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Research Collection Doctoral Thesis Functional Roles of Group II Metabotropic Glutamate Receptors in the Hippocampal CA1 Network Author(s): Rosenberg, Nadia Publication Date: 2015 Permanent Link: https://doi.org/10.3929/ethz-a-010553902 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection . For more information please consult the Terms of use . ETH Library
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Research Collection
Doctoral Thesis
Functional Roles of Group II Metabotropic Glutamate Receptorsin the Hippocampal CA1 Network
Author(s): Rosenberg, Nadia
Publication Date: 2015
Permanent Link: https://doi.org/10.3929/ethz-a-010553902
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
DISS. ETH NO. 23044
Functional Roles of Group II Metabotropic Glutamate Receptors in the Hippocampal CA1 Network
A thesis submitted to attain the degree of
DOCTOR OF SCIENCES of ETH ZURICH
(Dr. sc. ETH Zurich)
Presented by
NADIA ROSENBERG
MSc in Neurosciences, University of Geneva
born on 15.05.1982
citizen of
Sezegnin, GENEVE
accepted on the recommendation of
Prof. Dr. Hanns Ulrich Zeilhofer, examiner
Prof. Dr. med. Urs Gerber, co-examiner
Prof. Dr. Markus Rudin, co-examiner
2015
1
To my parents, who always gave me the right
cards to play this game…
2
1963 Sir John Carew Eccles.
Nobel prize in physiology or medicine. Photo credit: courtesy Australian national university
3
The brain represents the pinnacle of
sophistication in the realm of living
systems.
Molecular Neurobiology of the Mammalian Brain,
Patrick L. McGeer, Sir John C. Eccles, Edith G.
McGeer
4
Table of content
Summary .................................................................................................. 6
Résumé .................................................................................................... 8
List of abbreviations ................................................................................ 11
Chapter 1 ................................................................................................ 14
1. Introduction ............................................................................................................................................... 14
1.1 Ionotropic glutamate receptors .......................................................................................................... 16
1.1.1 The NMDA receptor: channel properties and function ................................................................ 16
1.2 Metabotropic glutamate receptors ..................................................................................................... 21
................................................................................................................................................................... 21
1.2.1 Group II mGlu receptors ............................................................................................................... 22
1.2.1.1 Structure ................................................................................................................................ 22
1.2.1.4 Localization ............................................................................................................................ 26
1.2.1.5 Regulation of synaptic transmission by mGlu II receptors in the hippocampus ................... 28
1.2.1.5.1 mGlu II receptors as presynaptic autoreceptors at glutamatergic synapses ................. 28
1.2.1.5.2 mGlu II receptors as presynaptic heteroreceptors at GABAergic synapses ................... 29
1.2.1.5.3 Group II mGlu receptors as postsynaptic receptors at glutamatergic synapses ............ 30
1.2.1.6 mGlu II receptors as therapeutic targets ............................................................................... 31
Chapter 2 ................................................................................................ 34
2.1 Abstract ............................................................................................................................................... 34
2.2 Introduction ......................................................................................................................................... 35
2.3 Material and methods ......................................................................................................................... 36
Tissue preparation ............................................................................................................................. 36
Electrophysiology ............................................................................................................................... 37
Western blots ..................................................................................................................................... 39
2.4 Results ................................................................................................................................................. 40
2.5 Discussion ............................................................................................................................................ 47
2.6 Figures ................................................................................................................................................. 51
2.7 Figure legends...................................................................................................................................... 56
2.7 References ........................................................................................................................................... 61
Chapter 3 ................................................................................................ 66
5
3.1 Introduction ......................................................................................................................................... 66
3.2 Material and methods ......................................................................................................................... 68
3.3 Results ................................................................................................................................................. 69
3.4 Discussion ............................................................................................................................................ 71
3.5 References ........................................................................................................................................... 73
Chapter 4 ................................................................................................ 75
4. General discussion and perspectives......................................................................................................... 75
Acknowledgments ................................................................................... 92
Summary
6
Summary
Background:
The hippocampal CA1 network plays a crucial role in the encoding of episodic and spatial
memories. CA1 pyramidal neurons, which serve as a relay between the hippocampus and the
neocortex, receive glutamatergic inputs from the CA3 region via the Schaffer-collaterals (SC), as
well as a large diversity of GABAergic interneuron inputs that regulate cell activity by providing
inhibition.
Information is relayed among neurons through fast synaptic transmission, via the ionotropic
receptors, or can be fine-tuned and modulated by metabotropic glutamate receptors. The
metabotropic glutamate receptors are a family of G-protein coupled receptors categorized into three
groups (mGlu I, mGlu II, mGlu III) according to their sequence homology, G-protein coupling, and
ligand selectivity.
mGlu II receptors (composed of mGlu2 and mGlu3 receptors subtypes) are good candidates for the
modulation of CA1 pyramidal neuron excitability as they have been shown to modulate NMDA
receptors (NMDAR) and are expressed in dendrites near SC-CA1 synapses. Furthermore,
evidence for their presence at presynaptic terminals of GABAergic interneurons suggest a role in
modulating network activity. In addition to their physiological roles, mGlu II receptors are implicated
in psychiatric disorders such as anxiety and schizophrenia. Clinical studies have shown that
glutamatergic modulation by activation of mGlu II receptors represent a promising target in the
treatment of schizophrenia.
Objectives: The aim of my thesis project was to characterize the function of mGlu II receptors in
the CA1 network.
Hypothesis 1: Selective activation of mGlu II receptors is sufficient to induce LTP in CA1 pyramidal
neurons.
Summary
7
Results 1: I present experimental data showing that selective pharmacological activation of mGlu II
receptors induces a postsynaptically-expressed form of LTP at Schaffer collateral CA1 pyramidal
cell synapses (chapter 2). This mGlu II receptor-mediated LTP is NMDA receptor-dependent,
involves the phosphorylation through PKC of serine 896 on the GluN1 subunit, and engages
synaptic and extrasynaptic NMDARs that are differentially modulated by mGlu2 and mGlu3
receptors.
Hypothesis 2: Priming NMDAR by mGlu II activation can induce metaplasticity at CA1 pyramidal
neurons.
Results 2: My experiments demonstrate that prior activation of mGlu II receptors has a priming
effect that facilitates subsequent induction of LTP. These findings elucidate a new metaplastic
mechanism for the modulation of synaptic function at Schaffer collateral inputs to CA1 pyramidal
cells.
Hypothesis 3: Selective activation of mGlu II induces LTD in interneurons of the CA1 region.
Results 3: In the second project (chapter 3), I demonstrated that selective activation of mGlu II
receptors induces LTD of GABAergic transmission onto CA1 pyramidal cells, which is mediated by
the mGlu3 receptor subtype. These results reveal that in addition to playing a role as postsynaptic
autoreceptors, mGlu II receptors can operate as heteroreceptors to modulate CA1 pyramidal
neuron excitability.
Conclusion:
In chapter four, I explore different scenarios regarding downstream events triggered by mGlu II
receptor activation which can underlie synaptic potentiation. I also discuss how the modulation of
interneuron activity by mGlu II receptors activation can shape synaptic integration of inputs arriving
at CA1 pyramidal neurons and how it this would affect CA1 network activity.
Résumé
8
Résumé
Background:
Le réseau neuronal de la région CA1 de l'hippocampe joue un rôle crucial dans l’encodage des
mémoires épisodiques et spatiales. Les neurones pyramidaux du CA1, qui servent de relais entre
l’hippocampe et le néocortex, reçoivent des afférences glutamatergiques de la région CA3 par les
collatérales de Schaffer, ainsi que des entrées synaptiques inhibitrices provenant d’une grande
diversité d’interneurones GABAergiques participant à la régulation de l’activité cellulaire.
L’information est relayée d’un neurone à l’autre soit par une transmission synaptique rapide via les
récepteurs ionotropiques, ou peut être affinée et modulée par les récepteurs métabotropes au
glutamate. Ces récepteurs appartiennent à une famille de récepteurs associés à des protéines G
subdivisée en trois groupes (mGlu I, mGlu II, mGlu III) selon l’homologie de leurs séquences, leur
association avec la protéine G et la sélectivité qu’ils ont pour leur ligand.
Selon plusieurs lignes d’évidences, les récepteurs mGlu II (comprenant les sous-types mGlu2 et
mGlu3) ont une grande chance d’être impliqués dans la modulation de l’excitabilité des neurones
pyramidaux de CA1. En effet, leur capacité à moduler les récepteurs NMDA (NMDAR) a été
démontré ainsi que leur expression dans les dendrites, proche des synapses des collatérales de
Schaffer et dans les terminaux présynaptiques des interneurones GABAergiques. En plus d’un rôle
purement physiologique, les récepteurs mGlu II sont impliqués dans des troubles psychiatriques
tels que l’anxiété et la schizophrénie. Des études cliniques ont montré que la modulation
glutamatergique par l’activation des récepteurs mGlu II est une cible prometteuse pour le traitement
de la schizophrénie.
Objectifs: Le but de mon projet de thèse était de caractériser la fonction des récepteurs mGlu II
dans le réseau neuronal de la région CA1.
Hypothèse 1 : L’activation sélective des récepteurs mGlu II est suffisante pour induire de
potentialisation à long terme (PLT) dans les neurones pyramidaux de la région CA1.
Résumé
9
Résultats 1: Je présente des données expérimentales montrant que l’activation pharmacologique
et sélective des récepteurs mGlu II induit une forme de PLT postsynaptique aux synapses des
collatérales de Schaffer (chapitre 2). Cette PLT médiée par les récepteurs mGlu II dépend des
NMDAR, implique la phosphorylation de la serine 896 de la sous-unité GluN1 par la PKC et engage
la contribution des NMDAR synaptiques et extrasynaptiques qui sont modulés de façon différentiels
par les récepteurs mGlu2 et mGlu3.
Hypothèse 2 : L’amorçage des récepteurs NMDA par l’activation préalable des récepteurs mGlu II
peut induire de la métaplasticité dans les neurones pyramidaux de la région CA1.
Résultats 2: Mes expériences ont démontré que l’activation préalable des récepteurs mGlu II a un
effet d’amorçage qui facilite l’induction ultérieure d’une PLT. Ces découvertes révèlent un nouveau
mécanisme métaplastique dans la modulation des fonctions synaptiques par les connections
provenant des collatérales de Schaffer sur les cellules pyramidales du CA1.
Hypothèse 3: L’activation sélective des récepteurs mGlu II induit une dépression à long terme
dans les interneurones de la région CA1.
Résultats 3: Dans le deuxième projet (chapitre 3), je démontre que l’activation sélective des
récepteurs mGlu II induit une dépression à long terme (DLT) de la transmission GABAergique sur
les cellules pyramidales du CA1, et que cette DLT est médiée par le récepteur mGlu3. Ces
résultats dévoilent qu’en plus de jouer un rôle en tant autorécepteur à la postsynapse, les
récepteurs mGlu II peuvent opérer en tant qu’hétérorecepteurs pour moduler l’excitabilité des
neurones pyramidaux du CA1.
Conclusion :
Dans le quatrième chapitre, j’explore les conséquences découlant de l’activation des récepteurs
mGlu II et dont certaines seraient à la base de la potentiation synaptique. Je traite également de la
modulation de l’activité des interneurones par les récepteurs mGlu II, et comment ces interactions
Résumé
10
pourraient sculpter l’intégration synaptique des afférences contactant les neurones pyramidaux du
CA1, ainsi que de leur impact sur l’activité du réseau neuronal CA1.
List of abbreviations
11
List of abbreviations
AC: adenylyl cyclase
ACPD: aminocyclopentane-1,3-dicarboxylic acid
ACSF: artificial cerebrospinal fluid
AMPAR: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
BINA: biphenyl-indanone A
CA1: cornu ammonis 1
CA3: cornu ammonis 3
CaMKII: Ca2+/calmodulin-dependent protein kinase II
cAMP: cyclic adenosine monophosphate
CNS: central nervous system
COOH: carboxyl group
CRD: cysteine-riche domain
DAG: diacyl-glycerol
D-AP5: D-(-)-2-Amino-5-phosphonopentanoic acid
DCG-IV: (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine
EC: extracellular
ECtx: entorhinal cortex
EPSP: excitatory postsynaptic potential
fEPSP: field excitatory postsynaptic potential
GABA: gamma-Aminobutyric acid
GRIP1: glutamate receptor-interacting protein 1
GRM2: glutamate receptor, metabotropic 2 (gene)
GRM3: glutamate receptor, metabotropic 3 (gene)
List of abbreviations
12
HFS: high frequency stimulation
IPSP: inhibitory postsynaptic potential
IP3: inositol trisphosphate
KO: knockout
L-CCG-I: (2S,1'S,2'S)-2-(Carboxycyclopropyl)glycine
LTD: long term depression
LTP: long term potentiation
MCPG: methyl-4-carboxyphenylglycine
mGlu I: the group I metabotropic glutamate receptors
mGlu II: the group II metabotropic glutamate receptors
mGlu III: the group III metabotropic glutamate receptors
mGlu: metabotropic glutamate receptor
NAAG: N-acetyl-aspartyl glutamate
NBQX: 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide
NAM: negative allosteric modulator
NEM: N-ethyl-maleimide
NMDAR: N-methyl-D-aspartate receptor
PAM: positive allosteric modulator
PICK1: protein interacting with C kinase-1
PKA: protein kinase A
PKC: protein kinase C
PP2C: protein phosphatase 2C
PSD: postsynaptic density
p-S896: serine 896 phosphorylated
RNA: ribonucleic acid
List of abbreviations
13
SEM: standard error of the mean
SNARE: Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor
SPET: single photon emission tomography
TM: transmembrane
VFD: venus flytrap domain
WT: wild type
Chapter 1
14
Chapter 1
1. Introduction
Since the late 19th century with Cajal’s careful descriptions of the micro-anatomy of the
nervous system, the nature of transmission from one neuron to another has been a matter
of debate. Because of technical limitations preventing the visualization of neuronal
ultrastructure, the central question was how transmission occurs between neurons. The
neuroanatomists Palay and Palade, and de Robertis and Bennett provided the first
anatomical evidence for the existence of a “neuronal junction” in 1954 by revealing the
ultrastructure of the synapse. They demonstrated the presence of presynaptic vesicles, the
pre- and postsynaptic components of the synapse, and a well-defined synaptic cleft by
using the high magnification power of the newly invented electron microscope (Eccles,
1963). A decade later, Streit et al., (1972) provided the first images of synaptic vesicles in
the process of fusing and releasing of neurotransmitter (Figure 1).
