Synaptic Vesicle Glycoprotein 2A (SV2A) as a Measure of Synaptic Plasticity in Animal Models of Depression

Saba Ali & Mariam Labrouzi

Masters in Biomedicine

June 2019

Supervisors

Professor, MD, DMSc, Jens D. Mikkelsen

Professor, Ph.D., M.Sc. Cathy Mitchelmore

Abstract

Depression is associated with a loss of synapses, which is brought on by a downregulation in important synaptic proteins involved in maintenance of synapses. The presynaptic protein, Synaptic Vesicle Glycoprotein 2A (SV2A) is a proposed indirect measure of synaptic density and has been shown to be inversely correlated to the severity of depressive symptoms exhibited in humans. Quantification of synapses through various methods using radiotracers targeting synaptic proteins, such as SV2A, may be a non-invasive method for diagnosing, monitoring and an in depth understanding of depression mechanisms. The overall aims of this thesis were to validate techniques detecting SV2A in the rat brain determined by in vitro autoradiography using the radiolabeled ligand [3H]UCB-J and immunoblotting using specific antibodies for SV2A and synaptophysin. Further; in order to determine the distribution of SV2A across brain regions and in depression, various animal models were employed, namely: Flinders Sensitive Line, chronic mild stress and electroconvulsive stimulation. The examination of the regional SV2A distribution across various brain regions, revealed that differences in the amount of SV2A are observed between age groups; and further, that the SV2A expression is higher overall compared to that of synaptophysin. In the diurnal cycle experiments we found that the SV2A binding and the corresponding blood corticosterone levels were correlated in the corticosterone treated animals. In the electroconvulsive stimulation rat model a significant difference was observed between the ECS and control group in two brain regions. A decrease in SV2A binding was observed in the amygdala and ventral hippocampus in the ECS group. In the chronic mild stress rat model, the SV2A binding was significantly increased in both the prefrontal cortex and the orbitofrontal cortex; as a response to agomelatine treatment. In conclusion, conducting similar in vitro experiments as presented here and in vivo and clinical studies is necessary for the proper understanding of the involvement of SV2A in synaptic plasticity and depression.

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Summary

Depression associeres med synapsetab grundet en nedregulering af vigtige proteiner involveret i opretholdelsen af synapser. Et foreslået indirekte mål for synapsedensitet er det præsynaptiske protein SV2A. Proteinet har vist sig at være omvendt korreleret til sværhedsgraden af symptomer på depression i mennesker. Kvantificering af synapser gennem forskellige metoder der bruger radioligander målrettet mod synapseproteiner, såsom SV2A, er mulige non-invasive tilgange til diagnosticering og monitorering af depression samt til at udvide den molekylærbiologiske forståelse af lidelsen. De overordnede formål med dette speciale var at validere teknikker til detektion af SV2A i rottehjernen ved brug af in vitro autoradiografi med radioliganden [3H]UCB-J og immunoblotting med antistoffer, der specifikt targeterer vesikelproteinerne SV2A og synaptophysin. Derudover, at bestemme fordelingen af SV2A henover hjerneregioner og i forskellige dyremodeller af depression, nemlig: Flinders Sensitive Line, elektrokonvulsiv stimulation og kronisk mild stres. Undersøgelsen af den regionale SV2A-fordeling i forskellige hjerneregioner viste forskelle i proteinmængden imellem aldersgrupper. Dette forsøg viste ydermere, at SV2A-ekspressionen overordnet set er højere end ekspressionen af synaptophysin. Døgnrytmeeksperimentet med administration af corticosteron viste, at SV2A-bindingen og de korresponderende blod-corticosteronniveauer var korrelerede i dyr behandlet med corticosteron. I rotterne behandlet med elektrokonvulsiv stimulation blev en signifikant forskel observeret mellem ECS- og kontrolgrupperne, specifikt i to hjerneregioner. Et fald i SV2A bindingen blev set i amygdala og ventral hippocampus i ECS-gruppen. I kronisk mild stress rottemodellen var SV2A bindingen signifikant opreguleret i prefrontal og orbitofrontal cortex som respons til agomelatinbehandling. Lignende in vitro-eksperimenter, som præsenteret her, samt in vivo- og kliniske studier er nødvendige for den dybdegående forståelse af SV2A’s rolle i synaptisk plasticitet og depression.

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Table of Contents

Abstract ...... 2 Summary...... 3 Table of Contents ...... 4 Abbreviations ...... 7

1 INTRODUCTION ...... 8

2 THEORETICAL BACKGROUND ...... 9 2.1 Major Depressive Disorder (MDD) ...... 9 2.2 Synapse Physiology and Glutamatergic Neurotransmission...... 12 2.3 Synaptic Plasticity ...... 13 2.4 Synaptic Plasticity and Depression ...... 14 2.5 Synaptic Vesicle Glycoprotein 2A (SV2A) ...... 15 2.6 Markers of Synaptic Density ...... 19 2.7 Animal Models of Depression ...... 20 2.7.1 Corticosterone ...... 21 2.7.2 Flinders Model...... 21 2.7.3 Electroconvulsive Stimulation (ECS)...... 22 2.7.4 Chronic Mild Stress (CMS) ...... 23 2.8 Theory behind the methods ...... 24 2.8.1 Autoradiography ...... 24 2.8.2 Western Blot ...... 25

3 STATE OF THE ART ...... 28 Main aims ...... 28

4 METHODS ...... 29 4.1 Animals and Brain Tissue for Autoradiography ...... 29 4.1.1 Tissue for Method Validation...... 29 4.1.2 Tissue for Studying the Effect of Corticosterone on the Diurnal Cycle...... 29 4.1.3 Flinders Resistant and Sensitive Line...... 30 4.1.4 Electroconvulsive Stimulation (ECS)...... 30 4.1.5 Chronic Mild Stress (CMS)...... 30

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4.2 In Vitro Autoradiography using [3H]UCB-J ...... 31 4.2.1 Data Analysis ...... 31 4.3 Animals and Brain Tissue for Western Blot Analyses...... 32 4.3.1 Tissue for Method Validation...... 32 4.3.2 Tissue for Localization Study...... 32 4.3.3 Flinders Resistant and Sensitive Line...... 32 4.3.4 Electroconvulsive Stimulation (ECS)...... 33 4.4 Western Blotting for the Detection of SV2A Expression...... 33 4.4.1 Sample Preparation and Protein Concentrations ...... 33 4.4.2 SDS Page and Immunoblotting...... 33 4.4.3 Gel electrophoresis and Blotting ...... 34 4.4.4 Immunodetection...... 34 4.5 Statistical Analyses ...... 35

5 RESULTS ...... 36 5.1 Autoradiography Method Validation ...... 36 5.2 Western Blot Method Validation ...... 37 5.3 Localization of SV2A-immunoreactivity in Various Brain Regions ...... 38 5.4 Effects of Diurnal Corticosterone ...... 39 5.5 Flinders Resistant and Sensitive Line ...... 41 5.6 Electroconvulsive Stimulation ...... 43 5.7 Antidepressant Treatment in Chronic Mild Stress Animals ...... 46

6 DISCUSSION ...... 48 6.1 Establishment of Methods ...... 49 6.1.1 Autoradiography Method Validation ...... 49 6.1.2 Western Blot Normalizations ...... 50 6.1.3 Troubleshooting ...... 50 6.1.4 Alternative Methods ...... 51 6.2 SV2A Distribution and Corticosterone Effects in Rats ...... 52 6.2.1 Localization of SV2A-Immunoreactivity ...... 52 6.2.2 Corticosterone Effect on Diurnal Cycle ...... 53 6.3 Models of Depression ...... 54 6.3.1 Flinders Sensitive Line Rats in Exhibition of Proteomic Changes in Depression...... 54 6.3.2 Electroconvulsive Stimulation and Synaptic Plasticity ...... 55 6.3.3 Antidepressant Effects of Agomelatine Increase SV2A Binding in CMS ...... 56 6.4 SV2A Regulation in Preclinical Depression Models ...... 56

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7 CONCLUSION ...... 58

8 REFERENCES ...... 60

9 APPENDICES ...... 65

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Abbreviations

[3H] Tritium AMPARs α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor BDNF Brain-derived Neurotrophic Factor Bmax Total Binding Site Density BSA Bovine Serum Albumin CMS Chronic Mild Stress EGTA Egtazic Acid ECS Electroconvulsive Stimulation ECT Electroconvulsive Therapy FRL Flinders Resistant Line FSL Flinders Sensitive Line HPA Axis Hypothalamic Pituitary Adrenal Axis HRP Horseradish Peroxidase Kd Radioligand Equilibrium Dissociation Constant kDa kilo dalton LBS Levetiracetam Binding Site LDP Long Term Depression LEV Levetiracetam LTP Long Term Potentiation MDD Major Depressive Disorder mPFC medial Prefrontal Cortex mRNA messenger RNA mTORC1 mechanistic target of rapamycin NMDARs N-methyl-D-aspartate receptor OFC/OFCtx Orbitofrontal Cortex PET Positron Emission Tomography PFC/PFCtx Prefrontal Cortex PTSD Post Traumatic Stress Disorder SDS Sodium Dodecyl Sulphate SNP Single Nucleotide Polymorphism SSRIs Serotonin Selective Reuptake Inhibitors SV Synaptic Vesicle SV2A Synaptic Vesicle Glycoprotein 2A SYN Synaptophysin TBS Tris Buffered Saline TBS-T Tris Buffered Saline Tween 20 TE Tissue Equivalent TH Tyrosine Hydroxylase TM Transmembrane UCB-J ((R)-1-((3-((11)C-methyl-(11)C)pyridin-4-yl)methyl)-4-)3,4,5-trifluorophenyl)pyrrolidin-2-one)

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1 INTRODUCTION Depression is a debilitating psychiatric disease and is a major issue on a global basis. In 2018, the World Health Organization stated that 300 million people are affected by this disease each year and that in 800,000 of these cases it is to such a degree that those suffering from it, resort to suicide. The most commonly prescribed antidepressant group is serotonin reuptake inhibitors (SSRIs); however, this treatment is far from effective in all patients. One third of patients have the desired response to the treatment, whilst one third are completely resistant to treatment with SSRIs (Hvilsom et al., 2019). Treatment resistant depression therefore presents as more challenging, as these patients are often resistant to conventional antidepressants, which makes it difficult to come up with a decent treatment regimen and make sure that these patients do not suffer the same fate as the before mentioned 800,000 people. Depression is associated with a loss of synapses, which is brought on by a downregulation in important synaptic proteins involved in maintenance of synapse number. Further, rodent models exhibit depressive-like symptoms and behavior as a result of loss of synapses in the medial Prefrontal Cortex (Holmes et al., 2019). An increase in synaptic plasticity and connectivity is correlated with an increase in control of emotions in depressed subjects. The same has been displayed in animal models with depressive behavior, who were reversed in this behavior as a result of the increase in synaptic connectivity. It has been suggested that one of the exacerbating reasons behind treatment resistant depression, is the loss in synapses which makes it more difficult to target the underlying neurobiological pathologies (Holmes et al., 2019). The presynaptic protein Synaptic Vesicle Glycoprotein 2A (SV2A) is a possible measure for synaptic number, since the number of vesicles, and therefore vesicle proteins, is restored quickly after neurotransmission in order to uphold the neurotransmission capacity of synapses. Furthermore, SV2A was shown to be inversely correlated to the severity of depressive symptoms exhibited in humans (Holmes et al., 2019). Based on the pathology and inefficiency of conventional treatment, the lack of knowledge on the underlying biology of depression, brings forth an urgency in the detailed understanding of these mechanisms (Hvilsom et al., 2019). Thus, quantification of synapses through various methods using radiotracers targeting synaptic proteins, such as SV2A, may be a non-invasive approach for diagnosing, monitoring and an in depth understanding of depression mechanisms.

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2 THEORETICAL BACKGROUND 2.1 MAJOR DEPRESSIVE DISORDER (MDD) Major depressive disorder is an impairing psychiatric disorder with a complex and not well- understood pathology. The disorder is under polygenic influence and is associated with interactions between genetic, epigenetic and environmental factors, among these stress and impaired neuroplasticity being the most significant (Kupfer et al., 2012, Hvilsom et al., 2019 and Zhao et al., 2008). These conditions make it difficult to identify single candidate genes that are associated with depression. However, identification of candidate genes, associated with known biological mechanisms and metabolic pathways for antidepressant drugs, has been more successful. Thus, enabling a way to predict the response to antidepressant treatment (Kupfer et al., 2012).

There are three primary groups of peripheral hormone-type factors, for which genetic variants are associated with depression, which are proposed to be involved in the pathophysiology of the disorder: (1) neurotrophic factors, such as Brain Derived Neurotrophic Factor (BDNF) in this case the Met allele of the Val66Met (Hajcak et al., 2009 and Kupfer et al., 2012), (2) SNPs in a number of proinflammatory cytokines (Kupfer et al., 2012) and (3) impaired regulation of the hypothalamic- pituitary-adrenocortical (HPA) axis due to SNPs associated with binding, etc. of glucocorticoids (Di lorio et al., 2017 and Kupfer et al., 2012). As an example, it has been shown that the level of serum and plasma BDNF is decreased in depressed subjects, which can be reversed by antidepressant treatment (Sen et al., 2008 and Bocchio-Chiavetto et al., 2010). Furthermore, the secretion and production of proinflammatory cytokines are increased in stressed and depressed individuals. Antidepressants can normalize the levels of cytokines by suppressing their synthesis (Dantzer et al., 2010 and Duman et al., 2016).

It is suggested that environmental events and different risk factors contribute to depression by converging molecular and cellular mechanisms, causing disruption of functional and structural connections. This results in dysfunction of the neuronal circuits that are essential for the regulation of mood and cognitive function (Duman et al., 2016). Evidence has been brought forth, suggesting that chronic stress leads to a loss of synapses in cognitive circuits. Specifically, in MDD, stress- induced changes cause cognitive and mood-related deficits (Holmes et al., 2019).

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Furthermore, the loss of neurogenesis in the hippocampus caused by stress is also associated with cognitive deficits in stress and depression (Duman et al., 2016) (see fig. 1).

Figure 1 - Decrease in synapse number due to chronic stress. A - Neurons in rat medial prefrontal cortex (mPFC), left pictures controls and right stress induced. The loss in dendrite density is apparent from the two-photon laser scanning microscopy pictures. The two pictures to the right, illustrate a magnification of the difference in dendrites before and after stress inducement. Figure from Duman et al.,2016. B - Lowered synapse number and neurotransmission, as a result of downregulation in BDNF/mTORC1 caused by stress. The subsequent downregulation in the AMPA subunit GluA1. Self-made figure with Biorender.

