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GENERALINTRODUCTIONINDEX

General introduction ........................................................................................................ 9 The brain: a very complex puzzle.................................................................................... 9 Brain puzzle pieces: the key players of brain activity ..................................................... 9 Inhibitory pieces: GABA receptors ............................................................................... 10 Excitatory pieces: Glutamatergic receptors ................................................................... 11

AMPA receptors ......................................................................................................... 11 The hippocampus ........................................................................................................... 13 Excitation and inhibition balance .................................................................................. 14 Synaptic plasticity ......................................................................................................... 15 More pieces of the puzzle: Auxiliary proteins............................................................... 18

TARPs .................................................................................................................. 20 Cornichons ........................................................................................................... 21 Shisas ................................................................................................................... 22

Aim of this thesis ........................................................................................................... 23 Bibliography .................................................................................................................. 24

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1GENERALINTRODUCTION

The brain: a very complex puzzle The brain is a fascinating and the most complex organ of the body. It has triggered many scientists to devote their time and energy to reveal its nature and how it functions. It was initially studied by anatomists such as the Spanish Nobel prize winner Ramón y Cajal (1906) and many others who were captivated by the beauty of its nature [1, 2]. Cajal’s drawings depict the complexity of the different brain areas, the variety of cell types, but at the same time, his drawings also showed certain order and structure inside an apparent chaos. The high complexity of this massive puzzle that is the brain underlies its capability of performing a multitude of tasks. At a macroscopic level, the brain puzzle pieces could be the different brain areas; visual cortex, motor cortex, hippocampus, amygdala, prefrontal cortex, thalamus, to name a few. Each brain area has its roles and functions; vision, motor movement, memory, fear, and all the rest of functions that are controlled by the brain. Zooming in at the microscopic level, we find smaller puzzle pieces; the diversity of cell types that exist in the different brain areas such as pyramidal neurons, purkinje neurons, and a variety of interneurons and glia cells, among others. The puzzle gets even more complex when we zoom in at the molecular level and we take into consideration the many different molecules that co-exist in each cell, and even more specifically, in each subcellular region. Synapses are of major interest since they are the sites where cells communicate with each other. Synapses undergo dynamic molecular changes that are responsible for the most relevant property of the brain: its plasticity. In this thesis I took on the challenge of investigating the role of several of these molecular puzzle pieces in neuronal functioning. A century ago, Ramon y Cajal was able to discern many anatomical and cellular details with a rudimentary microscopy. Nowadays, neuroscience techniques allow us to analyze the involvement of the several subunits that make up neurotransmitter receptors, discover the protein-to-protein interactions that occur inside the neurons, their effects on neuronal activity and on even higher level such as behavior, memory and learning. Using these modern techniques, we investigated the involvement of different molecular subunits and auxiliary proteins that make up and affect the communication between brain cells and plasticity in the brain. Brain puzzle pieces: the key players of brain activity Brain activity is tightly regulated by very complex molecular mechanisms. The way the brain generates and processes the information has an electrochemical origin. The neurons communicate with each other by means of neurotransmitters. The release and reception of the neurotransmitters is tightly regulated by intricate protein machineries that regulate neuronal activity. In a broader view, brain activity can be seen as the regulation between excitation and inhibition. There are excitatory neurons that, by definition, release excitatory neurotransmitters, such as glutamate, dopamine,

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acetylcholine, norepinephrine, epinephrine (adrenaline), and serotonin. Similarly, there are inhibitory neurons that release inhibitory transmitters, such as glycine and gamma-aminobutyric acid (GABA), the latter being the most abundant inhibitory neurotransmitter in the brain. In this thesis, I will focus on mechanisms underlying the fast excitatory neurotransmission mediated by AMPA receptors (AMPARs) and the fast inhibitory neurotransmission mediated by GABAA receptors (GABAARs). Inhibitory pieces: GABA receptors GABAARs are ionotropic inhibitory receptors that upon activation allow chloride influx into the cells, which in turn results in hyperpolarization of the adult neurons [3]. GABAA receptors are pentamers composed of a combination of subunits from α1-6, β1-3, γ1-3 and δ [4-7] (figure 1). There is another type of GABARs which are the metabotropic GABABRs, which are involved in intracellular signalling via G-protein coupled receptors linked to potassium channels. In the 80s, Barnard et. al. [8-12] identified the α- and β-subunit of the GABAARs. The binding sites for gamma-aminobutyric acid (GABA) agonists reside on the β-subunits, while the benzodiazepine binding sites reside on the α-subunits. Both subunits are strongly linked allosterically [8]. GABAAR composition varies from brain region and even between neurons within a given region [13-15]. In situ hybridization and immunocytochemical studies revealed that α1 & α2 are widely expressed throughout the brain [16-19] whereas others, like α6, are only expressed in specific populations of neurons, in particular on the cerebellar granule neuron [15, 20] and in particular the subunit α5 is located at extrasynaptic sites [17, 21]. Coexpression of α and β subunits in various combinations reproduced many properties of native GABAARs but not the sensitivity to benzodiazepines. Coexpression of the subunit γ was necessary in order to obtain GABAA-benzodiazepine-sensitive receptors [13, 22]. Moreover, coexpression of γ-subunit variants (γ1, γ 2, γ 3), together with α and β subunits, results in varying degrees of modulation by benzodiazepine receptor ligands [23-27] .

