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.

REFERENCES

Andersen P, Silfvenius H, Sundberg SH, Sveen O, Wigstrom H

(1978) Functional characteristics of unmyelinated fibres in the

hippocampal cortex. Brain Res 144:11–18.

Andreasen M, Lambert JD, Jensen MS (1989) Effects of new non-N-

methyl-D-aspartate antagonists on synaptic transmission in the

in vitro rat hippocampus. J Physiol 414:317–336.

Andy OJ (1962) Electrophysiological comparisons of the dorsal and

ventral hippocampus. In: Physiologie de l’Hippocampe. Paris:

CNRS. p. 411.

Angulo MC, Kozlov AS, Charpak S, Audinat E (2004) Glutamate

released from glial cells synchronizes neuronal activity in the

hippocampus. J Neurosci 24:6920–6927.

Anwyl R (1999) Metabotropic glutamate receptors: electrophysio-

logical properties and role in plasticity. Brain Res Brain Res Rev

29:83–120.

Ascher P, Nowak L (1988) Quisqualate- and kainate-activated

channels in mouse central neurones in culture. J Physiol

399:227–245.

Bali ZK, Budai D, Hernadi I (2014) Separation of

electrophysiologically distinct neuronal populations in the rat

hippocampus for neuropharmacological testing under in vivo

conditions. Acta Biol Hung 65:241–251.

Bannerman DM, Sprengel R, Sanderson DJ, McHugh SB, Rawlins

JN, Monyer H, Seeburg PH (2014) Hippocampal synaptic

plasticity, spatial memory and anxiety. Nat Rev Neurosci

15:181–192.

Bast T (2011) The hippocampal learning-behavior translation and the

functional significance of hippocampal dysfunction in

schizophrenia. Curr Opin Neurobiol 21:492–501.

Bell BD, Davies KG (1998) Anterior temporal lobectomy,

hippocampal sclerosis, and memory: recent neuropsychological

findings. Neuropsychol Rev 8:25–41.

62 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64

Bragdon AC, Taylor DM, Wilson WA (1986) Potassium-induced

epileptiform activity in area CA3 varies markedly along the

septotemporal axis of the rat hippocampus. Brain Res

378:169–173.

Brazier MA (1970) Regional activities within the human hippocampus

and hippocampal gyrus. Exp Neurol 26:254–268.

Castillo PE, Younts TJ, Chavez AE, Hashimotodani Y (2012)

Endocannabinoid signaling and synaptic function. Neuron

76:70–81.

Catania MV, Aronica E, Sortino MA, Canonico PL, Nicoletti F (1991)

Desensitization of metabotropic glutamate receptors in neuronal

cultures. J Neurochem 56:1329–1335.

Charpak S, Gahwiler BH, Do KQ, Knopfel T (1990) Potassium

conductances in hippocampal neurons blocked by excitatory

amino-acid transmitters. Nature 347:765–767.

Chernevskaya NI, Obukhov AG, Krishtal OA (1991) NMDA receptor

agonists selectively block N-type calcium channels in

hippocampal neurons. Nature 349:418–420.

Chevaleyre V, Takahashi KA, Castillo PE (2006) Endocannabinoid-

mediated synaptic plasticity in the CNS. Annu Rev Neurosci

29:37–76.

Chisari M, Zorumski CF, Mennerick S (2012) Cross talk between

synaptic receptors mediates NMDA-induced suppression of

inhibition. J Neurophysiol 107:2532–2540.

Chizhmakov IV, Kiskin NI, Tsyndrenko A, Krishtal OA (1990)

Desensitization of NMDA receptors does not proceed in the

presence of kynurenate. Neurosci Lett 108:88–92.

Colgin LL, Kubota D, Jia Y, Rex CS, Lynch G (2004) Long-term

potentiation is impaired in rat hippocampal slices that produce

spontaneous sharp waves. J Physiol 558:953–961.

Collingridge GL, Volianskis A, Bannister N, France G, Hanna L,

Mercier M, Tidball P, Fang G, Irvine MW, Costa BM, Monaghan

DT, Bortolotto ZA, Molnar E, Lodge D, Jane DE (2013) The

NMDA receptor as a target for cognitive enhancement.

