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Neurobiology of Learning and Memory 87 (2007) 510–521
www.elsevier.com/locate/ynlme
1074-7427/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nlm.2006.11.002
Electrical stimulation of the pedunculopontine tegmental nucleus
in freely moving awake rats: Time- and site-speciWc eVects
on two-way active avoidance conditioning
Raül Andero, Meritxell Torras-Garcia, María Fernanda Quiroz-Padilla,
David Costa-Miserachs, Margalida Coll-Andreu ¤
Institut de Neurociències, Departament de Psicobiologia i de Metodologia de les Ciències de la Salut, EdiWci B,
Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain
Received 28 July 2006; revised 2 November 2006; accepted 3 November 2006
Available online 13 December 2006
Abstract
The pedunculopontine tegmental nucleus (PPTg) is involved in the regulation of thalamocortical transmission and of several functions
related to ventral and dorsal striatal circuits. Stimulation of the PPTg in anesthetized animals increases cortical arousal, cortical acetylcholine
release, bursting activity of mesopontine dopaminergic cells, and striatal dopamine release. It was hypothetized that PPTg stimulation could
improve learning by enhancing cortical arousal and optimizing the activity of striatal circuits. We tested whether electrical stimulation (ES) of
the PPTg, applied to freely-moving awake rats previously implanted with a chronic electrode, would improve the acquisition and/or the
retention of two-way active avoidance conditioning, and whether this eVect would depend on the speciWc PPTg region stimulated (anterior vs
posterior) and on the time of ES: just before (pre-training) or after (post-training) each of three training sessions. The treatment consisted of
20 min of ES (0.2 ms pulses at 100Hz; current intensity: 40–80A). The results showed that (1) this stimulation did not induce either any signs
of distress nor abnormal behaviors, apart from some motor stereotyped behaviors that disappeared when current intensity was lowered; (2)
pre-training ES applied to the anterior PPTg improved the acquisition of two-way active avoidance, (3) no learning improvement was found
after either post-training ES of the anterior PPTg, or pre- and post-training ES of the posterior PPTg. The results give support to a role of
PPTg in learning-related processes, and point to the existence of functional PPTg regions.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Pedunculopontine tegmental nucleus; Mesopontine tegmentum; Two-way active avoidance; Electrical stimulation; Rat
1. Introduction
The pedunculopontine tegmental nucleus (PPTg) is con-
sidered a component of the ascending reticular activating
system, and as such modulates thalamocortical transmis-
sion during awake states and paradoxical sleep (Steriade,
Datta, Pare, Oakson, & Curro Dossi, 1990). It is also exten-
sively connected with striatal structures, and it has even
been posed whether it could be considered a striatal struc-
ture itself (Mena-Segovia, Bolam, & Magill, 2004). This
nucleus plays a role in several cognitive functions, probably
through its relationship with thalamic and striatal circuits,
as suggested by the fact that bilateral PPTg lesions disrupt
attentional mechanisms (Inglis, Olmstead, & Robbins,
2001; Kozak, Bowman, Latimer, Rostron, & Winn, 2005)
and impair several learning tasks (Winn, 2006). The nature
of the learning tasks that are disrupted after PPTg damage
is not clear, but tasks that depend on the activation of the
striatum or of cortico-striatal circuits could be specially
susceptible to this damage (Keating & Winn, 2002; Satorra-
Marin, Coll-Andreu, Portell-Cortes, Aldavert-Vera, &
Morgado-Bernal, 2001; Satorra-Marin, Homs-Ormo, Are-
valo-Garcia, Morgado-Bernal, & Coll-Andreu, 2005; Tay-
lor, Kozak, Latimer, & Winn, 2004). In turn, one might
*Corresponding author. Fax: +34 935812001.
E-mail address: Margalida.Coll@uab.es (M. Coll-Andreu).
R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521 511
expect that activation of this nucleus on certain periods of
the learning process could facilitate the acquisition and/or
the retention of several tasks.
At least from a theoretical point of view, activation of
the PPTg can be attained through electrical or chemical
stimulation. It has been found that electrical stimulation
(ES) of the PPTg in anesthetized rats (1) increases cortical
arousal (Detari, Semba, & Rasmusson, 1997; Dringenberg
& Olmstead, 2003; Rasmusson, Clow, & Szerb, 1994), an
eVect which seems mediated by a synergic action of this
nucleus on basal forebrain and thalamus (Dringenberg &
Olmstead, 2003), (2) enhances cortical acetylcholine release,
at least in part by activation of the nucleus basalis magno-
cellularis (Rasmusson et al., 1994), (3) stimulates dopamine
release into the striatum (Forster & Blaha, 2003), (4)
induces increased activity of substantia nigra cells of both
the pars compacta and pars reticulata (Scarnati, Campana,
& Pacitti, 1984), and increases burst Wring in those dopami-
nergic substantia nigra neurons that are already active
(Lokwan, Overton, Berry, & Clark, 1999). In turn chemical
stimulation of the PPTg through enhanced glutamatergic
transmission (1) increases either awakeness or paradoxical
sleep, in a dose-dependent manner (Datta, Patterson, &
Spoley, 2001a; Datta, Spoley, & Patterson, 2001b), and
increases c-fos expression in several thalamic nuclei (Ainge,
Jenkins, & Winn, 2004). Also chemical activation of the
cholinergic and glutamatergic PPTg neurons projecting to
dopaminergic systems (by means of bicuculline) increases
the frequency of bursting activity in ventral tegmental area
dopaminergic neurons (Floresco, West, Ash, Moore, &
Grace, 2003).
With regard to behavioral eVects, electrical stimulation
(ES) of the PPTg in decerebrate cats induces either stepping
or muscle tone suppression, depending on the frequency of
stimulation (Garcia-Rill, Skinner, Miyazato, & Homma,
2001; Lai & Siegel, 1990; Takakusaki, Habaguchi, Saitoh,
& Kohyama, 2004), as well as enhanced locomotion in
awake rats with no brain transection (Milner & Mogenson,
1988). In an awake macaque high frequency ES of the PPTg
reduced motor activity, while low frequency ES increased it.