Several neuronal systems are dysfunctional and display a reduced volume in important regions due to the loss of synapses and neurons in the depressive disorder (Holmes et al., 2019 and Kupfer et al., 2012). These systems include three main neuronal systems/regions: (1) subcortical systems involved in emotion and reward processing, (2) medial prefrontal and anterior cingulate cortical regions involved in emotional regulation, cognition and executive function, and (3) lateral prefrontal cortical systems, involved in cognitive control and regulation of emotion (Kupfer et al., 2012). There are some findings from neuroimaging studies, providing evidence of abnormalities in these neuronal systems. Such as, abnormally increased amygdala, ventral striatal and medial prefrontal cortical activity towards negative emotional stimuli (Surguladze et al., 2005 and Fales et al., 2008). This indicates that a low synaptic density is associated with neuronal network alterations and thus the symptoms of depression (Holmes et al., 2019).

Regional synaptic changes, such as synapse loss in the hippocampus and cerebral cortex have been demonstrated in patients with depression (Finnema et al., 2016). Among the altered brain structures and functions in depression, the most consistent is found to be reduced volume of the prefrontal cortex (PFC) and hippocampus (Duman et al., 2016). The structural changes in these regions involve loss of dendritic spines and synapses. Postmortem studies have shown reduced synapse number in the PFC,

Page 10 of 67 of depressed subjects (Castrén & Antila, 2017). The association between synapse loss and depression is based on the reduction of synapses in the PFC, caused by an inhibition of synaptic protein synthesis. Several pieces of evidence from human imaging, postmortem and animal studies emphasize that shrinkage of tissue in the hippocampus and/or PFC plays a vital role in the pathophysiology of depression and other mood disorders (Holmes et al., 2019). Recent, rodent model studies have also demonstrated that depression causes atrophy and loss of neurons and glial cells in the PFC and hippocampus, in depressed subjects (Duman et al., 2016). Regions such as the PFC, hippocampus and amygdala are all believed to be implicated in the pathology of depression (Højgaard et al., 2018), as synaptic loss and deficits in their functional connectivity is suggested to contribute to symptoms associated with MDD.

Genes involved in synaptic function were found to be down regulated in the dorsolateral PFC of depressed individuals (Holmes et al., 2019). Changes in synaptic proteins, specifically SV2A, have also been shown to be downregulated to an increasing degree, with the increase in severity of depressive symptoms exhibited in patients (Holmes et al., 2019). Further, an altered expression of SV2A has been seen in cognitive impairment (Löscher et al., 2016).

These changes in the brain can potentially be counteracted by antidepressants (Castrén & Antila, 2017). Current antidepressants such as selective serotonin reuptake inhibitors (SSRIs) produce subtle changes within weeks or months. This chronic antidepressant that blocks the reuptake and breakdown of serotonin has shown to increase synaptic plasticity and the generation of new neurons in the hippocampus and lastly, to upregulate neurotrophic factors. Chronic antidepressant treatments such as SSRIs, dopaminergic drugs, ECT and others depression-related treatment regiments, were shown to increase BDNF expression and the behavioral changes observed in mice models after these treatments were blocked in BDNF knockout mice (-/-) (Duman et al., 2016).

New knowledge and insights into the neurobiology of stress and mood disorders have provided an understanding of mechanisms underlying the vulnerability of individuals to depression. Ketamine and other NMDA receptor antagonists work acutely to increase rapamycin complex 1 (mTORC1) signaling via the kinase pathways and increase synaptic number and function in the PFC. There is evidence that the acute intravenous or nasal spray administration of ketamine can activate AMPA receptors which in turn stimulate mTORC1 signaling in cultured neurons via release of BDNF and activation of Akt and ERK signaling. This is also suggested, by the block of ketamine-induced antidepressant behavioral response in BDNF knockout mice (-/-). The rapid induction of mTORC1

Page 11 of 67 through the acute administration of ketamine could reverse the loss of connections in depressed patients and thereby reverse the depression pathology (Duman et al., 2016). Recent studies in the Flinders Sensitive Line rats, has shown that ketamine established normal dendritic spine conformation within one hour of administration in the rodent model of depression (Treccani et al., 2019). Lastly, ketamine has shown promising results in treatment-resistant depression. Though, ketamine has been marketed as the more tolerable esketamine in the form of a nasal spray, the drug is still sought out as a last-resort as the anesthetic agent still exhibits adverse effects in therapeutic doses (López-Díaz et al., 2019).

Thus, these new antidepressant agents have recently shown improvement in mood ratings as well as reversing the synaptic deficits caused by stress with an acute administration of the drug (Duman et al., 2016).

2.2 SYNAPSE PHYSIOLOGY AND GLUTAMATERGIC NEUROTRANSMISSION Synapses are fundamental components of neurons and play a role in the control of body functions, memory and emotions, by ensuring coordinated movement of information through the brain. A simultaneous firing of neurons strengthens the connection. In contrast, a repeated uncoordinated firing of neurons, weakens the connection between them. Hence, proper synaptic communication is required for an optimal brain physiology. Synapses can be divided into two groups; electrical synapses, in which there is a direct transfer of charged ions and small molecules through gap junctions and chemical synapses, in which the electrical activity is transferred unidirectionally from the presynaptic terminal to the postsynaptic terminal, through chemical mediators (Lepeta et al., 2016).

Chemical synapses convert either excitatory or inhibitory signals, hence increasing or decreasing the probability of neurons firing an action potential. Action potentials travel along axons to reach the end of the presynaptic cell, which causes a release of neurotransmitters from the presynaptic vesicles into the synaptic cleft. Neurotransmitters bind to and activate specific receptors transducing the chemical signal into an electrical impulse that depolarizes the postsynaptic cell. The release of neurotransmitter involves a sudden rise in intracellular Ca2+ levels as a result of presynaptic influx through voltage- dependent calcium channels (Lepeta et al., 2016). The activated receptors can either be metabolic receptors or ion-channels. Stimulation of a metabolic receptor, activates a cascade of second messengers, causing conformational changes of proteins within the cell. On the other hand, stimulation of an ion channel alters the flow of ions through it, causing a change in the voltage in the postsynaptic cell (Vose et al., 2017). The degree to which the voltage changes in the postsynaptic

Page 12 of 67 neuron, is a measure of the strength of a connection. Strengthening of synapses is known as long term potentiation (LTP), which means that the change in potential evoked by the presynaptic neuron will increase. The weakening of synaptic strength is known as long term depression (LTD), which means that the change in potential evoked by the presynaptic neuron will decrease (Vose et al., 2017 and Lepeta et al., 2016)).

Glutamate is the most abundant excitatory neurotransmitter in the brain. Glutamate activates a number of different receptors within the postsynaptic membrane of excitatory synapses, among which the two most important being AMPA receptors (AMPARs), that mediate the fast depolarizing currents, and NMDA receptors (NMDARs) that are important in modulating synaptic responses as a result of their voltage-dependent opening mechanism and their increased permeability to Ca2+. Presynaptic release of glutamate results in the binding of it to AMPARs at the postsynaptic neuron, causing the neuron to depolarize by which NMDARs are unblocked. Glutamate binds to the NMDARs, which leads to a large influx of calcium. However, voltage-dependent blockade of NMDARs by Mg2+ leads to a more moderate influx of calcium (Vose et al., 2017).

2.3 SYNAPTIC PLASTICITY LTP and LTD are two forms of long-term plasticity. LTP of synaptic transmission is an activity- dependent long-lasting increase in synaptic strength (Vose et al., 2017). LTP induction by repeated synaptic activity, high levels of paired presynaptic and postsynaptic activity, promotes the activation of NMDARs allowing a large Ca2+ influx. This influx causes activation of protein kinases, which results in gene transcription, protein synthesis, neurotrophic factor release, synaptic strengthening and growth of new synapses. LTD induction, low levels of paired presynaptic and postsynaptic activity, causes a weak activation of NMDARs, resulting in smaller influxes of calcium that further activate phosphatases and in removal of AMPARs from the dendritic spine, loss of neurotrophic factors and synaptic weakening (Vose et al., 2017).

Synaptic plasticity plays an important role in both LTP and LTD (Duman et al., 2016). It is one of the most important functions of the brain. It involves and ensures the ability to sense, assess and store complex information, as well as induce appropriate responses to the related stimuli (Finnema et al., 2016). Another parameter of synaptic plasticity is the efficient release of neurotransmitters. This means that synaptic plasticity is a function that allows modification of the structure of synapses after continuous electrical activity (Lepeta et al., 2016). Therefore, synaptic plasticity can be referred to as the strengthening and weakening of neurons, leading to changes in connections between them in the

Page 13 of 67 brain. Structural disruption or loss of synapses, triggered by alterations in synaptic molecular mechanisms and biochemical processes, results in network dysfunction with aberrant neuronal signaling (Finnema et al., 2016 and Lepeta et al., 2016). Synaptic loss, deficits and impaired synapse function are all features associated with brain disorders, such as Alzheimer’s disease, epilepsy, schizophrenia and depression (Finnema et al., 2016).

2.4 SYNAPTIC PLASTICITY AND DEPRESSION The mechanisms that cause changes in synaptic plasticity are linked to the pathophysiology of depression, as synaptic plasticity impairs the ability to form new connections between neurons in the depressive state (Duman et al., 2016). The formation of synapses between neurons in the nervous system, called synaptogenesis, is regulated by a complicated interaction of signaling pathways. Disruption of these vital pathways, such as loss of neurotrophic factor support and elevation of inflammatory cytokines, are involved in the susceptibility to depression (Duman et al., 2012). Furthermore, glia have been implicated in the regulation of synthesis and reuptake of glutamate and then affecting synaptic function and morphology (Popoli et al., 2011).

Glutamatergic synapses compose most of the excitatory synapses in the brain and connect brain regions, such as the PFC, hippocampus and amygdala. As these brain regions are relevant and important in depression and stress, glutamatergic signaling in the brain plays a vital role in the depressive state. Furthermore, glutamatergic afferents from the PFC control the HPA axis, which is a key regulator of stress (Vose et al., 2017). Stress is a significant factor in depression, as it causes disruption of neurotrophic factor signaling. Upon stress, activation of the HPA axis and increased levels of circulating glucocorticoids, occur. Chronic stress leads to a sustained increase of glucocorticoids levels, which affects the brain, among other organs. Dysregulation of the HPA axis and high levels of glucocorticoids, have been implicated in depression as they influence neuronal function and behavior (Duman et al., 2016 and Zhao et al., 2008). As an example, rodents chronically exposed to adrenal-glucocorticoids have shown a decrease in synaptic number and function. This leads to atrophy of neurons and disruption of connectivity in the PFC and hippocampus (Duman et al., 2016). Evidence from both rodent and human studies have shown that stress and elevated levels of glucocorticoids influence the expression of factors that play a negative role in the regulation of synaptic proteins (Duman et al., 2016). Interestingly, both acute and chronic stress have also shown to impair the induction of LTP within both the amygdala-PFC and hippocampal-PFC axes (Vose et al., 2017).

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Subtle alterations in proteins that are associated with the synaptic vesicle cycle, directly or indirectly, contribute to the pathophysiology seen in several neurological and psychiatric illnesses (Castrén & Antila, 2017). As mentioned among the diseases with synaptic plasticity implication, is depression, in which neurotrophic factors are vital in the normal function, regeneration and plasticity of neuronal tissue (Duman et al., 2016). Especially, BDNF is required for the maintenance of synaptic connections.

These connections are dependent on the communication and the synapses’ ability to carry and transfer a nerve impulse and this may not be possible without the presence of important synaptic proteins. BDNF has been linked to synaptic plasticity, through its regulation of the late protein synthesis- dependent stage of LTP (Duman et al., 2016).

Studies have further found that BDNF upregulates the release of neurotransmitters and LTP both in in vitro and in vivo models (Castrén & Antila, 2017). The molecular biological processes behind, are carried by kinases that are mediated by neurotrophic, but also endocrine and metabolic factors. The PI-3K-Akt and Raf-MEK-ERK pathways are suggested to be involved, by their downstream targets. The one proposed to by far be the most important in its involvement in synaptic plasticity and synthesis of synaptic proteins, is the before mentioned mTORC1 (Hoeffer & Klann, 2010). mTORC has been shown to be downregulated post-mortem in depressed subjects (Duman et al., 2016). The possible mediation of neurological and psychiatric diseases by endocrine and metabolic factors, makes the brain especially vulnerable to pathophysiologies in certain conditions. These could be hormonal changes brought on from chronic illnesses or by regular body systems. In the case of depression this means that not only can the synaptic plasticity be impaired, its cause can be from a dysfunction in ordinary regulation by neurotrophic factors or endocrine and metabolic factors (Duman et al., 2016).

Essentially, the physiological conditions can be risk factors contributing to dysregulation of mood and cognitive function through changes inflicted onto the neuronal circuitry and possibly also the dysregulation of proteins. Changes in synaptic proteins has therefore been implicated in the depressive state.

2.5 SYNAPTIC VESICLE GLYCOPROTEIN 2A (SV2A) Synaptic Vesicle Glycoprotein 2A (SV2A) is a transmembrane glycoprotein present in the neuronal presynaptic vesicles. SV2A is important for synaptic function and is one of the most monodispersed

Page 15 of 67 proteins, having a great uniformity in vesicles, which might provide an accurate measure of synaptic density (Mendoza-Torreblanca et al., 2013).

The human SV2A gene is located on the locus 21.2 on chromosome 1. It is approximately 14,565 bp long and of these 4353 bp are messenger RNA (mRNA) coding with 13 exons subsequently translated into an 82.6 kDa protein consisting of 742 amino acids. SV2A has a large conserved N-terminal, 12 transmembrane (TM) domains and one large conserved cytoplasmic loop between the 6th and 7th TM domain, a large intraluminal loop between the 7th and 8th TM domain, which is very glycosylated and lastly, a short C-terminal (Mendoza-Torreblanca et al., 2013) (see fig. 2).

