Effects of Distinct Nachrs on the Release of Noradrenaline and Acetylcholine in the Habenular-Interpeduncular System

Effects of distinct nAChRs on the release Of noradrenaline and acetylcholine in the habenular-interpeduncular system

Doctoral thesis at the Medical University of Vienna for obtaining the academic degree

Doctor of Philosophy

Submitted by

Farahnaz Beiranvand

Supervisor:

Ass.Prof. Priv.Doz. Mag. Dr, Petra Scholze

Center for Brain Research Medical University of Vienna

Vienna, August 2015

TABLE OF CONTENTS

TABLE OF CONTENTS ...... 1

DECLARATION ...... 3

LIST OF FIGURES AND TABLES ...... 5

LIST OF ABBREVIATIONS ...... 6

ACKNOWLEDGEMENT ...... 8

ABSTRACT ...... 9

ZUSAMMENFASSUNG ...... 9

1 INTRODUCTION ...... 11

1.1 THE CHOLINERGIC SYNAPSE ...... 11 1.2 THE CHOLINERGIC NEUROTRANSMISSION PATHWAY...... 13 1.3 NICOTINIC RECEPTORS ...... 14 1.3.1 nAChR structure overview ...... 15 1.3.2 Muscular nicotinic acetylcholine receptors...... 19 1.3.3 Neuronal nicotinic acetylcholine receptors...... 19 1.3.4 Distribution of nAChR in the CNS ...... 22 1.4 NICOTINE-INDUCED ACH RELEASE IN THE BRAIN ...... 24 1.5 NICOTINE-INDUCED NORADRENALINE RELEASE IN THE BRAIN ...... 25 1.6 PHARMACOLOGY OF NICOTINIC RECEPTORS...... 26 1.6.1 Agonists of nAChRs ...... 26 1.6.2 Antagonists of the nAChR channels ...... 28 1.6.3 Competitive antagonists ...... 28 1.6.4 Non-competitive antagonists of the nAChRs ...... 29 1.7 STRUCTURES OF INTEREST IN MY STUDIES ...... 30 1.7.1 Habenula ...... 30 1.7.2 Interpeduncular nucleus (IPN) ...... 33 1.7.3 Hippocampus (Hc) ...... 34 1.7.4 Cortex ...... 36 1.8 KNOCKOUT MICE USED IN MY EXPERIMENTS ...... 36 1.8.1 α5-KO ...... 36

1.8.2 β2-KO ...... 37 1.8.3 α7-KO ...... 37 1.8.4 β4-KO ...... 38

2 GOAL OF MY STUDIES ...... 39

3 MATERIALS AND METHODS ...... 40

3.1 ANIMALS ...... 40 3.2 EXPERIMENTAL SETUP ...... 40 3.3 TISSUE PREPARATION ...... 40 3.4 SUPERFUSION ASSAY TO INDUCE THE RELEASE OF [3H] -CHOLINE AND [3H] -NA ...... 41 3.5 REAGENTS ...... 42 3.6 EQUIPMENT ...... 43 3.7 TISSUE PREPARATION (FIGURE) ...... 44 3.8 DATA ANALYSIS ...... 46 3.9 STATISTICS ...... 46

4. RESULTS ...... 48

4.1 [3H]-ACH AND [3H]-NA RELEASE FROM THE RAT IPN ...... 48 4.2 [3H]-ACH AND [3H]-NA RELEASE FROM THE RAT HB...... 51 4.3 [3H]-ACH RELEASE IN THE RAT HIPPOCAMPUS AND CORTEX ...... 52 4.4 RELEASE OF [3H]-ACH AND [3H]-NA IN THE MOUSE IPN ...... 54 4.5 RELEASE OF [3H]-ACH AND [3H]-NA IN THE MOUSE HB ...... 56 4.6 RELEASE OF [3H]-ACH AND [3H]-NA IN THE MOUSE HIPPOCAMPUS AND CORTEX ...... 58

5 DISCUSSION AND CONCLUSIONS ...... 61

5.1 [3H]-ACH RELEASE IN THE IPN ...... 61 5.2 [3H]-ACH RELEASE IN THE HB ...... 63 5.3 [3H]-ACH RELEASE IN THE HC AND CORTEX ...... 63 5.4 [3H]-NA RELEASE IN THE HB AND IPN ...... 63 5.5 ROLE OF THE Α5 NACHR SUBUNIT...... 65

6 REFERENCES ...... 68

APPENDIX ...... 101

CURRICULUM VITAE ...... 101

DECLARATION

I hereby certify that this thesis submitted at the Medical University of Vienna in Austria is the result of my own research work for obtaining the academic degree Doctor of Philosophy and has not been submitted for any degree at other universities.

All the experiments were performed under the supervision of Univ. Ass Prof. Dr, Petra Scholze at the Department of Pathobiology of the Nervous System, Center for Brain Research, Medical University of Vienna (Austria).

The presented results have been published in peer-reviewed journal – British Journal of Pharmacology- as partial fulfilment of doctoral degree requirements.

Beiranvand, F., C. Zlabinger, A. Orr-Urtreger, R. Ristl, S. Huck and P. Scholze, 2014 Nicotinic acetylcholine receptors control acetylcholine and noradrenaline release in the rodent habenulo-interpeduncular complex. Br J Pharmacol 171: 5209-5224

Author contributions:

“1. F. Beiranvand (Medical University of Vienna): conception and design of release experiments, conduction of the experiments, collection and analysis of data and drafting and revising the article critically for important intellectual content.

2. C. Zlabinger (Medical University of Vienna): conduction of immunoprecipitation experiments and analysis of data.

3. A. Orr-Urtreger (Tel Aviv University): generation of α5, β4 and α7 KO mice, drafting and revising the article critically for important intellectual content.

4. R. Ristl (Medical University of Vienna): statistical advice and reanalysis of the entire dataset.

5. S. Huck (Medical University of Vienna): conception and design of the experiments, analysis and interpretation of data and drafting and revising the article critically for important intellectual content.

6. P. Scholze (Medical University of Vienna): breeding and dissection of all animals and drafting and revising the article critically for important intellectual content ”

The authors have no conflict of interest to declare.

Licence agreements provided by Copyright Clearance Center and/or Publishers

Figure 1 from the introduction was reprinted with permission from Springer: Histochem Cell Biol 130: 219-234, 2008.

Figure 2 from the introduction was adapted with permission from The American College of Neuropsycho-pharmacology (ACNP): The Fifth Generation of Progress, 2002.

Figure 3 from the introduction was adapted with permission from Nature Publishing Group: Nat Rev Neurosci 3: 102-114, 2002.

Figure 4 from the introduction was reprinted with permission from the Tocris Scientific Reviews: Nicotinic ACh Receptors, 2014.

Figure 5 from the introduction was reprinted with permission from ELSEVIER: Trends Neurosci 20: 92-98, 1997.

Figure 6 from the introduction was reprinted with permission from Cambridge University Press: CNS Spectr 13(6):484-9, 2008.

Figure 7 from the introduction was reprinted with permission from SfN: J Neurosci 28: 11825-11829, 2008.

Figures 10-15 and their legends were reprinted with permission from Wiley-Blackwell: Br J Pharmacol 171: 5209-5224, 2014.

LIST OF FIGURES AND TABLES

Figure 1: Scheme of the cholinergic synapse ...... 11

Figure 2: Major cholinergic pathways in the rat brain...... 14

Figure 3: Structure of the nicotinic acetylcholine receptor...... 17

Figure 4: Three main allosteric states of the nicotinic acetylcholine receptor ...... 18

Figure 5: Putative locations of nAChR...... 22

Figure 6: The habenular nuclei (Hb) and the dorsal diencephalic conduction system ...... 31

Figure 7: Most important afferent and efferent pathways of the Hb (habenula complex) ..... 33

Figure 8: Preparation of intact Hb and IPN nucleus ...... 44

Figure 9: Slice preparation of the rat hippocampus ...... 45

Figure 10: Chemically and electrically induced [3H]-ACh and [3H]-NA release from rat IPN . 50

Figure 11: Chemically and electrically induced [3H]-ACh and [3H]-NA release from rat Hb .. 52

Figure 12: Nicotine-induced and electrically induced [3H]-ACh release from rat hippocampal and parietal cortical slices ...... 53

Figure 13: Chemically and electrically induced [3H]-ACh and [3H]-NA release in mouse IPN ...... 55

Figure 14: Chemically and electrically induced [3H]-ACh and [3H]-NA release from the mouse Hb ...... 57

Figure 15: Chemically and electrically induced [3H]-ACh and [3H]-NA release from mouse hippocampal and parietal cortical slices ...... 59

LIST OF ABBREVIATIONS

α-BgTx α-Bungarotoxin α-CTX α-Conotoxins ACh Acetylcholine AChE Acetylcholinesterase AChBP Acetylcholine binding protein ANOVA Analysis of variance BFC Basal forebrain complex BChE Butyrylcholinesterase ChAT Choline acetyltransferase CHT Choline transporter CNS Central nervous system CPu Caudate and putamen DhβE Dihydro-β-erythroidine DBB Diagonal band of Broca BST Bed nucleus of stria terminalis DR Dorsal raphe MR medial raphe

EC50 Half maximal agonist concentration EPI Epibatidine FR Fasciculus retroflexus Hb Habenula Hc Hippocampus GPi Globus pallidus internal segment GABA γ-aminobutyric acid IPN Interpeduncular nucleus IP immunoprecipitation KO Knockout LDT Laterodorsal tegmental LH Lateral hypothalamus LHb Lateral habenula LPO Lateral preoptic area mAChRs Muscarinic acetylcholine receptor MCA Mecamylamine MHb Medial habenula

MLA Methyllycaconitine nAChR Nicotinic acetylcholine receptor NMJ Neuromuscular junction PB Pineal body PNS Peripheral nervous system SM Stria medullaris SNc Substantia nigra pars compacta PPT Pedunculopontine tegmental nucleus TM Transmembrane TTX Tetrodotoxin WT Wild type VTA Ventral tegmental area VAChT Vesicular acetylcholine transporter VDCC Voltage-dependant Ca2+ channels

ACKNOWLEDGEMENT

I would like to acknowledge all the participants who have guided and supported me in this study.

First of all I would like to express the deepest appreciation to Prof. Dr. Sigismund Huck for his excellent guidance, patience, mentoring and support throughout my PhD training. His profound knowledge made this work successful.

Special thanks and deep appreciation to Ass.Prof. Dr Petra Scholze for a fundamental role, continuous support and help with any kind of problems. Despite her busy schedule, she always made herself available for training and technical support.

I also gratefully acknowledge Karin Schwarz for the technical support and sharing her skills as well as Gabi Koth and Traude Mössner for supporting and creating a very nice working atmosphere.

Furthermore, I am grateful to my friends from the lab Anna, Fatma, Xenia, Christoph, Max, for the great time we spent together and friendly work atmosphere.

Finally, my heartfelt gratitude goes to my beloved family for their love, support and help. Without their help and understanding, this thesis would never have been written.

This project has been financially supported by the Austrian Science Fund (FWF) and Ministry of Health and Medical Education of Iran.

ABSTRACT

The Habenula-Interpeduncular (Hb-IPN) complex plays a critical role in nicotine intake and nicotine withdrawal. In my thesis work, I have focussed on the role of nicotinic acetylcholine receptors (nAChRs) on the release of two neurotransmitters: Acetylcholine (ACh) and Noradrenaline (NA). I found that nAChR activation caused major ACh release in the IPN (but not in the Hb) in both rats and mice, and that β4-containing presynaptic receptors are crucial for this effect. On the other hand, nAChR activation caused the release of NA in both the Hb and the IPN. Contrary to the release of ACh, this effect depended in mice on pre-terminal receptors containing the subunits α5, β2, and β4. Interestingly, the nicotine-induced NA release in the hippocampus (Hc) differed from the Hb-IPN complex by not being affected by the absence or presence of the α5 subunit, suggesting that in mice, the types of receptors differ between these structures. Neither the release of ACh nor of NA was noticeable affected in mice lacking the α7 nAChR subunit. My observations emphasize that hetero- oligomeric nAChRs at noradrenergic endings in the Hb-IPN complex could play a central role, particularly in symptoms of nicotine withdrawal and thus in nicotine dependence.

ZUSAMMENFASSUNG

Der Habenula-Interpeduncular (Hb-IPN) Komplex hat eine Schlüsselfunktion beim Konsum von Nikotin und bei der Symptomatik in dessen Entzug. Im Mittelpunkt meiner Dissertation stehen somit nikotinische Acetylcholinrezeptoren (nAChRn) und ihre Rolle in der Freisetzung von zwei zentralen Neurotransmittern: Acetylcholine (ACh) und Noradrenalin (NA). Meine in vitro Experimente haben gezeigt, dass bei Aktivierung von nAChRn im IPN (nicht aber in der Hb) sowohl bei Mäusen als auch bei Ratten in substantiellem Ausmaß ACh freigesetzt wird, und dass dieser Effekt auf der Aktivierung von präsynaptischen Rezeptoren, die die Untereinheit β4 enthalten, beruht. Bei Aktivierung von nAChRn wird außerdem NA freigesetzt. Zum Unterschied von der Freisetzung von ACh ist dieser Effekt allerdings sowohl im IPN als auch in der Hb nachzuweisen. Ich konnte weiters mittels Material von Mäusen zeigen, dass hier prä-terminale (und nicht präsynaptische) Rezeptoren von Bedeutung sind, wobei Rezeptoren, die die Untereinheiten α5, β2, und β4 enthalten, involviert sind. Hingegen waren – verglichen mit Kontrollmäusen – im Hippocampus von Mäusen, denen die α5 Untereinheit fehlt, keine Unterschiede in der Freisetzung von NA zu beobachten. Dies lässt folgerichtig schließen, dass sich die nAChRn, die zur Freisetzung von NA führen, im Hippocampus von denen im HB-IPN Komplex unterscheiden. Ich konnte

weiters keine Beeinträchtigung in der Freisetzung von ACh und NA in Mäusen beobachten, bei denen α7 nAChRn ausgeschaltet waren. Meine Versuche weisen darauf hin, dass hetero-oligomere nAChRn an noradrenergen Nervenendigungen im Hb-IPN Komplex eine wichtige Rolle im Nikotinentzug und somit für die Abhängigkeit von Nikotin spielen könnten.

1 INTRODUCTION

1.1 The cholinergic synapse

The term cholinergic synapse refers to a structure capable of producing acetylcholine (ACh) at the presynaptic site and uses it for neurotransmission by means of receptors located postsynaptically. Upon release, ACh is cleaved by the enzyme acetylcholinesterase (AChE) into acetate and choline, the latter being transported back into endings by the (high affinity) choline transporter (CHT1) (MEYER AND COOPER 1983; OKUDA AND HAGA 2000; AMENTA AND TAYEBATI 2008). The presynaptic cholinergic nerve terminal has all essential components for the synthesis, storage, and release of ACh. The enzyme choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) are produced in the soma and carried down to the terminals by axonal transport.

The synapse can be characterized by the landmarks ChAT, AChE, CHT1, VAChT, and ACh (ANGLADE AND LARABI-GODINOT 2010). Six sequential steps are required for cholinergic transmission (Fig. 1).

Figure 1: Scheme of the cholinergic synapse

Synthesis and recycling pathway of acetylcholine at a cholinergic nerve terminal. ACh: acetylcholine, AChE: acetylcholinesterase, BChE: butyrylcholinesterase, ChAT: choline acetyltransferase, CHT1: high-affinity choline transporter-1, M: muscarinic receptor, N: nicotinic receptor, VAChT: vesicular ACh transporter. The target cell may be of neuronal (forming a synapse) or non-neuronal origin. Figure adapted from KUMMER et al. (2008)

1) Acetylcholine synthesis

ACh was the first molecule identified as chemical neurotransmitter (LOEWI 1921). It is synthesized in the cytosol from choline and acetyl coenzyme-A by a reaction catalyzed by ChAT (ODA 1999).

2) Acetylcholine Storage

ACh is actively transported from the cytoplasm into presynaptic vesicles by means of the VAChT. The VAChT is located at the vesicle membrane and acts as an antiporter by coupling the efflux of protons with an influx of ACh (USDIN et al. 1995).

