Optogentic Neuromodulation in Animal Models Of

Optogenetic Neuromodulation in a Rodent Model of Depression

Inaugural-Dissertation

zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert–Ludwigs–Universität Freiburg im Breisgau

vorgelegt von Lisa-Marie Pfeiﬀer aus Kassel

Freiburg im Breisgau September 2019 Angefertigt in der Abteilung für Stereotaktische und Funktionelle Neurochirur- gie des Universitätsklinikums Freiburg unter der Leitung von Prof. Dr. med. Volker A. Coenen und PD Dr. Máté D. Döbrössy.

Dekan der Fakultät für Biologie: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Andreas Hiltbrunner

Betreuer der Arbeit: Prof. Dr. med. Volker A. Coenen Betreuer der biologischen Fakultät: Prof. Dr. Wolfgang Driever

Referent: Prof. Dr. Wolfgang Driever Koreferentin: Prof. Dr. Ilka Diester Drittprüferin: Prof. Dr. Carola Haas

Datum der mündlichen Prüfung: 28.11.2019 To Mom and Dad. “Rabbit’s clever," said Pooh thoughtfully. "Yes," said Piglet, "Rabbit’s clever." "And he has Brain." "Yes," said Piglet, "Rabbit has Brain." There was a long silence. "I suppose," said Pooh, "that that’s why he never understands anything.”

— A.A. Milne, Winnie-the-Pooh I Contents Contents

List of TablesVI

List of FiguresVII

ErklärungIX

AbstractX

Deutsche ZusammenfassungXII

1 Introduction1 1.1 Neuromodulation in psychiatric diseases ...... 1 1.2 Major Depression ...... 2 1.2.1 The Neurobiology of Depression ...... 3 1.2.1.1 (Epi-) Genetics and Environmental Factors . . . . .4 1.2.1.2 Monoamine Deficiency Theory ...... 5 1.2.1.3 Stress Hypothesis ...... 6 1.2.1.4 Dysbalanced Neurotrophins and Neurogenesis . . . .7 1.2.1.5 Inflammation Theory ...... 8 1.2.1.6 Gut Microbiota Theory ...... 8 1.2.1.7 Dysregulation of the Reward Circuitry ...... 9 1.2.2 The Neuroanatomy of the Reward Circuitry ...... 12 1.2.3 Therapy for Major Depression ...... 14 1.2.3.1 Pharmacotherapy ...... 15 1.2.3.2 Psychotherapy ...... 17 1.2.3.3 Electroconvulsive Therapy ...... 17 1.2.3.4 Transcranial Magnetic Stimulation ...... 18 1.2.3.5 Vagus Nerve Stimulation ...... 18 1.2.3.6 Deep Brain Stimulation ...... 18 1.3 Animal models of Depression ...... 20 1.3.1 The Chronic Mild Unpredictable Stress Protocol ...... 20 1.3.2 Learned Helplessness ...... 21 1.3.3 Early Life Stress ...... 21 1.3.4 Olfactory Bulbectomy ...... 22 1.3.5 Genetically Modified Rodents ...... 22 1.3.6 Selectively Bred Animals - The Flinder’s Sensitive Line Rat . 24 II Contents

1.3.7 Testing Depressive-like Phenotype ...... 26 1.3.7.1 Forced Swim and Tail Suspension Test ...... 26 1.3.7.2 Open-space Swimming Test ...... 26 1.3.7.3 Sucrose Preference Test ...... 27 1.3.7.4 Others ...... 27 1.4 Optogenetics ...... 28 1.4.1 Microbial Opsins ...... 28 1.4.2 Opsin Targeting Strategies ...... 30 1.4.2.1 Targeting with Viruses ...... 30 1.4.2.2 Projection Targeting ...... 31 1.4.2.3 Transgenic Animal Targeting ...... 31 1.4.2.4 The Long Evans TH::Cre Rat and the Cre/loxP Re- combination System ...... 32 1.4.3 Light Delivery ...... 34 1.4.3.1 Light Requirements ...... 34 1.4.3.2 Light Sources ...... 35 1.4.3.3 Optical Properties of Brain Tissue ...... 36 1.4.4 Validation/Readouts ...... 37 1.4.5 Optogenetics and Major Depression ...... 38

2 Aims 41

3 Materials and Methods 42 3.1 Chemicals and Equipment ...... 42 3.1.1 Solutions for Immunohistochemistry ...... 42 3.1.2 Antibodies ...... 43 3.1.3 Substances Applied to the Animals ...... 43 3.1.4 Viruses for Optogenetics ...... 44 3.1.5 Kits used for Molecular Analysis ...... 44 3.1.6 Primer ...... 45 3.1.7 Mastermix for PCR...... 45 3.2 Animals ...... 46 3.2.1 Breeding and Genotyping of Long EvansTH::Cre Rats . . . . 46 3.3 Establishment of the Virus Injection ...... 48 3.3.1 Stereotactic Surgery - Virus Injection ...... 48 3.3.2 Immunohistochemistry ...... 50 3.3.2.1 Transcardial Perfusion ...... 51 III Contents

