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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
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Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways
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
* Author for correspondence (e-mail: C.H.Brennan{at}qmul.ac.uk)
Accepted 11 March 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: nicotine, alcohol, conditioned place preference, drug dependency, zebrafish, gene expression
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Wise, 1996;
Wise and Bozarth, 1984).
Nicotine leads to elevated dopamine in the nucleus accumbens via
direct activation of nicotinic acetylcholine receptors present on the neurons
of the mesolimbic dopaminergic pathway
(Mansvelder and McGehee,
2002). Ethanol exposure has a broader range of effects that
include altering activity of glutamatergic, opioid and gamma amino butyric
acid (GABA)ergic neurons that interact with the mesolimbic system and
ultimately also results in increased levels of dopamine in the nucleus
accumbens (Tupala and Tiihonen,
2004).
How dopaminergic transmission and reinforcement is related to addiction is
not fully understood. However, from a cellular and molecular perspective it is
likely that repeated exposure to addictive drugs causes stable changes in gene
expression, posttranslational modification and/or synaptic plasticity that
have lasting effects on brain function and thus behaviour. In this context a
number of studies have identified lasting neuro-adaptations that are
associated with such addiction-related behaviours as compulsive drug taking
and persistent tendency to relapse
(Kalivas, 2004;
Shaham and Hope, 2005;
Weiss et al., 2001). These
neuro-adaptations include altered basal levels or sensitivity of dopaminergic,
serotonergic and glutamate neurotransmission
(Kalivas et al., 2003;
Tupala and Tiihonen, 2004;
Weiss et al., 2001) in
addition to dysregulation of neuro-endocrine systems
(Lovallo, 2006;
Weiss et al., 2001).
Similarly, expression analysis has identified components of a number of
neurotransmitter (glutamatergic, cannabinoid, monaminergic) and signal
transduction pathways [ERK (extracellularly regulated kinase), PI3K
(phosphatidylinositol 3-kinase) and NFkappaβ (nuclear factor kappa beta)]
that are altered in their levels or domains of expression in the brains of
animals demonstrating drug dependency (Lu
et al., 2006; Pollock,
2002; Rhodes and Crabbe,
2005; Yuferov et al.,
2005). Changes in the gene expression of many of these compounds
were identified using a hypothesis-driven or candidate-gene approach, based on
results of pharmacological analysis (Koob
et al., 2004; Nestler,
2004). However, more recently, microarray analysis has enabled the
simultaneous interrogation of expression levels of thousands of genes in
different brain regions of control and drug-treated animals. This approach has
identified further candidate molecules and pathways that may be the basis of
the neuro-adaptation that underlies drug addiction
(Lehrmann et al., 2006;
Yuferov et al., 2005).
The primitive nature of reward reinforcement pathways and the near
universal ability of drugs of abuse to target the same system allow
drug-associated reinforcement to be modelled in non-mammalian species. Indeed,
reinforcement pathways are strongly activated by drugs of abuse in several
model systems including rodents, fish, insects and nematodes
(Bretaud et al., 2007;
Darland and Dowling, 2001;
Mohn et al., 2004;
Ninkovic and Bally-Cuif, 2006;
Ninkovic et al., 2006;
Wolf and Heberlein, 2003).
Conditioned place preference (CPP), where drug exposure is paired with
specific environmental cues, is commonly used as a measure of drug reward or
reinforcement (Tzschentke,
1998). Persistent CPP that lasts following a period of abstinence
or in the face of an adverse stimulus is a model for dependency. Recently, by
virtue of its inherent suitability for forward genetic screens, the zebrafish
has become established as a valuable animal disease model
(Anderson and Ingham, 2003;
Berghmans et al., 2005;
Shin and Fishman, 2002). With
respect to studies of drug-induced reinforcement and addiction, anatomical
analyses have demonstrated that neurons expressing tyrosine hydroxylase (the
rate limiting enzyme in catecholamine synthesis) project from the posterior
tuberal nucleus to the basal forebrain in a manner reminiscent of the ventral
tegmental–nucleus accumbens connection of the mesolimbic system in
mammals (Rink and Wullimann,
2002). Zebrafish show CPP responses to cocaine
(Darland and Dowling, 2001),
amphetamine (Ninkovic and Bally-Cuif,
2006) and opiates (Bretaud et
al., 2007) and the amphetamine-induced response is modified by
pathways known to influence dopamine release in the nucleus accumbens in other
systems (Ninkovic et al.,
2006). These results demonstrate the existence of a conserved
drug-responsive `reward' or reinforcement pathway in zebrafish and suggest
that zebrafish may show adaptive changes and behavioural correlates of
addiction after prolonged exposure to addictive drugs. We use CPP and
microarray analysis to test this hypothesis with regard to nicotine and
ethanol exposure.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
They were kept on a constant 14 h:10 h light:dark cycle at 28°C. The
animals used in these experiments were 0.5–1 g, 4-month-old, sex and age
matched Tuebingen wild-type stock, bred in house.
