Post on 14-Feb-2017
Neuroscience 317 (2016) 47–64
MAJOR DORSOVENTRAL DIFFERENCES IN THE MODULATIONOF THE LOCAL CA1 HIPPOCAMPAL NETWORK BY NMDA, mGlu5,ADENOSINE A2A AND CANNABINOID CB1 RECEPTORS
S. KOUVAROS AND C. PAPATHEODOROPOULOS *
Laboratory of Physiology, Department of Medicine, School of
Health Sciences, University of Patras, 26504 Rion, Greece
Abstract—Recent research points to diversification in the
local neuronal circuitry between dorsal (DH) and ventral
(VH) hippocampus that may be involved in the large-scale
functional segregation along the long axis of the hippocam-
pus. Here, using CA1 field recordings from rat hippocampal
slices, we show that activation of N-methyl-D-aspartate
receptors (NMDARs) reduced excitatory transmission more
in VH than in DH, with an adenosine A1 receptor-
independent mechanism, and reduced inhibition and
enhanced postsynaptic excitability only in DH. Strikingly,
co-activation of metabotropic glutamate receptor-5
(mGluR5) with NMDAR, by CHPG and NMDA respectively,
strongly potentiated the effects of NMDAR in DH but had
not any potentiating effect in VH. Furthermore, the synergis-
tic actions in DH were occluded by blockade of adenosine
A2A receptors (A2ARs) by their antagonist ZM 241385 demon-
strating a tonic action of these receptors in DH. Exogenous
activation of A2ARs by 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofura-nuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic
acid hydrochloride (CGS 21680) did not change the effects
of mGluR5–NMDAR co-activation in either hippocampal
pole. Importantly, blockade of cannabinoid CB1 receptors
(CB1Rs) by their antagonist 1-(2,4-dichlorophenyl)-5-(4-iodo-
phenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide
(AM 281) restricted the synergistic actions of mGluR5–
NMDARs on excitatory synaptic transmission and postsy-
naptic excitability and abolished their effect on inhibition.
Furthermore, AM 281 increased the excitatory transmission
only in DH indicating that CB1Rs were tonically active in DH
but not VH. Removing the magnesium ions from the perfu-
sion medium neither stimulated the interaction between
http://dx.doi.org/10.1016/j.neuroscience.2015.12.0590306-4522/� 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Lab of Physiology, Department ofMedicine, University of Patras, 26 500 Rio, Patras, Greece. Tel: +30-2610-969117; fax: +30-2610-969169.
E-mail address: cepapath@upatras.gr (C. Papatheodoropoulos).Abbreviations: A2AR, adenosine A2A receptor; AM 281, 1-(2,4-dichlor-ophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide; CB1R, cannabinoid CB1 receptor; CGS, 4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]ben-zenepropanoic acid hydrochloride; CHPG, (RS)-2-chloro-5-hydroxyphenylglycine sodium salt; CPP, 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; DH, dorsal hippocampus; DMSO,dimethyl-sulfoxide; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine;EPSP, excitatory postsynaptic potential; Fv, fiber volley; mGluR5,metabotropic glutamate receptor-5; MTEP, 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride; NMDAR, N-methyl-D-aspartatereceptor; PS, population spike; VH, ventral hippocampus; ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol.
47
mGluR5 and NMDAR in VH nor augmented the synergy
of the two receptors in DH. These findings show
that the NMDAR-dependent modulation of fundamental
parameters of the local neuronal network, by mGluR5,
A2AR and CB1R, markedly differs between DH and VH. We
propose that the higher modulatory role of A2AR and
mGluR5, in combination with the role of CB1Rs, provide
DH with higher functional flexibility of its NMDARs, com-
pared with VH. � 2016 IBRO. Published by Elsevier Ltd. All
rights reserved.
Key words: hippocampus, dorsoventral, NMDA receptor,
mGlu5 receptor, A2A receptor, CB1 receptor.
INTRODUCTION
The segregation of functions along the longitudinal axis of
the hippocampus is a rather well established concept
(Small, 2002; Fanselow and Dong, 2010; Bannerman
et al., 2014). In addition, recently observed differences
in the functioning of the local neuronal circuitry between
the opposite poles of the hippocampus have attracted
the attention of researchers leading to a gradually accu-
mulating body of evidence and to the emergent concept
of diversification of the intrinsic neuronal network between
the dorsal (DH) and the ventral (VH) hippocampus. These
observations have been made at several levels of organi-
zation including cell properties (Liagkouras et al., 2008;
Dougherty et al., 2012; Honigsperger et al., 2015),
neurochemical markers (Gage and Thompson, 1980;
Verney et al., 1985; Sotiriou et al., 2005; Pandis et al.,
2006), synaptic transmission (Papatheodoropoulos
et al., 2002; Petrides et al., 2007; Georgopoulos et al.,
2008; Maggio and Segal, 2009), synaptic plasticity
(Papatheodoropoulos and Kostopoulos, 2000a,b; Maruki
et al., 2001; Colgin et al., 2004; Maggio and Segal,
2007; Grigoryan et al., 2012; Kenney and Manahan-
Vaughan, 2013; Keralapurath et al., 2014; Pofantis and
Papatheodoropoulos, 2014), gene expression profiles
(Thompson et al., 2008; Dong et al., 2009), and network
electrographic activity (Gilbert et al., 1985; Bragdon
et al., 1986; Papatheodoropoulos et al., 2005; Sabolek
et al., 2009; Mikroulis and Psarropoulou, 2012; Patel et al.,
2012; Papatheodoropoulos, 2015a). This diversification
is expected to have important implications for the
information processing and the functional roles performed
by the two hippocampal segments. Revealing functional
48 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
differences at the synaptic level, including the modulatory
actions of the plethora of receptors and their interactions,
between DH and VH will help us understand how the cir-
cuitries of the two hippocampal segments process infor-
mation. Consequently, we will be able to make links
between the operations of the small-scale circuit and the
large-scale functional segregation along the long axis of
the hippocampus.
The glutamate N-methyl-D-aspartate receptors
(NMDARs) and metabotropic receptors (mGluRs) as
well as the adenosine receptors are important
modulators of the neuronal activity (Anwyl, 1999;
Mukherjee and Manahan-Vaughan, 2013; Sebastiao
and Ribeiro, 2014). The family of mGluRs comprises
three groups of receptors (Sheffler et al., 2011), with
those belonging to the group I mGluRs having particularly
important role in modulating the activity of hippocampal
pyramidal cells (Charpak et al., 1990; Desai and Conn,
1991; Pedarzani and Storm, 1993; Gereau and Conn,
1995b; Mannaioni et al., 1999; Mannaioni et al., 2001).
NMDARs (Monaghan and Cotman, 1985) and group I
metabotropic glutamate receptor-5 (mGluR5) are espe-
cially abundant in the CA1 field of the hippocampus
(Shigemoto et al., 1992; Romano et al., 1995). Notably,
mGluR5 and NMDARs interact synergistically to potenti-
ate NMDAR-mediated responses (Doherty et al., 1997;
Anwyl, 1999; Mannaioni et al., 2001; Tebano et al.,
2005). Furthermore, recent observations have demon-
strated that this synergistic action of the two glutamate
receptors is under the control of adenosine A2A receptor
(A2AR) (Tebano et al., 2005). A2ARs have a prominent
modulatory role in the brain (Cunha et al., 2008;
Sebastiao and Ribeiro, 2009) and they are involved in
hippocampus-dependent processes (Costenla et al.,
2010).
In this study, using recordings of local field potentials
from the CA1 area of adult rat hippocampal slices we
show that pharmacological manipulation of the three
receptors, i.e. NMDAR, mGluR5 and A2AR, has
remarkably different actions on synaptic transmission,
postsynaptic excitability and paired-pulse inhibition
between DH and VH. In addition it is shown that a
considerable portion of these actions require the activity
of CB1 cannabinoid receptors.
EXPERIMENTAL PROCEDURES
Animals and slice preparation
Hippocampal slices were prepared from forty eight adult,
2–4 month-old male Wistar rats. All experimental
treatment and procedures were conducted in
accordance with the European Communities Council
Directive Guidelines (86/609/EEC, JL 358, 1, December,
12, 1987) for the care and use of Laboratory animals
and they have been approved by the Prefectural Animal
Care and Use Committee (No: EL 13BIO04). In
addition, all efforts have been made to minimize the
number and the suffering of animals used. Animals were
housed in our Institution under controlled conditions of
temperature (20–22 �C), light–dark cycle (12/12 h) and
free access to food and water. Hippocampal slices from
the DH and the VH were prepared as previously
described (Papatheodoropoulos and Kostopoulos,
2000a). Specifically, animals were decapitated after deep
anesthesia with diethyl-ether. The brain was removed and
placed in chilled (2–4 �C) standard artificial cerebrospinal
fluid where the two hippocampi were excised free. The
standard medium contained 124 mM NaCl, 4 mM KCl,
2 mM MgSO4, 2 mM CaCl2, 1.25 mM NaH2PO4, 26 mM
NaHCO3 and 10 mM glucose, equilibrated with 95% O2
and 5% CO2 gas mixture at pH = 7.4. Transverse slices
500–550-lm thick were prepared from the regions
extending more than 1 and less than 4 mm from the dor-
sal (septal) and the ventral (temporal) ends of the hip-
pocampus using a McIlwain tissue chopper. In order to
maintain an orthogonal cut plane during sectioning of
the two poles a turn of the plate supporting the structure
was required. From each animal 1–4 slices from each
pole were selected for experimentation. Immediately after
sectioning, slices were transferred and maintained to an
interface type recording chamber continuously perfused
with standard medium of the same composition as above
described and humidified with a mixed gas containing
95% O2 and 5% CO2 at a constant temperature of 31
± 0.5 �C. Slices were left to equilibrate for at least one
and a half hour after their preparation before starting
recordings. In a set of experiments, slices were bathed
in medium containing no magnesium ions (magnesium-
free medium, Mg2+ = 0 mM). Furthermore, in some
experiments, slices containing the CA1 but not the CA3
field, after disconnecting the two fields by a knife cut of
the Schaffer collaterals, were also used (specified in
Results).