3.3.2.2 Immunofluorescent Stainings ...... 51 3.3.2.3 Microscopical Analysis - Epifluorescent Microscopy . 52 3.4 Behavioural Characterization of Long Evans vs. Sprague Dawley Rats 52 3.4.1 Sucrose Preference Test ...... 53 3.4.2 Ultrasonic Vocalization ...... 53 3.4.3 Double-H Maze ...... 53 3.4.4 Forced Swim Test ...... 55 3.4.5 Elevated Plus Maze ...... 56 3.4.6 Open Field Test ...... 56 3.5 Establishment of the Chronic Mild Unpredictable Stress Protocol . . 57 3.5.1 Weight Measurements ...... 59 3.5.2 Corticosterone Measurements ...... 60 3.5.3 Sucrose Preference Test - New Protocol ...... 62 3.5.4 Social Interaction Test ...... 62 3.5.5 Object Recognition Test ...... 64 3.6 Optogenetic Stimulation of the Medial Forebrain Bundle in the Flinder’s Sensitive Line Rat Depression Model - 6-OHDA Lesioned vs. Unle- sioned Rats ...... 64 3.6.1 Stereotactic Surgery - 6-OHDA-Lesion ...... 65 3.6.2 Stereotactic Surgery - Virus Injection and Cannula Implantation 66 3.6.3 Laser Set Up and Light Parameters ...... 67 3.6.4 Behaviour Testing ...... 68 3.6.4.1 Activity measurements ...... 68 3.6.5 Immunohistochemistry ...... 68 3.6.6 Microscopical Analysis ...... 69 3.7 Dopamine-specific Optogenetic Stimulation of the Medial Forebrain Bundle in a Stress-induced Rat Depression Model ...... 70 3.7.1 Stereotactic Surgery - Virus Injection and Cannula Implantation 71 3.7.2 Chronic Mild Unpredictable Stress (CMUS) Protocol . . . . . 71 3.7.3 Behaviour Testing ...... 74 3.7.3.1 Open Space Swimming Test ...... 74 3.7.4 Immunohistochemistry and Microscopy ...... 75 3.8 Final Analysis and Statistics ...... 75

4 Results 76 4.1 Establishment of the Virus Injection ...... 76 IV Contents

4.2 Behavioural Characterization of Long Evans vs. Sprague Dawley Rats 78 4.2.1LE andSD Rats Differ in Weight But Not in Weight Growth Dynamics ...... 78 4.2.2SD Rats Show a Decreased Exploratory Behaviour Compared toLERats ...... 80 4.2.3SD Rats Show Deficits in Spatial Learning Compared toLE Rats ...... 82 4.2.4 No Strain and Gender Differences are Detected in EPM and FST...... 84 4.2.5 Sucrose Preference is Equally Pronounced in Both Strains . . 86 4.2.6LE Males Emit Less Calls in the High Band Compared toSD Males ...... 87 4.3 Establishment of the CMUS Protocol ...... 88 4.3.1 CMUS Had no Significant Effect on the Weight of the Animals 89 4.3.2 Levels of FCM Increased Significantly in Both Groups After CMUS...... 89 4.3.3 CMUS Did Not Have an Effect on the Rats Performance in EPM, SPT,OF, SIT, FST and USV...... 90 4.3.4 Rats That Underwent CMUS Spent Significantly Less Time with Novel Objects ...... 93 4.4 Optogenetic Stimulation of the Medial Forebrain Bundle in the Flinder’s Sensitive Line Rat Depression Model - 6-OHDA Lesioned vs. Unle- sioned Rats ...... 94 4.4.1 Mean Transfection Rate of VTA DA neurons Was Low . . . . 95 4.4.2 Amphetamine-induced Activity Was Decreased in Lesioned Rats 97 4.4.3 Optogenetic Stimulation Increased Homecage-Activity . . . . . 98 4.4.4 Testing of a Depressive-like Phenotype Did Not Show Signi- ficant Differences Between Groups ...... 99 4.4.5 Social Behaviour as Measured in the social interaction test (SIT) Did Not Differ Between Groups ...... 100 4.4.6 No Differences Were Seen in the Animals’ Exploratory Behaviour102 4.5 Dopamine-specific Optogenetic Stimulation of the Medial Forebrain Bundle in a Stress-induced Rat Depression Model ...... 102 4.5.1 The Applied Virus Injection Parameters Led to a Transfection Rate of over 50 % ...... 103 V Contents

4.5.2 The Weight Development in Stressed Animals Was Slightly Decreased ...... 105 4.5.3 Corticosterone Levels Decreased over the Length of the Ex- periment ...... 105 4.5.4 Optogenetic Stimulation Did not Lead to an Increase in Home- Cage-Activity ...... 106 4.5.5 Anxiety-Behaviour was Decreased in dopamine (DA)-Stim An- imals ...... 107 4.5.6 DA-Stim Rats Spent Less Time in the Center Zone of theOF Compared to CTRL Rats ...... 107 4.5.7 Social Behaviour is Increased in DA-Stim Rats ...... 108 4.5.8 Stimulation Rescued the Depressive-like Phenotype in the OSST109 4.5.9 Immobility, Sucrose Consumption and USVs Did Not Diﬀer Between Groups ...... 110

5 Discussion 113 5.1 Establishment of optogenetics ...... 114 5.2 The Long Evans TH::Cre Rat ...... 117 5.3 The CMUS Protocol ...... 118 5.4 Optogenetic Stimulation of the MFB in the FSL Depression Model - 6-OHDA Lesioned vs. Unlesioned Rats ...... 120 5.4.1 The FSL rat ...... 120 5.4.2 Optogenetic Stimulation of the MFB in the FSL Depression Model - Discussion of Results ...... 121 5.5 DA-speciﬁc Optogenetic Stimulation of the MFB in the CMUS-induced Depression Model ...... 123 5.6 Conclusions ...... 129 5.6.1 Potential Mechanism of Action of the Optogenetic Stimulation of the MFB ...... 129 5.7 Outlook ...... 131 5.7.1 Application of Optogenetics in Humans ...... 132