Figure 1. GABA receptor subunit composition and agonists binding sites. Taken from Jacob et. al. 2008 [3] There are pharmacological differences due to subunit composition of the receptors. GABAARs expressing the α1 subunit show higher affinity for GABA than those expressing α2 or α3 subunits [24]. Other pharmacological properties of the receptors

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might also be affected if the subunit composition is altered. An example would be the loss of zolpidem efficacy in the hippocampus from mutant mice lacking the γ2 GABAA subunit [28]. Moreover, receptors which are composed of α3 subunits, in combination with α1 and α2subunits, yield increased responses to benzodiazepines than do receptors that contain only α1 or α2 subunits. The role of the diverse GABAARs may vary depending on their subunit composition. For example, neurons expressing α1- GABAARs have been found to mediate sedation, whereas those expressing α2-GABAA receptors mediate anxiolysis. [17]. The described subunit heterogeneity of GABAA receptors that provides such diversity of function and pharmacology sensitivity has clinical implications. Deficits in the functional expression of GABAARs are critical in epilepsy, anxiety disorders, cognitive deficits, schizophrenia, depression, and substance abuse [3, 29-34]. Many of these diseases are treated with drugs that act on the GABAergic system. Excitatory pieces: Glutamatergic receptors. Glutamate is released from presynaptic neurons and binds to the ionotropic glutamate receptors (iGluRs) and to the metabotropic receptors (mGluRs). There are three types of iGluRs: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), N-methyl-D-aspartate (NMDA) and Kainate (KA) receptors [35, 36]. The metabotropic receptors (mGluRs) have a slower response than the ionotropic receptors and are coupled to GTP-binding proteins (G-proteins) that regulate the production of intracellular messengers [37]. NMDARs are tetrameric ion channels assembled from GluN1-3 subunits with splice variants GluN2A-D, and GluN3A-B [38-40]. NMDARs have a delayed activation that is determined by the coexistence of glutamate binding and a membrane depolarization status that would release the magnesium block that they have by default while at rest. They are permeable to calcium and sodium, contributing to the depolarization of the neuron. Kainate receptors (KARs) are tetramers formed from the subunits GluA5-7, KA1-2. KARs are also permeable to sodium and calcium like AMPAR but they have slower kinetics. Presynaptic KARs modulate GABA release and therefore, have an effect in inhibition. AMPA receptors AMPARs mediate fast excitatory neurotransmission. AMPARs are tetrameric ion channels assembled from GluA1, GluA2, GluA2L (a long splice variant of GluA2), GluA3, GluA4 and GluA4c (short c-tail splice variant of GluA4) subunits (formerly referred as GluR1-4 or GluRA-D) assembled in a dimer of dimer manner [41-43] to form either heteromeric or homomeric receptors. Heteromeric AMPARs typically consist of two GluA2 subunits and either two GluA1, GluA3 or GluA4 subunits. The homomeric receptors are less abundant; they consist of 4 subunits of the same type GluA1, GluA2, GluA3 or GluA4. Great understanding of the structure and assembly of the AMPAR subunits came from the first X-ray structure of a full-length homomeric GluA2 AMPAR [42-45] (figure 2).

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Figure 2. AMPAR structure. A) Topology of iGluR subunit. B) Structure of homomeric GluA2-AMPAR in the closed antagonist bound state. The four GluA2 subunits are colored differently. C) Model of the assembly of the four subunits. D) The ligand binding and transmembrane domanings (LBD-TMD) linkers. Taken from Sobolevsky 2015 [41] .