Behavioural assays
Fish were subject to treatment regimes as detailed in
Table 1.
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Assessing the reinforcing properties of ethanol or nicotine using conditioned place preference
Experiment 1: conditioned place preference assay following a single drug exposure
A balanced conditioning paradigm modified from Darland and Dowling
(Darland and Dowling, 2001)
was used to assess the reinforcing properties of ethanol or nicotine in
zebrafish. The testing apparatus was a 2 l rectangular tank (Aquatic Habitats,
Apopka, FL, USA) that could be divided in half with a Perspex divider. Each
end of the tank had distinct visual cues (1.5 cm diameter black spots
uniformly distributed on all sides versus vertical 0.5 cm wide black
and white stripes). After an initial 5 min settling period each fish was
tested for baseline preference by determining the time spent on a given side
of the tank over a 2 min period. Each fish was then restricted first to the
preferred side for 20 min using a Perspex divider so that the fish was
surrounded by either spots or stripes and then the fish was restricted to the
least preferred side and either nicotine, ethanol or fish-water added in a
volume of 10 ml so as to give the desired final drug concentration. Drug
concentrations used ranged from 0–300 µmol l–1 for
nicotine (0–50 mg l–1) and 0–264 mmol
l–1 [0–1.5% (v/v)] for ethanol. After 20 min the fish
were removed to fresh water in clean tanks and returned to the aquarium. To
determine the reinforcing effects of ethanol or nicotine, the place preference
of each fish was determined the following day by again, after a 5 min settling
period, determining the percentage time spent on each side of the tank over a
2 min test period. Any change in place preference was determined by
subtracting the baseline time spent on the drug-treatment side from the final
time spent on the drug-treatment side expressed in seconds. Fish that showed a
greater than 70% baseline preference for either side of the tank,
approximately 10% of fish tested, were not used further. Each drug
concentration was tested on 15–24 fish and two parallel groups of 20
control fish received fish-water only. All fish tracking was performed
manually with assessment of place preference performed by an observer blinded
to the treatment conditions.
Conditioned place preference following repeat exposure to nicotine or ethanol
Experiment 2: place preference following three consecutive conditioning sessions
Following determination of baseline preference, each fish was restricted
first to the preferred side for 20 min and then to its least preferred side
where it was exposed to either nicotine or ethanol for 20 min. Fish were
exposed to tank concentrations of nicotine ranging from 0–300 µmol
l–1 (0–50.0 mg l–1) for 20 min each
day for 3 days before determination of their place preference. Each drug
concentration was tested on 10–12 fish. As the results of these
experiments and others (Ninkovic and
Bally-Cuif, 2006) suggested that repeat exposure to the apparatus
leads to a slight change in the baseline preference that stabilizes over three
consecutive exposures, in all subsequent experiments fish were subject to
three conditioning sessions in the absence of any drug prior to the
determination of their baseline preference.
Experiment 3: place preference following 4 weeks of daily conditioning
Groups of 35 sex and age matched fish were subject to the conditioning
paradigm on the consecutive days in the absence of any drug to allow
familiarization to the apparatus and protocol. Baseline place preference for
each fish was then determined as described above. Any fish showing greater
than 70% baseline preference for either side of the tank was not used further;
5–10% of fish were excluded on this basis. Following determination of
baseline preference each fish was restricted first to the preferred side for
20 min and then to its least preferred side where it was exposed to either 30
µmol l–1 nicotine or 175 mmol l–1 ethanol
for 20 min. Conditioning sessions were repeated each day over a 4 week
period.
Conditioned place preference despite an adverse stimulus
Adverse stimulus test
Following determination of their basal preference, individual fish were
placed in the testing apparatus, allowed to settle for 5 min and then each
time the fish entered its preferred side it was punished by removal from the
tank to the air for 3 s. On return to the tank the fish was restricted to its
non-preferred side for 30 s to allow recovery. As a control, separate fish
were subject to the same procedure but without the 3 s punishment: they were
restricted to their least preferred side for 30 s each time they entered the
preferred side. After this time the divider was removed and the fish allowed
free access to the entire tank. In each case the number of returns to the
preferred side over a 10 min period was determined.