Chapter 1
15
Figure 1. Electron micrograph showing synaptic vesicles (sv) in the presynaptic terminal, the fusion of a
vesicle with the presynaptic membrane (double arrow) and the synaptic cleft (one arrow). (Streit, 1972).
Chemical synapses allow neuronal communication through the use of neurotransmitters.
The principal action of neurotransmitters was believed to be mediated solely by ligand-
gated ion channel receptors (ionotropic receptors) responsible for fast synaptic
transmission by opening ‘ionic paths’ in the postsynaptic membrane. However, in the late
1970s, Eccles et al., (1979) employed the term metabotropic to describe another form of
neurotransmission. The idea behind this principle was that slow and indirect metabolic
changes in the postsynaptic cell could be initiated by the binding of neurotransmitter, which
would not rapidly affect the membrane conductance.
The mechanisms of metabotropic signal transduction are now well established. In contrast
to ionotropic receptors, metabotropic receptors modulate and fine-tune synaptic
transmission. A major representative of this class is the metabotropic glutamate (mGlu)
receptor, which binds glutamate, the main excitatory neurotransmitter in the mammalian
central nervous system (CNS).
Chapter 1
16
1.1 Ionotropic glutamate receptors
In the CNS, Glutamate acts on ligand-gated ion channel to mediate fast excitatory
neurotransmission through the ionotropic glutamate receptors. These cation-permeable ion
channel receptors can be divided into three large classes based on pharmacology and
structural homology: α-amino-3-hydroxy-5-methyllisoxazolepropionic acid (AMPA)
receptors (AMPARs), kainate receptors and N-methyl-D-aspartate receptors (NMDARs).
Ionotropic glutamate receptors are integral membrane receptors that form a central ion
channel pore composed of four large subunits (Dingledine et al., 1999).
In this section on ionotropic receptors, I will focus on NMDA receptors because during my
research project I was interested in understanding the specific modulation of this ionotropic
receptor by group II mGlu receptors.
1.1.1 The NMDA receptor: channel properties and function
In the early 1960s, it was known that glutamate was an important metabolite in the brain,
but at that time the thought of glutamate being a transmitter acting on a single receptor was
considered unlikely because of its non-stereoselective action at excitatory synapses.
However, the idea that glutamate could function as a transmitter could not be completely
ruled out because specific antagonists were not yet available. This problem motivated
Jeffrey Watkins to characterize glutamate analogs with greater stereoselectivity, which
would allow distinguishing between different glutamate receptors subtypes. Among these
new glutamate analogs, one of them turned out to be an especially potent excitant, namely
NMDA (Watkins and Jane, 2006). Discovery of the NMDAR occurred in the 1970s,
followed by the cloning and characterization of complementary DNA encoding the first
NMDA subunit (Moriyoshi et al., 1991), now known as GluN1.
Chapter 1
17
Enormous breakthroughs have been made since then in characterizing NMDARs. They
have been shown to play a pivotal role in the regulation of synaptic transmission and
impaired function of NMDARs is implicated in diverse neurological and psychiatric
disorders (Dingledine et al., 1999; Lau and Zukin, 2007).
Functional NMDARs are heterotetramers. So far, three family subtypes of NMDAR subunit
have been identified: the GluN1 subunit, the GluN2 subunits (comprising GluN2A, GluN2B,
GluN2C and GluN2B) and GluN3 subunits (GluN3A, GluN3B) assembling in two
fundamental GluN1 subunits and two GluN2 subunits or in some cases a mixture of GluN2
and GluN3 subunits (Cull-Candy and Leszkiewicz, 2004).
Amongst the ionotropic glutamate receptors, NMDARs exhibit several remarkable features.
First is their ion channel voltage-sensitive block by extracellular Mg2+. To allow cations to
be conducted, glutamate must bind to a GluN2 subunit and the membrane has to be
depolarized to dislodge extracellular Mg2+ from the pore (Ruppersberg et al., 1994). Thus,
the NMDAR acts as coincidence detector of pre and postsynaptic activity; activated by
presynaptic glutamate release and postsynaptic depolarization, which represents the
foundation of the theory of learning and memory at the synaptic level (Bourne and Nicoll,
1993). As proposed by Hebb (1949), the repetition of activity in a presynaptic neuron
associated with the discharge of a postsynaptic neuron increases synaptic efficiency, which
results in a long lasting increase of synaptic strength called LTP (Bliss and T. LØmo, 1973).
Second, NMDARs are highly permeable to Na+, K+ and also to Ca2+ (Hollmann and
Heinemann, 1994), whose influx triggers intracellular cascades to initiate long-lasting
changes in synaptic efficacy. Third, the response mediated by NMDAR activation displays
unusually slow kinetics due to the slow unbinding of glutamate (Lester et al., 1990) and
finally, their activation requires, in addition to glutamate, the binding of the co-agonist
Chapter 1
18
glycine or serine (Johnson and Ascher, 1987; Dingledine et al., 1999; Cull-Candy and
Leszkiewicz, 2004; Paoletti et al., 2013).
The GluN2 subunits are a major determinant of the biophysical properties of the receptor
and show different spatiotemporal patterns of expression during development.
GluN1/GluN2B predominating in the cortex, hippocampus and cerebellum during the first
week of postnatal life, and then switches to GluN1/GluN2A as the neuron becomes mature,
but the replacement of GluN2B with GluN2A is not complete (Tovar and Westbrook, 1999;
Paoletti et al., 2013).
GluN3 seems important to maintain the synapse in an immature state, because deletion of
GluN3A accelerates the expression of synaptic maturation markers (Henson et al., 2012)
and increases dendritic spine density and growth (Das et al., 1998). GluN2C is mainly
restricted to the cerebellum, whereas GluN2D is primarily localized in thalamic and
hypothalamic nuclei (Monyer et al., 1994).
Every subunit provides specific permeation and gating properties to the channel. For
example, the GluN2A subunit confers to the receptor faster kinetics, a greater channel
open probability, and a shorter deactivation time constant than the GluN2B subunit does.
Also, GluN2C and GluN2D subunits generate low conductance and are less sensitive to
Mg2+ block as opposed to GluN2A and GluN2B subunits (Retchless et al., 2012; Paoletti et
al., 2013). Furthermore, NMDARs with different subunit composition have been shown to
be targeted to different somatodendritic locations in neurons (Köhr, 2006), as for example
in CA3 pyramidal cells of the hippocampus, where inputs arising from the fornix target
GluN2A, whereas associational/commissural inputs target GluN2B (Ito et al., 1997). They
also have different synaptic locations, the GluN2A subunit being synaptic and the GluN2B
subunit being mainly extrasynaptic (Steigerwald et al., 2000; Groc et al., 2006). Thus, the
Chapter 1
19
composition and location of NMDARs are determining factors of glutamatergic postsynaptic
transmission.
Besides the long extracellular N-terminal domain where the ligand binds, and the three
transmembrane segments (Figure 2), the C-terminal domain regulates receptor interaction
with numerous cytosolic proteins by short docking motifs (Kornau et al., 1995). These
protein-protein interactions influence NMDAR localization, precise membrane targeting,
and degradation. In addition, the C-terminal domain of NMDAR subunits expresses various
phosphorylation sites for post-translational modifications, which are important in regulating
mobility, localization, and activity of the receptor (Chen and Roche, 2007).
Figure 2. Model of the NMDA receptor
Because of its tremendous impact on postsynaptic regulation and synaptic plasticity,
NMDAR-dependent long-term potentiation (LTP) has been extensively studied in the
hippocampus, a brain region crucial for memories. Disruption of NMDARs in the
hippocampus has been shown to impair synaptic plasticity and leads to memory deficits
Chapter 1
20
(Bannerman et al., 1995; Tsien et al., 1996; Lee and Kesner, 2002). In addition, it has been
demonstrated that long-term depression (LTD) or LTP could inactivate or reactivate,
respectively, fear conditioning (an associative memory) in an NMDAR-dependent manner
(Nabavi et al., 2014). Therefore, NMDAR-dependent LTP is postulated to be the cellular
mechanism underlying memory encoding and learning.
Within the hippocampus, this from of LTP is conventionally induced by a brief and strong
pattern of electrical stimulation, which provokes Ca2+ influx through the NMDAR. The
increase in Ca2+ concentration activates a great number of intracellular mediators such as
protein kinases, which give rise to an enhancement of AMPA-mediated transmission in
three ways. First, Ca2+ triggers incorporation of AMPAR into the cell membrane (Shi et al.,
1999), which requires SNARE complexes (Lu et al., 2001) to bind with complexin, a protein
regulating neurotransmitter release when located presynaptically. However, the molecular
pathway underlying this mechanism still needs to be defined (Ahmad et al., 2012). Second,
it activates calmodulin, which in turn activates the calcium/calmodulin-dependent protein
kinase II (CaMKII) that further phosphorylates AMPAR leading to an increase of the
channel conductance. Third, Ca2+ influx leads to phosphorylation of the protein stargazing
by CaMKII, which brings AMPARs to the synapse and immobilizes them by binding to
postsynaptic density protein 95 (PSD95) (Lisman et al., 2012).
Chapter 1
21
1.2 Metabotropic glutamate receptors
mGlu receptors are members of the large family of G-protein-coupled receptors. Since their
discovery (Sladeczek et al., 1985) and the cloning of G-protein-coupled glutamate receptor
(mGlu1) in 1991 (Masu et al., 1991), seven other mGlu types have been described. They
are classified into three groups on the basis of their sequence homology, pharmacological
properties and preferred intracellular transduction pathway.
mGlu I receptors (mGlu1 and mGlu5 receptors) can couple to Gq-protein and activate
phospholipase C, which results in the production of inositol 1,4,5-triphosphate (IP3) and
diacylglycerol (DAG). mGlu II receptors (mGlu2 and mGlu3) and mGlu III receptors
(mGlu4, mGlu6, mGlu7 and mGlu8) can couple to Gi,o proteins and reduce cAMP levels via
inhibition of adenylyl cyclase (AC) (Conn and Pin, 1997). In addition, distinct mGlu
receptors may physically interact with each other as has been shown for the mGlu2/4
receptor heterodimer (Doumazane et al., 2011; Yin et al., 2014).
A family of mG/uRs (from an /des by Darryle D. Schoepp and P. Jeffrey Conn, 1993). From (Schoepp and Conn, 1993)
Chapter 1
22
In the next part of my dissertation, I will exclusively deal with mGlu II receptors because my
research is focused on understanding the functional contribution of mGlu II receptors to
synaptic plasticity and how they modulate NMDAR function.
1.2.1 Group II mGlu receptors
mGlu2 and mGlu3 are encoded by GRM2 and GRM3, respectively, and their transcripts
are expressed in the hippocampus, prefrontal cortex and cerebellum. Whereas GRM2 does
not undergo alternative splicing, mGlu3 RNA can generate at least four variants, where
GRM3∆4 (missing exon 4) appears to be the most abundant (Sartorius et al., 2006).
1.2.1.1 Structure
The structural architecture of mGlu receptors consists of constitutive dimers (two
protomers). Each protomer includes an extracellular (EC), a transmembrane (TM) and an
intracellular domain. The large N-terminal EC is divided in two regions: the glutamate
binding region, also called the Venus flytrap domain (VFD) and the cysteine-rich domain
(CRD), which steps in between the VFD and TM regions to propagate conformational
changes induced by ligand binding. The presence or absence of ligand modulates the
dynamic equilibrium of the structure, which can adopt a resting or active conformation
(Muto et al., 2007). The transmembrane heptahelical domain possesses the binding sites
for positive and negative allosteric modulators, as well as a site for regulation by kinases
(Figure 3).
Chapter 1
23
Figure 3. Structural model of a group II metabotropic glutamate receptor and it model of activation. Adapted
from (Muto et al., 2007).
The intracellular C-terminal domain is critical as it is involved in the modulation of G-protein
coupling. In addition, it is this region which is subject to alternative splicing, to regulation by
phosphorylation, and to protein-protein interactions (Fagni et al., 2004; Niswender and
Conn, 2010). Of particular interest with respect to the modulation of NMDARs are protein-
protein interactions. Studies using the yeast two-hybrid system have shown that the
signaling protein tamalin directly interacts with mGlu2 and mGlu3 (Kitano et al., 2002;
Fagni et al., 2004). On the other hand, the protein phosphatase 2C (PP2C) (Flajolet et al.,
2003) and scaffolding proteins PICK1 and GRIP binds selectively to mGlu3 (Hirbec et al.,
2002).
1.2.1.3 Pharmacology
L-glutamate causes depolarization of neurons by binding to a variety of receptors as
discussed above. Its interaction with a diversity of receptors can be explained by the great
Chapter 1
24
flexibility of the molecule (Gonzalez and Barreto, 2013), which allows glutamate to adopt a
three-dimensional structure or spatial conformation to recognize specific receptors and to
form a complex as a key in a keyhole. Small differences in the chemical structure of a
natural or synthetized analogue can exert a completely different behavior on the receptor.