Neuropharmacology 64:13–26.

Costenla AR, Cunha RA, de Mendonca A (2010) Caffeine, adenosine

receptors, and synaptic plasticity. J Alzheimer’s Dis 20(Suppl 1):

S25–34.

Cunha RA, Ferre S, Vaugeois JM, Chen JF (2008) Potential

therapeutic interest of adenosine A2A receptors in psychiatric

disorders. Curr. Pharm. Des. 14:1512–1524.

Cunha RA, Johansson B, van der Ploeg I, Sebastiao AM, Ribeiro JA,

Fredholm BB (1994) Evidence for functionally important

adenosine A2a receptors in the rat hippocampus. Brain Res

649:208–216.

Daw NW, Stein PS, Fox K (1993) The role of NMDA receptors in

information processing. Annu Rev Neurosci 16:207–222.

Desai MA, Conn PJ (1991) Excitatory effects of ACPD receptor

activation in the hippocampus are mediated by direct effects on

pyramidal cells and blockade of synaptic inhibition. J

Neurophysiol 66:40–52.

Dingledine R, Hynes MA, King GL (1986) Involvement of N-methyl-D-

aspartate receptors in epileptiform bursting in the rat hippocampal

slice. J Physiol 380:175–189.

Dixon AK, Gubitz AK, Sirinathsinghji DJ, Richardson PJ, Freeman TC

(1996) Tissue distribution of adenosine receptor mRNAs in the

rat. Br J Pharmacol 118:1461–1468.

Dobrunz LE, Stevens CF (1997) Heterogeneity of release probability,

facilitation, and depletion at central synapses. Neuron 18:995–1008.

Doherty AJ, Palmer MJ, Henley JM, Collingridge GL, Jane DE (1997)

(RS)-2-chloro-5-hydroxyphenylglycine (CHPG) activates mGlu5,

but no mGlu1, receptors expressed in CHO cells and potentiates

NMDA responses in the hippocampus. Neuropharmacology

36:265–267.

Dong HW, Swanson LW, Chen L, Fanselow MS, Toga AW (2009)

Genomic-anatomic evidence for distinct functional domains in

hippocampal field CA1. Proc Natl Acad Sci USA

106:11794–11799.

Dougherty KA, Islam T, Johnston D (2012) Intrinsic excitability of CA1

pyramidal neurones from the rat dorsal and ventral hippocampus.

J Physiol 590:5707–5722.

Etherington LA, Frenguelli BG (2004) Endogenous adenosine

modulates epileptiform activity in rat hippocampus in a receptor

subtype-dependent manner. Eur J Neurosci 19:2539–2550.

Fanselow MS, Dong HW (2010) Are the dorsal and ventral

hippocampus functionally distinct structures? Neuron 65:7–19.

Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G

(2004) Neuronal synchrony mediated by astrocytic glutamate

through activation of extrasynaptic NMDA receptors. Neuron

43:729–743.

Fitzjohn SM, Irving AJ, Palmer MJ, Harvey J, Lodge D, Collingridge

GL (1996) Activation of group I mGluRs potentiates NMDA

responses in rat hippocampal slices. Neurosci Lett

203:211–213.

Fontinha BM, Delgado-Garcia JM, Madronal N, Ribeiro JA, Sebastiao

AM, Gruart A (2009) Adenosine A(2A) receptor modulation of

hippocampal CA3–CA1 synapse plasticity during associative

learning in behaving mice. Neuropsychopharmacology

34:1865–1874.

Forsythe ID, Westbrook GL (1988) Slow excitatory postsynaptic

currents mediated by N-methyl-D-aspartate receptors on cultured

mouse central neurones. J Physiol 396:515–533.

Fotuhi M, Standaert DG, Testa CM, Penney Jr JB, Young AB (1994)

Differential expression of metabotropic glutamate receptors in the

hippocampus and entorhinal cortex of the rat. Brain Res Mol Brain

Res 21:283–292.

Freund TF, Buzsaki G (1996) Interneurons of the hippocampus.

Hippocampus 6:347–470.