3) Acetylcholine Release

The release of classical neurotransmitters such as ACh is initiated by a transient rise of intracellular calcium at the presynaptic active zone (SUDHOF 2012). Normally, an action potential upon entering a presynaptic terminal would open (voltage-gated) Ca2+ channels to pass Ca2+ from the extracellular space. However, Ca2+ may also gain access via ligand- gated ion channels such as nicotinic ACh receptors. As more detailed below, signalling by presynaptically-located receptors appears to be a major mechanism in the function of nicotinic receptors in the central nervous system (CNS)(ROLE AND BERG 1996; WONNACOTT 1997).

4) Acetylcholine receptors

Upon release, ACh may bind to two different kinds of pre- and/or post synaptic receptors in CNS and the peripheral nervous system (PNS): muscarinic and nACh receptors. Muscarinic acetylcholine receptors (mAChRs) are named by the water-soluble alkaloid from Amanita muscaria and belonged to the family of G-protein coupled receptors. To date, five distinct receptors M1, M2, M3, M4 and M5 have been cloned (FELDER 1995). M1, M3 and M5 bind to the Gq/G11-type of G-proteins to activate phospholipase pathways, whereas M2, M4 bind to the Gi/o-type of G-proteins to inhibit the enzyme adenylyl cyclase (BRANN et al. 1993;

BROWN 2010).

In the PNS, mAChRs are located at effector cells to modulate a variety of physiological functions such as smooth muscle contraction, glandular secretion, or bradycardia (BRANN et al. 1993; ROUX et al. 1998; DHEIN et al. 2001; WALCH et al. 2001).

The second types of cholinergic receptors are nAChRs which are primarily - though not exclusively (WESSLER AND KIRKPATRICK 2008) - located in the CNS, in autonomic ganglia,

and at the neuromuscular junction (NMJ) (ALBUQUERQUE et al. 1995; SKOK 2002; DANI AND

BERTRAND 2007). Considering that the main part of my thesis is based on these receptors, I will briefly review the structure and function of nAChRs as well as their distribution in the nervous system in more detail below.

5) Acetylcholine degradation

Due to the presence of AChE, signaling of ACh is extremely short-lived. With a turnover number of >10.000 sec-1 (WILSON AND HARRISON 1961; QUINN 1987; TAYLOR et al. 1994), AChE rapidly cleaves all ACh released into the synaptic or peri-synaptic space (reviewed in COLOVIC et al. 2013).

6) Recycling of choline

The degradation product choline is transported back into the nerve ending by the CHT1, whereas acetate dissipates by passive diffusion (BLACK AND RYLETT 2012)I). Blockade of CHT1 by hemicholinium-3, or loss of function of CHT1 (KRISHNASWAMY AND COOPER 2009) will eventually deplete ACh stored in presynaptic vesicles and thus cause reduction or loss of function of cholinergic transmission, a mechanism that has been studied at autonomic

synapses (BENNETT AND MCLACHLAN 1972; KRISHNASWAMY AND COOPER 2009).

1.2 The cholinergic neurotransmission pathway

In the CNS, cholinergic systems are capable of modulating neurotransmitter release, cell excitability, neuronal maturation, and plasticity (WONNACOTT 1997; HOHMANN AND BERGER- SWEENEY 1998; BRUEL-JUNGERMAN et al. 2011; reviewed in PICCIOTTO et al. 2012). Because of this, the activation of cholinergic systems contributes to processes such as pain, arousal, anxiety, sleep and cognitive functions (WOOLF 1991; WOOLF 1996; EVERITT AND ROBBINS 1997; GOTTI et al. 1997; CHANGEUX AND EDELSTEIN 2001; HOGG et al. 2003; PICCIOTTO et al. 2012). Three main cholinergic subsystems provide cholinergic input to almost all brain regions (see also Fig. 2): The basal forebrain complex, which is made of four major cholinergic cell groups (Ch1-Ch4) (HEDREEN et al. 1984; ZABORSZKY et al. 2008) and

projects throughout the cortex and the hippocampus (SAPER AND CHELIMSKY 1984; KOLIATSOS et al. 1988). Second, the brainstem cholinergic system which consist of four distinct nuclei (Ch5-Ch8) to innervate the thalamus, the midbrain, and the caudal pons (MESULAM et al. 1983; WOOLF AND BUTCHER 1986; WOOLF AND BUTCHER 1989). The third subsystem arises from cholinergic interneurons located in the striatum and provides dense local innervation throughout the striatum (reviewed in ZHOU et al. 2002; DANI AND BERTRAND

2007). In the PNS, a large portion of the autonomic nervous system is cholinergic, including α-motor neurons, preganglionic sympathetic and parasympathetic neurons, as well as postganglionic parasympathetic neurons (SMOLEN 1988; SCHAFER 1998; SKOK 2002).

Figure 2: Major cholinergic pathways in the rat brain

The basal forebrain complex (BFC) is the principal source of cholinergic input to the cerebral cortex, hippocampus, olfactory bulb, amygdala and habenula (Hb). The medial habenula nucleus (MHb) innervates interpeduncular nucleus (IPN). The pedunculopontine tegmental nucleus (PPT) and laterodorsal tegmental (LDT) areas innervate the brain stem at midbrain regions. Figure adapted from Picciotto, M.R. Neuropsychopharmacology (2002): The American College of Neuropsycho- pharmacology (ACNP): The Fifth Generation of Progress.

1.3 Nicotinic receptors

The first functional nicotinic receptor complex was isolated from the electric organs of the fishes Torpedo californica and Electrophorus electricus. The unique structure of the electric organ of torpedo with its high density of nAChR, combined with the availability of the high- affinity competitive antagonist, α-bungarotoxin (α-BgTx) which binds almost irreversibly to muscle-type nAChRs, allowed purifying the first nAChRs with affinity chromatography (DOLLY AND BARNARD 1984; KALAMIDA et al. 2007). These receptors have a high sequence homology with the nAChR at the NMJ (CHANGEUX et al. 1998). Introducing methods of reverse genetics led to the cloning and sequencing of the gene encoding the α1-subunit (NODA et al. 1983) and the subsequent cDNA cloning of the β1, γ, δ and ε subunits (LAPOLLA

et al. 1984; BOULTER et al. 1986b; BUONANNO et al. 1986). The isolation of the cDNA clone for the α4 subunit of neuronal nAChR followed soon (BOULTER et al. 1986a). To date, the gene sequences of 16 distinct nicotinic subunits have been identified in the mammalian genome (ALBUQUERQUE et al. 2009).

The discovery and crystallization of the acetylcholine binding protein (AChBP) from the snail Lymnaea stagnalis provided a milestone in understanding structural and functional properties of the nAChR at the atomic level (KALAMIDA et al. 2007; ALBUQUERQUE et al. 2009). AChBP, which is a structural and functional homolog to the extracellular ligand binding domain of the α7-nAChR, served as a valuable template for structure-function studies (DELLISANTI et al. 2007; KALAMIDA et al. 2007; ALBUQUERQUE et al. 2009; ZOURIDAKIS et al. 2009).

1.3.1 nAChR structure overview nAChR are ligand-gated ion channels and prototype members of the Cys-loop receptor superfamily, which also includes receptors for γ-aminobutyric acid (GABAA-ρ), serotonin (5- HT3) and glycine (LE NOVERE AND CHANGEUX 1995; CHANGEUX et al. 1998; KARLIN 2002). This superfamily shares a common structure (Cys-loop), consisting of a conserved sequence of 13-amino acids arranged as a loop by two cross-linked (disulfide bond) cysteines on the N-terminal domain (Fig. 3) (LESTER et al. 2004).

Nicotinic receptors are composed of five subunits. The basic structure of each nAChR subunit has four main parts. 1) A hydrophilic extracellular N-terminal domain consisting of 210–220 amino acids, which contains the agonist binding site. 2) Four highly hydrophobic α- helical transmembrane (TM) domains. 3) A long cytoplasmic loop between TM3 and TM4, which in size and amino acid sequence shows the highest variability between subunits. 4) A short C-terminal end with a variable amino acid sequence on the extracellular surface. The five subunits are arranged in a manner to form a central water-filled, cation-selective pore in the plasma membrane (Fig. 3). The second transmembrane (TM2) segment from each subunit lines the pore and determines physical aspects of receptor properties, such as ion selectivity and conductance (GALZI et al. 1992; GORNE-TSCHELNOKOW et al. 1994; MCGEHEE AND ROLE 1995; KARLIN 2002; KALAMIDA et al. 2007; ALBUQUERQUE et al. 2009; ZOURIDAKIS et al. 2009).

Generally, all subunits identified to date can be classified into two subgroups: α- and non-α (ALBUQUERQUE et al. 2009). α-Type subunits contain two adjacent cysteines (Cys192- Cys193) in their N-terminal part, which are homologous to the muscle α1 subunit. This pair

of vicinal cysteines is crucial for the principal ligand binding site (Fig. 3). The non-α subunits (i.e. β1–β4, γ, δ, and ε) lack the two vicinal cysteines and form the complementary binding site (KARLIN et al. 1986; LE NOVERE AND CHANGEUX 1995; CHANGEUX AND EDELSTEIN 1998; LUKAS et al. 1999). Currently, out of an overall of 17 subunits (α1–10, β1-4, γ, δ, ε), 16 subunits have been identified in the mammalian genome (KALAMIDA et al. 2007; ALBUQUERQUE et al. 2009; HURST et al. 2013).

Neuronal nAChRs heterologously expressed in Xenopus laevis oocytes revealed key features of receptor function. Hence, subunits combine in various compositions with distinct properties (ANAND et al. 1991; COOPER et al. 1991; LUETJE AND PATRICK 1991; LUKAS AND

BENCHERIF 1992; SARGENT 1993; MCGEHEE AND ROLE 1995). Accordingly, the subunits α7, α8, and α9 are capable of forming homo-oligomeric receptors (GOTTI et al. 1991; GERZANICH et al. 1994; MCGEHEE AND ROLE 1995; VETTER et al. 1999; ALBUQUERQUE et al. 2009). On the other hand, the subunits α2-α4 and α6 produce functional receptors only when co- expressed with either β2 or β4 (COUTURIER et al. 1990; LINDSTROM et al. 1995; MCGEHEE AND ROLE 1995; ALBUQUERQUE et al. 2009), whereas α10 combined with α9 greatly enhances the function of α9 (ELGOYHEN et al. 2001). Furthermore, α5 and β3 are considered accessory subunits, as they need the other essential subunits (both α and β) to assemble into functional receptors. These two subunits do not contribute to ligand binding but occupy a structural position in functional receptors (RAMIREZ-LATORRE et al. 1996; ALBUQUERQUE et al. 2009; GOTTI et al. 2009) (see also KURYATOV et al. 2008).

The agonist-binding site is formed at the interface between two adjacent α and β subunits, which contribute the primary and complementary part of the site, respectively (LUETJE AND PATRICK 1991; KARLIN 2002; GOTTI AND CLEMENTI 2004; ALBUQUERQUE et al. 2009). Whereas hetero-pentameric receptors have two binding sites, homo-pentameric receptors have five such sites. Hence, their α subunits contribute both the primary and complementary part (PALMA et al. 1996; GOTTI AND CLEMENTI 2004; KALAMIDA et al. 2007; ZOURIDAKIS et al. 2009). Binding-site occupancy by either ACh or exogenous agonists causes an allosteric change within the three-dimensional structure of the receptor, which in turn leads to opening of the ion channel and cation influx. This process may depolarize the cell membrane up to the threshold of activating voltage-gated Na+ – and in consequence – voltage-gated Ca2+ channels. Due to the high Ca2+ conductance of neuronal nAChRs, Ca2+ may, however, pass the plasma membrane even in the absence of a preceding action potential. The latter mechanism is of paramount importance for the action of nAChRs located at presynaptic sites (CHANGEUX et al. 1987; ROLE AND BERG 1996; WONNACOTT 1997; UNWIN et al. 2002; ALBUQUERQUE et al. 2009; ZOURIDAKIS et al. 2009).

Figure 3: Structure of the nicotinic acetylcholine receptor

Panel A. The basic structure of single receptor subunit indicating a large extracellular N-terminal region, four membrane-spanning domains (M1-M4), a large cytoplasmatic loop linking M3-M4 domains and the short C-terminal end. The N-terminal consist of a Cys-loop which is common to all nAChR subunits (marked in yellow connected circles). The vicinal Cys-Cys pairs is characteristic for α-type subunits (marked in red circles). The second transmembrane domain (TM2) which lines the pore of the ion channel determines physical aspects of receptor properties. Panel B. A top view of the nAChR structure showing the arrangement of five subunits around a central pore and TM2 the channel-lining region (marked in violet). Panel C. The schematic arrangement of five nAChR subunits in an assembled receptor. The arrows show ACh binding sites between α and the adjacent non-α subunit of the muscle type nAChR. Figure taken from KARLIN (2002).

Nicotinic receptors have three main functional states that include resting (closed), open, and desensitized at a given moment (WANG AND SUN 2005) (Fig. 4). Short exposure to agonists causes opening of the cation-selective pore. However, with the agonist still bound, receptors may enter a short-lived closed state. Long lasting or repetitive exposure to agonists leads to receptor desensitization, which is a non-conducting, high affinity agonist-bound state of the channel (OCHOA et al. 1989; PAPKE 1993; CORRINGER et al. 1998; WANG AND SUN 2005; DANI AND BERTRAND 2007; CIURASZKIEWICZ et al. 2013). The onset and duration of desensitization is in the millisecond-to-second range and highly dependent on the subunit composition of nAChR (VIBAT et al. 1995; FENSTER et al. 1997). Homomeric α7-nAChRs have the fastest rate of desensitization of any nAChRs. In heteromeric nAChRs, both the α and the β subunits contribute to the rate of desensitization. However, β2-containing nAChRs desensitize faster than β4-containing nAChRs (FENSTER et al. 1997; QUICK AND LESTER

2002; WANG AND SUN 2005).

Figure 4: Three main allosteric states of the nicotinic acetylcholine receptor

Resting channel closed: non-conducting state, active channel open: conducting state, desensitized state: non-conducting conformation with agonist bound. Figure taken from Wonnacott (2014): The Tocris Scientific Reviews: Nicotinic ACh Receptors.

1.3.2 Muscular nicotinic acetylcholine receptors

Due to the overall high sequence homology with nAChRs in the electric organs of Torpedo and Electrophorus, the muscle-type nAChR is the best-characterized member of the nAChR family. It is localized postsynaptically in the NMJ and consists of five different subunits: α1, β1, γ, δ (in embryonic or denervated muscle); or α1, β1, δ, and ε (in adult muscle) (TAKAI et al. 1985; ZOURIDAKIS et al. 2009). The interfaces between α subunits and an adjacent δ and γ (or ε) subunit form the two ligand-binding sites (Fig. 3). Because two different non-α subunits contribute, the two binding sites differ in their affinities for the agonist (BLOUNT AND MERLIE 1989). Activation of receptors is a prerequisite for the electrochemical transmission of impulses from motor nerves to muscle fibers, which thereafter leads to muscle contraction (KALAMIDA et al. 2007).

1.3.3 Neuronal nicotinic acetylcholine receptors

Neuronal nAChRs, which are homologous to muscle-type receptors, are widely distributed in the central and peripheral nervous systems (SARGENT 1993; MCGEHEE AND ROLE 1995; GOTTI AND CLEMENTI 2004). The different possible combination of known subunits (α2–α10 and β2–β4) has the potential to yield a wide array of nAChRs. Although only a small subset of possible combinations occurs naturally in the nervous system, individual receptors can contain up to four different subunits. Distinct receptors defined by their subunit composition are differentially expressed not only in different regions in the CNS but also between neurons within a specified region (see below) (LLOYD AND WILLIAMS 2000; GOTTI et al. 2006b). Neuronal nAChR are localized at postsynaptic, presynaptic or even axonal sites (WONNACOTT et al. 1990; LENA et al. 1993; HURST et al. 2013). In ganglia of the autonomic nervous system, postsynaptic nAChRs function by conveying fast synaptic transmission.