Experiment 4: place preference despite an adverse stimulus
Following 4 weeks of conditioning, the effect of punishment compared with
restriction on the number of returns made to the drug treatment side over a 10
min period was determined. Single fish were placed in the conditioning
apparatus, allowed a 5 min settling period and then each time the fish entered
the drug-treatment side it was restricted to the non-drug-treatment side for
30 s using a Perspex divider. After 30 s the divider was removed and the fish
allowed free access to the whole tank. The number of returns made over a 10
min period was determined. An hour later each fish was returned to the testing
apparatus, allowed 5 min to settle and then each time the fish entered the
drug treatment side it was removed from the tank to the air for 3 s. On return
to the tank, the fish was restricted to the non-drug-treatment side for 30 s
to allow recovery. After this time the divider was removed and the fish
allowed free access to the tank. Again the number of returns made over a 10
min period was determined. Tests were carried out on 18–20 fish for each
treatment group with two parallel control groups.
Conditioned place preference following a period of abstinence
Experiment 5: groups of 35 sex and age matched fish were used for each drug treatment with two parallel control groups
Following determination of their baseline preference, fish were exposed to
either 30 µmol l–1 nicotine or 175 mmol
l–1 ethanol for 20 min each day over a 4 week period. The day
after the last drug treatment each fish was tested for a change in place
preference by, following a 5 min settling period, determining the time spent
on each side of the tank over a 2 min test period. The change in place
preference was calculated as final time minus baseline time spent on the
drug-treatment side as previously. An hour later 10–12 fish from each
group were also tested for place preference in the face of an adverse stimulus
(see experiment 4) before being sacrificed. The remaining fish were then
returned to the aquarium for a period of 1 or 3 weeks where they experienced
no further drug treatment. At 1 or 3 weeks following the last drug treatment
the fish were again tested for their place preference and 10–12 fish
from each group also tested for place preference despite an adverse stimulus
before being sacrificed.
RNA extraction and microarray analysis
Brains from control fish or fish that had been conditioned to ethanol or
nicotine for 20 min each day over a 4-week period followed by 3 weeks of
withdrawal were homogenized using an Ultra Turrax T25 polytron homogenizer in
Trizol (Invitrogen, Carlsbad, CA, USA) and RNA extracted according to the
manufacturer's instructions. Total RNA (5 µg) from the zebrafish brain
tissue was used to synthesize double stranded cDNA according to the one-cycle
protocol from Affymetrix
(www.affymetrix.com/support/technical/manual/expression_manual.affx).
Eight cDNA synthesis reactions were performed, two for each drug treatment and
two for each parallel set of control animals. RNA from two brains was pooled
for each cDNA synthesis. An in vitro transcription was performed for
16 h at 37°C to generate biotinylated cRNA. Biotinylated cRNA (20 µg)
was fragmented at 94°C for 35 min and 15 µg of fragmented cRNA was
added to the hybridization cocktails. Zebrafish expression arrays were
hybridized for 16 h at 42°C and subsequently stained and scanned according
to the manufacturer's instructions. All microarray images were analysed by
Microarray Suite 5.0 (MAS 5, Affymetrix;
www.affymetrix.com).
Each microarray was initially multiplied by a scaling factor to make its mean
intensity equal to an arbitrary target intensity value (100 was used in our
experiment). The scaling factor for each array must be within threefold of
each other or they are not suitable for comparison. Following scaling,
microarray data were imported into GeneSpring 6.1 (Agilent Technologies,
Stockport, UK). Normalization of all imported data was performed in GeneSpring
according to the manufacturer's recommendations. Imported files were
normalized using the `per chip' (normalizes to a median or percentile) and
`per gene' (normalizes to median) function. GeneSpring first divides each raw
intensity value by the median of the chip. Then each value is further divided
by the median value of each gene across samples, resulting in the final
normalized value. The normalized data were then filtered to identify
differentially expressed genes between control and drug-treated zebrafish.
Data were initially `filtered on flags' eliminating genes called `absent' in
all samples. Subsequently genes called either present or marginal in 70% of
the arrays were used in statistical or fold-change comparisons. We used ANOVA
comparing control versus ethanol-treated and control versus
nicotine-treated animals to identify genes with statistically different levels
of expression in control and drug-treated groups. We also generated lists of
genes that were 1.5-fold increased or decreased in control versus
ethanol-treated, or control versus nicotine-treated animals. Venn
diagram analysis of the merged fold change and statistically significant lists
was then performed to identify genes showing at least a 1.5-fold significant
different change in expression in both ethanol- and nicotine-treated
animals.
