Cell- and input-specific expression of the α5-GABAAR in the CA1 area of the mouse hippocampus

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Cell- and input-specific expression of the α5-GABAAR in

the CA1 area of the mouse hippocampus.

Mémoire

Sona Amalyan

Maitrise en biochimie

Maître ès sciences (M.Sc.)

Québec, Canada

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Résumé

Dans l'hippocampe, les processus de mémoire et d'apprentissage dépendent fortement de l'inhibition GABAergique, qui est fournis par une population hétérogène d'interneurones (INs) via l'activation de sous-types spécifiques de récepteurs GABA. La sous-unité alpha5-GABAAR (α5-GABAAR) est fortement exprimée dans l'hippocampe de la souris, du singe et

du cerveau humain. Il a été rapporté que, dans les cellules pyramidales CA1, cette sous-unité est principalement localisée sur les sites extrasynaptiques, où elle est responsable de la génération de la conductance inhibitrice tonique. Si la sous-unité α5-GABAAR peut être

ciblée sur des types spécifiques de synapses dans des types cellulaires distincts reste inconnue. En utilisant l'immunohistochimie dans des coupes d'hippocampe de souris, nous avons étudié l'expression spécifique de la sous-unité α5-GABAAR dans les cellules et les

synapses de l’oriens/alveus de le région CA1. Nos résultats démontrent que la sous-unité α5-GABAAR est principalement exprimée dans les INs positifs à la somatostatine. De plus, la

densité de sous-unité était plus élevée dans les dendrites proximales et diminuait avec la distance par rapport au soma, ce qui correspond à une diminution de la densité des synapses inhibitrices dépendant de la distance. De plus, l'α5-GABAAR ciblait les synapses formées par

les entrées exprimant le peptide intestinal vasoactif (VIP+) et la calrétinine (CR+) et, dans une moindre mesure, celles produites par les projections exprimant de la parvalbumine (PV+). En résumé, nos résultats montrent que la sous-unité α5-GABAAR présente une

expression spécifique à la cellule et à la synapse dans l'hippocampe CA1. Comme la sous-unité α5-GABAAR a été impliquée dans plusieurs maladies, comprenant la maladie

d'Alzheimer et le syndrome de Down, les nouvelles connaissances sur la localisation de l'α5-GABAAR seront importantes pour le développement de la thérapie cellulaire spécifique.

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Abstract

In the hippocampus, memory and learning processes are highly dependent on the GABAergic inhibition, which is provided by a heterogeneous population of interneurons (INs) via activation of specific sub-types of GABA receptors. The alpha5-GABAAR subunit

(α5-GABAAR) is highly expressed in the hippocampus of the mouse, monkey and human brain.

It has been reported that, in the CA1 pyramidal cells, this subunit is predominantly located at extrasynaptic sites, where it is responsible for generation of tonic inhibitory conductance. Whether the α5-GABAAR subunit can be targeted to specific types of synapses in distinct

cell types remains unknown. Using immunohistochemistry and electophysiological approach in mouse hippocampal slices, we studied the cell- and synapse-specific expression of the α5-GABAAR subunit in the CA1 oriens/alveus INs. Our results demonstrate that the

α5-GABAAR subunit is mainly expressed in the somatostatin-positive INs. In addition, the

subunit density was higher in proximal dendrites and declined with distance from the soma, consistent with a distance-dependent decrease in the density of inhibitory synapses. Furthermore, the α5-GABAAR was targeted to synapses made by the vasoactive intestinal

peptide (VIP+)- and calretinin (CR+)-expressing inputs and to a lesser extent to those made by the parvalbumin-positive (PV+) projections. In summary, our results show that the α5-GABAAR subunit exhibits a cell- and input-specific expression in the CA1 hippocampus. As

the α5-GABAAR subunit has been implicated in several diseases, including Alzheimer’s

disease and Down syndrome, the new insights into the α5-GABAAR localization will be

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List of figures

Figure 1: Synthesis, release and uptake of GABA. ... 2

Figure 2: Neuronal diversity of GABAergic INs in the CA1 area of the hippocampus. 5 Figure 3: Mechanism of GABAergic inhibition. ... 7

Figure 4: Regulation of intracellular chloride by chloride co-transporters. ... 8

Figure 5: Synaptic and extrasynaptic localization of GABARs ... 10

Figure 6: GABAAR structure. ... 12

Figure 7: Trafficking and anchoring mechanisms of GABAAR. ... 15

Figure 8: Rdx and a5-GABAAR subunit coexpression... 16

Figure 9: Distribution of the GABAAR subunits in the brain and their pharmacological action. ... 17

Figure 10: Schematic representation of inhibitory currents. ... 18

Figure 11: Domain-specific distribution of GABAAR subunits. ... 19

Figure 12: The α5-GABAAR subunit distribution in the brain. ... 23

Figure 13: Rdx expression in SOM+ vs PV+ INs in the hippocampal CA1 area. ... 35

Figure 14: The α5-GABAAR subunit expression on the dendrites of mGluR1a+ cells. ... 38

Figure 15: Synaptic and extrasynaptic expression of α5-GABAAR subunit on mGluR1a+ dendrites in the hippocampal CA1 O/A layer. ... 39

Figure 16: Parameters of the LAS AF software automatic colocalization analysis. .... 42

Figure 17: Correlation of the threshold and background with colocalization rate. ... 43

Figure 18: The α5-GABAAR subunit expression at the VIP+, CR+ and PV+ inhibitory inputs. ... 45

Figure 19: The expression of the α5-GABAAR subunit at PV+ IN synapses. ... 46

Figure 20: The α5-GABAAR subunit expression in VIP+/CR+ IS3 cells. ... 47

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List of tables

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List of abbreviations

GABA : gamma-amino butyric acid IN(s) : interneuron(s)

GAD : glutamic acid decarboxylase GABA-T : GABA transaminase SSA : succinic semialdehyde

VGAT : vesicular GABA transporter GAT : GABA transporter

GABAR : GABA receptor

IPSC : inhibitory postsynaptic current IPSP : inhibitory postsynaptic potential CA : cornu ammonis

BGT-1 : betaine GABA transporter 1 DG: dentate gyrus SUB: subiculum EC : entorhinal cortex PC(s) : pyramidal cell(s) str. O : stratum oriens str. A : stratum alveus

str. PYR : stratum pyramidale str. RAD : stratum radiatum

str. LM : stratum lacunosum-moleculare O/A : stratum oriens/alveus

VIP(+) : vasoactive intestinal peptide-positive BC : basket cell

SOM(+) : somatostatin-positive O-LM : oriens lacunosum-moleculare

IS(1, 2, 3) : interneuron specific interneuron(1, 2, 3) CR(+): calretinin-positive

GTP : guanosine triphosphate NKCC : Na+-K+-Cl- co-transporter KCC2 : K+-Cl- co-transporter ER : endoplasmic reticulum PLIC1 : ubiquitin-like protein GA : Golgi apparatus

GABARAP : GABA receptor associated protein NSF : N-ethylmaleimide-sensitive factor

BIG2 : brefeldin-A-inhibited GDP/GTP exchange factor 2 GODZ : Golgi-specific DHHC zinc-finger-domain protein GRIF(s) : GABAAR-interacting factor protein(s)

PRIP(s) : phospholipase-C-related catalytically inactive protein(s) AP2 : clathrin-adaptor protein 2

CCV(s) : clathrin-coated vesicle(s) HAP1 : huntingtin-associated protein 1 Rdx : radixin

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α5-GABAAR : alpha5-GABAAR

mIPSC(s) : miniature IPSC(s) uIPSC(s) : unitary IPSC(s) sIPSC(s) : spontaneous IPSC(s) PV(+) : parvalbumin-positive CCK(+) : cholecystokinin-positive NMDA : N-methyl D-aspartate

EPSP(s) : excitatory postsynaptic potential(s) LTP : long-term potentiation

NAM(s) : negative allosteric modulator(s) MTM : Morris T-maze

ChR2 : Channelrhodopsin BiS : bistratified

DREADD : designer receptors exclusively activated by designer drugs PBS : phosphate buffered saline

PFA : paraformaldehyde

mGluR1a : metabotropic glutamate receptor 1A CNO : clozapine N-oxide

ROI : region of interest ChR2 : Channelrhodopsin-2

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Acknowledgements

I would like to express my deepest gratitude to Dr Lisa Topolnik for supervising me during my Master’s studies. Thank you for being such an incredible mentor. It has been both a pleasure and an honor to learn from the best. You have consistently guided me with encouragement, always provided constructive criticism to improve my skills and made this work environment conducive to learning, but also warm and pleasant all at once. Thank you for your availability and all the opportunities that you have created for me. This project would not be completed if it wasn’t for your daily motivation and scientific inspiration. I am forever grateful to have shared this experience with you. As a result, I understand what I want to pursue, I know who I am and where I want to go from here.