Recordings, data processing and analysis
Field potentials consisting of presynaptic fiber volley (Fv),
excitatory postsynaptic potential (EPSP) and population
spike (PS) were evoked by delivering electrical pulses
(of varying amplitude and stable duration of 100 ls) at
Schaffer collaterals using a bipolar platinum–iridium
electrode (25-lm wire-diameter, at an inter-wire
distance of 100 lm, World Precision Instruments, USA)
and recorded from the CA1 stratum radiatum (the EPSP
and Fv) and stratum pyramidale (the PS) using carbon
fiber electrodes (diameter 7 lm, Kation Scientific,
Minneapolis, USA). Stimulation and recording electrodes
were placed at a distance of 350 lm from each other.
Signals were acquired with a Neurolog amplifier
(Digitimer Limited, UK), band-pass filtered at 0.5 Hz–
2 kHz, digitized at 10 kHz and stored in a computer disk
using the CED 1401-plus interface and the Signal6
software (Cambridge Electronic Design, Cambridge, UK)
for off-line analysis. Single electrical pulses were
delivered at the frequency of 0.033 Hz. However,
stimulation frequency was increased to 0.01 Hz during
the short periods of drug-induced rapid changes in the
evoked response (noted in Results). Only slices which
displayed stable EPSP and PS for at least 10 min were
selected for further experimentation. Input/output curves
between stimulation intensity and evoked response
were made in control conditions and during drug
application. In conditions of strong drug-induced
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 49
suppression of the synaptic response input/output curves
could not be made. EPSP was quantified by the maximum
slope of its rising phase. Fv was quantified by its
amplitude measured as the difference between the
baseline and the peak negative voltage. PS was
quantified by its amplitude measured as the length of
the projection of the minimum peak on the line
connecting the two maxima peaks of the PS waveform.
Given that the size of Fv indicates the number of
activated afferent fibers (Andersen et al., 1978), we used
the EPSP/Fv ratio to quantify synaptic effectiveness. The
PS/EPSP ratio was always used as an index of postsy-
naptic excitability. The ratio was measured at the 40–
60% of maximum EPSP. The deviation in the value of
EPSP was due to the fact that during the short periods
of drug-induced rapid change in the synaptic response it
was not feasible to adjust the stimulation intensity so that
to obtain half-maximum response. However, comparisons
of the ratio between control and drug conditions were
always made at the same time points and using control
EPSPs of similar amplitude (obtained from I/O curves)
despite the fact that EPSP could be somewhat reduced
due to drug action. We also measured the facilitation of
EPSP and the potency of the inhibition in suppressing
PS by employing the experimental protocol of paired-
pulse stimulation according to which two pulses of identi-
cal intensity were delivered at the Schaffer collaterals in
rapid succession. Taking into account that the magnitude
of synaptic facilitation is inversely related to the probability
of transmitter release (Dobrunz and Stevens, 1997), we
used the phenomenon of paired-pulse facilitation in order
to establish whether presynaptic mechanisms are
involved in the drug effects observed under the various
pharmacological conditions. Paired-pulse facilitation of
EPSP was studied at the inter-pulse interval of 50ms
which corresponds to the interval that maximum facilita-
tion is induced in both DH and VH (Papatheodoropoulos
and Kostopoulos, 2000b). The magnitude of facilitation
was quantified by the ratio between the second and the
first response i.e. EPSP2/EPSP1. The strong recurrent
inhibition that controls the excitation of the hippocampal
principal neurons (Freund and Buzsaki, 1996) can be
examined and quantified by activating the local networks
of inhibitory interneurons through an orthodromic stimulus
(the first of the pair, conditioning stimulus) and observe
the inhibitory effect on the suppression of the response
evoked by the following second (test) stimulus. The
inter-pulse interval of 8 ms was chosen for the study of
recurrent paired-pulse inhibition on the basis of previous
observations showing maximum suppression of PS at
inter-pulse intervals of between 4 and 10 ms
(Papatheodoropoulos et al., 2002) and the fact that at
short interval there is minimum contamination of inhibition
by synaptic facilitation. The strength of paired-pulse inhi-
bition was quantified by the ratio between the conditioned
and the unconditioned response (PS2/PS1) with stimula-
tion intensity set to produce a half-maximum PS1. In order
to make possible the comparison of PS2/PS1 ratio
between control and drug conditions and taking into
account that this ratio strongly depends on the amplitude
of PS1, we required to briefly adjust the stimulation
intensity to obtain a half-maximum PS during the short
period of drug-induced rapid change in PS (described in
Results). Thus, in order to make possible the compar-
isons between control and drug conditions, the ratios
PS/EPSP and PS2/PS1 were always obtained under sim-
ilar conditions of postsynaptic activation (i.e. EPSP or
PS1). Whenever this was not possible, calculations of
PS/EPSP and PS2/PS1 were not made. Summarizing,
the variables EPSP/Fv, EPSP2/EPSP1, PS/EPSP and
PS2/PS1 quantified the synaptic effectiveness, synaptic
facilitation, postsynaptic excitability and paired-pulse inhi-
bition respectively.
Drugs
The following drugs were used: the NMDAR agonist N-methyl-d-aspartic acid (NMDA, 25 lM, 50 lM); the
NMDAR antagonist 3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 10 lM); the selective
mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycinesodium salt (CHPG, 50 lM); the selective mGluR5
antagonist 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine
hydrochloride (MTEP, 200 lM); the selective A1
adenosine receptor antagonist 8-cyclopentyl-1,3-dipropyl
xanthine (DPCPX, 30 nM); the A2AR agonist 4-[2-[[6-
amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride
(CGS 21680, 10 nM); the selective A2AR antagonist 4-(2-
[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, 100 nM); the selective
CB1 cannabinoid receptor antagonist 1-(2,4-dichloro-
phenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-
pyrazole-3-carboxamide (AM 281, 5 lM). Drugs were
purchased from Tocris Cookson Ltd, UK (NMDA, CPP,
CHPG, MTEP, DPCPX, CGS 21680, ZM 241385 and
AM 281) as well as from Sigma-Aldrich, Germany
(NMDA and CHPG). Drugs were first prepared as stock
solutions and then were dissolved in standard medium
and bath applied to the tissue. Stock solutions of
NMDA, CPP, CHPG and MTEP were prepared in
distilled water, whereas stock solutions of DPCPX, ZM
241385 and AM 281 were prepared in dimethyl-
sulfoxide (DMSO) at a concentration that when diluted
for bath application the final volume of DMSO was lower
than 0.05%. Stock solutions in water were maintained at
4 �C while solutions in DMSO were prepared in aliquots
and kept at �20 �C. Stock solutions were diluted in
standard medium to the desired concentrations the day
of the experiment.
Statistics
The non parametric tests Wilcoxon test and Mann–
Whitney U test were used for comparisons between
related and independent two groups of values
respectively. The values of the various parameters are
expressed as mean ± S.E.M. and ‘‘n” throughout the
text indicates the number of slices and animals (slices/
animals) used in the analysis. The detection of
statistically significant differences was made using the
number of slices.
50 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
RESULTS
Baseline measures in DH and VH
Field potentials (Fv, EPSP and PS) were evoked and
measured with varying stimulation strength in both DH
(n= 15/15) and VH (n= 15/15) (Fig. 1A–D). The
dorsal and ventral slices were obtained from the same
animals and constituted a sample of the whole
population of slices used in this study. In keeping with
previous observations (Papatheodoropoulos, 2015b) we
found that Fv was significantly higher in DH than in VH
at moderate to high stimulation intensities (Fig. 1A).
However, the relationship between presynaptic activity
(Fv) and postsynaptic depolarization (EPSP) was quite
similar between the two hippocampal poles (Fig. 1B).
Furthermore, neither EPSP nor PS did significantly
differ between DH and VH at any stimulation intensity
Fig. 1. Comparisons of baseline measures between DH and VH.
Cumulative input/output curves illustrating the relationships between
the various variables are shown in A-D diagrams for both DH (circle)
and VH (squares). Inset in graph shown in ‘‘D” illustrates the
relationship between stimulation strength and PS. Plots in E and F
show the values of paired-pulse facilitation and paired-pulse inhibition
quantified by the ratios EPSP2/EPSP1 and PS2/PS1 respectively.
