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C. Poch , L. Espana-Serrano
,
M. A. Jimenez-Arriero
and J. Hoenicka,,
Facultad de Psicologa, Universidad Complutense de Madrid,
Campus de Somosaguas, Madrid 28223, Spain; Hospital
Universitario 12 de Octubre, Servicio de Psiquiatra, Avda. de
Cordoba
s/n, Madrid 28041, Spain; Centro de Investigacion
Biomedica
en Red de Salud Mental (CIBERSAM), Spain
*Corresponding author: J. Hoenicka, Servicio de Psiquiatra,
Polymorphisms of DRD2 and ANKK1 have been associated with psychiatric syndromes where there is believed
to be an underlying learning process deficit such as
addiction, post-traumatic stress disorder and psychopathy. We investigated the effects of the DRD2 C957T and
ANKK1 Taq IA single nucleotide polymorphism (SNP),
which have been associated with psychopathic traits
in alcoholic patients, on fear conditioning and aversive
priming in healthy volunteers. We found that the DRD2
C957T SNP, but not the ANKK1 Taq IA SNP, was associated with both differential conditioning of the skin
conductance response and the aversive priming effect.
There were no differences between the genotype groups
with respect to the extinction of the skin-conductance
conditioned response. These results suggest that the
C957T SNP could be related to learning differences associated with the risk of developing psychiatric disorders
in individuals that are carriers of the C homozygous
genotype. Our genetic data raise the possibility that the
dopaminergic system functional variations determined
by this SNP could affect fear learning.
Keywords: ANKK1, aversive priming, cognitive bias, DRD2,
fear conditioning, polymorphism
The study of biological and environmental influences on psychopathic personality has indicated that individual differences
in antisocial behaviour are highly heritable (Fontaine et al .
2008; Viding et al . 2005). In clinical samples, the clearest link
between genetic variation and psychopathic traits has been
established with genes of the locus 11q22-q23 (Ponce et al .
2008). The most recent data show that the single nucleotide
polymorphisms (SNPs) of the D2 dopamine receptor gene
(DRD2 ) and the ankyrin repeat and kinase domain 1 (ANKK1),
doi: 10.1111/j.1601-183X.2009.00543.x
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Huertas et al.
Methods
Participants
Sixty-three healthy undergraduate students (32 females) at the Universidad Complutense de Madrid, Madrid, Spain, were recruited for
the study. Their age range was 1927 years and they received 20
as a monetary incentive. Data from one participant were excluded
because the genetic analysis could not be completed, and data from
a further two participants were excluded because of extreme values
on the recognition time task. The study was approved by the Ethics
Committee of Hospital 12 de Octubre, Madrid. Participants signed
two informed consent forms, one for the experimental procedure
and one for the genetic study.
Stimulus materials
During the conditioning phase, four pictures of neutral black-andwhite faces (2 male, 2 female) from the Eckman and Friesen set
(numbers 21, 41, 65 and 99) were presented to each participant. Half
of the participants were presented the two male faces as conditioned
stimuli (CS+ and CS) and the two female faces served as additional
faces to familiarize the participant with them. For the remaining
participants, the faces were presented in the opposite manner.
The face that was coupled to the CS+ was also balanced among
participants. During the priming phase, these four faces were used
as those to be recognized (old faces). In addition, 10 neutral faces
from the Eckman and Friesen set (numbers 6, 13, 28, 33, 47, 56, 72,
83, 92 and 110) were used as distractors (distractor-faces). The faces
were projected from the control room on a screen located about
110 cm in front of the participant. The image size was 22 30 cm.
Electric shocks were generated by a Mark 100 stimulator (Farrall
Instruments, Inc., Grand Island, NE, USA) fed by batteries and
optically isolated from the computer. The electric shocks were applied
to the internal surface of the forearm through a Tursky concentric
electrode. The intensity of the shock was fixed by the participant
following the instruction it must be clearly uncomfortable but not
painful. During the conditioning phase, shocks were applied to the
dominant forearm for 200 milliseconds, and during the priming phase
they were applied to the non-dominant forearm for 180 milliseconds.
