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RESEARCH ARTICLE
Conditioned increase of locomotor activity
induced by haloperidol
Luis Gonzalo De la CasaID*, Lucı
´a Ca
´rcel, Juan Carlos Ruiz-Salas, Lucı
´a Vicente,
Auxiliadora Mena
Department of Experimental Psychology, Universidad de Sevilla, Seville, Spain
*delacasa@us.es
Abstract
Dopamine antagonist drugs have profound effects on locomotor activity. In particular, the
administration of the D2 antagonist haloperidol produces a state that is similar to catalepsy.
In order to confirm whether the modulation of the dopaminergic activity produced by halo-
peridol can act as an unconditioned stimulus, we carried out two experiments in which the
administration of haloperidol was repeatedly paired with the presence of distinctive contex-
tual cues that served as a Conditioned Stimulus. Paradoxically, the results revealed a dose-
dependent increase in locomotor activity following conditioning with dopamine antagonist
(Experiments 1) that was susceptible of extinction when the conditioned stimulus was pre-
sented repeatedly by itself after conditioning (Experiment 2). These data are interpreted
from an associative perspective, considering them as a result of a classical conditioning
process.
Introduction
Pavlovian conditioning has been proposed as a fundamental process to explain how organisms
learn to respond adaptively in anticipation of the occurrence of environmental events [1,2]. A
relevant area in which the adaptive relevance of Pavlovian associations has been demonstrated
is related to the effects of repeatedly presenting a neutral stimulus accompanied by the effects
of a drug. This procedure has led to seemingly contradictory results, since while in some cases
the Conditioned Response (CR) that appears has been similar to that produced by the drug
[3,4], on other occasions the CR has been of an opposite nature to that induced by the drugs
[5,6]. Eikelboom & Stewart [7] have proposed that the origin of these differences could be
related to the effect of the drug on the nervous system: whilst on some occasions the Condi-
tional Stimulus (CS) is associated with an Unconditioned Response (UR) dependent on the
central nervous system, at other times the CS is associated with a peripheral UR that will
appear to compensate for the central effects of the drug. In the first case, the association
between the drug and the CS would lead to the appearance of a CR similar to the one that is
produced by the drug, while in the second case, the CR would be opposite to that produced by
the drug at the central level.
The first experimental evidence to highlight the effects of classical conditioning in the field
of drugs was described by Pavlov himself, who reported that the repeated administration of
PLOS ONE | https://doi.org/10.1371/journal.pone.0200178 October 3, 2018 1 / 14
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OPEN ACCESS
Citation: De la Casa LG, Ca
´rcel L, Ruiz-Salas JC,
Vicente L, Mena A (2018) Conditioned increase of
locomotor activity induced by haloperidol. PLoS
ONE 13(10): e0200178. https://doi.org/10.1371/
journal.pone.0200178
Editor: Vilfredo De Pascalis, La Sapienza University
of Rome, ITALY
Received: June 15, 2018
Accepted: September 14, 2018
Published: October 3, 2018
Copyright: ©2018 De la Casa et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Funding: This research was supported by
PSI2015-64965-P grant (MINECO, FEDER, UE).
Competing interests: The authors have declared
that no competing interests exist.
morphine in the presence of a given context gave rise to a CR similar to that produced by mor-
phine alone [8]. From these pioneering studies, which demonstrated that contextual cues can
be used as CSs that acquire the ability to induce physiological and behavioral states similar to
those produced by the drug, a number of studies have been developed to demonstrate the con-
ditioning of various responses produced by a wide range of drugs including, for example, mor-
phine-induced hyperthermia [9,10], stereotypy or hyperactivity induced by amphetamine,
cocaine, or apomorphine [11–14], amphetamine-induced hyperthermia [15], or haloperidol-
induced catalepsy [16–18]. The conditioning process supported by these drugs has been used
to identify the neurobiological bases of learning [13], and has been considered as a possible rel-
evant factor in the relapse of addicts [19,20], since it helps to explain the development of toler-
ance and the sensitization of drug-induced responses [21,22].
