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Development of DA and 5HT neurons involved in reward-
associated behavior in zebrafish
Su Guo, Ph.D.University of california, San Francisco
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Red: 5HT
Green: TH
3-day old zebrafish labeled with anti-TH and 5HT antibodies
Basal forebrainDA and 5HT
neurons
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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
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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
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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
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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.
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Choice chamber
drug water
draining
Pre-expose larvalZebrafish to the drug
Test their preference forthe drug in the choice chamber
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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)
*
*
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Morphine preference is dependent on opioid receptors
0
5
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20
25
30
0 0.8 0.8
*
NaloxoneMorphine
0 0 0.5
Drug concentration (M)
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The too few mutant
TH5HT
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The basal forebrain DA/5HT neurons are required for morphine preference behavior
-15
-10
-5
0
5
10
15
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25
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0 0.25 0.5 1
WTtfu *
*
a
Morphine pre-exposure concentration (mg/l)
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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
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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?
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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
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Mutations affecting the development of basal FB DA/5HT neurons
• too few
• motionless
• twin-of-motionless
• foggy
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The foggy mutant
48 hpf 48 hpf
WT foggy
DA 5HT
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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
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The importance of regulating transcription elongation during development
promoter
Transcription initiation
Transcriptionelongation
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foggy expression
28 hpf 48 hpf
DAPI FLAG-FOGGY
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The foggy mutation selectively disrupts its repressor activity
Repressor
Activator
Met Cys Ser Val Phe Leu Gln
Asp
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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.
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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.
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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)