I would like to thank my advisory committee members: Igor Timofeev and Frédéric Bretzner. Thank you for your advice, your suggestions and feedback.

To my lab members who became my friends, thank you for welcoming me with open arms and for sharing all your knowledge. Each of you has had a strong positive impact on my learning experience here. I am thankful to have met such a nice team: Ruggiero Francavilla, Xiao Luo, Olivier Camiré, Étienne Gervais and Émilie Pic. Your help has been tremendously appreciated and you have made this journey one filled with joyful memories that I will never forget.

And last but not least, I would like to thank my supportive, loving family; to my parents in Armenia and my aunt and uncle in Quebec. Thank you for all your encouragements during my studies and above all, thank you for believing in me.

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Avant-propos

Mon mémoire contient un article scientifique rédigé en anglais basé sur les données obtenus dans mon projet et de notre laboratoire. L'article a été soumis en Septembre 2018 dans le Journal of Neuroscience et il est maintenant en révision. Les auteurs de l'article sont présentés dans l'ordre: Elise Magnin*, Ruggiero Francavilla*, Sona Amalyan*, Etienne Gervais, Linda Suzanne David, Xiao Luo, Lisa Topolnik. * Ces auteurs ont également contribué à cette étude. Mon rôle dans cet article était de fournir des informations sur l'expression spécifique de la sous-unité alpha5-GABAAR dans les cellules et au niveau des synapses et d’examiner

les cellules qui peuvent être ciblées en utilisant l'approche pharmacogénétique dans l’hippocampe de la souris. Aussi j’ai fait l'analyse des données des expériences de comportement et l’analyse électrophysiologique.

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1. Introduction

1.1. GABA signaling in the brain

Synaptic transmission in the brain can occur through two major types of synapses: excitatory glutamate and inhibitory gamma-aminobutyric acid (GABA) synapses, which form a dynamic functional partnership. The balance between excitation and inhibition keeps the brain functioning normally. In the neocortex, inhibitory GABAergic neurons (interneurons, INs), which are also known as typically smooth nonpyramidal cells, represent 15-30% of total population of neurons (Fairen et al., 1984). Glutamate and GABA are the major excitatory and inhibitory neurotransmitters, respectively. The pyramidal and spiny stellate cells are excitatory and release glutamate, whereas non-pyramidal INs release GABA and are considered as inhibitory (Houser et al., 1984).

1.1.1. Synthesis and release

GABA is the major inhibitory neurotransmitter in the central and peripheral nervous system. From the molecular point of view, GABA is a non-protein amino acid, which was found to be highly concentrated in the mammalian brain (Roberts & Frankel, 1950). Synthesis of GABA is initiated from glutamine, which is supplied by astrocytes and transported to the neurons through glutamine transporter (Battaglioli & Martin, 1991). The excitatory neurotransmitter glutamate, which is derived from glutamine, is decarboxylated and transformed to GABA via glutamic acid decarboxylase (GAD). After synthesis, GABA can undergo two different processes: either degradation by GABA transaminase (GABA-T), yielding succinic semialdehyde (SSA), or loading in synaptic vesicles by vesicular GABA transporter (VGAT) (Fig. 1) (Rowley et al., 2012). All the GABA-releasing neurons express GAD for the synthesis of GABA.

Release of GABA neurotransmitter can be induced in two ways. The first way is the induction of action potential, which depolarize the presynaptic terminal, cause to opening of

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voltage gated calcium channels and influx of calcium ion. This serves as a signal for the fusion of vesicles with the presynaptic membrane, which leads to the exocytosis of neurotransmitter in the synaptic cleft (Nicholls, 1989; Rowley et al., 2012). The second way is due to the GABA transporter 1 (GAT-1), which is in normal conditions removes GABA from the synaptic cleft into presynaptic site. Under certain conditions, such as high-frequency firing, GAT-1 may dynamically change its direction and release non-vesicular GABA to the synaptic cleft (Rowley

et al., 2012). However, the prevalent mechanism of GABA release in normal conditions

remains the first type via action potential and calcium influx (Jonas & Buzsaki, 2007). Overall, release of GABA is a complex process involving also proteins controlling the calcium-dependent exocytosis (synaptotagmin-1) and vesicle fusion (SNARE complex) (Rizo & Rosenmund, 2008).

Synthesis of GABA is initiated by glutamine, which is synthetized in astrocytes. Glutamine transformation to glutamate is mediated by the glutaminase enzyme. Glutamate leads to synthesis of GABA, which is transported inside the vesicles by VGAT or transformed to SSA. GABA is released from the vesicles and then uptaken by GAT, located on the presynaptic site and on astrocytes. Adapted and modified from Rowley et al., 2012 (Rowley et al., 2012).

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1.1.2. Binding and Inactivation (recycling) of GABA

Once GABA is released from the presynaptic terminals, it binds to pre-, post- and extra-synaptic GABA receptors (GABARs). There are three known types of GABARs: ionotropic, bicuculline-sensitive GABAAR, which is directly activated by GABA binding; metabotropic

GABABR, which is a G-protein coupled receptor and ionotropic GABACR (Barnard et al.,

1998). Binding of GABA to ionotropic GABAAR directly opens its ion channel, which is

permeable to Cl- and bicarbonate. Flow of these ions inside the cell induces inhibitory postsynaptic currents (IPSCs), which typically lead to the hyperpolarization of the postsynaptic cell membrane (but please see Chapter 1.2.2.) (Nicoll et al., 1990). Activation of metabotropic GABABRs is induced by the binding of GABA, which indirectly activates the potassium

channels via G-proteins. GABACRs or GABAA-rho receptors have the same properties of

activation and provide the same inhibition as GABAARs but they are mainly expressed in the

retina, and less in hippocampal cornu ammonis (CA1) area (Enz et al., 1995; Alakuijala et al., 2006).

Neurotransmitter uptake as well as receptor desensitization are two important factors which may control synaptic transmission. Uptake of GABA from the synaptic cleft and extracellular space is performed by four types of GATs (GAT-1, GAT-2, GAT-3 and the betaine GABA transporter 1, BGT-1). Each of these transporters has a specific regional expression in the brain, thus suggesting their different regulation of GABA (Borden, 1996). GATs are activated by the high level of GABA in the synaptic cleft and extracellular space, which leads to the uptake of GABA, therefore decreasing its inhibitory effect. GABA can be transported to the presynaptic vesicles directly or indirectly, through transport via glial cells (Hertz, 1979). Reversed transport of GABA by GATs can have a protective role as a response to several disorders and conditions, such as seizures, during which it will release the GABA from the presynaptic part to the synaptic cleft (Szatkowski et al., 1990).