In our work, we will focus specifically on the conditioning of locomotor activity, using the
administration of the dopaminergic antagonist haloperidol as a US. The usual procedure
employed in this type of experiment involves a design that includes two groups that differ in
terms of the time at which the drug is administered [23]. For the first of the groups, which is
usually referred to as the Paired group, the drug is administered before introducing the animal
into the experimental context that will serve as the CS. After spending a period of time that
usually ranges between 30 and 60 min in the context CS, an innocuous solution is adminis-
tered and the animals are returned to their home cages. The second group, usually called the
Unpaired group, first receives the saline solution in such a way that exposure to the context
takes place in the absence of the drug, and the corresponding dose of the drug is administered
before returning the animal to its home cage. After a rest period of around two days without
receiving any type of drug or behavioral treatment, a test trial is carried out in which all ani-
mals of both groups are injected with the innocuous solution before introducing them to the
context CS to record the activity.
Using this basic procedure, results have consistently revealed the existence of the condition-
ing of locomotor responses using dopamine agonists such as amphetamine or apomorphine.
In particular, a significant increase in conditioned locomotor responses has been observed on
the test trial for the paired group in comparison with the unpaired group [23–28]. Less consis-
tent are the results that have been obtained when the US employed is the dopamine antagonist
haloperidol [29,30], possibly due to the fact that, depending on the dose administered, haloper-
idol can result in both an increase as well as a decrease in locomotor activity. More specifically,
when a low dose of haloperidol (less than 0.1 mg / kg) is administered repeatedly, a progressive
increase in the locomotor response is observed, which has been interpreted as the result of a
sensitization process due to the selective blockade of the presynaptic autoreceptors that results
in dopamine levels rising, leading to an increase in locomotor activity [30]. However, when a
higher dose is repeatedly administered (from 0.1 mg / kg), both pre and post-synaptic recep-
tors are blocked, resulting in a reduction in locomotor behavior. This can even induce a state
of catalepsy, in which the animals maintain unusual postures for prolonged periods of time
[29–31]. When, after repeated administration of the dopaminergic antagonist, a drug-free test
is carried out in the same context in which the drug was administered, different results emerge
depending on the dose of drug given during the conditioning trials. Thus, with doses of halo-
peridol that can be classified as high (specifically 0.1, 0.25 and 0.5 mg / kg), an increase in con-
ditioned catalepsy has been found in the Paired group with respect to the Unpaired control
group on the test trial without the drug [29]. However, Dias et al [30] found an increase in
locomotor activity in a group that had received ten pairings of the context-CS and a low dose
of haloperidol (0.03 mg / kg), although in this case the subjects had received 5 trials in which
2.0 mg / kg of apomorphine had been injected before the conditioning test.
Conditioned increase of locomotor activity induced by haloperidol
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In the present study we set out to analyze the conditioning of locomotor activity following
the repeated pairing of a context-CS and the effects of the administration of the dopaminergic
D2 antagonist haloperidol. The method most commonly used in the literature to evaluate such
behavioral patterns is either to observe movements in a limited space, generally an open field
cage where the total distance traveled, the number of turns, grooming, etc. are usually recorded
[32] or the so-called “bar test”, consisting in place the forepaws of the animal on a bar situated
at a height adequate to the animal tested and record the time elapsed until the animal put
down the paws on the floor [33]. However, in our case we recorded the percentage of time that
the animal remained in motion during each of the experimental sessions (60 min duration)
since we expected a reduction in motor activity both after drug administration and when test-
ing conditioning.
On the basis of previous findings, we anticipate that, with the concentrations of the drug we
have used, after pairing the context with a dopaminergic antagonist (haloperidol) it will be
observed a conditioned decrease in general activity [3,29–31].
