“Men ought to know that from nothing else but the brain come joys, delights, laughter and sports, and sorrows, griefs, despon-dency, and lamentations. And by this, in an especial manner, we acquire wisdom and knowledge, and see and hear and know what are foul and fair, what are bad and what are good, what are sweet and what are unsavoury …And by the same organ we become mad and delirious, and fears and terror assail us…All these things we endure from the brain when it is not healthy…In these way I am of the opinion that the brain exercises the greatest power over man.”

Hippocrates, On the Sacred Disease (Fourth century, B.C.)

Cover: Labelling of VGLUT2 (red), TH (green) and DAPI (blue), on a coronal section of an adult mouse at bregma -0.10 mm, illustrating the striatum and cortex.

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Wallén-Mackenzie Å, Nordenankar K, Fejgin K, Lagerström MC, Emilsson L,

Fredriksson R, Wass C, Andersson D, Egecioglu E, Andersson M, Strandberg J, Lindhe O, Schiöth HB, Chergui K, Hanse E, Långström B, Fredriksson A, Svensson L, Roman E, Kullander K. (2009) Restricted cortical and amygdaloid removal of Vesicular glutamate transporter 2 in preadolescent mice impacts do-paminergic activity and neuronal circuitry of higher brain function. J Neurosci., 29(7):2238 –2251.

II Birgner C#, Nordenankar K#, Lundblad M, Mendez JA, Smith C, le Grevès M, Galter D, Olson L, Fredriksson A, Trudeau LE, Kullander K, Wallén-Mackenzie Å. (2010) VGLUT2 in dopamine neurons is required for psy-chostimulant-induced behavioural activation. Proc Natl Acad Sci USA, 107:389-394. # Shared first authorship.

III Alsiö J, Nordenankar K, Arvidsson E, Birgner C, Mahmoudi S, Halbout B, Smith C, Fortin GM, Olson L, Descarries L, Trudeau LÉ, Kullander K, Lévesque D, Wallén-Mackenzie Å. (2011). Enhanced sucrose and cocaine self-administration and cue-induced drug seeking after loss of VGLUT2 in midbrain dopamine neurons in mice. J Neurosci., 31(35): 12593-603.

IV Nordenankar K#, Emilsson L#, Birgner C, Lagerström MC, Fejgin K, Jazin E, Roman E, Kullander K, Wallén-Mackenzie Å. (2012) Altered expression of Vglut2 affects behaviour in a gender-dependent manner. Manuscript. # Shared first authorship.

V Nordenankar K, Birgner C, Emilsson L, Ghelani T, Studer E, Bergfors A, Fejgin K, Kullander K, Wallén-Mackenzie Å. (2012) Targeted prenatal deletion of Vglut2 expression in the forebrain decreases anxiety-related behaviour of the adult mouse. Manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction....................................................................................................... 11 Neuronal circuits.......................................................................................... 11 Glutamate ..................................................................................................... 11

Ionotropic glutamate receptors .............................................................. 11 Metabotropic glutamate receptors (mGluRs)........................................ 12

Vesicular glutamate transporters (VGLUTs)............................................. 12 Expression of the Vgluts in the CNS .......................................................... 13 Mouse genetics ............................................................................................ 15

VGLUT1-deficient mice ........................................................................ 16 VGLUT2-deficient mice ........................................................................ 17 VGLUT3-deficient mice ........................................................................ 18

Dopamine ..................................................................................................... 18 DA receptors ........................................................................................... 19

Expression of Vglut2 in DA neurons.......................................................... 20 Functional properties of VGLUT2 in DA neurons............................... 21

The brain reward circuitry........................................................................... 22 The DA and glutamate hypotheses of schizophrenia ................................ 22 Drug addiction ............................................................................................. 24

DA receptors in addiction ...................................................................... 24 Circuitries implicated in addiction......................................................... 24

Materials and Methods..................................................................................... 26 Production of transgenic mice .................................................................... 26 Behavioural analysis.................................................................................... 27

Anxiety-related behaviour...................................................................... 27 Depression-like behaviour ..................................................................... 27 Social dominance.................................................................................... 28 Learning and memory............................................................................. 28 Locomotor functions .............................................................................. 28 Sucrose bottle choice test ....................................................................... 29 Operant self-administration.................................................................... 29

In situ hybridisation and immunohistochemistry ...................................... 30

Aims .................................................................................................................. 31 Specific aims................................................................................................ 31

Paper I...................................................................................................... 31 Paper II .................................................................................................... 31 Paper III ................................................................................................... 31 Paper IV................................................................................................... 31 Paper V .................................................................................................... 31

Results and Discussion..................................................................................... 32 Paper I .......................................................................................................... 32 Paper II ......................................................................................................... 34 Paper III........................................................................................................ 34 Paper IV ....................................................................................................... 36 Paper V......................................................................................................... 36

Closing remarks................................................................................................ 38

Acknowledgments ............................................................................................ 40

References......................................................................................................... 43

Abbreviations

alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid

AMPA

Acetyl choline ACh Beta-galactosidase β-gal Central nervous system CNS Cyclization recombinase Cre Dopamine DA Dopamine transporter DAT Elevated plus maze EPM Fast Analytical Sensing Technology FAST Fixed ratio FR Enhanced green fluorescent protein eGFP In situ hybridization ISH Multivariate concentric square field MCSF N-methyl-D-aspartate NMDA Nucleus accumbens NAcc Self-administration SA Positron emission tomography PET Postnatal P Pre-pulse inhibition PPI Progressive ratio PR Reference memory error RME Retrosplenial group RSG Schizophrenia SZ Serotonergic neurons 5-HT Single cell reverse transcriptase PCR RT-PCR Substantia nigra pars compacta SNc Substantia nigra pars reticulata SNr Tyrosine hydroxylase TH Ventral tegmental area VTA Vesicular glutamate transporter VGLUT Vesicular glutamate transporter 1 VGLUT1 Vesicular glutamate transporter 2 VGLUT2 Vesicular glutamate transporter 3 VGLUT3 Vesicular monoamine transporter VMAT Working memory error WME

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Introduction

Neuronal circuits The central nervous system (CNS) consists of billions of nerve cells, neu-rons, all having elaborate electrical and chemical signalling mechanisms. Each one of these neurons is interconnected via synapses to form neuronal circuits. The neuronal circuits determine all characteristics of our life, from sensory experience to motor coordination and higher brain functions such as learning and memory. A central goal in neuroscience is to link neuronal cir-cuits to behavioural outputs. The use of transgenic mouse models has con-tributed to our current understanding of many circuits in the brain. Causative factors of disorders such as addiction and schizophrenia have gained a tre-mendous understanding due to transgenic models [1]. However, much re-search remains to be done in order to get a more complete picture of the neurobiology behind these disorders.

Glutamate For more than two decades, it has been known that glutamate is the most abundant excitatory neurotransmitter [2]. Glutamatergic neurons and gluta-mate-mediated excitatory signalling are involved in all neuronal networks of the CNS [3]. In addition to its role in neurotransmission, glutamate is a ubiq-uitous amino acid; in order to work as a neurotransmitter it has to be trans-ported into vesicles and released at the axon terminal [4]. Glutamate binds to, and activate, pre- and postsynaptic membrane receptors whereupon the signal is transmitted [5]. There are two kinds of glutamate receptors, me-tabotropic G-protein-coupled receptors and ligand-gated ion channels. Membrane transporters localised both on pre- and postsynaptic neurons, as well as on surrounding glia cells, quickly remove excess glutamate from the synaptic cleft [6,7].

Ionotropic glutamate receptors There are three classes of ionotropic glutamate receptors, the NMDA (N-methyl-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid) and kainate receptors [5].

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The NMDA receptor forms a heterotetramer between two NMDA receptor 1 subunit (NR1) and two NMDA receptor 2 (NR2) subunits [8]. The AMPA receptor is composed of four types of subunits, GluR1, GluR2, GluR3 and GluR4 [9]. The AMPA and NMDA receptors are widely distributed in the CNS and play a role in pre- and postsynaptic neurons. They are crucial in neuronal development and mediating synaptic transmission in aspects of learning and memory [8,10-12]. Kainate receptors also have a function in both pre- and postsynaptic neurons. The distribution in the brain is more restricted compared to AMPA and NMDA, and although their function is not as well defined, they are implicated in synaptic plasticity [13].