Quantitative real-time PCR
Microarray results for each cDNA were validated for selected genes, chosen
from different groups when genes were sorted according to biological process,
using quantitative real-time PCR (Q-RT-PCR). Gria2
[
-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) ionotropic
glutamate receptor subunit GluR2] was chosen for validation as this gene has
been consistently reported to be upregulated in models of drug dependency.
Other genes were selected at random as we aimed to identify changes in the
expression of genes not previously associated with drug addiction. Primers
used for PCR were based on the array sequences and are given in
Table 2. Parallel 25 µl PCRs
were set up, each containing 1 µl (25 ng) cDNA and 300 ng each primer. PCR
was performed (50 cycles) at 55°C on a MX3000P QPCR system (Stratagene,
Cedar Creek, TX, USA) followed by a thermal dissociation step to allow
analysis of the product for purity. DNA synthesis was monitored using SYBR
green (Stratagene, Cedar Creek, TX, USA) and normalization of expression
against β-actin permitted comparison between cDNAs. Each measurement was
performed in duplicate from two different animals on each of three separate
days with reverse transcriptase-free samples for each treatment acting as
negative controls.
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Statistical analysis
CPP was analyzed using ANOVA followed by Tukey's post-hoc
comparison and by paired or two sample t-test as appropriate.
Conditioned place preference despite an adverse stimulus data were analyzed
using two-way ANOVA with a repeat measure over condition (restricted
versus punished) using Graphpad Prism 5, Instat (GraphPad, San Diego,
CA, USA), followed by post-hoc two-sample or paired t-test,
as appropriate, with Bonferroni adjustment. Microarray data were analyzed
using ANOVA parametric tests without multitask correction, variances not
assumed equal (Welch t-test). A P-value of 0.05 was
considered significant. This restriction tested 9201 genes. Approximately 460
genes would be expected to pass the restriction by chance.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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The aim of our study was to assess behaviour and gene expression changes in zebrafish following chronic exposure to nicotine or ethanol. As high concentrations of nicotine induced signs of toxicity in zebrafish and the rate of metabolism of nicotine in zebrafish is unknown, we were concerned that repeated exposure may lead to the toxic build up of the drug in the fish and influence the CPP response, or tolerance to the effects of nicotine may develop. We therefore tested the CPP response following 3 days of drug treatment. We detected a significant increase in preference for the treatment side in control fish after 3 days of treatment compared with before treatment (paired t-test, P<0.05; Fig. 2) suggesting that the place preference changes slightly as the fish become familiarized or habituated to the apparatus and handling procedure. Despite this habituation effect, fish exposed to either 6 or 30 µmol l–1 nicotine induced a significant increase in preference for the treatment side compared with the reaction of control, water-treated fish (two-sample t-test P<0.05; Fig. 2). Three repeat exposures to 300 µmol l–1 nicotine led to a significant decrease in place preference compared with either control fish, or to fish exposed to a single treatment of 300 µmol l–1 nicotine (two-sample t-test and paired t-test, respectively, P<0.05; Fig. 2).
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These results demonstrate that zebrafish show a dose-dependent acute reinforcement response to both nicotine and ethanol, consistent with the hypothesis that they may show lasting behavioural and gene expression adaptations following continued, repeated exposure to these drugs. Concentrations of 30 µmol l–1 nicotine and 175 mmol l–1 ethanol were chosen for such repeated drug treatments.
Repeat exposure to nicotine or ethanol induces conditioned place preference that persists despite prolonged drug abstinence
Following 4 weeks of repeated 20 min daily exposure to either 30 µmol
l–1 nicotine or 175 mmol l–1 ethanol,
zebrafish showed a significant (two-sample t-test,
P<0.05) increase in time spent in the treatment side: 50±6
s and 72±11 s for nicotine and ethanol respectively, compared with
7±7 and 3±6 s (mean ± s.e.m.) for each of the control
groups. This CPP response persisted for 3 weeks following the last drug
exposure for both nicotine-treated and ethanol-treated fish. However, after 7
or 21 days of drug abstinence the nicotine-treated fish showed a significant
reduction (two-sample t-test, P<0.05) in preference for
the treatment side when compared with the day after the last drug treatment
(Fig. 3A).