Experiment 1
The purpose of this experiment was to examine the conditioning of the locomotor response
induced by the effect of two different doses of a drug that acts as a dopaminergic antagonist
(haloperidol, 0.5 and 2.0 mg / kg). For this, the animals in the Paired condition received the
administration of haloperidol before exposure to an experimental context that was to serve as
a CS, whilst animals in the Unpaired condition received haloperidol after exposure to the
experimental context.
Based on the previous results we anticipate that with the selected doses (0.5 and 2.0 mg /
kg) there will be a decrease in the activity on a subsequent drug-free test trial in presence of the
conditioned context that probably will be more intense with the higher dose.
Subjects
32 male Wistar rats (n = 8) experimentally naïve, participated in this experiment. Mean weight
at the start of the experiment was 384 g. (range 292–490). Food and water were available ad
libitum throughout the experiment. Animals were individually housed and maintained on a
12:12 h light:dark cycle (lights on at 06:00 h). All behavioral testing was conducted during the
light period of the cycle. Four days before the start of the experimental sessions, each of the
animals was handled 5 min daily. All procedures were conducted in accordance with the
guidelines established by the European Union Council established by the Directive 2010/63/
EU, and following the Spanish regulations (R.D 53/02013) for the use of laboratory animals.
The ethical commission of University of Seville supervised and approved all the procedures
and all protocols used in the this specific study (report: CEEA-US2015-28/4)
Apparatus and materials
Four identical Panlab conditioning boxes (model LE111, Panlab/Harvard Apparatus, Spain)
were used, each measuring 26 x 25 x 25 cm (H x L x W). Each chamber was enclosed in a
sound-attenuating cubicle (model LE116. Panlab/Harvard Apparatus, Spain). The walls of the
experimental chambers were made of white acrylic. A loudspeaker located at the top of each
chamber produced a 70 dB 2.8-kHz noise used as background, and the floor consisted of stain-
less steel rods, 2 mm in diameter, spaced 10 mm apart (center to center). Each chamber rested
on a platform that recorded the signal generated by the animal movement through a high sen-
sitivity Weight Transducer system. Such signal was automatically converted into percent of
general activity, defined as the percentage of the total time that movement was detected on
Conditioned increase of locomotor activity induced by haloperidol
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2-min periods, by a commercial software (StartFear system software, Panlab/Harvard Appara-
tus, Spain). Sampling was performed continuously at a frequency of 50Hz.
Haloperidol (Pensa Pharma) dissolved in 0.1% ascorbate/saline (2.0 mg/ml) was injected
subcutaneously in the nape of the neck at a dose of 0.5 or 2.0 mg/kg. A 0.1% ascorbate/saline
solution was used as vehicle. A delay of 20 min was introduced from the drug administration
to the introduction of the animals in the experimental chambers.
Procedure
Four groups were arranged following a 2 x 2 factorial design, with main factors Conditioning
(Paired vs. Unpaired) and Dose (0.5 vs. 2.0 mg/kg of haloperidol). Regarding the Conditioning
factor, those animals in the Paired condition received an injection of the correspondent drug
before to be introduced in the experimental context, and an injection of vehicle before to be
returned to their home cages; those rats in the Unpaired condition received the vehicle before
experimental context exposure, and the drug after each session (and before to be returned to
the home cages).
The experimental treatment started with a single 60-min. baseline session intended to mea-
sure general activity of each animal without the effect of the drug, and to habituate the rats to
the new context (before this session each animal was injected with vehicle). The next day
started the context conditioning stage. This phase comprised four 60-min sessions conducted
on consecutive days. Those animals in the Paired/0.5, and Paired/2.0 groups were injected
with the correspondent haloperidol dose before being introduced on the experimental context.
Immediately after each session, each animal was injected with an equivalent dose of vehicle
before to return to the home cage; those animals in the Unpaired/0.5, and Unpaired/2.0 groups
received the Vehicle before context exposure, and the drug just before to be returned to their
home cages. Mean percent of activity was registered for each conditioning session as an index
of sensitization.