Gage FH, Thompson RG (1980) Differential distribution of

norepinephrine and serotonin along the dorsal-ventral axis of

the hippocampal formation. Brain Res Bull 5:771–773.

Georgopoulos P, Petrides T, Kostopoulos G, Papatheodoropoulos C

(2008) Varying magnitude of GABAergic recurrent inhibition

enhancement by different sedative/anesthetic agents in dorsal

and ventral hippocampus. Brain Res 1207:43–59.

Gereau RWT, Conn PJ (1995a) Multiple presynaptic metabotropic

glutamate receptors modulate excitatory and inhibitory synaptic

transmission in hippocampal area CA1. J Neurosci

15:6879–6889.

Gereau RWT, Conn PJ (1995b) Roles of specific metabotropic

glutamate receptor subtypes in regulation of hippocampal CA1

pyramidal cell excitability. J Neurophysiol 74:122–129.

Gereau RWT, Heinemann SF (1998) Role of protein kinase C

phosphorylation in rapid desensitization of metabotropic

glutamate receptor 5. Neuron 20:143–151.

Gilbert M, Racine RJ, Smith GK (1985) Epileptiform burst

responses in ventral vs dorsal hippocampal slices. Brain Res

361:389–391.

Grienberger C, Chen X, Konnerth A (2014) NMDA receptor-

dependent multidendrite Ca(2+) spikes required for

hippocampal burst firing in vivo. Neuron 81:1274–1281.

Grigoryan G, Korkotian E, Segal M (2012) Selective facilitation of LTP

in the ventral hippocampus by calcium stores. Hippocampus

22:1635–1644.

Guerineau NC, Bossu JL, Gahwiler BH, Gerber U (1997) G-protein-

mediated desensitization of metabotropic glutamatergic and

muscarinic responses in CA3 cells in rat hippocampus. J

Physiol 500(Pt 2):487–496.

Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice

KC (1991) Characterization and localization of cannabinoid

receptors in rat brain: a quantitative in vitro autoradiographic

study. J Neurosci 11:563–583.

Herreras O, Jing J (1993) Blockade of antidromic invasion of CA1

pyramidal cells during synaptic activation of NMDA receptors.

Brain Res 616:330–334.

Hoehn K, White TD (1990) N-methyl-D-aspartate, kainate and

quisqualate release endogenous adenosine from rat cortical

slices. Neuroscience 39:441–450.

Honigsperger C, Marosi M, Murphy R, Storm JF (2015) Dorsoventral

differences in Kv7/M-current and its impact on resonance,

temporal summation and excitability in rat hippocampal

pyramidal cells. J Physiol 593:1551–1580.

S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64 63

Ireland DR, Abraham WC (2002) Group I mGluRs increase

excitability of hippocampal CA1 pyramidal neurons by a PLC-

independent mechanism. J Neurophysiol 88:107–116.

Izumi Y, Zorumski CF (2012) NMDA receptors, mGluR5, and

endocannabinoids are involved in a cascade leading to

hippocampal long-term depression. Neuropsychopharmacology

37:609–617.

Jarvis MF, Williams M (1989) Direct autoradiographic localization

of adenosine A2 receptors in the rat brain using the

A2-selective agonist, [3H]CGS 21680. Eur J Pharmacol 168:

243–246.

Kano M (2014) Control of synaptic function by endocannabinoid-

mediated retrograde signaling. Proc Japan Acad Ser B Phys Biol

Sci 90:235–250.

Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M,

Watanabe M (2009) Endocannabinoid-mediated control of

synaptic transmission. Physiol Rev 89:309–380.

Kawamura Y, Fukaya M, Maejima T, Yoshida T, Miura E, Watanabe

M, Ohno-Shosaku T, Kano M (2006) The CB1 cannabinoid

receptor is the major cannabinoid receptor at excitatory

presynaptic sites in the hippocampus and cerebellum. J

Neurosci 26:2991–3001.

Kenney J, Manahan-Vaughan D (2013) NMDA receptor-dependent

synaptic plasticity in dorsal and intermediate hippocampus

exhibits distinct frequency-dependent profiles.

Neuropharmacology 74:108–118.