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Conditioned place preference persists despite adverse consequences
Drug seeking, despite adverse consequences, is an accepted model of drug
dependence in animal studies. Here we used a 3 s removal from the tank each
time the fish entered the drug treatment side as an adverse stimulus or
punishment for drug seeking. To establish the aversive effect of removal from
the tank we determined the number of returns separate control fish made to
their initially preferred side of the tank over a 10 min period in the face of
either 30 s restriction or 3 s removal from the tank followed by 30 s
restriction. 3 s removal from the tank led to a significant reduction
(two-sample t-test, P<0.05) in the number of returns
control fish made to the initially preferred side
(Fig. 4A). Following 4 weeks of
conditioning, 3 s removal from the tank significantly reduced the number of
returns made by control, water-treated fish, and ethanol-conditioned fish but
did not significantly alter the number of returns made by nicotine-conditioned
fish (Fig. 4B,C). There was a
significant interaction between drug and treatment (repeat-measures two-way
ANOVA; Fig. 4) such that 3 s
removal from the tank had a significantly reduced effect on decreasing the
number of returns made by nicotine- or ethanol-conditioned fish compared with
controls (post-hoc paired t-test, P<0.01;
Fig. 4B,C). Furthermore,
nicotine- or ethanol-conditioned fish continued to demonstrate increased drug
seeking despite punishment up to 21 days following the last drug exposure
(Fig. 4D,E). Thus zebrafish
show persistent dependency-related behaviour following a 4-week daily exposure
to either 30 µmol l–1 nicotine or 175 mmol
l–1 ethanol.
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Changes in gene expression for selected genes were confirmed by Q-RT-PCR of cDNA generated from the original RNA used for the microarray (Fig. 5B). The microarray results for ionotropic glutamate receptor subunit 2a (gria2a), Alport syndrome, mental retardation, midface hyperplasia and elliptocytosis chromosome region 1 (AMMECR1), calcineurin B (CalB) and peripheral benzodiazepine receptor (pBDZR) were validated by Q-RT-PCR.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Acute effects of ethanol treatment on zebrafish development and behaviour
in terms of swim behaviour and the startle response have been described
(Damodaran et al., 2006;
Dlugos and Rabin, 2003;
Gerlai et al., 2000;
Lockwood et al., 2004). Dlugos
and Rabin (Dlugos and Rabin,
2003) and Gerlai et al.
(Gerlai et al., 2006) have
also demonstrated adaptation of adult zebrafish after chronic exposure to
ethanol such that tolerance to the acute effects of the drug develops. By
contrast, studies of the effect of nicotine on zebrafish development and
behaviour are limited (Levin et al.,
2007; Levin and Chen,
2004; Levin et al.,
2006; Matta et al.,
2007; Svoboda et al.,
2002). Levin et al. have shown that 3 min exposure to low doses of
nicotine (38–77 µmol l–1 nicotine) improves memory
function in zebrafish (Levin and Chen,
2004) and that acute exposure to similar concentrations has an
anxiolytic effect (Levin et al.,
2007). Despite the emerging use of zebrafish for the study of
reinforcing effects of drugs of abuse
(Bretaud et al., 2007;
Darland and Dowling, 2001;
Ninkovic and Bally-Cuif, 2006)
this is the first report of reinforcing properties of ethanol or nicotine in
this species.
In mammals, reinforcing effects are seen at blood concentrations of around
30 mmol l–1 for ethanol and 0.05–0.6 µmol
l–1 for nicotine (Lewis
and June, 1990; Matta et al.,
2007; Rimondini et al.,
2002; Roberts et al.,
2000). Here, we obtained reproducible reinforcing effects at tank
concentrations of 175 mmol l–1 for ethanol and 30 µmol
l–1 for nicotine. Although we did not determine brain ethanol
or nicotine concentrations in our study, previous work of others suggests that
these tank concentrations are considerably higher than the brain
concentrations that would have been reached. Dlugos and Rabin
(Dlugos and Rabin, 2003) have
shown that a 15 min exposure of zebrafish to 88 mmol l–1
(0.5% v/v) ethanol in the tank water led to a brain ethanol concentration of
approximately 20 mmol l–1. Assuming a linear relationship
between tank concentration and brain concentration this suggests that the
brain alcohol concentration in our experiment may have reached 40 mmol
l–1, which is somewhat higher than the brain alcohol level
reached in mammals after consumption of alcohol doses that are reinforcing.
However, the precise relationship between tank concentration and brain alcohol
concentration and the nature of factors that may influence the rate of uptake,
such as temperature, age and activity, has yet to be established. Furthermore,
several additional factors, including the rate of metabolism and excretion,
influence the final brain concentration reached. Alcohol dehydrogenase is the
principle enzyme responsible for ethanol metabolism in mammals. Although a
zebrafish alcohol dehydrogenase has been identified, the details of its
distribution and kinetics have not been established. Thus, although further
work is required to establish the pharmacodynamics of ethanol in zebrafish,
the available data is consistent with the reinforcing effect of exposure to a
tank concentration of 175 mmol l–1 ethanol seen here.