Localization of nAChRs in the CNS

In the CNS, nAChRs primarily function by modulating the release of neurotransmitters (MCGEHEE et al. 1995; ROLE AND BERG 1996; WONNACOTT 1997). Still, nAChRs are also located on cell bodies and dendrites, where upon the release of ACh they give rise to excitatory postsynaptic potentials (EPSP) (ROLE AND BERG 1996; ALBUQUERQUE et al. 2009). The presence of postsynaptic nAChR has been shown, for example, in the substantia nigra pars compacta (SNc), medial habenula, ventral tegmental area (VTA), interpeduncular nucleus (IPN), hippocampus, and in retinal ganglion cells (SARGENT 1993; ALKONDON et al. 1998; SORENSON et al. 1998). Due to the widespread distribution of pre- and postsynaptic receptors, nAChRs affect a wide range of physiological and pathophysiological processes

associated with cognitive functions, learning and memory, reward, arousal, and motor control (MCGEHEE AND ROLE 1995; ROLE AND BERG 1996; PICCIOTTO et al. 2000; GOTTI AND

CLEMENTI 2004). The unique characteristics and modulatory effects of distinct nAChRs on neurotransmitter systems make them potential therapeutic targets for the treatment of various disorders (JENSEN et al. 2005).

Presynaptic and Preterminal nAChRs

Neurotransmitter release from presynaptic sites may be either positively or negatively regulated by auto- and hetero-receptors (LANGER 2008). However, activation of nAChRs consistently enhances transmitter release (WONNACOTT 1997). Depending on the proximity to the release site (the active zone), they can be classified as presynaptic if residing on terminal boutons, or as preterminal when located at some distance from the release site (see Fig. 5) (WONNACOTT 1997; VIZI AND LENDVAI 1999). Presynaptic and preterminal modulate neurotransmitter release in two alternative ways.

Presynaptic nAChRs: Due to their substantial Ca2+ permeability, activation of neuronal nAChRs might per se provide sufficient Ca2+ influx to trigger exocytosis, if close enough to the release machinery (MULLE et al. 1992; RATHOUZ AND BERG 1994; ROGERS AND DANI

1995; LENA AND CHANGEUX 1997). On the other hand, activation of preterminal nAChR may lead to membrane depolarization followed by action potentials with ensuing calcium influx (by voltage-dependant Ca2+ channels, VDCC) at the active zone and subsequent neurotransmitter release (LENA et al. 1993; RATHOUZ AND BERG 1994; SOLIAKOV et al. 1995;

SORIMACHI 1995). In fact, regardless of being presynaptic or preterminal, the overall outcome of nAChR activation leads to elevated calcium at the active zone and thereby facilities neurotransmitter release.

Tetrodotoxin (TTX) has been a valuable tool to differentiate between presynaptic and preterminal nAChRs (WONNACOTT 1997; KRISTUFEK et al. 1999; VIZI AND LENDVAI 1999; ALBUQUERQUE et al. 2009). TTX inhibits voltage-gated sodium channels and thus action potentials (CESTÈLE AND CATTERALL 2000). Nicotinic agonist-induced transmitter release from preparations containing just the nerve endings for the respective transmitter system (synaptosomes) is mostly regarded as being caused by presynaptic receptors (but see WONNACOTT 1997).

By now it is generally accepted that presynaptic nAChR can facilitate the release of most classical neurotransmitters, including GABA, glutamate, ACh, and the monoamines dopamine, NA, and serotonin (MCGEHEE et al. 1995; WONNACOTT 1997; ALBUQUERQUE et al.

2000; GOTTI et al. 2009). Of interest, different subtypes of presynaptic/preterminal nAChRs may be present in different regions of the CNS (WONNACOTT 1997; GOTTI et al. 2009). For example, two different presynaptic nAChR subtypes modulate dopamine release in the striatum and ACh release in the IPN (GRADY et al. 2001). Presynaptic receptors may even differ for one and the same transmitter system (e.g. NA in the hippocampus and the cortex) (KENNETT et al. 2012). In striatal synaptosomes, α6β2* and α4 (non-α6)β2* nAChRs each contribute about 50% of the effect of nicotine on DA release (CHAMPTIAUX et al. 2003). GABA release from axon terminals may be induced in different brain regions by α4β2* nAChR activation (with or without the α5 subunit) (WONNACOTT 1989; LU et al. 1998; AZAM et al. 2003; MCCLURE-BEGLEY et al. 2009).

It seems that glutamatergic axon terminals express presynaptic nAChR with less heterogeneity. In most sub-cortical areas, glutamatergic transmission is modulated by α7- containing nAChRs (MCGEHEE et al. 1995; ALKONDON et al. 1996; GRAY et al. 1996; GIROD et al. 2000; MANSVELDER AND MCGEHEE 2000; BARAZANGI AND ROLE 2001; MARCHI et al. 2002; KALAMIDA et al. 2007). However, evidence has been provided in the frontal cortex (ROUSSEAU et al. 2005) and the hippocampus (ZAPPETTINI et al. 2010) that β2-containing receptors also modulate glutamate release. As shown in my thesis, the cholinergic endings (arising from the medial habenula) require β4 as an essential subunit for the nicotine- induced release of ACh. Since these axon terminals co-release glutamate along with ACh (REN et al. 2011), the release of glutamate in response to nAChR activation probably depends on the β4 subunit as well (see also FOWLER et al. 2011).

The numerous physiological and behavioral effects of nicotine are likely a consequence of the multifaceted modulation of various neurotransmitter systems in the CNS. Since nicotinic modulation of NA and ACh release in Hb-IPN system, the cortex, and the hippocampus are the main topic of my study, I will briefly review them below.

Figure 5: Putative locations of nAChR.

Figure taken from WONNACOTT (1997), Reviews: Presynaptic nicotinic ACh receptors

1.3.4 Distribution of nAChR in the CNS

As mentioned above, there are numerous possible subunit combinations that might assemble into a pentameric receptor. However, only a small subset is actually expressed in the CNS. Due to the binding of the snake toxin α-BgTx, neuronal nAChRs can be grouped into two major classes: α-BgTx-sensitive, and α-BgTx-insensitive (MARKS AND COLLINS 1982; CLARKE et al. 1985; GOTTI AND CLEMENTI 2004; GOTTI et al. 2006b; KALAMIDA et al. 2007; GOTTI et al. 2009; ZOURIDAKIS et al. 2009).

α-BgTx-sensitive nAChRs

α-BgTx-sensitive receptors exist as homo- or hetero-pentamers. Hence, the subunits α7-α9 may form homo-pentameric receptors (COUTURIER et al. 1990; GERZANICH et al. 1994; GOTTI

et al. 1994; GOTTI AND CLEMENTI 2004). α7 is the predominant homo-pentameric receptor in the mammalian CNS; these receptors show a low affinity for nicotine (CHEN AND PATRICK 1997). α7-containing receptors are also widely expressed in the PNS (CUEVAS AND BERG 1998; BIBEVSKI et al. 2000; BERG AND CONROY 2002; GOTTI AND CLEMENTI 2004) and in non- neuronal tissues and cells (e.g. lung, macrophages, and skin epithelial cells) (ARREDONDO et al. 2002; GAHRING AND ROGERS 2005; DE JONGE AND ULLOA 2007). However, α7 may co- assemble with β2 (LIU et al. 2009), as does α9 with α10, to form α-BgTx-sensitive, hetero- pentameric receptors (KEYSER et al. 1993; GOTTI et al. 1994; GOTTI AND CLEMENTI 2004; GOTTI et al. 2006b). Whereas recombinant homomeric α9 receptors are functional, α10 homomeric receptors are not. However, α10 combined with α9 confers 100- to 1000-fold larger macroscopic currents (PLAZAS et al. 2005). α9/10-containing receptors are present in cochlear hair cells (ELGOYHEN et al. 1994) and in sensory ganglia (LIPS et al. 2002; MCINTOSH et al. 2009). The α8-containing receptor has only been found in the chick nervous system where it may form homomeric α8 receptors or heteromeric α7–α8 receptors (KEYSER et al. 1993; GOTTI et al. 1994).

Though α7 receptors are widely expressed throughout the rat brain, they are predominately found in the limbic system (hippocampus, amygdala, and hypothalamus), forebrain and the cortex (SEGUELA et al. 1993; HOGG et al. 2003; GOTTI et al. 2006b; GOTTI et al. 2009;

BADDICK AND MARKS 2011). α7 receptors exhibit particularly high Ca2+ permeability (SEGUELA et al. 1993; CASTRO AND ALBUQUERQUE 1995; GOTTI AND CLEMENTI 2004; ALBUQUERQUE et al. 2009) and have fast kinetics for activation, desensitization (COUTURIER et al. 1990;

CASTRO AND ALBUQUERQUE 1995), and recovery, even after long-term exposure (KAWAI AND BERG 2001). α7 receptors are mostly located at GABAergic and glutamatergic presynaptic terminals and contribute e.g. to increased neurotransmitter release in the hippocampus (GRAY et al. 1996; ALKONDON et al. 1997; RADCLIFFE AND DANI 1998). Additionally, α7 nAChRs have been detected at glutamatergic axon terminals in the human neocortex, the rat striatum (MARCHI et al. 2002), the olfactory bulb (ALKONDON et al. 1996), MHb (GIROD

AND ROLE 2001), the VTA (JONES AND WONNACOTT 2004), the IPN (MCGEHEE et al. 1995), and the rat frontal cortex (ROUSSEAU et al. 2005). α7 receptors furthermore mediate fast synaptic transmission in restricted brain regions (JONES et al. 1999; GOTTI AND CLEMENTI 2004). Upon activation, α7 nAChRs contribute to calcium-sensitive intracellular signaling, which possibly leads to neuronal plasticity (GHOSH AND GREENBERG 1995; BROIDE AND LESLIE 1999; BERG AND CONROY 2002), long-term potentiation (HUNTER et al. 1994; FUJII et al. 2000; MANSVELDER AND MCGEHEE 2000), and improved memory performance (BETTANY

AND LEVIN 2001).

α-BgTx-insensitive nAChRs

α-BTX-insensitive nAChRs in the brain mainly consist of the α subunits α2, α4 and α6, in combination with β2 and/or β4 (ALBUQUERQUE et al. 2009). The pharmacological and

biophysical properties of these receptors depend on both α and β subunits (MCGEHEE AND ROLE 1995; ALBUQUERQUE et al. 2009). Amongst those, α4β2-containing receptors are the most prevalent subtype in the CNS (GOTTI et al. 2006; GOTTI et al. 2009). By showing slower kinetics and a high affinity for nicotine, their functional profile is quite different from α7 nAChRs (KALAMIDA et al. 2007; GOTTI et al. 2009). Accordingly, β2 and α4-KO mice lose 3H- nicotine binding in most parts of the brain (PICCIOTTO et al. 1995; ZOLI et al. 1998; PICCIOTTO et al. 2001).

Quite the opposite, α3β4-containing receptors are the most abundant subtype in autonomic ganglia (PERRY et al. 2002; WANG et al. 2002b; GOTTI AND CLEMENTI 2004; DAVID et al. 2010), the adrenal medulla (GOTTI et al. 2009), and in some restricted brain regions such as the pineal gland (PERRY et al. 2002; HERNANDEZ et al. 2004) and the Hb-IPN system (GRADY et al. 2009; SCHOLZE et al. 2012). Moreover, α6-containing receptors are prominently expressed in the rodent visual pathway (GOTTI et al. 2005) and in striatal dopaminergic neurons (LE NOVERE et al. 1996; CHAMPTIAUX et al. 2002; GOTTI AND CLEMENTI 2004).

1.4 Nicotine-induced ACh release in the brain

nAChRs as components of the cholinergic system are localized on both pre- and postsynaptic sites (CLARKE 1993; SARGENT 1993). The release of ACh from cholinergic axon terminals is under positive control by nACh autoreceptors (see below) or inhibited upon activation of muscarinic autoreceptors (MARCHI et al. 1981; RAITERI et al. 1984; VANNUCCHI

AND PEPEU 1995). Whereas inhibitory effects of presynaptic muscarinic receptors are undisputed (MARCHI et al. 1981; RAITERI et al. 1984; VANNUCCHI AND PEPEU 1995), observations on facilitated ACh release by nAChR activation in different brain regions such as a cortex, hippocampus and striatum have been inconsistent.

Hence, nicotine-induced ACh release has been demonstrated by using both synaptosomes and slice preparations from rodent hippocampus (ARAUJO et al. 1988; WILKIE et al. 1996), cortex (ROWELL AND WINKLER 1984; MARCHI AND RAITERI 1996; MARCHI et al. 1999), and the

IPN (GRADY et al. 2001; GRADY et al. 2009). However, nicotine failed to induce ACh release in the rat striatum (ARAUJO et al. 1988; LAPCHAK et al. 1989), and no involvement of nAChRs

in the release of ACh from the rat cortex was reported in another study (IANNAZZO AND MAJEWSKI 2000).

To date, few studies aimed at characterizing the types of nAChRs, which modulated ACh release in brain. Based on the pharmacological profile, α4β2-containing autoreceptors have been proposed to trigger ACh release in the hippocampus (WILKIE et al. 1996). More recently, Grady et al. identified α3β4 and α3β3β4 receptors that facilitated ACh release in the mouse IPN. The latter results were based on using the entire range of mice lacking distinct nAChR subunits (α2, α4, α5, α6, α7, β2, β3, and β4) (GRADY et al. 2009).

In my current thesis work, I sought to investigate ACh release in response to chemical (nicotinic agonists and elevated KCl) and electrical stimulation from the Hb-IPN system. For comparison, I also measured ACh release from hippocampal and cortical slices in both mice and rats.

1.5 Nicotine-induced noradrenaline release in the brain

A variety of nuclei in the brainstem serve as source for noradrenergic projections throughout the brain, the locus coeruleus (LC) probably being best known (STJARNE 1966; SAMUELS AND SZABADI 2008; ROBERTSON et al. 2013).

NA is known to play a critical role in brain’s behavioral and physiological processes, including attention, learning and memory, mood, and stress responses (WISE 1978; BREMNER et al. 1996), all of which processes being relevant for psycho-stimulant addiction. For example, stress responses, which promote the risk for relapse of addiction, directly activate the LC-NA system (BELUJON AND GRACE 2011; BRUIJNZEEL 2012). Moreover, NA has been shown to play a role in tobacco and nicotine withdrawal (reviewed in BRUIJNZEEL 2012). Such findings support the idea that nicotine indirectly - through activation of nAChR located on noradrenergic axon terminals - contributes to mechanisms of tobacco addiction (reviewed in BRUIJNZEEL 2012).

Arqueros et al (1978) were the first to show that nicotine (>1 mM) induced NA release from preloaded striatal and hippocampal slices (ARQUEROS et al. 1978). Later, nicotine-evoked NA release in the Hc (BRAZELL et al. 1991; SACAAN et al. 1995; SCHOLZE et al. 2007) and frontal cortex (SACAAN et al. 1995; SUMMERS AND GIACOBINI 1995; ANDERSON et al. 2000; KENNETT et al. 2012) has been widely reported. Accordingly, distinct nAChR subtypes

modulate NA release in the rat and mouse hippocampus (CLARKE AND REUBEN 1996; AZAM AND MCINTOSH 2006; SCHOLZE et al. 2007). Interestingly, nicotinic receptors that modulate NA release in the rat Hc differ from receptors that modulate NA in the frontal cortex (Kennett et al., 2012).

To my knowledge, there has been no report on nicotine-induced NA release in the Hb-IPN system. In my thesis work I investigated NA release in the Hb-IPN complex by nAChR activation and characterize the types of receptors, which mediate this effect.

1.6 Pharmacology of nicotinic receptors

1.6.1 Agonists of nAChRs

Nicotinic agonists use the binding site of ACh to cause the conformational changes which leads to activation of the receptor. Besides naturally occurring substances, attempts have been made to synthesize compounds which may preferentially activate particular types of receptors. Here I mention some of the agonists which are of particular relevance and/or which I have used in my experiments.