A test session was conducted 48 hours after the last conditioning day, and consisted in
injecting the corresponding dose of vehicle for all rats and registering activity for 60 min. in
presence of the experimental context in periods of 10 min. The dependent variable used as an
index of conditioning was mean percent of activity.
Results
Mean percent activity during the baseline day collapsed across 60 min was 52.24% (range
28.99% - 73.94%). A 2 x 2 ANOVA (Conditioning x Dose) conducted on mean activity
revealed that neither the main effects nor the interaction was significant (all ps>.40).
Fig 1 shows mean activity across the four conditioning days as a function of groups. As can
be seen in the figure, those animals that were injected with haloperidol before context exposure
(Paired condition) showed a very low and stable percent of activity during all conditioning
days. Those animals injected with the drug after context exposure (Unpaired condition)
showed higher levels of activity that decreased across days, probably reflecting a habituation
process.
A 4 x 2 x 2 mixed ANOVA with main factors Days (within-subject), Conditioning (Paired
vs. Unpaired), and Dose (0.5 vs. 2.0) was conducted on mean percent activity collapsed across
each 60 min session. The main effects of Days and Conditioning were significant, F(3,84) =
9.21; p<.001; η
2
= .25, and F(1,28) = 357.90; p<.001; η
2
= .93, respectively. The main effect
of Days reflects a general reduction of activity across sessions, and the effect of Conditioning
was due to the overall lower levels of activity for those animals in the Paired as compared to
those in the Unpaired condition (Mean = 5.02%, SD = 3.35, and Mean = 35.74%, SD = 8.90,
Conditioned increase of locomotor activity induced by haloperidol
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respectively). The main effect of Dose was also significant, F(1,28) = 22.86; p<.001; η
2
= .45,
due to a higher level of activity for those animals that received the 0.5 mg/kg as compared to
those injected with the 2.0 mg/kg (Mean = 24.54%, SD = 19.31, and Mean = 16.22%,
SD = 13.56, respectively). Finally, the Days x Conditioning interaction was significant, F(3,84)
= 5.95; p<.01; η
2
= .18, reflecting a progressive reduction of activity across days that was
restricted to those animals that received the vehicle injection before context exposure. No
more interactions were significant (all ps>.06).
Fig 2 (panel A) shows mean percent activity during the test day collapsed across 10-min
periods as a function of Conditioning (Paired vs. Unpaired) and haloperidol Dose (0.5 vs. 2.0
mg/kg). Fig 2 (panel B) depicts mean activity collapsed across the entire session duration as a
function of Conditioning and Dose. As can be seen in the upper panel of the figure, mean per-
cent activity decreased across the test session, but it was higher for the animals in the Paired/
0.5 Group. Similarly, as can be seen in the bottom section of Fig 2, there was a general increase
in activity that was restricted to the group that had received the lower dose of the drug before
context exposure (Paired/0.5) as compared to the group that had received the drug after con-
text exposure (Unpaired/0.5).
A 6 x 2 x 2 ANOVA with main factors 10-min Periods (within-subjects), Drug, and Condi-
tioning performed on mean percent activity collapsed across 10 min periods revealed a signifi-
cant main effect of Periods, F(5,140) = 91.34; p<.001; η
2
= .77, due to the overall reduction of
activity across the session, and a significant Periods x Drug interaction, F(5,140) = 7.66; p<
.001; η
2
= .21, that reflects a faster decrease of activity across 10-min periods for the animals
Fig 1. Mean percent activity on conditioning days as a function of conditioning, and haloperidol dose. Percent activity
was collapsed across each 60 min session. The animals had received either 0.5 mg/kg or 2.0 mg/kg of haloperidol before (P:
Paired) or after (U: Unpaired) being introduced in the context-CS for 60 min.
