Muscarinic acetylcholine neurotransmission enhances the late-phase of long-term potentiation in the hippocampal-prefrontal cortex pathway of rats in vivo: a possible involvement of monoaminergic systems.
ABSTRACT The prefrontal cortex is continuously required for working memory processing during wakefulness, but is particularly hypoactivated during sleep and in psychiatric disorders such as schizophrenia. Ammon's horn CA1 hippocampus subfield (CA1) afferents provide a functional modulatory path that is subjected to synaptic plasticity and a prominent monoaminergic influence. However, little is known about the muscarinic cholinergic effects on prefrontal synapses. Here, we investigated the effects of the muscarinic agonist, pilocarpine (PILO), on the induction and maintenance of CA1-medial prefrontal cortex (mPFC) long-term potentiation (LTP) as well as on brain monoamine levels. Field evoked responses were recorded in urethane-anesthetized rats during baseline (50 min) and after LTP (130 min), and compared with controls. LTP was induced 20 min after PILO administration (15 mg/kg, i.p.) or vehicle (NaCl 0.15 M, i.p.). In a separate group of animals, the hippocampus and mPFC were microdissected 20 min after PILO injection and used to quantify monoamine levels. Our results show that PILO potentiates the late-phase of mPFC LTP without affecting either post-tetanic potentiation or early LTP (20 min). This effect was correlated with a significant decrease in relative delta (1-4 Hz) power and an increase in sigma (10-15 Hz) and gamma (25-40 Hz) powers in CA1. Monoamine levels were specifically altered in the mPFC. We observed a decrease in dopamine, 5-HT, 5-hydroxyindolacetic acid and noradrenaline levels, with no changes in 3,4-hydroxyphenylacetic acid levels. Our data, therefore, suggest that muscarinic activation exerts a boosting effect on mPFC synaptic plasticity and possibly on mPFC-dependent memories, associated to monoaminergic changes.
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ABSTRACT: Applications of acetylcholine (AcCho) to pyramidal cells of guinea pig cingulate cortical slices maintained in vitro result in a short latency inhibition, followed by a prolonged increase in excitability. Cholinergic inhibition is mediated through the rapid excitation of interneurons that utilize the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). This rapid excitation of interneurons is associated with a membrane depolarization and a decrease in neuronal input resistance. In contrast, AcCho-induced excitation of pyramidal cells is due to a direct action that produces a voltage-dependent increase in input resistance. In the experiments reported here, we investigated the possibility that these two responses are mediated by different subclasses of cholinergic receptors. The inhibitory and slow excitatory responses of pyramidal neurons were blocked by muscarinic but not by nicotinic antagonists. Pirenzepine was more effective in blocking the AcCho-induced slow depolarization than in blocking the hyperpolarization of pyramidal neurons. The two responses also varied in their sensitivity to various cholinergic agonists, making it possible to selectively activate either. These data suggest that AcCho may produce two physiologically and pharmacologically distinct muscarinic responses on neocortical neurons: slowly developing voltage-dependent depolarizations associated with an increase in input resistance in pyramidal cells and short-latency depolarizations associated with a decrease in input resistance in presumed GABAergic interneurons.Proceedings of the National Academy of Sciences 10/1985; 82(18):6344-8. · 9.74 Impact Factor
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ABSTRACT: The medial prefrontal cortex (mPFC) participates in several higher order functions including selective attention, visceromotor control, decision making and goal-directed behaviors. We discuss the role of the infralimbic cortex (IL) in visceromotor control and the prelimbic cortex (PL) in cognition and their interactions in goal-directed behaviors in the rat. The PL strongly interconnects with a relatively small group of structures that, like PL, subserve cognition, and together have been designated the 'PL circuit.' These structures primarily include the hippocampus, insular cortex, nucleus accumbens, basolateral nucleus of the amygdala, the mediodorsal and reuniens nuclei of the thalamus and the ventral tegmental area of the midbrain. Lesions of each of these structures, like those of PL, produce deficits in delayed response tasks and memory. The PL (and ventral anterior cingulate cortex) (AC) of rats is ideally positioned to integrate current and past information, including its affective qualities, and act on it through its projections to the ventral striatum/ventral pallidum. We further discuss the role of nucleus reuniens of thalamus as a major interface between the mPFC and the hippocampus, and as a prominent source of afferent limbic information to the mPFC and hippocampus. We suggest that the IL of rats is functionally homologous to the orbitomedial cortex of primates and the prelimbic (and ventral AC) cortex to the lateral/dorsolateral cortex of primates, and that the IL/PL complex of rats exerts significant control over emotional and cognitive aspects of goal-directed behavior.Neuroscience 10/2006; 142(1):1-20. · 3.12 Impact Factor
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ABSTRACT: In this Opinion article we describe a theory that the brain mechanisms underlying working memory for novel information include a buffer in parahippocampal cortices. Computational modeling indicates that mechanisms for maintaining novel information in working memory could differ from mechanisms for maintaining familiar information. Electrophysiological data suggest that the buffer for novel information depends on acetylcholine. Acetylcholine activates single-cell mechanisms that underlie persistent spiking of neurons in the absence of synaptic transmission, allowing maintenance of information without prior synaptic modification. fMRI studies and lesion studies suggest that parahippocampal regions mediate working memory for novel stimuli, and the effects of cholinergic blockade impair this function. These intrinsic mechanisms in parahippocampal cortices provide an important alternative to theories of working memory based on recurrent synaptic excitation.Trends in Cognitive Sciences 12/2006; 10(11):487-93. · 16.01 Impact Factor
MUSCARINIC ACETYLCHOLINE NEUROTRANSMISSION ENHANCES
THE LATE-PHASE OF LONG-TERM POTENTIATION IN THE
HIPPOCAMPAL–PREFRONTAL CORTEX PATHWAY OF RATS IN VIVO:
A POSSIBLE INVOLVEMENT OF MONOAMINERGIC SYSTEMS
C. LOPES AGUIAR,a1 R. NEVES ROMCY-PEREIRA,a1*
R. ESCORSIM SZAWKA,b O. YINETH GALVIS-ALONSO,c
J. APARECIDA ANSELMO-FRANCIb AND
J. PEREIRA LEITEa
aDepartment of Neurology, Psychiatry and Medical Psychology,
Ribeirão Preto School of Medicine, University of São Paulo, Av. Ban-
deirantes, 3900, Ribeirão Preto, SP 14049-900, Brazil
bRibeirão Preto School of Odontology, University of São Paulo, Av.