The first compound synthesized that acts as a selective mGlu receptor agonist was
(1S,3R)-1-AminoCycloPentane-1,3-Dicarboxylic acid (1S,3R-ACPD), an analog of
conformationally constrained glutamate (Hall et al., 1979). It was shown to activate mGlu II
receptors (Mistry and Challiss, 1996). However, 1S,3R-ACPD has a broad agonist effect
also extending to mGlu I receptors (Schoepp et al., 1999).
Next came the discovery of L-CCG-I, which is an agonist specifically for the mGlu2 and the
mGlu3 receptors but also activates subtypes of mGlu III including the mGlu6 and mGlu7
receptors (Brabet et al., 1998). The addition of a carboxylic group (COOH) to L-CCG-I
leads to the compound DCG-IV. Showing high selectivity for mGlu II receptors, DCG-IV
also displays agonist activity at the NMDAR (Ohfune et al., 1993) and has an antagonist
effect at mGlu III receptors (Brabet et al., 1998).
Figure 4. LY354740: a glutamate analog conformationally constrained resulting in a highly selective and
potent group II mGlu receptor agonist. Adapted from (Bechi et al., 2014).
Chapter 1
25
Later on, a new group of highly selective agonists for mGlu II receptors was developed.
The prototypic compound of this class is LY354740 (Monn et al., 1997) that was followed
by LY379268 and LY389795 (Monn et al., 1999). None of these potent mGlu II agonists
exhibit affinities for ionotropic glutamate receptors. Nevertheless, LY379268 and
LY389795, unlike their analog LY354740, induce weak agonist responses at mGlu III
receptor subtypes (Schoepp et al., 1999). Albeit highly selective for mGlu II receptors,
neither of these compounds can differentiate between mGlu2 and mGlu3 receptors.
It has been reported that the addition of a simple methyl group on the hexane ring system
of LY354740 results in a compound, LY395756, which can act as an mGlu2 agonist and
mGlu3 antagonist (Dominguez et al., 2005; Ceolin et al., 2011). Furthermore, selective
agonist action of a conformationally restricted cyclopropyl glutamate analog at the mGlu2
receptor has been reported (Nielsen et al., 2011), it has also been shown that NAAG, a low
affinity endogenous CNS peptide acts as a selective agonist for the mGlu3 receptor, but
has an additional effect as an NMDAR activator (Wroblewska et al., 1997; Neale, 2011).
On the other hand, β-NAAG acts as a reasonably selective antagonist for the mGlu3
receptor (Lea et al., 2001). A potent antagonist for mGlu II receptors is LY341495 (Ornstein
et al., 1998). However, LY341495 provides weak antagonist potency at group I and III
mGlu receptors (Schoepp et al., 1999). In addition to LY341495, another antagonist for
mGlu II receptors has been developed, MGS0039, which also has an antagonist effect at
the mGlu7 receptor (Chaki et al., 2004).
Because of the high structural conservation of mGlu2 and mGlu3 (mGlu2/3) receptors, the
development of a highly selective ligand is difficult. However, the development of selective
allosteric modulators has allowed increased selectivity for these receptors. Positive
allosteric modulators (PAMs) are compounds enhancing the response of the receptor to
Chapter 1
26
glutamate by binding to sites distinct from the orthosteric agonist-binding site. For instance,
LY487379 and BINA are highly selective PAMs for the mGlu2 receptor (Conn et al., 2009).
In contrast to PAMs, a family of negative allosteric modulators (NAMs) called MNI has
been characterized to selectively antagonize mGlu2 and mGlu3 receptors (Hemstapat et
al., 2007).
1.2.1.4 Localization
mGlu II receptors are broadly expressed throughout the CNS in nearly every major brain
area. In my dissertation project I have focused on characterizing functional roles of group II
mGlu receptors in the hippocampus.
Located in the limbic system, the hippocampus is a crucial structure for learning and
memory. The main input conveys sensory information from the entorhinal cortex (ECtx) to
the granule cells of the dentate gyrus via axons of the perforant path. In turn, granule cells
project, through the mossy fibers, to the CA3 region, which subsequently project their
axons, the Schaffer collaterals, to the CA1 area. A second perforant path emerging from
the ECtx, also called temporoammonic path, directly contacts distal apical dendrites of the
CA1 region. The CA1 region is the primary output of the hippocampus to the neocortex and
is of major interest because of its important role in spatial and episodic memory (Zola-
Morgan et al., 1986; Tsien et al., 1996).
Chapter 1
27
Figure 5. Wiring diagram of the hippocampus represented as a trisynaptic loop.
In the hippocampus, mGlu II receptors are localized at distinct presynaptic and
postsynaptic sites, which enable them to modulate a diversity of ionotropic receptors and
signaling proteins, and to alter neuronal excitability and synaptic transmission.
mGlu2 and mGlu3 receptors share some similarities; they are often localized in preterminal
and terminal parts of small unmyelinated axons. In the hippocampus they are found in the
stratum lacunosum moleculare of the CA1 region. Both receptors also show a somato-
dendritic localization in all regions of the hippocampus (Shigemoto et al., 1997; Tamaru et
al., 2001). However, selective immunoreactivity for mGlu2 receptor has been detected in
presynaptic structures of the mossy fibers and perforant path (terminating in CA3)
(Shigemoto et al., 1997), in particular in the preterminal section of axons remote from the
active zone. In contrast, mGlu3 is found in perisynaptic sites of granule cell spines, as well
as in their postsynaptic membrane specialization. In addition, the mGlu3 receptor is the
only group II mGlu receptor present in glial cells (Tamaru et al., 2001).
Chapter 1
28
At present there is no anatomical evidence available for the localization of group II mGlu
receptors on non-glutamatergic neurons of the hippocampus. However, mGlu3 receptors
have been detected at extrasynaptic sites of axon terminals in non-glutamatergic neurons
from other brain regions, such as the thalamus (Tamaru et al., 2001), and combined
electrophysiological/pharmacological studies have suggested their presence on CA3
GABAergic presynaptic terminals (Poncer et al., 1995; Anwyl, 1999).
1.2.1.5 Regulation of synaptic transmission by mGlu II receptors in the hippocampus
1.2.1.5.1 mGlu II receptors as presynaptic autoreceptors at glutamatergic synapses
The best established physiological role of mGlu II receptors in the hippocampus is their
ability to reduce synaptic transmission at glutamatergic synapses. This effect is mediated
by presynaptic mGlu receptors acting as autoreceptors to decrease transmitter release.
Suppression of synaptic transmission was demonstrated at mossy fiber-CA3 synapses by
pharmacological activation of mGlu II receptors with DCG-IV (Kamiya et al., 1996) and L-
CCGI (Manzoni et al., 1995), or by high-frequency stimulation of the mossy fibers, allowing
glutamate spillover to activate presynaptic mGlu II receptors (Scanziani et al., 1997). In
addition, several studies have demonstrated a physiological role for presynaptic mGlu II
receptors in the induction of LTD at mossy the fiber-CA3 synapse (Kobayashi et al., 1996),
specifically for the mGlu2 receptor (Yokoi et al., 1996). In addition to mossy fiber-CA3
synapses, enthorinal-CA1 synapses also exhibit this autoinhibitory response after burst
stimulation (Kew et al., 2000) In contrast, negative feedback of glutamate release is not
Chapter 1
29
observed at Schaffer collateral-CA1 synapses (Kamiya et al., 1996). These physiological
studies corroborate anatomical findings of the presynaptic localization of mGlu II receptors.
mGlu II receptor-mediated suppression of excitatory transmission has been attributed to a
reduction of calcium conductance by inhibition of calcium channels, including the N-type
channel in the dentate gyrus (Schumacher et al., 2000) and CA3 (Swartz and Bean, 1992)
and the P/Q type channel in the entorhinal cortex (Wang et al., 2012). A further mechanism
involves the blockade of the exocytotic machinery (Scanziani et al., 1995; Kamiya and
Ozawa, 1999), that could involve inhibition of adenylyl cyclase activity and subsequent
reduction of cAMP levels (Bellone et al., 2008; Wang et al., 2012).
1.2.1.5.2 mGlu II receptors as presynaptic heteroreceptors at GABAergic synapses
Activation of mGlu receptors with ACPD decreases the amplitude of GABA-mediated
IPSPs in the CA1 region (Desai and Conn, 1991). This reversible blockade of inhibition
was attributed to mGlu receptors located on axonal terminals of GABAergic interneurons
(Jouvenceau et al., 1995). In addition, ACPD induces LTD at GABAergic synapses in CA1
through a PKC and intracellular Ca2+ store independent process (Liu et al., 1993).
Consistent with these findings, Poncer et al. (1995) demonstrated in the CA3 area that
miniature IPSC frequency is reduced by DCG-IV, a selective mGlu II receptors agonist.
Moreover, in the same hippocampal region, GABAergic inhibition is differentially
modulated. Indeed, IPSPs originating from stratum radiatum are reduced by activation of
mGlu II receptors, whereas IPSPs from stratum oriens interneurons are not (Poncer et al.,
2000). GABA release at synapses formed by stratum radiatum interneurons was
suppressed by an N-type calcium channel blocker (Poncer et al., 2000). Thus activation of
mGlu II receptors may negatively modulate N-type calcium channels to reduce GABAergic
transmission at these synapses.
Chapter 1
30
1.2.1.5.3 Group II mGlu receptors as postsynaptic receptors at glutamatergic synapses
Until the early 2000s, most studies investigating the physiological roles of mGlu receptors
were conducted using ACPD as an agonist. Because of the non-specificity of this drug, the
interpretation of these early data is sometimes unclear.
LTP induction by ACPD was observed in the hippocampal CA1 area (Bashir et al., 1993;
Bortolotto et al., 1994; Bortolotto and Collingridge, 1995) and in the dentate gyrus
(O’Connor et al., 1994), and this form of plasticity was attributed to activation of mGlu I
receptors. Moreover, ACPD-induced LTP required depolarization of the postsynaptic
neuron to allow coactivation of NMDARs (O’Connor et al., 1995; Breakwell et al., 1996)
and was PKC-dependent (O’Connor et al., 1995). Activation of mGlu receptors with ACPD
has also been shown to have a priming effect on subsequent high frequency stimulation
(HFS)-induced LTP in CA1 (Mcguinness et al., 1991; Cohen et al., 1999). However, DHPG
(a selective agonist of group I) was shown to induce LTD at all synapses tested (Palmer et
al., 1997; Fitzjohn et al., 1999; Huber et al., 2000). Furthermore, MCPG (a non-selective
antagonist for mGlu I and II receptors), failed to inhibit LTP in the hippocampal CA1 area
(Manzoni et al., 1994). These discrepancies with regard to the functional role of
postsynaptic mGlu receptors in the hippocampus remain unresolved.
More recently, a number of studies have examined specific the roles for mGlu II receptors.
First, a study examining the function of mGlu3 in vivo in the dentate gyrus showed that the
expression of low frequency LTD-induced was inhibited by β-NAAG (an antagonist at the
mGlu3 receptor), and that this action was exerted postsynaptically in a cAMP-dependent
process (Pöschel et al., 2005). A further postsynaptic mechanism has been described in
which activation of mGlu2 by DCG-VI (an agonist of mGlu II receptors) induced an
inhibitory potassium current in the proximal dendrites of granule cells. The activated
Chapter 1
31
potassium current was mediated by GIRK channels. However, this outward current was not
observed in CA3 or CA1 pyramidal cells (Brunner et al., 2013).
In the CA3 region of the hippocampus, only one study has examined the somato-dendritic
role of mGlu II receptors. Ster et al. (2011) showed that activation of mGlu II receptors by
DCG-VI or LCCG-I (agonists for mGlu II receptors) induced an excitatory current in CA3
pyramidal cells and in CA3 stratum oriens interneurons. Experiments with knockout mice
revealed that the inward current was mediated by the mGlu3 receptor. The enhanced
excitability was associated with an increase in cationic conductance as well as a decrease
in potassium conductance, and promoted oscillatory activity at theta frequency in CA3
networks.
The only study examining the effects mediated by postsynaptic mGlu II receptors in the
CA1 region was done in acutely isolated CA1 neurons, where LY379268 (an agonist for
mGlu II receptors) enhanced NMDA-evoked currents. The proposed mechanism for the
increase of NMDAR function is an inhibition of the cAMP-PKA pathway and the activation
of src kinase via a decrease in Csk activity (Trepanier et al., 2013).
1.2.1.6 mGlu II receptors as therapeutic targets
The hippocampus and prefrontal cortex are regions displaying medium to high expression
of mGlu II receptors and deficits in mGlu II receptors function in these areas are associated
with a range of psychiatric disorders including anxiety and schizophrenia (Weinberger et
al., 1992). In addition, genetic studies of single-nucleotide polymorphisms have shown
several positive association of GRM3 with schizophrenia (Harrison et al., 2008).
A single photon emission tomography (SPET) study has revealed a significant reduction in
NMDAR binding in the hippocampus of schizophrenic patients (Pilowsky et al., 2006). In
addition, examination of schizophrenic post-mortem tissue revealed a sharp increase in
Chapter 1
32
neuroligin1-induced activation of the erbB receptor (Hahn et al., 2006), which decreases
NMDAR currents likely by enhancing GluN1 subunit internalization (Gu et al., 2005) and
inhibit LTP at Schaffer-collaterals (Huang et al., 2000). Hypofunction of NMDARs has long
been linked to the etiology of mental disorders but the underlying pathophysiology remains
unclear (Tsai and Coyle, 2002). In recent years, however, significant advances have been
made, with several studies identifying mGlu II receptors as targets for the treatment of
schizophrenia. Schizophrenia manifests as positive symptoms (delusions, hallucinatory
behavior, conceptual disorganization and other thought disorders), negative symptoms (as
for example blunted affection, emotional withdrawal, and lack of spontaneity) and
psychopathological symptoms (anxiety, poor attention, motor retardation, and depression)
(Peralta, 1994).