When the macaque was made Parkinsonian with MPTP,
unilateral low frequency ES of the PPTg led to signiWcant
increases in activity (Jenkinson, Nandi, Miall, Stein, & Aziz,
2004). Low-frequency ES of the PPTg has even been tried
in two human patients with advanced Parkinson’s disease,
which showed improved motor function and no complica-
tions after this treatment (Plaha & Gill, 2005).
On the grounds of the reported electrophysiological and
neurochemical eVects (cortical activation, increased cortical
acetylcholine and enhanced dopaminergic transmission) of
PPTg stimulation, as well as on the eVects of PPTg lesions
on learning tasks, we hypothetized that ES of the PPTg
might improve implicit learning tasks, and even would be
capable of reverting some of the deleterious eVects of brain
damage and/or aging. Given the fact that the integrity of
the nucleus seems specially required for the acquisition of
certain implicit learning tasks, such as two-way active
avoidance (Satorra-Marin et al., 2001), but do not seem
essential once learning has been achieved (Fujimoto, Ikegu-
chi, & Yoshida, 1992), it was expected that the facilitative
eVects would be higher with ES treatments applied in a
pre-training vs a post-training basis.
However, before testing this hypothesis, we had to Wnd
appropriate ES parameters feasible to be used with freely-
moving awake rats and devoid of overt motor eVects, as
well as of other undesirable side-eVects, such as convulsions
or signs of distress. This was required because PPTg ES and
chemical stimulation of the PPTg have been usually applied
in decerebrate and/or anesthetized animals, except for a few
works, where PPTg chemical stimulation (Ainge et al.,
2004) or ES (Milner & Mogenson, 1988) have been admin-
istered to freely moving rats. To that end, a pilot study was
undertaken. The results of this pilot study allowed us to
Wnd ES parameters devoid of overt side-eVects (apart from
occasional motor eVects that could be eliminated when the
current intensity was reduced), and, to our surprise, also
suggested the existence of putative site-speciWc eVects of
PPTg ES on learning. Thus the performance, on a two-way
active avoidance conditioning, of the rats that had the elec-
trode implanted within the nucleus at coordinates between
6.80 and 7.80 mm posterior to Bregma (a region here
deWned as “anterior PPTg”) tended to be better than that of
the animals that had the electrode at more caudal coordi-
nates (here deWned as “posterior PPTg”).
Therefore, the present work was aimed at studying (1)
whether ES of the PPTg, using parameters that are safe
when applied to awake rats, can facilitate the acquisition
and/or retention of two-way active avoidance conditioning,
an implicit task dependent on the striatal system; (2)
whether the eVects of PPTg ES depend on the time of
administration: before the training sessions (pre-training
ES), or after them (post-training ES); and (3) whether ES of
the anterior vs the posterior PPTg gives rise to diVerent
eVects on learning.
2. Methods
Two experiments were carried out. The aim of Experiment I was to test
the inXuence of pre-training ES of anterior and posterior PPTg on the per-
formance of two-way active avoidance conditioning, while the aim of
Experiment II was to test the eVects of post-training ES of anterior and
posterior PPTg on the same learning task.
All the procedures used in this work have been done in compliance
with the European Community Council Directive for care and use of labo-
ratory animals (86/609/EEC) and with the related Directive of the Auton-
omous Government of Catalonia (DOGC 2073 10/7/1995), and have been
approved by the Ethics Committee of our University.
2.1. Subjects
The subjects were naive male albino Wistar rats obtained from our
laboratory breeding stock. Seventy three rats, with a mean age of
103.49 days (SD D8.23) and a mean weight of 466.18 g (SD D30.90) at
the beginning of the experiment, were used in Experiment I, while the
number of animals used in Experiment II was seventy four, with a
mean age of 113.08 days (SD D13.47). and a mean weight of 442.65 g
(SD D38.83).
512 R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521
All the subjects were singly housed, always kept under conditions of
controlled temperature (20–23 °C) and humidity (40–70%) and subjected
to an artiWcial light–darkness cycle of 12–12 h (lights on at 8.00 A.M.).
Food and water were available ad libitum.
2.2. Experimental groups
Experiment I (pre-training condition): At the beginning of the experi-
ment, the subjects were randomly assigned to one of the following four
experimental groups: Anterior-ES, Anterior-Control, Posterior-ES, and
Posterior-Control groups. The ES groups received ES to either the ante-
rior (Anterior-ES group) or the posterior (Posterior-ES group) PPTg
before each of the three training sessions. The Control groups had elec-
trodes implanted at either the anterior (Anterior-Control group) or the
posterior PPTg (Posterior-Control group), but never received the ES treat-
ment.
Experiment II (post-training condition): The animals of Experiment II
were distributed into the same four experimental groups described for
Experiment I (Anterior-ES, Anterior-Control, Posterior-ES, and Poster-
ior-Control), but they received the ES or control treatments in a post-
training basis (immediately after each of the three training sessions), and
not in a pre-training basis as in Experiment I.
2.3. Handling procedures
Prior to surgery, all the animals were subjected to handling procedures,
in order to habituate them to being manipulated by the experimenters.
Handling consisted of weighing the animals and, thereafter, picking them
up with a gloved hand and placing them in an upright position. Rats were
picked up this way and returned to the home cage three times over a
period of 2 min. Those handling procedures were carried out on four
consecutive days.
2.4. Surgery
Stereotaxic surgery was carried out under general anesthesia with i.p.
ketamine chlorhydrate (ketolar®, Parke-Davis, 110 mg/kg) plus xylazin
(Rompun®, Bayer, 0.2 ml/kg). Stainless steel monopolar electrodes made
with insulated electrode wire (Plastics One, 0.15 mm diameter) welded to a
connector, were inserted aimed at the PPTg, in the right hemisphere, either
at the anterior half of the nucleus (Anterior-ES and Anterior-Control
groups) or at the Posterior part of the nucleus (Posterior-ES and Poster-
ior-Control groups). Electrodes were anchored to the skull with jeweller
screws and dental cement. In the rats that had to receive ES, a copper wire
welded to one of the screws as well as to the connector served as ground.
