Apresentação no II Simpósio "O Zebrafish Como Modelo Animal de Pesquisa"

2. Neurochemistry of defensive
behavior in zebrafish
Prof. Dr. Caio Maximino
Instituto de Estudos em Saúde e Biológicas
Universidade Federal do Sul e Sudeste do Pará

3. Adenosine and defensive
behavior in zebrafish
●
Racional: Efeito ansiogênico da cafeína
é consistente em diferentes modelos e
espécies
●
Cafeína também é psicoestimulante
●
Descrever alguns dos mecanismos
pelos quais a cafeína produz efeitos
sobre ansiedade, atividade, e
excitabilidade

4. Caffeine increases activity and melanophore
responses at intermediate doses, and scototaxis
and melanophore responses at high doses
Maximino et al., 2011

5. Participation of the A1
receptor, but
not A2
-like receptors, in scototaxis
Stewart et al., 2011

6. Participation of the A1
receptor, but
not A2
-like receptors, in scototaxis
Maximino et al., 2011
PACPX
DMPX

7. From Guimarães et al.,
2010

5-HT decreases defensive
withdrawal (escape / fear / panic) in
dlPAG, DMH, and Amy

5-HT increases defensive approach
(avoidance / anxiety) in dlPAG, Amy,
and SHS
Serotonin and anxiety

8. The serotonergic system of zebrafish
●
Non-conserved
neuroanatomy
From Lillesaar, 2011

9. Cluster 1 Clusters 2-4 Clusters 5-7 Cluster 8
+ + + -
+ + + -
+ + + -
+ + + +
+ + + -
- + + -
+ + + -
+ + + -
+ + + +
+ + + +
? ? ? ?
- + + +
- - + +
- - + +

10. Extra nuclei, extra genes
Cluster 1
(Pretectum)
Clusters 2-4
(Hypothalamus)
Clusters 5-7
(Raphe)
Cluster 8
(RF)
Cluster 9
(AP)
SC
Transcr.
factors
etv5b, fezf2 pet1 pet1 pet1
TPH isoform tph2 tph1a,tph1b, tph3 tph2 tph2 - tph1a
SERT isoform serta sertb serta n.d. sertb n.d.
Receptors htr1aa, htr2c htr1ab, htr2c htr1aa, htr1ab,
htr1bd
n.d.

11. Drug effects
Scototaxis Geotaxis AS effects on
scototaxis
5-HT levels
SSRI ↑ ↓ ↓ ↑
MAOIs 0 ↓ NT ↑
5-HTP ↑ NT NT ↑
pCPA ↓ ↑ NT ↓
Reserpine NT ↑ NT NT
Maximino et al., 2011; Stewart et
al., 2011; Iturriaga-Vásquez et al.,
2012; Maximino al., 2013; Kyzar et
al., 2013; Maximino et al., 2014;
Maximino and Herculano, In press

12. Brain 5-HT levels
Maximino al., 2013

13. 5-HT turnover and anxiety-
like behavior
Maximino et al., 2013
Maximino et al., 2014

14. Possible conclusions
●
In zf, 5-HT increases anxiety-like behavior and
decreases fear-like behavior AND/OR...
●
… in zf, 5-HT increases anxiety-like behavior and
induces serotonin toxicity
●
Whatever interpretation, the role of 5-HT in these
functions appears conserved

15. Which receptors are involved in
these responses?
Scototaxis Geotaxis
Buspirone ↓ ↓
WAY 100,635 ↓ ↓
m-PPF NT 0
SB 224,289 0 ↓
GR 55562 NT ↓
Ketanserin NT ↑
Ondansetron NT 0
1A
1B
2
3
Maximino et al., 2013; Nowicki et al., 2014

16. Glutamate and NO in scototaxis
Barbosa Jr. et al., 2012; Maximino et al.,
2015a

19. NO mediates the role of
5-HT1A
R and 5-HT1B
R
Maximino et al., 2015a; Maximino et al., 2015b

20. Proposed mechanism

21. The plot thickens...

26. Role of SERT in
AS-induced anxiety
Maximino et al., 2014

27. For the future
● In vitro studies
● Role of PKC in AS responses
● Participation of other 5-HT receptors
● Other downstream effectors
● Where in the brain? Microinjection, neuroanatomy, etc

28. More recently: PTSD?
– Neurobehavioral and endocrine respone to extreme stress
– Symptoms which appear after the exposure of the individual to
one or more aversive, traumatic events:
● Intrusive memories (“flashbacks”)
● Persistent avoidance
● Negative alterations in cognition and mood
● Arousal

29. A putative behavioral model of
PTSD in zebrafish (Lima)
Donor animal
Cold
anesthesia
Sacrifice
Club cell
damage
Washing cuts with
10ml ddH2
0
7ml “AS” solution
10'
Acclimation
n = 6
0cm 10cm
24hEXPOSURE
6'

30. Behavioral effects
● 24 h later:
– Increased geotaxis
– Increased scototaxis
– Increased startle
● Phenotype differences: Qualitatively and
quantitatively differences in effects on
scototaxis in lof and bsf

31. Behavioral Cutoff
Criteria (Face validity)
● ~25% EBR, ~20% MBR
● EBR: More scototaxis and
thigmotaxis, more erratic
swimming
● MBR: More thigmotaxis
and risk assessment in
relation to controls, but
not EBR

32. Neurochemistry of the alarm
response

33. Participation of NOS in
sensitization responses
● L-NAME 30' before stress: No effect
● L-NAME 30' after stress: Blocks sensitization of scototaxis
and risk asssessment
● L-NAME 90' after stress: Does not block sensitization of
scototaxis, but blocks sensitization of erratic swimming and
thigmotaxis
● AG 30' after stress: Blocks sensitization of scototaxis, risk
assessment and thigmotaxis

34. Theoretical model

35. For the future
● Electrophysiology
● Response localization (NADPHd, c-Fos, other
markers)
● Endocrine responses (mainly cortisol)
● Which isoforms? What downstream targets? What
upstream stimuli?