development of da and 5ht neurons involved in reward- associated behavior in zebrafish su guo, ph.d....

Development of DA and 5HT neurons involved in reward-

associated behavior in zebrafish

Su Guo, Ph.D.University of california, San Francisco

Red: 5HT

Green: TH

3-day old zebrafish labeled with anti-TH and 5HT antibodies

Basal forebrainDA and 5HT

neurons

Questions

• Do zebrafish have reward-associated behavior? – Experience-dependent morphine preference

• Do the basal forebrain DA/5HT neurons play a role in this behavior?– The too few mutant

• How is the development of basal forebrain DA/5HT neurons regulated?– The foggy mutant

Zebrafish: a vertebrate genetic model organism

QuickTime™ and aAnimation decompressor

are needed to see this picture.

Small-size

High fecundity

Easy to maintainIn large numbers

Rapid development

Optically transparent

In vivo tracing of biological processes

What is reward-associated behavior?

• A prior exposure to a substance elicits a preference behavior for the substance:– Spend more time in the substance-paired side

(CPP)– Self-administer the substance

Studying reward-associated behavior in larval zebrafish

• Larval zebrafish are amenable to genetic analysis and the study of cellular mechanisms

• Larval zebrafish are free-living (food hunting, escaping)

• The nervous system of larval zebrafish is more plastic than adult zebrafish.

Choice chamber

drug water

draining

Pre-expose larvalZebrafish to the drug

Test their preference forthe drug in the choice chamber

Larval zebrafish show experience-dependent preference for morphine

0

5

10

15

20

25

30

0 0.4 0.8 1..5

morphine pre-exposure concentration (M)

*

*

Morphine preference is dependent on opioid receptors

0

5

10

15

20

25

30

0 0.8 0.8

*

NaloxoneMorphine

0 0 0.5

Drug concentration (M)

The too few mutant

TH5HT

The basal forebrain DA/5HT neurons are required for morphine preference behavior

-15

-10

-5

0

5

10

15

20

25

30

0 0.25 0.5 1

WTtfu *

*

a

Morphine pre-exposure concentration (mg/l)

Functional analogy of zf basal FB to mammalian MB DA neurons

Mammals: adapted from Kandel’s Principles of Neural Science

Zebrafish: adapted from Rink and Wullimann, 2002

tfu

How is the development of basal FB DA and 5HT neurons regulated?

• Will the mechanisms be homologous to mammalian FB or MB neurons?

• What are the genes and pathways involved in the development of these neurons?

Mutagenesis screen

F2eggs

XG0

X

ENU mutagen

F1

One generation (EP) screen

+/m1 +/m2 +/m1

F1

XF2

+/m1 +/m1

m1/m125%

F3m1/m1

+/+ m1/m1 +/m1UV sperm

EP

3-50%+/+ +/m1

Two generation screen

Screen for mutants by TH immunostaining

o

Mutations affecting the development of basal FB DA/5HT neurons

• too few

• motionless

• twin-of-motionless

• foggy

The foggy mutant

48 hpf 48 hpf

WT foggy

DA 5HT

foggy encodes a regulator of transcription elongation

acidic region

conserved repeats c-ter

3 105 168 313 354 649 753698 998 1084 aa.

32% 28% 40%

57% 54% 62%

51% 32% 36%

95% 93% 95%

95% 94% 96%

34%

52%

86%

88%

1068 aa.

1208 aa.

1054 aa.

1082 aa.

1087 aa.

FOGGY (Zebrafish)

SPT5 (s. cerevisiae)

SPT5 homolog (C. elegans)

SPT5 homolog (Arabidopsis)

SPT5 homolog (Mouse)

DSIF-P160 (human)

Met Cys Ser Val Phe Leu Gln

Asp

The importance of regulating transcription elongation during development

promoter

Transcription initiation

Transcriptionelongation

foggy expression

28 hpf 48 hpf

DAPI FLAG-FOGGY

The foggy mutation selectively disrupts its repressor activity

Repressor

Activator

Met Cys Ser Val Phe Leu Gln

Asp

Question

• What genes are de-repressed that leads to defects in DA/5HT neuron development?– Micro-array study

• How is foggy’s repressor activity regulated during development? – Focus on the function of the C-terminal

domain, where the foggy mutation resides.

Summary

• Zebrafish is an attractive genetic model organism to study:– the development of specific neuronal types.– The developmental alteration that leads to

abnormal reward-associated behavior.

Acknowledgements

Sandrine Bretaud

Brent Lockwood

Reward-associated behavior in larval zebrafish

Foggy regulationJae-yeon Jeong

Foggy downstream tergetsKeerthi Krishnan

Arnon Rosenthal (Rinat Neuroscience)Mark Fishman (Novartis)Wolfgang Driever (University of Freiburg)