Bibliography 134

Acknowledgements 171

List of Publications 173 VI List of Tables List of Tables

1.1 Diagnostic Criteria for Major Depression ...... 3 1.2 Selectively Bred Depression Models ...... 25 1.3 Viral Promoters in AAVs for Speciﬁc Optogenetic Targeting . . . . . 31 1.4 Optogenetic studies in Depression ...... 40

3.1 Solutions for Histology ...... 42 3.2 Primary Antibodies ...... 43 3.3 Secondary Antibodies ...... 43 3.4 Substances Applied to the Animals ...... 44 3.5 Viruses for Optogenetics ...... 44 3.6 Kits for Molecular Analysis ...... 45 3.7 Primers ...... 45 3.8 Mastermix for PCR...... 45 3.9 Pilot Coordinates ...... 49 3.10 Final Coordinates ...... 50 3.11 Applied Stressors I ...... 57 3.12 CMUS protocol I ...... 58 3.12 CMUS protocol I - continuation ...... 59 3.13 ELISA Parameter ...... 61 3.14 NAc Coordinates ...... 65 3.15 Laser Properties ...... 70 3.16 Scanning Properties ...... 70 3.17 Applied Stressors II ...... 72 3.18 CMUS protocol II ...... 72 3.18 CMUS protocol II - continuation ...... 73 3.18 CMUS protocol II - continuation ...... 74

4.1 LE vs. SD – Summary of Results ...... 79 4.2 CMUS – Summary of Results ...... 88 4.3 6-Hydroxydopamine hydrochloride (6-OHDA)-Stim – Summary of Results ...... 94 4.4 DA-Stim – Summary of Results ...... 103

5.1 Controlled (+) Versus Non-Controlled (–) Potential Eﬀects of Genetic versus Light Control Groups ...... 114 VII List of Figures List of Figures

1.1 Schematic representation of the mesolimbic and mesocortical DAergic pathway...... 13 1.2 Possible genetic modiﬁcations using the Cre recombinase...... 33

3.1 Double-H Maze ...... 54 3.2 Elevated Plus Maze ...... 56 3.3 Social Interaction Test ...... 63 3.4 6-OHDA-Stim Project Design ...... 64 3.5 Laser Set Up ...... 67 3.6 DA-stim Project Design ...... 71

4.1 Virus Injection Site – AAV2-Ef1α-DIO-ChR2-EYFP ...... 77 4.2 Transfected Midbrain DA Neurons – AAV2-Ef1α-DIO-ChR2-EYFP . 78 4.3 LE vs. SD – Weight Development ...... 80 4.4 LE vs. SD – Open Field Test ...... 81 4.5 LE vs. SD – Double H – Latency ...... 82 4.6 LE vs. SD – Double H – Initial Errors ...... 83 4.7 LE vs. SD – Elevated Plus Maze ...... 84 4.8 LE vs. SD – Forced Swim Test ...... 85 4.9 LE vs. SD – Sucrose Preference Test ...... 86 4.10 LE vs. SD – Ultrasonic Vocalisation ...... 87 4.11 CMUS – Physiological Measurements ...... 89 4.12 CMUS – Elevated Plus Maze and Sucrose Preference ...... 90 4.13 CMUS – Open Field Test ...... 91 4.14 CMUS – Social Interaction Test ...... 92 4.15 CMUS – Forced Swim Test ...... 93 4.16 CMUS – Ultrasonic Vocalization and Object Recognition ...... 93 4.17 6-OHDA-Stim – Histology and Ultrasonic Vocalization ...... 95 4.18 6-OHDA-Stim – Evaluation of Transfection Rate ...... 96 4.19 6-OHDA-Stim – Evaluation of Cannula Position ...... 97 4.20 6-OHDA-Stim – Activity Measures ...... 98 4.21 6-OHDA-Stim – Forced Swim Test ...... 99 4.22 6-OHDA-Stim – Sucrose Preference Test ...... 100 4.23 6-OHDA-Stim – Social Interaction Test ...... 101 4.24 6-OHDA-Stim – Open Field Test ...... 102 VIII List of Figures

4.25 DA-Stim – Evaluation of Transfection Rate ...... 104 4.26 DA-Stim – Physiological Measurements ...... 105 4.27 DA-Stim – Activity and Anxiety Measurements ...... 106 4.28 DA-Stim – Open Field Test ...... 107 4.29 DA-Stim – Social Interaction Test ...... 108 4.30 DA-Stim – Open-Space Swimming Test and Ultrasonic Vocalization . 109 4.31 DA-Stim – Forced Swim Test ...... 110 4.32 DA-Stim – Sucrose Preference Test ...... 111 IX Erklärung Erklärung

Ich erkläre hiermit, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Drit- ter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der Quelle gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von Vermittlungs- beziehungsweise Beratungsdiensten (Pro- motionsberater oder anderer Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen. Die Arbeit wurde bisher weder im In- noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt. Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion zur Führung des Doktortitels nicht berechtigt bin.

Frankfurt,

Lisa-Marie Pfeiﬀer X Abstract Abstract

This thesis focused on an optogenetic stimulation strategy to investigate the role of the dopamine system on therapeutic stimulation of the medial forebrain bundle as a potential target for a treatment of Major Depression.

Major Depression is one of the most common psychiatric disorders worldwide. Nev- ertheless, the aetiology of this heterogeneous disorder remains unclear, although advances in understanding the neurobiology behind the disease have been made in the recent years. Several hypothesises are existing, including (epi-) genetic mechanisms, monoamine deficiencies, stress, inflammation and a dysregulation of the reward circuitry. The latter was the focus of this thesis, as one of the key symptoms of Major Depression, anhedonia, is very likely to be reflected by a dysfunctional reward circuit.