The incisor bar was set at ¡2.7 mm below the interaural line and the ste-
reotaxic coordinates used were the following ones, according to the stereo-
taxic atlas of Paxinos and Watson (1997): (1) Anterior location: AP: +1.3
from interaural line; L: +1.6 from midline; and P: ¡7.8 mm from the cra-
nium surface; (2) Caudal location: AP: +0.8 from interaural line; L: + 1.7
from midline; and P: ¡8.00 from the cranium surface.
2.5. Intracranial electrical stimulation (ES) treatment
The treatment was applied in a cage (26.5 £30.5 £36cm) constructed
of plexiglas and located inside an isolation wooden cage with a plexiglas
window that allowed the rats to be observed. The electrical current deliv-
ered by a stimulator (Model CS-20, Cibertec, Madrid, Spain) consisted of
square-wave pulses of 0.2 ms duration and a frequency of 100 Hz, that
were continuously administered during 20min. The intensity of current
ranged from 40 to 80 A, depending on the appearance of stereotyped
motor behaviors. Thus, the initial ES was 80 A, which was progressively
reduced if the animal showed abnormal reactions until those behaviors
completely disappeared. Control rats were placed in the same cage for
20 min, and the electrode clip was connected, but they never received any
ES. The ES or control sessions were administered immediately before each
of the three acquisition sessions (see below).
2.6. Two-way active avoidance task
Following surgical recovery (7–8 days), the animals were given three
sessions of two-way active-avoidance conditioning (30 trials each), sepa-
rated by 24 h. Prior to the Wrst training session, the rats were allowed to
freely ambulate in the shuttle-box for 10 min in order to become familiar-
ized with the training box. Active-avoidance testing was conducted in a
50 £24 £23 cm two-way automated shuttle-box (Letica L1-916) made of
plexiglas and subdivided into two virtual compartments with indepen-
dently electriWed stainless steel bars as Xoors, but without any physical
separation between them. The CS was a 60-dB, 1-kHz tone, of 3 s. When
the CS was on the animals had to cross to the other side of the shuttle-box
apparatus (avoidance response) in order to turn it oV and avoid the
appearance of the unconditioned stimulus (US). The US, which consisted
of a 100-Hz positive half-wave constant current of 0.5 mA intensity, was
turned oV when the animal made an escape response. The intertrial sched-
ule was a 1-min variable interval (ranging from 50 to 70 s). The shuttle-box
was connected to a computer that controlled the training schedule and
scored the number of avoidance and escape responses, the latency of
responses, and the number of crossings between the two sides of the shut-
tle box that the animals made. The overt behavior of the animals while in
the conditioning cage was also observed through a transparent red
plexiglas window on the front side of the isolation cage.
Twelve days after the third acquisition session, all the rats were sub-
jected to another session of two-way active avoidance in order to test the
level of long-term retention (LTR). This session was also composed of 30
trials and was preceded by a short period of free ambulation (1 min) before
the onset of the Wrst CS.
In Experiment I (pre-training condition), 20 min of ES or control treat-
ments were administered immediately before the 10-min habituation ses-
sion to the shuttle-box, that in turn was immediately followed by the Wrst
training session, as well as immediately before the second and third train-
ing sessions. In Experiment II (post-training condition), the ES or control
treatments were administered immediately after each of the three training
sessions. In contrast to the training sessions, the LTR session was neither
preceded nor followed by the ES or control treatments in any of the
groups.
2.7. Histology
At the conclusion of the experiment, the rats were killed with an over-
dose of sodium pentobarbital and perfused transcardially with 0.9% physi-
ological saline followed by 10% formalin. The brains were removed and
stored in 10% formalin before being sectioned (40 micron) on a cryostat
(CRYOCUT 1800, with microtome 2020, JUNG). The sections were then
processed for cresyl violet stain. The brain sections were examined onto
the microscope to determine the electrode tracks and the position of the
electrode tips.
2.8. Statistical analyses
The statistical analyses were made separately for the four pre-training
(Experiment I) and the four post-training groups (Experiment II). In both
experiments, the data from the three training sessions were analyzed sepa-
rately from that of the LTR session, given that the latter was not preceded or
followed by the treatment, and was separated by 12 days from the last train-
ing session. The data from the three training sessions were analyzed by
means of mixed multivariate analyses of variance (MANOVA; SPSS for
Windows) with a 2 £2£3 design: (1) Treatment [two categories: Electrical
stimulation (ES) and Control] £Electrode location (two categories: Ante-
rior and Posterior) £Session (three within-subject measures, corresponding
to the three acquisition sessions). The data from the LTR session were ana-
lyzed by means of multivariate analyses of variance (MANOVA; SPSS for
Windows) with a 2 £2 design (Treatment £electrode location). With regard
to the avoidance data on the LTR session, analyses of covariance were used,
with the data on the third training session as covariate. SigniWcance was set
at the level of p6.050. Other analyses were also carried out when needed.
R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521 513
3. Results
3.1. Histological analyses, general observations and body
weight
The histological preparations were examined separately
by two persons who were not aware either of the group or
of the performance of the subjects. Fig. 1 illustrates the
location of electrode tips for the subjects in each of the four
experimental groups subjected to pre-training treatments
(Experiment I), reconstructed onto Wgures of Paxinos and
Watson atlas (Paxinos & Watson, 1997). Fig. 2 illustrates
the location of electrode tips for the subjects in each of the
four experimental groups subjected to the post-training
treatments (Experiment II). Assignment of subjects to the
anterior or posterior groups was made taking into account
the actual location of electrode tips, not the intended
implanted area. Subjects with electrode tips outside the
PPTg were excluded from the statistical analyses. The Wnal
sample consisted of forty-eight rats in the pre-training con-
dition, distributed into the four groups described under
Procedures: Anterior-ES (nD13), Anterior-Control (nD9),
Posterior-ES (nD13), and Posterior-Control (nD13), and
forty-one rats in the post-training condition: Anterior-ES
(nD8), Anterior-Control (nD14), Posterior-ES (nD10),
and Posterior-Control (nD9) groups.
In either experiment, no diVerences among groups were
found in the body weight of the animals at any stage, either
before or after the surgical procedures.