Interest in mGlu II receptors with respect to schizophrenia was initiated by an early study
demonstrating that an mGlu II receptor agonist (LY354740) reversed the increase in
glutamate and the behavioral impairment produced by phencyclidine (PCP), an NMDAR
antagonist use to induce psychotic symptoms (Moghaddam and Adams, 1998). These
electrophysiological and behavioral effects have been further confirmed in rodent and
human studies (Cartmell et al., 1999; Krystal et al., 2005). Prior to this work, the major
hypothesis for the pathophysiology underlying schizophrenia was an alteration of
dopaminergic transmission. However, many of the drugs to treat schizophrenia that
modulate the dopamine system also act on serotonergic transmission, α-adrenergic,
histaminergic, and muscarinic acetylcholine receptors, provoking strong side effects such
as weight gain, akathisia or agitation (Patil et al., 2007). A randomized phase 2 clinical trial
provided evidence that an oral prodrug of LY404039 (LY2140023), a selective agonist for
mGlu II, greatly improved positive and negative symptoms after four weeks and did not
show the side effects observed with established medication (Patil et al., 2007).
Chapter 1
33
These preclinical and clinical studies provide strong evidence that glutamate modulation by
activation of mGlu II receptors represents a promising new avenue in the treatment of
schizophrenia.
Chapter 2
34
Chapter 2
Activation of Group II Metabotropic Glutamate Receptors Induces LTP at
Hippocampal SC-CA1 Synapses by Priming NMDA Receptors
Nadia Rosenberg, Urs Gerber, Jeanne Ster (in preparation)
2.1 Abstract
It is well established that activation of group I metabotropic glutamate (mGlu I) receptors
induces long term depression (LTD) at Schaffer collateral (SC)-CA1 synapses. In contrast,
application of ACPD, a mixed agonist at group I and group II mGlu receptors, induces long
term potentiation (LTP). Here we obtained whole-cell recordings from pyramidal cells and
field recordings in the hippocampal CA1 region to investigate the specific contribution of
mGlu II receptors to synaptic plasticity at SC-CA1 synapses in adult mice. Pharmacological
activation of mGlu II receptors with the specific agonist LY354740 induced
postsynaptically-expressed LTP. This form of plasticity is NMDA receptor-dependent and
involves the phosphorylation through PKC of serine 896 on the GluN1 subunit (GluN1 p-
S896). Both synaptic as well as extrasynaptic NMDA receptors, which are differentially
modulated by mGlu2 and mGlu3, the two mGlu II receptor subtypes, contribute to LTP
induction. LTP initiated by activation of mGlu II receptors was not occluded by LTP induced
with high frequency trains of stimuli (HFS). However, the phosphorylation of NMDA
receptors mediated by mGlu II receptor activation led to a priming effect that enhanced
subsequent HFS-induced LTP. These findings reveal a novel metaplastic mechanism for
the modulation of synaptic function at the SC input to CA1 pyramidal cells.
Chapter 2
35
2.2 Introduction
NMDA receptor dependent LTP is considered the dominant form in the hippocampal CA1
area (Collingridge et al., 1982; Tsien et al., 1996; Lee and Kesner, 2002). Several studies
however have provided evidence that mGlu receptors are also involved in plasticity.
Indeed, ACPD, a non-selective agonist for mGlu I and mGlu II receptors, has been
reported to induce LTP at Schaffer collateral synapses onto CA1 pyramidal cells (Bashir et
al., 1993; Bortolotto et al., 1994). Other studies have reported that the mGlu I receptors
mediate LTD (Palmer et al., 1997; Fritzjohn et al., 1999; Hubert et al., 2000). Whether
mGlu II receptors contribute to synaptic plasticity in the CA1 region has not yet been
investigated.
mGlu II receptors comprising the mGlu2 and the mGlu3 receptor subtype are present
throughout the brain with high expression in the hippocampus (Tamaru et al., 2001).
A well-established role for mGlu II receptors is in the negative feedback regulation of
glutamate release, as described at the mossy fiber-CA3 synapse where their activation at
presynaptic terminals depresses synaptic transmission (Kamiya et al., 1996; Scanziani et
al., 1997) and can induce LTD (Kobayashi et al., 1996; Yokoi at al., 2006). This function
has also been described at CA1 synapses onto subicular neurons (Kintscher et al., 2012),
at the lateral perforant path (Aksoy-Aksel et al., 2015) and at the medial perforant path in
between the entorhinal cortex and CA1 pyramidal cells (Macek et al., 1996; Capogna et al.,
2004). Besides their presence on presynaptic elements, mGlu II receptors also exhibit
postsynaptic localization (Shigemoto et al., 1997). In the prefrontal cortex, activation of
somato-dendritic receptors induces LTD involving a PKC and calcium IP3 - dependent
pathway (Otani et al., 2002). In Golgi cells of the cerebellum (Watanabe et al., 2003), as
well as in hippocampal granule cells (Brunner et al., 2013), postsynaptic mGlu2 receptor
activation induces a dendritic shunt via a G-protein-coupled inwardly rectifying potassium
Chapter 2
36
conductance. In contrast, somato-dendritic mGlu3 receptor activation in the hippocampal
CA3 area induces theta oscillations through an increase of neuronal excitability. This effect
is mediated by an inhibition of potassium conductance and an increase in cationic
conductance, as well as the release of intracellular calcium stores (Ster, 2011).
Furthermore, consistent with a postsynaptic location, activation of mGlu II receptors was
described to transiently potentiate NMDAR currents via a PKA-src-dependent pathway in
acutely isolated CA1 neurons (Trepanier et al., 2013), or via a PKC-dependent mechanism
in dissociated prefrontal cortex pyramidal neurons (Tyszkiewics et al., 2003). Because
calcium influx through activated NMDARs initiates intracellular signaling cascades to
trigger LTP (Lynch et al., 1983; Lüscher et al., 2012), modulation of NMDA receptors
function may contribute in the LTP.
Despite the dearth of information regarding their postsynaptic function in plasticity, it is well
established that mGlu II receptors play a crucial role in the regulation of various forms of
memory. Spatial memory encoding, in the adult rat, was impaired following a treatment
antagonizing mGlu II receptors (Altinbilek et al. 2009). In addition, working memory and
spatial novelty task, relying on the explorative drive of the animal, have been shown to be
impaired in double knockout (GRM2/3/) mice (Lyon et al., 2011).
To clarify the discrepancy the functional role of mGlu II receptors in hippocampal plasticity,
we characterized their physiological role at CA1-Schaffer collaterals using a combination of
selective pharmacological activation and knockout mice.
2.3 Material and methods
Tissue preparation
Acute slices were prepared from juvenile mice (P21-P27) following a protocol approved by
the Veterinary Department of the Canton of Zurich. Mice were decapitated and brains
Chapter 2
37
quickly removed in ice-cold artificial cerebrospinal fluid (ACSF) solution containing the
following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaCHCO3, 1 MgCl2, 2 CaCl2, 10
glucose (pH 7.4) and equilibrated with 95 % O2 and 5 % CO2. 350 µm (for field) or 300 µm
(for patch) thick sagital acute slices were prepared with a vibratome (HM 650V, Microm
International) in the same ice-cold ACSF. Sections were incubated in ASCF for 20 min at
34°C and then kept at room temperature for at least 1 hour before recording.
Electrophysiology
Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices). Acute
slices were transferred to a recording chamber mounted on an upright microscope
(Axioscope FS1; Zeiss). Slices were superfused continuously at 2 ml min-1 with ACSF at
28°C. Single test pulses were delivered to the Schaffer collaterals (stratum radiatum of
CA1) at 0.05 Hz via a monopolar electrode. Input-output curves were generated for each
slice, and the stimulation intensity was adjusted to 40% - 50% of the maximum response.
Following at least 10 min of stable baseline fEPSPs, LTP was induced either by bath-
application of LY354740 (1 µM for 10 min), a specific group II mGlu receptor agonist, or by
three trains of 100 Hz stimulation of 1 sec duration separated by 20 sec (high-frequency
stimulation; HFS). Alternatively, after a 5 min baseline, slices were pretreated with a group
II mGlu receptor antagonist (LY341495, 4 µM), a membrane permeable blocker of G-
proteins (NEM, 100 µM) or a NMDA receptor antagonist (D-AP5, 40 µM) for 10 min before
coapplication with LY354740. Slices were pretreated with a PKA antagonist (H89, 10 µM),
a GluN2A antagonist (NVP-AAMO77, 0.1 µM) or a GluN2B antagonist (Ro25-69813, 3 µM)
for 20 min before coapplication with LY354740, and were pretreated with PKC antagonists
(bisindolylmaleimide II, 1 µM; chelerytherine, 10 µM) or a protein tyrosine kinases inhibitor
(genistein, 30 µM) for 25 min before coapplication with LY354740. The use-dependent
NMDA receptor blocker (MK-801, 40 µM) was bath applied for 20 min while stimulating at
Chapter 2
38
0.05 Hz and then washed out for 10 min before applying LY354740 for 10 min. Evoked
responses were analyzed by measuring the slope of individual fEPSPs. The slopes from
three sequential sweeps were averaged. All slopes were normalized to the average slope
calculated during the predrug period (percentage of baseline).
Whole cell recordings were performed by using the blind patch-clamp method (Blanton
1989). Recordings from CA1 pyramidal neurons were obtained with patch pipettes (5-6
MΩ) filled with intracellular solution containing (mM) 135 K-gluconate, 5 KCl, 10 HEPES, 1
EGTA, 5 phosphocreatine (CrP), 2 MgATP and 0.4 NaGTP (pH 7.2). Membrane potentials
were corrected for junction potentials. EPSCs were recorded at -70 mV and EPSPs were
recorded in current-clamp mode at resting potential. Following at least 5 min of stable
baseline, LTP was induced by bath-application of LY354740 for 10 min and then washed
out for 30 min.
NMDA-mediated EPSCs were recorded at -70 mV with ACSF containing a low magnesium
concentration (0.1 mM) and in presence of an AMPA receptor antagonist (NBQX, 25 µM).
The amplitudes from three sequential sweeps were averaged. Input resistance was either
monitored continuously or calculated by measuring the amplitude of the steady-state
current evoked during a -5 mV voltage step delivered 50 ms before the test stimulation.
Cells were excluded if a change of > 20% occurred during the recording. All data were
analyzed offline using pClamp 9 (Molecular Devices) and are reported as the mean ± SEM.
Differences were considered significant at p < 0.05.
Drugs
D-AP5, NBQX disodium salt, LY341495, Ro 25-6981 maleate salt, MK-801 and genistein
were purchased from Abcam. LY354740, bisindolylmaleimide II, chelerytherine chloride
and H89 dihydrochloride were purchased from Tocris Cookson. NEM was purchased from
Chapter 2
39
Sigma-Aldrich and NVP-AAMO77-NX-6 was a generous gift from Novartis Pharma AG,
Switzerland.
Western blots
Acute slices were prepared as described in the tissue preparation section. Slices were
removed from the recording chamber 10 min after the application of LY354740 in the bath
or 5 min after HFS. The CA1 region was isolated under a binocular dissection microscope.
CA1 tissue was flash frozen in liquid nitrogen and stored at -80 °C before extraction in 50
µl of RIPA buffer (50 mM Tris-HCL pH 8.0, 150 mM NaCl, 0.1 %SDS, 1 % NP-40 and 0.5
% sodium deoxycholate) containing protease inhibitor cocktail (1:200), PMSF (1:500) and
phosphatase inhibitor 2 and 3 (1:200). After homogenization of samples, sonication for 10
min at 4 °C, and centrifugation for 10 min at 13000 G, 4 °C, the supernatant was collected
in Eppendorf tubes. Hippocampal proteins (20 µg) were resolved by electrophoresis on
an8% polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were
then incubated in a blocking solution containing 5% Western blocking solution (Roche) in
TBST 0.05 % (Tris-base 0.1 M, NaCl 1.5 M and 0.05 % Tween 20, pH 8.0) for 2 h at room
temperature and incubated in primary antibodies overnight at 4 °C. The following
antibodies were used; rabbit anti-GluN1 phospho S896 (1:1000, Abcam, ab75680) and
rabbit anti-GAPDH (1:1000, Cell signaling, #2118S). An antibody blocking peptide against
the rabbit anti-GluN1 phospho S896 antibody was used to confirm it specificity (1:100,
Abcam, ab203399). After four washes of 20 min in TBST, membranes were incubated for 1
h at room temperature with a secondary antibody (goat anti-rabbit, IRDye 680nm, 1:10000,
Li-Cor Biosciences). Protein signal and intensity were detected by infrared fluorescence
using an Odyssey IR scanner (Li-Cor Biosciences). Bands were quantified with Image J
software and normalized to GAPDH values.