As the main focus of this project is to study structural properties of the GABAAR in the

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1.2. GABAergic inhibition in the hippocampus

The hippocampal formation with amygdala, septal nuclei, cingulate cortex, entorhinal cortex, perirhinal cortex and parahippocampal cortex compose the limbic system of the brain (David & Pierre, 2009). This structure is located in the medial temporal lobe of each hemisphere of the brain. The hippocampal formation receives a vast amount of sensory information from the different cortical and subcortical structures, such as amygdala, thalamus etc. This structure consists from the hippocampus, dentate gyrus (DG), subiculum (SUB), and the entorhinal cortex (EC) (Squire et al., 2004; Schultz & Engelhardt, 2014). Hippocampus is involved in the initial memory formation, which was shown for the first time on the H.M. patient. He had epileptic seizures, which were resistant to antiepileptic drugs. After removal a part of the medial temporal lobe including hippocampus, seizures were reduced but H.M. patient became amnesic (Squire & Wixted, 2011).

Beside memory, the hippocampus plays an important role in navigation. John O’Keefe discovered that hippocampus is dedicated to spatial memory and allows the animal to navigate in familiar environments (O'Keefe, 1976). Subsequent studies on humans showed that hippocampus encodes a spatiotemporal context-dependent (episodic) memory, which is the ability to encode and store our daily personal experiences.

The hippocampus is composed of three regions: CA1, CA2 and CA3. The CA1 area represents the output region of the hippocampus, where a vast number of GABAergic INs (~21) controls the excitatory glutamatergic pyramidal cells (PCs), thus the flow of information (Fig. 2) (Buzsaki & Chrobak, 1995; Klausberger & Somogyi, 2008). CA1 area contains the principal cellular layer (stratum pyramidale, str. PYR), which is formed mainly by PCs’ bodies and includes different types of GABAergic INs; alveus, axons of pyramidal cells and INs; stratum

oriens (str. O) situated between the alveus and str. PYR, and containing the basal dendrites of

PCs and INs; stratum radiatum (str. RAD) and stratum lacunosum-moleculare (str. LM), containing the proximal and distal dendrites of the PCs and INs) (Spruston & McBain, 2007; David & Pierre, 2009). CA1 area receives five main glutamatergic inputs from the CA3 pyramidal cells, the entorhinal cortical pyramidal cells, the thalamic nucleus reuniens, the CA1 pyramidal cells and the amygdala (Somogyi & Klausberger, 2005). These excitatory inputs are controlled by INs located in the CA1 area, which can be classified based on expressed

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neurochemical markers, morphology and physiological properties (Freund & Buzsaki, 1996; Klausberger & Somogyi, 2008). For instance, vasoactive intestinal peptide-positive (VIP+) basket cells (BCs) make inhibitory synapses at the perisomatic part of PCs; other INs, such as somatostation-positive (SOM+) oriens lacunosum moleculare (O-LM) INs make their synapses on distal dendritic domains. The VIP+ interneuron specific interneurons of type 2 (IS2) and calretinin (CR) co-expressing type 3 (IS3) innervate other inhibitiory neurons, therefore controlling activity of PCs (Fig. 2) (Somogyi & Klausberger, 2005; Klausberger & Somogyi, 2008).

Figure 2: Neuronal diversity of GABAergic INs in the CA1 area of the hippocampus.

Schematic representation of hippocampal CA1 area. Neurons located in this area are characterized by their expressed neurochemical markers, morphology as well as their inhibitory and excitatory inputs. Each of these INs contact different domains of PCs, therefore controlling their spatiotemporal properties of input integration.

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1.2.1. Mechanisms of GABAergic inhibition.

GABAergic inhibition has important role in keeping the balance between the excitatory and inhibitory neurons, controlling input integration and generating rhythmic activity. Term “inhibition” describes the process involving GABA binding to ionotropic or metabotropic GABARs, changing the membrane potential and inducing inhibitory postsynaptic potential (IPSP), which typically hyperpolarize the postsynaptic cell membrane and inhibits the firing of a new action potential and excitation (but please see below). Inhibition mechanism occurring through ionotropic and metabotropic GABARs are different.

Metabotropic GABAB receptor is known to control calcium and potassium conductance

and be activated by spillover of GABA. Binding of GABA to the GABABR, which is coupled

to potassium channels by guanosine triphosphate (GTP)-binding proteins (G proteins), induces the opening of potassium channels and efflux of these ions (Fig. 3) (Gage, 1992; Jonas & Buzsaki, 2007). This brings neuron toward the equilibrium potential of potassium, reduction of action potential frequency and hyperpolarization (Rudolph & Knoflach, 2011; Jembrek & Vlainic, 2015). It is known that activated GABABR also can inhibit presynaptic voltage-gated

calcium currents, which blocks release of the neurotransmitter (Jonas & Buzsaki, 2007). In ionotropic GABAARs, triggered inhibition depends on chloride concentration gradient.

Typically, it is mediated by opening of chloride channels and Cl- influx. If the reversal potential is below the resting potential (~ -70 mV), the evoked inhibition will be hyperpolarizing. When the reversal potential is between the resting membrane potential and the action potential threshold, the inhibition will be “shunting” (Alger & Nicoll, 1979). Finally, during early development, when the reversal potential is above the action potential threshold, GABA triggers excitation instead of inhibition (Ben-Ari et al., 1989; Jonas & Buzsaki, 2007).

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After release of GABA neurotransmitter, it binds to GABAA and GABAB receptors. GABAAR being

an ionotropic receptor after binding opens its chloride channel and allows the flow of Cl-_{ion. Unlike}

GABAAR, GABAB is a metabotropic receptor, which induces inhibitory effect through protein.

G-protein being coupled with potassium channel, leads to its opening and potassium conductance provides suppression of action potential generation.

Adapted and modified from Herbison et al., 2011 (Herbison & Moenter, 2011)

1.2.2. Excitatory effect of GABA during development

and in several pathologies.

Chloride influx is the important determinant for the inhibition as its intracellular level determines influx or efflux of the anion and, consequently, inhibition or excitation processes. In normal conditions, the Cl−_{reversal potential is close to the membrane resting potential (Vm} = ~ -70 mV) and GABA generates hyperpolarization. The inhibitory properties of GABARs result from the fact that, the relatively small driving force for generating Cl− current (I) reduces the input resistance (R) of the neuron. Cl-_{influx produces a change in membrane voltage by}

decreasing the membrane potential, as described by Ohm's Law (Vm = I × R). These changes can be independent of the direction of the Cl−_{current, even though it may be in the depolarizing} direction (Smith, 2013).

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During brain maturation, intracellular chloride level is high, thus opening of chloride channels leads to the efflux of the Cl- from the cell, therefore increases membrane depolarization (Cherubini et al., 1991). Numerous studies showed that intracellular regulation of chloride ions is due to the chloride co-transporters, such as Na-K-Cl (NKCC) and K-Cl (KCC2), which are responsible for Cl-_{influx and efflux respectively. These transporters affect}

GABA signaling by the neuronal chloride homeostasis, which is the maintenance of the stable state and the equilibrium of the ion in the neuron. Before and immediately after birth (the first postnatal week) the level of KCC2 expression is low, which causes to the Cl-_accumulation

inside the cell (Ben-Ari et al., 1989). Up-regulation of KCC2 reduces the level of intracellular Cl- ions, which occurs later in development (Rivera et al., 1999; Yamada et al., 2004). During different disorders, such as epilepsy and autism, there can be inhibitory to excitatory shift in GABA (Fig. 4) (Khalilov et al., 2003; Dzhala et al., 2005; Ben-Ari et al., 2012; Cellot & Cherubini, 2014).

In immature neurons (the first postnatal week) GABA neurotransmitter has an excitatory effect (Ben-Ari et al., 1989). This is a result of the high chloride level inside the cell, where NKCC1 is upregulated and KCC2 is downregulated (left). Therefore, the opening of chloride channels leads to the efflux of chloride ions and induction of depolarization. During development (the second postnatal week) the level of NKCC1 is downregulated and KCC2 is upregulated (right), thus the chloride level in the cell is low and GABA binding to GABAAR will lead to the chloride influx, hyperpolarization, inhibition.

Excitatory effect of GABA can occur because of the several disorders and diseases. Adapted and modified from Watanabe et al., 2014 (Watanabe et al., 2014).

