1) The study examined the transgenerational effects of dopamine regulation and exploratory behaviour in guppies. 2) Male guppies exposed to stimulants showed increased exploratory behaviour, while females did not. 3) Offspring of exposed males (G2) showed decreased freezing and increased brain dopamine levels, indicating paternal effects on behaviour and physiology. No effects were seen maternally.

1. The trans‐generational link between
dopamine regulation and exploratory
behaviour in the Trinidadian guppy
Alex De Serrano, Helen Rodd
University of Toronto

2. Gene
Env
Gene x Env
2
Phenotype
Haemorhous mexicanus, Geoffrey E. Hill and David Bay; Daphnia lumholtzi, C.
Laforsch; Pheidole aberrans, Alex Wild

3. 3
PhenotypeEpigenetic
Mods
Haemorhous mexicanus, Geoffrey E. Hill and David Bay; Daphnia lumholtzi, C.
Laforsch; Pheidole aberrans, Alex Wild
G, E,
G x E

4. Alan Horsager, www.episona.com
= molecular processes
that regulate the genome
without alteration of
original DNA, and persist
through mitosis
Epigenetic modifications
4

5. 5
How long do epigenetic modifications persist?
G, E,
G x E PhenotypeEpigenetic
Mods
G, E,
G x E

6. 6
How long do epigenetic modifications persist?
G, E,
G x E
G, E,
G x E
When stimulus is no longer present?
Long term responses in:
• the individual
• offspring in utero
J
e.g. Weaver et al. 2004
PhenotypeEpigenetic
Mods
G, E,
G x E

7. 7
G1
G2
G3
Direct exposure includes germline
Adapted from Dunn et al. 2011

8. Image sources: http://www.bioseb.com
Via germ line exposure?
• Maternal (e.g. Burdge et al. 2011; Grissom et
al. 2014)
• Paternal (e.g. Rodgers et al. 2013; Vassoler et
al. 2012)
Stimuli include drugs, diet, stress
e.g. Paternal stress
How long do epigenetic modifications persist?
8

9. Image sources: http://www.bioseb.com
Via germ line exposure?
• However, studies have been
conducted in inbred lab rodents
• Studies of natural animal systems
and ecologically-relevant
behaviours are needed (e.g. Ledón-
Rettig et al. 2012)
e.g. Paternal stress
How long do epigenetic modifications persist?
9

10. Explore the potential epigenetic effects on an
ecologically-relevant behaviour across generations
10
My Goal:
PhenotypeEpigenetic
Mods
G, E,
G x E

11. • Willingness to explore novel
stimuli (Crusio 2001)
• Ecologically-relevant
behaviour
• Natural variation in degree
of exploration
• Some knowledge of
physiological pathway
Hopefully, the truth is out there
Exploratory behaviour
11

12. • High degree of exploration associated
with high levels of dopamine (Mallo et al.
2007)
• Stimulants increase exploration (acute
doses) (Carrey et al. 2000)
• Could persist via epigenetic
mechanisms(Csoka and Szyf 2009)
12
Physiological pathway: Dopamine Exploration

13. Explore the potential epigenetic effects of Dopamine
(DA) manipulation (via stimulants) on DA level and
exploratory behaviour
13
My Specific Goal:
G, E,
G x E PhenotypeEpigenetic
Mods
G, E,
G x E

14. Trinidadian Guppies
• Variation in exploration (Burns 2007)
• Tractable for large transgenerational experiments
• Live-bearers
• Dopamine pathway conserved throughout vertebrates
Lucy Hollis Z-Y. Huang David Sumpter
14

15. 1. Is exploratory behaviour in guppies affected by long-term
stimulant exposure via dopamine (G1)?
2. Does treatment of the parental generation affect offspring
behaviour (G2)?
• Maternal (in utero) vs. Paternal (germline) effects
Z-Y. Huang
Questions
15
Dopamine

16. Chronic Exposure
to stimulant (Ritalin)
3 months
1 month
Exploration assay
16
Control
G1 Overview

17. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
17
♂
Maternal line Paternal line

18. • Where
• Center vs. perimeter
• How
• Freezing
Exploratory trials: open field test
18 33 cm x 28 cm x 12 cm

19. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
19
♂
Maternal line Paternal line
1. Is exploratory behaviour in guppies affected by
long-term stimulant exposure via dopamine (G1)?

20. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
20
♂
Maternal line Paternal line
1. Is exploratory behaviour in guppies affected by
long-term stimulant exposure via dopamine (G1)?
Yes, in male guppies (no effect for females)

21. 2. Does treatment of the parental generation affect
offspring (G2) behaviour?
• Maternal (in utero) vs. Paternal (germline) effects
Lucy Hollis Z-Y. Huang David Sumpter
21
Dopamine
Questions

22. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
22
♂
Maternal line Paternal line

23. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
23
♂
Maternal line Paternal line
Multivariate: all behaviours
Univariate: Freezing

24. Z-Y. Huang
Control Treated
0
20
40
60
80
100
120
140
160
1
0
20
40
60
80
100
120
140
160
180
1
Control Treated
Rankedmeanresponse+/-SE
Daughters
Sons
No effect of Maternal treatment on
duration frozen
multivariate maternal effects: all n.s.
24
P = 0.64, n = 111 P = 0.49, n = 164

25. Z-Y. Huang
multivariate paternal effects: P = 0.058
Control Treated
0
20
40
60
80
100
120
140
160
180
1
*
Rankedmeanresponse+/-SE Paternal exposure reduced
duration frozen in (G2) daughters
25
Daughters
P = 0.02
n = 164

26. Z-Y. Huang
Control Treated
0
20
40
60
80
100
120
140
160
180
1
0
20
40
60
80
100
120
140
160
1
Control Treated
*
Rankedmeanresponse+/-SE
Daughters
Sons
P = 0.08
n = 111
Lsn = 138
Paternal treatment has marginal
effect on duration frozen in (G2) sons
multivariate paternal effects: P = 0.058
26
P = 0.02
n = 164

27. Z-Y. Huang
Control Treated
0
20
40
60
80
100
120
140
160
180
1
0
20
40
60
80
100
120
140
160
1
Control Treated
*
Rankedmeanresponse+/-SE
multivariate paternal effects: P = 0.058
27
Paternal treatment decreased
duration frozen in (G2) daughters
Daughters
P = 0.02
n = 164
Sons
P = 0.08
n = 111
Lsn = 138

28. Does parental treatment affect Brain
[Dopamine]?
• Subset of daughters (n = 40)
• Whole brain removed after open
field test
• [Dopamine] analyzed with ELISA
Guppy brain: rostral (top) to caudaul
28

29. Z-Y. Huang
Dopamine (squared) concentraLon (ng/mL)
0
1
2
3
4
5
6
1
Control Treated
Paternal
P = 0.037
*
29
Paternal treatment increased
[Dopamine] of daughters

30. Z-Y. Huang
Dopamine (squared) concentraLon (ng/mL)
0
1
2
3
4
5
6
1
Control Treated
Paternal
P = 0.037
*
30
0
1
2
3
4
5
6
1
Control Treated
Maternal
P = 0.34
No effect of Maternal treatment
on [Dopamine] of daughters

31. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
31
♂
Maternal line Paternal line

32. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
32
♂
Maternal line Paternal line
2. Does treatment of the parental generation affect
offspring (G2) behaviour?
• Maternal (in utero) vs. Paternal (germline) effects

33. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
33
♂
Maternal line Paternal line
Yes, Paternal treatment has an effect on behaviour and
[Dopamine] of daughters

34. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
34
♂
Maternal line Paternal line
Yes, Paternal treatment has an effect on behaviour and
[Dopamine] of daughters
• Effect of male behaviour (following: F1,104 = 7.24, P < 0.02)
on mothers during the mating period

35. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
35
♂
Maternal line Paternal line
Yes, Paternal treatment has an effect on behaviour and
[Dopamine] of daughters
• Effects on sperm or ejaculate

36. • DNA methylation
• Small non-coding
RNAs
• Histone tail
modifications
Some epigenetic modifications
36 Alan Horsager, www.episona.com

37. G1 Stimulant administration Stimulant administration
G2 In utero Germline exposure
G3 Germline exposure No direct exposure
G4 No direct exposure No direct exposure
♀
37
♂
Maternal line Paternal line
?
?

38. 38
PhenotypeEpigenetic
Mods
Haemorhous mexicanus, Geoffrey E. Hill and David Bay; Daphnia lumholtzi, C.
Laforsch; Pheidole aberrans, Alex Wild
G, E,
G x E

39. 39
PhenotypeEpigenetic
Mods
Haemorhous mexicanus, Geoffrey E. Hill and David Bay; Daphnia lumholtzi, C.
Laforsch; Pheidole aberrans, Alex Wild
G, E,
G x E

40. Acknowledgements
Z-Y. Huang
40