We also obtained reproducible reinforcing effects following exposure to 30
µmol l–1 nicotine in the tank water. In mammals
reinforcing effects of nicotine are observed at a blood nicotine concentration
of 0.05–0.6 µmol l–1. Again, although no data on the
pharmacodynamics of nicotine in zebrafish has been published, brief (3–5
min) exposure of zebrafish to 40–80 µmol l–1
nicotine in the tank water has a similar anxiolytic and memory enhancing
effect to that seen in humans with treatments that result in a blood nicotine
concentration in the range of 0.1–0.2 µmol l–1
(Marchant et al., 2007;
Rusted et al., 2005). These
results are consistent with the reinforcing effect of exposure to a tank
concentration of 30 µmol l–1 nicotine seen here. As
discussed by Matta et al. (Matta et al.,
2007), the rate at which nicotine reaches the central nervous
system and the concentration achieved in specific regions of the brain, are
determinant factors in eliciting reward and dependence in humans. Factors
influencing the pharmacodynamics of nicotine (or ethanol) in individual
species include the rate of uptake, efficiency of metabolism, potential
physiological effects of metabolites and the rate of excretion. In mammalian
species, nicotine is extensively and rapidly metabolised by the liver, with
70–80% of nicotine being converted to cotinine by the action of specific
cytochrome P450 enzymes, and approximately 5% being excreted unchanged.
Although several zebrafish cytochrome P450 enzymes have been characterized, a
zebrafish equivalent of the human CYP2A6 enzyme, the enzyme that is primarily
responsible for the metabolism of nicotine to cotinine in humans, has not been
identified. Further work is required to establish the rate of uptake,
metabolism, and clearance of nicotine or ethanol in zebrafish and how the
route of administration may effect final brain concentrations.
As 20 min exposure to high doses (600 µmol l–1; 100 mgl
l–1) of nicotine induced signs of toxicity in zebrafish (data
not shown) and the rate of metabolism of nicotine in zebrafish is not known,
we assessed whether exposure to nicotine on three consecutive days
significantly altered the results. Although zebrafish continued to show a
dose-dependent CPP response to nicotine exposure with maximal effect seen at
30 µmol l–1, there were important differences in the
results obtained. Control-treated fish showed a significant increase in
preference for the site of drug (in this case water) exposure after three
treatments compared with either their basal preference or following a single
treatment. The increase in preference shown by control fish suggests that
there was some habituation to the apparatus over the three test periods.
Similar habituation was seen by Ninkovic and Bally-Cuif
(Ninkovic and Bally-Cuif,
2006) when using a biased paradigm to study amphetamine induced
CPP in zebrafish. In the hands of Ninkovic changes in basal place preference
as a result of habituation to the apparatus stabilized after three exposures
suggesting that the results of our 3-day treatment may give a more reliable
measure of the basal preference of zebrafish to the conditioning apparatus
used. Thus consecutive conditioning sessions over 3 days in the absence of any
drug was performed prior to determination of basal preference in all
subsequent experiments. Importantly three exposures to tank concentrations of
either 6 or 30 µmol l–1 nicotine induced a significant
increase in time spent in the treatment side compared with control
water-treated fish, indicating a consistent reinforcement response to these
nicotine concentrations. Following three exposures to concentrations of 60
µmol l–1 or greater, nicotine-treated fish no longer
showed a significant increase in preference for the treatment side compared
with controls. Indeed after 3 days of exposure to 300 µmol
l–1 nicotine, zebrafish showed a significant decrease in
preference for the site of drug treatment compared with controls. This result
may reflect an inability of the fish to effectively clear such high doses of
nicotine from their systems between treatments. On binding nicotine, the
receptors responsible for many of the central effects of nicotine, including
activation of the `reward' circuit, are rapidly desensitized. In humans at
least 8 h of abstinence (overnight) may be required in order for nicotine
levels and associated tolerance to decline sufficiently to be able to detect
many of the physiological effects of nicotine. As neither the blood
concentration reached during the course of these experiments, nor the
clearance rate in zebrafish is known, the lack of reinforcement is consistent
with persistent desensitization and tolerance to the effects of acute
administration of the drug.