Chapter 2
40
2.4 Results
Group II mGlu receptor-dependent LTP at SC-CA1
As selective activation of mGlu I receptors in the hippocampus induces LTD (Palmer et al.,
1997; Fitzjohn et al., 1999), but co-activation of mGlu I and II receptors with the non-
selective agonist ACPD induces LTP (Bashir et al., 1993; Bortolotto et al., 1994), we
hypothesized that selective activation of II mGlu receptors would lead to LTP. Indeed, bath
application of LY354740 (1 µM, 10 min), a highly selective agonist of mGlu II receptors,
induced a significant potentiation of the fEPSP (to 138.1 ± 6.4%, p = 0.0004) evoked in
CA1 pyramidal cells by stimulation of the Schaffer collaterals in acute slices from adult
mouse. Confirmation that this form of LTP is initiated by mGlu II receptor activation was
obtained by application of the mGlu II receptor antagonist LY341495 (4 µM) (Schoepp et
al., 1999) bath applied for 20 min before and during LY354740 application, which
prevented LTP (94.3 ± 5.6%, p = 0.55 Fig. 1A).
mGlu II receptors can couple to Gi/o-proteins to decrease activity of adenylyl cyclase
(Niswender and Conn, 2010). We investigated whether perturbing this classical pathway
would inhibit LTP. Blocking G-proteins with bath-applied NEM (100 µM) prevented LTP
(101.1 ± 10.3%, p < 0.001), but the PKA inhibitor (H89, 10 µM) only partially reduced LTP
(112.5 ± 4.6%, p < 0.01; Fig. 1B), suggesting that an additional signaling pathway
contributes to mGlu II receptor-induced LTP. Previous work has shown that stimulation of
mGlu II receptors can also activate tyrosine kinases (Trepanier et al., 2013) and PKC
(Otani et al., 2002; Tyszkiewicz et al., 2004). We found that mGlu II receptor-induced LTP
was not affected by inhibiting tyrosine kinase with genistein (30 µM) (119.5 ± 4%, p > 0.05;
Fig. 1C), but was prevented in the presence of antagonists for PKC (bisindolylmaleimide, 1
µM, 101.8 ± 6.6%, p < 0.001; chelerythrine, 10 µM, 89.5 ± 5.4%, p < 0.0001; Fig. 1B, C).
Chapter 2
41
In the hippocampus mGlu II receptors exhibit both presynaptic as well as postsynaptic
expression (Petralia et al., 1996; Shigemoto et al., 1997; Tamaru et al., 2001). In our
experiments we observed no difference in the paired-pulse ratio recorded under control
conditions (2 ± 0.2) and during stable LTP at 60 min after wash-out of LY354740 (1.9 ±
0.2, P = 0.5363; Fig. 1D), ruling out a presynaptic mechanism of action. We thus conclude
that the mGlu II receptors mediating LTP in CA1 pyramidal cells are localized
postsynaptically.
mGlu II receptor-dependent LTP requires activation of NMDA receptors
In subsequent experiments we noted that pharmacological activation of mGlu II receptors
in the absence of Schaffer collateral stimulation at 0.05 Hz failed to induce LTP (with
synaptic stimulation: 134.1 ± 5.1 ; without: 96.9 ± 9.2%, p = 0.002; Fig. 2A,B). Furthermore,
this form of metabotropic plasticity was dependent on the activation of NMDA receptors, as
bath application of the specific antagonist D-AP5, 10 min before and during LY354740
application prevented LTP (98 ± 3.5, p = 0.0002; Fig. 2A).
The requirement for NMDA receptor activation was also apparent in experiments where the
effectiveness of mGlu II receptor activation on LTP induction was compared in CA1
pyramidal cells recorded either in current-clamp mode at resting potential or in voltage-
clamp mode at -70 mV. In current clamp, Schaffer collateral stimulation at 0.05 Hz evoked
sufficient depolarization to alleviate magnesium block from NMDA receptor channels
allowing LTP (128.8 ± 3%, p = 0.007). In contrast, voltage clamping the soma of CA1
pyramidal cells at -70 mV did not permit the Schaffer collaterals to depolarize the dendritic
NMDA receptors to a potential that was to adequate to induce LTP (99.6 ± 6.8%, p = 0.94;
Fig. 2C,D).
Chapter 2
42
Next we examined whether mGlu II receptor activation modulates NMDA receptor function.
NMDA receptor-mediated EPSCs were evoked in CA1 pyramidal cells voltage-clamped
at -70 mV in the presence of low magnesium (0.1 mM) and NBQX (25 µM) to block
AMPA/kainate receptors. Under these conditions, application of LY354740 (1 µM) resulted
in a significant increase in amplitude of NMDA EPSCs within two minutes of addition to the
bath (126.9 ± 7.2%, p = 0.0074), an effect which was, however, reversed within 2 minutes
of agonist wash out. This enhancement of the NMDA receptor-mediated EPSC was no
longer observed after blocking PKC (bisindolylmaleimide, 1 µM; 96.3 ± 5.1%, p = 0.51),
thus implicating enhanced NMDA receptor function as the mechanism through which mGlu
II receptors initiate LTP (Fig. 2E,F). Application of D-AP5 (40 µM) at the end of experiments
abolished EPSCs, confirming their mediation by NMDA receptors (Fig. 2E,F). A study in
prefrontal cortical neurons has shown that activation of mGlu II receptors increases NMDA
currents through a process involving phosphorylation of the GluN1 NMDA subunit at
Ser896 (Tyszkiewicz et al., 2004). We therefore tested whether a similar phosphorylation of
the GluN1 NMDA subunit in response to mGlu II receptor activation occurs in CA1
pyramidal cells. Indeed, application of LY354740 (1 µM, 10 min) increased phosphorylation
at GluN1 p-S896 in the CA1 region by about 70% (173.3 ± 18.5%, p = 0.02; Fig. 2D) as
compared to control tissue (Fig. 2G,H). This increase in phosphorylation was no longer
significant when mGlu II receptors were activated in presence of a PKC inhibitor (143 ±
15.7%, p = 0.10; bisindolylmaleimide, 1 µM). The efficacy of the antibody was tested by
incubating the nitrocellulose transfer membrane with both the primary antibody as well as a
blocking peptide, confirming its specificity for GluN1 p-S896.
LTP is mediated by both mGlu2 and mGlu3 receptors that differentially
activate synaptic and extrasynaptic NMDA receptors
Chapter 2
43
Postsynaptic metabotropic receptors are localized perisynaptically and extrasynaptically,
whereas NMDA receptors are found within and outside the synapse. We performed
experiments to determine the contribution to mGlu II receptor-mediated LTP of NMDA
receptors containing the GluN2B subunit, which in the hippocampus are primarily
expressed outside of the synapse, and of NMDA receptors containing the GluN2A subunit,
which are preferentially localized within the synapse (Steigerwald et al., 2000; Groc et al.,
2006). Under control conditions a similar reduction in mGlu II-mediated LTP was observed
with blockade of GluN2A subunit-containing NMDA receptors (NVP-AAMO77, 0.1 µM;
113.7 ± 2.8, p= 0.006) as with blockade of GluN2B subunit-containing NMDA receptors
(Ro25-6981, 3 µM; 118.3 ± 3.6, p= 0.013; Fig. 3A,C). However, after selective inhibition of
synaptic NMDA receptors with the use-dependent antagonist MK-801 (40 µM), which
reduced mGlu II-mediated LTP by more than half (118.4 ± 2.7%, p = 0.0107), additional
blockade of GluN2B subunit-containing NMDA receptors abolished LTP (Ro25-6981, 3 µM;
106.4 ± 2%, p = 0.0065; Fig. 3B,C). On the other hand, blocking GluN2A subunit-
containing NMDA receptors after MK-801 treatment, did not lead to a further reduction in
LTP (NVP-AAMO77, 0.1 µM; 116.5 ± 4.6%, p= 0.016). Thus, mGlu II receptors target both
synaptic and extrasynaptic NMDA receptors to trigger LTP.
mGlu II receptors comprise two types, the mGlu2 receptor and the mGlu3 receptor. We
used knockout mice to assess the contribution of these two receptors to LTP. LTP
mediated by mGlu II receptors was significantly reduced in mGlu3 KO mice (121.0 ± 1.7%,
p = 0.026) but not in Glu2 KO mice (136.1 ± 3.7%, p = 0.56) as compared to WT mice
(141.1 ± 6.7% Fig. 3D). However, when both receptors were blocked with LY341495 (4
µM), LTP was prevented. These findings indicate that activation of the mGlu3 receptor
alone can induce maximal LTP but the mGlu2 receptor alone is less effective. It has been
shown in several brain regions that postsynaptic mGlu2 receptors are located at
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44
extrasynaptic sites remote from the postsynaptic density, whereas postsynaptic mGlu3
receptors are expressed both synaptically as well as extrasynaptically (Tamaru et al.,
2001). This distinct distribution pattern suggests that mGlu2 and mGlu3 receptors may
differ in their ability to modulate synaptic versus extrasynaptic pools of NMDA receptors to
induce LTP. We tested this hypothesis by examining the effects of blocking either GluN2A
or GluN2B subunit-containing NMDA receptors in mGlu2 and mGlu3 KO mice. When the
GluN2B subunit was blocked (Ro25-69813, 3 µM), mGlu II receptor-mediated LTP was
reduced but still present in WT (118.3 ± 3.6%, p = 0.013) and mGlu2 receptor KO slices
(122.2 ± 2.8 %, p = 0.0203) but was completely blocked in mGlu3 receptor KO slices
(105.0 ± 2.7 %, p = 0.0008; Fig. 3E, F). When the GluN2A subunit was blocked (NVP-
AAMO77, 0.1 µM), mGlu II receptor-mediated LTP was reduced to a similar extent in WT
(118.9 ± 5.7 %, p = 0.027), in mGlu2 KO (121.1 ± 3.3 %, p= 0.018), and in mGlu3 KO mice
(120.9 ± 1.1 %, p= 0.0247; Fig. 3F). These results are consistent with the depicted
scenario (Fig. 3F, inset), in which the perisynaptically localized mGlu3 receptors can
access both synaptic as well as extrasynaptic NMDA receptors, whereas the more
remotely localized mGlu2 receptors can only modulate extrasynaptic NMDA receptors.
Comparing mGlu II receptor-mediated LTP with high frequency
stimulation-induced LTP (HFS-LTP)
As mGlu II-receptor mediated LTP and classical HFS-LTP in the hippocampus are both
NMDA receptor-dependent forms of plasticity, we investigated whether they share similar
mechanisms. We addressed this question by performing occlusion experiments. After
stable pharmacological induction of mGlu II- induced LTP (138.4 ± 6.6%; LY354540, 1 µM,
10 min), HFS-LTP was induced (3 trains at 100 Hz for 1 sec with an ISI of 20 sec). The
HFS protocol was repeated three times with a 15 min interval to obtain saturating LTP
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(274.8 ± 38.1%, p = 0.04; Fig. 4A). In experiments where the order was reversed, with
HFS-LTP (251.2 ± 28.9%), preceding mGlu II-receptor mediated LTP, a further significant
increase in LTP was again observed (283.3 ± 2%, p = 0.03; Fig. 4B). Moreover, as shown
in Fig. 4E and 4G inhibiting mGlu II receptors didn’t impair HFS-induced LTP (control:
165.4 ± 26.4%; LY341495: 164.2 ± 22.0%, p = 0.97), indicating that HFS doesn’t activate
mGlu II receptors. LFS of 80 pulses at 10 Hz induced an LTP (127.4 ± 5.2%, p = 0.006)
that was inhibited by mGlu II receptors antagonist (101.3 ± 4.6%, p = 0.006; Fig. 4F and
4G). These results indicate that mGlu II – LTP can be induced in physiological conditions,
and show significant differences in the induction process of in mGlu II-receptor mediated
LTP as compared to HFS-LTP even though certain mechanism may be common to both
responses.
Priming of NMDA receptors by mGlu II receptor activation mediates
metaplasticity
Our observation that activation of mGlu II receptors results in a rapid increase in the
amplitude of NMDA EPSCs (Fig. 2E) but a much slower induction of LTP (Figs. 1-4),
suggests a metaplastic response whereby the mGlu II receptor-mediated modulation of
NMDA receptors enhances the generation of synaptic plasticity. We further characterized
this phenomenon by inducing subsaturating LTP with one train of HFS (100 Hz, 1 sec) and
comparing the response with or without prior activation of mGlu II receptors (LY354740,1
µM, 10 min). For these experiments pharmacological activation of mGlu II receptors was
not accompanied by concomitant synaptic stimulation. Results were always compared in
recordings from different slices but from the same animal. Pharmacological priming
resulted in a significant increase in LTP (control: 132.8 ± 5.1%; priming: 165.4 ± 5.5%, p =
0.0014; Fig. 5A). We then checked whether priming differs in WT and mGlu II receptor KO
Chapter 2
46
mice. Priming, expressed as the value of HFS-LTP with LY354740 normalized to the value
of HFS-LTP without LY354740, had the greatest effect in WT (111.5± 9.8%) and in mGlu2
KO mice (86.3 ± 10.1%, p = 0.13), but was significantly reduced in mGlu3 KO mice (53.2 ±
15.5%, p = 0.013; Fig. 5C), which is in accordance with the decrease in LTP observed in
the absence of the mGlu3 receptor (Fig. 3D). To gain insight into the priming mechanism
we examined the phosphorylation status of Ser-896 from the GluN1 subunit of NMDA
receptors during these experiments. Activation of mGlu II receptors alone (LY354740, 1
µM, 10 min) led to almost a doubling of the phosphorylation level of GluN1 Ser896 (198.7 ±
36.3%, p = 0.027; Fig. 5D,E). Thus, mGlu II receptor-mediated phosphorylation does not
require synaptic stimulation, but on its own, that is, without synaptic stimulation, S896
phosphorylation is not sufficient to induce LTP (Fig. 2A). These findings are consistent with
a priming role for mGlu II receptors in which phosphorylation of the NMDA receptor renders
synapses more competent to undergo LTP.
We provide evidence that selective activation of mGlu II receptors induce LTP at CA1
pyramidal neurons by modulating synaptic and extrasynaptic NMDAR via PKC activation,
which has a mechanistically different signaling pathway as the HFS-mediated LTP.