3.2. EVects of electrical stimulation of the PPTg on gross
behavior
Several rats showed rotation or slight stereotyped
behaviors after initiation of the Wrst ES session. Rotation
consisted in contralateral pivoting, as described by Ikeda
et al. (2004): head-to-tail turns with very small diameter
and abnormal hindlimb stepping, characterized by the
sequential occurrence of a closing and an open step. The
stereotyped behaviors consisted in swing movements of the
hindlimb and, in a few occasions, oral stereotypies (mainly
biting). Both the rotation and the stereotyped behaviors
disappeared when the current intensity was lowered. Thus,
some animals could receive intensities of 80A, while oth-
ers had to receive lower intensities in order to avoid motor
eVects. The lower current intensity applied was 40 A. None
of the animals showed seizures or any sign of distress, as
well as any other anomalous behaviors.
SpeciWcally, in the pre-training condition rotation and/or
stereotypies were observed in two rats in Anterior-ES
group (which subsequently received current intensities of 40
and 60 A, respectively), and in 5 rats in Posterior-ES
group (the current intensities subsequently applied were
40 A in three of them and 60A in the remaining two). In
the post-training condition, rotation and stereotypies were
observed in three rats in Anterior-ES group (two of them
subsequently received current intensities of 40 A, while the
remaining one received an intensity of 60 A), and in one
rat in Posterior-ES group (that subsequently received a
Fig. 1. Electrode tip locations for Anterior-ES, Anterior-Control, Posterior-ES and Posterior-Control groups of Experiment I (pre-training treatment).
The electrode tips of one subject in Posterior-ES and three subjects in Posterior-Control groups were located into the PPTg between 8.30 and 8.50 mm pos-
terior to Bregma, and are not represented here. Coronal sections correspond to brain sites between ¡6.80 and ¡8.30 mm posterior to Bregma. Drawings
taken from (Paxinos & Watson (1997)).
514 R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521
current intensity of 50A). The intensity of current applied
to the remaining animals (that did not show stereotyped
behaviors) was 80 A.
3.3. Acquisition and LTR of two-way active avoidance:
Avoidance responses
Given the fact that the number of avoidance responses
followed a binomial distribution, a transformed variable
(arcsine square root of the proportion of avoidance
responses) was used for the statistical analyses in order to
normalize the variance.
3.3.1. Experiment I (pre-training conditions)
Fig. 3A depicts the mean proportion of avoidance
responses made by Anterior-ES and Anterior-Control
groups (left), and Posterior-ES and Posterior-Control groups
(right) on the acquisition and LTR sessions in Experiment I.
MANOVA analyses for the three training sessions indicated
that neither the treatment nor the electrode location had any
signiWcant eVect by themselves on avoidance proportion;
however, there was a signiWcant interaction between Treat-
ment and Electrode location [F(44,1) D5.87; pD.020]. The
session factor was also signiWcant [F(88,2) D52.58; p< .001].
Polynomial contrasts indicated that the evolution of avoid-
ance responses throughout the successive training sessions
Wtted a quadratic function [F(44,1)D4.62; pD.037], i.e. an
ascending evolution with one inXection, in all the groups.
To clarify the meaning of the signiWcant interaction
between Treatment and Electrode location, we carried out
analyses of nested eVects, which allow determining the
inXuence of the treatment separately for each electrode
location. Those analyses indicated that the treatment had a
signiWcant eVect on the number of avoidance responses
only when the electrode was located at the anterior PPTg,
but not when the electrode was located at the posterior
PPTg. SpeciWcally, the proportion of avoidance responses
was signiWcantly higher in Anterior-ES group than in Ante-
rior-Control group [F(45,1) D5.24; pD.027], while there
were not signiWcant diVerences between the Posterior-ES
and Posterior-Control groups.
We also calculated the proportion of subjects in each
group that reached a predeWned learning criterion (24 out
of 30 avoidance responses in at least one of the three train-
ing sessions or the LTR session). All the subjects in Ante-
rior-ES group reached this criterion, while this proportion
was 70% in Anterior-Control, 36.6% in Posterior-ES, and
76.92% in Posterior-Control groups. Fisher’s exact test
indicated that the diVerences in the proportions of subjects
that reached the learning criterion in Anterior-ES group and
Anterior-Control group did not reach signiWcance (Fisher’s
exact test, two-sidedD0.068). In contrast, the proportion of
subjects reaching the learning criterion in Posterior-ES group
was signiWcantly lower than that in Posterior-Control group
(Fisher’s exact test, two-sided D0.047).
The diVerence in the proportion of avoidance responses
between the third training session and the LTR session was
not signiWcantly aVected by any of the factors or their inter-
action. Analyses of covariance of the avoidance data on the
LTR session, with the arcsine square root of the proportion
Fig. 2. Electrode tip locations for Anterior-ES, Anterior-Control, Posterior-ES and Posterior-Control groups of Experiment II (post-training treatment).
Coronal sections correspond to brain sites between ¡6.80 and ¡8.30 mm posterior to Bregma. Drawings taken from Paxinos and Watson (1997).
R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521 515
of avoidance responses on the third session as covariate,
indicated that the covariate was signiWcant [F(43,1) D11.84;
pD.001], but none of the main factors or their interaction
was. Thus, the proportion of avoidance responses on the
LTR was inXuenced by that on the third training session,
without further inXuence of the experimental conditions.
3.3.2. Experiment II (post-training conditions)
Fig. 3B depicts the mean proportion of avoidance
responses made by Anterior-ES and Anterior-Control
groups (left), and Posterior-ES and Posterior-Control
groups (right) of Experiment II (post-training conditions)
on the three acquisition sessions and the LTR session. As in
Experiment I, for the statistical analyses a transformed var-
iable (arcsine square root of the proportion of avoidances)
was used to normalize variances given the binomial distri-
bution of avoidance responses. MANOVA analyses indi-
cated that neither the treatment nor the electrode location
or the interaction between those two main factors were sig-
niWcant. The session factor was signiWcant [F(74,2) D29.25;
p< .001], and Polynomial contrasts indicated that the evo-
lution of avoidance responses across the three training ses-
sions followed a signiWcant linear ascending trend [tD6.48;
p< .001] in all the groups.