Figure 2 - Schematic illustration of synaptic vesicle glycoprotein 2A (SV2A). The structure of SV2A consists of 12 transmembrane (TM) domains, with a large conserved N-terminal, a large conserved cytoplasmic loop between the 6th and 7th TM domain, a large intraluminal loop between the 7th and 8th TM domain and a short C-terminal. The first 57 amino acids of the N-terminus (yellow) is the site responsible for interaction of SV2A with synaptotagmin-1. There are 10 putative phosphorylation sites in the N-terminus (pink), associated with enhanced interaction between SV2A and synaptotagmin-1. The putative ATP-binding sites are shown in blue. The N‐terminus of SV2A has a tyrosine‐based endocytosis motif (Tyr46) that is required for trafficking of SV2 into synaptic vesicles (green). Between TM domain 2 and 3 Arg23, a canonical MFS transporter motif is found (green). Trp300 and Trp666 are essential for the SV2 action. Three putative glycosylation sites are essential for entry of botulinum toxin (BoNT/A and BoNT/E) into neurons (purple). Fourteen amino acids implicated in racetam binding have been reported (red). Figure modified from Mendoza- Torreblanca et al., 2013. The SV2 family of synaptic vesicle (SV) proteins is expressed in all vertebrates and consists of two major isoforms, SV2A and SV2B, and one minor isoform, SV2C. Out of the three, SV2A is by far the most abundant and is believed to be globally distributed in the brain (Bajjalieh et al., 1993).

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SV2A is vital for the normal function of neuronal synapses and neurotransmitter release. SV2A enhances action potential-dependent neurotransmitter release from the nerve terminals without altering the morphology or the number of synaptic vesicles. This neurotransmitter release is believed to primarily be glutamatergic (Löscher et al., 2016). A reduction in SV2A expression results in abnormal inhibitory neurotransmission in different brain regions of rats, which is likely to contribute to the seizure phenotype observed in epilepsy. The decrease in SV2A expression may contribute to the impairing of neuronal networks and therefore to the development of neuropathologies (Mendoza- Torreblanca et al., 2013).

In complete SV2A knockout mice (-/-) no changes were observed in SVs, which is an indication that SV2A is not a structural component of vesicles since its expression does not change the morphology of SVs (Janz et al., 1999). Reduction in neurotransmission in SV2A knockout animals is not caused by altered morphology of synapses or vesicles, which indicates that SV2A is not vital for vesicle biogenesis nor does it play a role in the structural integrity of vesicles (Crowder et al., 1999 and Janz et al., 1999). Due to the mentioned, it has been suggested that SV2A regulates exocytosis and the concentration of released Ca2+ into the synaptic cleft (Mendoza-Torreblanca et al., 2013).

SV2A might be involved in any one of the four stages of neurotransmitter release: filling of vesicles with the specific neurotransmitter, docking of vesicles, priming of vesicles at membrane and (fusion) exocytosis initiated by Ca2+ (Südhof & Rizo, 2011 and Abad-Rodriguez & Diez-Revuelta, 2015) (see fig. 3).

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Figure 3 - Illustration of vesicles at the presynaptic terminal. Overview of the vesicle cycle presynaptically; filling, docking, priming, exocytosis and endocytosis. The calcium stimulated neurotransmitter release into the synaptic cleft following exocytosis and the subsequent binding to and influx of neurotransmitters through receptors on the postsynaptic neuron. The magnification of a synaptic vesicle is illustrated in the top left corner and as seen here; important proteins are present on the vesicle surrounding the neurotransmitters. Among these vesicle proteins is SV2 (highlighted with a red circle). Figure modified from Löscher et al., 2016.

Though not understood in detail, SV2A is the target of the antiepileptic drugs Levetiracetam (LEV) and Brivaracetam. Discovered in the 1970’s, the hopes for LEV were for it to be the new addition in the treatment of cognitive disorders (Klitgaard & Verdru, 2007). The drug instead showed promising results in a mouse model of epilepsy, where it suppressed the seizure phenotype (Gower et al., 1992). LEV decreases the release of calcium from the presynapse into the synaptic cleft, which inhibits AMPA and calcium currents. Studies indicate that LEV alters short-term synaptic plasticity, which is the result of concurrent activation of enhancement (positive mechanisms) and depression (negative mechanisms) in the presynaptic terminal (Löscher et al., 2016).

LEV’s specific binding affinity for the Levetiracetam Binding Site (LBS) was much higher than any other antiepileptics or other agents before used in treating the brain associated disorders. The discovery of the LBS as being SV2A, in 2003, was done fairly late compared to the first use of LEV. SV2A as LEV’s target was further validated, as heterozygous SV2A knockout mice (+/-) responded less to LEV’s antiseizure effects and as SV2A was implicated in exocytosis control. UCB Pharma

Page 18 of 67 discovered the specific binding site through use of the radiolabeled version of LEV ([3H]Levetiracetam). The LBS was found to be localized on the synaptic plasma membranes and to be especially present in the hippocampus, neocortex and cerebellum (Noyer et al., 1995).

SV2A can be used to measure the number of nerve terminals, hence function as a prime marker of synaptic density. Multiple radiotracers binding SV2A have been developed. Some have shown more potential in their binding than others. For PET imaging two SV2A targeting ligands are highlighted [18F]UCB-H and [11C]UCB-J. The latter showing promising results as its biodistribution profile showed high uptake in the brains of non-human primates and the imaging quality in PET has been impeccable (Löscher et al., 2016). Furthermore, [11C]UCB-J showed high uptake in the cortex and striatum in humans, whilst the uptake into the cerebellum and thalamus were lower. The potential of radiotracers for use in SV2A distribution analyses through PET imaging is great, not only in the study of the epilepsy pathology but also in measuring synaptic density. The further investigation is vital, as there is great potential in exploring a non-invasive technique for the establishment of synaptic density in different neurological and psychiatric disorders. Such a technique could possibly aid in the diagnostics, progression monitoring, targeted treatment development and treatment monitoring of various neurological and psychiatric disorders (Löscher et al., 2016). The tritium labeled UCB-J ([3H]UCB-J) ligand has been developed for preclinical use, as this has a slower decay and in vitro experiments, using for example autoradiography, have been made more straightforward. In this study, we use the selective radioligand [3H]UCB-J targeting SV2A.

2.6 MARKERS OF SYNAPTIC DENSITY Similar to SV2A, synaptophysin is a synaptic vesicle protein located in the presynaptic neurons and is specifically present in the membranes of vesicles. Synaptophysin consists of four TM domains and is the most polydispersed vesicle protein, which reflects its variability among the vesicles of different neurotransmitter classes and ubiquity at the synapse. The calcium-binding glycoprotein is 38 kDa in size (Masliah et al., 1991) and has further been implicated in channel formation, exocytosis and endocytosis (Glantz et al., 2007). The ubiquity and variability of this protein has led to the use of synaptophysin immunostaining for quantification of synapses. Thus, being accepted as a marker of synaptic density (Finnema et al., 2016). Early immunolabeling studies revealed the involvement of synaptophysin in the organization of the part of the hippocampus that governs episodic memory/neurogenesis, the dentate gyrus (Masliah et al., 1991). This research group is among the first

Page 19 of 67 to address the potential of utilizing synaptophysin in synaptic quantification. In 1990, Masliah et al. used monoclonal antibody targeting synaptophysin on cortical sections to quantify presynaptic terminals. Since then the use of synaptophysin as a marker of synaptic density has been widely accepted (Finemma et al., 2016, Hamos et al., 1989 and Masliah et al., 1991). In 2016, Finemma et al. compared the established marker of synaptic density, synaptophysin, and the potential marker, SV2A, through western blotting and confocal microscopy. The experiments done on baboon gray matter showed very similar patterns in both protein expression and in the anatomical location of the two proteins. Upon western blot analysis, synaptophysin and SV2A showed linear correlation across all gray matter regions (Finemma et al., 2016). Furthermore, a study investigating synaptic density in 26 individuals with MDD, post-traumatic stress disorder (PTSD) or comorbid MDD/PTSD by PET with the SV2A radioligand [11C]UCB-J, observed a significant correlation between the severity of depressive symptoms and SV2A density. Individuals with severe depression showed a lower SV2A density (Holmes et al., 2019). This establishes SV2A as a marker of synaptic density at the same level as the accepted marker, synaptophysin.

Changes in synaptic proteins has been implicated in the depressive state. Thus, the in-depth examination of depression from a molecular biological viewpoint is necessary, for the proper understanding of the disorder. In order to obtain this knowledge, proper animal models exhibiting the behavior and pathophysiology of depression are vital.

2.7 ANIMAL MODELS OF DEPRESSION Animal models of depression are useful in providing insight into the neurobiology and pathophysiology of depression, further playing a key role in the discovery of new and improved antidepressants. The procedure for validating animal models of psychiatric disorders include consideration of predictive validity (the correspondence between drug actions in the model and in the clinic), face validity (phenomenological similarities between the model and the human disorder) and construct validity (consistency of the model with a theoretical rationale) (Nishi et al., 2009 and Mallei et al., 2015). There are three desirable features in an animal model of depression: (1) the model should respond appropriately to chronic treatment with antidepressant drugs, (2) the model should employ realistic inducing conditions and (3) the model should display a core symptom of the disorder (Willner, 1997).

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2.7.1 CORTICOSTERONE In drawing the connection between stress and depression, findings showing that the induced elevation in cortisol levels and increased activity in the HPA axis, in depressed individuals alter the normal cortisol release, have been brought forth. Thus, chronic administration of the rodent stress hormone, equivalent to cortisol in humans, corticosterone, is widely used to induce depression-like behavior in animal models. Under normal circumstances stress occurs as a result of naturally elevated blood cortisol levels. By administering corticosterone over a course of weeks, the same stress-inducement can be achieved in animals. Studies have shown that the repeated corticosterone administration in male rats may cause changes in their emotional behavior corresponding to the symptoms and molecular mechanisms observed in major depression. Among the physiological changes observed is the neuronal remodeling in the hippocampus, amygdala and mPFC (Zhao et al., 2008 and Johnson et al., 2006). There are multiple routes of corticosterone administration for mimicking the daily fluctuations seen in chronic stress. Subcutaneous injection is the administration ensuring the best control over the corticosterone levels (Zhao et al., 2008). However, the same effects can be obtained by implanting a pump with a set flow of the hormone into the brain. Both of these approaches are efficient in meeting the goal of inducing depression-like behavior in a controlled fashion in animal models, unlike the application of corticosterone pellet implantation or the addition of corticosterone to the drinking water accessible to the animals (Zhao et al., 2008). The enzyme tyrosine hydroxylase (TH) is involved in the regulation of the dopamine and noradrenergic system of the hippocampus and has been implicated in the pathogenesis of depression. The TH expression is further altered in multiple brain regions of rodents as a result of corticosterone administration (Zhao et al., 2008). Thus, the administration of corticosterone, through a pump inserted into the brain of rats, in the mimicking of stress-inducement seen in depression, is a vital methodology for the application in animal models for the subsequent examination of corticosterone-induced stress.

2.7.2 FLINDERS MODEL The Flinders Sensitive Line (FSL) of rats are descendants of Sprague-Dawley rats with psychomotor retardation. FSL rats have been proposed as a genetic rat model of human depression. The rat line is inbred over generations, based on the level of anxiety the rats present with, when exploring a boxed area. The FSL rats are more anxious than rats are normally, thus they will stay close to the edge and only move across the open field occasionally. The comparison group Flinders Resistant Line (FRL), will be resistant to feeling and exhibiting anxiety, they present with movement around the middle of the boxed area to a greater extent. The FSL acts as a model of depression, with increased anxiety,

Page 21 of 67 when compared to the FRL line (Nishi et al., 2009). This depression model is therefore based on the behavioral pattern of the animal and this is also reflected in the biology, since the genetics that underlie the behavior have been selected for, for generations. The FSL model is therefore considered a genetic model of depression.

Alterations in both presynaptic and postsynaptic functions of the serotonergic neurotransmission in the brain, are present in FSL rats. The molecular biological presentations are low 5-HT1A density but higher, compensatory, levels of 5-HT1B. This downregulation of 5-HT1A has also been proposed as the molecular mechanism in humans suffering from depressive symptoms (Nishi et al., 2009). Furthermore, the neurochemical profile of the FSL rats are altered, both pre-and postsynaptically, affecting both neurotransmission and receptor density and affinity (Shayit et al., 2003).

These neurochemical alterations are associated with human depression. Since FSL rats exhibit normal cognitive and hedonic function, but reduced psychomotor, sleep, immune functions and appetite, their presentations partially resemble the pathology seen in depressed humans (Overstreet et al., 2005).

In humans the equivalent to the presentations in the FSL rats, is clinical depression with lethargic presentations (tired, drowsy, etc.) (Nishi et al., 2009). The most pressing for the use of the FSL rats as a model of depression, is that the relief of their depressive symptoms requires a chronic administration of anti-depressant therapeutics, an acute treatment will not suffice. In the face of this being a requirement for a model mimicking the human disorder, one of the few animal models to uphold this is the Flinders model (Nishi et al., 2009).

2.7.3 ELECTROCONVULSIVE STIMULATION (ECS) Electroconvulsive stimulation (ECS) is an experimental animal model of electroconvulsive therapy (ECT), in which the stimuli are generated by electrical impulses induced on the head of rodents by electrodes (Jang et al., 2017). ECT is one of the most effective treatments of MDD (Kupfer et al., 2012). It has earlier been shown, that most rats exhibiting depression-like behavior and then treated with either acute or chronic ECS displayed a behavior comparable to sham (control) rats. Furthermore, repetitive administration of ECS has shown efficiency in treatment of drug-resistant depression.

Henceforth, ECT treatment has displayed improvement in 50% of patients, with an initial response after 3 ECT treatments in a week. They had at least a 50% reduction on the Hamilton Rating scale of depression (Husain et al., 2004).

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ECS is a model that globally elevates brain activity; hence it is widely used to examine activity- dependent alterations of synaptic proteins and their effects on synaptic strength (Jang et al., 2017). The specific neurobiological mechanism underlying the efficacy of the antidepressant effect in humans is unclear (Hvilsom et al., 2019). However, ECT treatment in humans has shown to normalize the HPA axis abnormalities (Maynard et al., 2018). Both acute and chronic ECS treatment in rodents has shown to improve reorganization of the neuronal network, contributing to positive progression in cognitive flexibility (Jang et al., 2017). It is suggested that ECS affects the brain by inducing synaptic changes that are considered to be important for the antidepressive effect. These changes include normalization of the neuroendocrine system and neurotrophic effects such as neurogenesis and synaptogenesis (Hvilsom et al., 2019).