Metabotropic glutamate receptors (mGluRs) The mGluRs consist of eight different types, mGluR1-8, and are subdivided into three groups; group I (mGluR1 and 5), II (mGluR 2 and 3) and III (mGluR4, 6, 7, 8). They are grouped based on structure, sequence homology and second messenger systems [9]. Group I mGluRs are often localised post-synaptically, and their activation leads to cell depolarisation and increased neuronal excitability [14]. In contrast, group II and group III mGluRs are often localised to presynaptic terminals, where they inhibit neurotransmitter release. This inhibition occurs at excitatory (glutamatergic), inhibitory (GABAergic) and neuromodulatory (monoamines, acetylcholine (ACh) and peptides) synapses [3,15]. The mGluRs are widely distributed throughout the CNS, however, by different expression patterns. For example, while group I mGluRs is the most abundant receptor type in the striatum, expression of all subtypes, except mGluR6, has been detected in the striatum [16-18].

Vesicular glutamate transporters (VGLUTs) Most information of glutamatergic neurotransmission is gained from phar-macological and gene targeting studies on the postsynaptic neuron. This includes cytoplasmic membrane transporters and the different glutamate receptors. However, these studies have not addressed the presynaptic gluta-mate-signalling neuron itself. This is due to the fact that, for long, specific markers for the neurons that use glutamate as a neurotransmitter were miss-ing. In 2000, a protein known as a brain-specific sodium-dependent phos-phate transporter [19] was found to fulfil the criteria for a vesicular gluta-mate transporter (VGLUT) [20,21]. This protein actively transports gluta-mate into vesicles within the presynaptic terminal and was named Vesicular glutamate transporter 1 (Vglut1). Soon after followed the discovery of two additional Vgluts, Vglut2 [22] and Vglut3 [23,24]. The Vgluts belong to the solute carrier family, the Slc family, and are also named Slc17a6 (Vglut2), Slc17a7 (Vglut1) and Slc17a8 (Vglut3). The isolation and characterisation of

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the VGLUTs permit the proper identification of cells that have the capacity to use glutamate as a neurotransmitter. VGLUT1 and VGLUT2 are localised in asymmetrical synapses, the characteristic structure for excitatory syn-apses. They are now considered the best available markers for glutamatergic neurons and also constitute an important gene family for the study of excita-tory neurons in vivo [25,26]. VGLUT3, on the other hand, is more sparsely distributed in the brain and found in neurons using transmitters such as GABA, ACh and serotonin [23,27].

VGLUTs have been identified in different organisms such as Drosophila, zebrafish and the frog [28-30]. Experiments performed in Drosophila indi-cate that VGLUT in the fruit fly determines both the quantal size and gluta-mate content of the synaptic vesicles [28]. The identification of VGLUTs in different species shows that the Vglut genes are evolutionarily conserved. In the mouse, the Vglut1 and Vglut2 genes are located close to each other on chromosome 7, whereas Vglut3 is located on chromosome 10 [31]. In the human genome, Vglut1 is located on chromosome 19, Vglut2 is located on chromosome 11, and Vglut3 is located on chromosome 12, according to Na-tional centre for technology (NCBI).

Outside the nervous system, the VGLUTs can be found in the glutamate-secreting non-neuronal cells of different organs, such as in the pineal gland [32], islets of Langerhans, intestine, stomach [33], testes [34] and hair folli-cles [35].

Expression of the Vgluts in the CNS Although it is not known why there are three different isoforms of VGLUT, it is recognised that there is a striking difference between their expression patterns in the adult rodent brain. Vglut1 and Vglut2 display a clear comple-mentary distribution as shown by in situ hybridisation histochemistry [25,26,36,37]. During mouse development, Vglut2 is widely expressed in the brain, but after birth it becomes more restricted to deep structures, including the thalamus, with low expression remaining in the forebrain. Vglut2 is also expressed in the brainstem and spinal cord. In contrast, Vglut1 has a very low expression before birth but is strongly upregulated soon after birth, mainly in forebrain regions, but also in the cerebellum (Figure 1 A).

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Figure 1. Distribution of Vesicular glutamate transporters in the rodent brain. (A) Illustration of a sagittal rat brain section showing the mRNA distribution of the three vesicular glutamate transporters (Vgluts). Vesicular glutamate transporter 1 (Vglut1) mRNA is found mostly in the cortex and cerebellum, while Vglut2 mRNA is found in the deep cerebral structures, including the thalamus and brain stem. Vglut3 mRNA has a more restricted distribution in serotonin neurons of the mid-brain rahpe nuclei, ACh neurons of the striatum and basal forebrain and a subset of GABA neurons in the cortex and hippocampus. (B) Illustration of coronal rat brain sections showing the protein distribution of VGLUT2 and VGLUT3 in axon termi-nals containing DA (yellow), serotonin (pink), ACh (green) or GABA (blue). Modi-fied from El Mestikawy et al, 2011, Nat Rev Neurosci [38].

The protein distribution, which reflects the presynaptic axon terminals, also displays a complementary pattern. VGLUT2 is localised in the telenchephal-ic region of the glomeruli in the olfactory bulb, layer IV and VI of the neocortex, striatum, and the granular layer of dentate gyrus (Figure 1 B). VGLUT1, on the other hand, is localised in the olfactory tubercle, layers I-III of the neocortex, piriform and entorhinal cortex, hippocampus, dentate gyrus and subiculum. In the lower brain stem region, both VGLUT1 and VGLUT2 are detected, with a more intense presence of VGLUT2. Both VGLUT1 and VGLUT2 are found in cerebellum, and again they are essen-tially distributed in a complementary pattern [25,26,36,37]. The complemen-tary localization of VGLUT1 and VGLUT2 proteins is however, not abso-lute. For example, there is evidence from both hippocampal and cortex neu-rons, that the two isoforms can co-exist in the same synaptic terminal [37,39]. Also, several studies describe co-localisation of both isoforms, in-

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cluding in cerebellum mossy fibers [40,41] and in the cortex [42]. There are only a few neurons in the brain that contain the VGLUT3 protein, mainly the 5-HT neurons in the dorsal and medial raphe nuclei as well as the ACh neu-rons in the striatum and nucleus accumbens (NAcc) (Figure 1 B) [23,24,27,43,44].

The differential distribution of VGLUT1, VGLUT2 and VGLUT3 ap-pears to correlate with the probability of release [26,45]. VGLUT1-positive synapses show a low probability of transmitter release while VGLUT2- and VGLUT3-positive synapses show a high probability of transmitter release [26]. Furthermore, the difference in release probability has been correlated to expression of endophilin A1, which acts as a positive regulator of exocytosis. VGLUT1 has a higher ability to bind to endophilin A1 compared to VGLUT2 and VGLUT3, and inhibits endophilin-induced enhancement of release probability [45].

In addition to the above mentioned areas, VGLUT proteins can also be detected in the retina [34], spinal cord and dorsal root ganglion [35] as well as in non-neuronal cells, such as astrocytes [46] in the CNS.

Mouse genetics The physiological importance of the VGLUT proteins can be revealed by studies of VGLUT-deficient mice. Homologous recombination in mouse embryonic stem cells has advanced our understanding of gene function in the mammalian brain. Yet, conventional mouse knockouts (KOs) may lead to embryonic lethality, thereby preventing the study of gene function later in life. Several methods have been developed during the last decades to study the activation and inactivation of genes of interest. The CreLoxP-system is a compelling approach that enables the deletion of any existing gene in the mouse genome in a temporally and spatially controlled manner. The Cycliza-tion recombinase (Cre) catalyses the recombination between two 34bp-long recognition sites, the so called LoxP sites [47]. The LoxP sites can be in-serted into the mouse genome by homologous recombination. The design is made so that the LoxP sites flank the gene sequence to be removed. That gene, or gene sequences, are then referred to as “flanked by lox sites”, or “floxed”. Mice homologous for the floxed gene can be bred with a trans-genic mouse line that expresses Cre. The most common method to restrict Cre expression is to use a promoter with the required temporal and spatial specificity. This generates a conditional knockout (cKO) mouse of the gene of interest in the specific region where Cre is expressed (Figure 2 A).

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Figure 2. Examples of the CreLoxP-system. (A) Conditional knockout mice are made in several steps where first a targeting construct containing one LoxP-site on each side of the exons to be deleted is combined with the wildtype locus through homologous recombination. After production of mice carrying the floxed gene, these are crossed with mice expressing Cre in a temporally/spatially restricted manner. When Cre is expressed, the floxed exons are deleted, resulting in a gene producing non-functional protein. (B) Another possibility for the CreLoxP-system is to use so-called reporter mice to visualise specific neurons. Cre-mediated excision of a LoxP-flanked transcriptional "stop" sequence enables the expression of the reporter gene, for example the enhanced green fluorescent protein (eGFP) expressed under control of the ubiquitous neuronal promoter tau .