Keralapurath MM, Clark JK, Hammond S, Wagner JJ (2014)

Cocaine- or stress-induced metaplasticity of LTP in the dorsal

and ventral hippocampus. Hippocampus 24:577–590.

Kotecha SA, Jackson MF, Al-Mahrouki A, Roder JC, Orser BA,

MacDonald JF (2003) Co-stimulation of mGluR5 and N-methyl-D-

aspartate receptors is required for potentiation of excitatory

synaptic transmission in hippocampal neurons. J Biol Chem

278:27742–27749.

Kuno M, Takahashi T (1986) Effects of calcium and magnesium on

transmitter release at Ia synapses of rat spinal motoneurones

in vitro. J Physiol 376:543–553.

Lea PMT, Movsesyan VA, Faden AI (2005) Neuroprotective activity

of the mGluR5 antagonists MPEP and MTEP against acute

excitotoxicity differs and does not reflect actions at mGluR5

receptors. Brit J Pharmacol 145:527–534.

Lee KS, Reddington M, Schubert P, Kreutzberg G (1983) Regulation

of the strength of adenosine modulation in the hippocampus by a

differential distribution of the density of A1 receptors. Brain Res

260:156–159.

Leterrier C, Laine J, Darmon M, Boudin H, Rossier J, Lenkei Z (2006)

Constitutive activation drives compartment-selective endocytosis

and axonal targeting of type 1 cannabinoid receptors. J Neurosci

26:3141–3153.

Liagkouras I, Michaloudi H, Batzios C, Psaroulis D, Georgiadis M,

Kunzle H, Papadopoulos GC (2008) Pyramidal neurons in the

septal and temporal CA1 field of the human and hedgehog tenrec

hippocampus. Brain Res 1218:35–46.

Lujan R, Nusser Z, Roberts JD, Shigemoto R, Somogyi P (1996)

Perisynaptic location of metabotropic glutamate receptors

mGluR1 and mGluR5 on dendrites and dendritic spines in the

rat hippocampus. Eur J Neurosci 8:1488–1500.

Maggio N, Segal M (2007) Unique regulation of long term potentiation

in the rat ventral hippocampus. Hippocampus 17:10–25.

Maggio N, Segal M (2009) Differential corticosteroid modulation of

inhibitory synaptic currents in the dorsal and ventral

hippocampus. J Neurosci 29:2857–2866.

Mameli M, Carta M, Partridge LD, Valenzuela CF (2005)

Neurosteroid-induced plasticity of immature synapses via

retrograde modulation of presynaptic NMDA receptors. J

Neurosci 25:2285–2294.

Mannaioni G, Attucci S, Missanelli A, Pellicciari R, Corradetti R,

Moroni F (1999) Biochemical and electrophysiological studies on

(S)-(+)-2-(30-carboxybicyclo(1.1.1)pentyl)-glycine (CBPG), a

novel mGlu5 receptor agonist endowed with mGlu1 receptor

antagonist activity. Neuropharmacology 38:917–926.

Mannaioni G, Marino MJ, Valenti O, Traynelis SF, Conn PJ (2001)

Metabotropic glutamate receptors 1 and 5 differentially regulate

CA1 pyramidal cell function. J Neurosci 21:5925–5934.

Manzoni OJ, Manabe T, Nicoll RA (1994) Release of adenosine by

activation of NMDA receptors in the hippocampus. Science

265:2098–2101.

Maruki K, Izaki Y, Nomura M, Yamauchi T (2001) Differences in

paired-pulse facilitation and long-term potentiation between dorsal

and ventral CA1 regions in anesthetized rats. Hippocampus

11:655–661.

Mayer ML, Vyklicky Jr L, Clements J (1989) Regulation of NMDA

receptor desensitization in mouse hippocampal neurons by

glycine. Nature 338:425–427.

Mikroulis AV, Psarropoulou C (2012) Endogenous ACh effects on

NMDA-induced interictal-like discharges along the septotemporal

hippocampal axis of adult rats and their modulation by an early life

generalized seizure. Epilepsia 53:879–887.