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1.2.3. Phasic and tonic inhibition

GABAR-mediated inhibitory conductance depends from the receptor location and also its subunit compositions, therefore one can differentiate two types of inhibition: phasic (wiring transmission) and tonic (volume transmission) (Agnati et al., 1995; Mody, 2001; Farrant & Nusser, 2005). Phasic inhibition occurs due to synaptically localized GABARs, which directly bind high millimolar concentration of released GABA from the presynaptic vesicles and induce fast (less than 100-millisecond), high-amplitude and short-lasting IPSCs (Davis, 2002; Farrant & Nusser, 2005). Fast IPSCs were found to occur via somatic and proximal dendritic synapses (Freund & Buzsaki, 1996). They control the frequency of action potentials and inhibit spike initiation (Farrant & Nusser, 2005). The subunit combination of GABAARs is important

determinant for surface localization (synaptic and/or extrasynaptic) and mediated inhibition. The α1, α2, α3 subunits containing GABAARs are mainly located at synapses and mediate

phasic inhibition. In the hippocampus, the role of phasic inhibition is very important in generation of rhythmic and synchronized activity in neuronal networks, such as inhibition provided from BCs to large population of PCs, which generates theta and gamma frequency network oscillations (Cobb et al., 1995; Somogyi & Klausberger, 2005). The same role of phasic inhibition was found in other brain regions, such as olfactory bulb (Laurent, 2002). Spillover of GABA activates extrasynaptically located GABAARs, such as α5, α4, α6,

γ or δ subunit containing receptors, which have high affinity to GABA and result in slow (more than 100-millisecond) long-lasting inhibition (Fig. 5) (Davis, 2002; Caraiscos et al., 2004; Glykys et al., 2008; Brady & Jacob, 2015). Tonic inhibition is thought to be continually present and provide low-amplitude inhibitory conductance, which influence on input resistance, synaptic efficacy and input integration (Farrant & Nusser, 2005; Scimemi et al., 2005; Osten

et al., 2007). Summarizing GABAergic inhibition in the hippocampus, in the next chapter

will be discussed the structure and functional role of the GABAAR.

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GABARs can provide kinetically different inhibition depending on their location and subunit composition. Phasic inhibition occurs through the GABAARs, which are located at the synapses,

whereas extrasynaptically located GABAAR has higher affinity, detects less GABA and mediates tonic

inhibition.

Adapted and modified from Jacob et al., 2008 (Jacob et al., 2008).

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1.3. Structure and function of GABA

A

R

1.3.1. GABA

A

R structure

GABAA ionotropic receptor is a heteropentameric protein composed of five subunits,

most frequently two α, two β, and one γ subunit (Prenosil et al., 2006; Olsen & Sieghart, 2009). It is encoded by 19 genes and different subunit isoforms (α1–6, β1–3, γ1–3, δ, ε, θ, π, ρ1–3) give rise to a considerable diversity of GABAARs (Fig. 6) (Baumann et al., 2001; Simon et al.,

2004). These receptors are differentially expressed in the brain, localized in different cell types and subcellular areas. For example, the α1 has highest expression in the lateral nucleus of the amygdala, whereas the α3 subunit in intercalated nuclei and the γ2 is highly expressed in cortical nuclei. The expression of the GABAAR δ subunit, was found to be high in the

cerebellum, thalamus and olfactory bulb, whereas the α5 subunit had prominent expression in the hippocampus, amygdala, cortical layer V and olfactory bulb (Fritschy & Mohler, 1995; McKernan & Whiting, 1996; Pirker et al., 2000a; Sieghart & Sperk, 2002a; Stefanits et al., 2018). The kinetic properties of IPSC are determined by different combinations of the GABAAR subunits. For example α1, α4 and α6 subunits containing GABAARs are associated

with a fast current, whereas receptors containing δ, α3 and α5 subunits give rise to slow current (Caraiscos et al., 2004; Osten et al., 2007; Glykys et al., 2008; Jacob et al., 2008). In addition, many GABAAR subunits are known to be sensitive to benzodiazepines including α1, α2, α5

and others (Rudolph & Möhler, 2006; Mohler, 2015). Also diazepam binds to the α1, α2, α3 and α5 subunit containing GABAARs and leads to therapeutically different effects, such as: α2

containing GABAARs have been found to mediate the anxiolytic-like action of diazepam, the

α1-containing GABAARs mediate sedative action, and, finally, the α5-containing GABAARs

inhibition by benzodiazepines enhances memory (Navarro et al., 2002b; Rudolph & Möhler, 2006; Rudolph & Knoflach, 2011).

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The inhibitory GABAAR consists of five subunits (two α, two β, and one γ subunits) that

together form a ligand-gated chloride channel. After GABA binding to the GABAAR, chloride

influx results in the hyperpolarization of the postsynaptic cell membrane. The GABAAR

subunits are also binding sites for psychoactive drugs, such as benzodiazepines. Adapted and modified from Jacob et al., 2008 (Jacob et al., 2008).

1.3.2. Scaffolding and membrane localization

The density and localization of GABAARs is important key mechanism in synaptic

strength, learning and memory processes (Kennedy & Ehlers, 2006; Hausrat et al., 2015). Concentration of receptors is regulated by two trafficking modes: first, receptor trafficking between cytosolic compartments and plasma membrane and the second, exchange of cell surface receptors between synaptic and extrasynaptic domains. These activity-dependent processes involving signaling cascades, include receptor insertion and internalization processes.

The synthesis of GABAAR subunits starts in the endoplasmic reticulum (ER) and it is

regulated by the ubiquitylation with subsequent ER-associated degradation by proteasome

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(Fig. 7B) (Yi & Ehlers, 2007). Ubiquitylated GABAAR can be modulated by ubiquitin-like

protein PLIC1, which interaction prevents GABAAR degradation and facilitates receptor

accumulation at the synapses (Fig. 7B) (Bedford et al., 2001). The trafficking of these receptors into the Golgi apparatus (GA) and to the plasma membrane involves guiding proteins, such as GABAAR-associated protein (GABARAP, binds to the γ2 subunit),

N-ethylmaleimide-sensitive factor (NSF) and brefeldin-A-inhibited GDP/GTP exchange factor 2 (BIG2, bind to the β subunit) (Fig. 7B). In the GA palmitoylation of γ subunits via the palmitoyltransferase Golgi-specific DHHC zinc-finger-domain protein (GODZ), presents a crucial step in the delivery of the receptor to the plasma membrane (Keller et al., 2004). Several other proteins, such as GABAAR-interacting factor proteins (GRIFs) and phospholipase-C-related

catalytically inactive proteins (PRIPs) also play an important role in the trafficking of GABAARs (Beck et al., 2002; Jacob et al., 2008).

The main mechanism of GABAAR internalization is the clathrin-dependent

endocytosis, which requires interaction of the intracellular loops of the GABAAR β and γ

subunits with the clathrin-adaptor protein 2 (AP2) complex (Fig. 7C) (Kittler et al., 2000). β or γ2 containing GABAARs binding to the AP2 µ2 subunit, can be inhibited by the

phosphorylation of certain residues in GABAAR, which will increase the receptor

concentration on the cell-surface and enhance the efficacy of inhibitory synaptic transmission (Fig. 7C). After the endocytosis via dynamin (enzyme responsible for the scission of newly formed vesicles from the membrane), the recycled receptors from the clathrin-coated vesicles (CCVs), fuse with endosomes. These GABAARs either interact with huntingtin-associated

protein 1 (HAP1) and are recycled to the plasma membrane or undergo degradation in lysosomes (Fig. 7C) (Kittler et al., 2000).