Neutral tones were 1000 Hz and the intensity was set by the
participant following the instruction you must hear it clearly, but it
must not be unpleasant at all. The tones were presented for 200
milliseconds during the conditioning phase and for 180 milliseconds
during the priming phase.
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Procedure
The experiment had four phases: presentation of distractors,
habituation, conditioning and aversive priming.
Upon arrival, the participants were reminded that they could leave
the experiment at any time. They were fitted with skin conductance
electrodes, and then they viewed each of the 10 faces that would
operate as distractors in the priming phase two times. Each picture
was presented for 8 seconds and the inter-trial interval (ITI) was
4 seconds. Next, the shock electrodes were fitted, and the intensity
of the shock and the tone were set. During the breaks in the
experiment, participants were asked again about the unpleasantness
of the stimuli and the intensity was modified when necessary. The
participant could also ask to change the intensity at any time.
Next, the participants were informed that they would view four
new faces several times each, and that one of the faces would
sometimes be paired with the shock (CS+) and the other would
sometimes be paired with the tone (CS). During the habituation
phase, the procedure included a non-reinforced presentation of
each of the CS+, CS and the two other old faces. During the
acquisition phase, the procedure included eight presentations of
the CS+ paired with the aversive shock and eight presentations
of the CS paired with the neutral tone (paired trials), three
presentations of each of the CS+ and the CS alone (test
trials), and four presentations of each of the other two old
faces.
The set habituation-conditioning was organized in six blocks with
a total of 34 trials and a single break after the 20th trial. The first
block was the habituation phase. The second block consisted of two
paired trials of the CS+ and two paired trials of the CS. Blocks 3, 4
and 5 consisted of two paired trials of each of the CS+ and the CS,
one test trial of each of the CS+ and the CS, and one presentation
of each of the other two faces. The sixth block consisted of one
presentation of each of these last two faces. Within the blocks,
the trials of each type were presented in semi-random order, with
the restriction that none of the faces appeared in more than three
consecutive trials. The presentation time for the faces was 8 seconds,
the interval between the appearance of the face and the onset of
shock or tone [stimulus onset asynchrony (SOA)] was 3 seconds and
the duration of the shock and the tone was 200 milliseconds. The ITI
varied randomly between 16 and 20 seconds.
Next, the aversive priming (Huertas-Rodrguez 1991) phase began.
Participants were informed that they would see several faces and
should indicate as quickly as possible, but trying not to make
mistakes, if the face was one of the four faces presented during
the conditioning phase or one of the faces seen at the beginning
of the experiment. It was therefore a recognition task, and not
only a familiarity-judgement task. Participants were warned that any
face could be preceded by the neutral tone, the aversive shock or
neither of them. The recognition decision was made by pressing the
corresponding buttons on the console. By pressing the button, the
face disappeared. When the participant did not press any button in a
period of 3 seconds after the face onset, it disappeared automatically.
The SOA was 190 milliseconds. The duration of the prime (shock or
tone) was 180 milliseconds. The ITI varied randomly between 19 and
21 seconds.
Participants underwent two presentations of the CS+ and two of
the CS, seven presentations of the other two old faces and 10
presentations of the distractors. The CSs were preceded once by the
aversive prime, and once by the neutral one. The rest of the faces
were preceded four times by the aversive prime, seven times by the
neutral prime and six times by neither of them. There was one presentation of each primeCS sequence, because the aversive-prime/CS
sequence and neutral-prime/CS+ sequence could operate as counterconditioning trials, thus reducing or eliminating the expected effect in
subsequent presentations. After trial 14, a break of 4 min was taken.