Another use for the CreLoxP-system is to evaluate the specificity of neurons that express Cre. The use of so called reporter mice enables the visualisation of the specificity of the Cre. This provides insight into the spatio-temporal pattern of the activity. The reporter is, for example, a molecule that can be visualised under a microscope, such as enhanced green fluorescent protein (eGFP). The reporter construct is inserted after a ubiquitously expressed promoter, for example the microtubule-associated protein tau, [48], and downstream of a floxed stop cassette. Mice homologues for the floxed pro-moter construct are bred with the transgenic mouse line that expresses Cre. In the neurons where Cre is expressed, the stop cassette gets excised allow-ing expression, and subsequent detection, of the reporter gene (Figure 2 B). Today, Cre and reporter mice are being produced in large-scale projects in order to establish valuable tools for the study of the gene functionality. They are also being produced in a small scale in laboratories around the world, as it is a reasonably easy and a straightforward method.

VGLUT1-deficient mice In 2004, two studies on a VGLUT1 full KO mouse were published [49,50]. The VGLUT1 KO mice were viable at birth, however, around two weeks

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after birth, the mice were unable to feed properly and had a poor ability to move. Wojick et al., showed that the expression level of the VGLUTs de-termines the amount of glutamate that is loaded into vesicles and released, thereby regulating the efficacy of neurotransmission [49]. Feameau et al., showed that VGLUT1 and 2 targets to distinct synaptic release sites formed by a single hippocampal neuron during development. They further specu-lated that developmental co-localization of VGLUT2 with VGLUT1 may occur at multiple synapses [50].

Behavioural studies performed on the heterozygous VGLUT1 mice indi-cated that these mice have a normal short-term memory but impaired long-term memory in addition to increased anxiety and depression-like behaviour [51-53]. A recent study showed that the VGLUT1 heterozygous mice exhibit behavioural impairments associated with schizophrenia [54].

VGLUT2-deficient mice The expression pattern of Vglut2 in many brainstem areas is connected to vital functions, e.g. cardio- and respiratory functions [55]. In light of this finding, which indicated that loss of Vglut2 expression might be lethal, Kul-lander and Wallén-Mackenzie took a conditional approach to enable a spa-tially and temporally controlled deletion of Vglut2 gene function [56]. For this, the CreLoxP-system was chosen. By homologous recombination, exons 4-6 of the Vglut2 gene were flanked by LoxP sites. After production of mice homozygous for the floxed Vglut2 gene (Vglut2flox/flox , or Vglut2f/f mice), these were mated with mice carrying Cre expressed after the phosphoglycer-ate kinase (PGK) promoter (PGK-Cre, or P-Cre), which is turned on as soon as the embryo forms [57]. VGLUT2 null (Vglut2f/f;PCre) mice and littermate controls (Vglut2f/f mice) were obtained in the offspring, and it was immedi-ately evident that complete loss of VGLUT2 is lethal. The null pups were born completely immobile and cyanotic, and died soon after birth. Subse-quent electrophysiological analyses showed that the VGLUT2 null mice suffered from a complete loss of the stable autonomous respiratory rhythm generated by the pre-Bötzinger complex, thereby leading to respiratory fail-ure. In contrast to the disturbed respiratory rhythm generation, the locomotor central pattern generator in the spinal cord of VGLUT2 null mice displayed normal rhythmic and coordinated activity [56].

The Vglut2f/f mice were subsequently crossed with other Cre-expressing mice, the focus of this thesis, and used to study the role of VGLUT2 in spe-cific forebrain areas from embryogenesis (Paper V) and from postnatal day 15 (Paper I, paper IV), and in midbrain dopaminergic neurons (Papers II and III).

Moechars et al. independently produced a full VGLUT2 KO mouse by deleting exon 2 of the Vglut2 by homologous recombination, leading to a disrupted open reading frame and a premature stop codon. Since the null KO

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mice were not viable, their behavioural analyses were performed on the het-erozygous (Vglut2+/-) mice. The results indicated no differences in motor function, learning, memory and acute nociception or inflammatory pain compared to littermate controls. However, the Vglut2+/- mice did show an altered response in tests aimed to detect neuropathic pain [58]. In 2011, an additional study showed that Vglut2+/- mice had an increased response to amphetamine and increased sensitivity to the startling disrupting-effect of MK-801, a NMDA receptor antagonist. The heterozygous mice showed no impairment in sensorimotor gating when assessing behaviours related to schizophrenia [59].

By using the CreLoxP-system approach to investigate the putative role of Vglut2 in ventromedial hypothalamic neurons, Tong et al. could show that mice lacking Vglut2 in this region displayed abnormal glucose homeostasis [60].

VGLUT3-deficient mice In 2008, two different studies described the phenotype of mice with dis-rupted VGLUT3 function. Both showed that the KO mice are viable but display deafness and seizures, as well as hyperactivity due to a lack of cho-linergic neurotransmission in the striatum [61,62].

Dopamine Arvid Carlsson et al. identified dopamine (DA) as a neurotransmitter in 1958 [63]. DA is part of the catecholamine family, which also includes noradrena-line and adrenaline. Tyrosine hydroxylase (TH) is the rate-limiting enzyme of catecholamine synthesis. DA is packed into vesicles by the vesicular monoamine transporters (VMAT2) located in the nerve terminal. When DA is released from the presynaptic vesicles, it binds to specific receptors lo-cated on both the pre- and postsynaptic neurons. The DA transporter (DAT) terminates the signalling by clearing the synaptic cleft and the transmitter is repacked into vesicles or degraded by monoamine oxidase [64].

The DA neurons are located in the substantia nigra pars compacta (SNc; A9), the ventral tegmental area (VTA: A10) and the retrorubral area (A8) [65,66]. Based on several studies, the DA system has been subdivided into the nigrostriatal, mesocortical, mesolimbic and tuberoinfundibular DA sys-tems [67] (Figure 3).

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Figure 3. Schematic overview of dopaminergic projections in the rat brain. Modified from Ungerstedt et al., 1974 [65].

The DA neurons in the SNc play an important role in control of voluntary movement, while the DA neurons in the VTA are more strongly associated with cortical and limbic functions, including reward-related responses, moti-vation, aspects of memory processing as well as stress, anxiety and fear re-sponse [68,69]. The VTA DA neurons project to the ventromedial striatum, which include the NAcc and olfactory tubercle. Projections also go to sub-cortical (amygdala, septum and habenula) and cortical (piriform, entorehinal prefrontal and anterior cingulated) areas [65,66]. DA neurons make up about 60-65 % of the cells in the VTA [70]. The neurons are highly heterogeneous and vary in location and morphology, target areas, firing properties, ion channels and receptors [71-73]. Non-DA neurons in the VTA are primarily GABAergic and make up approximately 30-35 % of the cells in the VTA. They are mostly considered interneurons, but evidence indicates that these cells project parallel to DA neurons [70,74]. The glutamatergic population in the VTA comprise around 2-3 % of the VTA neurons [70].

DA receptors The targets for the released DA are the DA receptors located on the GABAergic medium spiny neurons (MSN) in the striatum as well as on neu-rons in the prefrontal cortex and amygdala. DA receptors are also located in the hippocampus, neocortex, hypothalamus and thalamus, but at a lower level. There are five subtypes of the metabotropic G-coupled DA receptors D1-D5. The D1 and D5 are receptors of the D1-like family, whereas D2, D3 and D4 are receptors of the D2-like family, based on sequence homology and pharmacology [75,76]. The D1 and D2 receptors are the most abundant in the central nervous system, with expression levels 10-100 times that of the D3-D5 receptors. There is a wide distribution of the D1 and D2 receptors in the CNS, although different areas have a different distribution of the recep-

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tors [75,76]. Within the mesolimbic pathway, the two receptor classes exert opposing intracellular effects. The D1 receptors activate G-protein that sub-sequently activates adenyl cyclase, thereby increasing the intracellular levels of cyclic adenyl cyclase and increasing 3´-5´cyclic adenosine phosphate (cAMP). Activation of the D2 receptor activates G-protein that inhibits cAMP by inhibiting adenyl cyclase [76,77].

Expression of Vglut2 in DA neurons It is becoming increasingly clear that most, if not all, neurons of the CNS and peripheral nervous system use more than one neurotransmitter [78,79]. Neuropeptides were long considered to be the most frequently colocalised transmitters [80], but evidence shows that several neuronal populations not only signal with the ¨classical¨ transmitter, but signal with two different ¨classical¨ transmitters, such as dopamine (DA) and glutamate [38]. For some time, it was believed that VGLUT3 was the only VGLUT expressed in non-glutamatergic neurons, but studies have shown that VGLUT1 and VGLUT2 can also be expressed in neurons not initially characterised as glu-tamatergic [38].