The exchange of cell surface receptors between synaptic and extrasynaptic domains is occurred from the stable binding of receptors to the scaffolding proteins (Triller & Choquet, 2005). Gephyrin is a post-synaptic scaffolding protein interacting with the membrane receptors and cytoskeleton, which anchors inhibitory glycine and GABAARs (Fig. 7A) (Kneussel et al.,

1999). Interaction between gephyrin and GABAARs involves also GABARAP,

phosphatidylinositol (3, 4, 5) phosphates-anchored proteins. These proteins are important for the accurate targeting of gephyrin to the membrane and receptor (Kneussel & Betz, 2000; Kennedy & Ehlers, 2006). Gephyrin was found to anchor several GABAAR subunits, such as

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α1, α2, α3, β2 and β3 (Fig. 7A) (Tretter et al., 2012; Kowalczyk et al., 2013). In addition, there is a gephyrin-independent GABAAR clustering protein, called radixin (Rdx). Rdx is

actin-binding protein from ezrin/radixin/moezin (ERM)-family, which is highly specific to the alpha5-GABAAR subunit (α5-GABAAR) subunit (Fig. 8) (Loebrich et al., 2006). Rdx

anchoring of the α5-GABAAR subunit at extrasynaptic sites required Rdx phosphorylation,

whereas dephosphorylation of Rdx leads to the synaptic distribution of the α5 subunit-containing GABAARs (Loebrich et al., 2006; Hausrat et al., 2015).

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A: GABAAR trafficking from the extrasynaptic site to the synapses. Interaction of post-synaptic

scaffolding protein gephyrin with GABAAR involves different types of proteins and downstream

signaling for the accurate anchoring of GABAARs to the cytoskeleton. Gephyrin-independent anchoring

of the α5-GABAAR subunit to the membrane through Rdx. B: Synthesis and trafficking of GABAAR to

the plasma membrane. Synthesis of GABAAR subunits starts in the ER, subsequent ubiquitylation, Figure 7: Trafficking and anchoring mechanisms of GABAAR.

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binding of PLIC1, which prevents degradation of GABAAR by proteasome. Trafficking of GABAAR

from Golgi apparatus to the plasma membrane involves several important proteins, such as GABARAP, NSF, BIG2, GODZ, PRIP and GRIF. C: Internalization, recycling and degradation of GABAARs.

Adapted and modified from Jacob et al., 2008 (Jacob et al., 2008)

A1: Neuronal expression of Radixin-GFP and myc-α5-GABAAR (A1: scale bar, 15µm).

Radixin/α5-GABAAR clusters localized in VIAAT expressing inhibitory synapses (A2, A3:Scale bar 4µm).

Adapted and modified from Loebrich et al., 2006 (Loebrich et al., 2006)

1.3.3. GABA

A

R function and properties

GABAAR depending on its subunit combination displays different regional, cellular

and subcellular expression patters. GABAAR subtypes determine temporal dynamics at

heterogenous GABAergic INs, which control the spiking and neural oscillations of principal cells, as well as dendritic inhibition. Although theoretically there can be a huge diversity of GABAAR subunit combinations, it was shown that only some of them are present in the brain

(McKernan & Whiting, 1996; Olsen & Sieghart, 2009). For instance, the most abundant GABAAR subtype is α1β2γ2, which is present in most brain regions; the α2β3γ2 GABAAR is

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mainly expressed in olfactory bulb, DG, hypothalamus; whereas the α5β3γ2 subtype has prominent expression in CA1 PCs (Caraiscos et al., 2004; Uusi-Oukari & Korpi, 2010) .

The GABAARs display distinct pharmacological features, such as sedative, anxiolytic

and memory enhancing effect (Fig. 9) (Mohler, 2015). In addition, their location at level of synaptic and extrasynaptic sites is a key determinant for GABAAR-mediated inhibitory kinetics

and function (Banks et al., 1998). The IPSCs can further be subdivided into GABAA, fast and

GABAA, slow IPSCs based on kinetics and amplitude (Pearce, 1993). For instance, α1 and α4

subunits are mostly located at the synapses and GABAARs containing these subunits mediate

fast current, whereas δ, α3, α6 and α5-GABAAR subunits containing receptors are mainly

located extrasynaptically, therefore mediating slow IPSCs (Fig. 10) (Gingrich et al., 1995; Caraiscos et al., 2004; Barberis et al., 2007; Glykys et al., 2008). Some of GABAAR subunits

can provide both slow and fast kinetics, such as the α6 and/or α5-GABAAR subunit. The latter

was found mainly expressed extrasynaptically on hippocampal CA1 PCs mediating tonic inhibition (Caraiscos et al., 2004; Prenosil et al., 2006). However, its synapse-specific expression in the CA1 INs remains unknown.

The figure demonstrates benzodiazepine-sensitive GABAAR α subunits (α1, α2, α3, α5) with diverse

distribution in the brain and their distinct pharmacological effects derived from their location. Adapted and modified from Mӧhler, 2015 (Mohler, 2015).

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The schematic illustration of the inhibitory transmission. Depending on whether the α1 (synaptic) or the α6 (extrasynaptic) receptors are activated, the occurred IPSCs will be fast (direct) or slow (indirect), respectively. Depending on the combinations miniature (mIPSCs), spontaneous (sIPSCs), and evoked (eIPSCs) IPSCs are generated. The fast direct IPSC is mediated by α1 containing receptors (yellow trace and arrows). The slow indirect IPSC is mediated by α6 containing receptors (blue trace and arrows). The resultant eIPSC (green trace) is the sum of the slow and fast currents. The scale bar is 10 pA and 100 ms for eIPSC, 10 ms for sIPSC, and 1.5 ms for mIPSC.

Adapted and modified from Mapelli et al., 2014 (Mapelli et al., 2014).

1.3.4. Synapse-specific distribution

GABAergic INs made their synapses on PCs and other INs in a domain-specific manner. For instance, BCs make synapses at the perisomatic part of PCs; axo-axonic cells innervate axon initial segments; whereas other INs, such as VIP+, SOM+ O-LM INs make their synapses on proximal or on distal dendritic domains of PCs (Somogyi & Klausberger, 2005; Klausberger & Somogyi, 2008). GABAARs located in these synapses can mediate

kinetically different IPSCs based on subunit composition. Previous studies showed that certain subunits have synapse-specific expression, which defines inhibitory properties for the cell (Prenosil et al., 2006). GABAARs containing α1-3 subunits primarily localize at inhibitory

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synapses, which sense GABA transmitter released from presynaptic terminals to generate fast inhibitory synaptic responses (Jacob et al., 2008). More specifically, α1-GABAAR subunit was

found to be mainly expressed at synapses formed by parvalbumin-positive (PV+) INs (Fig. 11) (Nusser et al., 1996; Nyiri et al., 2001). Expression of the α2-subunit containing GABAAR was

found at the synapses of cholecystokinin-positive (CCK+)/VIP+ BCs and from chandelier neurons (Nyiri et al., 2001). GABAARs are also densely expressed as well as at extrasynaptic

or perisynaptic sites, which were found to contain the α5-GABAAR or δ subunits (Fig. 11)

(Möhler & Rudolph, 2017). The subunits, which are located extrasynaptically and mediate tonic currents, showed higher affinity for GABA and are able to bind even low concentration (1 µM) of ambient GABA, than those clustering at inhibitory synapses (Mtchedlishvili & Kapur, 2006; Jacob et al., 2008).

The schematic demonstration of GABAAR subunits domain-specific expression on PCs. The α1

subunit-containing GABAARs were found at the synapses formed by PV+ INs, whereas α2-containing

GABAARs at CCK+ synapses. The α5-GABAAR subunit extrasynaptic expression is demonstrated on

PCs.

Adapted from Mohler, 2012 (Mohler, 2012)

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1.3.5. Role of the GABA

A

R in synaptic plasticity and

network oscillations.

Hippocampus is playing a critical role in the episodic memory formation and in the activity-dependent synaptic plasticity (Neves et al., 2008). Activity-dependent synaptic plasticity was found to be the key mechanism underlying the memory formation. It is expressed through post- and presynaptic changes, which alters the efficacy of neurotransmitter release and biophysical properties of postsynaptic receptors (Bear & Malenka, 1994; Choquet & Triller, 2013). Previous studies showed that synaptically released GABA saturate GABAARs,

therefore the strength of synapses is changed due to postsynaptic GABAARs (Otis et al., 1994).