The diVerences in the proportion of avoidance responses
between the third training session and the LTR session
were not signiWcantly aVected by either factor or their inter-
action. Analyses of covariance of the avoidance data on the
LTR session, with the arcsine square root of the proportion
of avoidance responses on the third session as covariate,
indicated that the covariate was signiWcant
[F(36,1) D207.36; p< .001], but none of the main factors or
their interaction was. Thus, the proportion of avoidance
responses on the LTR was inXuenced by that on the third
training session, but was not inXuenced by either the treat-
ment or the electrode location.
The proportion of subjects that reached the learning cri-
terion (24 out of 30 avoidance responses in at least one of
the three training sessions or the LTR session) was 62.5% in
Anterior-ES, 46.15% in Anterior-Control, 66.67% in Poster-
ior-ES and 40% in Posterior-Control. Fisher’s exact
test (two-sided) indicated that there was no signiWcant rela-
tionship between the treatment and the proportion of sub-
jects reaching the learning criterion, either when comparing
Anterior-ES and Anterior-Control groups or when
comparing Posterior-ES and Posterior-Control groups.
3.4. InXuence of abnormal motor behaviors and current
intensity on the number of avoidance responses
ANOVA indicated that there were no signiWcant diVer-
ences in the proportion of avoidance responses on any of
the training sessions (again, the arcsine square root was
used for the analyses) between the rats that showed rota-
tion and/or stereotypies and those that did not, neither in
Experiment I or in Experiment II. However, in Experiment
I, a signiWcant correlation was found between the current
intensity applied (which, in turn, was inXuenced by the
presence or absence of abnormal motor behaviors), and the
proportion of avoidance responses on the Wrst training ses-
Fig. 3. Mean proportion of avoidance responses (+SEM) on each training
sessions in the pre-training (upper Wgures), and the post-training (lower
Wgures) conditions. The statistical analyses (made with the arcsine square
root of the proportion of avoidance responses) indicated, in the pre-train-
ing condition, the proportion of avoidance responses on the three training
sessions was signiWcantly higher in Anterior-ES group than in Anterior-
Control group (upper Wg. left). In contrast, no statistical diVerences were
found, on the pre-training condition, between Posterior-ES and Posterior-
Control groups (upper Wg., right). DiVerences were neither found on the
post-training conditions, either between Anterior-ES and Anterior-
Control groups (lower Wg., left), or between Posterior-ES and Posterior-
Control groups (lower Wg., right). *pD.027.
516 R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521
sion (Pearson correlation D0.428; pD.029), but not on any
other training session. No correlation between those vari-
ables was found in the post-training condition (Experiment
II).
3.5. Locomotion: Number of crossings during adaptation to
the shuttle-box and during training
3.5.1. Experiment I (pre-training conditions)
Analyses of variance indicated that the number of cross-
ings between the two compartments of the training box
during habituation to it (prior to the Wrst training session
and, thus, immediately after the Wrst ES or control treat-
ment) was not signiWcantly inXuenced by the main factors
Treatment or Electrode Location alone. However, there
was a signiWcant interaction between those two main fac-
tors [Treatment £Electrode location: F(44,1) D4.12;
pD.048]. Nested eVects analyses indicated that in control
animals, electrode location tended to have an inXuence on
the number of crossings during habituation (pD.058). Spe-
ciWcally in control animals the number of crossings was
higher (although not signiWcantly) when the electrode was
in the Posterior PPTg than when it was in the Anterior
PPTg.
With regard to the number of intertrial crossings in each
of the three training sessions, multivariate analyses of vari-
ance indicated that none of the main factors (Treatment,
Electrode location and Session) and none of the interac-
tions between them were signiWcant. Also, neither the main
factors, nor their interaction, were signiWcant with regard to
the number of intertrial crossings on the LTR session.
However, the diVerence between the number of crossings
on the LTR session and that on the third training session
indicated a signiWcant inXuence of electrode location
[F(44,1) D6.80; pD.012], with the animals with the elec-
trode tip into the anterior PPTg showing and increase in
the number of intertrial crossings on the LTR, while this
variable showed a decrease when the electrode was located
into the posterior PPTg. Thus, in spite of the lack of signiW-
cant between-group diVerences in this variable on the LTR
session, its evolution between the third training session and
the LTR session was diVerent depending on the implanted
PPTg area.
3.5.2. Experiment II (post-training conditions)
Analyses of variance indicated that the number of cross-
ings between the two compartments of the training box
during habituation to it (prior to the Wrst training session)
was not signiWcantly inXuenced by the main factors Treat-
ment or Electrode Location, and there was no interaction
among those two factors.
With regard to the number of intertrial crossings in each
of the three training sessions, multivariate analyses of vari-
ance indicated that neither of the main factors (Treatment,
Electrode location and Session) nor the interactions
between them were signiWcant. No signiWcant main factors
or interactions were found with regard to the number of
intertrial crossings on the LTR session, as well as with
regard to the diVerence in the number of intertrial crossings
between the last training session and the LTR session.
3.6. Latency of avoidance responses
3.6.1. Experiment I (pre-training conditions)
Fig. 4A shows the mean latencies of avoidance responses
for each experimental group on each of the training ses-
sions. Multivariate analyses of variance indicated that the
main factors Treatment and Electrode Location were not
signiWcant, but the interaction between those two factors
was [F(44,1) D6.03; pD.018]. The Session factor
[F(88,2) D14.83; p< .001], and the interaction between Elec-
trode location and Session [F(88,2) D5.08; pD.008] were
also signiWcant. To clarify those multiple interactions, we
performed nested analyses combined with simple eVects
analyses. Those analyses indicated that in control subjects
(but not in ES subjects) there was a signiWcant interaction
Fig. 4. Mean avoidance latencies (+SEM) for each experimental group on
each of the conditioning sessions, in the pre-training (A) and the post-
training (B) conditions. In Experiment I, avoidance latencies were signiW-
cantly higher in Anterior-Control than in Posterior-Control groups on the
Wrst and third training sessions. In contrast, on the LTR session, Anterior-
ES animals had signiWcantly lower latencies than Posterior-ES animals.