With the discovery of VGLUT2, early studies combining in situ hybridi-sation and immunohistochemistry identified Vglut2 mRNA in noradrenergic neurons of the A1 and A2 groups and in adrenergic neurons in the C1, C2 and C3 groups of the rat medulla, raising the possibility that glutamate might act as a co-transmitter in certain catecholamine neurons [55,81]. In 2004 Dal Bo et al [82], demonstrated the presence of VGLUT2 in cultures of DA neu-rons and provided a molecular explanation for their previously reported abil-ity to release glutamate [83,84]. A subsequent single cell reverse tran-scriptase (RT) PCR study suggested that Vglut2 expression might be devel-opmentally regulated in mesenchephalic DA neurons, with much higher yield at postnatal day 0 (P0) than P45 [85].

The expression of Vglut2 in DA neurons was subsequently shown to be specific to subpopulations of the VTA, the most prominent of which is the rostral linear nucleus, the caudal linear nucleus and the interfascicular nu-cleus. Low expression levels were demonstrated in DA neurons in the parab-rachial pigmented nucleus and paranigral nucleus (Figure 4, A and B) [86-88]. The rostral and caudal linear neurons project to limbic and cortical re-gions, while the interfascicular neurons projects only to limbic regions [86]. Notably, there are some discrepancies in the reported expression levels of Vglut2 in the VTA and SNc. This is likely due to the use of different detec-tion techniques [70,85,88-90].

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Figure 4. Expression of Vglut2 in midbrain DA cell populations. In (A) and (B) are shown two bregma levels where Vglut2-positive DA neurons (blue dots) are located according to Kawano et al. Vglut2 expressing DA neurons are located in the VTA, the rostral linear nucleus (RLi), the caudal linear nucleus (CLi), the interfas-cicular nucleus (IF) and low expression in the parabrachial pigmented nucleus (PBP) and paranigral nucleus (PN). These regions have been shown to project to the nu-cleus accumbens (NAcc) and constitute a part of the reward system (discussed be-low).

According to an immunocytochemical electron microscopic study in P15 rats, 28 % of all TH immunopostive axon terminals in the NAcc and 17 % in the neostriatum contain VGLUT2 [87]. At P90, no double labelling could be detected [89,91], which suggests an age-dependent regulation of VGLUT2 and TH co-localization. An additional study shows that none of the three VGLUTs are found in dopminergic terminals in the rat striatum [92]. A re-cent study show that in neither P15 nor adult mice could dually labelled axon terminals be detected [93]. The results indicate a difference in co-localization of VGLUT2 and TH between rats and mice.

Functional properties of VGLUT2 in DA neurons An electrophysiology study made in mouse mesoaccumbal slices showed that DA neurons possess a bimodal signalling capability, for which DA alone cannot be responsible. The initial signalling is excitatory and faster than DA-meditated signalling and the authors hypothesised that it was medi-ated by glutamate [94]. Optogenetics is a technology recently developed to enable light-driven control of depolarization events in specifically targeted neurons [95]. Recent optogenetic studies in adult mice have provided evi-dence for the release of glutamate by VTA DA neurons in vivo in the NAcc but not in the dorsal striatum [96,97]. Furthermore, targeted deletion of Vglut2 in the mesencephalic DA neurons completely abolished such light-dependent excitatory postsynaptic potentials [97]. The question then arises whether this glutamate release occurs in the axon terminal, which also re-leases DA. A possibility is that the DA neuron possesses different subsets of axon terminals; one group which contains only TH, a second, smaller group,

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containing both TH and VGLUT2, and a third containing only VGLUT2 but not TH. This potential phenotypical heterogeneity among the DA axon ter-minals might also explain the lack of double labelling of TH/VGLUT2 in the adult rat and mouse striatum [92,93,98].

In addition to the possible role in mediating co-release of glutamate, the role for the VGLUTs could have other important functions. For example, VGLUT can acidify the vesicular lumen, hence increasing the filling of the “primary” neurotransmitter. This kind of cooperation between two vesicular proteins on the same vesicle, as demonstrated for VGLUT3 in ACh or 5-HT neurons, has been termed vesicular synergy [62,99]. Vesicular synergy has been suggested to occur with VGLUT2 in DA neurons [100] and GABA neurons [101], but it has not yet been proven.

The brain reward circuitry The brain reward circuitry consists of DA projections from cell bodies in the VTA to the limbic structures (the mesolimibic pathway) including the NAcc, amygdala, ventral pallidum, hippocampus, as well as to cortical areas (the mesocortical pathway) including the prefrontal cortex (PFC), the orbifrontal cortex (OFC) and the anterior cingulate [102,103]. The brain reward system is important for motivation and for mediating rewarding properties of both natural stimuli, such as food and sex [104], and addictive drugs [105].

The DA and glutamate hypotheses of schizophrenia Schizophrenia (SZ) is a complex disorder with substantial heterogeneity between patients. There is no easy genetic or environmental explanation for the disorder, but a number of theories point to abnormal neurotransmitter systems as contributing factors [106]. However, it is unclear to what extent any neurochemical finding reflects primary rather than secondary pathology or compensatory mechanisms [107]. The symptoms of SZ are clinically of-ten divided into positive and negative symptoms and cognitive deficiency [106,108]. The positive symptoms include hallucination and paranoia while negative symptoms involve social retardation and apathy. The cognitive symptoms include disorganised thoughts, memory problems, and difficulties with concentration, following instructions and completing tasks.

In SZ there is a difference between male and female patients. SZ is more common in males, who also have an earlier onset and poorer prognosis in comparison to females. Male patients have more negative symptoms, while females are more prone to be depressed and display subtypes of positive symptoms [109-112].

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The classical DA hypothesis of SZ postulates a subcortical dopaminergic hyperactivity of the D2 receptors in the striatum. The DA hypothesis of SZ is based on the finding that drugs such as amphetamine increase levels of DA in the brain and can cause positive symptoms, resembling those present in SZ patients [113,114]. Most pharmaceutical intervention of SZ is aimed at decreasing the activity of the D2 receptor. Brain imaging studies have found an increase in the release of DA and in the density and occupancy of the D2 receptor in the striatum of patients with SZ [115]. The DA hypothesis, how-ever, does not provide a sufficient explanation for the negative symptoms. Nevertheless, some studies have shown that low levels of VTA-derived DA projections to cortical and limbic structures are implicated in the negative symptoms of SZ [116].

Another hypothesis of SZ is the hypoglutamate hypothesis, which is based on pharmacological studies. Antagonists such as phencyclidine and ketamine have been shown to noncompetitively block the glutamate NMDA-receptor and bring on a reaction in humans that resembles SZ, including positive and negative symptoms and cognitive dysfunction [117,118]. This hypothesis implies that there is a hypoactivity of glutamatergic signalling in SZ patients [119]. Presynpatic factors can also cause aberrant glutamatergic transmission in SZ [120]. For example, abnormal expression of several pre-synaptic proteins such as synaptophysin, syntaxin, synapsin, Rab3, synapto-tagamin and vesicular transporter have been found in post mortem brain studies of SZ patients [120-123]. These findings indicate that the presynaptic components of glutamatergic synapses may be abnormal in SZ.

Evaluating SZ by using animal models Modelling SZ in rodents is a difficult task. Just as patients with SZ do not manifest every possible symptom of the disease, an animal model of SZ will not necessarily display abnormalities in all SZ relevant behaviour [124]. The most common approach for developing animal models has been to use pharmacological treatments and by gene targeting in mice. In accordance with the widespread historical interest in the hyperdopaminergic hypothesis of SZ, many mouse models have been developed which address most com-ponents of the dopaminergic activity in the brain [114,125]. The release and regulation of extracellular levels of DA by DA transporters and catabolic enzymes, as well as the role of the five DA receptors has been investigated in different mouse strains. There are also a large number of studies on mu-tant models of glutamate function. For example, gene-targeting studies in mice with reduced levels of either of the different subtypes of the NMDA receptor, or the metabotropic glutamate receptors, have resulted in SZ-related symptoms [114,125].

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Drug addiction Addiction is a chronic relapsing disorder that causes compulsive use and drug-seeking behaviour. Addictive drugs “hijack” the brain reward system and disturb the way the nerve cells normally send, receive and process in-formation. The mesolimibic and mesocortical circuits operate in parallel, but may have some different roles in addiction. The amygdala and hippocampus play an important role in conditioned learning in the process of addiction, whereas the NAcc and ventral pallidum appear to be involved in the primary reinforcing effects of drugs of abuse. The mesostriatal pathway is now also recognised to contribute to drug reward and addiction [102,103,126,127].

A variety of increasingly sophisticated animal models have provided an invaluable means for understanding the neurobiology of addiction. Examples of such models include intracranial self-stimulation, conditional place pref-erence, behavioural sensitisation and various self–administration paradigms [128].

