10/26/2011 The Journal of Experimental Biology Distinct startle - - PDF document

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” Biologists

Launched as The British

Journal of Experimental Biology in 1923

Impact factor of 3.040 for

2010

February 2010 Issue

The Authors

Anna K. Greenwood Post-Doc in the Peichel Lab at the Fred

Hutchinson Cancer Research Center

B.S. Psychology (1996) Rutgers University Ph.D. Neuroscience (2004)

( ) Stanford University

Studies 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. Peichel Associate Member, FHCRC Division of

Human Biology

B.A. Molecular & Cell Biology (1991)

University of California, Berkeley y y

Ph.D. Molecular Biology (1998)

Princeton University

Studies the “genetic and neural

mechanisms that underlie the evolution

• f behaviors” in sticklebacks

The Authors

Steven J. Zottoli Professor of Biology at Williams College

since 1980

A.B. (1969) Bowdoin College Ph D (1976) University of Ph.D. (1976) University of

Massachusetts, Amherst

Studies focus on goldfish with spinal cord

injuries to determine the neuronal basis

• f startle response recovery

Overview

Escape behavior – Mauthner Cells M-Cell diversity and pufferfish Behavioral and neuroanatomical methods and

results results

Key points and areas for further study Questions?

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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.

Super whole- genome duplication

C-start Behavioral Response

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

C-shape C shape

Stage 2: tail stroke for forward propulsion Stage 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 3 Stage 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

input

The 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-start Electrical stimulation of M-cells can elicit response HOWEVER, M-cells are not necessary; a delayed

response is elicited after ablation. Why?

M-cell spike C-start response Stimuli

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 h

M-cell

response in goldfish and zebrafish

These reticulospinal

neurons have cell bodies and axons that are smaller than those

• f 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

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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 structure

Life history is related to M-cell variation Life 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 stimulus

Some pufferfish do not have M-cells, while others have

M-cells that are small Relationship??

Relationship??

Purpose of Study

Purpose: To identify a correlation between M-cell

anatomy and C-start response in two pufferfish species in sister families

No hypothesis was given, why?

Discovery Science

Description of nature through observation and analysis Examples Cajal’s observations of neurons using Golgi’s method Jane 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 nigroviridis Green spotted puffer

Family Tetraodontidae Habitat: Estuaries, freshwater streams around South

Asia

Predators: Birds and other fish (?)

Figure 1 (A)

Diodon holocanthus Porcupine puffer or balloonfish

Family Diodontidae Habitat: Marine, inshore and reef areas in the tropics Predators: Sharks and large bony fish

Figure 1 (B)

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Methods: Behavior

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

Responses not analyzed quantitatively

Acoustic: 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 Platform Tactile: 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 3 Stage 2 Stimulus

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 time Distance 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 tracing Silver stain and plastic sections

Methods: Neuroanatomy Retrograde 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

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Methods: Neuroanatomy How 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 cord A ons take p biotin de tran amine (BDA) and transport

Axons take up biotin dextran amine (BDA) and transport

it back to the cell body

Brains were removed, frozen, and were sectioned Discussion with Prof. Hopkins and http://www.vectorlabs.com/catalog.aspx?dpID=11&locID=14193

Methods: Neuroanatomy How 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

BDA The HRP bo nd to a idin catal es the breakdo n of

The HRP bound to avidin catalyzes the breakdown of

H2O2 to H2O and O2

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

Methods: Neuroanatomy Silver stain and plastic sections

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

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

Posterior Anterior Dorsal

Telencephalon Optic tectum Cerebellum Hindbrain

Sagittal Horizontal Transverse Figure 2 (A) Ventral

Diencephalon

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Results: Neuroanatomy Retrograde tracing shows distinct M-Cell in

• T. nigroviridis

Hindbrain segments Anterior Midline M-cell Figure 2 (B): Horizontal section, T. nigroviridis

100 µm

Posterior

Results: Neuroanatomy Retrograde tracing shows distinct M-Cell in

• T. nigroviridis

Optic tectum

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

1 mm Torus semicircularis

Ventral M-cell

100 µm

Results: Neuroanatomy Retrograde tracing does not show distinct M-Cell in

• D. holocanthus

Dorsal

Optic tectum

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

100 µm 1 mm

Ventral

Torus semicircularis

No M-cell in this or neighboring sections!

Results: Neuroanatomy Plastic sections show distinct M-cells in

• T. nigroviridis

Midline Dorsal Dorsal Midline

Lateral dendrite

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

50 µm 50 µm

Ventral M-cell Ventral

Axon hillock and initial segment Ventral dendrite

Results: Neuroanatomy Plastic section show distinct crossing of M-cells

Midline Dorsal Figure 3 (C): Transverse section, T. nigroviridis M-cells traced caudally where they cross

50 µm

Ventral

Results: Neuroanatomy Plastic 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

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Summary of Results

• D. holocanthus rarely exhibited fast-start behavior and lacked
• bvious M-cells candidates

There 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

• 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. Fetcho

Unique anti-predator adaptations Maybe these alternative mechanisms allowed for less

l ti t i t i M ll d f t t t selective pressure to maintain M-cells and fast-starts

Perhaps 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-cells

We don’t know enough about the natural behavior and We 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-starts

What 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. holocanthus

Do pufferfishes possess M-cell homologues found in

goldfish and zebrafish? If so do these cells also show goldfish 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