There are a number of criteria (see
DSM-IV 1994) that need to be
met before CPP is considered a model of dependence rather than reinforcement
(O'Brien and Gardner, 2005).
These include the persistence of the response despite prolonged abstinence and
CPP in the face of adverse consequences. We examined our model against these
criteria using 3 weeks as a period of abstinence and 3 s removal from the tank
as an adverse consequence. Removal from water has been shown previously to
induce stress in fish: cortisol levels are increased when trout are removed
from water for 30 s (Demers and Bayne,
1997). We confirmed that 3 s removal from the tank was an adverse
stimulus for zebrafish by comparing the number of returns previously
un-treated control fish made to a given region of the tank when punished or
not (Fig. 4). Both
ethanol-treated and nicotine-treated fish showed persistent CPP despite
punishment. This CPP despite punishment persisted for 3 weeks after the last
drug treatment consistent with it being a dependency-related behaviour.
To determine whether the drug-associated CPP persisted following a period of abstinence, CPP responses were determined after 7 and 21 days of abstinence. As can be seen from Fig. 3B the CPP shown by ethanol-treated fish did not alter significantly over this time. However, nicotine-treated fish showed a significant decrease in CPP after 3 weeks of abstinence compared with 24 h after the last drug treatment. As all fish were tested for their place preference on each occasion, the decline may reflect a tendency towards extinction of the preference by exposure to the conditioning cues in the absence of drug treatment. The basis for the difference in behaviour of ethanol-treated fish was not explored. Nonetheless, zebrafish treated with either ethanol or nicotine for 4 weeks showed the dependence-related behaviour of drug-induced CPP that persists over prolonged periods of abstinence and in the face of an adverse stimulus. Although these behaviours are consistent with the establishment of drug dependency in zebrafish, it is also possible that the establishment of new memories, extinction memory or the reversal of existing memories is impaired in fish pretreated with ethanol or nicotine. Further studies are required to address this possibility. As discussed below, the expression of several genes associated with synaptic plasticity, memory and learning, such as calcineurin identified here, were found to be altered following chronic exposure to alcohol or nicotine.
Homeostatic theories of drug dependence and relapse suggest that
long-lasting neuro-adaptations occur that underlie the change in behaviour. We
used microarray analysis and Q-RT-PCR to determine whether exposure to
nicotine or ethanol that induced dependence-related behaviour in zebrafish
induced similar changes in gene expression in this species as in mammalian
models of drug dependence. We focused on changes that were seen in common in
both treatment groups rather than in individual groups as these changes may
reflect conserved adaptations underlying dependency rather than a specific
response to the individual drug. We identified 153 genes that showed a
significant, 1.5-fold or greater, change in expression in the brains of both
nicotine-treated and ethanol-treated fish compared with controls. Several of
these shared genes are components of pathways that also show lasting
adaptation in the brains of mammalian models of dependence. These include
glutamate receptors [AMPA and NMDAR1 (N-methyl-D-aspartate
receptor 1) (Kalivas, 2004;
Noda and Nabeshima, 2004;
Sanchis-Segura et al., 2006)]
and the peripheral BDZR (Sanna et al.,
2004), and molecules associated with synaptic plasticity such as
NCAM (Abrous et al., 2002;
Miller et al., 2006;
Weber et al., 2006). Although
neuro-adaptations related directly to dopamine stimulation are thought to be
critical for the development of addiction, alterations in glutamatergic
neurotransmission are thought to be key to the relapsing nature of drug
addiction (Chao et al., 2002;
Gao et al., 2006;
Kalivas, 2004). In this
regard, repeated intermittent exposure to cocaine, amphetamine or ethanol (as
used here) has been reported to cause alterations in levels of AMPA and NMDA
glutamate receptor subunits in the ventral tegmental area
(Churchill et al., 1999;
Fitzgerald et al., 1996;
Nestler, 2001;
Nestler, 2004;
Ortiz et al., 1995). However,
at least in terms of cocaine-increased gria1 (AMPA GluR1) expression, protein
levels do not result from increased mRNA but seem to be due to
posttranscriptional mechanisms including trafficking to the cell surface
(Beitner-Johnson et al., 1992;
Borgland et al., 2006;
Gao et al., 2006;
Lu et al., 2002;
Ungless et al., 2001). We
detected an increase in whole brain gria2 mRNA expression. Such a
whole brain change in gria2 mRNA expression has not been reported for
mammalian models of dependency although gria2 mRNA is increased in
the nucleus accumbens both in animal models of dependency
(Boudreau and Wolf, 2005;
Lu et al., 2003) and in brains
of human cocaine users (Crespo et al.,
2002). The majority of brain AMPA receptors are either
gria1–gria2 (GluR1–GluR2) or gria2–gria3 (GluR2–GluR3)
oligomers although other subunit compositions also occur
(Wenthold et al., 1996;
Wolf et al., 2004).