Chronic stress-induced depression causes dendritic atrophy and deficiencies in BDNF, which as mentioned is an important component of neuronal morphology, including dendritic spine density and structure. ECS has shown to increase both BDNF expression and stimulate dendritic regeneration, thus displaying effective treatment of chronic stress-induced depression (Maynard et al., 2018).

2.7.4 CHRONIC MILD STRESS (CMS) Chronic mild stress (CMS) is a widely used behavioral animal model of depression that is suggested by many to be a model that has the greatest validity and translational potential (Willner, 2017). In studies of animal models of chronic stress, a lower synaptic density has been seen in the PFC and hippocampus (Holmes et al., 2019). The CMS model was developed based on an observation made in the late 1980s, showing that rats subjected to several serious stressors had a lack of ability to increase their fluid intake when substances such as sucrose or saccharin were added to their drinking water (Willner, 2017). Several studies have earlier been investigating the effects of CMS on different reward-related behavioral endpoints in order to examine the concept of stress-induced anhedonia (in this case decreased sucrose preference). Moreover, the examination of whether the CMS model can be utilized as a base, in which the mechanisms of action of antidepressant drugs can be investigated (Willner, 2017). In these experiments, rats were exposed to continuous varieties of mild stressors, such as periods of food and water deprivation and changes of cage mates. After a period of chronic exposure to stressors the rats showed a gradual reduction in their consumption of, as well as preference for, a sweet solution. However, it was possible to reverse this deficit by chronic treatment with antidepressant drugs (Willner, 2017).

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The CMS model is designed to mimic the chronic-low strain stress, which can be compared to the everyday life stress in humans. Agomelatine, which is a serotonin 2C receptor antagonist and a melatonin 1 and 2 receptor agonist (Højgaard et al., 2018), has shown a general favorable tolerability and efficacy. Agomelatine is believed to be an alternative for patients who do not respond to existing pharmacotherapies or who cannot tolerate their side-effects. Several studies have shown evidence for the positive efficiency of this drug as a treatment for depression (Kupfer et al., 2012). Furthermore, agomelatine has also shown to affect neuronal plasticity (Højgaard et al., 2018). An antidepressant- like effect of agomelatine in both humans and animal models, has been observed in previous studies (Højgaard et al., 2018; Taylor et al., 2014 and Dagyte et al., 2011). Agomelatine has shown to have an anxiolytic effect in clinical trials and induce fearless behavior in rats. (Millan et al. 2005 and Højgaard et al., 2018).

The CMS model responds to chronic, but not acute, administration of a variety of established antidepressant drugs and to electroconvulsive stimulation, which supports the predictive validity of the CMS model (Willner, 1997). Regarding the face validity, the model has shown a range of behavioral and physiological changes, that parallel those of depression. The theoretical rationale regarding the construct validity of the CMS model from a physiological perspective is that this procedure simulates anhedonia, a loss of responsiveness to pleasant events. The CMS model accounts for the psychobiological processes in depression and mimics the regular response to antidepressant action (Willner, 2017), further strengthening the construct validity of the model.

2.8 THEORY BEHIND THE METHODS In this section, the basic principles of the methods used in this study are described.

2.8.1 AUTORADIOGRAPHY Autoradiography is a bio-analytical technique used to determine binding characteristics of a radioactively labeled ligand to a target protein, in a biological sample. The method applies a radiolabeled ligand to bind its target, in order to determine the distribution of the protein of interest in a given tissue sample and furthermore, the binding affinity/selectivity of the ligand. The technology utilizes imaging plates with photostimulable phosphor crystals that are designed to take up radioactive irradiation and release it upon stimulation with light. The crystals along with an organic binder are able to store some of the energy emitted by the radioactive irradiation which can then be translated into binding units. The image plate will emit photo stimulated luminescence when exposed to visible

Page 24 of 67 or infrared light, the intensity of the photo stimulated luminescence then released is proportional to the amount of radiation observed from the samples (Amemiya and Miyahara, 1988).

The pharmacological profile of the radioligand is also determined through autoradiography, to name the two most applied: the density of receptors/binding sites, Bmax, and the dissociation constant, Kd (Griem-Krey et al., 2019). Investigating the binding characteristics of the radioligand to the target protein, at various increasing concentrations, by saturation binding studies, makes it possible to determine the two constants. The Kd value is a measure for the affinity of the ligand for its binding sites. Kd indicates the concentration at which half of the target proteins in the measured region is bound to the ligand. A low Kd value indicates that a low concentration of ligand is needed to bind half of the target proteins. Thus, Kd indicates the affinity of the ligand to the target protein, and a low

Kd therefore indicates a high affinity (Hulme & Trevethick, 2010).

Though, the traditional use of autoradiography was performed on tissue homogenates (Griem-Krey et al., 2019), in this study, in vitro autoradiography is applied in order to preserve the anatomical integrity of the tissue sample, which enables the analysis of the regional distribution of the protein of interest.

In the present study, brain sections from different rat models are utilized in in vitro autoradiography, for the characterization of the binding of [3H]UCB-J to the protein of interest, SV2A.

2.8.2 WESTERN BLOT The following description of western blot is based on the protocol used in this study. Western blot is an analytical technique used to detect specific proteins in a sample of tissue homogenates. The technique applies three different steps. The first step is separation of the proteins based on size through gel electrophoresis, followed by transfer of the proteins to a cellulose membrane to produce a band for each protein and lastly visualizing and identifying the target protein by using a primary antibody specific to the protein of interest and a secondary antibody specific to the primary antibody (Mahmood & Yang, 2012).

The secondary antibody is conjugated with the enzyme horseradish peroxidase (HRP). The conjugation with HRP ensures the detection of protein of interest by chemiluminescence, as the production of luminescence is proportional to the amount of HRP-conjugated secondary antibody that is bound to primary antibody. This means that it is an indirect measure of the presence of the target

Page 25 of 67 protein, which is detected by the signal it produces corresponding to the position of the target protein (Eslami & Lujan, 2010 and Mahmood & Yang, 2012).

SDS-page is a method used to separate proteins according to size (kDa). Smaller proteins move more easily, therefore first, through the acrylamide gel and then the larger proteins follow. The proteins are prepared for SDS-page gel electrophoresis, by treatment with sodium dodecyl sulphate (SDS) as well as boiling to denature the proteins by interruption of their secondary and tertiary structure, while sulfide bridges are disrupted by β-mercaptoethanol. Denaturing the higher-order structures of the proteins ensures that the protein is linearized and does not form aggregates. The denatured proteins are loaded onto the wells of a polyacrylamide gel. The binding of the SDS to the denatured proteins makes them move towards the positively charged anode through the acrylamide of the gel, as the SDS is negatively charged (Mahmood & Yang, 2012).

Along with the protein samples, a marker, containing a mixture of stained proteins with different, known molecular weights, is added into a well. The molecular weight of the protein samples can be estimated, by comparing their position on the gel to the different bands of the protein-ladder (Mahmood & Yang, 2012).

In this study, the stain-free method for protein detection in western blotting is used in order to skip the step between protein separation and the visualization and normalization of one´s protein of interest. The method applies a polyacrylamide in the gel itself containing a tri-halo compound (methane molecule in which three hydrogens have been changed for halogens) that will bind the protein´s tryptophan amino acids and therefore enhance the fluorescence of the tryptophan amino acids. This is done by activating the gel with UV irradiation, which means inducing a covalent reaction between the tryptophan residues and the halo-compounds that will then allow the detection of fluorescence. This method enables the visualization of loaded protein on the gel, the validation of successful transfer after blotting – therefore making staining obsolete and the use of full protein content per lane for normalization instead of housekeeping genes (Elbaggari et al., 2008).

After separation of the proteins, by gel electrophoresis, the proteins are transferred from the gel onto a polyvinylidene difluoride (PVDF) membrane by electroblotting. The gel is placed on top of the membrane and placed in a cassette. It is important that there is close contact of gel and membrane to ensure a clear image. Furthermore, the placement of the membrane between the gel and the positive electrode is important. Thus, the membrane must be placed facing the anode, so that the negatively

Page 26 of 67 charged proteins can migrate from the gel onto the membrane. The electroblotting utilizes an electric field oriented perpendicular to the surface of the gel, causing proteins to move out of the gel and onto the membrane (Mahmood & Yang, 2012).

The use of the stain free method instead of β-actin for normalization is done by quantifying the total density of protein in each lane of the blot for background and adjusting the band intensity of the proteins of interest to these values, instead of normalizing to the band intensity of β-actin, which is an inferior method (Gilda & Gomez, 2013). The normalization is done automatically by the ImageLab software using the rolling disk background subtraction algorithm to couple the backgrounds of each lane and subtracting these values from the bands of interest.

Washing and blocking before exposing the membrane to antibodies is important in order to prevent antibodies from binding non-specifically to other proteins than the protein of interest, reduce the background and remove unbound antibody (Eslami & Lujan, 2010).

The antibodies applied can either be monoclonal or polyclonal. Polyclonal antibodies (pAbs) are produced by injecting an immunogen into an animal. A polyclonal antibody is a collection of many immunoglobulins, each generated from different B cell clones in the body. These antibodies target different epitopes or binding sites on a single antigen. Monoclonal antibodies (mAbs) are produced ex vivo using tissue-culture techniques. A monoclonal antibody is generated by identical B cells which are clones from a single parent cell. Monoclonal antibodies only recognize the same epitope of an antigen and are therefore more specific (Wood, 2011).

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3 STATE OF THE ART MAIN AIMS Two major aims of this thesis were; firstly, to validate techniques detecting synaptic vesicle glycoprotein 2A (SV2A) in the rat brain determined by [3H]UCB-J radioligand binding and immunoblotting. Secondly, to determine the SV2A distribution across brain regions and in various animal models of depression.

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4 METHODS The present thesis presents several separate studies that aimed to determine the [3H]UCB-J binding to SV2A using in vitro autoradiography, as well as investigating the level of the two proteins (SV2A and SYN), in different rat brain regions, by western blot analyses. The individual experiments are described below in section 4.1 and 4.3

4.1 ANIMALS AND BRAIN TISSUE FOR AUTORADIOGRAPHY

4.1.1 TISSUE FOR METHOD VALIDATION. For the initial autoradiography study, characterizing binding properties and saturation of [3H]UCB-J, brain tissue samples from one female Sprague-Dawley rat (Charles River, Germany) giving birth to pups five days earlier, were used. The Sprague-Dawley rat was sacrificed at 12 weeks of age. The brain was subsequently removed and kept on -80℃ until use.

4.1.2 TISSUE FOR STUDYING THE EFFECT OF CORTICOSTERONE ON THE DIURNAL

CYCLE. The experimental animal study was conducted in the laboratory of Dr. Martin Rath, Institute of Neuroscience, University of Copenhagen. Briefly, brain tissue samples from 24 different male Sprague-Dawley rats (120g at the start of the experiment) [Taconic Farms (Ejby, Denmark)] were used. The rats were housed under controlled light conditions in a standard 12h light:12h dark schedule (12L:12D) with access to food and water ad libitum. Animals were sacrificed at specific Zeitgeber times (ZT). Half of the rats were sacrificed at ZT3 (light phase) and half at ZT15 (dark phase). The rats were further divided into three subgroups: sham, SCNx and SCNx + corticosterone.

All rats had an electrode positioned into the suprachiasmatic nucleus (SCN) of the hypothalamus. A lesion of the SCN was made, by turning on the electrode and burning the tissue, on the SCN of the SCNx group; sham group received no current. The last group had both the lesion and were administered with corticosterone through the inserted pump. The SCNx + corticosterone group was treated with corticosterone for 12 hours at a time with 12-hour intervals, until sacrificed.

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To mimic the daily corticosterone fluctuation; high levels of corticosterone were therefore administered during the night (2.5 mg/kg/12–5 µL/h) and in order to keep the micropump functioning a low level of corticosterone had to be administered during the day (1 µL/h). Blood corticosterone measured from blood samples taken before euthanasia.

4.1.3 FLINDERS RESISTANT AND SENSITIVE LINE. The animals were bred by Betina Elfving and Gregers Wegener, Department of Translational Neuropsychiatry, University of Aarhus. In this study, brain tissue samples from 10 female Flinders Sensitive Line (FSL) and 10 female Flinders Resistant Line (FRL) Sprague-Dawley rats were collected (approximately 10 weeks of age). These animals are inbred strains of Flinders rats as originally described (Nishi et al., 2009).

4.1.4 ELECTROCONVULSIVE STIMULATION (ECS). The experimental animal study was conducted by Søren Christiansen and associate professor David Woldbye, Department of Neuroscience, University of Copenhagen. For the ECS study brain tissue samples from 12 different male Wistar Han rats from Charles River, Germany were used. Six of which were electro stimulated (ECS) 14 times, by current given through the temples and six of which were control rats. The control rats (sham) had an ear-clip placement with no current. The rats weighed between 328-455g and were approximately 10 weeks of age at the time of tissue collection.

4.1.5 CHRONIC MILD STRESS (CMS). The experimental animal study was conducted by Kristoffer Højgaard, Department of Translational Neuropsychiatry, University of Aarhus. The brain tissue samples from 30 different male Wistar Han rats (120g) were used. These rats were obtained from [Taconic Farms (Ejby, Denmark)]. The rats were housed individually under controlled light conditions in a standard 12h light:12h dark schedule (12L:12D) with access to food and water ad libitum. These conditions were only changed as a part of stress inducement. The rats were treated two different ways for 10 weeks of CMS protocol. The control group of anhedonic vehicle (Anh-veh) were stress induced and received 1 mL/kg vehicle (1% hydroxyethyl cellulose suspension in distilled water) once a day for the last 5 weeks. The treatment group consisted of two different subgroups, agomelatine responder group (Ago-R) and agomelatine non-responder group (Ago-NR) that were stress induced and received 40 mg/kg of agomelatine i.p. daily for the last 5 weeks of the experiment. The rats were 16 weeks of age at the time of sacrifice (Højgaard et al., 2018).

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All brains used for autoradiography were harvested from sacrificed rats and immediately stored at - 80°C until use. The brains were sectioned in 20µm-thick slices (except from CMS experiment, that were cut in 12 µM thick slices) in coronal plane serial sections using a cryostat and placed onto pre- coated glass slides (SuperFrost PLUS cat.#J1800AMNZ, Thermo Scientific), and subsequently dried at RT for approximately one hour. Glass slides were kept at -20°C until use.