Monaghan DT, Cotman CW (1985) Distribution of N-methyl-D-

aspartate-sensitive L-[3H]glutamate-binding sites in rat brain. J

Neurosci 5:2909–2919.

Monory K,MassaF, EgertovaM, EderM,BlaudzunH,WestenbroekR,

Kelsch W, Jacob W, Marsch R, Ekker M, Long J, Rubenstein JL,

Goebbels S, Nave KA, During M, Klugmann M, Wolfel B, Dodt HU,

Zieglgansberger W, Wotjak CT, Mackie K, Elphick MR, Marsicano

G, Lutz B (2006) The endocannabinoid system controls key

epileptogenic circuits in the hippocampus. Neuron 51:455–466.

Mor A, Grossman Y (2007) High pressure modulation of NMDA

receptor dependent excitability. Eur J Neurosci 25:2045–2052.

Morris RG, Davis S, Butcher SP (1990) Hippocampal synaptic

plasticity and NMDA receptors: a role in information storage?

Philos Trans R Soc Lond B Biol Sci 329:187–204.

Moschovos C, Kostopoulos G, Papatheodoropoulos C (2012)

Endogenous adenosine induces NMDA receptor-independent

persistent epileptiform discharges in dorsal and ventral

hippocampus via activation of A2 receptors. Epilepsy Res

100:157–167.

Mukherjee S, Manahan-Vaughan D (2013) Role of metabotropic

glutamate receptors in persistent forms of hippocampal plasticity

and learning. Neuropharmacology 66:65–81.

Newcomer JW, Krystal JH (2001) NMDA receptor regulation of

memory and behavior in humans. Hippocampus 11:529–542.

Nikbakht MR, Stone TW (2001) Suppression of presynaptic

responses to adenosine by activation of NMDA receptors. Eur J

Pharmacol 427:13–25.

Ohno-Shosaku T, Hashimotodani Y, Ano M, Takeda S, Tsubokawa

H, Kano M (2007) Endocannabinoid signalling triggered by NMDA

receptor-mediated calcium entry into rat hippocampal neurons. J

Physiol 584:407–418.

Ohno-Shosaku T, Shosaku J, Tsubokawa H, Kano M (2002)

Cooperative endocannabinoid production by neuronal

depolarization and group I metabotropic glutamate receptor

activation. Eur J Neurosci 15:953–961.

Panatier A, Robitaille R (2015) Astrocytic mGluR5 and the tripartite

synapse. Neuroscience (in press).

Pandis C, Sotiriou E, Kouvaras E, Asprodini E, Papatheodoropoulos

C, Angelatou F (2006) Differential expression of NMDA and

AMPA receptor subunits in rat dorsal and ventral hippocampus.

Neuroscience 140:163–175.

Papatheodoropoulos C (2015a) Higher intrinsic network excitability in

ventral compared with the dorsal hippocampus is controlled less

effectively by GABAB receptors. BMC Neurosci 16:75.

Papatheodoropoulos C (2015b) Striking differences in synaptic

facilitation along the dorsoventral axis of the hippocampus.

Neuroscience 301:454–470.

Papatheodoropoulos C, Asprodini E, Nikita I, Koutsona C,

Kostopoulos G (2002) Weaker synaptic inhibition in CA1 region

of ventral compared to dorsal rat hippocampal slices. Brain Res

948:117–121.

Papatheodoropoulos C, Kostopoulos G (2000a) Decreased ability of

rat temporal hippocampal CA1 region to produce long-term

potentiation. Neurosci Lett 279:177–180.

64 S. Kouvaros, C. Papatheodoropoulos /Neuroscience 317 (2016) 47–64

Papatheodoropoulos C, Kostopoulos G (2000b) Dorsal–ventral

differentiation of short-term synaptic plasticity in rat CA1

hippocampal region. Neurosci Lett 286:57–60.

Papatheodoropoulos C, Moschovos C, Kostopoulos G (2005)

Greater contribution of N-methyl-D-aspartic acid receptors in

ventral compared to dorsal hippocampal slices in the expression

and long-term maintenance of epileptiform activity. Neuroscience

135:765–779.