Bandeirantes, 3900, Ribeirão Preto, SP 14049-900, Brazil
cDepartment of Molecular Biology, São José do Rio Preto School of
Medicine, Av. Brigadeiro Faria Lima, 5416, São José do Rio Preto, SP
Abstract—The prefrontal cortex is continuously required for
working memory processing during wakefulness, but is par-
ticularly hypoactivated during sleep and in psychiatric disor-
ders such as schizophrenia. Ammon’s horn CA1 hippocampus
subfield (CA1) afferents provide a functional modulatory path
that is subjected to synaptic plasticity and a prominent mono-
aminergic influence. However, little is known about the musca-
rinic cholinergic effects on prefrontal synapses. Here, we inves-
tigated the effects of the muscarinic agonist, pilocarpine (PILO),
on the induction and maintenance of CA1-medial prefrontal
cortex (mPFC) long-term potentiation (LTP) as well as on brain
monoamine levels. Field evoked responses were recorded in
urethane-anesthetized rats during baseline (50 min) and after
LTP (130 min), and compared with controls. LTP was induced 20
min after PILO administration (15 mg/kg, i.p.) or vehicle (NaCl
0.15 M, i.p.). In a separate group of animals, the hippocampus
and mPFC were microdissected 20 min after PILO injection and
used to quantify monoamine levels. Our results show that PILO
potentiates the late-phase of mPFC LTP without affecting either
post-tetanic potentiation or early LTP (20 min). This effect was
correlated with a significant decrease in relative delta (1– 4 Hz)
power and an increase in sigma (10 –15 Hz) and gamma (25–
40 Hz) powers in CA1. Monoamine levels were specifically al-
tered in the mPFC. We observed a decrease in dopamine, 5-HT,
5-hydroxyindolacetic acid and noradrenaline levels, with no
changes in 3,4-hydroxyphenylacetic acid levels. Our data,
therefore, suggest that muscarinic activation exerts a boosting
effect on mPFC synaptic plasticity and possibly on mPFC-de-
pendent memories, associated to monoaminergic changes.
© 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: prefrontal cortex, hippocampus, synaptic plastic-
ity, long-term potentiation, acetylcholine, monoamines.
The hippocampus plays an essential role in the establish-
ment of declarative memories encoding context represen-
tations of emotional and non-emotional experiences (Sco-
ville and Milner, 1957; Squire, 1992; Fanselow, 2000).
Mnemonic information processed in its circuitry has two
direct cortical outputs: the entorhinal cortex and the pre-
frontal cortex (Swanson and Cowan, 1977; Swanson,
1981; Ferino et al., 1987; Jay et al., 1989; Jay and Witter,
1991; Caballero-Bleda and Witter, 1994; Barbas and Blatt,
1995; Witter and Amaral, 2004). The prefrontal cortex, in
particular, lies on a privileged position to access online
information from the thalamus and primary sensory corti-
ces, body-state inputs from the brainstem and memory
traces encoded by the hippocampus (Groenewegen and
Uylings, 2000; Dalley et al., 2004; Vertes, 2006). In fact,
this particular merging of information in the prefrontal cor-
tex is thought to be essential to the development of adap-
tive behavioral strategies and prospective plans in rodents,
macaques and humans (Fuster, 1973; Funahashi et al.,
1989; Goldman-Rakic, 1995, 1996; Vertes, 2006). More-
over, both the hippocampus and the prefrontal cortex are
implicated in the expression of several psychiatric disor-
ders such as schizophrenia, bipolar disorder and major
depression, in which neurotransmitter unbalances are ob-
served (Goldman-Rakic, 1999; Lyons, 2002).
The cholinergic system is an important modulator of
memory performance involving the hippocampus and the
prefrontal cortex (Hasselmo, 1999; Gu, 2002; Chudasama
et al., 2004; Dalley et al., 2004). Cholinergic neurons from
the septum project to the hippocampus, whereas neurons
in the basal forebrain and laterodorsal tegmental nucleus
project to the medial prefrontal cortex (mPFC) (McKinney
et al., 1983; Mayo et al., 1984; Satoh and Fibiger, 1986).
The activity of cholinergic neurons is distinctively regulated
across the sleep–wake cycle, showing higher levels during
rapid-eye movement (REM) sleep, attention-demanding
tasks and learning, but reduced levels during slow–wave
sleep and quiet wakefulness (Detari et al., 1984). Besides,
the cognitive decline observed in Alzheimer’s disease,
schizophrenia and normal aging is known to be associated
with a reduction of cholinergic activity in subcortical and
cortical brain regions (Giacobini, 1990; Barnes et al., 2000;
Messer, 2002; Dunbar and Kuchibhatla, 2006).
1Authors contributed equally to this study.
*Corresponding author. Tel: ?55-16-3602-2556 or ?55-16-3602-4535.
E-mail address: email@example.com (R. Neves Romcy-Pereira).
Abbreviations: CA1, Ammon’s horn CA1 hippocampus subfield; DOPAC,
3,4-dihydroxyphenylacetic acid; fPSP, field post-synaptic potential;
HPLC-ED, high-performance liquid chromatography with electrochemical
detection; LFP, local field potential; LTP, long-term potentiation; mEsc,
methyl-scopolamine; mPFC, medial prefrontal cortex; PILO, pilocarpine;
REM, rapid-eye movement; 5-HIAA, 5-hydroxyindole-3-acetic acid.