Acetylcholine

ACh is the endogenous neurotransmitter at cholinergic synapses. It is synthesized presynaptically and upon release rapidly degraded by AChE. In ACh release experiments, atropine must be added if effects due to the activation of mAChRs are to be avoided. I furthermore used hemicholinium to prevent the rapid re-uptake of choline by the CHT1. ACh binds to α4β2* receptors with high affinity compared with α3β4* receptors, whereas it binds to α7 nAChRs with much lower affinity (MARKS et al. 1986; ANAND et al. 1993; GOPALAKRISHNAN et al. 1995; XIAO et al. 1998; JENSEN et al. 2003; XIAO AND KELLAR 2004).

Nicotine

Nicotine (Nic), a natural prototype for nAChR agonists, is the major alkaloid present in Nicotiana tabacum and the cause of tobacco dependency. Because of this, nicotine is the reference agonist in research related to nAChRs. Nicotine more potently binds to α4- containing than to α3-containing receptors, whereas α7 receptors showed the lowest sensitivity in response to nicotine (MARKS et al. 1986; ANAND et al. 1993; FENSTER et al. 1997; GOTTI et al. 1997; KEM et al. 1997; XIAO AND KELLAR 2004). Nicotine can easily cross

the blood–brain barrier (BENOWITZ 2009; TEGA et al. 2013). It is rapidly degraded to cotinine

(BENOWITZ 2009).

Epibatidine

Epibatidine (EPI) is an alkaloid that has initially been isolated from the skin of the Ecuadorian frog Epipedobates tricolor. From trace amounts of the substance, recovered from the skin and thereafter stored for many years until new analyzing techniques had become available, the structure of EPI could be resolved in 1992 (see BADIO AND DALY 1994). It binds to all nAChR subtypes, though with differing affinities. EPI binds most potently to α4β2 receptors, followed by α3β4 (both in the picomolar range), but less so to α7 (in the nanomolar range) (GERZANICH et al. 1995; XIAO et al. 2004). EPI has been widely used to characterize heteromeric nAChRs in the mammalian CNS (PERRY et al. 2002; BADDICK AND MARKS 2011).

ABT-594

ABT-594 [(R)-5-(2-Azetidinylmethoxy)-2-Chloropyridine], the synthetic analogue of EPI, is a potent ligand for α4β2 nAChRs. The substance differs from EPI by an increased selectivity for α4β2 receptors and a decreased affinity for α3-containing and α7 receptors (DONNELLY- ROBERTS et al. 1998; MEYER et al. 2000). ABT-594 is best known for its analgesic effect, which is even higher than morphine (BANNON et al. 1998; DECKER et al. 1998). The analgesic properties of ABT-594 as well as its high selectivity for distinct nAChR subtypes make it a promising therapeutic agent for pain relief.

A-85380

A-85380 [(3-(2(s)-azetidinylmethoxy) pyridine)] is a potent full agonist of high selectivity for α4β2 nAChRs. Hence, A-85380 binds to heterologously expressed β2-containing receptors with much higher affinity than to β4-cotaining nAChR expressed in HEK-293 cells (XIAO et al. 2004). Likewise, an autoradiography study revealed that in brain regions known to express α3-containing receptors, [3H]A-85380 binding is significantly lower than the binding of [3H]epibatidine (reviewed in RUETER et al. 2006). Due to this selectivity, A-85380 is a promising substance for the treatment of pain (ABREO et al. 1996; RUETER et al. 2006).

(-)-Cytisine

Cytisine (CYT) is the toxic plant alkaloid extracted from the golden rain tree (Cytisus Laburnum). Due to its rigid conformation, cytisine has been used as a template for developing novel nicotinic ligands (CASSELS et al. 2005). Cytisine binds with high affinity to β2-containing nAChRs where it also acts as a partial agonist (with predominantly antagonistic properties). However, cytisine is a full agonist at β4-containing receptors

(LUETJE AND PATRICK 1991; COVERNTON et al. 1994; PAPKE AND HEINEMANN 1994; DAVID et al. 2010; BADDICK AND MARKS 2011).

1.6.2 Antagonists of the nAChR channels

Compared with agonists, antagonists are more heterogeneous with no obvious primary structure similarity (DALY 2005). nAChR antagonists can be competitive or non-competitive. Competitive antagonists reversibly compete with agonists for the same binding site (α/β or α/α subunit interface). The inhibitory effect of antagonists is surmountable by increasing the concentration of the agonist. Non-competitive antagonists interact with nAChR by binding to a site distinct from the agonist binding. Effects of agonists are therefore not overcome by increasing the concentration of agonists (ARIAS et al. 2006; ALBUQUERQUE et al. 2009).

1.6.3 Competitive antagonists

Dihydro-β-erythroidine (DhβE)

DhβE is an alkaloid derived from the seeds and other plant parts of Erythrina sp. Leguminosae. DhβE is a competitive antagonist, which blocks β2-containing receptors with higher potency than β4-containing receptors. However, α subunits also contribute to the binding properties of DhβE (HARVEY AND LUETJE 1996; CHAVEZ-NORIEGA et al. 1997; CHAVEZ-NORIEGA et al. 2000).

α-bungarotoxin (α-BgTx)

α-BgTx is a member of the α-neurotoxin family obtained from the venom of the elapid snake Bungarus multicinctus. α-BgTx specifically inhibits not only muscle-type but also homomeric

α7 nAChRs in a competitive manner (CLARKE 1992; DALY 2005).

Methyllycaconitine (MLA)

MLA is a diterpenoid alkaloid isolated from several larkspur species (Delphinium). MLA is a competitive antagonist in the nanomolar range at α7 nAChRs but also inhibits hetero- oligomeric receptors at higher concentration. It is used as a molecular probe for distinguishing between neuronal α7, non-α7, and muscle nAChR subtypes (WARD et al. 1990; ALKONDON et al. 1992; PALMA et al. 1996).

α-Conotoxins

α-Conotoxins (α-CTX), small, disulfide-rich peptides, are a subset of conotoxins (CTX), a large family of peptides isolated from marine cone snails. α-CTXs target different isoforms of nAChRs in the NMJ and the CNS (reviewed in AZAM AND MCINTOSH 2009). Hence, α-CTx GI specifically inhibits muscular-type receptors and has widely been used for this purpose at in vitro and in vivo experiments.

A variety of other α-CTXs were, on the other hand, found to specifically inhibit distinct neuronal nAChRs. Hence, α-CTX MII potently and selectively blocks α3β2 (fully blocked by 100 nM toxin) (CARTIER et al. 1996; HARVEY et al. 1997) and α6-containing nAChRs

(MCINTOSH et al. 2004), whereas α-CTX AuIB selectively inhibits α3β4 receptors. Moreover, a series of α-CTx MII and α-CTX BuIA analogs are more selective for α6-containing than for α3β2 and thus discriminate between α6β4 and α6β2 receptors (reviewed in AZAM AND MCINTOSH 2009). The α-CTXs α-CTX MII, α-CTX AuIB and α-CTX BuIA have successfully been used to characterize nAChRs involved in dopamine and NA release from striatum and the Hc, respectively (KAISER et al. 1998; LUO et al. 1998; AZAM et al. 2010).

α7 homo-pentameric receptors are effectively inhibited by α-CTX ImII, obtained from the venom of Conus imperialis. α-CTx RgIA, on the other hand, is an efficient antagonist at

α9/α10 receptors (reviewed in AZAM AND MCINTOSH 2009).

1.6.4 Non-competitive antagonists of the nAChRs

Mecamylamine

Mecamylamine (MCA) is a synthetic compound, which acts as a classical non-competitive and nonselective antagonist for nAChRs. MCA inhibits heteromeric neuronal nAChRs in the low-micromolar IC50 values and completely blocks them in concentrations of approximately 10 μM. It is a less potent antagonist for homomeric α7 (CHAVEZ-NORIEGA et al. 1997). It was used as a ganglionic blocker for the treatment of hypertension (STONE et al. 1956), an application that has been abandoned due to ganglionic side effects. MAC it is widely used in behavioral experiments because it can readily pass the blood brain barrier (YOUNG et al. 2001). Comparison of its effects with the action of antagonists that do not pass the blood- brain barrier (such as chlorisondamine) allows to determine the site of action (the central or peripheral nervous system) of nicotinic agonists (RUETER et al. 2003). MCA at relatively low doses effectively blocks effects of nicotine and improves abstinence rates (SHYTLE et al. 2002).

1.7 Structures of interest in my studies

1.7.1 Habenula

The habenula (Hb) as part of the epithalamus are a pair of small nuclei and an evolutionary conserved structure in all vertebrate species. In mammals, the Hb is anatomically subdivided into two distinct parts: the lateral habenula (LHb) and the medial habenula (MHb) (Fig. 6). The Hb complex, together with its connection, forms the dorsal diencephalic conduction pathway (DDC), which connects forebrain structures with areas of the mid- and hindbrain (KLEMM 2004) (Fig. 6C). It outputs to - and modulates - dopaminergic, serotonergic, and

noradrenergic monoamine midbrain areas (SUTHERLAND 1982) and thus plays a pivotal role in cognitive functions such as learning, memory and attention (LECOURTIER AND KELLY 2007). The Hb also influences behavior such as sleep, pain, sexual behavior, reward, and maternal behavior (reviewed in KLEMM 2004).

The stria medullaris as the main input to the Hb is composed of two individual pathways for the MHb and the LHb. The fasciculus retroflexus (FR) forms the main output of the Hb (KLEMM 2004; LECOURTIER AND KELLY 2007; HIKOSAKA 2010). It consists of two distinct parts: the core part arises from the MHb and terminates at the IPN, whereas the outer part originates from the LHb and projects to brainstem monoaminergic nuclei (HERKENHAM AND NAUTA 1979; LECOURTIER AND KELLY 2007).

The lateral habenula (LHb)

The LHb with its connectivity is a key structure to inhibit monoaminergic output from the mid-

and the hindbrain to forebrain limbic and striatal structures (reviewed in LECOURTIER AND KELLY 2007). The major afferent to the LHb arise from parts of the basal ganglia and lateral hypothalamic neurons as part of limbic regions (reviewed in HERKENHAM AND NAUTA 1977; LECOURTIER AND KELLY 2007). Additionally, the LHb receives reciprocal projection fibers from its targets, such as the median raphe and the VTA (reviewed in KLEMM 2004; LECOURTIER AND KELLY 2007). LHb cholinergic afferents arise from the entopeduncular nucleus (LECOURTIER AND KELLY 2007). The LHb mainly uses the FR bundle as output for projections to the VTA, substantia nigra, raphe nuclei, and the LC (reviewed in KLEMM 2004; LECOURTIER AND KELLY 2007).

Figure 6: The habenular nuclei (Hb) and the dorsal diencephalic conduction system

The habenula (Hb) is located in the dorsal diencephalon on both sides of the third ventricle (A) and is composed of two distinct nuclei: a LHb (Lateral habenula) and a MHb (Medial habenula) (B). The Hb forms a part of the dorsal diencephalon conduction (DDC) system that carries information from some forebrain nuclei to ascending brainstem nuclei (C: indicated by red lines). The DDC system acts in parallel to the medial forebrain bundle (C: indicated by green lines). Figure adapted from GEISLER AND

TRIMBLE (2008).

The medial habenula (MHb)

The MHb is unique in the brain due to its high density and the wide variety of nicotinic receptors (CLARKE et al. 1985; MULLE et al. 1991), particularly of the α3β4* type (QUICK et al. 1999; GRADY et al. 2009; SCHOLZE et al. 2012). Three components: the MHb, the FR bundle, and IPN constitute a special cholinergic system in the mammalian brain. The main input to the MHb consist of two distinct pathways: descending afferent fibers from the diagonal band of Broca (DBB) and the septofimbrial and triangular nuclei; and ascending fibers derived

from raphe nuclei and the LC (KLEMM 2004; LECOURTIER AND KELLY 2007). A part of stria terminalis which terminates in the MHb consists of both cholinergic and substance P- containing neurons (LECOURTIER AND KELLY 2007). Cholinergic neurons arise prominently from septum nuclei (n. fimbrialis septi and n. triangularis septi) (HERKENHAM AND NAUTA 1977; LECOURTIER AND KELLY 2007). The stria medullaris moreover conveys a major GABAergic inhibitory input from the diagonal band of Broca to the MHb (CONTESTABILE AND FONNUM 1983; LECOURTIER AND KELLY 2007) (Fig. 7).

The MHb outputs almost exclusively terminate in the IPN (HERKENHAM AND NAUTA 1979; KLEMM 2004; LECOURTIER AND KELLY 2007). Projection fibers are predominately cholinergic (KLEMM 2004; GRADY et al. 2009), glutamatergic (MCGEHEE et al. 1995; GIROD et al. 2000) and even both cholinergic and glutamatergic (REN et al. 2011) (Fig. 7).

The types of nAChRs that occur in the MHb have been a focus of intense research in recent years. MHb neurons express a large variety of hetero-pentameric nAChRs, namely α3β4, α4β2, α3α4β4, α3β2 α4β3β2*, α3β3β4*, and α6β3β4* nAChR (GRADY et al. 2009; SCHOLZE et al. 2012). Additionally, assembly of the α5 accessory subunit with both β2- and/or β4- containing receptors forms another subtype of nAChRs (e.g α3α5β2β4) and leads to further diversity of nAChR in the MHb (GRADY et al. 2009; SCHOLZE et al. 2012)

A major part of the hetero-pentameric nAChRs in the MHb consist of β4-containing nAChRs (GRADY et al. 2009; SCHOLZE et al. 2012). They are relevant for anxiety-related behavior as well as for withdrawal symptoms in nicotine dependence (SALAS et al. 2003b; FONCK et al. 2009). α5-containing nAChRs, on the other hand, constitute a small subset in the MHb (SCHOLZE et al. 2012) where they play a critical role in nicotine dependence and nicotine self adminstration (SALAS et al. 2009; FOWLER et al. 2011). Though α5 and/or β4 containing nAChRs in the habenular-IPN complex are apparently involved in nicotine dependence, the underlying mechanisms are not clear. In my thesis work I studied the release of ACh and NA in both the Hb and the IPN in response to nicotinic agonists as well as to electrical pulse stimulation. My experiments included rats, wild type (WT) mice, and mice lacking distinct nAChR subunit genes.

Figure 7: Most important afferent and efferent pathways of the Hb (habenula complex)

Blue and green lines show the axonal connections associated with Lateral habenula (LHb) and the Medial habenula (MHb), respectively; black lines are connected to both. The strength of the connection is indicated by the thickness of the lines. For abbreviations: see abbreviation list. Figure taken from HIKOSAKA (2008).

1.7.2 Interpeduncular nucleus (IPN)

The IPN as main target of the MHb refers to a group of cells located below the interpeduncular fossa in the midline of the ventral midbrain tegmentum. The IPN is surrounded anteriorly and posteriorly by the mammillary bodies of the hypothalamus and the pontine nuclei and dorsally by the VTA and the median raphe.

The IPN receives a major part of its input through the FR bundle, which mainly consists of cholinergic (KLEMM 2004; GRADY et al. 2009) and glutamatergic fibers (MCGEHEE et al. 1995; GIROD et al. 2000). In fact, cholinergic fibers were found to co-release both ACh and glutamate (REN et al. 2011). The IPN also receives significant input from the median raphe and - to a lesser extent - from dorsal tegmental regions and the LC (KLEMM 2004). The IPN

outputs ascending fibers to limbic structures and descending fibers to the monoaminergic nuclei in the brainstem (GROENEWEGEN et al. 1986; KLEMM 2004). Given the rich cholinergic innervation from the MHb and from forebrain nuclei, and a high turnover of ACh - paralleled with a high density of cholinergic markers - the IPN is presumably more relevant for cholinergic neurotransmission than other brain regions (SASTRY et al. 1979; CONTESTABILE AND FONNUM 1983; FONNUM AND CONTESTABILE 1984; ALBANESE et al. 1985; KLEMM 2004; GRADY et al. 2009). Both functional muscarinic and nicotinic receptors are present in IPN (ROTTER AND JACOBOWITZ 1984; TAKAGI 1984; GRADY et al. 2009). nAChRs in the IPN are located at both presynaptic and postsynaptic sites (MULLE et al.