The four critical prime/CS sequences were presented in trials 9, 13,
17 and 20. There were four presentation orders for these sequences,
according to the eight possible permutations that fulfill the requirement for an alternate conditioned stimulus (CS+ and CS) and prime
(aversive and neutral) presentations. The presentation order for each
participant was random.
The priming phase finished with a presentation of each CS+
and CS without being preceded by shock or tone. The order of
presentation was counterbalanced between the participants.
Genes, Brain and Behavior (2010) 9: 103109
Genotyping
Samples from epithelial cheek cells were collected with buccal
swabs. Genomic DNA extraction and amplification were performed
using the illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Europe, Barcelona, Spain) following the manufacturers protocol.
Genotyping was performed by Taqman assays, which were designed
to run on an ABI 7900HT machine with Sequence Detection System
software (Applied Biosystems, Foster City, CA, USA). In a previous
work, we evaluated the non-random association of the two SNPs
and found a low degree of linkage disequilibrium between Taq IA
and C957T in the control population (D = 0.58, r 2 = 0.14) (Ponce
et al . 2008).
The sequence-specific primers for ANKK1 Taq IA (5 CTGCCTCGAC
CAGC 3 and 5 CTGCCTTGACCAGC 3 ) were designed for the
C (A2) and T (A1) alleles, respectively. A common reverse primer
(5 -GCAACACAGCCATCCTCAAAG-3 ) was used. Five samples for
each genotype were confirmed by direct sequencing analysis. The
resulting genotypes for ANKK1 Taq IA were clustered according to
the presence of at least one A1 allele (A1+ genotype, A1 allele
homozygous and heterozygous; A1 genotype, homozygous for
the A2 allele) as previously described (Blum et al . 1990). DRD2
C957T was analysed as previously described by Hoenicka et al .
(2006). Briefly, the sequence-specific primers for the Taqman assays
(5 -CTGTCGGGAGTGCTG-3 and 5 -CTGTCAGGAGTGCTG-3) were
used for the C and T alleles, respectively, as was the common
reverse primer 5 -GCCCATTCTTCTCTGGTTTGG-3 . The genotypes
obtained were grouped assuming a recessive model for the C957
allele: homozygous individuals for the C allele vs. heterozygous and
homozygous individuals for the T allele.
We performed the HardyWeinberg equilibrium test using the
Haploview software (version 3.2; Whitehead Institute for Biomedical
Research; http://www.broad.mit.edu/mpg/haploview/index.php) and
we found no deviation from either in ANKK1 Taq IA (P = 0.44) or in
DRD2 C957T (P = 0.83). In addition, we observed in this sample the
same ANKK1 and DRD2 genotype distributions that were previously
found for both Spanish (Ponce et al . 2008) and European (NCBI:
http://www.ncbi.nlm.nih.gov/) healthy populations.
Statistical analysis
All SCR magnitudes were range corrected, by dividing each value
by the mean of the participants three maximal responses and
multiplying the result by 100 (PC SCR). A square-root transformation
was also applied to help to normalize the distribution [SQRT (1 + PC
SCR)]. In the case of the RTs, data were log transformed. However,
means are presented as milliseconds to allow a better interpretation.
The incorrect recognition responses (4.6% of total data) and RT
values over the grand mean 2.5 SD (1.7% of total data) were
replaced by the mean of the participant.
A repeated-measures general linear model (SPSS 15.0 for
Windows, SPSS Inc., Chicago, IL, USA) was used for each SNP,
with SCR and RT as consecutive dependent variables. We used a
two-tailed t -test for post hoc comparisons between genotypes, and
a one-tailed t -test for the comparisons for which we had a specific
prediction a priori. Given the unequal size of the genetically defined
groups (C957T SNP: CC group = 9, CT/TT group = 51; Taq IA SNP:
A1 = 18, A1+ = 42), additional MannWhitney tests were used
for post hoc comparisons between genotypes. This analysis gave
rise to an identical pattern of results to that of the t -test.