Neuroscience 153 (2008) 1309–1319
0306-4522/08$32.00?0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
Several studies have shown that monosynaptic projec-
tions from both the dorsal and ventral Ammon’s horn CA1
hippocampus subfield (CA1) to the mPFC can undergo
paired-pulse facilitation, long-term potentiation (LTP) and
long-term depression (LTD) (Laroche et al., 1990; Jay et
al., 1995; Takita et al., 1999; Izaki et al., 2002; Kawashima
et al., 2006). A number of reports have also demonstrated
that mPFC synaptic plasticity is modulated by glutamater-
gic, dopaminergic and serotonergic neurotransmission and
can be altered by drugs such as cocaine and clozapine
(Gurden et al., 1999; Ohashi et al., 2002; Gemperle and
Olpe, 2004; Jay et al., 2004; Dupin et al., 2006; Chen et al.,
2007; Huang et al., 2007). Although much effort has been
focused on the monoaminergic control of CA1-mPFC syn-
aptic plasticity, very little is known about the cholinergic
modulation and its implications to psychiatric disorders and
learning. Recently, Couey et al. (2007) showed that nico-
tine increases the threshold for spike-timing-dependent
potentiation in layer V pyramidal neurons of the prefrontal
cortex. In addition, we have found indirect evidence for the
involvement of the cholinergic system in the bimodal reg-
ulation of hippocampal and prefrontal cortical synaptic
plasticity. We observed that short-term (4 h) REM sleep
deprivation impairs the late-phase maintenance of LTP in
the hippocampus but prolongs it in the projections from the
posterior dorsal CA1 to the prefrontal cortex (Romcy-
Pereira and Pavlides, 2004). Such changes could underlie
the dorsal hippocampus and the prefrontal cortex have prom-
inent roles in spatial memory processing. In the present
study, therefore, we investigated the effects of the muscarinic
cholinergic agonist, pilocarpine (PILO), on the induction and
maintenance of LTP in the projections from the posterior
dorsal CA1 to the mPFC, as well as its influence on mono-
amine levels in the hippocampus and prefrontal cortex. In
order to monitor the effects of PILO on brain activity before
and after LTP, we also analyzed the oscillatory patterns of
the hippocampus and mPFC after drug administration. Our
results show that PILO prolongs the maintenance of CA1-
mPFC plasticity in vivo but has no effect on its induction.
This modulatory function is accompanied by a clear
change in the spectral composition of CA1 and mPFC
neuronal oscillations toward high frequencies (?10 Hz).
Besides, PILO administration specifically changes mono-
amine levels in the mPFC, without affecting their levels in
Forty-two male Wistar rats (250–450 g) were housed in standard
rodent cages in a vivarium maintained at 24 °C under a 12-h
light/dark cycle with lights on at 07:00 h. Food and water were
freely available during all phases of the experiment. All proce-
dures were performed according to the Brazilian College of Animal
Experimentation (COBEA) guidelines for animal research, affili-
ated with the International Council for Laboratory Animal Science
(ICLAS), and approved by the Ethical Commission at the Univer-
sity of São Paulo. Experiments were designed to minimize the
number of animals used and their suffering.
Electrode implants and LTP
Rats were anesthetized with urethane (1.2–1.5 g/kg, i.p., in NaCl
0.15 M; Sigma-Aldrich, St. Louis, MO, USA) and placed in a
stereotaxic frame for the implant of electrodes with body temper-
ature maintained at 37?0.5 °C by using a heating pad. The level
of anesthesia was maintained stable by supplementary doses of
anesthetic (30% of the initial dose) after checking the tail pinch
reflex, respiratory rate and EEG signals. In brief, the skull was
exposed and small holes drilled on it to allow access to the mPFC
(3.0 mm anterior to bregma, 0.5 mm lateral to midline and 3.2 mm
ventral to dura mater) and CA1 (5.7 mm anterior to bregma,
4.6 mm lateral to midline and 2.5 mm ventral to dura mater),
according to Paxinos and Watson (1998). Teflon-insulated tung-
sten wires (60 ?m diameter; A-M Systems, Carlsborg, WA, USA)
were used to prepare electrodes, which were lowered into the
brain through holes made on the skull after removing the dura
mater. A twisted bipolar electrode (tip separation 500 ?m) was
used for constant current stimulation of CA1 and a monopolar
electrode was used to record field post-synaptic potentials (fPSP)
in the mPFC. A micro-screw was implanted over the parietal
cortex and served as the recording ground. The final position of
the electrodes was adjusted to obtain the highest negative-going
response in the mPFC with latency to the first negative peak
around 18 ms and amplitude of at least 250 ?V (Laroche et al.,
1990; Jay et al., 1995). Monophasic test pulses of 200 ms were
delivered every 20 s at increasing intensities (60–500 ?A) and
used to plot input–output curves. Based on input–output curves,
we calculated the minimum intensity necessary to produce max-
imum mPFC responses and used 50% of this intensity to stimulate
CA1 during baseline, LTP and post LTP recordings. Baseline was
recorded using test pulses (200 ?s duration; every 20 s) for 50 min
before LTP induction and was followed by continuous fPSP re-
cordings for additional 130 min (every 20 s). LTP was induced by
tetanic stimulation of CA1-mPFC projections with two series of 10
trains (50 pulses at 250 Hz, 200 ms duration) every 10 s, 10 min
apart from each other (Romcy-Pereira and Pavlides, 2004). Pre-
frontal fPSPs were amplified and filtered (?100, 0.3–10 kHz;
P55-AC pre-amplifier, Grass Instruments Co., West Warwick, RI,
USA) before digitization at 4 kHz (PowerLab/16S, ADInstruments,
Colorado Springs, CO, USA). Constant current square pulses
used throughout the experiments were delivered through an S88
stimulator (Grass Instruments Co.).