The number of GABAAR in the postsynaptic membrane and its subunit composition have a

crucial role in the efficacy of GABAergic synaptic transmission (Luscher et al., 2011). In this case, two trafficking modes can alter the efficacy of transmission, such as trafficking of GABAARs between intracellular compartments and membrane by altered receptor recycling,

either GABAAR diffusion between extrasynaptic and synaptic sites.

GABAAR-mediated slow inhibition was found to play an important role in the

modulation of synaptic plasticity by long-lasting inhibitory effect on N-methyl-D-aspartate (NMDA)-receptor excitatory postsynaptic potentials (EPSPs), through maintaining dendritic hyperpolarization and inhibition of action potential generation (Kanter et al., 1996). Tonic inhibition through GABAARs can also suppress calcium-dependent spikes and sodium spike

back-propagation from the soma to the dendrites, as well as modulate gamma frequency by shaping interneuronal synchronization (Miles et al., 1996; Tsubokawa & Ross, 1996; Mann & Mody, 2010). In particular, GABAAR is important for the generation of theta and gamma

frequency network oscillations due to phasic inhibition, with fast “point to point” inhibition phasing and synchronizing the activity of PCs (Cobb et al., 1995; Jonas et al., 2004; Somogyi & Klausberger, 2005). Theta wave is a low frequency oscillation (4-10 Hz), which occurs during spatial navigation, rapid-eye-movement sleep and memory tasks, indicating online state of the hippocampus while gamma oscillations (30-80 Hz) occur within theta and are related to the processes mentioned above (Grastyan et al., 1959; Csicsvari et al., 2003; Klausberger & Somogyi, 2008).

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1.3.6. GABA

A

R as a pharmacological target in

diseases.

Generation of rhythmic cortical network activity involves the balance of excitatory and inhibitory synaptic inputs in a given neural circuit. This plays a crucial role in maintaining proper processing of brain signals in time and space. Dysfunction of GABAAR signaling, such

as: loss or rearrangement of GABAergic INs, changes in subunit composition, ionic channels dysfunction alters the excitatory/inhibitory balance. This was found to be crucial in several neurological disorders, such as epilepsy, schizophrenia, Alzheimer’s disease, Down syndrome and Autism spectrum disorder (Alakuijala et al., 2006; Rissman & Mobley, 2011; Mohler, 2015; Staley, 2015). Epilepsy is a disorder which occurs mainly from the change in the balance between excitatory and inhibitory activity. Seizures in epilepsy were shown to occur due to several factors, such as altered GABAAR trafficking and subunit expression (Benarroch, 2007).

The enhanced endocytosis of GABAAR, which may occur because of the decreased

phosphorylation of certain subunits, result to higher binding of receptor to AP2. This will reduce the level of GABAARs at synapses (Goodkin et al., 2005; Terunuma et al., 2008).

Altered expression of GABAAR subunits was found also in schizophrenia. The lack of

the α3 and α5-GABAAR subunits showed deficits in prepulse inhibition and this phenomenon

was rescued by anti psychotic drug haloperidol (Hauser et al., 2005; Yee et al., 2005). In addition, it was demonstrated that modulation of GABAAR with benzodiazepines, such as

flunitrazepam can suppress the induction of long-term potentiation (LTP) (Seabrook et al., 1997). In contrast, GABAAR subunit negative allosteric modulators (NAMs), such as NAM

RO4938581 of the α5-GABAAR subunit regulate the synaptic plasticity, memory, as well as it

alleviates severity of symptoms in several disorders (Down syndrome, Alzheimer’s disease) (Block et al., 2017; Xu et al., 2018). Thus, understanding the structural and functional properties of GABAARs, which shape inhibition, is important for the future pharmacological

and therapeutic approaches in several diseases.

As this project reveals new insights into the structural properties of the α5- GABAAR

subunit, in the next chapter will be provided information about this subunit expression, its role and implication in memory and neurodegenerative disorders.

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1.4. α5-GABA

A

R subunit

1.4.1. Distribution in the brain and role in memory.

The α5-GABAAR subunit is one of the common subunits in the brain, which is encoded

by GABRA5 gene. Several studies showed that the α5-subunit have high expression in the certain regions of the brain, such as the hippocampus, amygdala and olfactory bulb (Fig. 12A) (Fritschy & Mohler, 1995; Pirker et al., 2000a). However, the highest expression of the α5-GABAAR subunit was revealed in the hippocampal CA1 area in human brain as well as in other

mammals (Fig. 12B). The expression of the α5-GABAAR subunit mostly was found on the

dendrites of CA1 PCs, as well as in cortical layer 5 (Fig. 12A) (Sperk et al., 1997; Prenosil et

al., 2006; Möhler & Rudolph, 2017; Stefanits et al., 2018). Although the α5-GABAAR subunit

mRNA was suggested to be present in different INs, recent studies performed on human brain showed the subunit infrequent expression on the dendrites of CA1 INs (Paul et al., 2017; Stefanits et al., 2018). Based on the expression site, the α5-GABAAR subunit can provide two

types of inhibition, tonic and phasic, which are defined by the IPSCs kinetics. Tonic inhibition is induced by extrasynaptically located α5-GABAAR subunits, mainly on CA1 PCs, where it

was found to be implicated in the neural oscillations, hippocampal memory, learning and cognitive behaviour (Caraiscos et al., 2004; Prenosil et al., 2006; Mann & Mody, 2010; Möhler & Rudolph, 2017). The α5-GABAAR subunit is an exceptional target for an enhancement of

learning and memory. It was discovered that inverse agonists of the α5-GABAAR subunit

enhance LTP in mouse and improve cognitive performance (Atack, 2010). Mice with a partial deficit of the a5-containing GABAARs were shown to improve performance in trace fear

conditioning and hippocampus-dependent memory task (Crestani et al., 2002). The lack of the α5-GABAAR subunit in mice improved performance in a spatial learning task in the water

maze (Collinson et al., 2002). In the same test, the α5-GABAAR subunit selective partial

inverse agonists enhanced the performance of rats, thus confirming the link between the modulation of α5-containing GABAARs and memory (Sternfeld et al., 2004). These results

position the α5-GABAARs as a key target for a pharmacological enhancement of

hippocampus-dependent learning and memory. L-655,708, which is NAM of the α5-GABAAR, was found to

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and induced gamma oscillations in hippocampal slices. However, the α5-GABAAR subunit

inhibition by L-655,708 was shown to induce anxiety-like behavior in mice (Navarro et al., 2002b; Möhler & Rudolph, 2017). Recent studies revealed that the NAM of the α5-GABAAR

subunit RO493851 enhanced not only LTP and memory but also rescued most of the molecular abnormalities in the hippocampus of Ts65Dn mouse model of Down syndrome (Block et al., 2017). Furthermore, the other study, using rTG4510 mice model of Alzheimer’s disease, demonstrated the important role of NAMs of the α5-containing GABAARs in psychomotor

agitation, as well as age- and tau-associated hyperactivity (Xu et al., 2018).

A: Immunohistochemical distribution of the α5-GABAAR subunit in mouse brain with false

color-coding. High expression is demonstrated in the hippocampus, cortical layer 5 and olfactory bulb. Adapted and modified from Mohler et al., 2017 (Möhler & Rudolph, 2017).

B: The α5-GABAAR subunit expression in the human hippocampus. It is highly expressed in the DG,

SUB and CA1 area (Stefanits et al., 2018).

1.4.2. Changes in the level of expression.

The expression of the α5-GABAAR subunit can be altered by different conditions, such

as the presence of other subunits, the animal age as well as pathology (e.g., temporal lobe epilepsy, schizophrenia, autism, Alzheimer’s disease) (Salesse et al., 2011; Zurek et al., 2016;

A _B

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Ghafari et al., 2017; Kwakowsky et al., 2018). It was shown that during multiple T maze (MTM) training for spatial learning, the level of the α5-containing GABAARs is increased.