With regard to the post-training condition, Anterior-ES rats had signiW-
cantly higher avoidance latencies than Anterior-Control rats on the three
training sessions. *p< .05.
R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521 517
between Electrode location and Session [F(90,2) D4.44;
pD.015]. This interaction was due to the fact that avoid-
ance latencies in sessions 1 [F(45,1) D9.71; pD.003] and 3
[F(45,1) D4.46; pD.040] were higher in Anterior-Control
than in Posterior-Control groups.
A signiWcant interaction between Treatment and Elec-
trode location was also found for the avoidance latencies
on the LTR session [F(44,1) D6.13; pD.017], while neither
the treatment nor the electrode location alone were signiW-
cant. Nested eVects analyses indicated that avoidance latencies
in the LTR session were signiWcantly lower in Anterior-ES
group than in Posterior-ES group [F(45,1) D7.89; pD.007].
The diVerence in avoidance latencies between the LTR and
the third training session was not inXuenced signiWcantly by
any of the main factors or their interaction.
3.6.2. Experiment II (post-training conditions)
Avoidance latencies for each experimental group in the
post-training condition on each of the training sessions and
the LTR are depicted in Fig. 4B. MANOVA analyses indi-
cated a signiWcant interaction between treatment and elec-
trode location [F(37,1) D4.29; pD.045], while none of the
remaining main factors and interactions was signiWcant.
Nested eVects indicated that the interaction was due to the
fact that, in ES groups, avoidance latencies were higher
when the electrode was located at the anterior PPTg than
when it was located at the posterior PPTg [F(38,1) D4.28;
pD.045], while those diVerences were not found in control
animals.
None of the main factors and interactions was signiW-
cant for avoidance latencies in the LTR session, as well as
for the diVerence in avoidance latencies between the third
training session and the LTR session.
4. Discussion
The present work shows that ES can be applied to the
PPTg in rats that are unrestrained, not anesthetized and not
subjected to decerebrate preparation, without causing any
adverse eVects, apart from certain motor eVects in some
instances that can be reverted by adjusting current inten-
sity. Thus, ES of the PPTg can be used in awake animals to
study the possible beneWts of deep brain stimulation on
motor and cognitive deWcits induced by several conditions,
such as brain damage or aging, as well as, for example, in
dopamine depleted rats with Parkinsonian symptoms.
The results also indicate that ES of this nucleus can have
a facilitative inXuence on the acquisition of two-way active
avoidance, although this eVect is time- and site-speciWc,
since it was only found with pre-training ES applied
through electrodes located into the anterior PPTg (between
6.80 and 7.80 mm posterior to Bregma). Thus, the rats that
received pre-training (but not post-training) ES of the ante-
rior PPTg made a higher proportion of avoidance
responses than their controls on the training sessions, and
all of them reached the predeWned learning criterion, in
contrast to the remaining groups in which there was a
certain number of animals that did not fulWl the criterion.
Neither pre-training ES applied through electrodes located
into posterior PPTg areas (8 or more mm posterior to
Bregma), or post-training ES at both anterior and posterior
PPTg had any signiWcant eVect on the proportion of avoid-
ance responses.
Pretraining ES of the anterior PPTg did not seem to
have, however, any signiWcant eVect on memory consolida-
tion. Thus, the evolution of avoidance responses between
the third acquisition session and the LTR was similar in all
the groups (i.e., all the groups retained a similar proportion
of the learned experience), and the analyses of covariance
indicated that the performance on the LTR session was sig-
niWcantly inXuenced by that on the last training session, but
not by any of the experimental conditions. This suggests
that the good retention of the animals that had received
pre-training ES of the anterior PPTg was a consequence of
the enhanced acquisition level induced by the treatment,
but not to any additional eVect of the treatment on memory
consolidation.
4.1. Time speciWc eVects
The facilitative eVects of pre-training ES on two-way
active avoidance acquisition were expected according to the
hypothesis, because it seems that PPTg activity plays a role
in the codiWcation of new experiences. Thus, this nucleus
would be involved in learning about the relationship
between reinforcers and behavior, but its activity does not
seem to be essential when those associations have already
been made (Winn, 2006). Those results are also in line with
the fact that pre-training lesions of the PPTg severely dis-
rupt the acquisition of active avoidance, while post-training
lesions do not have any detrimental eVects on the same
tasks (Fujimoto et al., 1992), except when the animals are
challenged with a change in the stimuli used and/or the
attentional requirements of the task are increased (Homs-
Ormo, Morgado-Bernal, & Coll-Andreu, 2003).
4.2. Site-speciWc eVects
The lack of facilitative eVects of pre-training ES of the
posterior PPTg (which may have even had a slight detri-
mental eVect, since the proportion of animals in this group
that reached the learning criterion was signiWcantly lower
than in the corresponding control group) conWrmed the
preliminary data found in a previous pilot study, but was
not predicted by our hypothesis. While there is a general
agreement in considering that the PPTg is composed of at
least two regions (pars compacta and pars dissipata),
whether they are diVerent entities or are integrated compo-
nents of a unitary structure is a matter of debate (Rye,
Saper, Lee, & Wainer, 1987; Winn, 2006), and the possible
existence of functional compartments is not clear.
Given that the pars dissipata, which contains cholinergic
and non-cholinergic cells, is mainly located medially and
rostrally, it is likely that anterior ES would have aVected a
518 R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521
higher proportion of the dissipata than the compacta zones.
On the other hand, the pars compacta, which contains a
great proportion of densely packed cholinergic neurons
and is mainly located at caudolateral PPTg regions, may
have been mainly aVected by ES through the posterior
electrodes. However, the concordance between dissipata/
compacta and anterior/posterior regions is only partial.