https://doi.org/10.1371/journal.pone.0200178.g001
Conditioned increase of locomotor activity induced by haloperidol
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that received 2.0 mg/kg as compared to those that received the 0.5 mg/kg haloperidol dose. No
more interactions involving the Periods factor were significant (ps>.06). The analyses involving
the between-subject factors revealed significant main effects of Dose and Conditioning, F(1,28) =
19.47; p<.001; η
2
= .41, and F(1,28) = 21.47; p<.001, η
2
= .43, respectively. The main effect of
Dose reflects a significant higher percent of activity for those animals injected with the 0.5 mg/kg
dose as compared to those injected with the 2.0 mg/kg dose (mean = 50.94%, SD = 15.38, and
mean = 35.73%, SD = 11.33, respectively). The main effect of Conditioning reflects an overall
higher level of activity for the rats in the Paired as compared to those in the Unpaired condition
(mean = 51.33%, SD = 16.36, and mean = 35.35%, SD = 9.2, respectively).
Importantly, the Conditioning x Dose interaction was significant, F(1,28) = 8.09; p<.01,
η
2
= .22. Post-hoc comparisons comparing groups (Bonferroni, p<.05) revealed that the asso-
ciation between the context and the effect of the drug resulted in an increased activity at testing
as indicated for a significant difference between Paired/0.5 vs. Unpaired/0.5 groups, However,
the effect of context conditioning did not appear when the 2.0 mg/kg dose was injected, since
there were no significant differences between Paired/2.0 vs. Unpaired/2.0 groups. Also, per-
cent of activity was higher in the Paired/0.5 as compared to the Paired/2.0 group, but there
were no differences between groups in the Unpaired condition.
Experiment 2
The results of Experiment 1 revealed that after four pairings of a 0.5 mg/kg dose of haloperidol
with an initially neutral context, the latter acquired the ability to induce an increase in the overall
activity of the animals on a drug-free test trial. A possible explanation for this result from a non-
associative perspective is that haloperidol in the Paired condition had impeded proper processing
of the context during conditioning stage. Therefore, the context would have been functionally
novel at time of testing and it would have elicited non-habituated exploration responses. How-
ever, such interpretation is unlikely considering that the same result should have appeared in the
animals injected with the higher dose of haloperidol before context exposure, but locomotor
activity was similar at testing when comparing Paired/0.2 vs. Unpaired/0.2 groups.
Since this result not only fails to support our initial hypothesis, but also goes in the opposite
direction, we designed an additional experiment with two main purposes: to replicate the
result from Experiment 1, and to evaluate if an extinction treatment affects to the expected
conditioned increase in locomotor activity. Therefore, in the following experiment, two groups
were used that received exactly the same treatment described for the Paired/0.5 and Unpaired/
0.5 groups in Experiment 1, but four free-drug test trials were programmed in order to evalu-
ate the effect of an extinction process on the CR. Considering the results of the first experi-
ment, we now expect to find a conditioned increase in locomotor activity in the test phase for
the Paired group when compared with the Unpaired group, and a decrease of such response
across extinction days.
Subjects
16 male Wistar rats (n = 8) experimentally naïve, participated in this experiment. Mean weight
at the start of the experiment was 339 g. (range 459–266). The animals were housed and main-
tained as described for Experiment 1.
Fig 2. Mean percent activity on the drug-free test day as a function of conditioning, and haloperidol dose. (A) Percent
activity is represented collapsed across 10-min periods, and (B) across the complete 60-min session. The animals had received
either 0.5 mg/kg or 2.0 mg/kg of haloperidol during the four days of the conditioning stage before (P: Paired) or after (U:
Unpaired) been exposed for 60 min to the context-CS. Test session was drug-free.
https://doi.org/10.1371/journal.pone.0200178.g002
Conditioned increase of locomotor activity induced by haloperidol
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Apparatus, materials and procedure
The apparatus, materials, and procedure were the same described for the groups Paired/0.5
mg/kg, and Unpaired/0.5 mg/kg in Experiment 1, except that four free-drug tests trials, instead
of one, were conducted after conditioning stage.