1991; GRADY et al. 2001; GRADY et al. 2009). The IPN expresses high levels and a great variety of nAChR subunits. Hence, the subunits α3, α4, β2, β3, β4, and to a lesser extent α2, α5, and α6 have been found by immunoprecipitation (IP) in the IPN (GRADY et al. 2009; BEIRANVAND et al. 2014).

Unlike in many other parts of the brain, β4-containing receptors make up a major share of hetero-pentameric receptors in the IPN (GRADY et al. 2009; BEIRANVAND et al. 2014). About 20 % of these receptors contain both β2 and β4 subunits (BEIRANVAND et al. 2014). Observations on β2 null mice suggest that the subunits α2 and α5 co-assemble into β2- containing receptors (BEIRANVAND et al. 2014).

Injections of the local unaesthetic lidocaine not only into the MHb but also into the IPN increased the consumption of nicotine in rats, suggesting that both structures play a role in constraining the intake (FOWLER et al. 2011). However, while local injections of the potent α3β4 nicotinic antagonist 18-methoxycoronaridine (18-MC) in the MHb decreased nicotine self-administration, nicotine self-administration increased upon injection of 18-MC in the IPN (GLICK et al. 2011). It is therefore of interest to investigate transmitter systems, which may be modulated by nAChRs, and the types of receptors involved in the IPN.

In my thesis work, I now re-examined and extended previous data on ACh release from the IPN (Grady et al., 2009). Moreover, I studied for the first time the nicotine-induced NA release in both the Hb and the IPN and the types of nAChRs involved.

1.7.3 Hippocampus (Hc)

The Hc receives a rich cholinergic innervation via the medial septum-diagonal band complex

(WOOLF 1991; ALONSO AND AMARAL 1995; YOSHIDA AND OKA 1995). Cholinergic synapses are found on principal neurons as well as on interneurons (FROTSCHER AND LERANTH 1985; ALKONDON et al. 1997; RADCLIFFE et al. 1999) where they modulate cellular excitability and

synaptic transmission by both muscarinic and nicotinic cholinergic receptors (PICCIOTTO et al. 2000; WESS 2003; MCKAY et al. 2007; DREVER et al. 2011). The Hc is also innervated by ascending noradrenergic projection fibers from the LC (MOORE AND BLOOM 1979; LOY et al. 1980).

The Hc expresses a high diversity of nAChR subunits (α3-7 and β2-4) (AZAM AND MCINTOSH 2006; GOTTI et al. 2006). At least three distinct types of nAChRs have been characterized in the rat Hc: α7, α4β2 and α3β4 (ALKONDON AND ALBUQUERQUE 1993; ALKONDON et al. 1994; ORR-URTREGER et al. 1997; ZAREI et al. 1999; ALKONDON AND ALBUQUERQUE 2004). However, the most prevalent functional nAChRs in the Hc are comprised of the α7 and α4β2 subtypes (WADA et al. 1989; SEGUELA et al. 1993; RUBBOLI et al. 1994; ZAREI et al. 1999;

SUDWEEKS AND YAKEL 2000; ALKONDON AND ALBUQUERQUE 2004). α4β2 receptors may furthermore contain the subunits α6 and β3 (AZAM AND MCINTOSH 2006). nAChRs modulate neurotransmitter release in the Hc. Hence, in response to receptor activation, the release of excitatory and inhibitory neurotransmitters such as glutamate (GRAY et al. 1996; BANCILA et al. 2009) or GABA (ALKONDON et al. 1997; ALKONDON AND

ALBUQUERQUE 2001) is modulated. There is also evidence that activation of nAChRs modulate ACh release (MARCHI et al. 1981; ARAUJO et al. 1988; WILKIE et al. 1996;

WONNACOTT 1997).

The release of NA in response to nAChR activation has been subject to a number of studies (CLARKE AND REUBEN 1996; AZAM AND MCINTOSH 2006; SCHOLZE et al. 2007; AZAM et al. 2010; KENNETT et al. 2012). In the mouse, but not in the rat Hc, α6-containing receptors of the α6α4β2β3 and/or α6α4β2β3β4 types control NA release (AZAM AND MCINTOSH 2006; AZAM et al. 2010). For the rat Hc, on the other hand, α3β4* receptors have been proposed to at least contribute to nicotine-induced NA release (CLARKE AND REUBEN 1996; LUO et al. 1998). Hence, different types of nAChRs appear to be involved in the release of NA in the two species (AZAM AND MCINTOSH 2006).

However, the question of whether α5-containing receptors contribute to nicotine-induced NA release in the mouse Hc has not yet been addressed. Given the clear differences I found in α5-KO mice on NA release in the Hb and the IPN I conducted experiments aimed at investigating a possible contribution of α5-containing receptors on NA release in the mouse Hc.

1.7.4 Cortex

The cortex is strongly innervated by cholinergic afferents (LYSAKOWSKI et al. 1989; MESULAM et al. 1992). Basal forebrain nuclei are the main source of cholinergic inputs to the entire cortex (MESULAM 1995; ZABORSZKY et al. 1999; WOOLF AND BUTCHER 2011). In fact, ACh

appears to be a critical neurotransmitter for normal cognitive function (HASSELMO AND BOWER 1993; HASSELMO AND GIOCOMO 2006; SARTER et al. 2009; PICCIOTTO et al. 2012;

LUCHICCHI et al. 2014). ACh, by targeting nAChRs in neocortical and subcortical areas (PERRY et al. 1992), acts as a broad modulator of cortical activity (SCHLIEBS AND ARENDT 2011).

It is thus not surprising that almost all nicotinic agonists enhance cognitive performance (LEVIN et al. 2006; SARTER et al. 2014). In cortical slices preserving thalamo-cortical connections, it was shown that nAChR amplify the sensory information at excitatory (GIL et al. 1997; DISNEY et al. 2007; KAWAI et al. 2007; KRUGLIKOV AND RUDY 2008) and inhibitory synapses (CHRISTOPHE et al. 2002; GULLEDGE et al. 2007; ARROYO et al. 2012). Still, direct evidence on the release of ACh by nicotinic agonists has been controversial. Enhanced release by nAChR activation has been reported by (ARAUJO et al. 1988; LAPCHAK et al. 1989), whereas no such release was seen in human or rat neocortical slices (AMTAGE et al. 2004). However, electrochemical detection has unequivocally shown the release of glutamate upon nAChR activation (reviewed in SARTER et al. 2014).

α4β2 and α7 predominate in the cortex (ALKONDON AND ALBUQUERQUE 2004; ISHII et al. 2005; GOTTI et al. 2006), though α5-containing receptors (e.g the α4β2α5 subtype) are expressed to a lesser extent as well (HAN et al. 2000; ALKONDON AND ALBUQUERQUE 2004; WINZER-SERHAN AND LESLIE 2005; MAO et al. 2008; BAILEY et al. 2010). Recent data based on the positive allosteric modulator NS9283 suggest that activation of (α4)3(β2)2 autoreceptors play a dominant role in the release of glutamate (GRUPE et al. 2013).

In the present study I investigated the modulatory effect of nACh autoreceptors in cortical

slices from wild type mice and rats.

1.8 Knockout mice used in my experiments

1.8.1 α5-KO

α5 is considered an “accessory subunit” as it requires another α in addition to either a β2 or a β4 subunit for making a functional receptor (RAMIREZ-LATORRE et al. 1996). Yet, the presence of α5 has distinctive effects on the properties of receptors (RAMIREZ-LATORRE et al.

1996; GROOT-KORMELINK et al. 2001). α5-containing receptors are relatively restricted and/or concentrated to few regions in the CNS, such as the striatum, Hc, VTA, SNc and the Hb-IPN system (WADA et al. 1990; KLINK et al. 2001; ZOLI et al. 2002; MAO et al. 2008; GRADY et al. 2009; SCHOLZE et al. 2012; BEIRANVAND et al. 2014; XANTHOS et al. 2015). Mice null for the α5 nAChR subunit are normal in general behavior and gross anatomy (WANG et al. 2002b; SALAS et al. 2003a). However, they show a decreased sensitivity to nicotine-induced seizures and nicotine-induced hypolocomotion (SALAS et al. 2003a), show less withdrawal symptoms (SALAS et al. 2009; GAO et al. 2014), and have decreased aversion to high doses of nicotine in a self-administration paradigm (FOWLER et al. 2011; GAO et al. 2014). α5-KO mice were provided by Avi Orr-Urtreger, Genetic Institute of Tel Aviv University, Israel, on grounds of a cooperation.

1.8.2 β2-KO

β2-containing receptors are the most widely distributed nAChR subtype in the CNS (SARGENT 1993; MCGEHEE AND ROLE 1995) and are the main mediators of nicotine dependence (PICCIOTTO et al. 1998; WALTERS et al. 2006). Deletion of the β2 subunit strongly decreases 3H-nicotine binding in most part of brain regions (PICCIOTTO et al. 1995; ZOLI et al. 1998). Though mice lacking the β2 nAChR subunit do not have any obvious physical or autonomic deficits, they shown impaired attention and associative memory (PICCIOTTO et al. 1995; GUILLEM et al. 2011). Our β2-KO mice were generously provided by J.P. Changeux from the Laboratoire de Neurobiologie Moléculaire, Institute Pasteur in Paris, France.

1.8.3 α7-KO

Homomeric α7 nAChRs are widely expressed throughout the brain and are particularly enriched in the limbic system (SEGUELA et al. 1993). Mice lacking the α7 subunit have no detectable α-BgTx binding sites in the brain but are normal in general appearance, growth, survival, and anatomy (ORR-URTREGER et al. 1997). α7 nAChRs enhance glutamatergic neurotransmission and improve memory and cognition (MCGEHEE AND ROLE 1995; GRAY et al. 1996; ROLE AND BERG 1996; ORR-URTREGER et al. 1997; WONNACOTT 1997; BROIDE AND

LESLIE 1999; LEVIN AND REZVANI 2000). Hippocampal neurons in α7-KO mice failed to show rapidly desensitizing nicotinic currents (ORR-URTREGER et al. 1997). Mice lacking the α7 subunit are largely normal regarding the nicotine reinforcement pathway (NAYLOR et al. 2005; WALTERS et al. 2006) and base-line behavioral responses (PAYLOR et al. 1998). However, the presence of the α7 subunit is necessary to produce a full effect of nicotine

action on midbrain function (MAMELI-ENGVALL et al. 2006). We bought our α7-KO mice from Jackson Laboratories.

1.8.4 β4-KO

β4-containing nAChRs show high expression levels in restricted brain regions, including the olfactory bulb, medial habenula, pineal gland, IPN, and inferior colliculus (QUICK et al. 1999; PERRY et al. 2002; SALAS et al. 2003b; GAHRING et al. 2004; BADDICK AND MARKS 2011; SCHOLZE et al. 2012; BEIRANVAND et al. 2014). However, they are also detectable in the hippocampus, amygdala, and cortex (DINELEY-MILLER AND PATRICK 1992; PICCIOTTO et al. 2000). Mice lacking the β4 subunit are apparently normal but are less sensitive to nicotine- induced seizures and hypolocomotion (KEDMI et al. 2004; SALAS et al. 2004a). These mice have reduced nicotine withdrawal symptoms (SALAS et al. 2004b) and reduced anxiety-like behaviors (SALAS et al. 2003b). Our β4-KO mice were provided by Avi Orr-Urtreger, Genetics Institute, Tel Aviv University, Israel.

2 GOAL OF MY STUDIES

Ample experimental evidence has shown that nicotinic ACh receptors in the Hb-IPN complex play a pivotal role in the motivational drives that control nicotine intake. In my thesis project I focussed on two transmitter systems that may play a key role in nicotine dependency: ACh and NA, and asked the question whether and which types of nAChRs may modulate the release of these transmitters in the Hb-IPN complex. Rather than synaptosomes I used slices or intact pieces of tissue which allowed me to study transmitter release not only by chemical but also by electrical stimuli.

Given that pharmacological tools for specifying the types of neuronal receptors are scarce in the nAChR field, experiments were performed not only in rats but also in mice lacking distinct nAChR subunit genes. For reasons provided in the introduction I paid particular attention to the role of α5-containing receptors. I furthermore did some experiments on hippocampal and cortical slices in order to compare these results with observations made in the Hb-INP complex.

3 MATERIALS AND METHODS

A piece of text shown by quotation marks has been taken from the article of which I am the first author (BEIRANVAND et al. 2014).

3.1 Animals

Experiments were performed using 4-6 week-old Sprague-Dawley female rats (Oncins France strain A, Institute of Biomedical Research, Medical University of Vienna, Himberg, Austria). Additionally, 4-8 week old female and male WT C57Bl/6J mice and mice lacking the nAChR subunits α5 (WANG et al. 2002a), β2 (PICCIOTTO et al. 1995), α7 (ORR-URTREGER et al. 1997), and β4 (KEDMI et al. 2004) were used. All animals were housed in thermo-stable rooms (21° C) at a light-dark schedule of 10:14 hours in group cages (4-6 per cage) with free access to food and water. All procedures and care are on agreement with the Austrian federal law governing animal experimentation and the European Communities Council Directive (86/609/EEC) (Tierversuchsgesetz TVG 501/1989).

3.2 Experimental setup

In our experiments, the technique for tissue preparation and loading with the radiolabelled tracers and subsequent superfusion experiment to measure [3H]-NA and [3H]-ACh were as previously established in our lab with minor modifications (SCHOLZE et al. 2007).

3.3 Tissue preparation

Animals were anesthetized with carbon dioxide and sacrificed by decapitation. The skull was exposed by removal of the skin and opened, and the brain was recovered and placed into

ice-cold (4°C) low calcium loading buffer (see Table 2), saturated with 95 %O2/5 %CO2. The Hb and the IPN were dissected first. Both structures are readily discernible on the dorsal and the ventral side of the brain (see Fig. 8). The Hc and the (parietal) neocortex were next dissected and cut into 300–400 μm thick slices by means of a McIlwain tissue chopper (see Fig. 9). Slices were put back into ice-cold low calcium loading buffer and carefully separated using a stereo microscope.

3.4 Superfusion assay to induce the release of [3H] -choline and [3H]

-NA

After preparing the brain tissues, samples were loaded with radiolabelled tracers by incubation at 37° C in low calcium (loading) buffer for 120 min (IPN and Hb) or 60 min (hippocampal and cortical slices). Incubation with [3H]-NA (0.05 µM) was done in the presence of the irreversible MAO-A inhibitor clorgyline hydrochloride (0.5 µM). For measuring ACh release, samples were incubated with the ACh precursor [3H]-choline (0.1 µM). At the end of the incubation period, the brain tissues were rinsed two times with superfusion (2.5 mM calcium) buffer.

Subsequently, the tissues (one IPN, two Hb, or two slices per chamber) were placed with fire-polished Pasteur pipette into pre-warmed superfusion chambers between two nets and washed for an additional 60 min period with superfusion buffer, continuously bubbled with 95

%O2/5 %CO2. Superfusion at washing and at sample collection was done at a temperature of 29° C and a flow rate of 1 ml min-1.

For the [3H]-ACh release assay, 10 µM of the choline reuptake inhibitor (JOPE 1979) hemicholinium and 0.5 µM of the muscarinic receptor antagonist atropine were added to superfusion buffer for the last 10 minutes of washing and throughout sample collection. Subsequent to the 60-minute washout period, eight samples per channel were collected directly into scintillation vials at 2 min intervals and a flow rate of 1 ml min-1. Following a 6 min baseline collection period, stimulated [3H] outflow was achieved by electrical stimulation (100 pulses, 10 Hz, 0.5 msec, 40 V cm-1, 40 mA, unless otherwise mentioned), a 30-sec pulse of nicotine or cytisine, or a 90-sec pulse of elevated KCl.

The following antagonists were tested by adding them 10 min in advance, during, and after the stimuli indicated in the result section: MCA (to inhibit nAChRs); bicuculline (an inhibitor of

GABAA receptors); yohimbine (an inhibitor of α2 adrenoceptors); and tetrodotoxin (TTX) (an inhibitor of voltage-gated sodium channels). In order to probe transmitter release in the absence of calcium, the tissues were superfused with calcium-free buffer 10 min before and throughout the experiment. At the end of the experiment, the radioactivity retained by the tissue was recovered by extraction with 1 % sodium dodecyl sulfate (SDS) following 5 sec sonication using an ultrasonic homogenizer (Bandelin Sonopuls UW2200). Samples were mixed with scintillation cocktail (LSC-universal cocktail) and counted (TRI-CARB 2100TR, Packard).