A functional dissociation between anterior and posterior
PPTg has been described by Alderson and colleagues
(Alderson, Latimer, & Winn, 2006), who showed that pos-
terior PPTg lesions altered intravenous self-administration
of nicotine, while no eVects were found after lesions to the
anterior PPTg. This dissociation could be attributable to
the fact that the cholinergic and glutamatergic PPTg pro-
jections to nigral dopamine neurons are originated mainly
in the anterior PPTg (Lavoie & Parent, 1994), while the
projections to the ventral tegmental area are originated
mainly in the posterior PPTg (as well as in neurons of the
laterodorsal tegmental nucleus) (Oakman, Faris, Kerr,
Cozzari, & Hartman, 1995). Thus, the anterior PPTg could
play a crucial role in driving the activity of substantia nigra
projections to the dorsal striatum, and in modulating dor-
sal striatal functions. In contrast, the posterior PPTg could
be involved in functions related to the ventral striatum,
such as the incentive properties of drugs. However, it must
be noted that the anterior/posterior delimitations deWned in
the present work are a bit diVerent to the ones used by
Alderson et al. (2006), who subdivided the rats into those
having the lesions placed rostral vs caudal to the decussa-
tion of the cerebellar peduncles. Moreover, the comparison
between the extent and location of a given lesion and the
extent of tissue inXuenced by ES is not straightforward,
since the spread of ES around the electrode tip depends on
multiple factors (Gimsa et al., 2006; McIntyre & Grill,
1999). In spite of those caveats, both works point to the
existence of diVerent functional regions within the PPTg.
Further work is needed to examine the existence of func-
tional regions within the PPTg, and to clarify which are the
speciWc areas, or even the speciWc neurochemical pools of
neurons, aVected by the ES applied through electrodes
located at anterior vs. posterior PPTg regions.
4.3. Mechanisms mediating the facilitatory eVect of
pre-training electrical stimulation of the anterior PPTg
4.3.1. Locomotion and velocity of the conditioned responses
PPTg ES has been shown to induce stepping or muscle
atonia (depending on current intensity), in decerebrate ani-
mals, (Lai & Siegel, 1990; Takakusaki et al., 2004), and
there is a report showing enhanced locomotion in freely-
moving rats without brain transection after PPTg ES (with
higher current intensities than the ones used by us) (Milner
& Mogenson, 1988). Some of the animals in the present
work also displayed motor eVects (contralateral pivoting
and/or motor stereotypies) upon initial stimulation, but
those eVects disappeared when the current intensities were
lowered. Also a marginal eVect of electrode location (but
not of ES treatment) was found, in Experiment I, on the
number of crossings during adaptation to the shuttle-box
(immediately after the Wrst ES sessions and before the Wrst
training session). However, the ES treatment did not have
any signiWcant inXuence, in either experiment, on the num-
ber of crossings during adaptation to the shuttle-box or on
the number of intertrial crossings during the conditioning
sessions, although the evolution of this variable between
the third acquisition session and the LTR session was inXu-
enced by electrode location. Taking into account those
data, it seems unlikely that the eVects of PPTg ES on the
performance of the task may have been signiWcantly inXu-
enced by changes in locomotor activity during training.
Nevertheless, some of the experimental conditions may
have aVected avoidance latencies. Thus, in the pre-training
experiment, control rats (but not ES rats) had signiWcantly
higher avoidance latencies, on the Wrst and third training
sessions, when their electrode was located into the anterior
compared to the posterior PPTg. With regard to the ES ani-
mals, in the pre-training experiment they had signiWcantly
lower latencies when the electrode was located into the
anterior PPTg vs the posterior PPTg, although this eVect
was only found on the LTR session. In contrast, in the post-
training experiment ES animals had higher avoidance
latencies when the electrode was located into the anterior
PPTg than into the posterior PPTg. Although the meaning
of those data is not clear, they suggest that the small tissue
damage induced (to the anterior PPTg or to dorsal struc-
tures) by the electrode track may have slowed down the
velocity of avoidance responses. Apparently, this eVect was
reverted by pre-training (but not by post-training) ES.
Given that lower avoidance latencies might be related to a
more eYcient stimulus processing or to a better stimulus-
response association, and that the presumed positive eVect
of pre-training ES of the anterior PPTg on avoidance laten-
cies was maintained (or even increased) on the LTR session
(that was not preceded by any PPTg stimulation), the
eVects found on the learning task do not seem to reXect any
state-dependency, but an improvement of some learning-
related process which was maintained on the retention test.
4.3.2. InXuences on forebrain acetylcholine mechanisms
related to cortical desynchronization, processing of sensory
stimuli, and attention
The PPTg sends projections to the thalamus and the
nucleus basalis magnocellularis, and is also connected with
serotonergic and noradrenergic elements of the ascending
reticular activating system (Reese, Garcia-Rill, & Skinner,
1995). Intra-PPTg infusions of carbachol (Kinney, Vogel, &
Feng, 1998) or glutamate (Datta et al., 2001a, 2001b) induce
prolonged increases of cortical desynchronization. PPTg
ES (applied with similar frequency and duration of pulses,
but with diVerent duration of the ES sessions) enhanced
cortical desynchronization, an eVect that seemed mediated
through the joint inXuence of the PPTg on the thalamus
and the basal forebrain (Dringenberg & Olmstead, 2003),
and increased cortical acetylcholine release, probably
R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521 519
through the activation of the cholinergic and glutamatergic
eVerences of the PPTg to the nucleus basalis magnocellu-
laris (Rasmusson et al., 1994). It is known that increased
cortical acetylcholine (by iontophoretic application, ES of
the basal forebrain or by other means) can induce long-last-
ing eVects on neuronal excitability (the reported durations
of those eVects range from several minutes to one or even
several hours). SpeciWcally, this neurotransmitter strongly
potentiates the eVects produced by other excitatory inputs,
provided that the amount of acetylcholine exceeds a given
threshold level. In this way, increased acetylcholine release
facilitates the response to sensory stimuli, and can enhance
signal-to-noise ratio and improve selective attention,
although the eVects of this neurotransmitter on responses
to sensory stimuli vary depending on the speciWc sensory
modality (see Rasmusson, 2000; for a comprehensive
review on the inXuence of enhanced acetylcholine release
on neural plasticity). Interestingly enough, pre-training ES
of the nucleus basalis magnocellularis, which increases ace-
tylcholine release, can also facilitate the acquisition of two-
way active avoidance (Montero-Pastor, Vale-Martinez,
Guillazo-Blanch, & Marti-Nicolovius, 2004), and of a
socially transmitted food preference, and it also induces a
bilateral enhancement of c-fos expression in several cortical
and hippocampal areas (Boix-Trelis, Vale-Martínez, Guil-
lazo-Blanch, Costa-Miserachs, & Martí-Nicolovius, 2006).