Interestingly, the gria3 subunit, which forms a complex with gria2 in calcium
impermeable AMPA receptors, is upregulated in rats during alcohol abstinence.
Furthermore targeted gria3 gene knock out leads to a blunted
cue-induced reinstatement response to alcohol implying a role for this
subunit/AMPA receptor subtype in alcohol relapse
(Sanchis-Segura et al., 2006).
Gria2 loss-of-function mice display multiple behavioural
abnormalities (Gerlai et al.,
1998; Mead and Stephens,
2003) that have limited the use of this line in addiction studies.
Nonetheless, Gria2 loss-of-function mice show reduced
amphetamine-induced conditioned reinforcement of reward seeking
(Mead and Stephens, 2003).
Thus our finding that gria2 receptors are altered in their level of expression
in the brains of zebrafish showing persistent alcohol (or nicotine)-induced
CPP is consistent with the generally accepted model that alterations in
glutamate neurotransmission are critical for the expression of addiction
related behaviour.
In addition our microarray identified a number of genes that are implicated
in drug dependency but have not previously been reported to have altered
levels of expression. These include calcineurin B and the hypocretin receptor.
The calcineurin B gene was significantly increased (3.5-fold) in the brain of
both nicotine- and ethanol-treated fish. Although increased expression of
calcineurin in brain tissue from mammalian models of addiction has not been
reported previously, the involvement of calcineurin in synaptic plasticity and
neurotransmission related to drug dependence has been suggested. A line of
memory-deficient mice that overexpress calcineurin fail to demonstrate
amphetamine-induced CPP (Biala et al.,
2005). Additionally calcineurin regulates the release of dopamine
from presynaptic terminals such that high levels of calcineurin activity
inhibit dopamine release (Iwata et al.,
1997). This suggests that calcineurin levels may be increased in
nicotine- or ethanol-treated fish as an adaptive response to repeated dopamine
release. As elevated calcineurin also appears to have a negative effect on
short-term memory and learning (Genoux et
al., 2002; Malleret et al.,
2001; Mansuy et al.,
1998), the enhanced level of expression of this gene in the brains
of the drug-treated fish may have contributed to their continued drug seeking
in the face of punishment.
There have been a number of microarray analyses of gene expression changes
following either acute or chronic exposure to drugs of abuse (e.g.
Boudreau and Wolf, 2005;
Hemby, 2006;
Li et al., 2004;
Rimondini et al., 2002;
Toda et al., 2002;
Walker et al., 2004). Direct
comparison between these is difficult because of variation in the treatment
paradigms used and the length of time after drug exposure. Nonetheless
microarray analyses consistently report changes in expression of factors
associated with altered synaptic plasticity as well as components of the
dopaminergic and glutamate neurotransmitter and signal transduction pathways
as seen here (supplementary material Tables S1 and S2). Although we found a
number of changes in gene expression reminiscent of those seen in mammalian
models suggesting conservation of adaptive pathways, a number of novel genes
were also identified. The majority of published microarray analyses compare
frontal cortex or nucleus accumbens in control and drug-treated animals
(Li et al., 2004;
Rimondini et al., 2002) (for
reviews, see Pollock, 2002;
Rhodes and Crabbe, 2005;
Sommer et al., 2005;
Yuferov et al., 2005). The
rationale for this approach is twofold: (1) these are the primary brain
regions shown to be involved in mammalian reward responses and (2) the
complexity of the mammalian brain may lead to subtle differences being
obscured if whole brain tissue were used. We chose not to limit our study in
this way. This decision was based, in part, on the premise that the reduced
complexity of the zebrafish brain may allow pathways to be identified that are
obscured by the complexity of the mammalian brain or that had been excluded by
the choice of tissue. Additionally, the small size of the zebrafish brain
would have necessitated either the pooling of tissue from a large number of
animals, or a pre-amplification step in order to obtain enough cDNA for array
analysis.
In summary, we have demonstrated that zebrafish show the dependency-related behaviour of persistent CPP despite an adverse stimulus on repeated exposure to two of the most commonly abused drugs, nicotine and ethanol, and identified conserved changes in gene expression that may contribute to the change in behaviour. These findings add to the body of evidence validating the use of zebrafish as a model system for the study of the genetic basis of reward and addiction.
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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