In the same study, it was revealed that deletion of the α1 subunit increased the expression level of the α5-GABAAR subunit during MTM training, possibly as a compensation, as they

are mainly expressed at the same synapses and both are important for spatial learning (Araujo

et al., 1999; Ghafari et al., 2017). Interestingly, other studies showed the absence of tonic

currents in the mice lacking the α5-GABAAR subunit in the CA1 PCs and INs without

compensatory changes (Glykys et al., 2008).

The next factor, which can alter the expression level of the α5-GABAAR subunit is the

animal age. The α5-GABAAR subunit has the highest expression at postnatal day 10-30,

whereas with aging and in adult brain its level is decreased (Yu et al., 2006).

In several pathologies, the level of the α5-GABAAR subunit was altered, such as in

epilepsy, Alzheimer’s disease, schizophrenia. Previous studies from our laboratory revealed that the expression of the α5-GABAAR subunit and its anchor protein Rdx is decreased in the

hippocampal CA1 O/A INs in the animal model of temporal lobe epilepsy (Magnin, 2014). Recent study showed that in Alzheimer’s disease the α5-GABAAR subunit expression is

increased in the hippocampal CA1 O/A and PYR layers in the human brain (Kwakowsky et

al., 2018). Overall, changes in the level of the α5-GABAAR subunit expression in different

conditions provide insights about its indispensable role in proper functioning of the brain. Furthermore, the alterations can be crucial from the pathological point of view as well as in synaptic plasticity with further impact on memory formation.

1.4.3. Expression at inhibitory synapses onto INs

.

Preliminary work from our laboratory revealed the expression of the α5-GABAAR

subunit with its anchor protein Rdx at the level of synapses in O/A INs, with its preferential targeting to the inhibitory synapses formed by the CR+ cells (Magnin, 2014). In particular,

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optogenetic experiments, where the CR+ cells expressing the light-sensitive Channelrhodopsin 2 (ChR2) were stimulated, showed that light-evoked IPSCs in O/A INs were sensitive to the α5-GABAAR subunit inhibitors (Magnin, 2014). Another work of our team discovered that the

local inputs of CA1 O/A O-LM INs contain the α5-GABAAR subunit, as its eIPSCs were

sensitive to the α5-GABAAR subunit inverse agonist drug L-655,708 (Salesse et al., 2011).

However, it remains unknown whether the synaptic expression of the α5-GABAAR subunit is

cell- and/or input-specific, as well as whether it’s located in proximal or distal dendrites.

1.4.4. Implication of the α5-GABA

A

R subunit in

different disorders.

The α5-GABAAR subunit gene locus was found to be associated with several

psychiatric disorders, such as schizophrenia, autism spectrum disorders and Down syndrome. The ability of the α5-GABAAR subunit tonic inhibition to alter the activity of hippocampus

and prefrontal cortex, suggested that this subunit could be involved in schizophrenia (Hauser

et al., 2005; M Gill & A Grace, 2014). Previous studies showed that genetic deletion of the

α5-GABAAR subunit or its level reduction in mice induced behavioral abnormalities, which

were found in schizophrenia, such as prepulse inhibition and impaired latent inhibition (Hauser et al., 2005; Gerdjikov et al., 2008). Positive modulation of the α5-GABAAR subunit

rescued the normal hippocampal activity and improved cognitive symptoms in schizophrenia (M Gill & A Grace, 2014). In autism spectrum disorder, the involvement of the α5-GABAAR

subunit was shown by its reduced expression (Mendez, 2013).

Down syndrome is a disorder involving cognitive dysfunction, with a possible mechanism involving excessive GABAergic transmission (Belichenko et al., 2004). In the hippocampus of Ts65Dn mouse model of Down syndrome, the α5-GABAAR subunit

inhibition with the NAM RO493851, improved not only LTP, but also memory formation as well as rescued most of the molecular abnormalities (Block et al., 2017).

In temporal lobe epilepsy the mRNA expression of the α5-GABAAR subunit as well as

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increased excitability (Rice et al., 1996; Houser & Esclapez, 2003). Furthermore, studies on human brain showed that during neuropathological progression of Alzheimer’s disease GABAAR subunits are modestly affected. However, unlike α1-, β1- and β2- GABAAR

subunits, the α5-GABAAR subunit showed significantly reduced expression in the CA1, CA2

and CA3 during the progression of Alzheimer’s disease (Rissman et al., 2003). In contrast to this, a recent study performed on the human brain in Alzheimer’s disease, showed the α5-GABAAR subunit increased expression within CA1 O/A and PYR layers (Kwakowsky et al.,

2018). These controversial findings can be explained by different methods used in the studies for examining the expression of the α5-GABAAR subunit. Increased expression of this

subunit in the hippocampus may be responsible for memory deficits in Alzheimer’s disease (Wang et al., 2012; Kwakowsky et al., 2018).

In summary, the α5-GABAAR subunit can be a therapeutic target for the cognitive

enhancement in neurodegenerative, as well as in psychiatric disorders with cognitive dysfunction, such as schizophrenia, autism, temporal lobe epilepsy, Alzheimer’s disease and Down syndrome. However, from the structural point of view the cells and the synapses that may contain the α5-GABAAR subunit within the hippocampal CA1 O/A layer remains

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2. Hypothesis of the work

Recent studies showed the strong expression of the α5-GABAAR subunit in the human

hippocampal CA1 area (Stefanits et al., 2018). Previous work of our laboratory showed that the IPSCs evoked in CA1 O-LM cells by minimal electrical stimulation applied within PYR, which were generated by the activation of local inhibitory sources, were sensitive to the α5-GABAAR inverse agonist (Salesse et al., 2011). Given that sIPSCs generated via activation of

a pool of inhibitory synapses were not sensitive to the α5-GABAAR inverse agonist in all IN

types examined, we propose that the α5-GABAAR subunit is not expressed at all synapses and

it has an input- and cell-specific expression within CA1 O/A INs. In addition, given that α5-GABAAR subunit infrequent expression was found in neuronal dendrites of human

hippocampal CA1 area (Stefanits et al., 2018), we suggest that this subunit may show a differential distribution in CA1 O/A IN dendrites. Furthermore, as VIP+ INs provide inhibitory control to CA1 O/A INs, pharmacogenetic silencing of VIP+ cells can be used to study the functional role of the α5-GABAAR subunit expressed at those synapses. Considering the

heterogeneity of VIP+ cells in the hippocampus (Acsady et al., 1996a), we hypothesize that pharmacogenetic silencing may target different VIP+ cells, which requires to be investigated.

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3. Specific objectives

1. To examine the expression of the α5-GABAAR subunit anchoring protein Rdx in SOM+

and PV+ O/A INs, and to investigate the subcellular location of the α5-GABAAR subunit.

2. To evaluate the automatic analysis tool of LAS AF software for the synaptic α5-GABAAR

subunit colocalization analysis.

3. To examine the input-specific localization of the α5-GABAAR subunit at VIP+, CR+ and

PV+ synaptic inputs.

4. To study the efficacy of viral vector AAV8-DIO-hM4Di:mCherry transduction in different VIP+ cells, including IS3 (VIP+/CR+) vs BC (VIP+/CCK+) cells (Acsady et al., 1996a).

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4. Materials and methods

4.1. Animals

All experiments involving animals were carried out in accordance with the animal welfare guidelines of the Animal Protection Committee of Université Laval. For the immunohistochemical studies VIP-IRES-Cre, VIP-Cre:Ai9 and PV-Cre:Ai9 mice from both were used.