Classical treatment for Major Depression is pharmacotherapy with antidepressants, often combined with psychotherapy. A challenge is that approximately 50 - 60 % of the patients are treatment-resistant. Therefore the development of new treatment options was urgently needed. Deep Brain Stimulation, originally developed to treat Parkinson’s disease, showed promising results in clinical trials as a treatment for depression. In accordance with the hypothesis of a dysregulated reward pathway, researchers targeted the cortico-limbic network, stimulating areas like subgenual cingulate cortex and Nucleus accumbens. A more recent trial targeted the supero- lateral branch of the medial forebrain bundle for the stimulation, a brain structure that connects many of the previously targeted stimulation areas. Deep Brain Stimulation of this ﬁbre bundle resulted in a rapid antidepressant response with a high response rate amongst subjects.

The further examination of the stimulation of the medial forebrain bundle is in the spotlight of this thesis. For evaluating specifically the role of the dopamine system, the elegant technique of optogenetics was chosen. Two different rodent models of depression were used, the Flinder’s Sensitive Line rat and the Chronic Mild Unpredict- able Stress protocol, i.e. a stress-induced depression model. The use of optogenetics and a genetically modified rat line to specifically target dopamine neurons, the Long Evans TH::Cre rat, were established in several pilot experiments. Finally two experiments were conducted. First, the photostimulation of the medial forebrain bundle in the Flinder’s Sensitive Line rat was examined. To get a better understanding of the role of the dopamine system, animals with a depleted dopamine system were XI Abstract

compared to animals with an intact dopamine system. In the second experiment, TH::Cre rats were used to specifically target the dopamine neurons with optogenetic stimulation. Again, the medial forebrain bundle was the target area for stimulation. For both experiments, a battery of behavioural tests served as readout. In the first experiment, behaviour tests displayed no significant differences between stimulated and unstimulated rats. But, stimulated rats showed tendencies of increased activity and increased social behaviour. However, due to technical complications, these results would need to be verified in further experiments. In the second experiment, stimulated rats showed a significant decrease in depression- and anxiety-related behaviour, but other tests displayed conflicting results.

The present thesis increases our understanding of stimulation of the medial forebrain bundle, especially giving new insights in the role of the dopamine system in two rodent models of depression. Overall, the medial forebrain bundle seems to be a promising target for a potential treatment of major depression, especially for treatment-resistant patients. The results of this thesis, in particular those of the ﬁnal project, provide important new aspects of the role of the dopamine system in Major Depression. XII Deutsche Zusammenfassung Deutsche Zusammenfassung

Der Schwerpunkt dieser Dissertation lag auf der Untersuchung des Mechanismus der optogenetischen Stimulation des medialen Vorderhirnbündels als potentieller Angriﬀspunkt für eine Behandlung von Unipolarer Depression. Dabei wurde insbesondere untersucht, welchen Einﬂuss das Dopaminsystem auf die Auswirkungen der Stimulation hat.

Unipolare Depression ist eine der häuﬁgsten psychiatrischen Erkrankungen weltweit. Obwohl in den vergangenen Jahren Fortschritte in der Erforschung der Neurobiologie dieser heterogenen Erkrankung erzielt wurden, ist die Ätiologie der Krankheit bis heute unklar. Mehrere Hypothesen zur Entstehung von Depression existieren.Unter anderem werden Veränderungen der (epi-) genetischen Faktoren, Verringerung der Monoaminlevel, Stress, Entzündungen und ein dysfunktionales Belohnungssystem in Betracht gezogen. Letzteres ist der Schwerpunkt dieser Doktorarbeit, da eines der Kernsymptome von Unipolarer Depression, Anhedonie, vermutlich auf eine solche Dysfunktion zurückzuführen ist.

Die Pharmakotherapie mit Antidepressiva, oft kombiniert mit Psychotherapie, ist der klassische Behandlungsansatz für Unipolare Depression. Eine große Herausfor- derung hierbei ist, dass etwa 50 - 60 % der Patienten behandlungsresistent sind. Aufgrund dessen war die Entwicklung neuer Therapiemöglichkeiten dringend er- forderlich. Die Methodik der Tiefen Hirnstimulation wurde ursprünglich zur Be- handlung von Morbus Parkinson entwickelt, zeigte jedoch auch in klinischen Stud- ien zur Behandlung von Depression vielversprechende Ergebnisse. In diesen Studien haben Wissenschaftler, in Übereinstimmung mit der Theorie eines dysregulierten Belohnungssystems, verschiedene Areale des kortiko-limbischen Systems als Stimula- tionsort gewählt, wie etwa den subgenualen cingulären Kortex oder den Nucleus accumbens. Eine kürzlich durchgeführte Studie hat den supero-lateralen Arm des medialen Vorderhirnbündels als Angriﬀspunkt ausgewählt, da diese Faser- struktur viele wichtige Areale des Belohnungssystems verbindet, die in früheren Studien für die Tiefe Hirnstimulation genutzt wurden. Die elektrische Stimulation dieses Faserbündels resultierte in einem schnellen antidepressiven Eﬀekt mit einer hohen Ansprechrate der Studienteilnehmer.