In order to investigate the cholinergic modulation of mPFC syn-
aptic plasticity, rats were grouped according to drug treatment
during baseline recordings. (1) Veh-Veh animals received two
injections of vehicle (Veh; NaCl 0.15M, i.p.), one at 10 min and
one at 30 min after baseline recording onset; (2) methyl-scopol-
amine (mEsc) –Veh animals received an injection of the periph-
erally specific muscarinic antagonist mEsc (15 mg/kg, i.p., in NaCl
0.15 M; Sigma-Aldrich) at 10 min and an injection of vehicle at 30
min after baseline recording onset, and (3) mEsc-PILO animals
received an injection of mEsc (15 mg/kg, i.p., in NaCl 0.15 M) at
10 min and an injection of the muscarinic agonist PILO (15 mg/kg,
i.p., in NaCl 0.15 M; Sigma-Aldrich) at 30 min after baseline
recording onset. Baseline recordings were identified as BL1: 0–10
min; BL2: 10–30 min and BL3: 30–50 min. Since, mEsc is a
muscarinic antagonist that does not cross the blood–brain barrier
(Hughes, 1982; van Haaren and van Hest, 1989), mEsc pre-
treatment prevented the peripheral actions of PILO, such as lethal
parasympathetic effects even at the low doses (1 mg/kg) during
urethane anesthesia. After tetanization, LTP was calculated as
percentage of baseline levels measured during the first 50 min for
Veh-Veh and mEsc-PILO groups, or during the last 20 min of
baseline for mEsc-Veh group. Differences in baseline and LTP
decay were evaluated by comparing 10-min segments of aver-
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–13191310
aged fPSP amplitude values across baselines and between dif-
ferent time-points after LTP induction for all groups.
Local field potential (LFP) recordings
In order to monitor the brain activation state associated to the
cholinergic modulation, we recorded LFPs (or deep EEG) in CA1
and mPFC throughout the experiment, from the same electrodes
used to induce and record LTP. LFPs were amplified and filtered
(?100, 0.3–300 Hz; P55-AC pre-amplifier, Grass Instruments Co.)
before digitization at 1 kHz (PowerLab/16S, ADInstruments). They
were recorded at several time points during the LTP paradigm: (1)
before Veh or mEsc, at BL1; (2) before Veh or PILO, at BL2; (3)
before LTP induction, at BL3 and (4) 10 min after LTP induction.
For each animal, power spectra were calculated on 10 s-epoch
LFPs at all four time-points. Delta (1–4 Hz), theta (4–10 Hz),
sigma (10–15 Hz) and gamma (25–40 Hz) relative powers were
calculated and compared across the time to evaluate the effect of
mEsc and PILO injections on CA1 and mPFC oscillations. After
the electrophysiological procedures, a brief current pulse (1 mA/1
s) was delivered through the stimulating and recording electrodes
to mark their tip placements.
Three new groups of animals were prepared for monoamine quan-
tification. Rats were anesthetized with urethane (1.2–1.5 g/kg, i.p.,
in NaCl 0.15 M) and received an injection of either Veh or mEsc
(15 mg/kg, i.p., in NaCl 0.15 M) 1 h later. After 20 min, they
received an injection of either Veh or PILO (15 mg/kg, i.p., in NaCl
0.15 M) and were quickly decapitated 20 min later. All rats were
treated in a similar way as the animals used for electrophysiology,
including urethane anesthesia and the timing for drug injection
and decapitation. After decapitation, the brains were removed,
immediately frozen on dry ice and labeled as Veh-Veh, mEsc-Veh
or mEsc-PILO, according to drug treatment. They were stored at
?70 °C until sectioning.
Thick coronal brain sections were cut in cryostat set to ?10 °C
with controlled micrometric advance of the specimen (Microm HM
505, Mikron Instruments Inc., San Marcos, CA, USA). According
to rat brain atlas, three 1000 ?m thick sections were sliced from
the frozen brain and mounted onto chilled glass for micro-dissec-
tion with a 1.0-mm diameter needle by the punch technique
(Palkovits, 1973; Paxinos and Watson, 1998). Hippocampal and
mPFC punches were collected from the posterior dorsal CA1 and
prelimbic mPFC regions, corresponding to the stimulating and
recording sites used in the LTP experiment, respectively. Parietal
cortex punches were also obtained and used as a control. The
mPFC was dissected from a first section extending from 3.7–
2.7 mm anterior to bregma, in two punches vertically placed
between 3.0 and 5.0 mm ventral from dura mater. The parietal
cortex was dissected from a second section, extending from 1.8–
2.8 mm posterior to bregma, in two punches placed laterally at
2.0 and 4.0 mm ventral from dura mater. The dorsal posterior
CA1 region was dissected from a third section, extending from
4.8–5.8 posterior to bregma, in two punches placed laterally at
2.6 and 2.8 mm ventral from dura mater. With a micro-ultra-
sonic cell disrupter, punches were homogenized in a solution
containing 0.2 M perchloric acid (Merck, Darmstadt, Germany),
0.1 mM EDTA (Merck) and 8 ng/mL of 3,4-dihydroxyben-
zylamine (Aldrich, Milwaukee, WI, USA). The homogenates
were centrifuged for 20 min at 12,000?g. The supernatant was
removed, filtered through a 0.22-?m filter (Millex PVDF, Milli-
pore, Belford, MA, USA) and placed into auto-injector vials.
Protein content was determined in the remaining pellet by the
Bradford method (Bradford, 1976).
High-performance liquid chromatography with
electrochemical detection (HPLC-ED)
Tissue concentrations of dopamine, 3,4-dihydroxyphenylacetic
acid (DOPAC), 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) and
noradrenaline were measured by HPLC-ED. Fifty microliters of
each sample were injected by an auto injector (SIL-10Advp, Shi-
madzu, Kyoto, Japan). Separation was performed at 35 °C on a
250?4-mm reversed-phase C18 column (Purospher Star, 5 ?m,
Merck), preceded by a 4?4-mm C18 guard column (Lichrospher,
5 ?m, Merck). The mobile phase consisted of 100 mM sodium
dihydrogen phosphate monohydrate, 10 mM sodium chloride,
0.1 mM EDTA, 0.20 mM sodium 1-octanesulfonic acid (Sigma-
Aldrich) and 15% methanol (Merck). The pH was adjusted to 3.5
with phosphoric acid. The flow rate was set at 0.6 mL/min,
pumped by a dual piston pump (LC-10Advp, Shimadzu). The
detector potential was 0.60 V versus in situ Ag/AgCl (Decade,
VT-03 electrochemical flow cell; Antec Leyden, Netherlands).
