Lab at the Fred Hutchinson Cancer Research CenterB.S. Psychology (1996) Rutgers UniversityPh.D. Neuroscience (2004)( )Stanford UniversityStudies the “anatomical, developmental, and genetic basis for evolution of anti-predator morphology and behavior”Spent the summer of 2007 as a Grass Fellow at the Marine Biological Laboratory

The Authors

Catherine (Katie) L. PeichelAssociate Member, FHCRC Division of Human BiologyB.A. Molecular & Cell Biology (1991)University of California, Berkeleyy yPh.D. Molecular Biology (1998)Princeton UniversityStudies the “genetic and neural mechanisms that underlie the evolution of behaviors” in sticklebacks

The Authors

Steven J. ZottoliProfessor of Biology at Williams College since 1980A.B. (1969) Bowdoin CollegePh D (1976) University ofPh.D. (1976) University of Massachusetts, AmherstStudies focus on goldfish with spinal cord injuries to determine the neuronal basis of startle response recovery

Overview

Escape behavior – Mauthner CellsM-Cell diversity and pufferfishBehavioral and neuroanatomical methods and resultsresultsKey points and areas for further studyQuestions?

10/26/2011

2

Whole Genome Duplication in Teleosts Allow for Diversification

rclass

WGD =

Sequencing of Tetraodon nigroviridis (green spotted puffer) genome.Mulley, J. & Holland, P. (2004). Comparative genomics: Small genome, big insights. Nature 431, 916-917.

Supe

r

whole-genome duplication

C-start Behavioral Response

Mauthner cells (M-cells) initiate fast-start escape responseC-type fast-start (C-start)

Stage 1: contraction of muscles on one side of the body to form C-shapeC shapeStage 2: tail stroke for forward propulsionStage 3: gliding or a burst swim

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.

Stage 1 Stage 3Stage 2

Stimulus

Video: Escape Behavior in Zebrafish

C‐start video from Fetcho Lab

For other videos, check out the websites of Jimmy Liao and George Lauder

http://evolution.berkeley.edu/evolibrary/news/060201_zebrafish

The Mauthner Cell (Dorsal View)

Mauthner somas receive auditory and other sensory inputThe Mauthner axon synapses on contralateral motoneurons in the spinal cord

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.

Anterior Posterior

M-cell Activity is Unnecessary for C-start

M-cell activity precedes C-startElectrical stimulation of M-cells can elicit responseHOWEVER, M-cells are not necessary; a delayed response is elicited after ablation. Why?

M-cell spike C-start responseStimuli X

M-cell Activity is Unnecessary for C-start

Homologous reticulospinal neurons in the fifth and sixth hindbrain segments are involved with startle

i ldfi hM-cell

response in goldfish and zebrafishThese reticulospinal neurons have cell bodies and axons that are smaller than those of M-cells

MiD2 cm

MiD3 cm

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.

100 µm

10/26/2011

3

M-cell Diversity is Apparent in Teleosts

Many teleosts lack obvious M-cells, have small M-cells, M-cells with small axon diameter, or altered axon cap structureLife history is related to M-cell variationLife history is related to M-cell variation

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

Previous Studies with Pufferfish do not Link Cell Diversity and Behavior

Two previously-examined pufferfish species do not perform the fast-start in response to a tactile stimulusSome pufferfish do not have M-cells, while others have M-cells that are smallRelationship??Relationship??

Purpose of Study

Purpose: To identify a correlation between M-cell anatomy and C-start response in two pufferfish species in sister familiesNo hypothesis was given, why?

Discovery Science

Description of nature through observation and analysisExamples

Cajal’s observations of neurons using Golgi’s methodJane Goodall’s qualitative and quantitative observations of chimpanzee behavior

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 nigroviridisGreen spotted puffer

Family TetraodontidaeHabitat: Estuaries, freshwater streams around South AsiaPredators: Birds and other fish (?)

Figure 1 (A)

Diodon holocanthusPorcupine puffer or balloonfish

Family DiodontidaeHabitat: Marine, inshore and reef areas in the tropicsPredators: Sharks and large bony fish

Figure 1 (B)

10/26/2011

4

Methods: Behavior

Trials of tactile and acoustic stimuliTactile: touched body of fish with a plastic rod

Responses not analyzed quantitativelyAcoustic: hit a rubber mallet on the side of the platform holding the tank (several parameters)

Methods: Stimuli Setup

Aquarium 45º

High Speed Camera

Mirror

Acoustic: Rubber mallet at constant height

PlatformTactile: Plastic rod

Methods: Behavior - Acoustic

Latency: time between impact of mallet to axial movement of the head or tail

Probability: proportion of total trials evoking fast-start# fast start / total trials

Duration (of stage 1): stage 1 ended when fish began to straighten tail

Stage 1 Stage 3Stage 2Stimulus

Methods: Behavior - Acoustic

Angle: line extended along midline of anterior portion of fish; measured change in angle of this line

Time

Peak angular velocity: change in angle and timeDistance moved: minimum straight-line distance that the fish moved using a point on the midline in between the pectoral fins

*Response Start

End

Startle response + swim

Methods: Neuroanatomy

Retrograde tracingSilver stain and plastic sections

Methods: NeuroanatomyRetrograde Tracing

Tracing neural connections from the point of termination (synapse) to the source (cell body) using retrograde 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