4.2 IN VITRO AUTORADIOGRAPHY USING [3H]UCB-J The radioligand used in this study was UCB1537649 ([3H]UCB-J). [3H]UCB-J was synthesized at UCB (Braine 1’Alleud, Belgium) and radiolabeled by Quotient Bioresearch Ltd. (United Kingdom). The specific activity of [3H]UCB-J was 24 Ci/mmol at the time of synthesis. The radiochemical concentration was 1 mCi/mL. The specific activity left at the time of experiment was determined for each experiment individually.

All slides were treated with 6nM [3H]UCB-J (except for the initial autoradiography where various concentrations were applied) radioligand for the detection of binding to the SV2A protein in brain tissue.

The glass slides were pre-incubated (twice) in 50 mM Tris-HCl buffer (pH 7.4) with 0.5% bovine serum albumin (BSA) (#1706404, Bio-Rad Labs) for 10 minutes at room temperature, using a washing chamber. The slides were then incubated in 1 mL 50 mM Tris-HCl buffer (pH 7.4) containing 3 5 mM MgCl2, 2 mM EGTA, 0.5% BSA and 6nM of [ H]UCB-J pr. slide at room temperature for 1h on a RotaMax machine. The slides were washed twice in ice-cold pre-incubation buffer for 10 minutes and then dipped once in ice-cold distilled water for 10 seconds, using a washing chamber. The slides were thoroughly dried in the fume hood and subsequently exposed to paraformaldehyde overnight. The slides were taken out of the paraformaldehyde chamber and left to dry in the fume hood. The dry slides with brain sections bound by the radioligand were exposed to a Fuji imaging plate along with two tritium standard slides ([3H] microscales, 50-micron multi-level reference strips RPA 510, Batch 21A, GE Healthcare, UK and ART 0123B, (American Radiolabeled Chemicals, Inc.) for 48 hours, at 4°C.

4.2.1 DATA ANALYSIS The Fuji imaging plate was scanned using the FUJIFILM Bio-imaging Analyzer BAS-2500 to create an image. The image obtained from the autoradiography scan was analyzed using the ImageJ software (ImageJ, V 1.52d29, NIH, USA). Each section of the standard was measured for its radioactivity and

Page 31 of 67 background was also measured in the vicinity of the standards. From this, the standard curve was made, using the known concentrations corresponding to the standards. Further, for each brain section measured, three measurements were made for the binding (technical replicates) of the radioactive ligand [3H]UCB-J. The brain sections were marked and measured for their gray value in the program, ImageJ.

4.3 ANIMALS AND BRAIN TISSUE FOR WESTERN BLOT ANALYSES Rats were utilized to perform western blot studies. Brains were harvested from the sacrificed animals and subsequently dissected into different regions and stored at -80°C until use.

4.3.1 TISSUE FOR METHOD VALIDATION. The first western blot analysis was carried out on tissue from the frontal cortex of one single Sprague-Dawley female rat and liver from one Long Evans male rat. The Sprague-Dawley rat was sacrificed at 12 weeks of age and the Long Evans rat was sacrificed at 7 weeks of age and weighed 200g.

4.3.2 TISSUE FOR LOCALIZATION STUDY. To investigate the regional distribution of SV2A in rat brain and further to determine the protein levels of SV2A and SYN in different regions of the rat brain, western blot analyses were conducted. The first western blot analysis was performed on five different brain regions (hippocampus, striatum, brainstem, cerebellum and cortex) from a male Long- Evans rat. The rat was 1 year and 3 months old and weighed 868g when sacrificed. The second analysis was done on four different Long-Evans male rats that were 7 weeks and weighed 200g when sacrificed. They were utilized when switching over to the stain-free western blot analysis method. This analysis was conducted for the same purpose, investigation of the regional distribution of SV2A in rat brain. Three different brain regions (frontal cortex, brainstem and cerebellum) of the four rats were used.

4.3.3 FLINDERS RESISTANT AND SENSITIVE LINE. In order to investigate the level of SV2A in the Flinders Sensitive Line of rats, descendants of Sprague-Dawley rats, western blot analysis was conducted. The Flinders Resistant Line of rats was used as controls for comparison. Brain hemispheres from 10 female Flinders Sensitive Line (FSL) rats and 10 female Flinders Resistant Line (FRL) rats were used. The animals were approximately 10 weeks of age at the time of sacrifice, by guillotine.

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4.3.4 ELECTROCONVULSIVE STIMULATION (ECS). This animal study was conducted by Søren Christiansen and associate professor David Woldbye, Department of Neuroscience, University of Copenhagen. For the ECS study brain hemispheres from 4 different male Wistar Han rats from Charles River, Germany, were used. Two of which were electrostimulated (ECS) by current given through the temples and two of which were control rats. The control rats (sham) had an ear-clip placement with no current. The rats weighed between 328-455g and were approximately 10 weeks of age at the time of sacrifice.

4.4 WESTERN BLOTTING FOR THE DETECTION OF SV2A EXPRESSION

4.4.1 SAMPLE PREPARATION AND PROTEIN CONCENTRATIONS The tissue samples were weighed (in mg ± SD) and subsequently homogenized in 2xlysis buffer containing 10 mM Tris (pH 7.4), 25 mM EDTA, 100 mM NaCl, 1% Triton-x 100, 1% NP-40, 1M NaF, protease inhibitor cocktail (P8340, Sigma-Aldrich) and phosphatase inhibitor cocktail 2 (P5726, Sigma Aldrich). The lysates were centrifuged (5000g, 30min, 4ºC), supernatants collected, aliquoted and stored at -80ºC until further use. The protein concentrations of the obtained supernatant from the homogenized tissues were determined by Bio-Rad DC™ protein detection assay by making a standard curve. The standard curve was created by preparing a series of seven standard dilutions of bovine serum albumin (BSA- cat. #500-0007, Bio-Rad Labs) with known protein concentrations (ranging from 0.0219 mg/mL to 1.4 mg/mL) and a control of H2O. The samples (in triplicates) and BSA standards (in triplicates) were incubated with detection reagents (reagent A, B and S) for 15 minutes in the dark before the absorbances were measured at OD=655 using the iMark™ Microplate Absorbance Reader (BioRad). The protein concentrations of the samples were subsequently determined, by using the standard curve based on the BSA standard measurements.

4.4.2 SDS PAGE AND IMMUNOBLOTTING

Based on the protein concentrations determined above the sample stocks were diluted in H2O to obtain a concentration of 4 µg/µL of protein in the samples used for SDS-page. Samples were prepared by adding 4xSDS sample buffer (for 1 mL: 900µL of 2xLaemmeli Sample buffer and 100µL of 10% β- mercaptoethanol) in a 1:1 ratio. In order to linearize the proteins in the sample, the sample solutions containing the sample buffer were boiled, then cooled down, centrifuged and vortexed before loading 10µL of each sample along with 5 µL of Bio-Rad precision plus protein all blue standard ladder (cat#:

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161-0373), onto the pre-cast gel (Criterion TGX Stain-FreeTM Precast Gels; Bio-Rad – 15μL, 1.0 mm). The total amount of protein loaded in each well was 20µg. The electrophoresis chamber (Criterion Vertical Electrophoresis Cell) was run at 170V for approximately one hour in cold 1xTGS (Tris/Glycine/SDS) buffer (cat. #161-0772, Bio-Rad Labs).

4.4.3 GEL ELECTROPHORESIS AND BLOTTING The gel was activated in the ChemiDoc imager to enable the fluorescence of the sample proteins. The separated proteins were blotted from the pre-cast gels onto PVDF membranes (cat#: 1704157, Bio-Rad Labs) by electroblotting using a Trans-Blot Turbo Transfer system (BioRad Labs). In order to confirm that a successful blotting was performed, both the gel and membrane were imaged. The resulting membrane image was used for total protein normalization of the immuno-detected bands.

4.4.4 IMMUNODETECTION The membranes were washed 4x5 minutes in Tris-Buffered Saline Tween (TBS-T) under gentle shaking and were then incubated for one hour in blocking buffer (5% Blotting-Grade Blocker nonfat dry milk in TBS buffer – cat. #1706404, Bio-Rad Labs) for 1 hour, at RT. The membranes were then cut at the appropriate band size to enable the simultaneous incubation with two different antibodies targeting SV2A and SYN. The primary antibodies were prepared in blocking buffer in a 1:10000 ratio. The membrane sections were incubated in primary antibody solution ON (covered in parafilm to avoid evaporation), under gentle shaking at 4℃. The antibodies included in these studies are shown below (see table 2).

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Table 2. Schematic overview of primary and secondary antibodies applied in the western blot analyses. SV2A Synaptophysin

Primary Polyclonal Rabbit anti-SV2A, Polyclonal Rabbit anti- antibody 1:10000. Synaptophysin, 1:10000. (SYnaptic SYstems -Heidelberg, (SYnaptic SYstems - Heidelberg, Germany - #119002) Germany - #101002)

Specificity Specific for SV2A - Binds to SV2A (not to SV2B and SV2C)

Secondary Polyclonal Goat anti-Rabbit Immunoglobulins/ HRP, 1:20000. (Dako DK antibody #P0448)

The following day, the membranes were washed 4x5 minutes in TBS-T at gentle shaking RT, and subsequently incubated in the designated secondary antibody, targeting the specific primary antibody, in a 1:20000 ratio. The membranes were then washed in TBS-T (for 15 min. and then 4x5 min). Signals from antibody-protein interactions were visualized by incubating in the Western Lightning ECL Pro (Enhanced Chemiluminescence Substrate PerkinElmer) solution for 15 minutes and captured by the Bio-Rad ChemiDoc. The band intensities corresponding to the size of the specific proteins were detected, using the ImageLab software 6.0.

4.5 STATISTICAL ANALYSES All statistical analyses were performed using GraphPad Prism version 7.0d (San Diego, United States). Data was expressed as mean ± S.E.M. Between group comparisons were carried out by using either a standard un-paired t-test with Welch’s correction or one-way ANOVA. Correlation analyses were done using Pearson’s correlation coefficient for parametric data. We accept our significance based on a 95% confidence interval (CI), i.e. a p value below 0.05.

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5 RESULTS 5.1 AUTORADIOGRAPHY METHOD VALIDATION The binding of [3H]UCB-J to SV2A in rat cortex and striatum, was reversible and displayed dissociation kinetics as shown in fig. 4C and D. The total concentration (Bmax) defined as the total density of SV2A protein bound by radioligand, was estimated to be 270 fmol/mg TE in the cortex and 267 fmol/mg TE in the striatum. Low dissociation constants (Kd) were observed in both the cortex (3.4 ± 1.53 nM) and the striatum (6.16 ± 1.64 nM). In order to analyze the optimal concentration of the radioligand [3H]UCB-J to use in quantitative autoradiography experiments in this work, a saturation study was performed. As seen in fig. 4A, a series of concentrations ranging from 0-12 nM were applied to rat coronal brain sections. A standard curve was created by using the RPA510 21A radiolabeled standard and utilizing it to determining the binding of [3H]UCB-J to SV2A in the cortex and striatum from the gray values measured (see fig. 4B). Notably, the standard curve was not represented at high concentration, so an interpolation was created. Upon analysis, the optimal concentration was determined to be around 3-6 nM, and 6 nM was chosen for application in future experiments, since the radioligand reaches saturation at 12 nM as apparent in fig. 4C and to a lesser extent in D. The observed non-specific binding was low and linear. For future experiments we therefore only measure total binding, with the addition of 6nM of the radiolabeled ligand.

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Figure 4 - Autoradiography method validation. A - Representative 20 µm coronal sections of the cortical and striatal area from a female Sprague-Dawley rat brain, incubated for 48 hours with increasing concentrations (0nM-12 nM) of [3H]UCB-J. Three brain sections were measured per slide, all from one rat for all the different concentrations of the radioligand, thus technical triplicates were done. B - standard curve based on the interpolation with the measurements on the brain sections and the RPA 510 reference strip standard and their corresponding radioactive concentrations at the time of the experiment. Hyperbola plot with an 2 2 estimated Bmax of 26504, a Kd of 3.3 nM and an R of 0.99. The Kd is in the range that is expected and the R shows a 3 2 desired accuracy. C - Binding kinetics of [ H]UCB-J in cortex, with a Bmax estimated to 270, a Kd of 3.44 and an of R 0.94. X-axis represents the concentration of the added radioligand ([3H]UCB-J). Y-axis represents the level of 3 radioligand bound to the tissue. D - Binding kinetics of [ H]UCB-J in striatum, with a Bmax estimated to 267, a Kd of 6.2 and an R2 of 0.98. X-axis represents the concentration of the added radioligand ([3H]UCB-J). Y-axis represents the level of radioligand bound to the tissue.

5.2 WESTERN BLOT METHOD VALIDATION In order to determine the validity of our western blot method, initial experiments were performed on rat cortex and liver. Specifically, to exhibit specificity of the antibodies used and to establish the quality of the protocol applied. As seen in fig. 5, in the frontal cortical samples all three proteins (SV2A, SYN and β-actin) are visualized and give an intensity high enough to measure, meaning that all three proteins are present in the brain tissue isolated, as expected. In our western blots, we do see the right band sizes (100 kDa for SV2A and 40 kDa for SYN). The SV2A protein has some smear on top of the band (see Appendix 1 fig. 14), likely because of the degree to which the protein is glycosylated. No bands were observed in the immunodetection of SV2A and synaptophysin in the liver samples. The liver is a negative control, as we do not expect the two proteins, that are believed to exclusively be found in brain tissue,

Page 37 of 67 to be found in the liver. While β-actin is a housekeeping protein found in all tissue-types, also in hepatocytes. We conclude: that our SV2A and SYN antibodies are specific and selective, that we do see the expected band sizes and that the protocol can be used in future experiments.

Figure 5 - Western blot method validation. Frontal cortex from one female Sprague-Dawley rat and liver from one male Long Evans rat were used in the initial ordinary western blot analysis, for the validation of the method. The cellulose membrane (post immunoblotting) was incubated with SV2A, synaptophysin and β-actin by cutting the membrane according to the band size expected for SV2A and β-actin and a second incubation after stripping with synaptophysin antibody (see the full membrane in Appendix 1 fig. 14+15). As seen on the membrane picture, technical triplicates (three lanes for both frontal cortex and liver for each protein) are represented. All three proteins are present in the frontal cortex samples, but only β-actin is present in the liver samples. All three proteins are observed at the kDa expected, SV2A around 100, synaptophysin around 40 kDa and β-actin at 37 kDa.