3.5 Reagents

Table 1: Reagents

Chemical Supplier Molecular weight [g/mol]

Atropine Sigma 694.83

Ascorbic acid Sigma 176.12

Bicuculline Sigma 367.35

Choline chloride [Methyl-3H] ARC 139.6

Clorgyline hydrochloride Sigma 308.63

Disodium ethylenediaminetetraacetate Merck 372.2

Fumaric acid Merck 116.1

Glucose Merck 180.16

Hemicholinium Sigma 574.35

Levo-[ring-2, 5, 6-3H] ]-noradrenaline PerkinElmer 169.2

(-) -Nicotine Sigma 162.20

Mecamylamine hydrochloride Sigma 203.75

Monopotassium phosphate (KH2PO4) Merck 136.09

Magnesium sulfate (MgSO4) x 7 H2O Merck 246

Potassium chloride (KCl) Merck 74.55

Sodium chloride (NaCl) Merck 58.44

Sodium pyruvate Merck 110.04

Sodium bicarbonate (NaHCO3) Merck 84

Tetrodotoxin HCl (TTX) Latoxan 319.27

Yohimbine Merck 390.90

Table 2: Composition of loading and superfusion buffer*

Reagent Molarity (mM) MW (g/mol)

NaCl 118.00 58.44

KCl 4.80 74.55

CaCl2 x 2 H2O 0.2 147.02 Loading buffer

CaCl2 x 2 H2O 2.5 147.02 Superfusion buffer

MgSO4 x 7 H2O 1.20 246

NaHCO3 25.00 84

KH2PO4 1.20 136.08

Na2-EDTA 0.03 372.2

Glucose x H2O 11.00 198.17

Ascorbic acid 0.57 176.12

Fumaric acid 0.50 116.1

*Bubble with carbogen (95% O2 + 5%CO2) for 45min in advance

3.6 Equipment

For measuring transmitter release I used a semi-automated superfusion device made of identical 12 closed chambers. The superfusion buffer was pumped through tubing supplying these chambers by means of a multi-channel peristaltic pump (Ismatec). Superfusates leaving the chambers were collected by means of a fraction collector (Pharmacia LKB- SuperFrac). For chemical stimulation (nicotinic agonists or elevated KCl), the influx to chambers contained the respective stimulus with or without prior addition of an antagonist. Electrical stimulation was achieved by applying rectangular electrical pulses (programmable pulse generator from Hugo Sachs Elektronik) to platinum electrodes, located above and below tissue samples.

3.7 Tissue preparation (Figure)

The preparation of tissue is described in Fig. 8 and Fig. 9.

B C

Rat brain, dorsal view Rat brain, ventral view

Figure 8: Preparation of intact Hb and IPN nucleus A: The rat brain was removed from the skull and immediately submerged in ice-cold low-calcium uptake buffer B: Dorsal view of the rat brain to dissect habenular nuclei. C: Using a ventral view of the rat brain to dissect IPN (Interpeduncular nucleus).

A B

C D

Figure 9: Slice preparation of the rat hippocampus

A and B: brain hippocampi were dissected C: the tissue was chopped D: slices were separated using a light microscope.

3.8 Data analysis

The radioactive content of fractions was calculated by dividing their radioactivity by the total radioactivity of tissue at the beginning of the corresponding 2 min collection period. The total radioactivity at a given time was calculated by adding up the residual radioactivity (retained by tissue) with the radioactivity of all fractions collected onwards from this point according to the following equation:

Fn = [Rn / Rt + (R1+……. +Rn-1)] *100

Where

Fn = the radioactive outflow of a fraction n

Rn = mean of [3H]-radioactive content of 2 min in the n position in d.p.m

Rt = total radioactivity retained by tissue

R1+……. +Rn-1= radioactivity of all fractions collected onwards from n position

I usually collected three fractions under basal conditions (before stimulation-evoked release), and calculated the mean of these fractions for setting a baseline. Stimulation-induced release was calculated by normalizing to this (it est release in response to a stimulus divided by baseline). Stimulation was started at the beginning of the 4th fraction, and radioactivity was usually collected for four additional fractions. The stimulation-evoked release was then calculated as the area-under-the-curve (AUC) using GraphPad Prism version 5.00 for Windows (GraphPad Software) (see Fig. 10A).

3.9 Statistics

All data are presented as the means ± SEM (standard error of the mean) of usually at least six replicates per group. The unpaired Student’s t-test was used for assessing differences between two related data sets, whereas ANOVA (one-way analysis of variance) was applied when comparing multiple sets of data. When ANOVA showed a significant difference (P <0.05), post-hoc multiple comparison tests were applied: Tukey's test for a pairwise comparison; or Dunnett’s test for comparison of a datasets with reference group.

“Datasets consisting of three groups were compared pairwise using Fisher’s least significant difference (LSD) test: when comparing only three groups, no further adjustment for multiple testing is required when the global null hypothesis was rejected. Dose-response curves for the agonists were fitted using non-weighted non-linear regression to the dose-response curve (GraphPad Prism). An F test was used to test for different versus shared (identical)

EC50 parameter values. Differences are considered significant if P < 0.05 (BEIRANVAND et al. 2014) ”.

4. Results

4.1 [3H]-ACh and [3H]-NA release from the rat IPN

In a first step, I examined ACh ([3H]-ACh) and NA ([3H]-NA) release from intact rat IPN by applying electrical and chemical stimuli (Figure 10). ACh release has previously been investigated in synaptosomes of the mouse IPN (GRADY et al. 2001; FOWLER et al. 2011) However, transmitter release from synaptosomes can only be induced by chemical but not by electrical stimuli. Nicotine dramatically increased the release of [3H]-ACh in a

concentration-dependent manner (EC50 = 47.9 µM) and with a maximum release of 5.5 AUC (see Methods for technical details) (Figure 10B, D). Cytisine, which is a full agonist for β4- containing but a predominantly (partial) antagonist at β2-containing receptors (PAPKE AND HEINEMANN 1994), increased [3H]-ACh release to the same extent nicotine did (Figure 10B), suggesting that β4-containing receptors are the main population of nicotinic receptors which causes the [3H]-ACh release from presynaptic endings. MCA, a non-selective antagonist of nAChRs, potently inhibited the release induced by nicotine (Figure 10B). The nicotine- induced release was TTX-insensitive and thus not depending on action potentials. It was, however, dependent on extracellular calcium (Fig. 10B).

Quite the opposite, electrical pulses (100–300 pulses at 10 Hz) had only a small effect (AUC: 0.23), and this release was not significantly inhibited by TTX (Figure 10C). According to a previous report (REN et al. 2011), signaling by nAChR activation in the IPN requires a sustained train of stimuli applied to the cholinergic afferents (the fasciculus retroflexus). I thus modified our regular stimulation protocol by increasing the number and frequency of pulses to 1000 and 50 Hz, respectively. By applying this protocol I could measure a moderately increased [3H]-ACh outflow over baseline (1.2 ± 0.2 AUC), and this release was partially TTX-sensitive (Fig. 10C). Consistent with these results, ACh release in response to elevated KCl in the superfusion buffer (from 4.8 to 25 mM) was relatively low (AUC: 1.4). The KCl-induced [3H]-ACh release was dependent on extracellular calcium but not inhibited in the presence of TTX (Fig. 10C).

The effects of nicotine strikingly differed when measuring the release of [3H]-NA release in the IPN. Hence, the nicotine-induced [3H]-NA release was not only much smaller than nicotine-induced [3H]-ACh release (AUC 0.56 ± 0.08 at 100 µM nicotine), it was also fully inhibited by TTX (Fig. 10E). The partial β2 agonist cytisine was as effective as nicotine at 100 µM, whereas [3H]-NA outflow due to 10 µM cytisine was significantly smaller than

release induced by 10 µM nicotine (P < 0.01, unpaired Student’s t-test; Fig. 10E). I found 3 nicotine induced [ H]-NA outflow with an EC50 of 2.9 µM and an efficacy of 0.61 (Fig. 10F).

Unlike [3H]-ACh, electrical pulses induced significantly more [3H]-NA outflow than nicotine (P < 0.01, unpaired Student’s t-test; comparison between the [3H]-NA outflow in response to 10 μM nicotine and 100 pulses). The electrically induced [3H]-NA release was fully inhibited by TTX (Fig. 10E).

As mentioned above, I found electrically induced [3H]-ACh to be relatively low. In order to exclude that simultaneously released NA or GABA exert an inhibitory effect on this outflow I tested yohimbine (an α2 adrenoceptor antagonist) and bicuculline (a GABA-A receptor antagonist). However, application of neither substance led to an enhanced [3H]-ACh outflow in the IPN (Fig. 10C). Of interest, yohimbine was also ineffective in enhancing the electrically stimulated outflow of [3H]-ACh in the hippocampus (Fig. 10A), whereas it enhanced the electrically induced [3H]-NA outflow in the IPN by inhibiting α2 autoreceptors, as expected (Figure 10E).

Figure 10: Chemically and electrically induced [3H]-ACh and [3H]-NA release from rat IPN

A) [3H]-ACh release in response to a 30 s pulse of 30 μM nicotine (indicated by the horizontal bar at the 4 min time point). Atropine (0.5 μM) and hemicholinium (10 μM) were present throughout the experiment. Each data point represents the mean ± SEM of three separate IPN tissue samples. The shaded area under the curve (AUC) for this experiment was 2.2. (B) [3H]-ACh release in response to

nicotine or cytisine. (B1) TTX had no significant effect on the release induced by 30 μM nicotine (P >

0.05, Student’s t-test). (B2) Data were analyzed using ANOVA (significant with F6, 49 = 12.94 and P < 0.0001) and Dunnett’s post hoc test with 100 μM nicotine as the reference data (designated by #). (C) 3 [ H]-ACh release in response to high potassium or electrical pulses. (C1) Data were analyzed using

ANOVA (significant with F2, 20 = 3.58 and P = 0.047) and Fisher’s least significant difference (LSD) test for pairwise group comparisons. (C2) Data were analyzed by one-way ANOVA (significant with F6,

59 = 12.60 and P < 0.0001), followed by Tukey’s post hoc test for pairs of datasets. (D) Dose– 3 response curve of nicotine-induced [ H]-ACh release. Nicotine EC50: 47.9 μM, maximum release effect (AUC): 5.5. Each data point represents ≥9 separate measurements. (E) [3H]-NA release in response to the indicated stimuli. (E1)Student’s unpaired t-test was applied for a comparison of data with equal agonist concentrations. (E2) Release data were analyzed by one-way ANOVA (significant with F2, 24 = 16.70 and P < 0.0001) and pairwise group comparisons using Fisher’s LSD test. (F) 3 Dose–response curve of nicotine-induced [ H]-NA release. Nicotine EC50: 2.9 μM; maximum effect (AUC): 0.61. Each data point represents ≥6 separate measurements. All data are presented as the mean ± SEM. All concentrations are in μM, and the numbers in parentheses indicate the number of measurements. Bicuc, 30 μM bicuculline; Cyt, cytisine; MCA, 10 μM mecamylamine; Nic, nicotine; TTX, 1 μM tetrodotoxin; Yohim, 2 μM yohimbine; 100 P, 100 pulses (0.5 ms, 10 Hz, 40 mA). ns, P >

0.05; ***P < 0.001; **P < 0.01; *P < 0.05. Figure and legend taken from BEIRANVAND et al. (2014).

4.2 [3H]-ACh and [3H]-NA release from the rat Hb

I was next interested whether in the Hb, [3H]-ACh release and [3H]-NA behaved similar to the IPN and thus measured [3H]-ACh outflow by chemical and electrical stimulation in this structure. Unlike the IPN, nicotine-induced release of [3H]-ACh was hardly detectable (AUC: 0.03; Fig. 11A). However, similar to the IPN, [3H]-ACh outflow in response to electrical pulses was also low (AUC: 0.2), though TTX-sensitive (Figure 11A), whereas [3H]-ACh outflow by 25 mM KCl appeared slightly smaller than in the IPN (AUC: 0.56, Fig. 11A), was calcium-dependent, but TTX-insensitive (Figure 11A).

3 Nicotine concentration-dependently induced [ H]-NA release with an EC50 of 8.3 µM and a maximal effect of 0.57 (AUC), which was similar to the IPN (Fig. 11B, C). Nicotine responses were fully blocked in the presence of MCA and TTX (Fig. 11B). As in the IPN, the electrically-induced [3H]-NA release (by 100 pulses) was larger than nicotine-induced release (by 30 µM) (P < 0.001, unpaired Student’s t-test). Moreover, cytisine was equally effective with nicotine at 100 µM, but was significantly less efficacious at 10 µM (P < 0.001, unpaired Student’s t-test; Fig. 11B).

Figure 11: Chemically and electrically induced [3H]-ACh and [3H]-NA release from rat Hb

3 A) [ H]-ACh release in response to the indicated stimuli. (A1) Due to low levels, release in response to nicotine was not investigated further for TTX sensitivity or dependence on calcium. (A2) Difference was analyzed by Student’s unpaired t-test. (A3) Release data were analyzed by one-way ANOVA

(significant with F2, 24 = 4.25 and P = 0.026) and Fisher’s least significant difference test for pairwise 3 group comparisons. (B) [ H]-NA release in response to the indicated stimuli. (B1) Student’s unpaired t- test was applied for a comparison of data with equal agonist concentrations. Note that 10 μM nicotine was less efficacious than 10 μM cytisine, whereas the effect of 100 μM did not differ significantly from

100 μM cytisine. (B2) Difference was analyzed by Student’s unpaired t-test. (C) Dose–response curve 3 of nicotine-induced [ H]-NA release. Nicotine EC50: 8.3 μM, maximum release (AUC): 0.57. Each data point represents ≥6 separate measurements. All data are presented as mean ± SEM. All concentrations are in μM; the numbers in parentheses indicate the number of measurements. Cyt, cytisine; Nic, nicotine; MCA, 10 μM mecamylamine; TTX, 1 μM tetrodotoxin; 100 P, 100 pulses (0.5 ms, 10 Hz, 40 mA); 25 KCl: 25 mM KCl. ns, P > 0.05; ***P < 0.001; **P < 0.01; *P < 0.05. Figure and legend taken from BEIRANVAND et al. (2014).

4.3 [3H]-ACh release in the rat hippocampus and cortex

In the next step I examined [3H]-ACh release in the parietal cortex and the hippocampus for comparison with data from the IPN and the Hb. Nicotine-induced [3H]-ACh release was hardly detectable in the hippocampus (AUC 0.18; Fig. 12A) and also low in the cortex (AUC: 0.05; Fig. 12B). However, electrical field stimulation with our standard electrical pulse protocol caused a large outflow of [3H]-ACh in both the cortex and the hippocampus (AUC: 1.4 and 1.8 respectively; Fig. 12A, B), almost 10 times more than either in the Hb or the IPN.

Increasing the pulse number between 10 and 100 increased [3H]-ACh release approximately linearly (Fig. 12A). The electrically-induced release of [3H]-ACh was fully inhibited by TTX in both the hippocampus and the cortex (Fig. 12A, B). Similar to the IPN, yohimbine failed to increases the electrically-induced [3H]-ACh release in the Hc (Fig. 12A). However, excluding both the choline reuptake inhibitor hemicholinium and the muscarinic receptor antagonist atropine from the superfusion buffer reduced the [3H]-ACh released upon electrical stimulation by approximately 50% (P < 0.01; one-way ANOVA with Dunnett’s post hoc test; Fig. 12A).