Chromatographic data were plotted using Class-VP software (Shi-
madzu). Noradrenaline, dopamine, DOPAC, 5-HT and 5-HIAA
were identified by their peak retention time and quantified by the
internal standard method based on the area under the peak. Intra
and inter-assay coefficients of variation were less than 5% and
8%, respectively, for all measured compounds. Neurotransmitter
levels (dopamine, 5-HT and noradrenaline) were considered to
estimate neurotransmitter stocks in synaptic vesicles, whereas
dopamine and 5-HT metabolites (DOPAC and 5-HIAA, respec-
tively) were considered to reflect the amount of transmitter re-
leased in the samples. Metabolite to neurotransmitter ratio
(DOPAC/dopamine and 5-HIAA/5-HT) was taken as a measure of
Histology for electrode positioning determination
After electrolytic lesions at CA1 and mPFC electrode tip positions,
the animals received an additional dose of urethane (0.5 g/kg, i.p.
in NaCl 0.15 M) and were transcardiacally perfused with 100 ml of
NaCl 0.15 M followed by 250 ml of 10% formaldehyde in NaCl
0.15 M. Their brains were removed, post-fixed in the formalde-
hyde solution for 14 h at 4 °C and cryoprotected for 48 h in 20%
sucrose solution. After freezing in dry ice-chilled isopentane,
brains were cut in 30 mm slices, mounted on gelatinized slides
and processed for Cresyl Violet staining. Electrode tip positions
were determined after analysis of the slides under the microscope
using bright field (BX-60 Olympus, Center Vally, PA, USA).
The analysis of group differences after CA1-mPFC LTP was car-
ried out using a mixed model two-way ANOVA for repeated mea-
sures (group: fixed factor vs. time: repeated measures). Baseline
fPSP data and power spectrum differences in the mPFC and CA1
were analyzed using one-way ANOVA for repeated measures in
each group. Monoamine level differences were evaluated using
one-way ANOVA. Newman-Keuls post hoc tests were used fol-
lowing ANOVAs when necessary. The results are expressed as
mean?S.E.M. (standard error of the mean) and significance level
was set to P?0.05.
Effects of PILO on hippocampo–prefrontal cortex
LTP and brain oscillations
All animals included in our analysis had the stimulating
electrode positioned in the dorsal aspect of the temporal
CA1 subfield of the hippocampus and the recording elec-
trode positioned in the medial wall of the prefrontal cortex,
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–13191311
most of the time corresponding to the prelimbic area of the
mPFC (Fig. 1). Reliable field evoked responses in the
mPFC were obtained after CA1 stimulation and consisted
of a negative deflection with average latency of 16.8?0.6
ms, amplitude of 250–400 ?A and slope of ?37.5?2.4
?V/ms. They were consistent with mPFC fPSPs previously
reported in the literature and a result of hippocampal
monosynaptic projections to the mPFC (Laroche et al.,
1990; Degenetais et al., 2003; Romcy-Pereira and Pav-
As shown in the experimental schedule of Fig. 2 (Fig.
2A), baseline fPSPs were recorded during BL1 (before
mEsc), BL2 (before PILO) and BL3 (after PILO) and com-
pared within each group in order to monitor mEsc and PILO
effects on basal mPFC responses. Veh-Veh and mEsc-PILO
animals did not show any difference across baselines (fPSP
amplitude: Veh-Veh, F(2,7)?3.15, P?0.05, mEsc-PILO,
showed a slight increase in the amplitude of BL3 fPSPs
when compared with BL1 (fPSP amplitude: 14.60?2.20%;
F(2,12)?4.94, P?0.05). For this reason, post-LTP-evoked
responses in Veh-Veh and mEsc-PILO groups were nor-
malized against the average of BL1, BL2 and BL3, while
mEsc-Veh responses were normalized against BL3 val-
LTP in the mPFC was induced by the application of
high frequency trains of stimulation to CA1 and was con-
tinuously monitored for 130 min. Fig. 2B shows the effect
of PILO treatment on the late-phase of mPFC LTP. A
significant group effect was observed in the evoked re-
sponses after tetanization (fPSP amplitude: F(2,18)?3.96,
P?0.05). Although similar levels of post-tetanic potentia-
tion (at 0 min) were induced in all three groups (fPSP
amplitude, F(2,18)?1.87, P?0.05), LTP decayed faster in
Veh-Veh and mEsc-Veh animals as compared with mEsc-
PILO animals. In the mEsc-PILO group, evoked fPSPs
were enhanced at 130 min after LTP induction when com-
pared with mEsc-Veh group (148.59?1.06%, mEsc-PILO
vs. 122.42?3.32%, mEsc-Veh; F(2,18)?4.26, P?0.01). No
differences were observed in the fPSP amplitude when we
compared Veh-Veh and mEsc-Veh animals, at any time-
point after LTP induction.
LFPs were recorded in CA1 and mPFC in order to
monitor their oscillatory patterns before and after PILO
injection. Slow-wave oscillations in the delta band (1–4 Hz)
were dominant throughout the experiment in Veh-Veh and
mEsc-Veh animals, but they were reduced after PILO ad-
ministration, particularly in the hippocampus (Fig. 3A, top
panels). Twenty minutes after PILO injection (mEsc-PILO
animals), the relative delta power in CA1 decreased
Fig. 1. Electrode placement. Left, schematic representation of final electrode positions in the mPFC and CA1. Right, a typical lesion at electrode tips
in the mPFC and hippocampal histological sections. Antero-posterior coordinates are shown in relation to bregma.