AMPARs are expressed in both excitatory and inhibitory neurons. The subunit composition of the AMPAR varies between cell types, and brain regions. The most abundant AMPAR in the adult hippocampus are made of GluA1/GluA2 (~80%) and GluA3/GluA2 (~16%) dimers [46-48], whereas GluA4 is present in the immature hippocampus in early postnatal development [49], as well as in other mature brain regions, such as the thalamus and the cerebellum [50-52]. The existence of different AMPARs will then generate channels with different electrophysiological and trafficking properties. AMPAR heterotetramers GluA1/2 and GluA2/3 allow sodium and potassium fluxes but not calcium influx. They are therefore called calcium-impermeable AMPARs (CI-AMPARs). The GluA2 subunit is responsible for preventing the Ca2+ influx [53-55]. Homomeric or heteromeric assemblies of GluA1, A3, or A4, also known as GluA2-lacking AMPARs are Ca2+-permeable (CP-AMPARs), and subject to voltage-dependent block by intracellular polyamines, resulting in a characteristic rectifying current-voltage (I–V) relation [50, 56]. Heteromeric AMPARs containing GluA2 subunits are insensitive to polyamine block and they display a linear I-V profile. Each AMPAR GluA subunit contains a ligand-binding domain (LBD) where agonists such as glutamate bind, an extracellular N-terminal domain (NTD) that modulates receptor assembly, also known as amino-terminal domain (ATD), three transmembrane domains (M1, M3, and M4), one membrane-associated re-entrant loop/hairpin (M2) with a Q/R site that lines the pore, two intracellular loops, and one cytoplasmic domain (CTD) also termed C-terminal tail that can be either short or long. In addition, each subunit undergoes alternative splicing in the extracellular domain to produce the flip(i) and flop(o) variants [41, 42, 44, 57]. The C-terminal tail is involved in the protein to protein interactions that regulate AMPAR properties, phosphorylation and trafficking

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[46]. Long-tailed AMPARs are composed from the GluA1, GluA2L, and GluA4 subunits and short-tailed AMPARs are composed from GluA2, GluA3, and GluA4c. Those AMPARs composed of short C-terminal subunits (GluA2/3) cycle continuously cycling in and out of the synapse, whereas those containing long C-terminal subunits (GluA1/2 and GluA2/4) are delivered to synapses upon synaptic activity [58, 59]. Glutamate and other agonists like AMPA, bind to the AMPAR at the ligand-binding domain (LBD) causing opening of the pore. In addition, there are binding regions at the interface between subunits, within the pore, and in the region between the extracellular domain and the transmembrane segments. Ligands that target these sites can be classified into four categories: (1) competitive agonists and antagonists, (2) positive allosteric modulators, (3) negative allosteric modulators and (4) pore blockers. The kinetics of the AMPARs are determined by the different kinetics and interactions of the ligands. Moreover, it was recently discovered that the kinetics of the AMPARs are also tuned by the different AMPAR auxiliary proteins [60-63] . AMPARs are synthesized in the endoplasmic reticulum (ER) inside the neurons. They travel through the cytosol to reach the synapses where they are needed to mediate fast excitatory transmission. Export of AMPARs from the ER is thought to require the interaction of the cytoplasmic C-terminal domain of the AMPAR subunits with other auxiliary proteins. The GluA2 C-terminus has a PDZ motif that interacts with several PDZ domain-containing proteins, including PICK1 [64, 65], which is thought to be necessary for GluA2 exit from the ER. The GluA1 C-terminus also contains a PDZ domain which interacts with SAP97 [66, 67]. Finally, AMPAR exit from the ER and the acquisition of mature glycosylation at the Golgi complex is assisted by a family of transmembrane AMPAR auxiliary proteins [68, 69]. Further traveling and membrane localization of AMPARs at the synapses is also assisted by a protein machinery [70]. AMPARs get inserted into the membranes by exocytosis and get back into the cytosol by endocytosis where they will be further degraded and recycled [71-74]. AMPARs are very mobile receptors that undergo lateral diffusion to move from extrasynaptic sites to the synaptic sites where they are required to mediate fast excitatory transmission. Imaging studies using fluorescent recovery after photo bleaching and quantum dots technique, revealed that approximately half of the postsynaptic AMPARs were exchangeable with extrasynaptic receptors [75-78]. The availability of AMPAR at the synapse will determine the synaptic strength. Thus, AMPAR status, biophysical properties and AMPA trafficking are key mechanisms for synaptic plasticity. The hippocampus The hippocampus is a brain area responsible for memory formation and spatial navigation. It’s particularly well organized anatomy makes it especially suitable for network experiments. The hippocampus is divided into the following areas: Dentate Gyrus (DG) and the Cornu Ammonis 1, 2 and 3 (CA1,2,3) areas. The hippocampus is connected to multiple brain areas, including several cortices, amygdala and thalamus [79, 80], reflecting it’s great importance. The principal connections within the hippocampus are the mossy fibbers, which connect DG axons with CA3 neurons, the Schaffer collateral fibbers, which connect CA3 axons with CA1 neurons and the perforant path fibers that connect the entorhinal cortex (EC) with the DG neurons. There is a feedback loop hippocampus-EC created with the axons that project from CA1 and CA3 back to the EC.