Results
Mean percent of activity on the baseline day was 47.37% (Range: 25.82% - 65.67%). A one-way
ANOVA conducted on mean percent activity as a function of Groups revealed that the differ-
ences were non-significant (F<1).
Fig 3 depicts mean percent of motor activity collapsed across the 60 min for the four condi-
tioning days as a function of Groups (Paired vs. Unpaired). As can be seen in the figure, the
rate of activity was low and constant across the conditioning days for the Paired Group. The
animals in the Unpaired Group showed a high percentage of motor activity that decreased
across conditioning days reflecting the habituation to the contextual cues.
A 4 x 2 mixed ANOVA with main factors Days (within-subject) and Group (Paired vs.
Unpaired) was conducted on mean percent activity for each day (collapsed across the 60 min
of each trial duration). The main effect of Group was highly significant, F(1,14) = 288,64; p<
.001; η
2
= .95, due to the decrease in activity for those animals in the Paired as compared to
those in the Unpaired Group. This result confirmed the effectiveness of haloperidol to reduce
locomotor activity. The main effect of Days, and the Days x Groups interaction were also sig-
nificant, F(3,42) = 13.20; p<.001; η
2
= .49, and F(3,42) = 10.65; p<.001; η
2
= .43. The main
effect of Days reflects the overall decrease of activity across days, and the interaction was due
to a progressive reduction of activity across days for the animals in the Unpaired Group (due
to habituation) that contrast with the low and constant activity for the rats in the Paired
Group.
The top section of Fig 4 shows mean percent activity during the first test day collapsed
across 10-min periods as a function of Conditioning (Paired vs. Unpaired), and the bottom
section of the figure depicts mean percent of activity collapsed across the four 60 min free-
drug extinction days for the Paired and the Unpaired Groups. An inspection of the upper sec-
tion of the figure reveals that motor activity remained higher during all the 10-min periods in
the Paired as compared to the Unpaired Group. In addition, and as can be seen in the lower
section of Fig 4, there was a progressive decrease of locomotor activity across days in the Paired
Group that can be interpreted as a result of the extinction process.
A 6 x 2 mixed ANOVA with main factors Periods (within-subjects) and Group performed
on mean percent activity collapsed across 10 min periods for the first free-drug test day
revealed a significant main effect of Period, F(5,70) = 69.87; p<.001; η
2
= .83, due to the over-
all reduction of activity across the session, and a significant Period x Group interaction, F
(5,70) = 2.77; p<.05; η
2
= .17, due to a faster decrease of activity across 10-min periods for the
animals in the Unpaired as compared to those in the Paired Group. The main effect of Group
was also significant, F(1,14) = 10.95; p<.01; η
2
= .44, reflecting the higher level of activity for
the rats in the Paired as compared to those in the Unpaired condition (mean = 49.39%,
SD = 10.46, and mean = 31.49%, SD = 11.17, respectively). This result replicates the condi-
tioned increase of locomotor activity obtained in the Paired/0.5 Group from Experiment 1.
Additionally, a 4 x 2 mixed ANOVA with main factors Days and Group was performed on
mean percent activity in order to test whether the extinction procedure was effective in reduc-
ing the CR. The analysis revealed a significant main effect of Group, F(1,14) = 5.24; p<.05; η
2
= .27, due to the overall conditioned increase in activity showed for those animals in the Paired
as compared to the Unpaired Group. The main effect of Days was also significant, F(3,42) =
Conditioned increase of locomotor activity induced by haloperidol
PLOS ONE | https://doi.org/10.1371/journal.pone.0200178 October 3, 2018 8 / 14
9.82; p<.001; η
2
= .41, due to a general decrease of locomotor activity across days. Finally, the
2-way interaction was significant, F(3,42) = 3.50; p<.05; η
2
= .20, due to the progressive
decline in locomotor activity for the Paired Group reflecting the extinction of locomotor con-
ditioning across days.