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47
2.5 Discussion
We provide evidence that selective activation of mGlu II receptors induces LTP at Schaffer
collateral synapses onto CA1 pyramidal neurons by modulating synaptic and extrasynaptic
NMDARs via PKC activation. Furthermore, this metabotropically form of LTP differs from
classical HFS-mediated LTP, as these responses do not occlude each other.
Selective activation of mGlu II receptors with LY354740 induced LTP at CA1 - Schaffer
collateral synapses. Paired pulse ratio analysis showed this form of LTP is expressed
postsynaptically, in line with morphological data showing a postsynaptic localization of
mGlu II receptors in CA1 pyramidal cells (Shigemoto et al., 1997).
The principal intracellular transduction pathway utilized by mGlu II receptors involves the G
protein-dependent inhibition of adenylyl cyclase (Niswender and Conn, 2010). Accordingly,
we observed that mGlu II mediated LTP was prevented by blocking G proteins with NEM, a
membrane-permeable inhibitor. Previous studies have demonstrated that activation of
mGlu II receptors can also increase activity of PLC (Klein et al., 1997; Otani et al., 2002)
and PKC in the prefrontal cortex (Otani et al., 2002; Tyszkiewicz et al., 2004), probably via
the βγ complex of the Gi/o protein (Rebecchi and Pentyala, 2000). In line with these data,
inhibition of PKC completely blocked mGlu II–mediated LTP. A similar transduction
mechanism has been described at the parallel fiber – Purkinje cell synapse, where
activation of the mGlu4 receptor inhibits transmission through a Gi,o, and PLC-PKC-
dependent pathway (Abitbol et al., 2012). Nevertheless, in our experiments blockade of the
adenylyl cyclase - PKA pathway partially reduced LTP. Thus, mGlu II receptors appear to
signal both through the inhibition of the canonical adenylyl cyclase-PKA pathway, as well
as through activation of a PLC-IP3-PKC -dependent pathway.
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Recent studies suggest that the main role of the adenylyl cyclase-PKA pathway is to
modulate the functional expression of mGlu II receptors. Flajolet et al., (2003)
demonstrated that protein phosphatase 2C interacts only with a sequence of amino acids
in the C-terminal tail of mGlu3 receptor, in which the Ser-845 can be phosphorylated by
PKA. In addition, phosphorylation at this serine inhibits the interaction with PP2C. Inhibition
of PP2C by PKA-Ser845-phosphorylation may therefore disrupt the interaction between the
mGlu3 receptor and the endocytic machinery to stabilize the receptor in the membrane as
described for other receptors (Chen and Roche, 2007), or may enhance mGlu3 receptor-
triggered signal transduction (Uematsu et al., 2015).
mGlu II receptor-mediated LTP is activity dependent and unlike classical NMDA receptor-
dependent LTP exhibits a slow time course of potentiation. We postulate two mechanisms
to account for this difference involving a slow increase in intracellular calcium
concentration. First, stimulation of mGlu II receptors activates PKC, which increased
phosphorylation at ser-896 of the GluN1 subunit (Figure 2). PKC activation increases the
open probability of NMDA channels and relieves magnesium block (Chen and Huang,
1992), and promotes NMDA receptor trafficking to the synapse (Lan et al., 2001; Groc et
al., 2004), which together will enhance calcium influx. Subsequent synaptic simulation of
NMDA receptors will then lead to a gradual increase in intracellular calcium concentration,
directly through channel influx as well as indirectly through calcium dependent calcium
release from the endoplasmic reticulum (De Camilli et al., 1996; Nishiyama et al., 2000).
Second, activation of mGlu II receptors mobilizes intracellular Ca2+ stores (Ster et al.,
2011) via IP3 transduction (Otani et al., 2002). Both of these processes that increase
intracellular calcium concentration will facilitate the induction of LTP (Raymond and
Redman, 2002; Cavazzini et al., 2005).
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49
Our results show that mGlu II receptor-mediated LTP was significantly decreased in mGlu3
receptor but not in mGlu2 receptor KO mice. This finding is likely to be related to the
differential somato-dendritic localization of these receptors. Expression of mGlu2 receptors
is not observed in the vicinity of the synapse (Luján et al., 1997), such that these receptors
may preferentially modulate an extrasynaptic pool of NMDARs. In contrast, mGlu3
receptors are present both in close proximity to the PSD as well as extrasynaptically
(Tamaru et al., 2001), which would allow these receptors to act also on synaptic NMDARs.
Our experiments validate this scenario as LTP mediated by synaptic GluN2A subunit-
containing NMDARs was induced only after activation of mGlu3 receptors whereas LTP
mediated by extrasynaptic GluN2B subunit-containing NMDARs was observed after
activation of both by mGlu2 and mGlu3 receptors. The distinct spatial organization of mGlu
II receptor subtypes may reflect specific interactions with scaffolding proteins (Pawson and
Scott, 1997; Kim and Sheng, 2004). At present, however, this question remains to be
addressed with little information available except for the interaction of the mGlu3 receptor
with protein phosphatase 2C and PICK1 (Chung et al., 2000; Hirbec et al., 2002).
After applying a high frequency stimulation protocol to induce saturated NMDAR-
dependent LTP, we observed that subsequent mGlu II-mediated LTP was not occluded,
suggesting two separate mechanisms of induction. A common effector in multiple forms of
LTP is calcium, which has several different sources depending on the employed LTP
induction protocol (Raymond, 2007).
The increase in intracellular calcium initiated by mGlu receptors involves the activation of
IP3 receptors (Nakamura et al., 2000). However, as this study utilized the mixed agonist
ACPD, it is not clear which class of mGlu receptor was responsible. A later investigation
showed that in the perirhinal cortex the induction of LTD by mGlu I receptors require co-
activation of mGlu II receptors to allow sufficient release of calcium to induce plasticity
Chapter 2
50
(Cho et al., 2000). Together with our previously published results (Ster et al., 2011), these
findings confirm a role for mGlu II receptors in the release of intracellular IP3-dependent
calcium stores.
Chapter 2
51
2.6 Figures
Figure 1
Chapter 2
52
Figure 2
Chapter 2
53
Figure 3
Chapter 2
54
Figure 4
Chapter 2
55
Figure 5
Chapter 2
56
2.7 Figure legends
Figure 1: Characterization of transduction pathways mediating group II mGlu receptor-
induced LTP at the Schaffer collateral – CA1 pyramidal cell synapse
A, fEPSPs evoked in CA1 pyramidal cells by stimulation of Schaffer collaterals (0.05 Hz)
undergo LTP following activation of group II mGlu receptors with bath-applied LY354740 (1
µM, 10 min, n = 12). LTP is prevented by LY341495 (4 µM, n = 6) a selective group II mGlu
receptor antagonist. The inset shows representative traces (averages of 6 sweeps) before
(1, in gray) and after drug application (2). B, Induction of LTP is blocked by NEM (100 µM),
a membrane permeable blocker of G-proteins, and by bisindolylmaleimide (1 µM), a PKC
antagonist. Inset: representative traces (averages of 6 sweeps) before (1, in gray) and after
drug application (2). C, Quantification of fEPSP slope changes under a variety of
pharmacological conditions. (LY341495: p = 0.0014, n = 6, two-tailed unpaired Student’s t
test). ****P<0.0001, *P<0.05 significant versus control, only treated with LY354740 (LY35)
mice (ANOVA followed by Dunnett’s post hoc test). Statistics: F (5, 33) = 9.311 (LY354740
n = 9; NEM: p = 0.0023, n = 4; bisindolylmaleimide: p = 0.0012, n = 6; chelerytherine: 10
µM, p = 0.0001, n = 6; H89: 10 µM, p = 0.0067, n = 8; genistein: 30 µM, p = 0.05, n = 6. All
antagonists were applied in combination with LY354740. Values represent mean ± SEM.
D, Paired-pulse ratio (PPR) of evoked fEPSPs (interstimulus interval = 50 ms, averages of
6 sweeps) after LTP induction. Calibration bars for all traces: 0.5 mV and 10 ms. E, PPR is
not modified significantly after application of LY354740, consistent with a postsynaptic
mechanism of LTP (p = 0.54, n = 6). Values represent mean ± SEM (two-tailed paired
Student’s t test).
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57
Figure 2: Induction of LTP by group II mGlu receptor activation requires NMDA receptor
activation
A, fEPSPs recorded in the stratum radiatum express LTP only if group II mGlu receptor
activation (LY354740, 1 µM) occurs concomitant with repetitive Schaffer collateral
stimulation (0.05 Hz). LTP is prevented by blocking NMDA receptors (D-AP5, 40 µM).
Insets: representative traces (averages of 6 sweeps) before (1, in gray) and after drug
application (2). B, Quantification of fEPSP slope changes under the conditions presented in
A (no stim: p = 0.0023, n = 6; D-AP5: p = 0.0002, n = 6, two-tailed unpaired Student’s t
test). C, Whole-cell patch-clamp recordings of responses evoked by Schaffer collateral
stimulation show potentiation by activation of group II mGlu receptors during experiments
in current-clamp but not in voltage-clamp at -70 mV, a potential at which NMDA receptors
are blocked by magnesium. Insets: representative averaged traces. Calibration: 10 mV/20
pA and 5 ms. D, Quantification of EPSPs and EPSCs showing LY354740-induced LTP
only in current-clamp mode (I-clamp: p = 0.019, n = 8, V-clamp: p = 0.95, n = 5; two-tailed
paired Student’s t test). E, Pharmacologically isolated NMDA currents (25 µM NBQX)
evoked by Schaffer collateral stimulation at a holding potential of -70 mV with low
extracellular magnesium (0.1 mM) are potentiated in response to activation of group II
mGlu receptors (LY354740, 1 µM). NMDA currents are not potentiated if PKC is blocked
(bisindolylmaleimide, 1 µM). The insets shows representative traces (averages of 6
sweeps) before (1), after drug application (2), and after 40 µM D-AP5 (3) to confirm the
selective activation of NMDA receptors. Calibration: 20ms and 20 pA. F, Quantification of
EPSCs before, during, and after application of LY354740, in control conditions and when
PKC is blocked (control: p = 0.007, n = 8; bisindolylmaleimide: p = 0.51, n = 6, two-tailed
paired Student’s t test). G, Western blots showing phosphorylation at serine 896 of the
Chapter 2
58
GluN1 subunit (GluN1 p-S896) in control conditions (n = 7), after application of 1 µM
LY354740 alone (n = 5), or in combination with 1 µM bisindolylmaleimide (n = 7). The
specificity of the primary antibody against GluN1 p-S896 was confirmed by addition of an
antibody blocking peptide. The protein ladder indicates molecular weight markers (M) in
kDa. H, Protein quantification reveals increased phosphorylation at GluN1 p-S896 after
activation of group II mGlu receptors. This effect is blocked by the PKC antagonist
(LY354740: p = 0.02; bisindolylmaleimide: p = 0.10, two-tailed unpaired Student’s t test).
Figure 3: Characterization of NMDAR subunits that are targeted by group II mGlu receptor
subtypes to induce LTP in CA1 pyramidal cells
A, Blocking either the GluN2A (NVP-AAMO77, 0.1 µM) or the GluN2B NMDAR subunit
(Ro25-69813, 3 µM) significantly diminishes LTP. B, Following bath-application of the use-
dependent blocker MK-801 (40 µM, n = 7) to block selectively the synaptic NMDA
receptors, subsequent block of GluN2A subunits (NVP-AAMO77) has no further effect,
whereas block of GluN2B subunits (Ro25-6981) leads to an additional pronounced
reduction in LTP. C, Quantification of fEPSP slope changes under the conditions presented
in A and B (NVP-AAMO77: p = 0.027, n = 7; Ro25-69813: p = 0.013, n =7; MK-801 and
NVP-AAMO77: p = 0.73, n = 7; MK-801 and Ro25-69813: p = 0.007, n = 6, two-tailed
unpaired Student’s t test). Inset: Proposed scheme showing the two different pools of
NMDARs involved in the induction of LTP, one synaptic including mainly GluN2A subunits
and the other extrasynaptic including mainly GluN2B subunits. D, A comparison of group II
mGlu receptor-induced LTP in wild-type (n = 10), in mGlu2 KO, and in mGlu3 KO mice
shows that only the absence of the mGlu3 receptor reduces LTP. When mGlu2 and mGlu3
receptors are both blocked (LY341495 4 µM) LTP is prevented (n = 3). E, After blockade of
the GluN2B NMDAR subunit (Ro25-69813), LTP was prevented in mGlu3 KO mice, and
Chapter 2
59
reduced in wild-type (n = 8) and mGlu2 KO mice. F, Quantification of fEPSP slope changes
under the conditions presented in D and E as well as after blocking the GluN2A NMDAR
subunit (NVP-AAMO77) in KO mice (mGlu2 and mGlu3), which had no further effect
(mGlu2 KO: p = 0.56, n = 6; mGlu3 KO: p = 0.026, n = 6; mGlu2 KO and NVP-AAMO77: p
= 0.42, n = 8; mGlu3 KO and NVP-AAMO77: p = 0.38, n = 6; mGlu2 KO and Ro25-69813:
p = 0.41, n = 8; mGlu3 KO and Ro25-69813: p = 0.016, n = 6, two-tailed unpaired
Student’s t test). Inset: Working model illustrating how distinct mGlu2 and mGlu3 receptor
localization leads to preferential modulation of different pools of NMDARs, with only the
mGlu3 receptor able to modulate both synaptic and extrasynaptic NMDARs.
