First published online May 2, 2008
Journal of Experimental Biology 211, 1623-1634 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014399
Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways
Layla J. M. Kily1,
Yuka C. M. Cowe1,
Osman Hussain1,
Salma Patel1,
Suzanne McElwaine2,
Finbarr E. Cotter2 and
Caroline H. Brennan1,*
1 School of Biological and Chemical Sciences, Queen Mary, University of London,
Mile End, London E1 4NS, UK
2 Centre for Haematology, Institute of Cell and Molecular Science, Barts &
The London, Queen Mary's School of Medicine, 4 Newark Street, London E1 2AD,
UK
View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1. Conditioned place preference following a single 20 min treatment with
nicotine or ethanol. (A) Exposure to 3–300 µmol l–1
(0.5–50 mg l–1) nicotine induced a significant change
in preference compared with the control treatment (ANOVA,
*P<0.05). (B) 175 mmol l–1 (1% v/v)
ethanol induced a significant change in preference (ANOVA,
*P<0.05) compared with the control. Water-treated
control fish also showed a significant change in preference after treatment
compared with before treatment (paired t-test,
**P<0.05). Change in preference (s) is calculated as
time spent on treatment side after drug exposure minus `baseline' time spent
on treatment side before drug exposure. Place preference was determined over a
120 s period.
|
|
View larger version (7K):
[in this window]
[in a new window]
|
Fig. 2. Conditioned place preference (CPP) following a single exposure or three
consecutive exposures to nicotine. Fish showed a concentration-dependent
change in preference for the treatment side following both a single exposure
(grey bars) and three repeat exposures to nicotine on each of three
consecutive days (black bars). The CPP response to 6 µmol
l–1 nicotine after a single exposure was not determined.
Following exposure to 0, 3, 6, 30 and 150 µmol l–1
nicotine for 20 min on each of three separate days fish showed a significant
increase in place preference for the treatment side compared with before
treatment (**P<0.05). Fish subject to three treatments
with 6 or 30 µmol l–1 nicotine showed a significantly
greater change in place preference for the treatment side than control,
water-treated fish (*P<0.05). Three exposures to 300
µmol l–1 nicotine induced a significant decrease in place
preference compared with water-treated controls
(*P<0.05).
|
|
View larger version (9K):
[in this window]
[in a new window]
|
Fig. 3. Conditioned place preference persists over a 3-week period of abstinence
from nicotine or ethanol. (A) Following 4 weeks of daily 20 min exposure to 30
µmol l–1 nicotine (black bars) fish showed a significantly
greater change in place preference for the treatment side compared with
control water-treated fish (grey bars; paired t-test
*P<0.05). The change in preference exhibited by
nicotine-treated fish was significantly greater than the change in preference
exhibited by control, water-treated fish 24 h, 7 or 21 days after last drug
exposure (two-sample t-test, P<0.05): control,
water-treated fish showed no significant change in preference. The place
preference for the treatment side after 21 days of abstinence was
significantly less than the preference after 24 h of abstinence (two-sample
t-test, **P<0.05). (B) 4 weeks of daily 20 min
exposure to 175 mmol l–1 ethanol (black bars) induced a
significant change in preference compared with control, water treatment (grey
bars; *P<0.05, two-sample t-test). This
preference persisted over 3 weeks of abstinence.
|
|
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4. Conditioned place preference despite an adverse stimulus. Fish were
punished by 3 s removal from the tank each time they entered the
treatment-paired side: (A) punished versus unpunished/restricted
control fish; (B,D) nicotine-treated and paired control fish; (C,E)
ethanol-treated and paired control fish. (A) Fish that were punished by
removal from the tank for 3 s made significantly fewer returns to the
treatment side compared with unpunished/restricted fish (two-sample
t-test *P<0.01). (B,C), Number of returns made
to the drug-paired side in the face of restriction or punishment. Data were
subject to two-way repeat-measures ANOVA analysis followed by
post-hoc, paired or two-sample, t-test, as appropriate,
followed by Bonferroni adjustment. Following Bonferroni adjustment comparisons
were significant at the P<0.01 level. (B) Fish that had been
conditioned for 4 weeks with 30 µmol l–1 nicotine made
more returns to the drug-paired side than control fish when either restricted
(two-sample t-test, P=0.03) or punished (two-sample
t-test, *P<0.01). 3 s removal from the tank
caused a significant reduction in returns made by control fish (paired
t-test, restricted compared with punished,
**P<0.01) but not nicotine-treated fish.
Repeat-measures two-way ANOVA analysis showed there to be a significant
interaction between drug treatment and punishment (punishment plus drug
interaction F1,34=8.74, P=0.006). (C) 3 s removal
from the tank caused a significant reduction (paired t-test,
restricted compared with punished, *P<0.01) in number
of returns made by both control fish and fish that had been conditioned for 4
weeks with 175 mmol l–1 ethanol. Fish that had been
conditioned for 4 weeks with 175 mmol l–1 ethanol made
significantly more returns to the drug-paired side when punished (two-sample
t-test **P<0.01) but not restricted. Repeat
measures two way ANOVA analysis showed there to be a significant interaction
between drug treatment and punishment (punishment plus drug interaction
F1,34=7.24, P=0.011). (D,E) Significantly
increased drug seeking despite punishment persisted over 21 days of abstinence
(two-sample t-test, *P<0.05 drug-treated
compared with control).
|
|
View larger version (60K):
[in this window]
[in a new window]
|
Fig. 5. Microarray analysis. (A) Cluster analysis of genes identified as
differentially expressed in brains from control, nicotine- and ethanol-treated
fish. All Zebrafish data were imported into GeneSpring 6.1, analytical
software for microarray analysis. There was a significant 1.5-fold or greater
change in expression of 545 genes between control and treated animals. Using
these 545 genes an experiment tree was generated using a Spearman correlation.
Subsequently, a gene tree was produced using a Pearson correlation. The
resulting tree is shown. Data are coloured based on how far the gene is over-
or underexpressed (relative to a normalized expression level of 1), in terms
of the standard error of the measurement. The colour bar ranges from +3
to –3. The standard error is based on the standard deviation of
the replicate data for a particular gene and condition. Note that the samples
cluster according to their experimental treatment either control, ethanol or
nicotine treated. (B) Quantitative real-time PCR (Q-RT-PCR) was used to
validate the microarray data. Individual genes with different cellular roles
(see Table 3) were selected for
validation. The four genes selected showed similar expression changes when
assessed by Q-RT-PCR as determined by microarray analysis. EtOH, ethanol; Nic,
nicotine; Cal B, calcineurin B; AMMECR1, Alport syndrome, mental retardation,
midface hyperplasia and elliptocytosis chromosome region; GRIA2a, AMPA
glutamate receptor 2a; pBDZR, peripheral benzodiazepine receptor.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2008