5.3 LOCALIZATION OF SV2A-IMMUNOREACTIVITY IN VARIOUS BRAIN REGIONS The detection of SV2A and SYN in different brain regions was done using western blotting. Further, in order to determine whether SV2A correlated to the gold standard presynaptic marker SYN, we compared regional densities of SV2A and SYN using selective antibodies for the two proteins. Lastly, two experiments were included, one for five brain regions: hippocampus, striatum, brain stem, cerebellum and cortex from one rat of 1 year and 3 months of age (see fig. 6B) and another for three brain regions: cortex, brainstem and cerebellum from four rats of 7 weeks of age (see fig. 6A). SV2A and SYN signals were strong and specific in all predominantly gray matter regions such as cortex and hippocampus, but absent or weak in regions known to be denser in white matter such as the brainstem and cerebellum (see fig. 6A, B, C and D). As seen from the band intensities, of the samples from the older rat, in fig. 6D, the normalization of SV2A to SYN, shows that their expression pattern is very similar, apart from in the cerebellum where we see a large difference between the two protein band intensities. However, as seen on fig. 6B it may be due to the very faint band of SYN measured. The highest expression of SV2A (see fig. 6B) is seen in the hippocampus, cortex and cerebellum. In the younger rats, the expression pattern of SYN and SV2A was similar in all examined brain regions

Page 38 of 67 including the cerebellum (see fig. 6C), whereas the brainstem contained less SV2A. This may be due to the very faint band of SYN in the brainstem seen in fig. 6A. By far the highest expression of SV2A in the young rat, is seen in the cortex (see fig. 6A).

Figure 6 - Localization of SV2A-immunoreactivity in various brain regions. A - The cortex (ctx), brainstem (st) and cerebellum (cb) of four Long Evans rats of 7 weeks applied in stain free immunoblotting for the full protein normalization for SV2A and synaptophysin proteins (see the full membrane in Appendix 2). Representative bands are shown on the figure for each brain region and protein - strong for SV2A and fainter for synaptophysin. B - The hippocampus (hp), striatum (str), brainstem (st), cerebellum (cb) and cortex (ctx) from one male Long Evans of 1 year and 3 months, applied in ordinary immunoblotting protocol for the two synaptic vesicle proteins SV2A and synaptophysin. Representative bands are shown from each brain region and protein - stronger for SV2A than for synaptophysin. C - Analysis of results from experiment corresponding to the membrane shown in ‘’A’’. The normalization of the SV2A levels to the synaptophysin levels in three brain regions. Each point seen on the graph is based on the average of a technical triplicate. As seen on the graph each brain region has a higher level of SV2A in comparison to the level of synaptophysin. The brainstem relationship between the two proteins is very high, that is likely due to the faint synaptophysin band seen in ‘’A’’. D - Analysis of results from experiment corresponding to the membrane shown in ‘’B’’. The normalization of SV2A to synaptophysin levels in all five brain regions. The three points represented for each brain region are technical replicates. Across the brain regions, the SV2A levels are higher than the synaptophysin levels. In the cerebellum, the SV2A is many folds higher in expression than synaptophysin, however this may be due to the high intensity of the SV2A band compared to the synaptophysin band, as seen on the membrane picture on ‘’B’’.

5.4 EFFECTS OF DIURNAL CORTICOSTERONE In order to examine the influence of stress on synaptic plasticity, we studied the effect of circulating corticosterone on SV2A, using [3H]UCB-J binding. The gray value measurements were calibrated into SV2A binding, using the radioligand standard RPA510 21A. As can be seen on fig. 7A and D, the SV2A binding is similar in the cortex area of the six different treatment groups. However, the

Page 39 of 67 hippocampal SV2A binding is fluctuating a bit in comparison, as the binding of SV2A is higher in the two corticosterone treated groups (see fig. 7A and C).

Figure 7 - The effects of corticosterone on Diurnal Cycle corticosterone. A - 24 male Sprague-Dawley rats were applied in, in vitro autoradiography studies of corticosterone’s effect on the SV2A levels and therefore the binding to [3H]UCB- J. Two different euthanasia timepoints were applied in the prior studies done by Dr. Martin Rath, during the light phase (ZT3) and during the dark phase (ZT15). Further three groups are shown on the figure: the control group Sham, veh (that did not have the lesion or treatment), SCNx, veh (had lesion of the SCN, but not treatment) and the last group SCNx, cort (both had lesion done and received corticosterone treatment). The corticosterone was administered during the rats’ awake phase (the dark phase). The sections shown are coronal sections of 20 µm, with cortex and hippocampus visual. In the present study the sections were treated with 6 nM radioligand ([3H]UCB-J) and exposed to a fuji imaging plate for 48 hours before imaging. B - standard curve based on the interpolation with the measurements on the brain sections and the RPA 510 reference strip standard and their corresponding radioactive concentrations at the time of the experiment. 2 Hyperbola plot showing an R -value of 0.99, an estimated Bmax of 27013 and a Kd of 4.68 nM. The Kd is in the range that is expected and the R2 shows a desired accuracy. C - UCB-J binding in hippocampal area of brain sections from rats treated with and without corticosterone and with and without SCN lesion. Two groups are used as controls, one without any interventions (Sham, veh) and one with the lesion but no treatment (SCNx, veh). The three groups at the two different euthanasia timepoints are represented on the graph. No significant difference is seen between the different groups following one-way ANOVA. D - UCB-J binding in cortical tissue from rats treated with and without corticosterone and with and without SCN lesion. Two types of controls are utilized. Two groups are used as controls, one without any interventions (Sham, veh) and one with the lesion but no treatment (SCNx, veh). The three groups at the two different euthanasia time points are represented on the graph. No significant difference is seen between the different groups following one-way ANOVA.

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Correlation studies were performed for the SV2A binding measured and corresponding blood corticosterone measured before sacrifice for all animals. The two groups of animals that had the SCN lesion and were treated with corticosterone, both showed correlation for the SV2A binding and the blood corticosterone level. For the animals sacrificed during the light phase (ZT3) the SV2A binding decreases with the increase in corticosterone levels measured (see fig. 8A). Inversely, the SV2A binding seen in the animals sacrificed during the dark phase (ZT15) is directly correlated with the corticosterone levels (see fig. 8B).

Figure 8 - Correlation of SV2A vs. Corticosterone Levels. Correlation studies were performed on all the different groups of the corticosterone diurnal study (see the rest of the correlation analyses made, in Appendix 3), between SV2A binding and the blood corticosterone measured before euthanasia. Correlation was only observed in the following: A and B - In the cortical measurements of SV2A binding to [3H]UCB-J, a correlation was found to the corresponding blood corticosterone levels measured before euthanasia, for each animal, in the rats euthanized both during the light phase (ZT3) and the dark phase in the SCNx corticosterone treated group. p-value of ZT3 group’s correlation was 0.0341. P- value of Z15 groups’ correlation was 0.0091.

5.5 FLINDERS RESISTANT AND SENSITIVE LINE The detection of SV2A expression/binding in FRL and FSL rats, Flinders model of depression, was done through the use of western blotting and in vitro autoradiography with [3H]UCB-J radioligand. The SV2A expressions measured in the four FRL and four FSL rats, were not significantly different from each other (see fig. 9C). The SYN expression in the FRL and FSL rats was similar as well, even though the levels may seem to differ in figure 9D. These 6 low values included are those of animal 9 and 10 (triplicates from each). The lack in difference is apparent from the bands (see fig.9A). The difference in SV2A and SYN expression in the different rats, was not significant either (see fig. 9B). The difference seen in animal 9 and 10 in the normalization of SYN to SV2A (see fig. 9B) may be

Page 41 of 67 acknowledged as non-significant biological variation, as the synaptophysin levels in these two rats are very low compared to the others.

Figure 9 - Flinders Resistant and Sensitive Line Western Blot. A - Brain hemispheres from four Sprague-Dawley Flinders Sensitive and Resistant rats applied in the stain free immunoblotting for SV2A and synaptophysin proteins. As seen on the picture, representative SV2A and synaptophysin bands from the two different rat lines. Strong SV2A bands and fainter synaptophysin bands. B - SV2A normalized to synaptophysin in FRL and FSL rats, with technical triplicates applied. The levels observed in the SV2A and synaptophysin levels seem to be somewhat equal from the normalization. The synaptophysin levels for two of the FSL animals (ID 9 and 10) is much higher than the level of SV2A; however, this may be because of the very faint bands of synaptophysin seen in this study. C - SV2A levels observed in the two groups of depression model rats (FRL and FSL). Technical replicates applied for all the four animals in each group. No significant difference was observed between the two groups when applying an ordinary t-test. D - Synaptophysin levels observed in the two groups of rats in the model of depression (FSL and FRL). Technical replicates applied for all the four animals in each group. A significant difference was observed with Welch’s t-test, with a p-value of 0.0001, between the protein expression levels in the FRL and the FSL rats, as the expression was higher in the comparison group FRL compared to the FSL depression rats. However, the biological variation in synaptophysin, especially by FSL animal 9 and 10, may increase the significance unjustly.

The quantification of the SV2A binding to the radioligand [3H]UCB-J was done in three brain regions: neocortex, dorsal and ventral hippocampus. Using a standard curve made from the measurements of the radioligand standards RPA510 21A and ART0123B (added in order to avoid interpolation for high values) (see fig. 10B), the three brain regions were quantified for SV2A binding in six FRL and six FSL rats. As is visual on figure 10A, the SV2A binding did not fluctuate significantly in the two different rat types, nor did it differ in the three brain regions. The [3H]UCB-J binding to SV2A was very equal across the resistant and sensitive Flinders rats and across brain regions (see fig. 10C).

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Thus, no significant differences in expression nor binding, were observed in between the FRL and FSL rats.

Figure 10 - Flinders Resistant and Sensitive Line Autoradiography. A - Representative 20 µm coronal sections from neocortex, dorsal and ventral hippocampal sections from six FSL and six FRL rats applied in the binding studies of SV2A to the radioligand [3H]UCB-J. Three brain slices were measured per slide, all from one rat (technical triplicates). All slides were treated with 6nM [3H]-UCB-J radioligand and exposed to the imaging plate for 48 hours for the detection of binding to SV2A protein in the brain tissue. B - standard curve of binding from two tritium radioligand standards, the RPA 510 and the ART0123B and their corresponding radioactive concentrations at the time of the experiment. Hyperbola 2 2 plot showing an R -value of 0.98, a Bmax of 33066 and a Kd of 3.98. The Kd is in the range that is expected and the R shows a desired accuracy. C - UCB-J binding to SV2A in the neocortex, ventral and dorsal hippocampus from FRL and FSL rats. No significant differences were observed between the FRL and FSL animals when analyzing with t-test, one- way ANOVA and multiple comparison tests.

5.6 ELECTROCONVULSIVE STIMULATION The detection of SV2A binding and expression in the brain from rats exposed to repetitive ECSs. The tissue was analyzed by western blotting and autoradiography. The level of SV2A protein in hemispheres from two sham and two ECS rats revealed no significant difference between the two groups (see fig. 11B). The levels of the synaptic vesicle protein, synaptophysin, used in the comparison to SV2A are however differing in the sham and the ECS rats. The expressed levels of synaptophysin observed in the ECS rats is significantly higher than those observed in the sham rats (see fig. 11C). In the comparison between synaptophysin and SV2A in expression patterns, SV2A is expressed to a greater

Page 43 of 67 extent in both sham and ECS rats as seen on the band intensities (see fig. 11A). The fold difference between SV2A and SYN expression was significantly higher in sham rats compared to ECS rats (see fig. 11D).

Figure 11 - Electroconvulsive Stimulation Western Blot. A - Brain hemispheres from two of each Wistar Han ECS treated (14 stimulations - study done by Søren Christiansen) and Sham (control) rats of 10 weeks applied in the stain free immunoblotting for SV2A and synaptophysin proteins. As seen on the picture, representative SV2A and synaptophysin bands from the two different rat groups. Strong SV2A bands and faint synaptophysin bands. B - SV2A levels measured in the two sham and two ECS treated rats, both brain hemispheres were run and for each, technical triplicates were applied - thus for each animal 6 points are visible on the graph. No significant difference was observed in the SV2A expression between the ECS and the Sham rats. C - Synaptophysin levels measured in the two sham and two ECS treated rats, both brain hemispheres were run and for each, technical triplicates were applied - thus for each animal 6 points are visible on the graph. A significant difference was observed in the SYN expression between the ECS and the Sham rats when applying Welch’s t-test with a p value <0.0001. D - For the comparison of SV2A and Synaptophysin expression the two were normalized. As seen on the graph, SV2A compared to SYN in the Sham rats is much higher than in the ECS rats. A significant difference was observed in the fold difference between SV2A and SYN in the ECS and Sham rats when applying Welch’s t-test with a p-value of 0.0112.

Measurements of the SV2A binding to the [3H]UCB-J radiolabeled ligand was done in five brain regions, in six control rats (sham) and six ECS treated rats. Two standard curves were made, based on RPA510 21A and ART0123B, one for each of the separate experiments with half of the brain regions. The PFCtx and striatum were run together and the designated standard curve for these areas is shown in fig. 12C and the standard curve for the amygdala, dorsal hippocampus and ventral hippocampus, that were run on the same imaging plate, is seen on figure 12B. Overall, as is visual on fig. 12A, the differences in binding between the ECS and Sham brain sections, if at all present, are minute. However, significant differences were observed in two brain regions, the amygdala (see fig.

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12F) and the ventral hippocampus (see fig. 12H). In both regions, the SV2A binding is lower in the ECS rats compared to the binding seen in the sham rats. Thus, no significant differences were observed in the PFCtx, striatum and dorsal hippocampus between the sham and ECS rats.