Die Stimulation des medialen Vorderhirnbündels wurde in dieser Doktorarbeit tieferge- hend untersucht. Die elegante Methodik der Optogenetik wurde ausgewählt, um speziﬁsch die Rolle die das Dopaminsystem einnimmt, zu evaluieren. Zwei unter- XIII Deutsche Zusammenfassung

schiedliche Depressionsmodelle im Nager wurden für die Projekte ausgewählt, die "Flinder’s Sensitive" Rattenlinie und ein weiteres Modell, "Chronic Mild Unpredict- able Stress", mit stressiduziertem depressionsähnlichem Phänotyp. Sowohl die Ver- wendung der Optogenetik, als auch einer genetisch modifizierten Rattenlinie, der TH::Cre Ratte, welche die spezifische Stimulation des Dopaminsystems ermöglicht, wurden in mehreren Pilotexperimenten etabliert. Schließlich wurden zwei Hauptex- perimente durchgeführt. Im ersten Experiment wurde die Stimulation des medialen Vorderhirnbündels im "Flinder’s Sensitive Line" Rattenmodell erforscht. Um den Einfluss des Dopaminsystems auf den Stimulationseffekt besser verstehen zu können, wurden Tiere mit und Tiere ohne eine Dopaminläsion des Nucleus accumbens ver- glichen. Im zweiten Experiment wurde die optogenetische Stimulation des medialen Vorderhirnbündels mittels einer dopaminspezifischen Stimulation in TH:Cre Ratten untersucht. In beiden Experimenten wurde eine Reihe Verhaltenstests durchgeführt, um die Auswirkungen der Stimulation sichtbar und messbar zu machen. Die Verhaltenstests in der ersten Studie zeigten keinerlei signifikante Unterschiede zwischen den Gruppen, in stimulierten Ratten konnte jedoch ein Trend zu einer Verbesserung ihres sozialen Verhaltens beobachtet werden. Aufgrund verschiedener experimenteller Schwierigkeiten müssten diese Ergebnisse allerdings in weiter- führenden Experimenten bestätigt werden. In der zweiten Studie konnte in Ratten, die mit optogenetischer Stimulation behan- delt wurden ein robuster und signifikanter Rückgang in depressivem und ängstli- chem Verhalten beobachtet werden. Andere Verhaltenstests zeigten jedoch auch im Widerspruch stehende Ergebnisse.

Die vorliegende Dissertation verbessert unser Verständnis der Stimulation des medialen Vorderhirnbündels und erlaubt neue Einblicke in die Funktion des Dopamin- systems in zwei verschiedenen Depressionsmodellen. Abschließend lässt sich feststel- len, dass das mediale Vorderhirnbündel ein vielversprechender Angriﬀspunkt für die Behandlung von Unipolarer Despression ist, besonders für behandlungsresistente Pa- tienten. Die Ergebnisse dieser Doktorarbeit, besonders die des ﬁnalen Experimentes, bringen interessante neue Aspekte der Rolle des Dopaminsystems in Unipolarer De- pression hervor. XIV Abbreviations

Abbreviations

5-HT serotonin / 5-hydroxytryptamine

5-HTT 5-HT transporter

6-OHDA 6-Hydroxydopamine hydrochloride

AAV adeno-associated virus

ABC avidin-biotin-complex

ACTH adrenocorticotropin

A.D. anno domini

ALIC aterior limb of the internal capsule

AMPA α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

AP anterior-posterior

ATP adenosine triphosphate

BAC bacterial artiﬁcial chromosome

B.C. before christ

BDNF brain-derived neurotrophic factor

BO maximum binding wells

BR bacteriorhodopsin

BS blocking solution

BSA bovine serum albumine

BSL biological safety level

CaMKIIα calcium/calmodulin-dependent protein kinase type II alpha chain

Cg25 subgenuale cingulate cortex / Brodman Area 25

CMUS Chronic Mild Unpredictable Stress XV Abbreviations

CNS central nervous system

ChR channelrhodopsin

CREB cAMP response element-binding protein

CRF corticotrophin-releasing factor

CS carrier solution

CTRL control

CZ center zone

DA dopamine

DAB 3,3’-diaminobenzidine

DAPI 4’,6-diamidino-2-phenylindole

DBS deep brain stimulation

DH double-H maze

DIO double-ﬂoxed inverse open reading frame

DNA deoxyribonucleic acid

DOPAC 3,4-dihydroxyphenylacetic acid

DV dorso-ventral

ECT electroconvulsive therapy

Ef1α eukaryotic translation elongation factor 1 alpha

ELISA enzyme-linked immunoabsorbent assay

EPM elevated plus maze

EYFP enhanced yellow ﬂuorescent protein

FCM fecal corticosterone metabolite

FSL Flinder’s Sensitive Line fSST fugu somatostatin XVI Abbreviations

FST forced swim test

GABA gamma-aminobutyric acid

GECI genetically encoded Ca2+ indicator

GFP green ﬂuorescent protein

HCN hyperpolarization-activated cyclic nucleotide-gated cation

HDRS Hamilton Depression Rating Scale hGFAP human glial ﬁbrillary acidic protein

HPA hypothalamic-pituitary-adrenal

HR halorhodopsin hSynI human synapsin I

ICSS intracranial self-stimulation

IE initial error

IFN-α interferon-α

IL interleukin i.p. intraperitoneal i.v. intravenous

LE Long Evans

LED light-emitting diode

LV lenti virus

MADRS Montgomery-Åsberg Depression Rating Scale

MAO monoamine oxidase

MD Major Depression

MFB medial forebrain bundle

ML medio-lateral XVII Abbreviations

Mrgprd mas-related G-protein-coupled receptor D

MRI magnetic resonance imaging mRNA messenger ribonucleic acid

N north

NA noradrenaline

NAc nucleus accumbens

NE north-east

NMDA N-methyl-d-aspartate

NpHR halorhodopsin from Natronomonas pharaonis n.s. not signiﬁcant

NSB non-speciﬁc binding

NW north-west

OD optical density

OF open ﬁeld test

OR object recognition test

OSST open-space swimming test o.w. one-way

PFA paraformaldehyde

PCR polymerase chain reaction

PBS phosphate–buﬀered saline

PET positron emission tomography

PFC prefrontal cortex

PND post-natal day

RE repetitive error XVIII Abbreviations

REM rapid eye movement rm repeated measurements

RT room temperature

S south s.c. subcutaneous

SD Sprague Dawley

SE south-east

SIT social interaction test slMFB supero-lateral branch of the medial forebrain bundle