DA receptors in addiction The D1 receptor mediates reward learning in response to palatable food, and D1 deficient mice have reduced operant responding for sucrose [129]. Both D1 and D2 receptors have been shown to mediate behavioural and bio-chemical effects of acute and repeated cocaine administration [130-132]. Furthermore, a study showed that D2 receptor antagonists reduce sucrose preference [133]. Cocaine, methamphetamine, and heroine addicts, alcohol-ics and severely obese persons, show a small but persistent decrease in dor-sal striatal D2 receptors [134-138]. Studies in non-human primates show similar decrease following self-administration of cocaine, indicating a role of the D2 receptor in addiction [139,140]. However, studies in rodents show mixed results with increased [141,142], decreased [143] or no change [144,145] of D2 receptor levels. This could be due to different densities of D1 and D2 receptors in the striatum or the different administration routes of the drug, dosing regime and length of treatment/withdrawal period.

Circuitries implicated in addiction Elevated DA transmission in the NAcc is thought to be a primary mediator of addiction. However, repeated exposure, craving and relapse to cocaine are associated with the recruitment of glutamate transmission from the PFC and OFC [146] as well as activation of the dorsal striatum [147]. The PFC, OFC and anterior cingulate cortex regulate the emotional response, cognitive and executive functions with repeated drug exposure. The cellular adaptations of the prefrontal-NAcc glutamatergic pathways lead to diminished cognitive control and hyper-responsiveness to drug-associated stimuli

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[102,103,146,148,149]. The GABAergic MSNs in the NAcc and dorsal stria-tum receive both glutamatergic input from limbic and cortical regions and dopaminergic input from VTA. The MSNs integrate the information, and project to either motor regions that execute goal-directed behaviour or to limbic structures that execute emotional response [150]. The released DA modulates glutamate inputs and is dependent not only on DA receptor type but also on the glutamate receptor activated. Thus DA, via the D1 receptor, enhances NMDA receptor mediated responses, whereas via D2 receptors, DA reduces AMPA receptor mediated responses [151,152].

Drug-evoked synaptic plasticity occurs in the striatum and outlasts the presence of the drug in the brain, contributing to the reorganisation of neu-ronal circuits. Supporting this is the increased number of AMPA receptors in the NAcc for several weeks during withdrawal following repeated cocaine administration [153]. The NMDA receptors have also been found altered in experiments triggering cocaine-seeking, an experimental behavioural model of craving and relapse. Systemic administration of an NMDA antagonist blocks synaptic plasticity [154], as does ablation of the NR1 subunit of the NMDA receptor [155]. Overall these findings suggest that craving and re-lapse involves DAergic and glutamatergic interactions in the ventral mid-brain, striatum, and cortical and limbic structures such as hippocampus, amygdala, OFC and PFC. In conclusion, addiction is a complex disorder involving, and recruiting, several neurotransmitter systems and neuronal populations. Although much research has focused on revealing the mecha-nisms of this brain disease, much work remains to be done before we can fully understand and take control of its devastating nature.

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Materials and Methods

“Nevertheless the difference in mind between man and the higher animals, great as it is, certainly is one of degree and not of kind. We have seen that the senses and intuitions, the various emotions and faculties, such as love, memory, attention,

curiosity, imitation, reason, etc., of which man boasts, may be found in an incipient, or even sometimes in a well-developed condition, in the lower animals.”

Charles Darwin, The Descent of Man, page 86 (1871)

The experimental procedures used in this thesis work are described in detail in the materials and methods section of each paper. In this section, I discuss the methods I have used the most.

Production of transgenic mice The mice used for this thesis were kept according to the guidelines of Swed-ish regulation and European Union legislation. The Uppsala animal ethical committee has approved all studies. The production of the Vglut2f/f mouse, which is used in Paper I-V, is described in detail elsewhere [56]. In short, exons 4-6 are flanked by LoxP-sites and when the Vglut2f/f mouse is crossed with a Cre-expressing mouse, VGLUT2 expression is abolished in the Cre-specific region. The Cre mice described in this thesis are the CaMKIIα-Cre mice (Paper I and IV) [156], the DAT-Cre mice (Paper II-III) [157] and the Emx1-Cre mice (Paper V) [158]. The reporter mice used in this thesis are Z/AP, a double reporter mouse (Paper I) [159], and the TaumGFP reporter mouse [48](Paper I-II, Paper IV-V). The Z/AP reporter construct allows visualisation of cells that do not express Cre by beta-galactosidase (β-gal) expression, while cells that do express Cre are detected by their expression of alkaline phosphatase. The TaumGFP reporter is driven by the tƒau pro-moter, which enables localisation of cell nuclei that express Cre by β-gal expression and the projections of these cells by expression of GFP.

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Behavioural analysis Behavioural phenotyping is complex and challenging, but a number of ro-bust and reproducible methods have been established and validated to enable such analyses in the mouse.

Anxiety-related behaviour Tests of anxiety-related behaviour of the mouse are based on the approach-avoidance conflict. Mice have an innate drive to explore a new environment, but this drive is in conflict with exposure to open areas where the mice may be targets for predators [160,161]. The elevated plus maze (EPM) and the multivariate concentric square field (MSCF), used in Paper I-V, are based on this conflict [162,163].

The EPM is situated 52 cm above the floor and consists of four narrow arms, two of the arms are closed and two are open. The mouse is free to ex-plore the maze over a 10-minute period during which the latency to enter each arm, the number of entries and the duration of stay are scored.

The MCSF is a multivariate test arena for the mouse to explore freely over a 20 minute long session. Using the MCSF, we can evaluate behaviour associated with exploration, avoidance of open spaces, risk assessment, risk taking and shelter seeking. The arena contains a shelter, open and elevated areas, a hole board device, a bridge illuminated from underneath, slopes and areas with different light conditions. The dark corner room of the apparatus provides a shelter for the mouse. The bridge can be climbed from the slope, and therefore, the slope provides an assessment of risk-taking behaviour since the mouse can enter the bridge area. The bridge, the centre and central circle are regarded to be risky. However, the bridge can also be perceived as a possibility of escape for the mouse. The hurdle and corridors are explor-ative parts of the maze [163,164].

Depression-like behaviour In Paper I-II and Paper IV-V, the forced swim test was performed. The forced swim test is widely used to evaluate depression-like behaviour in rodents [165,166]. This is based on the assumption that an animal normally wants to escape from an aversive stimulus, but when the aversive stimulus is inescapable the animal will stop trying to escape (floating instead of swim-ming, in the forced swim test). Treatment with an antidepressant, especially serotonin-selective drugs [167], reduces the time spent floating.

The test apparatus consists of a clear Plexiglas cylinder with a diameter of 20 cm, which is filled with water up to 25 cm of height, a depth that prevents the animals from touching the bottom with their tail. The water is maintained at 25oC. The mouse is placed in the water and the time spent swimming is

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scored. Behaviour was recorded and analysed blindly in two trials performed 12 hours apart. In Paper I and IV, the scoring period was 12 minutes per trial, whereas in Paper II and V, it was 6 minutes per trial. A mouse was considered inactive when floating and making only the movements neces-sary to keep its nostrils above the water.

Social dominance The dominance tube test described in Paper I was used to evaluate the social behaviour of the mice. In the tube test, a control littermate and a KO mouse of the same sex were placed at opposite ends of a 30cm long and 3.5cm di-ameter tube and released. A subject was declared a “winner” when its oppo-nent backed out of the tube. Each pairing was performed twice.

Learning and memory Spatial learning and memory was examined in Paper II and Paper IV by using an eight-armed radial maze [168]. The apparatus consists of 8 arms with a central platform. The same four arms were baited with a hidden small piece of food pellet. For initial radial-arm maze learning, food deprived ani-mals were placed individually in the centre of the maze once each day for 5 days. The animals explored the maze for 6 minutes on the first trial day and thereafter the animals were allowed to remain in the apparatus until all pel-lets were obtained or a maximum time of 6 minutes. The optimal strategy for obtaining all the food in the least amount of time is to visit each arm only once during the trial. For each trial, a reference memory error (RME) was defined as a visit into an unbaited or incorrect arm. A working memory error (WME) was defined as a re-entry into an arm in which the reward was al-ready obtained during the session. The total number of entries, and the per-centage of correct responses in each arm were scored. 16 days after the last acquisition session, a retention test was performed to evaluate long time memory. The same arms were baited and the same procedure was used as described above.