Figure 4: Two modes of LTP induction, either by HFS or by group II mGlu receptor
activation, do not mutually occlude each other
A, LTP induced by group II mGlu receptor activation (LY354740, 1 µM) reached a peak
steady-state level after 45 min. At this point HFS (3 trains at 100 Hz for 1 s separated by 20
s) repeated three times leads to a significant further increase in LTP. B, Quantification of
fEPSP slope changes under the conditions presented in A (p = 0.0375, n = 4). Left display:
means for all slices; right display: responses for each slice. C, In reverse order
experiments, HFS was applied three times to achieve saturating LTP (n = 4). Subsequent
pharmacological activation of group II mGlu receptor activation induced a further increase
in LTP. D, Quantification of fEPSP slope changes under the conditions presented in C (p =
0.027, n = 4). Left display: means for all slices; right display: responses for each slice. E,
LTP induction by HFS (3 x 100 Hz, ISI: 20 sec, n = 6) is not impaired by group II mGlu
receptor antagonist (LY341495 (4 µM, n = 4). F, LTP induced by 80 pulses at 10 Hz (n = 5)
is blocked by group II mGlu receptor antagonist (LY341495 (4 µM, n = 4). G, Quantification
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60
of fEPSP slope changes under the conditions presented in E and F (10 Hz, 80 pulses,
control vs LY34: p = 0.009).
Figure 5: Pharmacological priming of group II mGlu receptors enhances HFS-induced LTP
A, LTP induced by high frequency stimulation (HFS, 3 trains at 100 Hz for 1 s separated by
20 s) was potentiated by prior priming of group II mGlu receptors (HFS, without LY354740:
n = 6; HFS + LY354740: 1 µM, n = 7). Insets show representative traces (averages of 6
sweeps) before (1, in gray) and after LTP induction (2). B, Quantification of fEPSP slope
changes under the conditions presented in A (p = 0.0014, two-tailed unpaired Student’s t
test). C, Net increase in LTP following priming with LY354740 in WT (n = 6), in mGlu2 KO
(n = 6), and in mGlu3 KO mice (n = 6). LTP is significantly reduced in mGlu3 KO mice
(53.1 ± 15.4%, p = 0.013) compared to WT (111.5 ± 19%) and mGlu2 KO mice (86.2 ±
8.7%). D, Western blots showing phosphorylation at serine 896 of the GluN1 subunit
(GluN1 p-S896) under different conditions (no stimulation, no LY LY354740: n = 7; no
stimulation + LY354740). The protein ladder shows molecular weight markers (M) in kDa.
E, Protein quantification indicates greater levels of phosphorylation at GluN1 p-S896 when
group II mGlu receptors are activated even in the absence of stimulation. (no stim + LY
LY354740: p = 0.0265, two-tailed unpaired Student’s t test), indicating that both
phosphorylation as well as activation of NMDA receptors are required to initiate plasticity.
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Chapter 3
Activation of mGlu II receptors induces long-term depression at GABAergic
synapses onto CA1 pyramidal cells
3.1 Introduction
CA1 pyramidal cells encode representation of episodic (Quiroga et al., 2005) and spatial
memories (O’Keefe, 1976). In addition to receiving glutamatergic synaptic inputs from
Schaffer collaterals and providing glutamatergic inputs to other cortical areas, CA1
excitatory neurons receive a large diversity of GABAergic interneuron inputs, which provide
inhibition and temporally regulate cell activity (Klausberger and Somogyi, 2008).
A type of parvalbumin interneuron, the basket cells, which belong to local circuits, are of
major interest because of their potential in modulating input to the CA1 area by targeting
somata and proximal dendrites of CA1 pyramidal cells (Klausberger, 2009). Basket cells
are located in or close to the CA1 pyramidal layer, and a single cell is able to innervate
more than 1500 pyramidal neurons (Sik et al., 1995). However, bistratified cells, another
type of interneurons, which show a similar localization as the basket cells, target mainly
dendrites in the stratum radiatum and stratum oriens (Halasy et al., 1996).
Modulation of neuronal activity by cells occurs largely through activation of G-protein-
coupled receptors (Hille, 1992), which exert different actions in cells depending on their
localization. Whereas somatodendritic receptors can modulate synaptic excitability by
altering postsynaptic channel properties, receptors located on the presynaptic terminal can
regulate neurotransmitter release mechanisms (Anwyl, 1999). mGlu II receptors are good
candidates for mediating glutamatergic modulation of CA1 cell excitability because they
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display both presynaptic and postsynaptic expression in the CA1 region. mGlu2 and mGlu3
receptors exhibit somatodendritic localization in CA1 pyramidal neurons (Shigemoto et al.,
1997; Tamaru et al., 2001), where we showed that their activation induces LTP at Schaffer-
collaterals synapses by modulating synaptic and extrasynaptic NMDARs (Rosenberg et al.,
in preparation). mGlu II receptors are not present on presynaptic terminals of CA3
pyramidal cells (Tamaru et al., 2001). Although there are no studies providing direct
anatomical evidence of their presynaptic expression in interneurons, the few
electrophysiological data addressing the role of mGlu receptors in GABAergic transmission
indicate that activation of presynaptic heteroreceptors reduces neurotransmitter release.
For example, activation of mGlu receptors with ACPD (mGlu I and mGlu II agonist) has
been shown to reversibly decrease the amplitude of GABAergic inhibitory postsynaptic
potentials (IPSPs) in CA1 neurons (Desai and Conn, 1991; Desai et al., 1994) via a
presynaptic mechanism (Jouvenceau et al., 1995). This presynaptic inhibitory effect is
consistent with a study showing the same effect in CA3 pyramidal cells, where activation of
mGlus with DCG-IV (a selective mGlu II receptor agonist) induced a reversible reduction of
miniature IPSP frequency (Poncer et al., 1995). Only one study, which was performed in
rabbit, has reported that activation of mGlu receptors with ACPD induces long-term
depression (LTD) in CA1 neurons (Liu et al., 1993).
In addition to these studies, immunoreactivity for NAAG, an endogenous peptide activating
mGlu3 receptors, has been observed in interneurons, thus colocalizing with GABA (Moffett
and Namboodiri, 1995). Furthermore, NAAG in combination with DCG-IV has been shown
to decrease GABA release in cultures of cortical neurons via a mechanism involving cAMP,
PKA and L-type calcium channel dependent (Zhao et al., 2001).
In this study we tested the hypothesis that mGlu II receptors act as heteroreceptors on
interneurons to induce long term inhibition of GABA release, which would represent an
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additional modulatory mechanism affecting CA1 pyramidal cell activity. Our investigation
with mGlu3 KO using LY354740, a highly selective mGlu II agonist, provides evidence that
mGlu3 activation induces LTD of IPSPs recorded in CA1 pyramidal neurons.
3.2 Material and methods
Tissue preparation
Acute slices were prepared from juvenile mice (P21-P27) following a protocol approved by
the Veterinary Department of the Canton of Zurich. Mice were decapitated and brains
quickly removed in ice-cold artificial cerebrospinal fluid (ACSF) solution containing the
following (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaCHCO3, 1 MgCl2, 2 CaCl2, 10
glucose (pH 7.4) and equilibrated with 95 % O2 and 5 % CO2. 350 µm (for field) or 300 µm
(for patch) thick sagittal acute slices were prepared with a vibratome (HM 650V, Microm
International) in the same ice-cold ACSF. Sections were incubated in ASCF for 20 min at
34°C and then kept at room temperature for at least 1 hour before recording.
Electrophysiology
Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices). Acute
slices were transferred to a recording chamber mounted on an upright microscope
(Axioscope FS1; Zeiss). Slices were superfused continuously at 2 ml min-1 with ACSF at
28°C. Single test pulses were delivered at the border of stratum pyramidal close from the
recording electrode (approximately 100 microns from the CA1 cell recorded), at 0.05 Hz via
a monopolar electrode.
Whole cell recordings were performed by using the blind patch-clamp method (Blanton et
al., 1989). Recordings from CA1 pyramidal neurons were obtained with patch pipettes (5-6
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MΩ) filled with intracellular solution containing (mM) 135 K-gluconate, 5 KCl, 10 HEPES, 1
EGTA, 5 phosphocreatine (CrP), 2 MgATP and 0.4 NaGTP (pH 7.2). Membrane potentials
were corrected for junction potentials. IPSCs were recorded at -70 mV in voltage-clamp
mode in presence of an AMPA receptor antagonist (NBQX, 25 µM). Following at least 5
min of stable baseline, LTD was induced by bath-application of LY354740 for 10 min and
then washed out for about 30 min.
The amplitudes from three sequential sweeps were averaged. Input resistance was
calculated by measuring the amplitude of the steady-state current evoked during a -5 mV
voltage step delivered 50 ms before the test stimulation. Cells were excluded if a change of
>20% occurred during the recording. All data were analyzed offline using pClamp 9
(Molecular Devices) and are reported as the mean ± SEM. Statistical comparisons were
made using two-tailed unpaired or paired Student’s t tests. Differences were considered
significant at p < 0.05.
3.3 Results
mGlu II receptor-dependent LTD at GABAergic-CA1 synapses is mediated by the
mGlu3 receptor subtype.
We hypothesize that the reduction of IPSPs induced by ACPD, a non-selective mGlu I and
mGlu II receptor agonist, previously described at CA1 pyramidal cells (Poncer et al., 1995),
is mediated by group II mGlu receptors.
We therefore recorded from CA1 pyramidal cells and positioned the stimulating electrode in
the stratum pyramidal, close to the recorded neuron, in order to stimulate interneurons
innervating the soma and proximal dendrites of CA1 neurons (Sik et al., 1995). Bath
application of LY354740 (1 µM, 10 min), a highly selective agonist for mGlu II receptors,
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induced significant LTD of IPSCs (84.44 ± 2.9%, p = 0.0032) which lasted till the end of the
experiment (77.34 ± 4.5%, p = 0.0043).
We next examined whether this effect was mediated by the mGlu3 receptor as
hypothesized. Indeed, in mGlu3 receptor KO mice, LY354740 no longer induced significant
LTD (104.9 ± 8.1%, p = 0.013 in compared to WT mice).
Figure 1. Induction of LTD at GABAergic-CA1 synapses by group II mGlu receptors activation is mediated by
the mGlu3 receptor subtype. A, Schematic of electrode placement to investigate LTD induction of IPSCs. B,
Whole-cell patch-clamp recordings in CA1 pyramidal neurons show that evoked IPSCs undergo LTD in
response to bath-application of LY35474 (1 µm), a selective agonist of group II mGlu receptors in WT mice
but not in mGlu3 receptor KO mice. Insets: representative averaged traces. Calibration: 100 pA and 10 ms C,
Quantification of IPSC amplitude during control, at 10 min and at 30 min after application of LY354740, in WT
and mGlu3 KO mice (WT: n = 6, mGlu3 KO: n = 5; two-tailed paired and unpaired Student’s t test).
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3.4 Discussion
These results demonstrate that selective activation of mGlu II receptors induces
heterosynaptic LTD of IPSCs evoked in CA1 pyramidal cells.
Comparable with the reported decrease of GABA release mediated by activation of mGlu3
receptor by NAAG in cortical neurons (Zhao et al., 2001), we show that LTD of IPSCs
evoked in CA1 pyramidal cells is mediated by the mGlu3 receptor subtype.
We did not determine the location of the mGlu receptors mediating LTD. However, a
previous study using ACPD to non-selectively activate mGlu receptors concluded that the
responsible mGlu receptors expressed on presynaptic GABAergic terminals (Jouvenceau,
1995). Moreover, Poncer et al. (1996) described a reduction of mIPSC frequency after
selective activation of mGlus II in CA3, consistent with a presynaptic site of action. We
therefore suggest that mGlu3 receptors mediate LTD of GABAergic responses at CA1
pyramidal cells through a presynaptic mechanism.
In future studies it will be interesting to determine the physiological conditions under which
presynaptic mGlu3 receptors are activated to control basal levels of GABA release.
Because of the high affinity of mGlu II receptors for glutamate (Cartmell et al., 2000), one
hypothesis would be that group II mGlu receptors are activated tonically by the ambient
glutamate present in the extracellular space (Baker et al., 2002). Such a process has been
described in the chicken cochlear nucleus where suppression of spontaneous GABA
release is mediated by presynaptic mGlu II receptors activated by ambient glutamate (Tang
et al., 2013). Another potential source of endogenous glutamate for mGlu II receptor
activation could be through spillover from neighboring glutamatergic synapses.
CA1 interneurons expressing parvalbumin have been shown to be required in spatial
working memory (Murray et al., 2011), a faculty impaired in schizophrenia (Glahn et al.,
2003). Moreover, the observation that interneurons are selectively damaged in
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schizophrenia (Zhang and Reynolds, 2002), a disorder frequently associated with mGlu3
receptor impairment (Harrison et al., 2008), suggests that understanding the mechanisms
underlying the regulation of these microcircuits will provide new insights into the
pathophysiology of this mental disorder.
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3.5 References
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Desai M a, 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.
Desai MA, McBain CJ, Kauer J a, Conn PJ (1994) Metabotropic glutamate receptor-induced disinhibitionis mediated by reduced transmission at excitatory synapses onto interneurons and inhibitory synapses onto pyramidal cells. Neurosci Lett 181:78–82.
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Halasy K, Buhl EH, Lörinczi Z, Tamás G, Somogyi P (1996) Synaptic target selectivity and input of GABAergic basket and bistratified interneurons in the CA1 area of the rat hippocampus. Hippocampus 6:306–329.
Harrison PJ, Lyon L, Sartorius LJ, Burnet PWJ, Lane T a (2008) The group II metabotropic glutamate receptor 3 (mGluR3, mGlu3, GRM3): expression, function and involvement in schizophrenia. J Psychopharmacol 22:308–322.
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Jouvenceau a., Dutar P, Billard JM (1995) Presynaptic depression of inhibitory postsynaptic potentials by metabotropic glutamate receptors in rat hippocampal CA1 pyramidal cells. Eur J Pharmacol 281:131–139.