Figure 12 - Electroconvulsive Stimulation Autoradiography. A - Representative 20 µm coronal sections from prefrontal cortex (PFCtx), amygdala, striatum, dorsal (D-hip) and ventral hippocampal(V-hip) sections from six ECS treated (14 stimulations - study done by Søren Christiansen) and six sham (control) rats applied in the binding studies of SV2A to the radioligand [3H]UCB-J. Three brain slices were measured per slide, all from one rat (technical triplicates). All slides were treated with 6nM [3H]-UCB-J radioligand and exposed to the imaging plate for 48 hours for the detection of binding to SV2A protein in the brain tissue. B - Standard curve, for amygdala, dorsal and ventral hippocampus, of binding from two tritium radioligand standards, the RPA 510 and the ART0123B and their corresponding radioactive concentrations 2 at the time of the experiment. Hyperbola plot showing an R -value of 0.98, a Bmax of 30250 and a Kd of 4.6. The Kd is in the range that is expected and the R2 shows a desired accuracy. C - Standard curve, for PFCtx and striatum, of binding from two tritium radioligand standards, the RPA 510 and the ART0123B and their corresponding radioactive 2 concentrations at the time of the experiment. Hyperbola plot showing an R -value of 0.98, a Bmax of 31483 and a Kd of 2 3.8. The Kd is in the range that is expected and the R shows a desired accuracy. D - Ligand bound to SV2A in the PFCtx in ECS and Sham rats. No significant difference was observed in the binding in this region between the two groups of rats. E - Ligand bound to SV2A in the striatum in ECS and Sham rats. No significant difference was observed in the binding in this region between the two groups of rats. F - [3H]UCB-J bound to SV2A in the amygdala in ECS and Sham rats. A significant difference was observed in the binding in this region between the two groups of rats with the application of an unpaired t-test with Welch’s correction, p-value of 0.027. G - Ligand bound to SV2A in the dorsal hippocampus in ECS and Sham rats. No significant difference was observed in the binding in this region between the two groups of rats. H - Amount of ligand bound to SV2A in the ventral hippocampus in ECS and Sham rats. A significant difference was observed in the binding in this region between the two groups of rats with the application of an unpaired t-test with Welch’s correction, p-value of 0.047.

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5.7 ANTIDEPRESSANT TREATMENT IN CHRONIC MILD STRESS ANIMALS In the study of the stress model rats, two groups were applied: vehicle anhedonic (control group), n=8 and agomelatine treated, n=16. The neocortex area was measured for the SV2A binding to the radiolabeled ligand. Figure 13A presents all three groups as differing in their binding of [3H]UCB-J. A higher binding in the treated group is observed, compared to the control group. Based on the standard curve, based on RPA510 21A and ART0123B, (fig. 13B) the gray values were interpreted into the concentration of ligand bound to the brain sections. The difference in binding to the brain sections shown in fig. 13A is also illustrated in fig. 13C and D. For both the OFC and PFC a significant difference is observed between the control vehicle and the treated groups (see fig. 13C and D). An increase observed in the OFC with more than 100 fmol/mg TE (see fig. 13C) with a significance above 0.0001 and in the PFC by approximately 50 fmol/mg TE (see fig. 13D) with a significance of 0.0115. The treated groups seem to have higher levels of SV2A, as the binding of [3H]UCB-J to the protein is higher in this group compared to the control group (see fig. 13C and D).

Figure 13 - Chronic Mild Stress Autoradiography. A - Representative 12 µm coronal sections with prefrontal cortex (PFCtx) and orbitofrontal cortex (OFCtx) visual on the sections from eight vehicle anhedonic (veh, CMS), eight non- responders and eight responders (the two latter treated with agomelatine (Ago-treated)). The CMS rats were applied in the binding studies of SV2A to the radioligand [3H]UCB-J. Three brain slices were measured per slide, all from one rat (technical triplicates). All slides were treated with 6nM [3H]-UCB-J radioligand and exposed to the imaging plate for 48 hours for the detection of binding to SV2A protein, in the brain tissue. B - Standard curve of binding from two tritium radioligand standards, the RPA 510 and the ART0123B and their corresponding radioactive concentrations at the time 2 of the experiment. Hyperbola plot showing an R -value of 0.97, a Bmax of 32121 and a Kd of 4.4. The Kd is in the range that is expected and the R2 shows a desired accuracy. C - [3H]UCB-J binding to SV2A in the orbitofrontal cortex. The treated (both responders and non-responders) were pooled for the analysis and compared to the vehicle CMS groups,

Page 46 of 67 which did not receive agomelatine treatment. A significant difference was observed between the treated and vehicle group, as the vehicle CMS group exhibits lower SV2A levels than the agomelatine treated group. Significance found with an unpaired t-test with Welch’s correction with a p-value below 0.0001. D - [3H]UCB-J binding to SV2A in the prefrontal cortex. The treated (both responders and non-responders) were pooled for the analysis and compared to the vehicle CMS groups, which did not receive agomelatine treatment. A significant difference was observed between the treated and vehicle group, as the vehicle CMS group exhibits lower SV2A levels than the agomelatine treated group. Significance found with an unpaired t-test with Welch’s correction with a p-value below 0.0115.

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6 DISCUSSION The aims of the present study were to validate techniques detecting SV2A in the rat brain determined by [3H]UCB-J radioligand binding and immunoblotting and furthermore, to determine the distribution of SV2A across brain regions and in various animal models of depression. The major part of the study was to investigate the radioligand binding to SV2A; the expression of SV2A in different animal models of depression; the effect of antidepressant treatment on the binding and whether synaptic density can be determined by [3H]UCB-J binding. In this section, we will discuss how suitable the used methods are for the purpose of highlighting the association of synaptic density to network alterations and symptoms of depression. Furthermore, the results from the conducted experiments will be compared to previous studies. Our final goal is to establish the potential of SV2A being a marker of synaptic density and its involvement in depression.

We show that: the use of autoradiography and western blot are useful methods to investigate the regional density of SV2A in different brain regions in animals; in vitro autoradiography is a strong method for measuring binding affinity of [3H]UCB-J to SV2A in different brain regions; using a tritium labeled ligand yields a high spatial resolution, ensuring that the density of the protein of interest (Bmax) in different brain regions can be measured easily; and the stain free western blotting method using protein and isoform specific antibodies is a valid method for studying the expression patterns of SV2A and synaptophysin in different brain regions.

The number of synapses in the brain changes in response to different stimuli during a lifetime (Finemma et al., 2016). Synaptogenesis and synaptic degeneration are important physiological and pathological processes in the regulation of synaptic density. By far, the evaluation of synaptic density in humans has been limited to the examination of brain tissue obtained from surgery or autopsy (Finemma et al., 2016). For the proper understanding, diagnosis and treatment of brain disorders associated with synaptic pathology, it is important to be able to quantify synaptic densities in the living human brain. As part of the preliminary studies we present methods for quantification of SV2A in different brain regions, implicated in the depressive disorder, using the radioligand [3H]UCB-J for targeting SV2A. [3H]UCB-J is chosen as the radiolabeled ligand based on a previous study (Nabulsi

Page 48 of 67 et al., 2016), where it was assumed that the specific binding of UCB-J detected was only to SV2A and not to the other two members of the protein family (SV2B and SV2C). The study showed selectivity of UCB-J to SV2A, as the binding affinity of UCB-J was 10-fold and 100-fold less potent for SV2C and SV2B, respectively.

6.1 ESTABLISHMENT OF METHODS

6.1.1 AUTORADIOGRAPHY METHOD VALIDATION Saturation binding studies showed that [3H]UCB-J bound with high affinity to SV2A in both rat cortex and striatum. Analyses of the binding kinetics of [3H]UCB-J gave a dissociation constant of 3.4 ± 1.53 nM in cortex and 6.16 ± 1.64 nM in striatum. The dissociation constant for cortex, indicates that a low concentration of ligand was needed in the cortex to bind 50% of the SV2A protein. In contrast, the dissociation constant seen in the striatum indicates that a higher concentration of ligand is needed in the striatum to bind 50% of the SV2A protein, compared to in the cortex. This observation might indicate that the radioligand has a lower binding affinity for SV2A in the striatum compared to in the cortex (see fig.4).

The Bmax value for both regions are approximately 270 fmol/mg TE, which is an indication that the SV2A density in the two regions is similar. No similar saturation binding studies, by autoradiography, with the UCB-J ligand labelled with tritium have previously been reported in the literature. However, the binding characteristics of the UCB-J ligand labelled with carbon 11, in homogenized brain tissue from a baboon, by liquid scintillation, has previously been investigated by Finnema et al., in 2016. Different brain regions of the baboon were analyzed in order to determine the binding of [11C]UCB-

J. The mean Kd value was determined to be approximately 3.9 nM. Another study, Nabulsi et al., 2016, also investigated the binding affinity of [11C]UCB-J in brain tissues from rhesus monkeys, by in vivo PET. The Kd value was found to be approximately 3.4 nM.

The Kd values from the two studies (Finnema et al., 2016 and Nabulsi et al., 2016) are highly comparable, since they are in the low nM-range, with the Kd values determined in our study, specifically in rat cortex. However, it is important to consider that the methods used to determine the

Kd values, in the two studies were different from those used in the present study; and that the values were found in different species.

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6.1.2 WESTERN BLOT NORMALIZATIONS The housekeeping protein, β-actin, can be used as a measure of the overall cell density, as it is a structural component of the cytoskeleton of all cells. Meanwhile, synaptophysin is a measure of the synapses, as synaptophysin is considered to be present and equally expressed in synapses of all brain regions (Tarsa & Goda, 2002). By normalizing SV2A to β-actin, the purpose is to relate the amount of the protein to the number of cells overall. Further, to normalize synaptophysin and SV2A in each protein sample loaded to the same value - the amount of β-actin found in that specific protein sample. When normalizing SV2A levels to synaptophysin, it is a measure of relating the amount of SV2A to the number of vesicles and synapse number (Finemma et al., 2016). Synaptophysin is an accepted marker of synaptic density, which is why the normalization of SV2A to SYN could clarify whether the proteins are evenly distributed in the synapses of different regions.

In the initial western blots done in this thesis, β-actin was used for normalization, as mentioned (see fig.5). However, the β-actin detection on the membranes was not a reliable measure, as it was not visible at every western blot carried out. It is suspected that it is due to a fragility of the antibody, that makes it unstable to thaw and freeze repeatedly for each experiment. As the housekeeping protein was no longer a viable option for normalization, a new method, using stain free gels, was employed for the duration of the thesis. The stain free method of western blotting was utilized in order to normalize the synaptophysin and SV2A protein band measurements; to the full amount of protein loaded in the specific lane. In this way, the protein found in a sample loaded, is normalized to that specific sample’s full protein amount.

6.1.3 TROUBLESHOOTING Western blot is a technique that is useful for protein detection, as it allows quantification of the protein expression. Even though the procedure of western blot is simple, a number of problems can arise, which can lead to unexpected results. These problems include unusual or unexpected bands; no bands or faint bands; high background and uneven and patchy spots on the blot. In this section, we will highlight the issues that arose during our western blot procedures and the possible solutions. If the protein seems to be in too high of a position, then reheating the sample can help to break the quaternary protein structure and ease the migration of the protein through the gel. Though, protein sizes can be different from their theoretical size due to post-translational modifications such as phosphorylation or glycosylation. One N-linked glycosylation will add 2.5 kDa to the protein’s

Page 50 of 67 weight (Ansari et al., 2006). In our case, SV2A has three N-linked glycosylation sites, which means that its size goes from the theoretical 82.6 kDa to approximately 90 kDa. This could explain the higher positioning of SV2A seen in the western blots carried out in this study. Detection of SV2A showed bands at approximately 100 kDa for all western blots carried out in this study. All SV2A bands detected in the present study showed smears located above the bands. Membrane lipids attached to the protein disturbs migration of the protein through the gel. This could possibly be a reason why the smear occurs. In previous studies, band profiles of SV2A in general also show this smear (Bajjalieh et al., 1994, Lambeng et al., 2005, van Vliet et al., 2009 and Finnema et al., 2016). Another problem, blurry bands, is caused by high voltage at the separation or air bubbles present during transfer. In this case, it is important to ensure that the gel is run at a lower voltage as too fast of a travel through the gel can result in curved bands, due to low resistance. In our case, we ran our gel electrophoresis at 170V, which is in between the low and the high runs seen in literature (Eslami & Lujan, 2010 and Mahmood and Yang, 2012). Problems involving the used antibody, antigen or buffer can result in no bands and high background. If the used antibodies are inefficient, it will lead to no bands showing. It is therefore advisable to change the antibody utilized when this situation arises. In the case of ECS, we experienced that the band corresponding to synaptophysin was not visible the first couple of western blots; however, another antibody was not tried. Another reason for no visible bands or weak signals is a low concentration or absence of the antigen, this was to the best of our ability avoided by measuring the specific protein concentrations for each experiment; however, the damage or absence of the specific SV2A-antibody binding site cannot be ruled out. Increasing exposure time can help make the band clearer if the binding to the antigen is weak. Patchy and uneven spots on the blot are usually caused by improper transfer. Uneven spots can also result from aggregation of the secondary antibody. This problem can be resolved by centrifugation and filtration of the secondary antibody. In the case of the ECS western blot this was somewhat of an issue; however, the information on secondary antibody aggregation did not come to light until after the experiment was concluded.

6.1.4 ALTERNATIVE METHODS Alternative methods that could be utilized in order to validate our experiments are (1) Q-PCR to look at the expression of different candidate gene targets for antidepressant drugs in the selected depression models. A down-regulation would show a lesser potential effect of the antidepressants and therefore, a high severity of the disorder. Further, looking at the expression patterns of proteins implicated in depression and synaptic plasticity, such as BDNF. We would then expect an upregulation in BDNF

Page 51 of 67 with the increase of synaptic plasticity as a results of antidepressant treatments, agomelatine and ECS. Further, we would expect a downregulation of BDNF in the depression models and in the corticosterone treated rats. (2) Confocal microscopy to visualize changes in both synaptophysin and SV2A. The co-distribution and overlap of the two proteins earlier seen in the study by Finemma et al. from 2016 is a fine basis for examining the same distributions in depression models. (3) Gold labeling and Electron microscopy the gold labeling of specific proteins, can aid in the study of the specific micro-location of the two proteins, for the comparison of the two and for the understanding of their structural positions. (4) PET imaging using the radiolabeled ligand [11C]UCB-J for in vivo studies of the SV2A distribution and localization for the accurate measurement of the spatial distribution in the comparison between controls and the animal models of depression.