Paired-pulse facilitation of half-maximum EPSP was measured at the
inter-pulse interval of 50ms. Paired-pulse inhibition was measured at
two stimulation intensities producing half-maximum PS (50%) and
90% of the maximum PS respectively. Data were collected from
fifteen dorsal and fifteen ventral slices obtained from the same fifteen
animals. Asterisks denote statistically significant differences between
DH and VH at *p< 0.05, **p< 0.005 or #p< 0.001 (Mann–Whitney
U test).
used (Fig. 1C and inset in Fig. 1D) confirming previous
results (Maggio and Segal, 2007; Pofantis and
Papatheodoropoulos, 2014). Plotting PS against EPSP
we found that postsynaptic excitability was also compara-
ble between DH and VH (Fig. 1D). Yet, paired-pulse facil-
itation (EPSP2/EPSP1) and paired-pulse inhibition (PS2/
PS1) were significantly stronger in DH (n= 15/15) com-
pared with VH (n= 15/15), (Fig. 1E, F), corroborating
previous studies (Papatheodoropoulos and Kostopoulos,
2000b; Papatheodoropoulos et al., 2002; Petrides et al.,
2007).
Divergent effects of NMDARs’ activation between DHand VH
The effects of NMDA on synaptic transmission (EPSP/
Fv), postsynaptic excitability (PS/EPSP) and paired-
pulse inhibition (PS2/PS1) were examined at the
concentrations of 25 lM and 50 lM (Fig. 2). These
concentrations were similar to those used by others in
hippocampal slices (Sah et al., 1989; Papatheodoropoulos
et al., 2005; Xue et al., 2011). In general, the effects of
NMDA were higher at 50 than 25 lM in both DH
and VH. At 25 lM the drug significantly increased
the EPSP/Fv and EPSP2/EPSP1 ratios in DH (by 7.37 ±
1.9% and 10.2 ± 2.5% respectively, n= 7/5, Wilcoxon
test, p< 0.05) but not VH (n= 7/4). Furthermore, in
DH the ratio PS2/PS1 but not PS/EPSP was significantly
increased under 25 lM NMDA (by 27.1 ± 11.6%,
n= 7/5, Wilcoxon test, p< 0.05). At 50 lM the drug sig-
nificantly reduced synaptic transmission (i.e. EPSP/Fv) in
DH (by 18.7 ± 2.1%, n= 12/9, Wilcoxon test, p< 0.005)
and produced an even higher reduction in VH (by 28.9
± 4.8%, n= 13/7, Wilcoxon test, p< 0.005; comparison
between DH and VH by Mann–Whitney U test, p< 0.05).
Also, 50 lM NMDA increased synaptic facilitation only in
DH (by 7.0 ± 2.0%, n= 7/5, Wilcoxon test, p< 0.05).
Strikingly, 50 lM NMDA produced a robust increase in
the PS/EPSP (by 33.6 ± 11.9%, n= 12/9, Wilcoxon test,
p< 0.01) and PS2/PS1 (by 220 ± 53%, n= 7/5,
Wilcoxon test, p< 0.05) in DH, but it did not affect these
parameters in VH (PS/EPSP, n= 13/7 and PS2/PS1,
n= 5/4). Furthermore, EPSP2/EPSP1 did not change in
VH (n= 8/5). These experiments showed that activation
of NMDARs reduced synaptic transmission in VH more
than DH but enhanced postsynaptic excitability and
reduced inhibition only in DH.
The action of NMDAR does not require the activity ofA1 adenosine receptors
One of the mechanisms proposed to underlie the
reduction in the synaptic transmission induced by
activation of NMDARs points to adenosine A1 receptors.
Thus, acute activation of NMDARs results in release of
adenosine (Hoehn and White, 1990) which can activate
presynaptic A1 adenosine receptors (A1Rs) and reduce
glutamate release (Yoon and Rothman, 1991; Manzoni
et al., 1994). We therefore examined whether blockade
of A1Rs by their antagonist DPCPX (30 nM), (Rombo
et al., 2015), would occlude the actions of NMDA. As
Fig. 2. The effects of activation of NMDARs in dorsal and ventral hippocampal slices. (A) Examples of field recordings from CA1 stratum radiatum
and stratum pyramidale (traces in the left panel) obtained from one dorsal and one ventral hippocampal slice before and during application of 25 lMNMDA. The effects of paired-pulse stimulation on PS are shown in the stratum pyramidale recordings (lower traces). The graph on the right panel
illustrates the time-course of EPSP/Fv change under 25 lM NMDA for DH (n= 7/5) and VH (n= 7/4). (B) Examples of single-pulse (traces in the
first and second row) and paired-pulse evoked potentials (third row) from one dorsal and one ventral slice illustrating the reversible effects of 50 lMNMDA. Note that NMDA reduced EPSP/Fv in VH more than DH and increased PS in DH but not VH. Also, note that NMDA reversed paired-pulse
inhibition of PS into robust facilitation in DH but not VH. (C) The time-course of EPSP/Fv change illustrating the higher NMDA-induced depression in
the excitatory synaptic transmission in ventral (n= 6/4) compared with dorsal slices (n= 6/4). (D) Collective results of the effects of NMDA on
synaptic transmission (EPSP/Fv), paired-pulse facilitation (EPSP2/EPSP1), postsynaptic excitability (PS/EPSP) and paired-pulse inhibition (PS2/
PS1) in DH and VH (patterned blue and clear red columns respectively). Asterisks denote statistically significant drug actions at *p< 0.05 or**p< 0.005 (Wilcoxon test). Differences in the drug effects between DH and VH are also indicated at #p< 0.05 (Mann–Whitney test). Artifacts in the
example traces in this and the following figures are truncated. DH data at 25 lM were collected from 7 slices/5 animals for all parameters, while at
50 lM were obtained from 12 slices/9 animals for EPSP/Fv and PS/EPSP, and from 7 slices/5 animals for PS2/PS1 and EPSP2/EPSP1. VH data at
25 lM were collected from 7 slices/4 animals for all parameters, while at 50 lM were obtained from 13 slices/7 animals for EPSP/Fv and PS/EPSP,
from 5 slices/4 animals for PS2/PS1 and from 8 slices/5 animals for EPSP2/EPSP1. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 51
Fig. 3. The depression of synaptic transmission by NMDA does not
involve A1Rs. Examples of field potentials from stratum radiatum and
stratum pyramidale from one dorsal and one ventral slice (A) and
collective data (B) illustrating the absence of effect of blockade of
A1Rs by their antagonist DPCPX (30 nM; DH, n= 5/5 and VH, n= 5/
5) on the depressive effect of NMDA on synaptic transmission. Data
obtained under 50 lM NMDA that are presented in Fig. 2 are also
shown here for comparison reasons (bars on the left). Asterisks
denote statistically significant drug effects at *p< 0.05 or **p< 0.005
(Wilcoxon test).
52 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
shown in Fig. 3 DPCPX did not prevent the effects of
NMDA on EPSP/Fv in either DH (n= 5/5) or VH (5/5).
Co-activation of mGluR5 with NMDARs enhancesNMDAR-mediated actions in DH but not VH
It has been previously shown that interaction between
mGluR5 and NMDAR potentiates NMDAR-mediated
responses (Fitzjohn et al., 1996; Mannaioni et al., 2001;
Tebano et al., 2005). Thus, we first examined whether
exogenous activation of only mGlu5Rs can foster
NMDARs’ activity and then we studied the effects of co-
activation of mGluR5 with NMDAR. We applied the selec-
tive agonist of mGluR5 CHPG at the concentrations of
50 lM and 200 lM, which were similar to those previously
used in in vitro hippocampal preparations (Kotecha et al.,
2003; Sarantis et al., 2015), in DH (n= 6/5 and n= 5/3
at 50 lM and 200 lM respectively) and VH (n= 4/3 and
n= 5/3 at 50 lM and 200 lM respectively). Neither drug
concentration had significant effects on any of the param-
eters studied in either DH or VH. In particular, the effects
(percent change) of 50 lM CHPG on EPSP/Fv, PS/EPSP
and EPSP2/EPSP1 were 12.7 ± 4.8, 2.6 ± 3.8 and 1.4
± 1.7 in DH (n= 6/5), and 3.2 ± 2.1, 3.9 ± 2.6 and
3.7 ± 0.6 in VH (n= 4/3). Likewise, the effects of
200 lM CHPG in EPSP/Fv, PS/EPSP, EPSP2/EPSP1
and PS2/PS1 were 0.8 ± 3.4, 6.6 ± 7.0, 15.0 ± 16.9
and 5.3 ± 5.5 in DH (n= 5/3), and 7.3 ± 3.0, 3.4
± 3.0, 26.8 ± 16.4 and 7.4 ± 4.1 in VH (n= 5/3).
Then, we investigated the effects of co-activation of
mGluR5 with NMDAR comparatively between DH and
VH by concurrently applying NMDA (50 lM) and CHPG
(50 lM) in dorsal and ventral slices. Remarkably, co-
activation of mGluR5 with NMDARs significantly
enhanced the effects of NMDAR’s activation on EPSP/
Fv, PS/EPSP and PS2/PS1 in DH but not VH (Fig. 4).
In particular, we found that co-application of NMDA with
CHPG in DH consistently produced a sequence of time-
dependent effects that consisted of three phases. The
first phase was short, developed during the first 3–5 min
from the start of application of the drug cocktail and was
characterized by a strong reduction in the paired-pulse
inhibition (increase in the PS2/PS1 ratio by 530 ± 88%,
n= 11/5, Wilcoxon test, p< 0.005) accompanied,
during the next 20–40 s, by an abrupt very intense
increase in the postsynaptic excitability (increase in the
PS/EPSP ratio by 641 ± 171%, n= 15/7, Wilcoxon
test, p< 0.005). Thus, the combined action of the
agonists of the two receptors in DH far surpassed the
corresponding effects of 50 lM NMDAR in PS/EPSP
(Mann–Whitney U test, p< 0.001) and PS2/PS1
(Mann–Whitney U test, p< 0.05). It is noted that the
large increase in the postsynaptic excitability occurred
concurrently with a gradual reduction in the excitatory
synaptic transmission (EPSP/Fv ratio) which was
significantly reduced during the first phase in both DH
(by 32.2 ± 3.6%, n= 19/10, Wilcoxon test, p< 0.001)
and VH (by 16.5 ± 3.7%, n= 10/7, Wilcoxon test,
p< 0.05). Notably, the reducing effect of mGluR5-
NMDAR co-activation on EPSP/Fv was significantly
higher in DH than VH (Mann–Whitney U test, p< 0.05).