Extinction
The CS genotype interaction analysis showed no significant
differences between groups in the extinction of the SCR
for the C957T SNP (P = 0.782) (Fig. 1b) or for the Taq I-A
(P = 0.147) (Fig. 1c).
Results
Discussion
Relationship between the C957T and TaqIA SNPs,
and fear conditioning
Acquisition
The SCR analysis showed a significant CS test-trial C957T
interaction (F2.116 = 3.154; P = 0.046). This interaction effect
was subsequently parsed into orthogonal linear and quadratic
contrasts to evaluate the effect of the test-trial on the differential response to the CS+ and the CS depending on the
Genes, Brain and Behavior (2010) 9: 103109
We found that DRD2 C957T, but not ANKK1 Taq IA, was
associated with both SCR differential conditioning and
aversive priming in healthy participants. Specifically, during
acquisition, the CT/TT group exhibited a continued decrease
in the SCR to the CS+ with repeated presentation of the
stimulus, whereas the response of the C homozygous
carriers was maintained or tended to increase. On the
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Huertas et al.
Figure 1: Conditioning of the skin conductance response as a function of genotype. (a) The differential conditioning paradigm;
(bc) Relationship between the genotypes (DRD2 C957T SNP ; ANKK1 Taq IA SNP) and the magnitude of the SCR to the CS+ and
CS in the three acquisition test trials and the extinction trial. Asterisks indicate significant differences: *P < 0.05; **P < 0.01. SQRT
[1 + PC SCR]: squared root of 1 plus the percentage of the skin conductance response respect to the mean of the participants three
maximal responses; 1,2,3: test trials of acquisition; e: extinction.
106
implications. Indeed, our results are in line with data and theories pertaining to psychiatric disorders associated to the C957
homozygous genotype such as the PTSD (Voisey et al . 2009),
schizophrenia (Hoenicka et al . 2006; Lawford et al . 2005)
and substance abuse (Perkins et al . 2008). For instance,
individuals with PTSD seem to be more conditionable than
trauma-exposed individuals without PTSD (Orr et al . 2000).
Further, several studies suggest that there is an increased
likelihood of flashbacks and intrusive phenomena related
to the traumatic experience in these patients as a consequence of a subsequent increase in arousal. This retroactive
effect occurs even if the arousal is artificially triggered [e.g.
(Bremner et al . 1997; Nixon and Bryant 2005)]. A similar phenomenon has been found in schizophrenia where stressful
situations raise the likelihood of hallucinations (Freeman and
Garety 2003). In the case of drug abuse, situations leading
to the increment of the arousal also increase the likelihood
of images and thoughts related to the substance of abuse
(Sinha 2008). Indeed, in the case of nicotine addiction, the
smokers homozygous for the C957 allele took more cigarette
puffs during negative vs. positive mood (Perkins et al . 2008).
On the contrary, our results in the C957 homozygous, a risk
genotype for psychopathic traits (Ponce et al . 2008), seem
not to be consistent with one of the most replicated findings
Genes, Brain and Behavior (2010) 9: 103109
Figure 2: Recognition time in the priming task as a function of genotype. (a) The priming procedure in the four conditions:
aversive-prime/CS+ (Av/CS+), aversive-prime/CS (Av/CS), neutral-prime/CS + (Ne/CS+) and neutral-prime/CS (Ne/CS); (bc)
Relationship between the genotypes (DRD2 C957T SNP; ANKK1 TaqIA SNP) and the recognition time of the CSs in the Av/CS+,
Av/CS, Ne/CS+ and Ne/CS sequences. Asterisks indicate significant differences: *P < 0.05.
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Huertas et al.
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Acknowledgments
This work has been supported by the Fondo para Investigaciones
Sanitarias (FIS), Madrid, Spain, grants no. 05/0731 and 08/0529.
CIBERSAM is an initiative of the Instituto de Salud Carlos III.
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