SN substantia nigra

SNP single nucleotid polymorphism

SNRI serotonin-noradrenaline reuptake inhibitor

SPT sucrose preference test

SSRI selective serotonin reuptake inhibitor

SW south-west

TA total activity

TAE tris-acetate-EDTA

TCA tricyclic antidepressant tDCS transcranial direct current stimulation

TH tyrosine hydroxylase

TMS transcranial magnetic stimulation

TNF-α tumor necrosis factor-α

TST tail-suspension test

USV ultrasonic vocalization XIX Abbreviations

VGAT vesicular GABA transporter

VGluT2 vesicular glutamate transporter 2

VMAT2 vesicular monoamine transporter 2

VNS vagus nerve stimulation

VTA ventral tegmental area

WT wild-type 1 1 Introduction 1 Introduction

This thesis focused on an optogenetic stimulation strategy to investigate and shed light on the role of the dopamine (DA) system in therapeutic stimulation of the medial forebrain bundle (MFB) as a potential target for a treatment of Major De- pression (MD).

1.1 Neuromodulation in psychiatric diseases

Psychiatric or mental diseases are characterized by alterations in mood, thinking and behaviour. They are a common cause for long-term disabilities worldwide and so represent a great burden for the patients, their families and also the economy (World Health Organization 2008). The therapy of patients suﬀering from psychiatric disorders classically includes pharmacotherapy and / or psychotherapy. However, some of the patients are treatment-resistant to these approaches or develop intolerable side eﬀects. In the attempt to treat patients refractory to treatment, alternatives had to be introduced, and neuromodulation is one of the experimental options (Andrade et al. 2017).

Since ancient times, electricity has been used to modulate the nervous system. Already in 46 A.D., the electric ray was applied to the head of patients suffering from headache and later it was also used to treat gout, haemorrhoids, epilepsy and depression (Kellaway 1946). Big steps of progress were made after Micheal Faraday’s experimental research on electricity started and after he invented an electrical generator in the beginning of the 19th century. In 1870, Fritsch and Hitzig debunked the theory of the "in-excitable" cortex by applying electricity to the head and ob- serving reactions from small eye or arm movements to seizures, depending on the intensity of the current applied (Fritsch and Hitzig 1870). In the early and mid 20th century, patients suffering from psychiatric diseases often underwent ablative surgery and experimental electric stimulation, but due to a lack of documentation and some major side effects, as well as with the rising field of medical ethics, these "psy- chosurgery" programs were almost stopped completely (Schwalb and Hamani 2008). Further progress was made with the invention of stereotactic surgery. In the 1950s, first temporary electrode implantations into various brain regions were described for treating chronic pain and in the 1970s there were first reports of chronic deep brain stimulation (DBS) with electrodes implanted into the thalamus to treat chronic pain 2 1 Introduction

(Schwalb and Hamani 2008). Promising results in treating pain led to the application of DBS in other ﬁelds, especially neuropsychiatric disorders, where it is a promising tool to treat diseases likeMD, obsessive compulsive disorder, tourette syndrome, epilepsy and dementia (Hardenacke et al. 2013; Pepper et al. 2015; Salanova et al. 2015; Schlaepfer and Lieb 2005; Schrock et al. 2015). Besides DBS, there are also other neuromodulation-based therapies that are used in treating psychiatric disorders, including electroconvulsive therapy (ECT), vagus nerve stimulation (VNS), transcranial magnetic stimulation (TMS) and transcranial direct current stimulation (tDCS) (see section 1.2.3).

1.2 Major Depression

The existence ofMD as a medical condition was already known since ancient times. Hippocrates was the ﬁrst to describe a condition named "melancholia" (greek for black bile) in around 400 B.C., but it was not until the mid of the 19th century that the attention for understanding the pathophysiology ofMD shifted to the brain (Nestler et al. 2002a). Today,MD is one of the most common psychiatric disorders worldwide with a lifetime prevalence of 10.8 % between 1994 and 2014 (Lim et al. 2018). The prevalence is 1.5 – 2.5 times higher in women than in men and nearly 50 % of the risk to developMD is contributed by inherited genetic factors (Fava and Kendler 2000). Other risk factors are environmental like stress or emotional trauma and also suﬀering from divers medical conditions like viral infections, Par- kinson’s disease and diabetes increases the risk to developMD (Nestler et al. 2002a). Nevertheless, the aetiology of the disease remains unclear. Several theories on how depression might develop are discussed in the following sections. Depression is diagnosed asMD based on the diagnostic criteria in the "Diagnostic and Statistical Manual" (DSM-V, 2013) with symptoms including depressed mood, low self esteem and feelings of hopelessness, worthlessness and guilt (see Table 1.2 on the following page).

MD is the leading cause for disability in the U.S. and the third-leading cause in Europe (World Health Organization 2017), which makes it a high socio-economic burden, and being the most costly brain disease in Europe (DiLuca and Olesen 2014). An additional problem, besides the high prevalence and the high ﬁnancial burden, is that treatment ofMD is almost entirely symptomatic. Therapy includes amongst others pharmacotherapy with antidepressants, psychotherapy, ECT, VNS, 3 1 Introduction

Table 1.1: Diagnostic Criteria for Major Depression According to DSM-V

Depressed mood Diminished interest and pleasure in activities (anhedonia) Change in weight or appetite Insomnia or hypersomnia Psychomotor agitation or retardation Fatigue or loss of energy Feelings of worthlessness and inappropriate guilt Diminished concentration Recurrent thoughts about death or suicide

MD is diagnosed, when at least 5 of the above symptoms are reported for longer than a 2 week period of time with two of the symptoms being 1 and 2.