The phase of intense increase in excitability and
reduction in inhibition observed in DH was immediately
followed by a second phase consisted of an abrupt
suppression in the excitability (decrease in the PS/
EPSP ratio by 74.4 ± 14.4%, n= 13/7, Wilcoxon test
p< 0.05) accompanied by a recovery in the PS2/PS1
ratio to control levels (change 9.6 ± 30% relative to
control values, n= 11/5, Wilcoxon test, p> 0.05). The
effects of this phase maximized at 6–7 min from the
beginning of the drugs application. In order to obtain
appropriate measurements of PS during the short
phases of rapid and transient changes in postsynaptic
excitability, the frequency of stimulation and field
potential recording was increased from 0.033 Hz to
0.01 Hz. It was also required to transiently adjust the
stimulus intensity, in order to obtain half-maximum PS
and render the comparison with control data feasible.
Notably, this second phase was not observed under
activation of NMDARs only. During the second phase
the EPSP/Fv ratio continued to decline in both DH (by
46.5 ± 4.3%, n= 9/4, Wilcoxon test p< 0.01) and VH
(by 31.1 ± 5.7%, n= 9/7, Wilcoxon test p< 0.01), with
the higher effect observed again in DH (Mann–Whitney
U test, p< 0.05). It is noted that during this second
phase of co-application of NMDA with CHPG (i.e. 6–
7 min), VH did not show significant change in paired-
pulse inhibition (n= 5/4), but it did show significant
reduction in postsynaptic excitability (n= 5/4).
Fig. 4. Co-activation of mGluR5 with NMDAR produces synergistic effects on synaptic transmission, postsynaptic excitability and paired-pulse
inhibition in DHbut not VH.NMDARandmGluR5were activated by 50 lMNMDAand 50 lMCHPG respectively. (A). In each of the two panels (DHand
VH) are shown field potential traces exemplifying the effects of the drug cocktail on the synaptic transmission (EPSP, top row), neuronal excitability (PS,
middle row) and paired-pulse inhibition (bottom row). Recordings from different time points during drug application are shown. Note that during the first
3–5 min co-activation of the two receptors produceda strong enhancement in neuronal excitability (evidencedby the increase inPSand theappearance
of secondary spikes) and a reduction in paired-pulse inhibition of PS which reversed into facilitation. In the next 6–7 min there was a considerable
reduction in excitability and a recovery in paired-pulse inhibition. Importantly, these sequential changes in excitability and inhibition occurred in DH but
notVH.Also, note that thedrug cocktail produced aprogressive reduction in the synaptic transmissionwhichwas stronger inDH than inVH. (B)Graph of
time-course of EPSP/Fv illustrating the higher reduction produced by the drug cocktail in DH comparedwith VH. Note that the effects of coactivity of the
two receptors contrasted so strikingly against the effects of only NMDARs’ activation (compare with the graph of Fig. 2C). (C) Time-course graph of the
change of EPSP/Fv showing thatCPPabolished the synergistic potentiating effect of CHPG-NMDAdrug cocktail in DH. (D)Graphs of collective data on
the drug effects on synaptic transmission (EPSP/Fv), paired-pulse facilitation (EPSP2/EPSP1), postsynaptic excitability (PS/EPSP) and paired-pulse
inhibition (PS2/PS1) in DH andVH (patterned blue and clear red columns respectively). Results obtained from three time points during co-application of
NMDA with CHPG are shown in order to illustrate the transient large changes in PS/EPSP and PS2/PS1 ratios. However, in PS/EPSP and PS2/PS1
graphs data for DH at 12–15 min are not presented because PS could not be evoked. Measures under pharmacological conditions with the presence of
receptor antagonists were collected at the time of the maximum effect of CHPG-NMDA drug cocktail (i.e. 12–15 min for EPSP/Fv and EPSP2/EPSP1
and 3–5 min for PS/EPSPandPS2/PS1). Asterisks denote statistically significant drug actions at *p< 0.05 or **p< 0.005 (Wilcoxon test). Differences
in the drug effects between DH and VH are indicated at #p< 0.05 or ##p< 0.005 (Mann–Whitney test). Data were collected as follows: For EPSP/Fv,
and the three timepoints,n= 19/10, 9/4 and20/10, inDH; andn= 10/7, 9/7 and12/9 in VH.ForEPSP2/EPSP1, and the three timepoints,n= 13/7, 8/
4 and7/4, inDH; andn= 4/4, 6/4 and5/4 inVH. ForPS/EPSP inDH, n= 15/7 (3–5 min) andn= 13/7 (6–7 min); inVH, n= 10/5 (3–5 min) andn= 5/
4 (6–7 min and 12–15 min). For PS2/PS1, n= 11/5 (3–5 min and 6–7 min) in DH and n= 5/4 (3–5 min, 6–7 min and 12–15 min) in VH. For all the
parameters and in each of the remaining conditions (MTEP, MTEP+ NMDA+ CHPG, CPP and CPP+ NMDA+ CHPG), n= 5/2 (only DH). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 53
A
B
C
Fig. 5. Effects of co-activation of mGluR5 with NMDAR in CA1
minislices. (A) Field potentials recorded simultaneously from the
dendritic and somatic layer (upper and lower traces respectively) of a
dorsal hippocampal slice containing the CA1 field disconnected from
the CA3 field by knife-cutting the Schaffer collaterals (CA1 minislice),
exemplifying the reversible effects of the cocktail containing 50 lMCHPG and 50 lM NMDA on synaptic transmission and postsynaptic
excitability. (B) Graph made with data obtained from a different CA1
minislice illustrating the reversible suppression of EPSP/Fv by the
CHPG-NMDA drug cocktail. (C) Changes produced in EPSP/Fv
(n= 9/2) and PS/EPSP (n= 6/2) ratios by the CHPG–NMDA drug
cocktail. Measures under drugs were obtained at the two different
time points indicated. Asterisks indicate statistically significant differ-
ences between control and drug conditions at *p< 0.05 or**p< 0.005 (Wilcoxon test).
54 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
The third phase was characterized by a further decline
in the EPSP/Fv ratio. More specifically, we found that the
robust reduction in EPSP/Fv ratio produced by the co-
application of NMDA with CHPG reached maximum
values at 12–15 min in both DH (84.5 ± 5.1%,
n= 20/10, Wilcoxon test, p< 0.001) and VH (44.7
± 8.2%, n= 12/9, Wilcoxon test, p< 0.005).
Importantly, the suppressive effect of the drug cocktail
on EPSP/Fv in the DH significantly exceeded that
observed under NMDAR’s activation (by 350%, Mann–
Whitney test, p< 0.001). In contrast, the reduction in
EPSP/Fv observed in VH was similar to that observed
under activation of NMDARs only (Mann–Whitney Utest, p> 0.05). Consequently, the suppressive effect of
co-activation of the two receptors on the excitatory
synaptic transmission was significantly stronger in DH
than in VH (Mann–Whitney U test, p< 0.005). During
this last phase the postsynaptic excitability continued to
decline in DH, without considerable change in paired-
pulse inhibition, until was not practically feasible to
record PS. In VH, either excitability or inhibition
displayed no further significant change (n= 5/4). These
results indicated that activation of mGluR5 potentiated
the effects of NMDAR on synaptic transmission,
postsynaptic excitability and paired-pulse inhibition in
DH but not VH. Application of NMDA and CHPG in the
presence of the antagonist of mGluR5 MTEP (200 lM),
(Lea et al., 2005; Sarantis et al., 2015), prevented the
synergistic effects of the two agonists (five dorsal slices
from two animals), (Fig. 4D). Similarly, the drug cocktail
NMDA–CHPG had no significant effects on any of the
parameters measured when applied in the presence of
the antagonist of NMDARs CPP (five dorsal slices from
two animals), at the concentration of 10 lM that blocks
NMDAR-mediated currents in CA1 neurons (Provini
et al., 1991), (Fig. 4C, D). Application of either MTEP or
CPP alone did not produce any significant effect on any
of the parameters (n= 5/2). Furthermore, the EPSP2/
EPSP1 ratio did not significantly change under any of
the pharmacological conditions used and in either hip-
pocampal pole (DH, n= 13/7 under NMDA–CHPG, and
n= 5/2 under MTEP or CPP; VH, n= 4/4 under
NMDA–CHPG).
The synergistic effects of NMDAR and mGluR5 occurin the isolated CA1 field
In order to examine whether activity in the CA3 field might
affect the drug actions obtained from the CA1 circuitry we
applied CHPG (50 lM) and NMDA (50 lM) in dorsal
slices containing the CA1 but not the CA3 field. As shown
in Fig. 5, the co-activation of mGluR5 with NMDAR in the
CA1 minislices produced a pattern of changes similar to
that observed in intact dorsal hippocampal slices. In
particular, the drug cocktail produced an early, robust and
transient increase in the PS/EPSP ratio by 932 ± 392%
(Wilcoxon test, p< 0.05, n= 6/2), followed by a
reduction of 80.2 ± 19.8% (Wilcoxon test, p< 0.01,
n= 6/2). Furthermore, the drug cocktail produced a
time-dependent suppression in the EPSP/Fv ratio
reaching a maximum effect of 96.5 ± 3.5% (Wilcoxon
test, p< 0.01, n= 9/2).