10/26/2011

5

Methods: NeuroanatomyHow retrograde tracing works

Spinal cords were cut Biotin dextran amine (BDA) was applied to the cut to identify all cells that project from the brain into the spinal cordA ons take p biotin de tran amine (BDA) and transportAxons take up biotin dextran amine (BDA) and transport it back to the cell bodyBrains were removed, frozen, and were sectioned

Discussion with Prof. Hopkins and http://www.vectorlabs.com/catalog.aspx?dpID=11&locID=14193

Methods: NeuroanatomyHow retrograde tracing works

To visualize BDA, incubated sections in avidin-horseradish peroxidase (avidin-HRP)Avidin binds to biotin—this locates the cells that took up BDAThe HRP bo nd to a idin catal es the breakdo n ofThe HRP bound to avidin catalyzes the breakdown of H2O2 to H2O and O2

Add DAB, which is oxidized (by HRP) and turns blackSo 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

Methods: NeuroanatomySilver stain and plastic sections

Silver stainingBrains removed and prepared for stainingMorse’s modification of Bodian’s silver technique

Embedding in plastic for thin sectioningSections were mounted on slides and stained with Toluidine BlueSections were mounted on slides and stained with Toluidine Blue

Results: Fast-start behavior detected in both Species

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 D. holocanthus

Green-spotted puffer

Porcupinefish

Table 1

Results: Neuroanatomy

PosteriorAnterior

DorsalTelencephalon Optic tectum

Cerebellum

Hindbrain

SagittalHorizontal

Transverse

Figure 2 (A)

VentralDiencephalon

10/26/2011

6

Results: NeuroanatomyRetrograde tracing shows distinct M-Cell in T. nigroviridis

Hindbrain segments

AnteriorMidline

M-cell

Figure 2 (B):Horizontal section, T. nigroviridis

100 µmPosterior

Results: NeuroanatomyRetrograde tracing shows distinct M-Cell in T. nigroviridis

Optic tectumDorsal

Figure 2 (C) and (D):Transverse section, T. nigroviridis

1 mm

Torus semicircularis

Ventral M-cell100 µm

Results: NeuroanatomyRetrograde tracing does not show distinct M-Cell in D. holocanthus

Dorsal Optic tectum

Figure 2 (E) and (F):Transverse section, D. holocanthus

100 µm1 mmVentral

Torus semicircularis

No M-cell in this or neighboring sections!

Results: NeuroanatomyPlastic sections show distinct M-cells in T. nigroviridis

Midline

Dorsal DorsalMidline

Lateral dendrite

Figure 3 (A) and (B):Transverse section, T. nigroviridis

50 µm 50 µmVentral

M-cell

Ventral

Axon hillock and initial segment

Ventral dendrite

Results: NeuroanatomyPlastic section show distinct crossing of M-cells

MidlineDorsal

Figure 3 (C):Transverse section, T. nigroviridis

M-cells traced caudally where they cross

50 µmVentral

Results: NeuroanatomyPlastic sections show distinct M-cells in only T. nigroviridis

Dorsal

Figure 4 (A) and (B):Transverse sections

T. nigroviridis:M-axons easily identified Ventral D. holocanthus:

Can’t see any M-axons!

50 µm 50 µm

10/26/2011

7

Summary of Results

D. holocanthus rarely exhibited fast-start behavior and lacked obvious M-cells candidatesThere is a correlation between M-cell presence or absence and fast-start behavior in related species

Presence correlated with normal fast-starts (T. nigroviridis)ese ce co e a ed o a as s a s ( g o d s)Absence correlated with abnormal fast-starts (D. holocanthus)

Why does D. holocanthus not have normal M-cells and fast-starts?

“Being eaten alive abruptly ends all chances of future reproduction and is not favourable from an evolutionary view point.” –Prof. FetchoUnique anti-predator adaptationsMaybe these alternative mechanisms allowed for less

l ti t i t i M ll d f t t tselective pressure to maintain M-cells and fast-startsPerhaps D. holocanthus uses these alternative mechanisms more than T. nigroviridis, so it has experienced even less selective pressure to maintain M-cells and fast-starts

http://www.flmnh.ufl.edu/fish/gallery/Descript/Balloon/Balloon.htm

Weaknesses

The acoustic stimulus: is this really just an auditory stimulus? Maybe water movements occur?M-cells weren’t observed in D. holocanthus, but this isn’t definitive evidence of lack of M-cellsWe don’t know enough about the natural behavior andWe don t know enough about the natural behavior and predators of these species to make a strong conclusion about why D. holocanthus lacks normal M-cells and fast-startsWhat about homologous neurons in the fifth and sixth hindbrain segments?

Questions for Future Studies

M-cells could not be seen—maybe they are just atypical? Use molecular markers to confirm the lack of M-cells in D. holocanthusDo pufferfishes possess M-cell homologues found in goldfish and zebrafish? If so do these cells also showgoldfish and zebrafish? If so, do these cells also show anatomical differences between pufferfish species?These species have morphological differences (musculature, body shape, body stiffness)—how do these factors contribute to fast-start responses?What is the relative use of fast-starts and inflation in pufferfish species in the wild? (Both behaviors cannot be performed at same time.)

The End