General discussion
The results of Experiment 1, in which two different doses of the dopaminergic antagonist halo-
peridol were administered, were not consistent with the hypothesis of the conditioning of
drug-induced locomotor activity from which we anticipated a decrease in activity in the pres-
ence of the CS that had been paired with the dopaminergic antagonist [29–31]. Firstly, none of
the doses given to the Paired groups produced an effect of sensitization to the drug, since for
these animals the percentage of activity remained at low and constant levels from the first day
(perhaps a floor effect can have masked any sensitization effect). Also, it is possible that our
dependent variable (the general activity of the animal) was not sufficiently sensitive to repeated
administrations of the drug, since in the experiments in which this sensitization effect has
been observed, other indices of activity as the bar test [29] have been used. In contrast, in the
test phase a significant increase in locomotor activity was observed for those animals in the
Paired condition that had received the lowest dose of haloperidol (0.5 mg / kg) with respect to
the Unpaired control group. This same result was replicated in Experiment 2 that also revealed
that the conditioned increase of activity was affected by an extinction treatment.
Fig 3. Mean percent activity on conditioning days as a function of conditioning. Percent activity was collapsed across each
60 min session. The animals had received 0.5 mg/kg of haloperidol before (P: Paired) or after (U: Unpaired) being introduced
in the context-CS for 60 min.
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In view of these results, we can conclude that the repeated pairing of a neutral stimulus (in
our case the experimental context) with the administration of a 0.5 mg / kg dose of haloperidol
produces a conditioned increase in locomotor activity during a subsequent test phase con-
ducted in the experimental context. Some authors have proposed that this type of response
could be the result of a non-associative process, since the administration of dopaminergic ago-
nist drugs prior to exposure to the context could hinder the processing of the latter, so that the
context would be functionally new at the time of testing and would thus elicit the same orienta-
tion responses that would be expected in response to a novel context [34,35]. However, this
possibility is unlikely attending to the results of the groups that received a 2.0 mg/kg dose of
haloperidol in Experiment 1, since from this perspective the higher dose of haloperidol should
have induced a similar o even a bigger increase in locomotor activity at testing as that observed
in the Group that received the 0.5 mg/kg dose. However, there were no significant differences
between the Paired vs. the Unpaired Group that received the highest antagonist’s dose, indicat-
ing that the hypothetical reduced processing of the context during the conditioning stage can
not explain the increased activity observed during testing.
However, an interpretation of our results in terms of reduced context processing cannot be
completely discarded since, at testing period, activity simultaneously could have tended to
increase due to impaired context processing induced by haloperidol, and could have tended to
be reduced due to conditioning. One of these mechanisms might have been dominant for the
group that received the lower haloperidol dose, while the other might have gained expression
in the animals that received the higher dose. Further research is warranted to evaluate this
possibility.
A second possible account of the origin of the increase in locomotor activity observed after
pairing the context with a drug can be established in strictly Pavlovian terms, based on the
assumption that the CS is a stimulus that acquires the same properties as the US and, therefore
evokes the same type of responses after the conditioning process [36,37], but see [38]. This the-
ory of stimulus substitution [8] seems to be at a first glance difficult to conciliate with our
results, since the observed CR (an increase in locomotor activity) is opposite to the UR (a
reduction of locomotor activity). However, the fact that the repeated administration of a low
dose of haloperidol has proved effective in inducing an increase in locomotor activity [30]
makes it possible to reconcile our results with this classical conditioning perspective.