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–13191312
approximately 35% (74.87?10.71% before PILO vs.
37.24?7.28% after PILO; F(3,12)?4.84, P?0.05) from pre-
PILO levels. This pattern could still be observed after LTP
induction. The desynchronization of CA1 neuronal activity
could also be seen by the concurrent increase in theta,
sigma and gamma relative powers (Fig. 3A, middle and
lower panels). Significant increases in sigma (?100%;
3.36?1.47% before PILO vs. 6.16?0.41% after LTP;
F(3,12)?3.24, P?0.05) and gamma (?200%; 4.48?1.95%
before PILO vs. 15.70?1.80% after PILO; F(3,12)?7.73,
P?0.001) powers were detected 20 min after PILO, which
lasted at least until 10 min after LTP induction. Although
less prominent than in CA1, the cholinergic activation of
mPFC promoted a decreasing trend in delta power that was
associated with an increasing trend in theta and a significant
boosting in gamma power (?400%; 1.32?0.56% before
mEsc vs. 6.36?1.93% after PILO; F(3,12)?4.43 P?0.05) in
the mPFC. We also detected small changes in sigma and
gamma powers in the Veh-Veh group, which were proba-
bly related to the level of anesthesia at the moment of LFP
recording. Importantly, mEsc did not affect EEG spectral
composition in any frequency band analyzed. Fig. 3B
shows illustrative examples of LFP segments recorded in
the CA1 and mPFC of control and PILO-treated rats.
Effects of PILO on monoamine levels in CA1
Fig. 4A shows the experimental paradigm used to prepare
animals for monoamine quantification. Dopamine, 5-HT,
and their metabolites DOPAC and 5-HIAA, as well as
noradrenaline, were quantified by HPLC-ED in mPFC, hip-
pocampus and parietal cortex micro-dissections. Fig. 4B
depicts the micro-dissection sites where samples were
collected from Veh-Veh, mEsc-Veh and mEsc-PILO
groups. Fig. 4C–E shows the results of monoamine quan-
tification. In the mPFC, PILO injection promoted a de-
crease in dopamine (?43.6%; F(2,18)?3.95, P?0.05),
Fig. 2. Experimental paradigm and LTP. (A) Experimental paradigm. Evoked fPSP responses in the mPFC were recorded during baseline (BL1–BL3)
and monitored for 130 min after CA1 tetanization (HFS). mEsc-PILO rats received mEsc (15 mg/kg, i.p.) followed by PILO (15 mg/kg, i.p.) before LTP
induction. Control rats (Veh-Veh, mEsc-Veh) received sterile saline instead of PILO and mEsc. (B) LTP. PILO treatment did not affect LTP induction
but significantly potentiated the late-phase of mPFC evoked fPSP amplitude after HFS. mEsc-PILO rats showed a significant enhancement of the
amplitude of mPFC evoked responses 130 min after LTP induction compared with mEsc-Veh animals (see bar graph). Bar graphs show statistical
differences in 10 min-epoch (short bar) and 30 min-epoch (long bar) recordings. Top tracings, representative fPSP responses in the mPFC after CA1
stimulation (dashed line, Veh-Veh; gray line, mEsc-Veh; black line, mEsc-PILO). Trace responses are aligned to the time scale in the x axis of the
graph. Data shown as mean?S.E.M. Differences between experimental groups were determined by two-way ANOVA with repeated measures
followed by Neuman-Keuls post hoc test: a P?0.05 compared with mEsc-Veh; b P?0.05 compared with Veh-Veh. Scale bars?20 ms (horizontal), 250
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–1319 1313
Fig. 3. Oscillatory brain activity after PILO treatment. (A) Relative power spectrum changes in CA1 and mPFC oscillations before and after drug
treatment (see also Fig. 2A). Top, PILO injection significantly decreased delta (1–4 Hz) power in CA1 (?35%, P?0.05) and set a trend toward
reduction in the mPFC (mEsc-PILO group). mEsc did not affect delta oscillatory activity either in CA1 or mPFC (mEsc-Veh group). Middle-top, PILO
produced a non-significant increase in theta (4–10 Hz) power in both areas. Middle-bottom, PILO significantly increased sigma (10–15 Hz) power
in CA1, and promoted a similar trend toward augmentation in the mPFC. Drifts in sigma power were seen in the mPFC of Veh-Veh rats, and
in CA1 of mEsc-Veh rats. Bottom, PILO strongly increased gamma (25–40 Hz) power in CA1 (?200%, P?0.05) and mPFC (?400%, P?0.05).
Importantly, mEsc did not affect gamma oscillatory activity either in CA1 or mPFC. (B) Representative mPFC and CA1 EEG tracings from mEsc-Veh
and mEsc-PILO animals. Data shown as mean?S.E.M. Differences between time-points were determined by one-way ANOVA with repeated
measures followed by Neuman-Keuls post hoc test: a, P?0.05 compared with before Veh/mEsc; b, P?0.05 compared with before Veh-PILO. Scale
bars?1 s (horizontal), 400 mV (vertical).
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–1319 1314
5-HT (?47.2%; F(2,18)?4.34, P?0.05), 5-HIAA (?24.3%;
F(2,17)?6.96, P?0.01) and noradrenaline (?33.6%;
F(2,18)?7.42, P?0.01) levels when compared with the con-
trol mEsc-Veh group. DOPAC levels were unchanged in
the mPFC (F(2,18)?2,52, P?0.11). As a result, dopamine
turnover displayed a trend toward an increase in mEsc-
PILO animals (?27.9% vs. mEsc-Veh; F(2,18)?3.30,
P?0.06) and 5-HT turnover did not change (F(2,17)?1.73,
P?0.21). In the hippocampus, on the other hand, we did
not detect any change in 5-HT, 5-HIAA or noradrenaline
levels (ANOVA, P?0.05). PILO also did not alter 5-HT
turnover in the hippocampus (F(2,18)?0.56, P?0.58). Do-
pamine and DOPAC levels were below the assay detection
limit (approximately 20 pg). In the parietal cortex, however,
PILO induced a distinct pattern of neurochemical changes
from that observed in the mPFC. There was a reduction in
5-HT (?67.5%; F(2,17)?9.13, P?0.01) and noradrenaline
(?31.2%; F(2,17)?5.72, P?0.05) levels, and an increase in
5-HIAA (?16.9%; F(2,17)?4.54, P?0.05) as compared with
mEsc-Veh animals. PILO also significantly increased 5-HT
turnover as compared with controls (?250.1% vs. mEsc-
Veh, ?555.3% vs. Veh-Veh; F(2,17)?21.00, P?0.001). Do-
pamine and DOPAC levels were below detection limit in
the parietal cortex.