4.2. Slice preparation

For in vitro electrophysiological analysis, acute hippocampal slices were prepared following at least 2 weeks after viral injection. Mice were deeply anesthetized with ketamine-xylazine (0,1mL/10g, i.p.). When the animal was completely anaesthetized and did not respond to the small pinch to the paw, it was fixed for the perfusion. The ribcage was opened, to get access to the heart and after making a little cut in the right atrium, the solution of ice-cold sucrose (20mL) containing (in mM): 250 sucrose, 2 KCl, 1.25 NaH2PO4, 26 NaHCO3, 7

MgSO4, 0.5 CaCl2, and 10 glucose, was injected into the left ventricle. The brain was quickly

removed into the ice-cold sucrose cutting solution (in mM: 250 sucrose, 2 KCl, 1.25 NaH2PO4,

26 NaHCO3, 7 MgSO4, 0.5 CaCl2, and 10 glucose) continuously aerated with carbogen gas

mixture (5% CO2, 95% O2). Transversal 300-µm thick hippocampal slices were cut using a Microm vibratome (Fisher Scientific) in ice-cold cutting solution and transferred to recovery artificial cerebrospinal fluid (ACSF, in mM: 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3,

3 MgSO4, 1 CaCl2, and 10 glucose; mOsm/L 295, pH 7.4 when aerated with carbogen) for 30

min at 33–37 °C. They were kept in the same carbogen-aerated solution at room temperature for at least 1 h before recordings.

For immunohistochemical analysis, coronal slices of the hippocampus were prepared from the male and female mice with the age P25-90. Before extracting the brain, mice were anaesthetized with ketamine-xylazine (0,1mL/10g, i.p.). When the animal was completely anaesthetized, it was perfused with the solution of cold sucrose (20mL) and then

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paraformaldehyde (PFA) 4% (20mL) were injected into the left ventricle carefully to not break the blood circulation. The perfused mouse was decapitated, the brain was gently extracted and placed in PFA 4% for 24 hours at 4°C. Fixed brain was washed with phosphate buffered saline (PBS) and kept in sodium azide as a preservative at 4°C.

For the sectioning, the brain was embedded in 4% agar and sectioned with thickness 50 µm using a vibratome (PELCO easiSlicer).

4.3. Electrophysiology

PV-Cre mice with injections of adeno-associated pAAV-Ef1a-DIO-ChR2:mCherry virus were used for whole-cell patch clamp recordings. The recording chamber was perfused with carbogenated 32 °C recording ACSF (in mM: 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 MgSO4, 2CaCl2, and 10 glucose; mOsm/L 295–300, pH 7.4) at a rate of 1.5–2 ml/min. Patch pipettes had 3–5 MΩ resistance when filled with intracellular solution (in mM: 130 CsMeSO4, 2CsCl, 10 diNa- phosphocreatine, 10 HEPES, 2 ATP-Tris, 0.2 GTP-Tris, 0.3% biocytin, 2 QX-314, pH 7.2–7.3, 280–290 mOsm/L). Hippocampal CA1 O/A INs were identified using a Zeiss AxioScope microscope with DIC-IR and 40x/0.8N.A objective, and Channelrhodopsin-2 (ChR2)-expressing INs were identified by mCherry expression. The IPSCs were evoked by optical activation of ChR2-expressing terminals using optical activation of ChR2-expressing terminals with 5-ms pulses of blue light (up to 10 mW optic power; filter set, 450–490 nm) by a wide-field stimulation through microscope objective. Synaptic latency was measured as the time between the onset of blue-light pulse and the onset of synaptic current. Resting membrane potential was measured immediately after forming whole-cell configuration at a holding potential of 0 mV. Currents were elicited at 20-30-s intervals between successive trials, filtered at 2–3 kHz (Multiclamp 700B amplifier), digitized at 10 kHz (Digidata 1440A, Molecular Devices) and acquired by pCLAMP10 software (Molecular Devices). The α5-GABAAR subunit inverse agonists MRK-016 (10 µM, Tocris) and L-655,708

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4.4. Immunohistochemistry

The slices were washed in PBS, incubated with 0.3% hydrogen peroxide (H2O2) in

distilled water for 30 minutes and after incubated with 0.2% Triton X-100 (PBS-Tri), 10% normal donkey serum (Jackson Immunoresearch), 4% bovine serum albumin and PBS-Tri for 2 h at room temperature. After the slices were incubated at 4°C overnight with primary antibodies (1:300 rabbit radixin; Abcam, ab52495; 1:200 rat somatostatin; Millipore, 2885355; 1:1000 mouse parvalbumin; Sigma, P3088; 1:200 rabbit GABA A receptor alpha5; Abcam, ab10098; 1:1000 goat calretinin; Santa Cruz Biotechnology, sc-11644; 1:500 goat metabotropic glutamate receptor 1A, mGluR1; Frontier Institute, Af1220; 1:500 mouse vesicular GABA transporter; Synaptic Systems, 131011; 1:800 rabbit cholecystokinin; Sigma; C2581; 1:500 rabbit mCherry; Biovision, 5993-100) in the solution containing 4% bovine serum albumin, 10% normal donkey serum, 0.2% Triton and PBS. After four consecutivePBS washes, the slices were incubated with secondary-antibodies in blocking solution for 2h at room temperature (1:200 Alexa 647 donkey anti-rabbit; Thermo Fischer Scientific; 1:200 Alexa 647 donkey anti-mouse; Life technologies; 1:250 Alexa 488 donkey anti-rat; Jackson Immuno Research; 1:200 FITC-cojugated, donkey anti-mouse; Jackson Immuno Research; 1:1000 Alexa 488 donkey anti-goat; Jackson Immuno Research; 1:1000 Cy3 donkey anti-goat; Jackson Immuno Reasearch, 1:1000 Alexa 546 donkey anti-rabbit; Thermo Fischer Scientific; 1:250 Dylight 650 donkey anti-goat; Thermo Fischer Scientific). Sections were washed in PBS and mounted with Dako Fluorescence Mounting Medium (Daco) for image acquisition.

Slices containing the cells filled with biocytin were fixed in 4% PFA overnight, rinsed in PB and kept at 4 °C in PB sodium azide. Biocytin was revealed using Streptavidin-Alexa-488 (1:200, Invitrogen).

Quantification analysis were performed for each experiment bilaterally at least on 6 sections per animal from 3 mice for each condition. For immunohistochemical experiments, images were obtained using Leica TCS SP5 imaging system. The controls were made without primary antibodies for each condition.

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4.5. Confocal microscopy

Confocal images were obtained using a Leica TCS SP5 imaging system coupled to a 488 nm Argon, 543 nm and 633 HeNe lasers. Z-stacks were acquired using a 20× (NA, 0.8) and 63× (NA, 1.4) oil-immersion objectives (Leica Microsystems). Z-series images were taken with a 1-µm step for the cell quantification and 0,2-µm for the subcellular and synaptic quantifications.

4.6. Data analysis

Cells expressing specific markers were counted in different CA1 layers. The cells were considered immunopositive when the corresponding fluorescence intensity was at least twice higher that of the background. The overall brightness and contrast of images were adjusted manually for representations. Portions of images were not modified separately. All the analyses were performed by LEICA LAS AS software.

For SOM/Rdx vs PV/Rdx colocalization analyses, images were manually analyzed by counting the total number of SOM+ and PV+ INs in the CA1 O/A and then the fraction of SOM+ vs PV+ cells which were coexpressing Rdx. Statistical analysis was done using Mann-Whitney U test with Clampfit 10.5 software. For the subcellular expression of VGAT and the α5-GABAAR subunit, the number of the α5-GABAAR subunit puncta and VGAT were counted

throughout the dendrites of SOM+/mGluR1a+ O-LM cells. The presence of synapses and expression of the α5-GABAAR subunit were counted per 10µm from perisomatic to distal

dendritic (~120 µm from the soma) regions.

The input-specific expression of the α5-GABAAR subunit at the VIP+, CR+ and PV+

synapses was first analyzed using the automatic colocalization tool of LAS AF software. Second, the analyses were done manually by counting the number of synaptic boutons (0.8 - 1.5µm; diameter of colocalizing with VGAT) containing the α5-GABAAR subunit puncta.

The recorded cells were analyzed using Clampfit 10.5 software. The IPSCs for each cell were compared before and after drug application in the bath.

Cell- and input-specific expression of the α5-GABAAR in the CA1 area of the mouse hippocampus