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Glutamatergic and gabaergic transmission has been extensively studied in the hippocampus. The molecular and functional properties of both ionotropic glutamatergic and gabaergic receptors have been characterized [39, 81-84]. The different neuronal subtypes of the hippocampus have been classified depending on their morphology, molecular contents and neurotransmitter release In particular, the hippocampus CA1 region contains up to 21 subtypes of interneurons and 2-3 types of pyramidal neurons (figure 3) [81].

Figure 3. Neuronal types in the hippocampal CA1 region. (Image taken from Klausberger Science 2008 [81]) The hippocampus has served as a model to study plasticity for decades. Its layered organized anatomy makes it very suitable for in vitro studies where specific cell types and anatomical regions can be easily identified. Moreover, in vitro studies provide the possibility to control pharmacology applications and understand neuronal function. Excitation and inhibition balance When excitation and inhibition are in balance, the overall activity of the brain fluctuates creating electromagnetic waves known as brain oscillations. Brain oscillations reflect a specific synchrony of the underlying neural network activity. Brain oscillations are classified in frequency bands as: delta (δ, 0.1 ~ 3 Hz), theta (θ, 5 ± 3 Hz ), alpha (α, 10 ± 3 Hz), beta (β, 13 ~ 35 Hz), gamma (40 ~ 100 Hz) and fast ripples (100 ~ 200 Hz). Human brain oscillations were described for the first time by Hans Berger in 1924 [85-87]. He described the alpha rhythm as the 10 Hz oscillation observed in the EEG measurements performed on his son while he was sitting quietly with closed eyes. He already suspected that the observed brain oscillations had their origin inside the cellular and molecular machinery of the recorded cortical tissue. In the 1990’s, Salmelin and Hari [88-90] recorded electromagnetic brain signals from healthy humans and characterized the observed alpha (~10 Hz) as well as the beta oscillations

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(13 ~ 35 Hz), which were associated with conscious awake activity and motor function. Higher frequency termed gamma oscillations (40–100 Hz) were firstly studied in the cat visual cortex [91] and were found to be associated with higher cognitive tasks and attention [92-96]. A specific oscillation was found in the hippocampus and entorhinal cortex associated with exploratory behavior and rapid eye movement during sleep [97-99] and defined as the theta rhythm (~5 Hz). AMPA and GABA receptors are both essential for the occurrence of brain oscillations, proved by the fact that blocking any of them resulted in the extinction of the brain network oscillations [100]. Oscillatory activity relies on AMPAR-mediated fast synaptic excitation for both the pyramidal cells and the interneurons [101-103] and specially on the feedback loop involving perisomatic inhibition onto the pyramidal neurons [100, 104-106]. Several hippocampal GABAergic neurons have been identified as key players of specific oscillations (figure 3) [81, 107-111]. Hippocampal interneurons were classified according to their protein expression, their morphology, their localization inside the hippocampus, and their firing correlation to the different oscillatory activity [81, 105, 111-119]. Among all interneurons, a sub-type of interneuron plays a key role in generating and maintaining the specific frequency range of beta oscillations: the perisomatic-targeting interneurons which are mainly parvalbumin positive (PV+). In particular, in the hippocampus, the most abundant PV+ interneuron are the basket cells [100, 110, 120, 121]. Another prominent type of interneurons are the cholecystokinin (CCK+) interneurons which are also involved in oscillatory network activity [122-125]. Not only the different receptors determine the overall neuronal activity, but their particular subunit composition also plays a key role on tuning brain activity. Depending on the subunit composition, different activity patterns and oscillations can take place. For example, if the GABAA receptors lack the β3 subunit, hippocampal theta and gamma oscillations get weaker [126]. On the contrary, higher expression of the GABAergic subunits β2 and β3 resulted in faster synaptic kinetics and faster oscillations [109]. In particular in CA1, PV+ basket cells mainly contain α1 GABAARs, while CCK+ interneurons contain α2/α3 GABAARs [127, 128]. Therefore, altering GABAAR activity by modifying receptor subunits would produce changes in oscillatory activity such as the frequency adaptation alteration seen when the α1-GABAR subunit is missing [129, 130]. To have a proper functioning of the brain, all these brain pieces, i.e. excitatory and inhibitory receptors, must be very well fine-tuned. The importance of keeping a proper balance between excitation and inhibition is clearly shown when such balance is perturbed. Disruption of the excitatory-inhibitory balance might lead to dramatic malfunctioning of the neuronal network. The network and behavioral consequences of a pharmacological or pathological excitation/inhibition misbalance can cause severe alterations. The misbalance might translate into an hyper-excited network, epileptic-like network [131-134]. Therefore, it’s of great importance to discern the role of each tiny brain puzzle piece, cause disruption or alterations in any brain piece could potentially have malfunctioning consequences. Synaptic plasticity