Figure 12: Nicotine-induced and electrically induced [3H]-ACh release from rat hippocampal .and parietal cortical slices

(A and B) [3H]-ACh release from hippocampal slices (A) and cortical slices (B) in response to the indicated stimuli. Nicotine-induced release was small in both the hippocampus and the cortex and was therefore not analyzed further. The data for electrically induced [3H]-ACh release in panel (A) were analyzed by one-way ANOVA (significant with F5, 57 = 5.07 and P = 0.0007), followed by Dunnett’s post hoc test (referenced to 100 pulses; indicated by #). (B) TTX fully prevented electrically induced release in cortical slices. The data are presented as mean ± SEM. All nicotine concentrations are in μM; the numbers in parentheses represent the number of measurements. Atr, atropine; HMC, 10 μM hemicholinium; Nic, nicotine; TTX: 1 μM tetrodotoxin; Yohim, 2 μM yohimbine; 100 P, 100 pulses (0.5 ms, 10 Hz, 40 mA). ns, P > 0.05; **P < 0.01. Figure and legend taken from BEIRANVAND et al. (2014).

4.4 Release of [3H]-ACh and [3H]-NA in the mouse IPN

The availability of mice with targeted deletion of distinct nAChR subunits enabled me to investigate the effect of these subunits on the nicotine-induced [3H]-NA and [3H]-ACh release in the mouse IPN and Hb. Nicotine concentration-dependently stimulated [3H]-ACh outflow

with an EC50 of 8.3 µM and an efficacy of 3.5 AUC (Fig. 13C) in wild type mice. Hence, nicotine induced [3H]-ACh release at lower concentration but with higher efficacy in the

mouse compared to the rat IPN (P < 0.05 and P < 0.01 for EC50 and efficacy, respectively; F- test). However, I observed several similarities in the [3H]-ACh release between the rat and the mouse IPN. (i) [3H]-ACh release induced by nicotine was calcium-dependent and TTX- insensitive (Figure 13A); (ii) our standard electrical pulse protocol produced extremely low responses (AUC: 0.15 with 100 pulses; 1.3 with 1000 pulses; Fig. 13B); and (iii) the response upon raising extracellular KCl from 4.8 to 25 mM was TTX-insensitive and partially calcium-dependent (Figure 13B). Moreover, elevating extracellular KCl to 15 mM induced only a marginal, TTX-insensitive release in the IPN (Figure 13B). I found the same KCl concentration was clearly more efficacious in inducing TTX-sensitive [3H]-ACh release in the Hc (Fig. 13A).

I did not see differences in the nicotine-induced [3H]-ACh release between wild type mice on one hand and mice lacking the α5, the α7, or the β2 subunit genes. However, [3H]-ACh release was abolished in mice with targeted deletion of the β4 subunit (Figure 13A).

Overall, [3H]-NA release in the mouse was similar to the rat IPN. Nicotine concentration- 3 dependently induced [ H]-NA with an EC50 and a maximum release of 1.34 μM and 0.54 AUC, respectively (Figure 13E), and this effect was inhibited by both MCA and TTX. Likewise, the [3H]-NA release in response to electrical pulses was significantly larger (AUC: 2.5) than the effects of nicotine (Fig. 13D).

Interestingly, the nicotine-induced [3H]-NA release in the IPN was reduced in the β2- and the β4-KO to nearly undetectable levels (Figure 13D). This suggests that each of the two subunits is necessary - but not sufficient - for the nicotine-dependent [3H]-NA release in the mouse IPN. Quite differently, deleting the α5 subunit increased the Hill coefficient (from 1.3

to 4.4) and caused a 50-fold shift in the EC50 (Figure 13E), indicating that nACh receptors in α5-KO mice were rendered less sensitive to nicotine. In contrast, deletion of the α7 subunit had no effect on the nicotine–induce [3H]-NA release from IPN (Figure 13D). Compared to WT, all three KO mice (α5-, β2- and β4-KO) had furthermore similar levels of electrically-induced [3H]-NA release (Figure 13D), suggesting that gene deletions did not affect the transmitter release machinery.

3 3 Figure 13: Chemically and electrically induced [ H]-ACh and [ H]-NA release in mouse IPN

(A) [3H]-ACh release in response to nicotine. Differences were analyzed by one-way ANOVA

(significant with F7, 87 = 6.44 and P < 0.0001), followed by Dunnett’s post hoc test (referenced to WT 3 30 μM nicotine; indicated by #). (B) [ H]-ACh release in response to the indicated stimuli. (B1)

Differences were analyzed by one-way ANOVA (significant with an F4, 25 = 62.37 and a corresponding

P value < 0.0001), followed by Tukey’s post hoc test for pairs of datasets. (B2) Differences were analyzed by one-way ANOVA (significant with F2, 29 = 18.36 and P < 0.0001) and Fisher’s least significant difference test for pairwise group comparisons. (C) Dose–response curve of nicotine- 3 induced [ H]-ACh release. Nicotine EC50: 8.3 μM, maximum effect (AUC): 3.5. Each data point 3 represents ≥6 separate measurements. (D) [ H]-NA release in response to the indicated stimuli. (D1)

Data were analyzed by one-way ANOVA (significant with F8, 112 = 8.42 and P < 0.0001), followed by

Dunnett’s post hoc test (referenced to WT 30 μM nicotine; indicated by #). (D2) Data in response to electrical stimuli were analyzed using one-way ANOVA (significant with F4, 30 = 4.01 and P = 0.010), followed by Dunnett’s post hoc test (referenced to 100 pulses; indicated by #). (E) Dose–response 3 curve of nicotine-induced [ H]-NA release. Data from WT mice are shown: nicotine EC50: 1.34 μM (confidence interval 0.38–4.71 μM); maximum effect: 0.54; Hill coefficient 1.3. With data from α5-KO mice: nicotine EC50: 69.24 μM (confidence interval 52.25–91.76 μM); maximum effect: 0.48; Hill coefficient: 4.4. Although conspicuous, the shift in the dose–response curve is not significant (F1,113 = 1.218 and P = 0.272). Each data point represents ≥6 separate measurements. The data are presented as mean ± SEM. All concentrations are in μM; the numbers in parentheses represent the number of measurements. MCA, 10 μM mecamylamine; Nic, nicotine; TTX, 1 μM tetrodotoxin; 100 P, 100 pulses (0.5 ms, 10 Hz, 40 mA); 15 KCl and 25 KCl: 15 mM and 25 mM KCl respectively. WT: C57BL/6J mice; α5-KO, α7-KO, β2-KO and β4-KO: α5-knockout, α7-knockout, β2-knockout and β4- knockout mice respectively. ns, P > 0.05; ***P < 0.001; **P < 0.01; *P < 0.05. Figure and legend taken from BEIRANVAND et al. (2014).

4.5 Release of [3H]-ACh and [3H]-NA in the mouse Hb

Similar to the rat, the electrically-induced [3H]-ACh release (AUC: 0.86) was larger than [3H]- ACh overflow in response to nicotine, which was nearly undetectable (AUC: 0.04) (Figure 14A). Based on the very low efficacy of nicotine I did not probe for TTX or calcium sensitivity and whether nicotine effects could be blocked by MCA. However, [3H]-ACh release by electrical stimulation was inhibited by TTX (Figure 14A).

[3H]-NA release from the mouse Hb quite closely resembled observations in the IPN. Hence, nicotine induced release in a TTX-sensitive manner, and this release was inhibited by MCA (Figure 14B). Besides, [3H]-NA release in response to nicotine was much reduced in β2- and β4-KO mice, whereas the release in α7-KO mice was quite comparable to WT (Figure 14B).

I found that nicotine induced release in the wild type mouse Hb with an EC50 and maximum release 11.87 μM and 0.8 AUC, respectively (Fig. 14C). However, deletion of the α5 subunit increased both the Hill coefficient (from 1.1 to 3.3) and the EC50 (to 77.3 μM) (Figure 14C), similar to the IPN. Likewise, the electrically-induced [3H]-NA release was quit large in comparison with the response of nicotine (AUC: 3.1 with 100 pulses in WT mice) and comparable in size in the different genotypes (Fig. 14C).

Figure 14: Chemically and electrically induced [3H]-ACh and [3H]-NA release from the mouse Hb

A) [3H]-ACh release in response to the indicated stimuli. Nicotine-induced release was small and therefore not analyzed further. The release data in response to electrical stimuli were analyzed using one-way ANOVA (significant with F3, 23 = 4.05 and P = 0.019), followed by Dunnett’s post hoc test (referenced to 100 pulses; indicated by #). (B) [3H]-NA release in response to the indicated stimuli.

(B1) Differences were analyzed using one-way ANOVA (significant with an F8, 98 = 6.16 and P <

0.0001), followed by Dunnett’s post hoc test (referenced to WT 30 μM nicotine; indicated by #). (B2)

Differences were analyzed using one-way ANOVA (significant with F4, 28 = 12.11 and P < 0.0001), followed by Dunnett’s post hoc test (referenced to WT 100 pulses; indicated by #). (C) Dose– 3 response curve of nicotine-induced [ H]-NA release. With data from WT mice: nicotine EC50: 11.87 μM (confidence interval: 2.93– 48.07 μM); maximum effect: 0.80; Hill coefficient: 1.1). With data from α5-

KO mice. Nicotine EC50: 77.3 μM (confidence interval: 55.2–108.4 μM); maximum effect: 0.87; Hill

coefficient: 3.3). The shift in the dose–response curve is not significant (F1, 112 = 0.79 and P = 0.375). Each data point represents ≥6 separate measurements. The data are presented as mean ± SEM. All concentrations are in μM; the numbers in parentheses represent the number of measurements. MCA, 10 μM mecamylamine; Nic, nicotine; TTX, 1 μM tetrodotoxin; 100 P, 100 pulses (0.5 ms, 10 Hz, 40 mA). WT: C57BL/6J mice; α5-KO, α7-KO, β2-KO and β4-KO: α5-knockout, α7-knockout, β2-knockout and β4-knockout mice respectively. ns, P > 0.05; **P < 0.01; *P < 0.05. Figure and legend taken from

BEIRANVAND et al. (2014).

4.6 Release of [3H]-Ach and [3H]-NA in the mouse hippocampus and cortex

I observed some subtle differences between mice and rats in that [3H]-ACh release in response to 30 μM nicotine was larger in mice in the cortex (AUC: 0.3) than in the hippocampus (AUC: 0.07; Fig. 15A,B). Quite differently, electrical pulse stimulation (100 electrical pulses) was more effective in the hippocampus (AUC: 3.0) than in the cortex (AUC: 0.96; Fig. 15A, B). The release induced by elevating KCl to 15 mM was partially TTX- and calcium-dependent, whereas release in response to 25 mM KCl was TTX-insensitive but greatly reduced in the absence of calcium (Fig. 15A).

As mentioned above, deletion of α5 had a major impact on the nicotine-induced release of [3H]-NA in the mouse Hb and IPN. I thus added experiments to study [3H]-NA release in the hippocampus of wild type and α5-KO mice. Interestingly, and unlike Hb and IPN, the nicotine-induced release did not differ significantly between wild type and α5-KO mice (Figure 15D). Moreover, electrical stimulation and even more so elevated KCl triggered a large overflow of [3H]-NA which in case of 15 mM KCl was TTX- sensitive and fully dependent on the presence of calcium in the superfusion buffer (Fig. 15).

Figure 15: Chemically and electrically induced [3H]-ACh and [3H]-NA release from mouse hippocampal and parietal cortical slices

A and B) [3H]-ACh release from hippocampal (A) and cortical slices (B) in response to the indicated stimuli. Differences in KCl-induced release (panel A) were analyzed using one-way ANOVA

(significant with F5, 33 = 22.75 and P < 0.0001), followed by Tukey’s post hoc test for pairs of datasets. 3 (C) [ H]-NA release from hippocampal slices in response to the indicated stimuli. (C1) Data were analyzed using Student’s t-test (100 pulses; WT vs. α5-KO). (C2) Data were analyzed using one-way

ANOVA (significant with F5, 33 = 29.85 and P < 0.0001), followed by Tukey’s post hoc test. The graph shows statistical comparisons of interest. (D) Dose–response curve of nicotine-induced [3H]-NA release. With data from WT mice: nicotine EC50: 5.60 μM (confidence interval 0.94–33.14 μM); maximum effect: 0.58; Hill coefficient: 1.0). With data from α5-KO mice: nicotine EC50: 4.21 μM

(confidence interval 1.07–16.59); maximum effect: 0.49; Hill coefficient: 1.0. EC50 values do not differ significantly (F2, 55 = 0.42 and P = 0.66). The data are presented as mean ± SEM. All nicotine concentrations are in μM; the numbers in parentheses represent the number of measurements. Nic, nicotine; TTX, 1 μM tetrodotoxin; 100 P, 100 pulses (0.5 ms, 10 Hz, 40 mA); 15 KCl, 25 KCl: 15 mM KCl and 25 mM KCl respectively. WT: C57BL/6J mice; α5-KO: α5-knockout mice. ns, P > 0.05; ***P <

0.001; **P < 0.01. Figure and legend taken from BEIRANVAND et al. (2014).

5 DISCUSSION AND CONCLUSIONS

In the introduction I have briefly summarized observations stating that the Hb-IPN system serves as an important connector between the forebrain and structures of the mid- and hindbrain - particularly the dopaminergic, serotonergic, and noradrenergic monoamine nuclei located there. I also mentioned that both the Hb and the IPN express high levels and a great variety of nAChRs. It is unlikely that the presence of these receptors just happens by chance. In fact, α2- α5-, and β4-containing nAChRs in the Hb-IPN system play a critical role in nicotine withdrawal in mice (SALAS et al. 2004b; SALAS et al. 2009); α5-containing receptors control nicotine intake in mice (FOWLER et al. 2011) and possibly also in men (BIERUT et al. 2008); and α5-, β4-containing nAChRs may modulate anxiety-related behavior in mice (SALAS et al. 2003b), possibly in a progesterone-dependant manner during the estrous cycle (GANGITANO et al. 2009). As mentioned before, nAChRs in the Hb-IPN system occur at both pre- and postsynaptic sites.

In my thesis work I have now focussed on the role of nAChRs on the release of two neurotransmitters: ACh and NA. Free ACh may activate both muscarinic and nicotinic receptors, whereas free NA may modulate the neuronal activity in these structures by acting on α and/or β receptors. ACh release in the IPN has previously been studied with a synaptosomal preparation (GRADY et al. 2009). However, by using intact tissue of both Hb and IPN I was able to induce transmitter release with electrical field stimulation and to compare these results with nicotine-induced transmitter release. Electrical pulses generate action potentials and by this mechanism mirror more closely the (physiological) mechanisms of transmitter release from discharging afferents (or interneurons).

5.1 [3H]-ACh release in the IPN

In line with previous observations (GRADY et al. 2001; GRADY et al. 2009), substantial nicotine-induced [3H]-ACh release was detected in the IPN in both rats and mice. [3H]-ACh release was prevented completely by the non-selective nAChR antagonist MCA and was also entirely calcium-dependent. TTX had no effect on nicotine-induced [3H]-ACh release, suggesting that presynaptic nAChRs are the unique source of calcium entry (KULAK et al. 2001) and that voltage-gated calcium channels “activated in consequence of action potentials” are not involved (Wonnacott, 1997). My results are also consistent with Grady et al. (2001, 2009) by showing that β4- (but not β2-) containing nAChRs are essential for the formation of nAChRs which trigger the release of ACh. By taking advantage of β3-KO mice, Grady et al. in 2009 could furthermore demonstrate that receptors containing this subunit

significantly also contribute. ACh release was, on the other hand, not affected in mice lacking the subunits α2, α4, α6, or β2 (Grady et al., 2001; Grady et al., 2009).

I have indirect evidence that β4-, but not β2-containing nAChRs are also critical for ACh release in the rat IPN. Since the release of ACh is entirely independent of TTX (meaning independent of action potentials), all Ca2+ required for exocytosis enters the nerve endings by nAChRs at (or close to) the active zone. Given that cytisine in these experiments had an efficacy like nicotine and thus acted as a full agonist, I can conclude that β4- and not β2- containing receptors play the major role not only in mice but also in rats. Like me, Grady at all (2001) already noticed that relatively high KCl concentrations (in comparison with the release GABA or dopamine from cortical and striatal synaptosomes, respectively) were required to induce the release of ACh.