Locomotor functions On the rotarod (Paper II), the mouse must continually walk forward on an accelerating rod to prevent it from falling off. The mouse is placed on the rod and the speed increases from 0 RPM to 45 RPM over one minute. Each mouse performed the test three times per day for three days. On the beam walk (Paper II), the mouse was given one minute to cross the one-meter long (12 mm in diameter) rod. Each mouse performed the test three consecutive times. Mice that did not complete the task were excluded from the trial.

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Sucrose bottle choice test Sucrose preference was tested using a two-bottle choice test with water, as rodents are very attracted to the taste of sugar [169]. The mice had access to a sucrose solution in one bottle and tap water in a second one. Each concentration was tested for 48 h and presented in an increasing order: 0.3%; 1.0%; 3.0%; 10% and 30%. Sucrose preference was defined as significantly higher consumption of sucrose than water.

Operant self-administration Operant self-administration (SA) methods are widely used in preclinical drug abuse research and are considered to be good and valid models of hu-man drug consumption [128]. In our experiments, mice were allowed to self-administer grain-based food pellets and sucrose pellets as well as intrave-nously delivered cocaine (Paper III). The operant responding was studied in MED-PC operant chambers (Med Associates inc., St. Albans, VT, USA) placed inside sound attenuating boxes with separate ventilation fans masking external sounds (Figure 5). Nose-poke devices were situated on each side of a food pellet receptacle.

Figure 5. A mouse during a trial in the operant self-administration box. (A) Responding in the active nose-poke hole causes illumination of the stimulus light above the nose-poke hole, and the delivery of a reinforcer in the food cup. (B) Col-lection of reinforcer in the in the food cup.

Fixed ratio (FR) and progressive ratio (PR) On a FR schedule, the mice need to nose-poke a fixed number of times to receive each delivery of the reinforcer. High FR schedules (e.g. FR5) are suitable for strong reinforcers, such as psychostimulants and sucrose [170]. The PR schedule is used to measure motivation/craving. The PR schedule exponentially escalates nose-poke response requirements within sessions [171].

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Operant self-administration of food/sucrose Following acqusition of the nose-poke task, the mice were analysed in a two-phase paradigm in order to reveal differences in consumption of sweet ver-sus non-sweet food. In the first phase, mice were food-restricted and ana-lysed for their consumption of non-sweet food pellets. In the second phase, non-food-restricted mice were analysed for consumption of sugar pellets. When an animal had acquired the task on the FR5 schedule, the program was switched to the PR schedule. The number of nose-pokes of both the active and the inactive devices were recorded.

Operant self-administration of cocaine Following acquisition of the nose-poke task, the mice underwent anaesthesia and surgery for the implantation of an intravenous catheter into the right jugular vein. Upon recovery, acquisition of cocaine responding was made on one initial dose. Thereafter the mice were allowed to self-administer differ-ent doses of cocaine according to a Latin square design. In a last step, all animals responded for the lowest dose. After a 21-day long forced absti-nence in home cage environment, the mice were allowed to respond to the presentation of visual and auditory cocaine-associated cues in the absence of cocaine delivery, and the cocaine-seeking behaviour was scored and ana-lysed.

In situ hybridisation and immunohistochemistry For detection of gene expression in brain tissue, non-radioactive in situ hy-bridisation (ISH) histochemistry was used in Paper I-II and Paper IV-V. By using a digoxigenin-labelled in vitro transcribed RNA probe, the comple-mentary mRNA expressed in the tissue can be detected. In a subsequent step, an enzyme-coupled antibody recognises the digoxigenin molecule. The cells with the transcription of the desired gene are visualised by the addition of a substrate that becomes coloured by the enzyme-coupled antibody. Since the mRNA is located in the cell soma, this area becomes visible. Detected sig-nals indicate that there is gene expression in the cell, but it does not show where and when the protein is localised.

Immunofluorescence (IF) histochemistry was used to detect proteins in brain tissue. An antibody recognises a protein epitope and binds to where it is situated in the cell. An advantage with IF is the possibility to combine several antibodies (Paper I, Paper II, Paper IV and Paper V) and also the possibility to combine it with ISH (Paper II).

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Aims

The overall aim of this thesis is to increase the understanding of how neu-ronal circuits using glutamatergic and dopaminergic signalling regulate dif-ferent kinds of behaviour. For this purpose, I used the floxed Vglut2 mouse as my primary research tool. Three different Cre-mice were used to delete VGLUT2 in specific neuronal circuits in order to study the consequences on an anatomical, histological and behavioural level.

Specific aims Paper I Analyse if Vglut2 expression in the preadolescent forebrain, which is re-stricted to certain subpopulations, has a functional role in cognitive and emo-tional aspects of behaviour.

Paper II Analyse if Vglut2 expression in midbrain DA neurons plays a role in basal behaviour and psychostimulant-induced locomotion.

Paper III Investigate if Vglut2 expression in midbrain DA neurons plays a role in re-ward consumption and drug-induced seeking.

Paper IV Study if the altered behavioural responses described in Paper I were similar in male and female mice.

Paper V Evaluate if Vglut2 expression in the forebrain during development is impor-tant for cognitive and emotional aspects of adult behaviour.

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Results and Discussion

Paper I We wished to investigate the role of Vglut2 expression in restricted popula-tions of the cortex (the retrosplenial group (RSG), layers III and V and piri-form cortex), hippocampus (subiculum and presubiculum) and parts of the amygdala. The expression of Vglut2 in these areas changes during develop-ment and at preadolescent shows this restricted mRNA expression pattern [50]. We bred CaMKIIα-Cre mice with Vglut2f/f mice to generate Vglut2f/f;CaMKII-Cre cKO mice, in which Vglut is deleted from P15. We evalu-ated several aspects of behaviour of the cKO mice by comparing them with control littermates. The phenotype of the Vglut2f/f;CaMKII-Cre mice was striking considering that the deletion of Vglut2 was restricted to subpopulations of the amygdala, cortex and hippocampus. The loss of Vglut2 lead to resulted in SZ-like behaviour (as assessed in mice) such as increased locomotor activity, altered social dominance, decreased sensorimotor gating and impaired long-term memory.

To be able to reveal the neurocircuitry affected by our deletion, we ana-lysed the target regions of the neurons that had lost Vglut2 expression. We observed reduction of VGLUT2-positive varicosities in the NAcc, probably due to the loss of Vglut2 expression in the subiculum, which is the major output region of the hippocampus, sending projections to the NAcc, cortex and amygdala. The amygdala also sends projections to the NAcc and could be responsible for the reduced number of varicosities. The cortical RSG and the hippocampal subicular region could be responsible for the observed re-duction of VGLUT2-positive varicosities in the dorsolateral striatum. Other parts of the cortex also send projections to the striatum, which can contribute to the reduction of VGLUT2-positive terminals observed there. Both the dorsolateral striatum and the NAcc are regions that showed significant re-duction of VGLUT2-positive terminals. These areas are also target areas for the nigrostriatal pathway and mesocorticolimbic dopaminergic pathway; pathways involved in spontaneous hyperlocomotion (Figure 6).

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Figure 6. A schematic overview of the VGLUT2-mediated signalling that is af-fected in the Vglut2f:fCaMKII-Cre mice. The loss of VGLUT2-mediated signalling in the cortex, hippocampus and amygdala leads to severe behavioural changes. The effect on the midbrain DA system is mainly from loss of VGLUT2-signalling pro-jections from the amygdala and cortex. Abbrevations: RSG; retrosplenial group, SNc; Substantia nigra pars compacta, VTA; ventral tegmental area.

The DA neurons are controlled by glutamatergic cortical projections either directly onto DA neurons or indirectly via GABAergic spiny neurons, acting like an accelerator or a brake, respectively [119]. If the brake were no longer functional, due to the reduction of glutamate, this would lead to an increase of DA release. Using a positron emission tomography-scan, we showed that the hypoglutamatergic state increased the number of available dopaminergic D2 receptor binding sites in the striatum. Further strengthening our hypothe-sis that the DA system is altered is the observation that the cKO mice showed a partial attenuation of the deficits in sensorimotor gating by the DA-stabilising antipsychotic drug aripiprazole and an increased sensitivity to amphetamine.

In conclusion, this paper showed a functional role of VGLUT2 in higher brain function and emotion. Further, the Vglut2f/f;CaMKII-Cre mice display ele-ments from all three main categories of symptoms of SZ. In addition, we showed a link between the induced hypoglutamatergic state and hyperdopa-minergia, which may be of relevance for disorders such as SZ.