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Synapses are highly specialized areas where neurotransmitters (NTs) are released into the synaptic cleft from the presynaptic neuron by means of an extensive molecular machinery of vesicle exocytosis involving many proteins [135]. At the postsynaptic neuron sites, the NTs are received and bound to the corresponding receptors. The information is thereby transferred from the presynaptic neuron to the postsynaptic neuron by means of electrochemical communication. The postsynaptic neuron will integrate the incoming inputs and act accordingly; i.e. depolarizing and firing specific patterns of action potentials if mainly excitatory inputs were received, or hyperpolarizing and inhibiting the postsynaptic firing if inhibitory inputs govern. Synapses undergo plasticity. They are capable of changing their efficacy and strength by means of altering their molecular contents. In 1949, Donald Hebb proposed a set of rules for synaptic plasticity [136]. Hebbian theory postulated that “when an axon of cell A is near enough to excite a cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A' s efficiency, as one of the cells firing B, is increased” . Or in short: “Cells that fire together, wire together”. Hebb set the basis for what we now call spike-timing-dependent plasticity (STDP). In line with Hebb’s predictions, synapse strength can be modified depending on the millisecond timing of action potential firing and the sign of synaptic plasticity depends on the spike order of presynaptic and postsynaptic neurons [137-140]. Bi and Poo [141] investigated the specific timing requirements between pre- and postsynaptic neurons in STDP. By varying the timing and order of pre and postsynaptic spiking, they found that critical time windows exist for synaptic modification on the order of tens of milliseconds. The relative spike timing between two neurons will determine the direction of the plasticity, either towards potentiation or towards depression [142, 143]. Plasticity is classified depending on the duration of the changes occurred. If plasticity changes last few to several hundreds of milliseconds, plasticity would fall into the category of short-term plasticity (STP), and if plasticity lasts over several dozens of minutes, hours or even days, it would then be considered long term plasticity. In 1973, Bliss and colleagues [144] reported long-lasting activity-dependent synaptic strengthening in hippocampal synapses, defined then as long-term potentiation (LTP). Interestingly, plasticity changes are bidirectional and reversible. The opposite synaptic process can also occur, causing long-term depression (LTD) [145, 146]. Similarly, for the case of short-term plasticity, the paired-pulse-ratio (PPR) quantification will determine the synaptic change: the augmentation of the synaptic strength would then be termed paired-pulse facilitation (PPF) and on the contrary, the decrease in synaptic strength will be reflected as paired-pulse depression (PPD). The frequency of the synaptic activity determines the direction of the plasticity. High frequency stimulation (HFS) induces LTP, while low frequency stimulation (LFS) induces LTD, i.e. 1 Hz during 15 minutes [142, 147, 148]. Typically, HFS, also known as tetanic stimulation, requires activation of the presynaptic fibres at 100 Hz 3-4 times in a minute. Another broadly used LTP induction protocol is the theta burst stimulation which consists of short trains of 5 pulses at 100 Hz, repeated 10 times at 5 Hz (theta frequency) and repeated 3-4 times. Theta stimulation is thought to be more physiological-like than the tetanus stimulation, based on the fact that hippocampal pyramidal neurons fire bursts of action potentials at theta frequency when animals are engaged in learning and exploring the environment [97, 99].

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All these plasticity changes rely on complex molecular machineries involving many membrane receptors, vesicle release proteins, synaptic density proteins, kinases and other molecules such as ions. All of these pieces are required players for the synapse to undergo plasticity. To highlight a few, calcium is a key regulator [149, 150] at both the pre- and post-synaptic sites since it regulates vesicle release as well as the intracellular signaling cascade on the postsynaptic site. The amount of neurotransmitter released determines the degree of synaptic strength presynaptically. On the postsynaptic side, ionotropic NMDARs and AMPARs play a key role in long term plasticity. Their number, availability and functionality determine the degree of synaptic strength postsynaptically [151, 152]. It is well established that NMDARs are essential for LTP induction and maintenance over time [153, 154]. Upon glutamate activation of AMPARs the postsynaptic neuron depolarizes, which in turn will cause the removal of the magnesium block at the NMDARs, and therefore calcium will enter the cells activating multiple signaling cascades intracellularly [151, 152, 155-157]. The amount of AMPARs at the synapses was shown to vary during plasticity: potentiated synapses contained more AMPARs and depressed synapses showed less AMPARs (figure 4) [148, 151, 152]. These studies indicated that AMPARs were crucial in determining long lasting plasticity.