A2ARs control the synergistic effects of mGluR5 andNMDAR in DH but not VH
It has been previously shown that the potentiating effect of
mGluR5 on NMDAR-induced reduction in excitatory
synaptic transmission in the hippocampus is controlled
by the endogenous permissive action of A2ARs on the
synergy between mGluR5 and NMDAR (Tebano et al.,
2005). Thus, we asked whether the potentiating effects
of CHPG observed in DH on excitatory synaptic transmis-
sion, postsynaptic excitability and paired-pulse inhibition
could be abolished after blockade of A2ARs. Therefore,
we applied 50 lM CHPG and 50 lM NMDA in the pres-
ence of the selective antagonist of A2ARs ZM 241385
(100 nM), (Pugliese et al., 2009). As shown in Fig. 6,
ZM 241385 occluded the synergistic effects of mGluR5
and NMDAR on synaptic transmission (n= 12/11),
neuronal excitability (n= 7/5) and paired-pulse inhibition
A B C
DE
F G
Fig. 6. Tonic activity of A2AR in DH but not VH controls the synergistic effects between mGluR5 and NMDAR. (A) Examples of field potentials from
the stratum radiatum (EPSP, upper traces) and stratum pyramidale (PS, lower traces) of one dorsal and one ventral hippocampal slice illustrating
the effects of the A2A receptor antagonist ZM 241385 (100 nM). Note that blockade of A2ARs occluded the effects of NMDAR–mGluR5 synergy
(compare with the corresponding traces in Fig. 4A). (B) Time-course of the change of EPSP/Fv showing that co-application of CHPG with NMDA in
the presence of ZM 241385 produced a similar reduction in the synaptic transmission in DH (n= 8/8) and VH (n= 8/8) by preventing the
synergistic potentiating effects in DH. (C) Time-course of the change of EPSP/Fv illustrating the absence of effects of the agonist of A2AR CGs
21680 on the action of co-activation of mGluR5 with NMDAR either in DH or VH. Data were obtained from one dorsal and one ventral hippocampal
slice. (D) Field potentials (traces of EPSP and PS in upper and lower lines) from one dorsal and one ventral hippocampal slice exemplifying the
effects of CHPG-NMDA drug cocktail in the presence of CGs 21680 (10 nM). (E)–(G). Cumulative data on the effects of ZM 241385 and CGS 21680
on synaptic transmission (E), postsynaptic excitability (F) and paired-pulse inhibition (G), in DH and VH. For reason of comparisons, data obtained
under CHPG-NMDA are also shown in the left part of graphs. Asterisks denote statistically significant drug actions at *p< 0.05 or **p< 0.005
(Wilcoxon test). Differences in the drug effects between DH and VH are indicated at #p< 0.05 or ##p< 0.005 (Mann–Whitney test). Data were
collected as follows: In ‘‘ZM” condition, DH, n= 12/11 for EPSP/Fv, n= 7/5 for PS/EPSP and n= 6/4 for PS2/PS1; VH, n= 12/11 for EPSP/Fv
and n= 5/4 for PS/EPSP and PS2/PS1. In ‘‘CGS” condition, DH, n= 6/3 for EPSP/Fv and PS/EPSP and n= 5/3 for PS2/PS1; VH, n= 5/4 for all
parameters.
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 55
(n= 6/4) in the dorsal hippocampal slices. In particular,
blockade of A2ARs in DH significantly reduced the syner-
gistic action of CHPG–NMDA drug cocktail on EPSP/Fv
by 70% (from �84.5 ± 5.1%, n= 20/10 to �25.4
± 6.0%, n= 12/11, Mann–Whitney U test, p< 0.001).
The reduction of EPSP/Fv induced by the CHPG–NMDA
drug cocktail under blockade of A2ARs became similar to
that observed under activation of NMDARs only. Further,
ZM 241385 prevented the synergistic effects of CHPG-
NMDA drug cocktail on the postsynaptic excitability and
paired-pulse inhibition. More specifically, blockade of
A2ARs in the dorsal slices reduced the transient
CHPG–NMDA-induced increase in the PS/EPSP ratio
by more than twelve times (from 641 ± 171%, n= 15/7
to 43.2 ± 11.4%, n= 7/5, Mann–Whitney U test,
p< 0.005). Similarly, ZM 241385 restricted the transient
CHPG–NMDA-induced increase in the PS2/PS1 ratio
from 530± 88% (n= 11/5) to 219.4 ± 78.6% (n=6/4),
(Mann–Whitney U test, p< 0.05). Then we asked
whether the absence of potentiating action of mGluR5
on NMDAR-mediated effects in the VH may result from
a low endogenous activity of A2ARs in this hippocampus
segment. We applied CHPG (50 lM) and NMDA (50 lM)
in the presence of the selective agonist of A2ARs CGS
21680 (10 nM), (Rodrigues et al., 2014). As shown in
Fig. 6C–G, CGS 21680 did not facilitate the synergic
actions of mGluR5 and NMDAR in either DH or VH. Thus,
under CGS 21680 the effects of the CHPG–NMDA drug
cocktail on PS/EPSP and PS2/PS1 were similar to those
obtained without exogenous manipulation of A2ARs, i.e.
the effects of drug cocktail in DH were strong (increase
in PS/EPSP and PS2/PS1 by 152 ± 38%, n= 6/3 and
56 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
397 ± 142%, n= 5/3, respectively). In VH the effects
were lower and non significant (10.1 ± 13% and 67.9
± 50.6% for PS/EPSP and PS2/PS1 respectively,
n= 5/4). Yet, EPSP/Fv was affected similarly by the drug
cocktail in DH and VH (-58.8 ± 9.6% and -39.8 ± 8.8% in
DH, n= 6/3 and VH, n= 5/4 respectively). CGS 21680
alone did not significantly affect any of the parameters
either in DH or VH and the EPSP2/EPSP1 ratio did not
change at any condition, either in DH or VH. These results
demonstrated that both A2AR and mGluR5 powerfully
contribute in facilitating NMDAR-mediated actions in the
DH but not the VH circuitry.
The synergistic effects of mGluR5 and NMDAR in DHrequire cannabinoid CB1 receptors
Activation of postsynaptic mGluR5 or NMDAR might led
to release of endocannabinoid substances that act at
presynaptic CB1 cannabinoid receptors (Ohno-Shosaku
et al., 2007). Activation of cannabinoid CB1 receptors
(CB1Rs) can suppress the neurotransmitter release from
both excitatory and inhibitory terminals (Chevaleyre
et al., 2006; Castillo et al., 2012; Kano, 2014) and reduce
excitatory and inhibitory synaptic transmission (Kano
et al., 2009). Thus, CB1Rs could potentially be involved
in the synergistic effects of mGluR5 and NMDARs. This
kind of thinking prompted us to examine whether block-
ade of CB1Rs could affect the actions of mGluR5 and
NMDAR co-activation. We then co-applied CHPG and
NMDA in the presence of the antagonist of CB1Rs AM
281 (5 lM), (Leterrier et al., 2006), in five dorsal slices
obtained from three animals. Fig. 7 shows that blockade
of CB1Rs significantly limited the synergistic effects of
NMDA and CHPG. Specifically, the NMDA–CHPG drug
cocktail applied in the presence of AM 281 significantly
reduced EPSP/Fv (by 57.8 ± 8.1%, Wilcoxon test,
p< 0.05), and increased PS/EPSP (by 174.4 ± 36.0%,
Wilcoxon test, p< 0.05) and PS2/PS1 (by 169.5
± 89%, Wilcoxon test, p< 0.05). However, these actions
were significantly lower than those observed with the
application of NMDA–CHPG only (comparison between
the two conditions for all three parameters, Mann–Whit-
ney U test, p< 0.05). Furthermore, the synergistic effects
of mGluR5 and NMDAR under AM 281, on EPSP/Fv and
PS/EPSP, were significantly stronger as compared with
the effects of NMDA in standard medium (comparison
between the two conditions, Mann–Whitney U test,
p< 0.005). On the contrary, the effect of drug cocktail
on paired-pulse inhibition (PS2/PS1) under AM 281
(169.5 ± 89%) was similar to the effect of NMDA in stan-
dard medium (220 ± 53%, see Fig. 2), (Mann–Whitney Utest, p> 0.05). Notably, the effect of NMDA–CHPG on
EPSP/Fv in DH under blockade of CB1Rs became similar
to the effect of the two drugs in VH under standard condi-
tions (i.e. without blocking CB1Rs). Theoretically, the
effects of blockade of CB1R could be interpreted as
mediating the actions of NMDAR rather than those of
synergy between mGluR5 and NMDAR. Thus, in order
to conclude whether the CB1R preferentially participates
in mediating the synergistic effects of mGluR5–NMDAR
or it is mainly involved in mediating the effects of only
NMDAR, we applied 50 lM NMDA in the presence of
5 lM AM 281. As shown in Fig. 7C, under this condition
the effects of activation of NMDAR were similar to those
produced by NMDA in standard medium. Specifically,
application of NMDA under blockade of CB1Rs signifi-
cantly reduced EPSP/Fv (by 26.7 ± 6.5%, n= 5/3, Wil-
coxon test, p< 0.05), and enhanced PS/EPSP (by
48.8 ± 12.9%, Wilcoxon test, p< 0.05) and PS2/PS1
(by 194.4 ± 56.6%, Wilcoxon test, p< 0.05). Given that
the suppressive effect of NMDAR’ activation on EPSP/
Fv was greater in VH (see Fig. 2), we applied NMDA
under the presence of AM 281 also in ventral slices. We
observed that as in DH, blockade of CB1R in VH did not
affect the action of NMDA on EPSP/Fv (-36.9 ± 6.4%,
n= 5/3). It is also of note that the CB1Rs did not affect
the delayed abrupt suppression in excitability accompa-
nied by the recovery of inhibition that constituted the third
phase of NMDA–CHPG actions. Thus, at 6–7 min of
NMDA–CHPG application in the presence of AM 281,
PS/EPSP was suppressed by 74.8 ± 5.4% (n= 5/3, Wil-
coxon test, p< 0.05) and PS2/PS1 returned to the levels
under AM 281 (0.6 ± 35.5%, n= 5/3).These results
demonstrated the important role that CB1R plays in pref-
erentially mediating the synergistic effects of mGluR5–
NMDAR co-activation in DH, without being implicated in
the effects of only NMDAR, either in DH or in VH. Interest-
ingly, blockade of CB1Rs produced an increase in basal
excitatory synaptic transmission in DH but not VH
synapses. Specifically, application of AM 281 significantly
increased EPSP/Fv in DH (by 10.9 ± 3.8%, n= 10/5,
Wilcoxon test, p< 0.05) but not in VH (-3.0 ± 2.1%,
n= 5/3; comparison between DH and VH, Mann–Whitney
U test, p< 0.05), (Fig. 7C). This tonic CB1R-dependent
control of synaptic transmission in DH is consistent with
previous findings (Slanina and Schweitzer, 2005), while
its absence in VH is conspicuous considering the impor-
tant role that CB1Rs play in modulating synaptic function.