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Paper II In this study we wished to evaluate the functional role of VGLUT2 in DA neurons. First, we showed that Vglut2 mRNA is expressed in multiple re-gions in the embryo, including the ventral midbrain where DA neurons de-velop. We could detect expression of Vglut2 mRNA in TH-positive DA neu-rons in the midbrain. To further analyse the function of Vglut2 expression in DA neurons, we crossed the Vglut2f/f mice with DAT-Cre mice, generating Vglut2f/f;DAT-Cre cKO mice. Our analysis of basal behaviour did not show any differences between the Vglut2f/f;DAT-Cre cKO and littermate control mice in motor coordination and memory function. However, the cKO mice had an increased latency of movement when assessed in EPM. To evaluate the pos-sible role of VGLUT2 in psychostimulant-induced responses, the cKO mice were administrated amphetamine at three different doses in a home-cage environment. The cKO mice showed a significant difference in behaviour with an overall lower total activity at all doses analysed compared to control mice. However, both horisontal (locomotion) and vertical (rearing) behav-iour was higher in the cKO mice than in controls, suggesting that the higher total activity in the control mice were due to stereotypies, and that cKO mice were more insensitive to these. To confirm that the loss of Vglut2 in DA neurons does not affect pure glutamatergic neurons, we used a highly sensi-tive in vivo amperometry method called the Fast Analytical Sensing Tech-nology (FAST) and could confirm that the overall glutamate release is not affected in the cKO mice.

We hypothesised that the blunted response of the cKO mice to ampheta-mine results from reduced mesoaccumbens DA transmission. If so, this could be due to loss of co-release of glutamate or loss of vesicular synergy between VGLUT2 and VMAT2. Another possibility is that it is the result of developmental defects in the midbrain DA system [172] and that the loss of VGLUT2 in the cKO mice could lead to dysfunctional wiring.

In summary, we showed that the loss of VGLUT2 in DA neurons leads to a blunted behavioural response to the DA-releasing drug amphetamine.

Paper III The finding that VGLUT2 in DA neurons (referred to as VGLUT2/DA) is required for psychostimulant-induced behavioural response (Paper II) was further investigated by studying voluntary consumption of rewarding sub-stances in the Vglut2f/f;DAT-Cre cKO mice. By performing the sucrose bottle test, we could show that the cKO animals displayed a preference for sucrose, at the low dose, which the control mice did not prefer over water. We also found that the cKO mice consumed more sucrose reinforces in an operant SA paradigm, in a non-hunger-driven manner, indicating a role for

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VGLUT2/DA in palatability-stimulated feeding. We wished to determine whether the VGLUT2/DA also plays a role in the operant responding for cocaine and cocaine-associated cues. When the mice nose-poked for cocaine over a range of concentrations, the cKO mice displayed a significantly in-crease in SA at the lowest dose compared to controls. Further, in a cocaine-seeking analysis, it was revealed that the cKO displayed as much as 76% increased cocaine-seeking in response to drug-associated cues. Since expo-sure to drug-associated cues and memories is implicated in the vulnerability to relapse to drug use, this result implies a role for VGLUT2 in this behav-iour [173].

We addressed striatal function to reveal whether biochemical alterations had occurred that could explain this behavioural phenotype. The cKO dis-played increased D1 receptor binding in the dorsal striatum, while there was an up-regulation of D2 in the NAcc shell. We further analysed expression of the immediate early genes c-fos and Nur77. c-fos and Nur77 are both in-volved in acute and repeated psychostimulant treatment [174,175]. The analysis in the striatum of the cKO mice showed that the basal level of DA-regulated c-fos and Nur77 was increased. The basal levels were increased in both the NAcc and dorsal striatum in such a potent way that cocaine could not further increase the levels. By using FAST analysis we showed that DA release was decreased in both the NAcc and dorsal striatum of the cKO mice.

The increased D1 receptor and D2 receptor and IEG levels observed could lead to the increased responsiveness to palatable food and cocaine. Another plausible explanation is directly related to the reduced DA release. It has been speculated that DA hypofunction stimulates drug taking in order to normalise DA levels [176,177]. However, the deficiency is typically asso-ciated with reduced DA receptor binding [178,179] and a general (non-dose dependent) increase in self-administration [180]. In summary, although DA hypofunction may be correlated to the behaviour of the cKO, a sensitised phenotype may be a more plausible explanation for this relationship than reward deficiency mechanisms. While initial reward behaviours are con-nected to the NAcc, the dorsal striatum is involved when an addict or ex-perimental animal makes the transition from a short-term use of drugs to the compulsive phase [147]. Studies have revealed that inactivation of the dor-sal, but not ventral striatum, reduces cocaine seeking after abstinence in rats [181]. The up-regulation of the D1 receptor and the elevated levels of IEG suggest an important modulatory role for VGLUT2 in dorsal striatal DA neurons in cue-induced relapsing behaviour, manifested in the mice as in-creased cocaine-seeking.

Taken together, we showed that loss of VGLUT2 from DA neurons leads to increased consumption of both natural (sugar) and drug (cocaine) rein-forcers and also promotes cocaine-seeking in response to cocaine-associated

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cues. Further, our findings showed that loss of VGLUT2 in DA neurons leads to biochemical neuroadaptations in the reward system.

Paper IV We performed a gender-dependent analysis of our previously reported be-havioural data from Vglut2f/f;CaMKII-Cre cKO mice described in Paper I, com-plemented with analysis of Vglut2 transcriptional levels in a cohort of human brain autopsies from female and male SZ patients and control individuals.

The basal behavioural tests revealed gender differences in the cKO mice. The female cKO showed reduced anxiety and impaired social behaviour compared to controls, something that was not observed in the cKO males.We investigated the effect of the conditional gene targeting of Vglut2 on the DA system, as seen in Paper I, by challenging the mice with amphetamine. The amphetamine treatment significantly increased the locomotor activity of the cKO female mice compared to the controls, an effect which was not seen in the male mice. Additionally, we analysed the DA levels by HPLC and de-tected a significant increase in DA in the striatum and prefrontal cortex in the female cKO mice, but not in the male cKO mice, compared to control mice. These results strengthen our hypothesis that the DA system was af-fected and plays a vital role to the behavioural changes seen in the female but not in the male cKO mice.

We analyzed mRNA levels of Vglut2 in Brodman area 8 and 9 of the cor-tex of brains from deceased patients diagnosed with SZ. The mRNA levels were significantly increased in the female SZ subjects compared to female controls, but no such difference was noted in male patients. This might sug-gest a different response to medication between the genders. Alternatively, women diagnosed with SZ may have higher endogenous levels of Vglut2 mRNA than do controls.

In conclusion, we demonstrated that in both mice and humans, altered ex-pression of Vglut2 levels is correlated to alterations in behaviour, with a more prominent phenotype in females than in males.

Paper V The expression profile of VGLUT2 is highly interesting, as it is expressed both as mRNA and protein already at midgestation, long before synapto-genesis occurs in the brain. The SZ-like phenotype of the Vglut2f/f;CaMKII-Cre cKO mice lead us to investigate the putative importance of the temporal aspect of VGLUT2-mediated signalling in the forebrain. For this, we used an Emx1-Cre transgenic mouse, where Cre-expression is turned on at E11.5 in the cortex, amygdala and hippocampus. Compared with the spatial expres-

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sion of CaMKIIα-Cre, the Emx1-Cre showed the same expression in the hippocampus and cortex, while there was some variation in the amygdala. The Emx1-Cre mouse was cross-bred with the Vglut2f/f mice to generate Vglut2f/f;Emx1-Cre mice. We confirmed the deletion of Vglut2 by using in situ hybridisation for Vglut2 mRNA expression. Subpopulations of the cortex and hippocampus were identified to have lost their expression of Vglut2 mRNA in Vglut2f/f;Emx1-Cre cKO mice compared to controls. The expression of Vglut2 mRNA in enthorinal and piriform cortex in the cKO mice was not deleted as seen in the Vglut2f/f;CaMKII-Cre cKO mice.We analysed the presynap-tic terminals in the respective projection target areas to determine whether there were any detectable changes in the neurons that had lost the Vglut2 mRNA expression. We could not detect any significant differences in VGLUT2 protein levels in projecting target areas in the striatum, cortex and hippocampus, suggesting that when Vglut2 is removed during embryonic development, mechanisms to replace the lost expression are activated. The same battery of behavioural tests as for the Vglut2f/f;CaMKII-Cre mice was per-formed on these mice. The Vglut2f/f;Emx1-Cre mice cKO mice displayed normal cognitive behaviour and normal sensorimotor gating.