Figure 4. LTD and LTP trigger the removal and insertion, respectively, of AMPAR at the synapses. (Image taken from Fleming & England review 2010 [151]) It is not only the ionotropic receptors themselves, but their subunit composition that is crucial in determining the degree of plasticity at the synapses. For example, the long subunits of AMPARs (GluA1, GluA2-long, GluA4) are inserted into the synaptic sited in an activity-dependent manner [46, 49, 58, 158]. In particular, the GluA1 subunit plays a crucial role in plasticity processes like LTP [159, 160]. In the hippocampus, GluA1 lacking mice showed impaired LTD and LTP related to an impaired insertion of AMPARs-GluA1 in the synapses. Specific mutations at the phosphorylation sites of GluA1 produced impaired LTP and LTD [161-163]. Moreover, these plasticity mechanisms require activation of several protein phosphatases and protein kinases in the downstream signalling. LTP in CA1 depends on phosphorylation of GluA1 [164], Ca2+-calmodulin-dependent protein kinase II (CaMKII) [165, 166] and cAMP-dependent protein kinase (PKA) [155, 167-169]. LTD, on the other hand, is accompanied by a dephosphorylation of GluA1 [164], phosphorylation of GluA2 [65, 170]. Altering AMPAR phosphorylation regulates synaptic transmission by either changing single channel properties or receptor trafficking [58, 171, 172]. It is thought that those intracellular signaling pathways control the insertion of AMPAR into the synaptic sites [152, 173]. Overall, AMPARs have been proven to be crucial for LTP and LTD plasticity processes [174].

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Similarly to glutamatergic synapses, GABAergic synapses undergo plasticity changes [175]. Changes of the GABAAR availability at the synaptic sites as well as changes on the GABAARs’ subunit composition modulate the synaptic strength of GABAergic synapses postsynaptically, whereas the amount of GABA released regulates GABAergic plasticity presynaptically. It has been found that altering α1-GABAAR subunit expression in the brain, alters synaptic plasticity and excitability [176]. Such α1-mediated plasticity has been observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. More pieces of the puzzle: Auxiliary proteins To make the puzzle even more complex, and therefore more exciting and challenging to disentangle, the above described ionotropic glutamate receptors do not exist in isolation in the membrane. It has been found that several membrane receptors are surrounded by a variety of cytoplasmic and transmembrane proteins which interact with several receptors. In particular, the recent discovery that AMPAR are surrounded by a complex network of proteins changed our vision of the synapse into a much more complex one. It is now well established that these auxiliary proteins interact with AMPARs in such a way that they are capable of modulating several biophysical and physiological properties of the AMPARs [61, 72, 177-182]. According to S. Tomita [63], the following criteria have to be fulfill in order to fall into the category of ligand-gated ion channel auxiliary subunit (also termed auxiliary protein): (1) to be a non-pore-forming protein and therefore do not have ion-channel conductance, (2) to have a direct and stable interaction with a pore-forming subunit of the ion channel, (3) to modulate channel properties and/or trafficking in heterologous cells, (4) to be necessary for in vivo native ion channel functioning. Three main families of AMPARs’ auxiliary proteins have been discovered and characterized: transmembrane AMPAR regulatory proteins (TARPs), cornichon proteins and cystine-knot AMPAR auxiliary protein (CKAMP) (figures 5 & 6). The first AMPA auxiliary protein discovered was stargazin (γ2) by Letts et. al. in 1998 [183]. The cerebellar granule cells of the stargazer mouse displayed loss of functional AMPARs at the surface. The mouse that genetically lacked TARP-γ2 suffered from ataxia and epileptic seizures [184, 185]. To date, the following specific AMPA auxiliary proteins have been discovered: stargazin (TARP γ2), TARPs γ3-γ5,γ7-γ8, cornichons 1-4, Shisas 6-7, and Shisa 9 which was initially referred as CKAMP44 (Cys-knot AMPA auxiliary protein with a molecular weight of 44 kDa) [60]. Shisa 4 and 8 are also expressed in the brain and their functionality remains to be studied.