Fig. 4. Monoamine and metabolite levels in the mPFC, hippocampus and parietal cortex (ParCx) 20 min after PILO injection. (A) Rats were
anesthetized with urethane (1.5 g/kg, i.p.) and received Veh, mEsc (15 mg/kg, i.p.) or PILO (15 mg/kg, i.p.) injections according to the paradigm.
(B) Punch samples (1000 mm thick?1.0 mm diameter) from the mPFC, hippocampus and ParCx (control) were obtained and prepared for HPLC-ED
quantification of DA, 5-HT, noradrenaline and metabolites. Antero-posterior coordinates are shown in relation to bregma. (C–E) DA, 5-HT and NA
quantification results. Metabolite to neurotransmitter ratios for DA and 5-HT are shown in the lower graphs of C and D, respectively. Data are shown
as mean?S.E.M. Differences between experimental groups were determined by one-way ANOVA followed by Newman-Keuls test. a P?0.05
compared with Veh-Veh group; b P?0.05 compared with mEsc-Veh group. DA levels in the hippocampus and ParCx were not quantified as they were
below detection limit (approximately 20 pg). DA, dopamine; NA, noradrenaline.
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–1319 1315
Cholinergic projections from the septum, basal forebrain
and laterodorsal tegmental nucleus to limbic and neocor-
tical areas modulate a variety of brain processes including
arousal, attention and memory. Despite the importance of
hippocampo-prefrontal cortex communication to emotional
and cognitive processing, very little has been reported
about the cholinergic control of CA1-mPFC synaptic trans-
mission. In this study, we demonstrated that the musca-
rinic agonist PILO enhances synaptic plasticity in the hip-
pocampal inputs to mPFC in intact animals, which is also
associated with cortical monoaminergic changes and fast
oscillatory brain rhythms. Our main findings show that (1)
PILO strengthened mPFC LTP late-phase without affect-
ing its induction phase. PILO sustained mPFC LTP above
control levels at 130 min after LTP induction; (2) the effects
of PILO on mPFC LTP were paralleled by a decrease in
the relative power spectrum of delta oscillations and an
increase of sigma oscillations in CA1, as well as an in-
crease of gamma oscillations in CA1 and mPFC; (3) PILO
specifically altered monoamine levels in the mPFC com-
pared with control groups. It decreased dopamine levels
without changing the levels of its metabolite, DOPAC;
decreased both 5-HT and its metabolite, 5-HIAA tissue
levels; and it also decreased noradrenaline levels; but (4)
PILO did not affect monoamine levels in the hippocampus.
The cholinergic enhancement of mPFC LTP observed
in our study is consistent with previous reports using intact
animals showing a muscarinic potentiation of LTP mainte-
nance in the motor (Boyd et al., 2000), somatosensory
(Verdier and Dykes, 2001) and visual cortices (Dringen-
berg et al., 2007), as well as in CA1 (Iga et al., 1996) and
dentate gyrus (Frey et al., 2003) of the hippocampus.
Although muscarinic agonists were also shown to enhance
LTP induction in brain slices of CA1, dentate gyrus (Blitzer
et al., 1990; Abe et al., 1994) and, piriform and perirhinal
cortices (Hasselmo and Barkai, 1995; Cheong et al.,
2001), we did not observe any effect of PILO on mPFC
LTP induction. This discrepancy could either reflect a dif-
ference in the animal preparation used (brain slice vs.
intact animal), a particular response to our LTP protocol or
a specific property of the muscarinic modulation of CA1-
mPFC synaptic plasticity. On the other hand, we could also
consider the possibility that a small amount of mEsc may
have leaked across the blood–brain barrier following sys-
temic mEsc pre-treatment, counteracting PILO central ef-
fects (Pakarinen and Moerschbaecher, 1993). However, in
spite of the small effect of mEsc pre-treatment on baseline
mPFC responses in mEsc-Veh animals, it is unlikely that
mEsc leakage inhibited a hypothetical PILO-induced in-
crease in LTP induction. In support of this, we found that
the dose of mEsc used in our study did not alter LTP levels
in mEsc-Veh animals when compared with Veh-Veh ani-
mals, as would be expected after mild muscarinic blockade
(Dringenberg et al., 2007). Therefore, these findings indi-
cate that PILO promotes strengthening of mPFC LTP by
sustaining its late-phase without directly affecting its
Three important findings suggest that mPFC is partic-
ularly sensitive to cholinergic inputs and support the effects
of PILO on mPFC plasticity. First, mPFC cells have a
particularly strong fast-hyperpolarizing response to acetyl-
choline, which is followed by a long-lasting depolarization
(Gulledge et al., 2007). Second, pyramidal prefrontal neu-
rons treated with PILO lack the initial fast-hyperpolarizing
response and have a very potent slow depolarization pos-
sibly associated to the activation of M1 muscarinic post-
synaptic receptors (McCormick and Prince, 1985). Third,
M1 receptor activation promotes tonic firing and enhances
the temporal integration of dendritic excitatory fPSPs in
mPFC neurons, attenuating isolated single fPSPs and po-
tentiating fPSPs generated by spatially and temporally
coherent inputs (Carr and Surmeier, 2007).
In addition, we also observed that the PILO-enhance-
ment of mPFC LTP was associated with a desynchroniza-
tion of LFPs recorded in the hippocampus and cortex.