DBS and TMS (for a detailed description see section 1.2.3). Problematic concernig pharmacotherapy is, that an estimated 44 % of the patients does not respond to two consecutive therapies with antidepressants and an estimated 33 % does not respond to four (Bergfeld et al. 2018; Rush et al. 2006). Treatment-resistance makesMD even worse, as the suicide risk (to attempt suicide at least once in life) rises from 8.4 – 15.9 % in non-resistant patients (Bernal et al. 2007; Chen and Dilsaver 1996) to approximately 30 % in resistant patients (Hantouche et al. 2010). Thus,MD is a life- threatening disease, causing enormous decreases in life quality of patients and their families as well as a high ﬁnancial socio-economic burden. The lack of understanding the aetiology of the multi-factorial disease and the needed improvement of treatment makes it necessary to shed more light onto this matter.

1.2.1 The Neurobiology of Depression

Although the aetiology ofMD remains unclear, advances in understanding the neurobiology behind the disease have been made in the recent years. Several hypothesises have been formulated on the level of genes (e.g. candidate genes associated with MD, epigenetics), molecules (e.g. growth factors, proinﬂammatory cytokines) and neural circuits (e.g. reward network). Additionally, environmental factors like stress 4 1 Introduction

seem to play an important role and can act as the trigger for a subpopulation of MD patients. A selection of theories is discussed in the following sections.

1.2.1.1 (Epi-) Genetics and Environmental Factors

ForMD a heredity of 37 % was estimated in twin studies (Sullivan et al. 2000), but despite this robust evidence for genetic heredity it was impossible to find the specific genes involved in the disease for a long time. For finding these specific genetic variants, genome-wide association studies compared allele frequencies at millions of single nucleotid polymorphisms (SNPs) across the genome between disease and control groups, but early studies could not find any SNPs that reached the significance threshold (Sullivan et al. 2013). One of the reasons could be thatMD is thought to be a polygenic disease (Sullivan et al. 2013), but recently some genetic factors have been identified, that were more directly implicated inMD. Mullins and Lewis summarize in their review from 2017 five studies that were conducted between 2013 and 2016 and were able to identify significantly altered SNPs inMD (Mullins and Lewis 2017). One study identified two loci on chromosome 10, one near the SIRT1 gene (involved in biogenesis of mitochondria) and one in the LHPP gene (Cai et al. 2015). The Social Science Genetic Association Consortium could identify two additional genes in 2016, the KSR2 (kinase suppressor of ras 2) and the DCC (encodes a transmembrane receptor involved in axon guidance)(Okbay et al. 2016). The genetic testing company 23andMe could detect 17 independent SNPs in 15 regions, but only two of them were significant in meta-analysis and replication sample (Hyde et al. 2016). MEF2c, which codes for a transcription factor that plays a role in learning and memory and NEGR1, which is involved in neurite growth, were identified. For increasing the sample sizes the last two studies were combined, resulting in finding one significant SNP in the FHIT gene. This gene encodes a tumor suppressor protein which is also involved in oxidative stress and the circadian clock (Direk et al. 2017). Thus, genetic factors seem to play an important role, nevertheless, the disease cannot be named a "genetic" disease, as roughly two thirds of the factors are non-genetic. Important seems also to be the interaction between genes and the environment. One example emphasizing the gene-environment-interaction model is the influence of stress on the gene of the 5-HT transporter (5-HTT) on the SLC6A4 gene (Klengel and Binder 2013). The polymorphic 5-HTTLPR region can be either expressed as a long allele (16 repeat units of 22bp) associated with a high transporter function, or a 5 1 Introduction

short allele (14 repeat units of 22bp) associated with low transporter function. Caspi et al. could show in their study, that exposure to stressful life events correlated with an increased risk of depressive episodes or suicidality in short allele carriers, but not in the long allele carriers (Caspi et al. 2003). Thus, the individuals’ susceptibility to environmental stress may vary due to the possible expression of genetic risk factors. Other genes that were examined included BDNF and COMT, being involved in regulating neuroplasticity and neurotransmitter signalling. Anyhow, results have been inconsistent. A potential mechanism explaining the gene-environmental-interaction is epigenetics, as epigenetic processes are able to regulate the activity of deoxyribonucleic acid (DNA), including gene expression. Two well-studied epigenetic mechanisms are histone-acetylation and histone-methylation. Hyperacetylated histones are associated with actively transcribed chromatine regions, whereas hypoacetylated histones are found in transcriptional silent regions (Allfrey et al. 1964). Histone-acetylation seems to be a highly dynamic mechanism with half-lives in the range of minutes (Sun et al. 2013). Histone-methylation can be observed at arginine, lysine and his- tidine residues, and silencing or activation by methylation is depending on the exact residues on the N-terminal tails (Izzo and Schneider 2010). Covington et al. could show that after chronic social defeat stress histone acetylation ﬁrst decreased and then persistantly increased in the nucleus accumbens (NAc) (Covington et al. 2009). In addition, inhibitors of histone deacetylase infused directly into the NAc have a robust antidepressant eﬀect. Regarding histone-methylation inMD, results have been published in a chronic social defeat model (Covington et al. 2011). After this stress protocol, the levels of several histone methyltransferases were decreased in the NAc of susceptible mice, whereas in resilient mice they were upregulated. So highly prob- able, both histone-acetylation and histone-methylation play an important role in the pathophysiology ofMD.