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Figure 5. Schematic structures of a ionotropic glutamate receptor and three transmembrane auxiliary proteins families. Image adapted from Haering et. al 2014 [186]

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Figure 6. Diagram of AMPARs and their interacting proteins. AMPARs interact with several transmembrane proteins as well as with post-synaptic intracellular proteins creating a complex network that modulates AMPAR functioning. Adapted from Choquet et. al. 2010 [71] TARPs TARPs are non-pore-forming integral membrane proteins with four transmembrane domains that interact with AMPARs. The stargazin TARP, also named γ2, has more family members: γ3, γ4, γ5, γ7 and γ8. The proteins γ1 and γ6 are not considered TARPs because they do not modulate AMPAR activity. TARPs are classified into two types depending on their PDZ domain: Type I TARPs (γ2, γ3, γ4, γ8) have a typical PDZ binding domain at the C-terminus, whereas Type II TARPs (γ5 & γ7) have a non-canonical PDZ binding domain at the C-terminus [63, 187, 188]. TARPs are widely expressed in the central nervous system. They are expressed differentially all over the brain, and particularly, γ2 is expressed in every type of neuron [189]. Almost all tissues and cell types in the brain express more than one type I TARP subunit, with the exception of cerebellar granule cells, which express only one type I TARP, γ2, and one type II TARP, γ7 [189]. Most AMPARs in the cerebellum have at least one associated TARP [187]. Since the initial discovery of stargazin protein, it has been shown that TARPs play a role in AMPAR maturation [190, 191], AMPAR trafficking [192-197] to the cell surface, AMPAR clustering and subcellular localization [188, 198, 199]. In particular, type I TARPs modulate both trafficking and functional channel properties of AMPARs [200], whereas Type II TARPs modulate only channel properties of AMPARs [181, 201]. Moreover, TARPs also modulate the pharmacology of AMPARs [200, 202-204]. For example, stargazin γ2 lowers the blocking ability of the quinoxalinedione-based glutamatergic antagonists CNQX and NBQX [202, 205, 206]. With respect to the functional properties of AMPARs, TARPs affect several properties of AMPAR functioning like: AMPARs’ kinetics, i.e. changes in the ESPCs’

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deactivation times, the desensitization times, the recovery from desensitization, and single channel conductance [61, 207-212]. TARPs have been found to be involved in synaptic plasticity [180, 193, 199, 210, 213, 214]. TARPs affect short-term plasticity as well as long term plasticity as it has been been found for TARP γ8. Lack of TARP γ8 resulted in reduced STP and also reduced LTP in the hippocampus [199, 210]. It is then well established that TARPs are indeed AMPAR auxiliary proteins and that they take part in AMPARs’ life cycle and functioning. Cornichons Cornichons are a distinct family of transmembrane AMPAR auxiliary proteins. Mammalian cornichons are homologous to the cornichons found in drosophila and yeast [215]. Cornichons also fulfill the criteria set by S. Tomita [63] to be auxiliary proteins of ionotropic receptors; they are non-pore-forming proteins, they do not have any ion-channel conductance, they have a direct and stable interaction with the AMPAR, and they have been found to modulate the biophysical properties and trafficking of AMPAR [62, 186, 215-218]. Therefore, cornichons are necessary for in vivo native AMPAR functioning. In particular, the isoforms cornichons-2 and cornichons-3, but not the cornichons-1 slow the deactivation and desensitization of AMPAR-mediated currents, independently of the subunit composition of the receptors (GluA2 containing or not). Cornichons increase surface expression of AMPARs [215] Moreover, cornichon-2 and cornichon-3 also affect the functional properties of AMPARs, They enhance the sensitivity of AMPARs to glutamate, they enhance single-channel conductance, they alter channel gating by slowing the deactivation and desensitization times, and they also enhance calcium permeability of CP-AMPARs. Cornichon-2/3 decrease the block by intracellular polyamines of CP- AMPARs [62]. It was initially thought that the functional role of cornichons in neurons was limited to intracellular trafficking of AMPARs from the ER to the Golgi apparatus [216]. It was later found that in hippocampus and in cerebellar neurons, cornichons were also involved in the trafficking of AMPARs to the synapses but in a collaborative manner together with TARP-γ8. Hippocampal AMPAR-mediated synaptic transmission was reduced in the cornichon-2/3 conditional knockout mice. This deficit was thought to be because of the selective loss of surface GluA1-containing AMPARs [218]. Neurons lacking cornichon-2/3 showed faster kinetics, attributed to the loss of GluA1/2 receptors, which deactivate more slowly than the remaining GluA2/3 receptors. A direct role of cornichons in plasticity has not yet been described. Cornichons might affect plasticity indirectly by competing with TARPs on their binding site at AMPARs. It was found that cornichon-2/3 interaction with non-GluA1 subunits was prevented by TARP-γ8 [218]. Thus, the current view of the AMPAR functioning and trafficking includes interactions between the different AMPAR subunits with TARPs, cornichons, and most probably also with the newly characterized Shisa proteins.