Patel J, Fujisawa S, Berenyi A, Royer S, Buzsaki G (2012) Traveling

theta waves along the entire septotemporal axis of the

hippocampus. Neuron 75:410–417.

Pedarzani P, Storm JF (1993) PKA mediates the effects of

monoamine transmitters on the K+ current underlying the slow

spike frequency adaptation in hippocampal neurons. Neuron

11:1023–1035.

Petrides T, Georgopoulos P, Kostopoulos G, Papatheodoropoulos C

(2007) The GABAA receptor-mediated recurrent inhibition in

ventral compared with dorsal CA1 hippocampal region is

weaker, decays faster and lasts less. Exp Brain Res

177:370–383.

Pofantis H, Papatheodoropoulos C (2014) The alpha5GABAA

receptor modulates the induction of long-term potentiation at

ventral but not dorsal CA1 hippocampal synapses. Synapse

68:394–401.

Provini L, Ito S, Ben Ari Y, Cherubini E (1991) L-homocysteate

preferentially activates N-methyl-D-aspartate receptors to CA1 rat

hippocampal neurons. Eur J Neurosci 3:962–970.

Pugliese AM, Traini C, Cipriani S, Gianfriddo M, Mello T, Giovannini

MG, Galli A, Pedata F (2009) The adenosine A2A receptor

antagonist ZM241385 enhances neuronal survival after oxygen–

glucose deprivation in rat CA1 hippocampal slices. Brit J

Pharmacol 157:818–830.

Rebola N, Srikumar BN, Mulle C (2010) Activity-dependent synaptic

plasticity of NMDA receptors. J Physiol 588:93–99.

Rodrigues TM, Jeronimo-Santos A, Sebastiao AM, Diogenes MJ

(2014) Adenosine A(2A) Receptors as novel upstream

regulators of BDNF-mediated attenuation of hippocampal

Long-Term Depression (LTD). Neuropharmacology 79:

389–398.

Romano C, Sesma MA, McDonald CT, O’Malley K, Van den Pol AN,

Olney JW (1995) Distribution of metabotropic glutamate receptor

mGluR5 immunoreactivity in rat brain. J Comp Neurol

355:455–469.

Rombo DM, Newton K, Nissen W, Badurek S, Horn JM,

Minichiello L, Jefferys JG, Sebastiao AM, Lamsa KP (2015)

Synaptic mechanisms of adenosine A2A receptor-mediated

hyperexcitability in the hippocampus. Hippocampus 25:

566–580.

Sabolek HR, Penley SC, Hinman JR, Bunce JG, Markus EJ, Escabi

M, Chrobak JJ (2009) Theta and gamma coherence along the

septotemporal axis of the hippocampus. J Neurophysiol

101:1192–1200.

Sah P, Hestrin S, Nicoll RA (1989) Tonic activation of NMDA

receptors by ambient glutamate enhances excitability of neurons.

Science 246:815–818.

Sarantis K, Tsiamaki E, Kouvaros S, Papatheodoropoulos C,

Angelatou F (2015) Adenosine A2A receptors permit mGluR5-

evoked tyrosine phosphorylation of NR2B (Tyr1472) in rat

hippocampus: a possible key mechanism in NMDA receptor

modulation. J Neurochem 135:714–726.

Schlicker E, Kathmann M (2001) Modulation of transmitter release via

presynaptic cannabinoid receptors. Trends Pharmacol Sci

22:565–572.

Sebastiao AM, Ribeiro JA (1992) Evidence for the presence of

excitatory A2 adenosine receptors in the rat hippocampus.

Neurosci Lett 138:41–44.

Sebastiao AM, Ribeiro JA (2009) Tuning and fine-tuning of synapses

with adenosine. Curr Neuropharmacol 7:180–194.

Sebastiao AM, Ribeiro JA (2014) Neuromodulation and

metamodulation by adenosine: impact and subtleties upon

synaptic plasticity regulation. Brain Res.

Sheffler DJ, Pinkerton AB, Dahl R, Markou A, Cosford ND (2011)

Recent progress in the synthesis and characterization of group II

metabotropic glutamate receptor allosteric modulators. ACS

Chem Neurosci 2:382–393.