In view of those data, the eVects of pre-training PPTg ES
found in the present work could have been at least partially
mediated by increased cortical desynchronization and cor-
tical acetylcholine release as a result of enhanced activation
of nucleus basalis magnocellularis and of certain thalamic
nuclei. This, in turn, may have induced plasticity changes
and increased attention for a prolonged time, so as to be
capable of having an inXuence during the subsequent train-
ing sessions.
Other data indicating that the PPTg plays a role in sen-
sory Wltering to structures higher in the neuraxis are the
involvement of this nucleus in pre-pulse inhibition of the
startle response (Diederich & Koch, 2005; Koch, Kungel, &
Herbert, 1993), and in the modulation of event-related
potentials induced by auditory, visual and possibly somato-
sensory stimuli, such as P1 or P13 responses, which in turn
trigger cortical arousal responses (Reese et al., 1995). The
cholinergic projections of the PPTg to the nucleus reticu-
laris pontis oralis are also involved in the induction of hip-
pocampal theta rhythm during paradoxical sleep (Kinney
et al., 1998), and in driving type 2 hippocampal theta
rhythm in awake animals, a rhythm related to the process-
ing of sensory stimuli relevant to the initiation and mainte-
nance of voluntary motor behaviors (in rats, type 2 theta is
observed, for example, before jump avoidance responses
and in aversive classical conditioning paradigms) (see
Bland & Oddie, 2001; for a review on the inXuence of theta
rhythm on sensorimotor integration). In humans, bursts of
transcranial magnetic stimulation on the theta range
applied to the motor cortex induced consistent, long-lasting
plasticity changes (Huang, Edwards, Rounis, Bhatia, &
Rothwell, 2005). Therefore, increased theta rhythm may
have also contributed to the facilitative eVects found with
pre-training ES of the PPTg.
In view of those considerations, the lack of facilitatory
eVects of pre-training ES of the posterior PPTg is intrigu-
ing, since the cholinergic cells of the PPTg, which play a
central role in the functions described above, are more
numerous in the pars compacta (i.e in posterolateral PPTg)
than in the pars dissipata. The reasons for the diVerences
between ES of the anterior vs the posterior PPTg could be
diverse. For example, the parameters used in the present
work may have been inappropriate, when applied to the
posterior PPTg, to adequately activate the neurons in this
region. Given the positive correlation found, in Experiment
I, between the current intensity applied and the proportion
of avoidance responses on the Wrst training session, the lack
of facilitative eVects of pre-training ES of the posterior
PPTg could also be inXuenced by the higher frequency of
animals that showed motor eVects upon initial ES in Pos-
terior-ES group (Wve out of 13), compared to the Anterior-
ES group (2 out of 13), and that had to receive lower cur-
rent intensities. Finally, the diVerences between the eVects
of anterior and posterior PPTg on learning might be related
to still unknown electrophysiological and/or functional
diVerences between anterior and posterior PPTg regions.
4.3.3. Modulation of dopaminergic activity involved in
processing of sensory stimuli
The PPTg neurons responsive to the onset of sensory
stimuli seem to relay this information to the mesopontine
dopaminergic cells, which would in turn integrate sensory
information with information about reward and about the
appropriate motor response (Pan & Hyland, 2005). PPTg
ES in anesthetized rats induces prolonged (around 30min)
increases of nigral dopamine eZux (Forster & Blaha, 2003),
and enhances bursting activity in nigral dopaminergic neu-
rons (Lokwan et al., 1999). Given that burst Wring in dopa-
minergic neurons is correlated with the occurrence of
salient stimuli, PPTg stimulation may have induced
prolonged changes in dopamine neurons that might have
facilitated their subsequent responsiveness to the training-
related stimuli.
The diVerential eVects found after anterior vs posterior
PPTg ES could be related, at least in part, to the fact that
cholinergic neurons in the anterior PPTg send their projec-
tions mainly to the dopaminergic cells of the substantia
nigra, which modulate the activity and functions of the dor-
sal striatum, while those in the posterior PPTg send their
projections mainly to the dopaminergic cells of the ventral
tegmental area. Two-way active avoidance is an implicit
learning task sensitive to manipulations of dorsal striatal
systems (Da Cunha et al., 2001). Therefore, ES of the ante-
rior PPTg could have improved two-way active avoidance
at least in part by increasing dopaminergic activity in the
dorsal striatal system. In contrast, the possible inXuences of
ES of the posterior PPTg on dopaminergic neurons project-
ing to ventral striatal circuits would not have a decisive
520 R. Andero et al. / Neurobiology of Learning and Memory 87 (2007) 510–521
inXuence on tasks that mainly depend on the dorsal stria-
tum.
In summary, the data described in this paper point to the
existence of functional regions within the PPTg, and indi-
cate that ES of the PPTg can be safely administered, under
certain circumstances, to freely-moving awake rats. This
renders it a suitable procedure to study the inXuence of the
PPTg on motor and cognitive functions. This procedure
could also become a promising tool to examine the feasibil-
ity of deep brain stimulation of the PPTg to revert motor
and cognitive eVects of degenerative diseases, such as Par-
kinson disease.
Further works are needed to determine which are the
electrophysiological and neurochemical eVects induced by
the speciWc parameters of ES used by us, whether enhanced
cortical arousal, cortical acetylcholine release, induction of
hippocampal theta rhythm, and modulation of dopaminer-
gic activity have contributed to the facilitative eVects found,
and whether the site-speciWc eVects observed after pre-
training ES are related in part to the diVerential involve-
ment of anterior and posterior PPTg regions in diVerent
dopaminergic systems or are accounted for by other mech-
anisms.
Acknowledgment
This research was supported by funds from the Ministe-
rio de Ciencia y Tecnología and FEDER (BS02003-04113).
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