Mg2+-free medium does not affect the synergybetween mGluR5 and NMDARs
The absence of synergistic effect of mGluR5 and
NMDARs in VH stimulated us to examine whether relief
of magnesium block of NMDARs could influence this
synergistic action. We then applied NMDA and CHPG in
slices perfused with medium containing no magnesium
ions (Mg2+-free medium, Mg2+ = 0 mM). Slices were
perfused with Mg2+-free medium for 30 min and then the
drug cocktail of NMDA and CHPG was applied in the
medium. Under this condition, the effects of co-activation
of mGluR5 with NMDARs on excitatory transmission
(EPSP/Fv) and postsynaptic excitability (PS/EPSP) were
significantly potentiated only in VH (Fig. 8). Specifically,
co-application of NMDA (50 lM) and CHPG (50 lM)
significantly reduced excitatory transmission (EPSP/Fv)
and enhanced excitability (PS/EPSP) in both DH (by
60.4 ± 4.8%, and 494.0 ± 242%, n= 5/3, respectively,
Wilcoxon test, p< 0.05) and VH (by 79.0 ± 9.3%, and
123.8 ± 31.0%, n= 5/3, respectively, Wilcoxon test,
p< 0.05). In VH but not DH, these effects were
significantly greater compared with those observed in
normal medium (comparisons in VH, Mann–Whitney Utest, p< 0.05). Mg2+-free medium did not significantly
A B
C
Fig. 7. The synergistic actions of mGluR5 and NMDAR in DH require CB1Rs. (A) Examples of field potentials obtained from one dorsal
hippocampal slice illustrating the effects of the co-activation of mGluR5 and NMDAR in the presence of the antagonist of CB1Rs AM 281 (5 lM).
Traces were collected at the times of drug maximum effects. (B) Time-course of the change of EPSP/Fv following co-application of CHPG with
NMDA in the presence of 5 lM AM 281 (n= 5/3, dark blue symbol). For comparison reasons, the time courses of EPSP/Fv changes induced by
NMDA–CHPG or NMDA alone in the absence of CB1R blockade are also shown (gray symbols). Note, that blockade of CB1Rs restrains the
synergistic action between mGluR5 and NMDAR but is stronger than the effect of NMDAR activation only (compare with data shown in Fig. 4A).
Values in this graph were calculated as the percentage change of those obtained during the last 5 min of AM 281 application, when drug effects
were stabilized; hence, the enhancing effect of AM 281 on EPSP/Fv does not appear in this graph. (C) Graphs of collective data of the drug effects
on synaptic transmission (EPSP/Fv), postsynaptic excitability (PS/EPSP) and paired-pulse inhibition (PS2/PS1). Asterisks denote statistically
significant actions at *p< 0.05 (Wilcoxon test). Significant differences between different drug conditions are indicated at #p< 0.05 or ##p< 0.005
(Mann–Whitney U test). Data were collected from five slices obtained from three animals. The synergistic effects of NMDA–CHPG and NMDA alone
in standard medium are also shown for comparisons (bars on the left), but significant differences among these data are not shown. Note that the
mGluR5–NMDAR synergistic action on inhibition (PS2/PS1) but not excitatory transmission (EPSP/Fv) or postsynaptic excitability (PS/EPSP),
under blockade of CB1Rs, was similar to that found with application of NMDA only (see also the main text). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 57
affect the actions of the NMDA or the drug cocktail on
inhibition (PS2/PS1) in either pole. These effects of
Mg2+-free medium could be interpreted either as an
enhancement of the synergy between mGluR5 and
NMDAR or simply as an augmentation of the activity of
NMDAR induced in the absence of Mg2+. In order to
distinguish between the two possible alternatives, we
applied only NMDA in a set of slices perfused with
Mg2+-free medium. We observed that the effects of
activation of only NMDAR were similar to those of
co-activation of NMDAR and mGluR5 (compare the two
drug conditions shown in the last two bars in each
graph). In particular, application of NMDA significantly
reduced EPSP/Fv and enhanced PS/EPSP in DH (by
53.1 ± 4.6%, and 130.8 ± 22.2%, n= 5/3, respectively,
Wilcoxon test, p< 0.05) and VH (by 77.0 ± 7.0%, and
130.0 ± 18.3%, n= 5/3, respectively, Wilcoxon test,
p<0.05). These observations showed that removing the
magnesium ions neither stimulate the interaction
between mGluR5 and NMDAR in VH nor significantly
augment the synergistic action of the two receptors in
DH. Lastly, we applied only CHPG in five ventral slices,
obtained from three animals, bathed in Mg2+-free
medium. None of the parameters displayed any
considerable change (EPSP/Fv, PS/EPSP and PS2/PS1
changed by 0.7 ± 2.5%, 2.5 ± 3.2% and -4.7 ± 15.3%
respectively). Interestingly, removing Mg2+ from the
perfusion medium significantly increased synaptic
transmission and postsynaptic excitability only in DH.
Specifically, in DH the EPSP/Fv increased by 51.7
± 9.3% (n= 10/5, Wilcoxon test, p< 0.005) and PS/
EPSP increased by 19.0 ± 5.1% (n= 10/5, Wilcoxon
test, p< 0.05). On the contrary, in VH, Mg2+-free
medium did not significantly affect either EPSP/Fv (8.7
± 6.6%) or PS/EPSP (3.4 ± 4.9%), (n= 10/5). PS2/
PS1 did not significantly change in either pole (9.9
± 11.9% in DH and 22.5 ± 10.2% in VH). It is
interesting that the increase in the excitatory synaptic
A D
CB
Fig. 8. The synergy between mGluR5 and NMDAR does not change in Mg2+-free medium. Collective results on the effects of Mg2+-free medium
on the actions of either NMDA–CHPG drug cocktail or NMDA (A–C) and the time course of EPSP/Fv change induced by the NMDA–CHPG drug
cocktail in Mg2+-free medium (D) are shown. Asterisks denote statistically significant actions at *p< 0.05 or **p<0.005 (Wilcoxon test). Significant
differences between different drug conditions are indicated at #p< 0.05 or ##p< 0.005 (Mann–Whitney U test). Data were collected from five dorsal
and five ventral slices obtained from three animals. For comparison reasons, drug effects observed in normal medium are also shown (bars on the
left), but significant differences among these data are not indicated. Note that the enhancing outcome of Mg2+-free medium on the actions of the
drug cocktail (NMDA–CHPG) on excitatory transmission (EPSP/Fv) and postsynaptic excitability (PS/EPSP) was due to the augmenting actions of
NMDAR. Also, notice that Mg2+-free medium increased basal excitatory synaptic transmission and excitability in DH but not VH.
58 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
transmission by Mg2+-free medium in DH but not VH is
consistent with the hypothesis that VH compared with
DH displays higher basal transmitter release probability
(Papatheodoropoulos and Kostopoulos, 2000b; Pofantis
and Papatheodoropoulos, 2014). This is because condi-
tions that enhance probability of transmitter release, such
as increased Ca2+/Mg2+ratio, will affect more strongly
synapses with relatively low basal transmitter release
probability (Wernig, 1972; Kuno and Takahashi, 1986).