Electrical field stimulation may also excite GABAergic and/or monoaminergic neurons with inhibitory effects on the release of ACh. In addition to GABAergic interneurons (KAWAJA et al. 1989; LENA et al. 1993; HSU et al. 2013), the IPN receives significant serotonergic input from the median raphe and - to a lesser extent - from dorsal tegmental regions, as well as noradrenergic input from the locus coeruleus (GROENEWEGEN et al. 1986). I tested for possible inhibitory effects by using the α2 adrenoceptor antagonist yohimbine and the GABA-A receptor antagonist bicuculline but could not find any difference in electrically- induced ACh release in the presence of these substances.

It appeared that the electrically-induced [3H]-ACh release in the IPN is quite different from Hc or cortex. With our standard pulse protocol (100 pulses, 10 Hz), ACh release in rats was about 6 times larger in either cortex or Hc than in the IPN, whereas in mice it was 6 times larger in the cortex and even 20 times larger in the Hc than in the IPN. It thus required more pulses at higher frequency (1000 pulses at 50 Hz) to detect noteworthy ACh release in the rat and mouse IPN. A similar observation has previously been made by REN et al. (2011) who reported that - unlike glutamatergic transmission - tetanic (optogenetic) light pulses (≥ 20 Hz for 20 sec) were required to release enough ACh to trigger slow nicotinic transmission. MHb cells tonically fire at a frequency of about 4-5 Hz (KIM AND CHANG 2005; GORLICH et al. 2013) and increase their discharge rate by up to 2-fold upon the application of nicotine (KIM AND CHANG 2005). It is thus unclear whether cholinergic afferents to the IPN fire at a frequency sufficient for nicotinic transmission.

5.2 [3H]-ACh release in the Hb

Quite the opposite to the IPN, nicotine-induced release of [3H]-ACh was hardly detectable in the rat and mouse Hb, whereas electrical field stimulation had a small but significant effect, particularly in mice. These observations show that presynaptic/preterminal nAChRs do not play a major role in the release of ACh in this structure.

5.3 [3H]-ACh release in the Hc and cortex

I measured the release of ACh in rat and mouse hippocampal and cortical slices for comparison with observations in the Hb and the IPN. Three types of stimuli: nicotine, electrical field, and increased KCl were applied. [3H]-ACh release in response to electrical pulses and KCl was readily detectable and TTX-sensitive. However, similar to the Hb, nicotine–induced [3H]-ACh release was hardly detectable, though all these regions receive cholinergic input from the basal forebrain complex (WOOLF et al. 1984; BIANCO AND WILSON 2009).

Evidence has been provided that activation of nicotinic autoreceptors directly triggers ACh release in both cortex (ROWELL AND WINKLER 1984; BEANI et al. 1985; ARAUJO et al. 1988; LAPCHAK et al. 1989) and the Hc (ARAUJO et al. 1988; WILKIE et al. 1996; WONNACOTT 1997). However, in a different study, no ACh release could be observed in slices from human and rat cortex in response to nAChR activation (AMTAGE et al. 2004), and nicotinic autoreceptors seem to absent in the striatum (ARAUJO et al. 1988; LAPCHAK et al. 1989). Nicotinic autoreceptor activation enhanced ACh release in cortical slices stimulated by electrical pulses at low (0.3 Hz) frequency (MARCHI AND RAITERI 1996) or in cortical synaptosomes stimulated by elevated KCl (MARCHI et al. 1999). In view of these variable observations, nAChR activation consistently caused robust release of ACh in both synaptosomal preparations (GRADY et al. 2009) and intact tissue pieces (BEIRANVAND et al. 2014) of the IPN.

5.4 [3H]-NA release in the Hb and IPN

Previous investigations have shown that noradrenergic nerve endings from the LC innervate Hb (GOTTESFELD 1983) and the IPN (BATTISTI et al. 1987). However, so far there have been no reports on the effect of nAChR activation regarding [3H]-NA release from either the IPN or the Hb. I thus focused on two related issues: assessment of nicotine-induced [3H]-NA release in the rat and mouse Hb and IPN; thereafter I determined the subunits which are essential for nicotine-induced [3H]-NA release in these structures. Hence, these experiments

were performed on intact tissue pieces taken from rats and wild type mice as well as from mice lacking the α5, α7, β2, or β4 subunit genes.

Nicotine caused substantial release of [3H]-NA from the IPN and Hb in both rat and mouse. This effect was specific for nAChR activation as it was blocked by co-application of the non- selective and non-competitive nAChR antagonist MCA. The [3H]-NA release differed in some important aspects from [3H]-ACh release. First, the nicotine-induced [3H]-NA release in both IPN and Hb was TTX-sensitive, showing that pre-terminal rather than presynaptic nAChRs were involved (Wonnacott, 1997). Second, the [3H]-NA release was much less than the nicotine-induced [3H]-ACh release in the IPN. Moreover, the [3H]-NA release in response to electrical field stimulation (with our standard protocol) was easily detectable in both IPN and Hb and greatly exceed the nicotine-induced [3H]-NA release (see Fig. 10E2 and Fig. 13D2).

Interestingly, deletion of either β2 or β4 had a large effect on the nicotine-induced [3H]-NA release in both mouse IPN and Hb, demonstrating that these subunits are essential - but not sufficient - for mediating the effect. GRADY et al. (2009) have previously shown that ACh- induced 86Rb+ efflux was lost in Hb synaptosomes from β2-KO mice. 86Rb+ efflux is a valuable technique to investigate transmitter release in response to nAChR activation. It does, however, not distinguish between the types of neurotransmitter involved. My experiments now reveal that pre-terminal β2-containing nAChR on noradrenergic nerve endings may significantly contribute to the effects observed by GRADY et al. (2009). In the very same publication, deletion of β2 reduced (but did not eliminate) 86Rb+ efflux induced by ACh in the IPN. We may thus conclude that 86Rb+ efflux which remains in β2-KO mice might be due to activation of nAChRs in cholinergic endings, whereas the reduction in β2-KO mice may be due to (at least in part) receptors on noradrenergic nerve endings. This does not exclude that presynaptic or preterminal nAChRs may also reside on nerve endings of other neurotransmitter systems. We and others have shown that β2- and β4-containing receptors occur in both the Hb and the IPN of rats and mice (GRADY et al. 2009; SCHOLZE et al. 2012; BEIRANVAND et al. 2014).

α7-containing presynaptic or preterminal receptors do not play a major role in either [3H]- ACh or [3H]-NA release, as the nicotine-induced radioactive overflow was unaffected in α7- KO mice. I will discuss the more complex effects of the α5 subunit on [3H]-NA release below.

Since rats with null mutation of nAChR subunits are not available, contributions of β2- and β4-containing receptors could only be investigated with pharmacological tools. I thus found that cytisine at low concentration (10 µM) induced less [3H]-NA release than nicotine, whereas effects were similar at 100 µM. As mentioned above, [3H]-NA release is mediated

by preterminal receptors and thus depends on action potentials. Hence, cytisine depolarized nerve endings to the threshold of voltage-gated sodium channels mainly by (the less- sensitive) β4-containing receptors, whereas nicotine at lower concentration could also act on (the more sensitive) β2-containing receptors. These observations support the idea that both β2- and β4-containing nAChRs contribute to [3H]-NA release not only in mice but also in rats.

5.5 Role of the α5 nAChR subunit

I investigated whether the presence or the absence of the α5 subunit affected [3H]-ACh or [3H]-NA release in response to nicotine. The results have shown that in the IPN, the nicotine- induced [3H]-ACh release was not affected in α5-KO mice, whereas in these mice, nicotine was clearly less potent (but equally efficacious) in inducing the release of [3H]-NA. A set of experiments run in parallel with my thesis work has shown that most α5-containing receptors are lost in β2-KO mice, whereas levels of α5 were not affected in β4-KO animals (BEIRANVAND et al. 2014). Hence, α5 primarily co-assembles with β2 to form functional receptors. Whether these receptors also contain β4, or whether α5 co-assembles with β4 into a separate entity could not be resolved in these experiments. Since α4 was also reduced in the β2-KO animals, receptors consisting of α4β2α5* seems to be a likely - though not exclusive - combination in the mouse IPN.

It is thus of interest to compare the properties of α4β2α5* receptors with recombinant receptors of known subunit composition. Hence, and in support of my observations, chick α4β2 expressed in Xenopus laevis oocytes were found to be more potently activated than α4β2α5 receptors (RAMIREZ-LATORRE et al. 1996; FUCILE et al. 1997). The impact of α5 on (human) α4β2 receptors depends on the stoichiometry: (α4β2)2α5 receptors expressed in human embryonic kidney (HEK) cells were more sensitive to nicotine than (α4β2)2α4 but less sensitive than (α4β2)2β2 receptors (KURYATOV et al. 2008). In a less likely combination, human α5 combined with α3β2 in oocytes was found to be more potently activated by the agonists nicotine and ACh than just α3β2, whereas α5 combined with α3β4 had little effect on the potency on agonists (FUCILE et al. 1997; GERZANICH et al. 1998; FISCHER et al. 2005). My observation that [3H]-NA release in Hb and IPN was less potently induced by nicotine in α5-KO than in WT mice thus fits best to observations made on chick nAChRs expressed in oocytes (RAMIREZ-LATORRE et al. 1996; FUCILE et al. 1997) and the results for (α4β2)2α4 receptors (KURYATOV et al. 2008) .

Again for the matter of comparison with my data from Hb and IPN, I studied the impact of the α5 subunit on nicotine-induced [3H]-NA release in the mouse hippocampus. Based on the use of the α-CTX MII, BuIA and PIA, and of α4, β2, β3, and β4 null mutant mice, a previous

publication proposed that two populations of nAChRs contribute to agonist-induced [3H]-NA release in mouse hippocampal synaptosomes: a novel α6(α4)β2β3β4 subtype and an α6(α4)β2β3 receptor (AZAM et al. 2010). In line with these results, our group has previously published that nicotine-induced [3H]-NA release was missing in β2-KO mice (SCHOLZE et al. 2007). It is also noteworthy - and in agreement with my observations - that according to their observations, AZAM et al. (2010) did not imply the α5 subunit to contribute to receptors which trigger [3H]-NA release in mice. Since close to 20% of nAChRs contain α5 (XANTHOS et al. 2015) this subunit must play a role in the Hc different from modulating the release of [3H]- NA.

It has generally been assumed that noradrenergic input to the habenular complex (KLEMM 2004; LECOURTIER AND KELLY 2007; BIANCO AND WILSON 2009), hippocampus (OLESKEVICH et al. 1989; ROBERTSON et al. 2013) and the prefrontal cortex (BERRIDGE AND WATERHOUSE 2003; ROBERTSON et al. 2013) all arise from locus coeruleus as the main source. However, as shown in my thesis, nAChRs at noradrenergic nerve endings differ between the Hc and the habenular complex by the role of α5 in triggering transmitter release. Along these lines, nAChRs triggering [3H]-NA release reportedly also differ between the (rat) hippocampus and prefrontal cortex (KENNETT et al. 2012). There are two possible mechanisms that might explain why nAChRs at noradrenergic endings differ: Assuming that the population of noradrenergic neurons was homogenous, the expression of receptors may be modulated by the target region. Alternatively, different subpopulations of noradrenergic cells may project to distinct brain regions. The latter hypothesis is supported by recent findings that noradrenergic neurons in the brainstem are subdivided into four genetically separable groups (ROBERTSON et al. 2013). Based on this new classification, future experiments may allocate distinct populations of brainstem noradrenergic cells with different properties - including various types of nAChRs - to distinct projection patterns in the brain.

It is well-know that brain NA is one of the main mediators of stress responses (MORILAK et al. 2005) and that stressors facilitate the initiation of smoking, reduce the motivation to quit, and increase relapse risk of addiction. Likewise, dysregulation of noradrenergic transmission at least partially mediates withdrawal symptoms associated with nicotine abstinence (reviewed in BRUIJNZEEL 2012). Accordingly, NA reuptake inhibitors decrease relapse rates and withdrawal symptoms related to quit smoking. Moreover, activation of α2-adrenoceptor receptors or inhibition of β-adrenoceptors reduces somatic signs related to nicotine withdrawal (reviewed in BRUIJNZEEL 2012). Given that nAChRs in the Hb-IPN complex play a critical role in nicotine intake and nicotine withdrawal (SALAS et al. 2004b; SALAS et al. 2009; FOWLER et al. 2011), my findings that receptors containing the subunits α5, β2, and β4

contribute to nicotine-induced NA release puts the noradrenergic system into a key role in tobacco dependence.

Taken together, I have shown that і) receptors containing the β4 (but not receptors containing the subunits β2 or α5) are crucial for nicotine-induced [3H]-ACh release from the IPN ii) contrary to nicotine, electrical pulses induce little release of [3H]-ACh in the IPN iii) deleting the α5 subunit rendered nAChRs less to responses to nicotine in inducing NA release in the mouse Hb-IPN complex V) receptors containing the subunits β2 and/or β4 are crucial - though not sufficient - in inducing NA release in the mouse Hb-IPN complex.

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APPENDIX

CURRICULUM VITAE

PERSONAL DATA

Farahnaz Beiranvand

Building 5-1, Boostan Ave, Golstan St, Baghery Town, Hemmat Highway,Tehran, Iran

e-mail: [email protected] Tel: +989124376103

DATE OF BIRTH: 21/08/1971

EDUCATION

Since November 2010: PhD student Center for Brain Research, Medical University of Vienna, Austria Effects of distinct nAChRs on the release Of noradrenaline and acetylcholine in the habenular-interpeduncular system Supervisor: Petra Scholze, Ass. Prof.

2000-2004: MSc in Physiology Faculty of Physiology, Iran University of Medical Sciences, Tehran, Iran Specialization: Human Physiology Thesis title: Noscapine antagonizes vasoconstrictor action of bradykinin in isolated human umbilical artery Supervisor: Massoumeh Shafiei, Ass. Prof

1990-1995: Bachelor of Science in Nursing Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran Specialization: Bachelor of Nursing

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CAREER HISTORY

2004-2009: University of Medical Sciences and Health Services Head nurse (Charge Nurse) of Plastic Surgery Ward and member of research group, Shohada Hospital, Khorramabad, Lorestan, Iran

1995-2000: University of Medical Sciences and Health Services Nurse at Intensive Care Unit (ICU) ward, Shohada Hospital, Khorramabad, Lorestan, Iran

AWARDS

2009: PhD scholarship to study abroad, the Ministry of Health and Medical Education, Iran

2000: Scholarships for master's degree courses, Medical University of Iran, Tehran, Iran

2006: Winning the occupational competition for nursing knowledge and skills award from University of Medical Sciences and Health Services, Khorramabad, Lorestan, Iran

TECHNICAL SKILLS

Computer skills: SigmaPlot V12 GraphPad Prism Microsoft Office (Word, Excel, PowerPoint) CorelDRAW 12 IBM SPSS (Statistics) EndNote X3 and Reference Manager

Technical skills: Measurement of neurotransmitter release in vitro Isometric tension recording technique Intensive Care Unit skills (ICU – CCU) Electrocardiograph

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LANGUAGE SKILLS

Language

Persian Native speaker English Advanced

LEISURE ACTIVITIES

Traditional music, psychology, literature

CONFERENCES

Poster presentation at the 19th Scientific Symposium of APHAR and 13th ANA Meeting, September 16-19, 2013, Vienna Noradrenaline and acetylcholine release in the IPN and habenula of mouse and rat

Poster presentation at 9th YSA PhD-Symposium, June 19–20, 2013, Vienna Nicotinic receptors modulate acetylcholine release in the CNS

Poster Presentation at the 8th YSA-PhD-Symposium, June 13–14, 2012, Vienna Transmitter released by nicotinic receptors in the spinal cord

Poster presentation at 2th Seminar on Medical Error Prevention, May 16-17, 2007, Mashehad, Iran The Role of nuclear technology in reducing diagnostic errors in heart patients

Talk at 3th international Congress on Health, Medication and Crisis, December 21-23, 2007, Tehran, Iran The effect of post traumatic stress disorder (PTSD) in the children of Durood earthquake survivors

LIST OF PUBLICATIONS

Mahmoudian, M., N. Aboutaleb, F. Beiranvand, A.-A. Moazzam and M. Shafiei, 2011 Noscapine antagonizes vasoconstrictor action of bradykinin in isolated human umbilical artery. Medical Journal of the Islamic Republic Of Iran 25: 82-86.

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