Klausberger T (2009) GABAergic interneurons targeting dendrites of pyramidal cells in the CA1 area of the hippocampus. Eur J Neurosci 30:947–957.
Klausberger T, Somogyi P (2008) Neuronal diversity and temporal dynamics: the unity of hippocampal circuit operations. Science 321:53–57.
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Liu YB, Disterhoft JF, Slater NT (1993) Activation of metabotropic glutamate receptors induces long-term depression of GABAergic inhibition in hippocampus. J Neurophysiol 69:1000–1004.
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Murray AJ, Sauer J-F, Riedel G, McClure C, Ansel L, Cheyne L, Bartos M, Wisden W, Wulff P (2011) Parvalbumin-positive CA1 interneurons are required for spatial working but not for reference memory. Nat Neurosci 14:297–299 Available at: http://dx.doi.org/10.1038/nn.2751.
O’Keefe J (1976) Place units in the hippocampus of the freely moving rat. Exp Neurol 51:78–109.
Poncer JC, Shinozaki H, Miles R (1995) Dual modulation of synaptic inhibition by distinct metabotropic glutamate receptors in the rat hippocampus. J Physiol 485 ( Pt 1:121–134.
Quiroga RQ, Reddy L, Kreiman G, Koch C, Fried I (2005) Invariant visual representation by single neurons in the human brain. Nature 435:1102–1107.
Shigemoto R, Kinoshita a, Wada E, Nomura S, Ohishi H, Takada M, Flor PJ, Neki a, Abe T, Nakanishi S, Mizuno N (1997) Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. J Neurosci 17:7503–7522.
Sik a, Penttonen M, Ylinen a, Buzsáki G (1995) Hippocampal CA1 interneurons: an in vivo intracellular labeling study. J Neurosci 15:6651–6665.
Tamaru Y, Nomura S, Mizuno N, Shigemoto R (2001) Distribution of metabotropic glutamate receptor mGluR3 in the mouse CNS: Differential location relative to pre- and postsynaptic sites. Neuroscience 106:481–503.
Tang Z-Q, Liu Y-W, Shi W, Dinh EH, Hamlet WR, Curry RJ, Lu Y (2013) Activation of synaptic group II metabotropic glutamate receptors induces long-term depression at GABAergic synapses in CNS neurons. J Neurosci 33:15964–15977 Available at: http://www.ncbi.nlm.nih.gov/pubmed/24089501.
Zhang ZJ, Reynolds GP (2002) A selective decrease in the relative density of parvalbumin-immunoreactive neurons in the hippocampus in schizophrenia. Schizophr Res 55:1–10.
Zhao J, Ramadan E, Cappiello M, Wroblewska B, Bzdega T, Neale JH (2001) NAAG inhibits KCl-induced [3H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance. Eur J Neurosci 13:340–346.
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Chapter 4
4. General discussion and perspectives
For my PhD research project, I described a new form of plasticity at CA1-Schaffer collateral
synapses that is initiated by activation of mGlu II receptors. In contrast to previous studies
performed in other hippocampal regions, where dentate granule cells exhibited an
inhibitory response (Brunner et al., 2013) and CA3 pyramidal cells an excitatory response
(Ster et al., 2011) mediated by postsynaptic mGlu II receptors, we show that in CA1
pyramidal cells activation of mGlu II receptors does not alter membrane potential but rather
modulates the properties of synaptic and extrasynaptic NMDARs to induce LTP.
Model. Simplified diagrams of the proposed mechanisms involved in the induction of mGlu II-mediated LTP
in mGlu2 and mGlu3 KO mice. A. In mGlu2 KO mice, activation of mGlu3 receptors could trigger (1) the
upregulation of synaptic NMDAR, (2) the mobilization of intracellular Ca2+ stores, (3) the diffusion of
extrasynaptic NMDARs to the PSD, or (4) the intracellular trafficking of extrasynaptic NMDARs to the
synaptic membrane. Because of the synaptic and extrasynaptic location of mGlu3 receptors, LTP induction
would lead to a normal LTP. B. LTP would be impaired in mGlu3 KO mice because mGlu3 receptors are not
expressed at the perisynapse, thus not allowing synaptic NMDAR modulation.
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mGlu II receptor-mediated LTP could result from various mechanisms. First, as discussed
in Chapter 2, mGlu II receptors located close to the synapse may directly increase the
open time or conductance of synaptic NMDARs to increase Ca2+ influx into the cell and
trigger LTP. Second, extrasynaptic mGlu II receptors could mobilize intracellular Ca2+
stores through a G protein-dependent PLC pathway. Third, extrasynaptic NMDARs could
be recruited to the synapse by lateral diffusion (Groc et al., 2006). Fourth, extrasynaptic
NMDARs could be internalized and trafficked to the synaptic membrane as shown in the
model on the previous page.
However, further investigations will be needed to validate these hypotheses. First, single
channel recording (Neher and Sakmann, 1976) could provide information regarding the
kinetic behavior of NMDARs after the activation of mGlu II receptors. Second, blockade of
sarcoplasmic reticulum Ca2+ -ATPase with cyclopiazonic acid (CPA) could confirm the
involvement of Ca2+ stores in this form of LTP. Third, single-particle tracking (Saxton and
Jacobson, 1997) would allow the visualization of extrasynaptic NMDAR migration to the
synapse when mGlu II receptors are activated. Fourth, extensive studies on mGlu II
receptors-protein interaction, as well as experiments leading to the disruption of the
exocytotic machinery could provide information about a possible role of intracellular
NMDAR trafficking. Finally, activation of mGlu II receptors may trigger a combination of
mechanisms to strengthen the synapse.
mGlu II-mediated LTP was reduced in mGlu3 but not in mGlu2 KO mice. As discussed in
Chapter 2, this form of metabotropic LTP involves a synaptic and extrasynaptic component.
In contrast to the mGlu2 receptor, which is expressed exclusively in extrasynaptic domains,
the mGlu3 receptor is present both extrasynaptically as well as at the perisynapse close to
the postsynaptic density. Thus, synaptic NMDARs are not modulated in neurons lacking
mGlu II receptors in the vicinity of the synapse. As a result, we suggest that the residual
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LTP remaining in mGlu3 KO mice represents an extrasynaptic component of LTP, which
involves extrasynaptic modulation of NMDARs by mGlu2 receptors.
A further mechanism may be related to changes in neuronal excitability. For each
experiment performed with KO animals, an input-output curve was generated to adjust
stimulation intensity to obtain 40% - 50% of the maximum response. Interestingly, we
observed a greater excitability response as indicated by increased slope of the fEPSPs in
mGlu3 KO mice, as compared to mGlu2 KO and WT mice (Figure 6). Thus, this increased
excitability most likely is the result of higher expression of ionotropic receptors in dendritic
spines, but could also be the result of a greater proportion of large spines or an increase in
dendritic branching.
Figure 6. Input-output curves showing increased synaptic transmission in mGlu3 KO mice. The graph shows
f-EPSP slopes as a function of stimulus intensity (mean ± SEM) in the CA1 stratum radiatum for WT (n = 7),
mGlu2 KO (n = 5) and mGlu3 KO (n = 6). Sensitivity to electrical stimulation was similar between mGlu2 KO
and WT mice, while responses were on average higher in mGlu3 KO, with the maximal difference observed
at 50 µA (mGlu3 KO vs WT: 0.13 ± 0.04 mV/ms, p = 0.01; mGlu3 KO vs mGlu2 KO: 0.11 ± 0.04 mV/ms, p =
0.04, two-tailed unpaired Student’s t test).
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Moreover, activation of the mGlu3 receptor, the only mGlu II receptor subtype expressed in
astrocytes (Petralia et al., 1996), has been shown to increase glial glutamate transporter
expression (Aronica et al., 2003). Thus, mGlu3 KO mice may exhibit higher extracellular
levels of glutamate and potentially display a higher level of LTP.
In the chapter 3, we showed that mGlu II-mediated LTD at GABAergic synapses onto CA1
pyramidal cells was mediated by activation of mGlu3 receptors.
Apical tufts of CA1 pyramidal neurons receive inputs from the entorhinal cortex, whereas
basal and proximal dendrites receive mainly inputs from CA3 pyramidal cells (Amaral and
Witter, 1989). As distinct populations of GABAergic interneurons target specific dendritic
domains on pyramidal cells, it would be interesting to examine which class of interneurons
is involved in mGlu3 mediated-LTD to clarify how they influence synaptic integration. On
the basis of the placement of the stimulating electrode, it is likely that mainly interneurons
located in the stratum pyramidal were activated, which are also those receiving inputs from
CA1 pyramidal cells (Knowles and Schwartzkroin, 1981), namely the basket cells (Finch et
al., 1983). However, it is possible that bistratified cells, another type of interneuron located
in stratum pyramidal close to stratum oriens, were also stimulated (Halasy et al., 1996).
Thus, several scenarios regarding the modulation of CA1 pyramidal cell inputs can be
envisaged depending on the class of interneuron involved. First, mGlu3-LTD may be
mediated by bistratified cells. Because their axons connect CA1 dendrites in the stratum
radiatum, the synaptic disinhibition provoked by the reduction of GABAergic transmission
could provide an additional effect to boost mGlu II-mediated LTP at the CA1-Schaffer
collateral synapses. However, further studies blocking inhibitory afferents to stratum
radiatum while inducing LTP should be performed to provide evidence for this hypothesis.
Second, if mGlu3-mediated LTD is mediated by basket cells, which mostly target the
perisomatic area of CA1 neurons, we suggest a function for mGlu3 in theta oscillations
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(Buzsáki, 2002), as was shown in the CA3 area (Ster et al., 2011). Theta activity is
observed during numerous locomotor activities such as spatial exploration (Buzsáki, 2002)
and is generated via an interaction between a phasic excitation powered by the dendrites,
and a phasic shunt of perisomatic inhibition generated by interneurons (Losonczy et al.,
2010). We speculate that a phasic rise of glutamate in the extracellular space, generated
by an overall increase of network activity, could activate mGlu3 receptors located
extrasynaptically on the axon terminals (Tamaru et al., 2001) and phasically reduce GABA
release, which would shift the inhibitory phase and attenuate oscillations providing a
regulatory mechanism to decrease excitability. In line with this idea, genetic and clinical
studies have provided evidence for the involvement of mGlu3 in schizophrenia, in which
abnormal theta oscillations between different brain regions (Ford et al., 2007), as well as
increased theta rhythm in the left temporal lobe has been reported during auditory
hallucinations (Ishii et al., 2000).
Finally, we suggest in the first study that activation of mGlu II receptors may have a priming
effect on NMDARs that facilitates LTP. In addition, mGlu3 KO mice showed impairment in
this form of metaplasticity. Interestingly, it has been demonstrated that exposure to a novel
environment facilitates LTP by reducing its induction threshold, a phenomenon that is
dependent on theta oscillations (Li et al., 2003). Testing of these hypotheses will require in
vivo experiments where mGlu3 receptors could be conditionally knocked down in CA1
pyramidal cells or in different population of interneurons.
The “glutamate hypothesis of schizophrenia” postulates an impairment of NMDAR
regulation as the underlying cause of the pathophysiology of certain forms of schizophrenia
(Gao et al., 2000; Tsai et al., 2002; Hahn, 2000). The finding that mGlu II receptors,
specially of the mGlu 3 subtype, can act as postsynaptic autoreceptors to increase NMDAR
function, and as an heteroreceptor to reduce inhibition, suggests that drugs acting on mGlu
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II receptors may be powerful agents to restore the balance of disrupted glutamate networks
underlying the negative and positive symptoms of the disorder.
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Acknowledgments
First of all, I would like to truly thank Urs Gerber for giving me the immense opportunity of
working in his lab, and for introducing me to the challenging field of electrophysiology. I
also deeply appreciate the availability and knowledgeable advice he always gave me
throughout my PhD, as well as for all of the help and energy he brought during the last
steps of my thesis.
I sincerely thank Uli Zeilhofer for accepting me as his student and being my committee
supervisor. Moreover, I want to thank him for having the door open every time I needed
advice or had questions. In the same vein, I would like to thank Markus Rudin for having
been my initial supervisor in this institution, for the kindness you showed towards me, and
now for being part of my thesis committee.
A special thank you to Jeanne alias Djannette for teaching me the secrets of
electrophysiology, for being tough but fair, for sharing your passion for science, for your
eternal support “tu peux trébucher mais pas tomber!”, for being a second mentor to me but
above all for being a friend. Also, I thank Fede for his electrophysiology tips and for sharing
all of my lab adventures, and Hansjörg, the master of trouble-shooting, without whom it
would have been impossible to accomplish my work.
The biggest thank you is dedicated to all of my lab mates, with whom I shared countless
hours of lab life. Elo, Momo and Anissa, for all the moments spent together on the balcony
rebuilding the world. Koki my PhD survival kit provider. Safa, who thought me how to
“shine”, Greg for his moments of musical procrastination. Megan who calmed me down
during panic attacks. Ali my desk and fruit mate. Laitue my nark. The Spanish crew, Dayra,
Acknowledgments
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Saray, Anahi y Gabi. The Fritjof lab, especially Ladan, Simon, Alex, Maria, Andreas and all
the HIFO people who provided the best work atmosphere ever.
A tremendous thank you to Stefoufou for hours of phone calls and Alex for rescheduling
and sharing meetings... To Coco, Lola and Lilly my distractors. To Danny for his quote
“you will be fine” and his goodies. Thank you for your unconditional support. Also thank
you to Wolfi, Arwen,Quangi, and to Kimoun who, even while far away, remained close. To
Dani and his oversight, Al and Lolo.
Last but not least, I want to thank the people without whom I would not be here: my
parents. Los quiero mucho.
THANK YOU all for your amazing support which helped me to push through in hard
moments and which never failed.