DISCUSSION
This study demonstrates strikingly large differences
between DH and VH in the effectiveness of NMDARs,
mGluR5, A2ARs and CB1Rs in modulating synaptic
transmission, neuronal excitability and inhibition at the
local hippocampal microcircuitry. The main findings of
the study are: (a) the lower suppressive effect on
synaptic transmission but higher enhancing effects on
neuronal excitability of NMDAR activation in DH
compared with VH; (b) the strong synergistic effects of
mGluR5 and NMDAR observed in DH but not VH; (c)
the tonic modulatory action of A2ARs on the mGluR5–
NMDAR synergy in DH but not VH; (d) the involvement
of CB1Rs in the synergistic actions of mGluR5–
NMDARs in DH but not VH.
Interpretation of NMDA actions in DH and VH
We found that exogenous activation of NMDARs reduces
synaptic transmission and paired-pulse inhibition and
increases postsynaptic excitability. Qualitatively, these
effects are in keeping with previous observations having
shown that activation of NMDARs suppresses excitatory
synaptic transmission (Chernevskaya et al., 1991;
Manzoni et al., 1994; Tebano et al., 2005), enhances neu-
ronal excitability (Sah et al., 1989; Mor and Grossman,
2007) and reduces GABAergic inhibition (Stelzer and
Shi, 1994; Chisari et al., 2012) in the network of hip-
pocampal pyramidal cells. In addition, we found that the
action of NMDARs in reducing synaptic transmission
was stronger in VH than DH while the effects on postsy-
naptic excitability and paired-pulse inhibition were much
higher in DH than VH.
These results suggested that different mechanisms
underlie the effects of NMDAR on synaptic transmission
and postsynaptic excitability with different contribution of
each mechanism between DH and VH. It has been
shown that suppression of the excitatory synaptic
transmission in CA1 by NMDAR can be induced through
increase in the activation of presynaptic adenosine A1
receptors resulting from elevation of exctracellular levels
of adenosine (Chernevskaya et al., 1991; Manzoni
et al., 1994). In addition, taking into account that
the action of A1 receptors is higher in DH than VH
(Lee et al., 1983) it would be expected that this action of
NMDARs would be higher in DH than in VH. However,
in the present study we found that A1 receptors were
not involved in this action of NMDARs. Even more unex-
pectedly, while the NMDAR-induced reduction in the exci-
tatory synaptic transmission in DH was accompanied by
increase in the synaptic facilitation, suggesting a presy-
naptic mechanism of action (Dobrunz and Stevens,
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 59
1997), the facilitation in the VH, the hippocampus seg-
ment that displayed even larger NMDAR-induced reduc-
tion in transmission, did not significantly change,
suggesting that other than presynaptic mechanisms con-
tribute to the reduction in the excitatory synaptic transmis-
sion in this hippocampal segment. A mechanism through
which NMDARs can decrease the excitatory synaptic
transmission engages the very recently described postsy-
naptic mechanism of Ca2+-induced SK channels (Wang
et al., 2014). We should also note that low concentration
of NMDA induced augmentation instead of reduction in
the synaptic transmission in DH. This action might be
mediated by the recently revealed presynaptic NMDARs
(Mameli et al., 2005; Suarez et al., 2005).
The enhancing action of NMDARs on neuronal
excitability can more straightforwardly be interpreted by
the direct contribution of these receptors to the
postsynaptic depolarization (Forsythe and Westbrook,
1988; Andreasen et al., 1989). Activation of NMDARs
enhances excitability of CA1 principal cells and induces
spontaneous network discharges (Dingledine et al.,
1986; Sah et al., 1989; Herreras and Jing, 1993; Mor
and Grossman, 2007; Bali et al., 2014). An additional
mechanism contributing to increased postsynaptic
excitability could involve changes in GABAergic inhibition.
Indeed, activation of NMDAR can induce reduction in the
GABAergic inhibition in hippocampal pyramidal cells
through a Ca2+-dependent interaction between NMDAR
and GABAA receptors (Stelzer and Shi, 1994; Chisari
et al., 2012). The strong reduction in paired-pulse inhibi-
tion observed in DH could then apparently result from this
disinhibitory action of NMDARs leading to increase in
pyramidal cell excitability. As described more thoroughly
in the next section, an alternative and recently studied
mechanism that could potentially explain the suppressive
action of NMDARs on inhibitory as well as excitatory
synaptic transmission implicates endocannabinoid-
mediated retrograde signaling at inhibitory and excitatory
synapses (Kano et al., 2009). However, we showed that
blockade of CB1R does not affect the actions of only
NMDAR activation. Overall, though the NMDAR-induced
effects on excitability and inhibition can be understood
on the basis of previously shown mechanisms, the mech-
anisms underlying the suppression of excitatory transmis-
sion following activation of NMDARs remain rather
unclear.
Interpretation of the effects of mGluR5–NMDARco-activation in DH and VH
Hippocampal pyramidal neurons strongly express
mGluR5 (Shigemoto et al., 1992; Fotuhi et al., 1994)
prevalently located postsynaptically (Romano et al.,
1995; Lujan et al., 1996; Mannaioni et al., 2001; Tebano
et al., 2005) where they interact with NMDARs and
enhance NMDAR-mediated responses (Fitzjohn et al.,
1996; Doherty et al., 1997; Mannaioni et al., 2001;
Tebano et al., 2005). Furthermore, mGluR5 reduces inhi-
bition in CA1 pyramidal neurons (Desai and Conn, 1991;
Gereau and Conn, 1995a; Mannaioni et al., 2001).
Accordingly, we found that concomitant activation of
mGluR5 and NMDAR greatly facilitated the effects of
NMDAR on excitatory synaptic transmission, postsynap-
tic excitability and synaptic inhibition in DH. Remarkably,
however, the VH was proven quite insensitive to modula-
tion of mGluR5. It has been recently revealed a specific
time-dependent mGluR5-induced phosphorylation of
NMDAR that requires Src tyrosine kinases activity and
fully matches the time course of changes in excitability
and synaptic transmission during co-activation of mGluR
with NMDAR (Sarantis et al., 2015). It could be possible
that this molecular interaction between mGluR5 and
NMDAR does not occur in VH under exogenous activation
of these receptors used in the present study.
The modulatory role of A2ARs on central synapses it is
well established (Cunha et al., 2008; Sebastiao and
Ribeiro, 2009). Though A2ARs in the (dorsal) hippocam-
pus have a rather limited distribution (Jarvis and
Williams, 1989; Sebastiao and Ribeiro, 1992; Dixon
et al., 1996) they are nevertheless functional, displaying
excitatory action (Sebastiao and Ribeiro, 1992; Cunha
et al., 1994; Rombo et al., 2015). It has been recently
demonstrated that the synergistic interaction between
postsynaptic mGluR5 and NMDAR is under the permis-
sive control of adenosine A2ARs (Tebano et al., 2005).
Consistently, we found that blockade of A2ARs in DH pre-
vented the facilitatory effects of co-activation of mGluR5
with NMDAR on NMDAR-depended responses on all
parameters studied, suggesting that endogenous tonic
activity of A2ARs control the synergistic effects of mGluR5
and NMDAR. Furthermore, exogenous activation of A2AR
by its selective agonist did not promote the effects of co-
activation of mGluR5/NMDARs in either hippocampal
pole. These results suggest that endogenous activity of
A2ARs is sufficient for the synergistic action of mGluR5
and NMDAR to be expressed in DH and that the particular
permissive implication of A2AR in VH is either absent or
very low to be detected with the experimental design in
this study. Despite the lack of neurochemical data on
the levels of A2AR in VH, previous observations have indi-
cated that there are functional A2AR in VH and they are
involved in epileptogenesis (Moschovos et al., 2012). It
is likely that the functional implication of A2AR differs
under different activity states of the hippocampal circuitry
(Nikbakht and Stone, 2001; Sebastiao and Ribeiro, 2014).
An important and recently revealed player in the
modulation of inhibitory and excitatory synaptic
transmission is the endocannabinoid CB1R (Castillo
et al., 2012; Kano, 2014). Presynaptic CB1Rs
(Kawamura et al., 2006) are expressed in the hippocam-
pus (Herkenham et al., 1991), and they can suppress
transmitter release from glutamatergic and GABAergic
terminals (Schlicker and Kathmann, 2001; Wang, 2003)
after being activated by postsynaptically Ca2+-induced
released endocannabinoids (Kano et al., 2009). The
reduction in GABAergic transmission can then cause
enhancement in postsynaptic excitability (Kano et al.,
2009). In consistency with this scheme of CB1Rs actions,
we found that blockade of CB1Rs curtailed a portion of the
potentiating effects of co-activation of mGluR5 with
NMDAR on excitatory transmission and postsynaptic
excitability and totally blocked their effects on inhibition.
Fig. 9. Schematic drawing summarizing the mechanisms underlying
the synergistic effects of co-activation of mGluR5 and NMDARs.
These effects were observed in DH but not VH. Intense co-activation
of mGluR5 with NMDARs first leads to an abrupt and profound
reduction in inhibition (1) immediately followed by an abrupt and
strong increase in excitability of CA1 pyramidal cells (2). After the
delay of a few minutes a strong reduction in the excitatory transmis-
sion occurs (3). These actions were gated by the tonically active
A2ARs and were partly mediated by the activity of presumably
presynaptic CB1Rs which induce reduction in both inhibitory and
excitatory transmission. The activation of CB1Rs can occur by
endocannabinoids (eCB) whose synthesis might be triggered by the
interaction of mGluR5 and NMDARs. The interaction between
mGluR5 and NMDARs could directly contribute to the increased
excitability through enhancement of NMDAR-mediated depolariza-
tion, while interaction between NMDAR and GABAARs might con-
tribute to reduced inhibition and subsequently to increased excitation.