More specifically, there is ample evidence to suggest that the main pharmacological action
of haloperidol consists of the blockade of D2 receptors, some of which are autoreceptors
located in terminals and dopaminergic dendrites, while others are located postsynaptically in
the soma, dendrites, and terminals [39]. Haloperidol at medium or high doses, by blocking
presynaptic D2 receptors (autoreceptors), increases the release of dopamine [40], but the
increase in dopaminergic transmission is nullified by the blockade of post-synaptic D2 recep-
tors. However, at low doses, haloperidol exerts its antagonist action only in the autoreceptors,
and not by blocking the post-synaptic receptors, since the concentration of drug required to
produce antagonist action in the post-synaptic site would be greater [41]. As we have indicated
above, Dias et al [30] showed that the repeated administration of a very low dose of haloperidol
(0.03 mg / kg) produces an increase in locomotor activity that could be related to the selective
blockade of presynaptic autoreceptors. In the same study, a high dose of haloperidol (1.0 mg /
Fig 4. Mean percent activity on the drug-free test day as a function of conditioning, and haloperidol dose. (A) Percent
activity is represented collapsed across 10-min periods, and (B) across the complete 60-min session. The animals had
received 0.5 mg/kg of haloperidol during the four days of the conditioning stage before (condition P: Paired) or after
(condition U: Unpaired) been exposed for 60 min to the context-CS. Test session was drug-free.
https://doi.org/10.1371/journal.pone.0200178.g004
Conditioned increase of locomotor activity induced by haloperidol
PLOS ONE | https://doi.org/10.1371/journal.pone.0200178 October 3, 2018 11 / 14
kg) caused an inhibitory effect on locomotion that could be related to the blockade of post-syn-
aptic D2 receptors.
Albeit speculative, based on the fact that in our experiments with haloperidol we used only
4 pairings of the context-CS and the drug-US, compared to the 8 pairings in the experiments
of Schmidt & Beninger [23], or the 10 employed by Banasikowski y Beninger [29] or Dias et al.
[30], and taking into account that the CR is usually of a lower intensity than the UR [42,43],
we suggest that in our experiments the presentation of the context associated with the 0.5 mg /
kg dose of haloperidol may have led to a low intensity CR that would have been functionally
equivalent to the response induced by a low dose of haloperidol. This CR could have blocked
the presynaptic dopamine autoreceptors, preventing the feedback mechanism that would limit
the release of the neurotransmitter, and leading to an increase in dopamine. However, the
post-synaptic blocking would have been less effective and the excess of dopamine could have
caused the conditioned increase in locomotor activity [36].
Supporting information
S1 File. Mean percent of freezing along sessions for Experiment 1 (SPSS file).
(SAV)
S2 File. Mean percent of freezing along sessions for Experiment 2 (SPSS file).
(SAV)
Acknowledgments
The authors thank to Fe
´lix Hermoso and Francisco Jose
´Pe
´rez Dı
´az for their useful comments
and help in running the experimental sessions.
Author Contributions
Conceptualization: Luis Gonzalo De la Casa, Lucı
´a Ca
´rcel, Juan Carlos Ruiz-Salas, Lucı
´a
Vicente, Auxiliadora Mena.
Formal analysis: Luis Gonzalo De la Casa, Lucı
´a Ca
´rcel, Juan Carlos Ruiz-Salas, Lucı
´a Vicente,
Auxiliadora Mena.
Funding acquisition: Luis Gonzalo De la Casa.
Investigation: Luis Gonzalo De la Casa, Lucı
´a Ca
´rcel, Juan Carlos Ruiz-Salas, Lucı
´a Vicente,
Auxiliadora Mena.
Methodology: Luis Gonzalo De la Casa, Lucı
´a Ca
´rcel, Juan Carlos Ruiz-Salas, Lucı
´a Vicente,
Auxiliadora Mena.
Project administration: Luis Gonzalo De la Casa.
Resources: Luis Gonzalo De la Casa.
Software: Juan Carlos Ruiz-Salas.
Supervision: Luis Gonzalo De la Casa.
Writing – original draft: Luis Gonzalo De la Casa, Lucı
´a Ca
´rcel, Juan Carlos Ruiz-Salas, Aux-
iliadora Mena.
Writing – review & editing: Lucı
´a Vicente.
Conditioned increase of locomotor activity induced by haloperidol
PLOS ONE | https://doi.org/10.1371/journal.pone.0200178 October 3, 2018 12 / 14
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