PILO significantly shifted the oscillations from a high-am-
plitude low-frequency pattern (delta oscillations) to a low-
amplitude high-frequency pattern with higher contribution
of sigma and gamma oscillations in CA1 and mPFC. These
results confirm that PILO affected brain oscillatory state by
the time LTP was induced and suggest that the late-phase
of mPFC LTP is favored during desynchronized states, as
seen in behaving animals during wakefulness and REM
sleep (Bramham and Srebro, 1989; Leung et al., 2003). In
fact, CA1-mPFC evoked responses are modulated by the
behavior state in rats (Romcy-Pereira and Pavlides, un-
published observations). Recent evidence also indicate
that mPFC neuronal firing is entrained by the cholinergic-
associated theta oscillation of the hippocampus and in-
creases its synchrony with CA1 neurons during working
memory tasks (Jones and Wilson, 2005; Siapas et al.,
2005). Such hippocampo-cortical interactions may be im-
portant mechanisms for gating information flow and pro-
moting plastic changes thought to underlie the storage of
information within this network.
It is possible that the muscarinic enhancement of CA1-
mPFC LTP may have been influenced by monoaminergic
changes induced by PILO at the time of LTP induction, 20
min after injection. The trend toward an increase in
DOPAC/dopamine ratio detected in the mPFC suggests
that PILO induced an enhancement of dopamine release.
This is in agreement with studies showing that systemic
and midbrain administration of muscarinic agonists, includ-
ing PILO, augment the release of dopamine in the mPFC
(Gronier et al., 2000; Stanhope et al., 2001; Ichikawa et al.,
2002). It is also possible that PILO modulates a supposed
dopamine release induced by the electrical stimulation of
the hippocampus, since NMDA stimulation of the ventral
hippocampus increases DA release in the mPFC (Peleg-
Raibstein et al., 2005). It was shown that although dopa-
mine inhibits normal synaptic transmission, it enhances
early and late mPFC LTP in vivo (Jay et al., 1996; Gurden
et al., 1999). In contrast to what we observed in the mPFC,
in the hippocampus and parietal cortex, the undetected low
levels of dopamine suggest that it does not play a major
influence on the PILO-induced mPFC LTP strengthening.
C. Lopes Aguiar et al. / Neuroscience 153 (2008) 1309–13191316
As for 5-HT, the simultaneous decrease in neurotransmit-
ter and metabolite levels observed in the mPFC indicates
that PILO may have inhibited raphe neurons. In fact, ion-
tophoretic application of acetylcholine into the dorsal raphe
of anesthetized rats inhibits about one-half of its neurons
(Koyama and Kayama, 1993), which could indicate a re-
duction in cortical 5-HT release. Considering that a single
dose of fluvoxamine treatment seems to have no effect on
CA1-mPFC LTP in vivo (Ohashi et al., 2002), we do not
believe that the serotonergic changes observed here af-
fected PILO-induced mPFC LTP enhancement. Besides,
PILO did not alter 5-HT turnover in the mPFC. In the
parietal cortex, however, PILO altered 5-HT and 5-HIAA
levels as compared with mPFC and hippocampus. 5-HT
turnover was strongly enhanced in the parietal cortex as
compared with the other areas. These data suggest that
the serotonergic transmission may undergo specific mus-
carinic modulation, presumably associated to particular
distributions of cholinergic receptors in cortical areas. Al-
though we observed specific noradrenaline changes in the
mPFC, noradrenaline also tended to be reduced in the
Considering that LTP is a synaptic correlate of memory
storage, our result of muscarinic strengthening of mPFC
LTP agrees with reports that prefrontal-dependent memo-
ries are enhanced by muscarinic activation. Muscarinic
agonists enhance the performance in tasks requiring at-
tentional and working memory capacities in rats, monkeys
and humans (Granon et al., 1995; Sarter and Bruno, 1997;
Ragozzino and Kesner, 1998; Gill et al., 2000; Chudasama
et al., 2004; Hasselmo and Stern, 2006). Besides, working
memory tasks induce an increase in acetylcholine efflux in
the prefrontal cortex of rats (Hironaka et al., 2001). Our
findings also support the idea of an important cholinergic
modulation of the synaptic plasticity in prefrontal circuits
involved in cognitive and emotional processes underlying
normal and psychiatric behaviors (Sarter et al., 2005). In
one case, it was recently reported that REM sleep, a
cholinergically-driven behavioral state, has an enhancing
effect on hippocampal LTP but a dampening effect on
mPFC LTP (Romcy-Pereira and Pavlides, 2004). In agree-
ment to that, REM sleep is correlated with a reactivation of
temporal lobe structures along with a deactivation of the
prefrontal cortex (Maquet et al., 1996). In addition, the mus-
iology of schizophrenia (Raedler et al., 2007). The atypical
antipsychotic drug clozapine, which possibly exerts its cog-
nitive-enhancing effects by increasing acetylcholine re-
lease in the prefrontal cortex (Ago et al., 2006), promotes
an enhancement of synaptic plasticity in the mPFC (Gem-
perle et al., 2003). It has also been shown that acetylcho-
line levels specifically increase in the hippocampus and
prefrontal cortex of rats during an inescapable stress
model of depression (Mark et al., 1996). Therefore, the
muscarinic neurotransmission has an important modula-
tory influence on prefrontal cortex functions. Further inves-
tigations should address the role of specific muscarinic and
nicotinic receptor modulators on synaptic plasticity at con-
nections between the mPFC and other relevant cortical
and subcortical structures involved in cognitive and neuro-
Acknowledgments—We would like to thank Renata Scandiuzzi
and Renato Meirelles for their excellent technical support. We
would also like to thank Ana Claudia Zanetti, Graziela Bachiega
and Raquel do Val for valuable discussions and advice. The work
was supported by grants from CAPES-PRODOC, FAPESP and
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(Accepted 26 February 2008)
(Available online 29 February 2008)
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