Activation of mGluR5 in astrocytes can trigger the release of
glutamate which acting at NMDARs can contribute to increased
excitability in CA1 pyramidal cells (CA1 PC).
60 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64
Hence, the synergistic actions of mGluR5–NMDAR on
inhibition can be fully explained by the activity of CB1Rs
at GABAergic terminals (Schlicker and Kathmann, 2001;
Kano et al., 2009), while the effects of mGluR-NMDAR
on excitatory transmission and postsynaptic excitability
can partly be explained by the activity of CB1Rs. It should
be noted that thought it is known that activation of CB1Rs
can occur following activation of NMDARs (Ohno-
Shosaku et al., 2007) or mGluR5 (Ohno-Shosaku et al.,
2002) and that some kind of interaction between mGluR5
and NMDARs has been proposed to only underlie long-
term depression of excitatory transmission in the CA1
field (Izumi and Zorumski, 2012), this is the first time, to
our knowledge, to show that CB1Rs-dependent short-
term effects occur following co-activation of mGluR5 with
NMDARs.
Increase in postsynaptic excitability could be a direct
effect of the interaction of mGluR5 with NMDAR that
leads to enhanced NMDAR-mediated depolarization and
increased firing in CA1 pyramidal cells (Mannaioni et al.,
1999). Furthermore, it has been shown that mGluR5-
dependent release of glutamate from astrocytes can
enhance postsynaptic excitability by increasing NMDAR-
mediated currents in CA1 pyramidal cells (Angulo et al.,
2004; Fellin et al., 2004; see review by Panatier and
Robitaille, 2015). This regulation of synaptic transmission
by astrocytes could potentially contribute to the enhance-
ment of excitability that is induced by co-activation of
mGluR5 with NMDAR and remains after blockade of CB1
Rs. The absence of CB1R-dependent signaling, either
tonic or induced by mGluR5–NMDAR co-activation in VH
constitutes a striking specialization in the way that the local
neuronal circuitry handles information along the hippocam-
pus. An abstract picture of the actions of the various recep-
tors on synaptic transmission and neuronal excitability
observed in DH but not VH is presented in Fig. 9.
A particular observation in the present study was
the rapid transition from the strong enhancement to the
suppression in postsynaptic excitability induced by the
co-activation of mGluR5 with NMDAR. This transition
was very fast (less than 1 min in most experiments) and
it could be attributed to agonist-induced desensitization
of mGluR5 (Catania et al., 1991; Gereau and
Heinemann, 1998) with a possible contribution from
NMDARs (Ascher and Nowak, 1988; Mayer et al., 1989;
Chizhmakov et al., 1990). It is interesting that the time-
course of the rapid changes in excitability observed in
the present study was similar to that of mGluR desensiti-
zation (Guerineau et al., 1997).
Implications for dorso-ventral diversification
NMDARs play fundamental roles in the functioning of
neuronal networks and information processing (Daw
et al., 1993; Grienberger et al., 2014) with crucial involve-
ment in hippocampus-dependent synaptic plasticity and
learning-memory processes (Morris et al., 1990;
Newcomer and Krystal, 2001; Collingridge et al., 2013).
Expectedly, there are several mechanisms that partici-
pate in the modulation of NMDARs including the group I
mGluR5 (Doherty et al., 1997; Mannaioni et al., 2001)
and adenosine A2AR (Tebano et al., 2005; Sebastiao
and Ribeiro, 2009, 2014). The mGluR5 is involved in
NMDAR-dependent synaptic plasticity phenomena
(Rebola et al., 2010). In addition, blockade of A2AR pre-
vents LTP in the DH (Fontinha et al., 2009). The different
dorsoventral modulation of NMDARs by mGluR5 and
A2ARs may therefore have important implications for
phenomena of synaptic plasticity, including long-term
potentiation. Accordingly, the previously demonstrated
remarkably lower ability of VH vs DH to sustain induction
of NMDAR-dependent LTP (Papatheodoropoulos and
Kostopoulos, 2000a; Maruki et al., 2001; Colgin et al.,
2004) may be related to the lower functional state of these
modulatory receptors in VH compared with DH.
It has been known for a long time that the VH in
rodents and the corresponding anterior hippocampus in
human is the more susceptible segment of the structure
to epileptic or epileptic like network activity (Andy, 1962;
Brazier, 1970; Gilbert et al., 1985; Bragdon et al., 1986;
Bell and Davies, 1998; Moschovos et al., 2012). The
activity of all three receptors, NMDARs, mGluR5 and
A2ARs increase excitability in the hippocampus and could
potentially lead to abnormal cellular and network dis-
charges (Stasheff et al., 1989; Ireland and Abraham,
2002; Etherington and Frenguelli, 2004; Zeraati et al.,
2006; Moschovos et al., 2012). In the present study, we
intensively activated NMDARs and mGluR5 using exoge-
nous agonists. This condition might mimic situations of
high network activity. At first, the higher activity of A2AR
S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 61
and mGluR5 in DH seems incompatible with the resis-
tance of DH to network hyperactivity. However, DH dis-
plays CB1R activity after mGluR5–NMDAR co-activation
that contributes to robust reduction in excitatory transmis-
sion that follows the period of increased excitability. What
is more, CB1Rs tonically control excitatory synaptic trans-
mission in DH indicating that they play an important role in
regulating network excitability in this part of the hippocam-
pus even under basal conditions. Accordingly, CB1Rs in
glutamatergic neurons are crucially implicated in protect-
ing DH from epileptiform seizures (Monory et al., 2006).
Thus, in states of intense network activity DH is prevented
from entering into a state of exacerbated neuronal dis-
charges. In contrast, the absence of CB1R activity in VH
might contribute to its susceptibility to epileptiform
network discharges. Several other mechanisms might
contribute to this susceptibility of VH, including the
higher excitability of principal neurons (Dougherty et al.,
2012), the lower synaptic GABAergic inhibition
(Papatheodoropoulos et al., 2002; Petrides et al., 2007)
or the different composition of synaptic receptors
(Sotiriou et al., 2005; Pandis et al., 2006). However, in
the absence of factors that could abnormally increase
the network excitability, the VH circuitry appears to work
physiologically suggesting that the propensity of VH to
hyperexcitability is counterbalanced by adaptive alter-
ations that dampen this property. Indeed, recent results
suggest that the a5GABAA receptor-mediated transmis-
sion is higher in VH than in DH (Sotiriou et al., 2005;
Pofantis and Papatheodoropoulos, 2014). Similarly, the
considerably lower ability of the ventral hippocampal
synapses to undergo NMDAR-dependent long-term
potentiation (Papatheodoropoulos and Kostopoulos,
2000a; Maruki et al., 2001; Colgin et al., 2004; Maggio
and Segal, 2007) might contribute to prevent epilepitform
network activity given that synaptic strengthening favors
epileptogenesis (Sutula and Steward, 1987). Thus, the
significant dorsoventral differences in the actions of
NMDARs, mGluR5 and A2ARs and CB1Rs observed in
this study could be viewed as specializations of the local
microcircuitries in order to effectively support the specific
functional demands attributed to each hippocampal segment.
The recently strengthened concept of functional
segregation along the longitudinal axis of the
hippocampus is perhaps more conspicuously and
concisely expressed by the diptych of cognition and
emotionality. Accordingly, DH is preferentially implicated
in spatial memory while VH has a deeper involvement in
anxiety (Fanselow and Dong, 2010; Bast, 2011;
Bannerman et al., 2014). Given this idea, the greater
modulatory potential of NMDARs in DH compared with
VH, is consistent with the evidence showing that
NMDARs in the hippocampus are implicated in cognitive
contextual integration and not in emotion-based context-
shock association (Fanselow and Dong, 2010).
CONCLUSIONS
The present results demonstrate very conspicuous
differences in the actions of the interacting receptors
NMDAR, mGluR5 and A2AR, on the functioning of the
local neuronal microcircuitry between the dorsal and the
ventral segment of the hippocampus. Thus, activation of
NMDARs suppresses excitatory synaptic transmission in
VH more than DH while enhances postsynaptic
excitability only in DH. Even more strikingly, the positive
modulation of NMDAR by mGluR5, the permissive role
of A2AR and the involvement of CB1Rs to this synergy
were strong in DH but absent in VH. It could be that,
under conditions of intense neuronal activity, the
inherently higher excitability of the VH compared with
DH has been homeostatically compensated by an
adaptive constrain in the contribution of some
excitability-enhancing mechanisms such as mGluR5 and
A2AR. We propose that at states of highly activated
network, while the local network activity in DH is
dynamically tuned by the modulatory actions of
interacting A2AR and mGluR5 on the NMDAR with the
intervening role of CB1Rs, the involvement of NMDAR in
the functioning of VH is more vaguely and
stereotypically adjusted. It is expected that this will have
important implications for the information processing
performed by DH and VH.
Acknowledgments—This research has been co-financed by the
European Union (European Social Fund–ESF) and Greek
national funds through the Operational Program ’Education-an
d-Lifelong-Learning’ of the National Strategic Reference Frame-
work (NSRF)–Research Funding Program: Thales. Investing in
knowledge society through the European Social Fund; (# MIS:
380342). The authors are grateful to Reviewers for their con-
structive comments and suggestions. None of the authors have
any conflict of interest to disclose.
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(Accepted 30 December 2015)(Available online 5 January 2016)