Vglut2 is expressed in the anterior cortical (ACo), the posterolateral corti-cal (PLOc), the basomedial amygdaloid (BMA) and medial amygdaloid (MeA) nuclei together with the bed nucleus of the accessory olfactory tract (BAOT) in control mice. The four amygdala nuclei lacked Vglut2 expression in the Vglut2f/f;CaMKII-Cre cKO mice. In the Vglut2f/f;Emx1-Cre cKO mice, how-ever, the expression of Vglut2 in the MeA and BAOT was normal, while in the ACo and BMA it was strongly reduced. We wished to address this dis-crepancy in deletion of the four amygdala nuclei between the Vglut2f/f;CaMKII-

Cre and Vglut2f/f;Emx1-Cre cKO mice. The amygdala is highly involved in emo-tional behaviours and therefore we used the MCSF to investigate the emo-tional state of the cKO mice. The lack of avoiding open areas, increased risk-taking and decreased shelter-seeking were significantly changed for the two mouse lines compared to controls. However, possibly due to the partial dele-tion in the amygdala of the Vglut2f/f;Emx1-Cre mice, they did not show the in-creased exploratory activity and risk assessment that was seen in the Vglut2f/f;CaMKII-Cre mice.

In summary, this study shows that targeted deletion of VGLUT2 in the forebrain at midgestation does not significantly alter cognitive function or the level of locomotion, possibly due to redundancy mechanisms, but does lead to aspects of altered emotional behaviour.

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Closing remarks

Despite its restricted expression in forebrain neurocircuitry and in subpopu-lations of midbrain DA neurons, the gender-dependent cognitive and affec-tive behaviour displayed by the Vglut2f/f;CaMKII-Cre cKO mice alongside with the increased consumption of cocaine and sweet food in the Vglut2f/f;DAT-Cre cKO mice, indicate that the functional role of VGLUT2 is quite profound.

Figure 7. Overview of the neuronal circuitries and the different Cre-LoxP lines described in Paper I-V. More specific Cre lines such as SNc-Cre, VTA-Cre, cor-tex-cre, amygdala-cre and hippocampus-Cre in combination with the floxed VGLUT2 mouse could be instrumental in further elucidation of the role of VGLUT2 in neuronal circuits of motor and limbic behaviour. Vglut2-expressing areas are shown in blue.

In light of the findings described in this thesis, some considerations for fu-ture studies might be worthwhile. With the use of the CreLoxP-system, it would be highly appealing to, in an even more restricted spatial and temporal manner than shown here, delete Vglut2 in the circuitries analysed in this thesis (Figure 7). For example, transgenic mice expressing Cre under con-trol of an amygdala-specific promoter (“amygdala-Cre” in Figure 7) would be useful for further analysis of the described alterations in emotional behav-iour in the Vglut2f/f;CaMKII-Cre and Vglut2f/f;Emx1-Cre mice, while Cre expressed

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under control of nigra-specific (“SNc-Cre” in Figure 7) versus VTA-specific (“VTA-Cre” in Figure 7), would be useful for assessing, for example, the role of the glutamate/DA co-phenotype in psychostimulant-induced locomo-tion versus drug-seeking behaviour.

An interesting observation is that VGLUT2 is expressed early in devel-opment, already at midgestation. Future studies will likely address if VGLUT2 might play a developmental role, such as in promoting neuronal survival or synapse formation, which would be expected to have a major impact on adult behaviour. Moreover, we will need to address the fate and role of the glutamate, which is released from dopaminergic axon terminals. It is still not clear whether this glutamate is sufficient to produce a downstream effect, a feature which is of major functional significance and which should be addressed shortly.

The results in this thesis show the importance of novel ways of studying neuronal circuits in the brain. We have addressed the role of the presynaptic glutamatergic neuron and shown that the temporal and spatial distribution of VGLUT2 is of great significance for normal brain function.

40

Acknowledgments

Finally, here we are and I could not have made it this far if it would not be for all the great people I have had around me through the years. Firstly and mostly, I would like to thank my supervisor Åsa Mackenzie. I am so grateful you gave me the opportunity to be in your group, you are a true inspiration. Thank you for always showing me support and always keeping your door open whenever help is needed in any way. I am also very thankful that you have given me the chance to go to scientific meetings and for in-cluding me in all the collaborations you have initiated. Klas Kullander, my co-supervisor, thank you for spreading a very friendly and open atmosphere around the lab. Of course I would not be where I am today if it wasn’t for all the people in the awesome team ÅsaM who have helped me along the way: Casey, the most pedantic and tidiest person in the lab, but maybe a wee bit crazy other-wise, thank you for always being helpful. Carolina, always fun to work to-gether, as well as a really nice friend. Emma, my sweet co-Phd, we work really well together and are great friends, we were on the best vaca-tion/conference in San Diego! Ernesto came along just before Christmas, full of ideas and brought a lot of energy to our group. Johan who I had the pleas-ure of working with for a short and sweet J intense productive period, al-ways helping me in any way possible, thank you for the collaboration, it was great, and you’re a really good friend as well. Anna, thank you for guiding me to through all the steps of Q-PCR and being so nice. Emelie, thank you for being ever helpful and helping with the behavioural experiments. Also, I would like to thank the other students that I have taught; Assar, Emily, Li Li, Nadine and Tina. Erik, who came and worked with us one semester and did great work on the Emx-story and is a fun person to hang with. So, I think this concludes that I have an awesome environment at my working place. Anders E, my go-to-guy whatever the deal was, if it concerned in situ, TV-shows or anything in between you always helped and were a great friend. Fatima, that has almost been in the lab as long as I have, the kindest and sweetest person. Kasia, that hangs in the animal house as much as I do, it’s always nice with the company down there. Bejan, that has the best taste in

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the lab since she as well, is a big fan of the all time best show, Buffy, which no one else seems to be?! Nicole, my roommate for a very short time, but we succeeded in becoming really good friends. Thank you, Lill-Anders for help-ing me with the digigate and perfusions, it is very much appreciated and also for being a good friend. My nice roommates; Christiane that knows every-thing about immunos, but has the messiest desk and Hanna that has the best laugh in the lab that always makes you laugh back. Martin with the ironic/sardonic humour that always puts you in a good mood, Kali that is always up for playing beach volleyball (and good at it) and Chetan that is genuinely super interested in everything whether it is some behavioural set up or anything else. Malin, my room mate for many years, I still wonder if I am going to hear the whole song..ja det var på kapris, or maybe that’s all I am going to get ;). Kia, Johan, Sharn, Björn, Pavo, Sanja and Martina thank you for making the lunches and the hours in the lab much more enjoyable and fun. Richardson, thank you for always having a good idea around the corner. Old members of the Kullander group; Nadine, Daniel, Anna, Henrik G, Henrik B and Smitha, thank you for creating such a nice atmosphere dur-ing my first couple of years here. Maria, thank you for being my sparring partner in the gym and my fika-partner, always a sweet friend. All the great collaborators I have had the opportunity to work with; the Nor-dic co-transmission network team; Guillaume, Louis-Éric, Alfredo, Laurent, Daniel and Noemie, Montreal, hopefully I will come and visit your lab in the future! Briac, thank you for performing the surgeries in Paper III. Madeleine, you taught me the eight arm radial maze, thank you so much for taking the time. Marita and Erika that have taught me a lot about behavioural studies when I was all new to it. Kim, thank you for the time in Göteborg, when we did all the dissections and you were a great host. Thank you Lina, for the joint paper that we did. Thanks to all the time I spent in the BMC, I have had met other nice friends as well such as Greta, Josefin, Sahar, Markus, Jonathan and Mathias. Thanks to Birgitta, Emma, Mariana, Ulla, Cecilia, Lena and Ted who do all the really important things so the place is running smoothly.

Susanne, Sussie, Eva, Jana, Joanna, Jenny thank you for taking care of our mice, without you we could do practically nothing in our lab ;)

My lovely beach playing friends, without you life would not be so meaning-ful, all the summers I spent with you and all the beach camps, all the hours in the car going to tournaments, Anna D, my latest partner, Agnes, my first partner, and Emma and Maria in between, thank you that you all shared the dedication to practise and the will to win. Vera, thank you for all the times

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we won and all the good times together! Then all you other sparring people, Sofia, Elin, Malin, Evelina, Anna N, Sara, Linnea, Hanna and Emma E; the Fyrishov crew. I love that we all share the same passion and that you all are the best people to hang with. Then we have the Biolog-familjen; Emma, my first friend in Uppsala and one of the reasons why I stayed, Elisabet, Hanna, Hanna and Anna thanks for all the great fikas, the laughs and the good old times. Ulrika, the person who made me move to Uppsala, thank you! Klara and Anna, the ones that are like family, I’ve known you forever and hopefully we always stay close.

Mamma, pappa, Johan and Erika, thank you for being ever present in my life. Last but not least, I would like to thank Pelle for always being my greatest support and biggest fan, but mostly for being the one person I care for most in the whole world, at least until our baby comes ;).

“There is one more thing, it’s been emotional.”

Big Chris; Lock, Stock and two Smoking Barrels

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