Shigemoto R, Nakanishi S, Mizuno N (1992) Distribution of the mRNA

for a metabotropic glutamate receptor (mGluR1) in the central

nervous system: an in situ hybridization study in adult and

developing rat. J Comp Neurol 322:121–135.

Slanina KA, Schweitzer P (2005) Inhibition of cyclooxygenase-2

elicits a CB1-mediated decrease of excitatory transmission in rat

CA1 hippocampus. Neuropharmacology 49:653–659.

Small SA (2002) The longitudinal axis of the hippocampal formation:

its anatomy, circuitry, and role in cognitive function. Rev Neurosci

13:183–194.

Sotiriou E, Papatheodoropoulos C, Angelatou F (2005) Differential

expression of gamma-aminobutyric acid—a receptor subunits in

rat dorsal and ventral hippocampus. J Neurosci Res 82:690–700.

Stasheff SF, Anderson WW, Clark S, Wilson WA (1989) NMDA

antagonists differentiate epileptogenesis from seizure expression

in an in vitro model. Science 245:648–651.

Stelzer A, Shi H (1994) Impairment of GABAA receptor function by N-

methyl-D-aspartate-mediated calcium influx in isolated CA1

pyramidal cells. Neuroscience 62:813–828.

Suarez LM, Suarez F, Del Olmo N, Ruiz M, Gonzalez-Escalada JR,

Solis JM (2005) Presynaptic NMDA autoreceptors facilitate axon

excitability: a new molecular target for the anticonvulsant

gabapentin. Eur J Neurosci 21:197–209.

Sutula T, Steward O (1987) Facilitation of kindling by prior induction

of long-term potentiation in the perforant path. Brain Res

420:109–117.

Tebano MT, Martire A, Rebola N, Pepponi R, Domenici MR, Gro MC,

Schwarzschild MA, Chen JF, Cunha RA, Popoli P (2005)

Adenosine A2A receptors and metabotropic glutamate 5

receptors are co-localized and functionally interact in the

hippocampus: a possible key mechanism in the modulation of

N-methyl-D-aspartate effects. J Neurochem 95:1188–1200.

Thompson CL, Pathak SD, Jeromin A, Ng LL, MacPherson CR,

Mortrud MT, Cusick A, Riley ZL, Sunkin SM, Bernard A, Puchalski

RB, Gage FH, Jones AR, Bajic VB, Hawrylycz MJ, Lein ES (2008)

Genomic anatomy of the hippocampus. Neuron 60:1010–1021.

Verney C, Baulac M, Berger B, Alvarez C, Vigny A, Helle KB (1985)

Morphological evidence for a dopaminergic terminal field in the

hippocampal formation of young and adult rat. Neuroscience

14:1039–1052.

Wang K, Lin MT, Adelman JP, Maylie J (2014) Distinct Ca2+ sources

in dendritic spines of hippocampal CA1 neurons couple to SK and

Kv4 channels. Neuron 81:379–387.

Wang SJ (2003) Cannabinoid CB1 receptor-mediated inhibition of

glutamate release from rat hippocampal synaptosomes. Eur J

Pharmacol 469:47–55.

Wernig A (1972) The effects of calcium and magnesium on statistical

release parameters at the crayfish neuromuscular junction. J

Physiol 226:761–768.

Xue JG, Masuoka T, Gong XD, Chen KS, Yanagawa Y, Law SK,

Konishi S (2011) NMDA receptor activation enhances inhibitory

GABAergic transmission onto hippocampal pyramidal neurons via

presynaptic and postsynaptic mechanisms. J Neurophysiol

105:2897–2906.

Yoon KW, Rothman SM (1991) Adenosine inhibits excitatory but not

inhibitory synaptic transmission in the hippocampus. J Neurosci

11:1375–1380.

Zeraati M, Mirnajafi-Zadeh J, Fathollahi Y, Namvar S, Rezvani ME

(2006) Adenosine A1 and A2A receptors of hippocampal CA1

region have opposite effects on piriform cortex kindled seizures in

rats. Seizure 15:41–48.

(Accepted 30 December 2015)(Available online 5 January 2016)