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Shisas The third family of AMPAR auxiliary proteins is the Shisa family. The first Shisa to be discovered and characterized was Shisa9, initially named CKAMP44 from Cystine-knot AMPAR auxiliary protein with a molecular weight of 44 kDa [60]. There are currently 9 identified shisa family members, but only the Shisas 4,6-9 are expressed in the brain. In particular, Shisa9 is more abundant in the dentate gyrus (DG) in the hippocampus and in cerebellum, and is also present in the cortex [60]. Shisa6 is specifically expressed in cerebellum and co-expressed in the hippocampus together with Shisa7 [219]. The latter is also broadly expressed through cortical regions and not in cerebellum. Shisa 4 and 8 are also expressed in the brain and its functionality remains to be studied. The characterization of Shisas as AMPAR auxiliary proteins is very recent. To date, three family members (Shisa6, Shisa7 and Shisa9) have been found to fulfill the criteria to be ion-channel auxiliary proteins. . The initial report on Shisa9 (CKAMP44) [60] showed that Shisa9 interacts with GluA1 and GluA2, and that overexpression of Shisa9 lowers AMPAR steady-state currents. Shisa9 also affected the functional properties of AMPARs by increasing receptor desensitization, as well as the recovery from desensitization. Moreover, Shisa9 is also involved in short-term plasticity since overexpression of Shisa9 results in lower paired-pulse ratio from the AMPAR-mediated EPSCs in hippocampal pyramidal CA1 neurons. Very recently, another study came up [210] showing that similarly to cornichons, Shisa9 acts in a collaborative manner with TARP γ8. The coexpression of both auxiliary subunits is necessary for the efficient targeting of AMPARs to the cell surface of DG granule cells. Shisa9 and TARP γ-8 are proposed to be contained in the same AMPAR complex based on electrophysiological and biochemical evidence [210]. With respect to plasticity, it was found that TARP-γ8 but not Shisa9 is required for LTP [210]. Another Shisa family protein, Shisa6, has been very recently characterized as an AMPAR auxiliary protein [219, 220]. Shisa6 has been shown to interact with GluA1-3 receptors and affect AMPAR functioning in several ways: lack of Shisa6 in hippocampal pyramidal CA1 neurons results in faster AMPAR kinetics and it results in impaired short term plasticity. Moreover, Shisa6 protects AMPARs from desensitization. Similarly to Shisa9, Shisa6 interacts with postsynaptic density proteins by its C-terminal PDZ domain, which results in confinement of AMPARs at the postsynaptic sites. The newly discovered Shisa proteins have opened up a new vision of AMPARs functioning and tuning by showing that AMPARs’ functioning depends on a complex network of interacting proteins.

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Aim of this thesis This thesis focuses on specific features of excitatory and inhibitory hippocampal synaptic transmission. In particular, this thesis contains two main topics: 1) the role of the GABAergic α1 and α2 subunits on synaptic transmission and hippocampal network oscillations and 2) the role of the two newly discovered AMPAR auxiliary proteins Shisa7 and Shisa9 in excitatory synaptic plasticity and network activity. The following research questions were addressed:

1. What is the role on synaptic and network activity of the α1 and α2 GABAAR subunits?

2. Do interactions of the C-terminal of the Shisa9 protein affect AMPAR functioning and synaptic properties? Do such interactions affect the fast network oscillations?

3. Is Shisa7 an AMPAR auxiliary protein? Does Shisa7 affect AMPAR functioning? Does Shisa 7 affect the synaptic properties? Does Shisa7 affect glutamatergic plasticity? Does Shisa7 have a role in memory?

To address these questions we used a multidisciplinary approach combining molecular assays, electrophysiological techniques of patch-clamp and multi-electrode arrays for single cell and field potential recordings, respectively, on in vitro mice hippocampal brain preparations. The study was supported by molecular assays and completed with mice behavior paradigms such as fear conditioning in order to test if AMPAR auxiliary proteins have a role in hippocampal memory formation. The combined performed studies gave us a broader overview of the role of the AMPAR auxiliary proteins Shisa9 and Shisa7 as well as the differential role of the GABAergic subunits α1 and α2.

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Chapter2

Alpha2‐containingGABAARsexpressedinhippocampal

regionCA3controlfastnetworkoscillations.Tim S. Heistek, Marta Ruiperez-Alonso, A. Jaap Timmerman, Arjen B. Brussaard and Huibert D. Mansvelder

Dept. Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands.

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