6.2 SV2A DISTRIBUTION AND CORTICOSTERONE EFFECTS IN RATS

6.2.1 LOCALIZATION OF SV2A-IMMUNOREACTIVITY In studying the distribution of SV2A and comparing this to that of synaptophysin, two age-groups of rats were used. Firstly, the five regions (hippocampus, cortex, striatum, brainstem and cerebellum) were examined in one old rat of 1 year and 3 months, whilst three of these regions (cortex, brainstem and cerebellum) were also examined in younger rats, of 7 weeks. In experimental set-ups, of two separate experiments, we are then able to compare the distribution expression of SV2A across age classes and regions. Starting the comparison within the age groups, the older rats seem to have a somewhat equal distribution of the two proteins, across brain regions and a slightly higher level of SV2A compared to synaptophysin. However, the cerebellum and brainstem of both age groups, seem to have a much higher expression ratio between SV2A and synaptophysin. This high ratio is most likely due to the fainter bands of synaptophysin seen for the cerebellum and brainstem compared to the other regions (see fig. 6A and B). This is in line with what Finnema et al. found in 2016, that the cerebellum and brainstem compared to several other regions were lower in synaptophysin expression compared to SV2A. This can be explained by the makeup of the cerebellum, since it is a structure with many synapses and white matter substance; it makes it different in composition to other regions. This is also the case for the brainstem which contains many glia and is therefore a mixture of myelinated and unmyelinated cells. When not taking the results of the cerebellum into account, the two different age groups of rats are different in their overall expression of SV2A and synaptophysin. The expression ratio between SV2A and synaptophysin in the younger rats is higher than that seen in the old rat. This is in accordance with the literature, as an increase in synapses and their associated

Page 52 of 67 proteins occurs until the rats are approximately one month of age. Then a drop of more than half of the synapses, occurs between two and six months, which then ends with an overall decline in neurogenesis in the old rats beyond 1 year (Petralia et al., 2014). The experiments done in studying the SV2A localization across brain regions; and two age groups and the validation of the methods, is a further indication that SV2A is a potential marker of synaptic density as previously seen in multiple studies (Finemma et al., 2016; Holmes et al., 2019 and Mendoza-Torreblanca et al., 2013).

6.2.2 CORTICOSTERONE EFFECT ON DIURNAL CYCLE In order to examine the influence of stress on synaptic plasticity, the effect of administered circulating corticosterone on SV2A binding in hippocampal and cortical tissue, was investigated. The SV2A binding was shown to be close to equal in the two regions in all four vehicle groups. However, both corticosterone treated groups are higher in SV2A binding in the hippocampus than in the cortex, when compared to the vehicle groups, (fig. 7C and D) for both euthanasia timepoints (ZT3 and ZT15). However, this difference was not significant and high levels of SV2A are also seen in the groups not treated with corticosterone, which might be caused by endogenous stress-hormone levels as seen in fluctuations in blood corticosterone levels (data not shown). This indicates that no changes in synapses and thus SV2A binding occurred as a response to the chronic stress state of the animals, induced by administration of corticosterone. This could suggest that stress hormones are not the factor inducing the depressive state or the LTD in synaptic plasticity. Especially, studies highlighting the influence of inflammatory cytokines on the volume of brain regions important in depression, emphasize that high stress hormone levels are not necessarily the causative factor in depression (Baumann et al., 1997 and Hendrie & Pickles, 2013). Correlation studies performed for the SV2A binding measured and the corresponding blood corticosterone for all animals; showed a conspicuous correlation in the corticosterone treated rats with the SCN lesion for both euthanasia timepoints (ZT3 and ZT15). For the animals sacrificed during the light phase (ZT3) the SV2A binding was inversely correlated with the SV2A binding (see fig. 8A). In contrast, the SV2A binding seen in the animals sacrificed during the dark phase (ZT15) was shown to be directly correlated with the blood corticosterone levels (see fig. 8B). This might indicate that there is a variation between the two corticosterone treated groups depending on their euthanasia timepoints. The rats’ suprachiasmatic nucleus (the clock of the brain) is lesioned, which means that biologically they cannot distinguish between day and night; they do however see the changes in light and dark, but do not behave accordingly. Whether these are factors that have an influence on the biological mechanisms IS difficult to state and no evidence exists supporting this and the mechanism

Page 53 of 67 behind the results presented, are not known. Furthermore, the study is only performed on four rats, which is too few to draw a proper conclusion. It is therefore necessary to repeat the study with a larger number of subjects, in order to examine changes in SV2A binding in different brain regions, as a response to high levels of administered stress hormone.

6.3 MODELS OF DEPRESSION In this thesis, three major models of depression have been employed: the genetic depression model Flinders Sensitive Line, the electroconvulsive stimulation model that represent one of the most effective antidepressant and synaptic plasticity stimulating treatments and lastly, the chronic mild stress model of depression. The hypotheses proposed for the depression models, were a downregulation in SV2A expression and binding in the depression-like behaviors compared to the controls. In the ECS and agomelatine treated CMS animals, the hypotheses proposed were that the treated groups would increase in SV2A expression and binding. These hypotheses are based on the findings that loss in synapses and therefore synaptic proteins is implicated in depression-pathology. As seen by Holmes et al. in 2019, the depression severity is inversely correlated to the SV2A levels seen.

6.3.1 FLINDERS SENSITIVE LINE RATS IN EXHIBITION OF PROTEOMIC CHANGES IN DEPRESSION In the FSL depression model a significantly lower SYN expression was observed, in comparison SV2A stayed somewhat similar in expression levels across the board. SYN and SV2A were expected to follow each other in their expression patterns; however, this was not the case. The lack of difference observed in SV2A (see fig.9C) may be due to the sample being of complete hemisphere homogenates. In order to ensure a sensitivity high enough to observe the differences occurring in the depression- model, it may be necessary to have dissections of regions known to be involved in depression- pathology, such as the hippocampus and PFC. In the binding studies; however, no differences were seen between the FSL and FRL rats despite the inclusion of regions specific for depression associated changes (see fig.10). We are comparing the two lines inbred from Sprague-Dawley rats, where one shows depressed symptoms and one does not. Both are genetically selected for; for generations. However, we are not comparing the FSL (depression-like) to a group of ordinary Sprague-Dawley rats. This may be a limitation as previous studies have found a difference in other brain-associated markers in the FRL (non-depression-like) and Sprague-Dawley rats. Nishi et al. found, in a study from 2009, that there was a significant difference in the 5-HT1A between FRL and Sprague-Dawley

Page 54 of 67 rats. The comparison restricted to FRL rats in the present study is thus, not necessarily equivalent to the comparison between healthy and depressed subjects. This would have been on a stronger basis if compared to a wild type Sprague-Dawley rat. In this fashion, the comparison of the SV2A in the FSL to the measured in a group of wild type Sprague-Dawley, might have been a basis on which a significant difference would have been observed.

6.3.2 ELECTROCONVULSIVE STIMULATION AND SYNAPTIC PLASTICITY The levels of SV2A binding to [3H]UCB-J in the different brain regions of the ECS compared to the control rats, was not expected based on the hypothesis proposed. ECS has been shown to increase synaptic plasticity, protein expression and synapse number. However, in the present study we find that the prime brain regions highlighted by multiple studies when addressing depression and the morphological/physiological changes, are namely PFC, hippocampus and amygdala (Duman et al., 2016; Finemma et al., 2016; Højgaard et al., 2018 and Holmes et al., 2019). As observed in the present study, significant differences were observed between the ECS and control (sham) group in the amygdala and ventral hippocampus (fig. 12 F and H). The significant differences are observed in the expected brain regions; however, the control groups exhibit higher SV2A binding than the ECS treatment groups, this is in turn not expected. A possible explanation for the lack in increase in the ECS groups is that synaptic plasticity, the prime result of ECS treatment, happens at the dendrites and not presynaptically. As SV2A is a presynaptic vesicle protein, this might be the reason that we do not observe any significant increases in the SV2A binding in the ECS animals. The reason this experiment was conducted was to observe whether synaptic plasticity occurring, mainly, at the postsynapse has an influence on the presynaptic plasticity as well. This might be the case as increases in postsynaptic density will simultaneously strengthen the post- to presynaptic connection and therefore also cause an upregulation in proteins associated with the presynaptic neurons. However, studies have so far only found evidence, suggesting that synaptic plasticity-associated proteins such as BDNF, increase the synapse density at the presynapse (Sanchez et al., 2006). Further investigations using similar methods as employed in this current study; are therefore, necessary for the better understanding of the connectivity between postsynaptic plasticity and its influence on the presynapse and its associated proteins.

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6.3.3 ANTIDEPRESSANT EFFECTS OF AGOMELATINE INCREASE SV2A BINDING IN CMS The CMS model has been applied in the examination of the relationship between the depressive-like behavior exhibited by the animal model and a change in synaptic density. Thus far; chronic stress has been linked to a loss in synaptic connections in mood-regulating circuitry and destabilizing changes in plasticity control, due to the effect of stress on homeostasis (Holmes et al., 2019). However, the CMS model of depression has been criticized for lacking reliability. There has been a frequently expressed assumption that the CMS procedure is unreliable or difficult to replicate (Willner, 2017). However, data from many different laboratories reporting depressive-like and antidepressant-reversible effects of CMS across a variety of depression-relevant endpoints, have been summarized. As seen in our CMS experiments, it was possible to establish an increase in SV2A binding in both the OFC and the PFC as a result of the agomelatine treatment administered (see fig. 13C and D). This in accordance with what was expected and hypothesized. Antidepressants have shown to increase synapse number in chronically stressed and depressed individuals, thus an increase in synaptic vesicles and synaptic vesicle proteins is also to be expected (Duman et al., 2016). Therefore, based on the antidepressant-like effects of agomelatine on the CMS animals observed by Højgaard et al. in 2018, an increase in SV2A binding in the two cortical areas by [3H]UCB-J was also expected in this study.

6.4 SV2A REGULATION IN PRECLINICAL DEPRESSION MODELS As seen in the previous sections, the depression-models employed in the experiments conducted in the course of this thesis; have been inconsistent in their success of portraying the expected changes in expression and binding of SV2A, previously seen in literature. The Flinders and the ECS models did not yield the expected results, while the CMS model did show that agomelatine increases the SV2A binding in the PFC and in the OFC, after CMS protocol making the rats chronically stressed. Despite the results shown here, previous studies have shown that SV2A changes are exhibited in both humans and in preclinical models of depression. Moreover, the antiepileptic drug targeting SV2A, LEV, has been implicated in altering short-term synaptic plasticity (Löscher et al., 2016). We therefore suggest, that SV2A is key in synaptic plasticity as it is the specific target of the drug. These are grounds to further investigate SV2A and its involvement in plasticity and depression. Therefore, we propose that further studies on the SV2A expression and binding in the same and similar models

Page 56 of 67 utilized here; need to be conducted, in order to conclude definitively on the SV2A regulation in depression models and potentially in synaptic plasticity.

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7 CONCLUSION The overall aims of this thesis were to validate techniques detecting SV2A in the rat brain determined by [3H]UCB-J radioligand binding and immunoblotting and furthermore, to determine the distribution of SV2A across brain regions and in various animal models of depression. The establishment of the two methods: in vitro autoradiography using the radioligand [3H]UCB-J targeting SV2A and stain free western blotting using selective antibodies for SV2A and synaptophysin, were successful. The examination of the regional SV2A distribution across various brain regions, revealed that differences in the amount of SV2A are observed between age groups; and further, that the SV2A expression is higher overall compared to that of synaptophysin. Brain regions dense in gray matter, such as the cortex, show higher expression of SV2A compared to regions, such as the brainstem and cerebellum, constituting of white matter as well. In the diurnal cycle experiment we found that the SV2A binding and the corresponding blood corticosterone levels were correlated in all the corticosterone treated animals. An inverse correlation was found for the animals sacrificed during the light phase (ZT3) and a direct correlation was found for those sacrificed during the dark phase (ZT15). In the Flinders model experiments, no significant differences in SV2A binding or expression were observed between the sensitive and resistant line of genetically depressed rats. This may be due to a lack of a wild type Sprague-Dawley control for thorough comparisons. In the electroconvulsive stimulation rat model a significant difference was observed between the ECS and control (sham) group in two brain regions, a decrease in SV2A binding was observed in the amygdala and ventral hippocampus in the ECS group, which was not expected based on the hypothesis proposed. This may be due to the synaptic plasticity, being induced by the treatment, primarily being of postsynaptic nature; and does therefore, not affect the presynapse directly. In the chronic mild stress rat model, the SV2A binding was significantly increased in both the prefrontal cortex and the orbitofrontal cortex; with treatment utilizing the antidepressant agomelatine. Based on the inconsistency between some of the results presented in this thesis and those from previous studies, we propose that the SV2A expression and binding must be further investigated applying similar models utilized here, in order to conclude definitively on the SV2A dysregulation in depression models and potentially in synaptic plasticity.

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In conclusion, studies; firstly in vitro in a similar fashion as presented here; secondly in in vivo animal models of depression and lastly clinical studies of SV2A expression in MDD, are necessary for the proper understanding of the involvement of SV2A in synaptic plasticity and depression.

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9 APPENDICES Appendix 1

Validation of Western Blot Method

Appendix Figure 14 - Full Membrane from Validation of Western Blot Method (SV2A and β-actin). Full membrane post-immunodetection of SV2A and β-actin, triplicates with frontal cortex tissue from Sprague-Dawley rat and triplicates with liver tissue from Long Evans rat. SV2A only visible in cortical tissue at approximately 100 kDa as expected, whilst β-actin visible in both cortex and liver tissue at approximately 37 kDa, as expected.

Appendix Figure 15 - Full Membrane from Validation of Western Blot Method (Synaptophysin). Full membrane post- immunodetection of synaptophysin (SYN), triplicates with frontal cortex tissue from Sprague-Dawley rat and triplicates with liver tissue from Long Evans rat. Synaptophysin only visible in cortical tissue at approximately 40 kDa, as expected, whilst not visible in liver tissue samples loaded.

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Appendix 2

Localization Study

Appendix Figure 16 - Full membrane from Localization Study. Post immunodetection of SV2A and synaptophysin (SYN) using the stain free western blotting method for full protein normalization. Rat tissue from cortex (ctx), brainstem (st) and cerebellum (cb) of four Long Evans rats run in triplicates. SV2A and synaptophysin observed at the expected 100 and 40 kDa, respectively.

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Appendix 3

Correlations in Corticosterone Study Hippocampus

Appendix Figure 17 - Hippocampus/Blood Corticosterone Correlations made in Corticosterone Study. In the process of finding the two correlations seen in figure 8 of the results section, all other groups and ZT times were also run for correlation between hippocampal SV2A binding with their corresponding blood corticosterone levels. No correlations were found between the blood corticosterone level measured in the rats before euthanasia and the SV2A binding in the hippocampal measurements.

Cortex

Appendix Figure 18 - Cortex/Blood Corticosterone Correlations made in Corticosterone Study. In the process of finding the two correlations seen in figure 8 of the results section, all other groups and ZT times were also run for correlation between their cortical SV2A binding and corresponding blood corticosterone levels. The only correlations found between the blood corticosterone level measured in the rats before euthanasia and the SV2A binding in the cortical measurements, were the two presented in figure 8 in the results section.

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