1.2.1.2 Monoamine Deﬁciency Theory

One of the classic theories to explain the aetiology ofMD is the so called monoamine deﬁciency theory, which focuses on alterations in the monoamine neurotranmitters noradrenaline (NA), DA and serotonin / 5-hydroxytryptamine (5-HT). The role of these monoamines was discovered unintentionally in the 1950s from clinical observations that were made when treating schizophrenia and non-psychiatric conditions like tuberculosis and high blood pressure (Krishnan and Nestler 2008). Reserpine, a 6 1 Introduction

natural indole alkaloid, originally used as antipsychotic and antihypertensive drug, induced depressive-like symptoms in a subset of patients. The drug blocks the uptake and storage of DA,NA and 5-HT by inhibiting the vesicular monoamine transporter 2 (VMAT2), which leads to a depletion of the mentioned monoamines. In contrast to that, imipramine and iproniazid, originally designed for treating schizophrenia and tuberculosis, showed a robust antidepressant effect in humans. Imipramine belongs to the so called tricyclic antidepressants (TCAs) with the main mechanisms of strong reuptake inhibition ofNA and 5-HT, which leads to an increased level of these monoamines. Iproniazid, originally a medication for tuberculosis, inhibits the monoamine oxidase (MAO). The MAO normally breaks down all of the three monoamines mentioned above by catalysing their oxidation. When inhibited, the levels of these monoamines increase, which again leads to an antidepressant effect. The majority of today’s antidepressant medications are based on these findings. They either block the reuptake of the monoamines, like selective serotonin reuptake inhibitors (SSRIs), or their metabolism, like the MAO inhibitors. Nevertheless, today it is known that the monoamine deficiency theory does not show the whole picture, as in fact none of the single theories are standing alone. There are some evidences, that the deficiency of monoamines is not the single reason for develop- ingMD and one is, that although the effect of antidepressant drugs on the levels of monoamines acts within hours, the antidepressant effect in patients is not seen until weeks after the beginning of the treatment. When experimentally depleting NA, 5-HT and DA, a moderate decrease in mood could be observed in formerly untreatedMD patients, but no mood alterations were detected in healthy controls (Ruhé et al. 2007). Additionally, a high subset of patients is treatment-resistant to antidepressants. These indications render the monoamine theory overly simplistic and other factors need to be taken into account to explain the aetiology ofMD.

1.2.1.3 Stress Hypothesis

MD is characterized by several alterations in the main neurobiological systems that mediate stress responses. One of them is the neuroendocrine system consisting of the hypothalamic-pituitary-adrenal (HPA) axis, which has shown to be altered inMD patients (Holsboer 2000). The HPA axis is activated by stress, leading to a release of glucocorticoids (cortisol in humans and corticosterone in rodents) into the blood. Once activated, neurons of the paraventricular nucleus of the hypothalamus release the corticotropin-releasing factor, which stimulates the production and secretion of 7 1 Introduction

adrenocorticotropin (ACTH) from the anterior pituitary. ACTH then activates the synthesis and secretion of glucocorticoids from the adrenal cortex. The HPA axis is mediated by several brain regions, including hippocampus and amygdala. A feedback loop is present through glucocorticoids regulating neurons of the hippocampus and the paraventricular nucleus. Regarding the role of the HPA axis inMD, elevated levels of glucocorticoids have been detected in the serum of patients suﬀering from the disease and in rodent experiments, the chronic administration of glucocorticoids induced depression-like symptoms (Raison and Miller 2003). These ﬁndings are supported by clinical observations made from patients with Cushing’s syndrome. Those patients have very high levels of circulating cortisol and show depressive-like symptoms and hippocampal atrophy (McEwen 2007). Other pathological alterations of metabolism that are associated withMD, like insulin resistance and abdominal obesity might also be explained by a hyperactive HPA axis (Brown et al. 2004).

1.2.1.4 Dysbalanced Neurotrophins and Neurogenesis

Brain-derived neurothrophic factor (BDNF) is part of the neurotrophic family of growth factors and is expressed in several brain areas, including hippocampus, and also in other types of tissues (Bathina and Das 2015). It plays an important role in differentiation and maturation of neurons as well as in their survival (Acheson et al. 1995; Binder and Scharfman 2004; Huang and Reichardt 2001). Additionally to being involved in neuroplasticity, BDNF is also implicated in neurogenesis, as it controls the development from neural stem cells to neurons (Zigova et al. 1998). There are several points that speak for a role of BDNF in the aetiology ofMD. First, the time delay of three to four weeks for the classic pharmacological medications such as SSRIs to show efficacy goes hand in hand with the duration of the formation of synaptic connections between new-born neurons. Second, it has been shown that stress results in impaired adult hippocampal neurogenesis (Van Bokhoven et al. 2011) and also decreases BDNF levels. This is supported by findings already made before the research on neurotrophins started, showing that stressful events can lead to damage and atrophy of hippocampal neurons (McEwen 1999). Included in the hippocampal functions is also the regulation of the HPA axis, which is altered inMD, as described above. Additionally, the hippocampus has connections to the amygdala and the prefrontal cortex (PFC), structures that are directly involved in mood and emotion and thus possibly contribute to the symptoms ofMD. Third, in contrast to the action of stress, the treatment with several classes of antidepressants, including 8 1 Introduction

SSRIs and MAO inhibitors, increases the level of BDNF in the hippocampus (Nibuya et al. 1995). Thus, neurogenesis seems to contribute to the eﬀect of antidepressant medication and it should be kept in view concerning the aetiology ofMD.

1.2.1.5 Inﬂammation Theory

The inflammation hypothesis is one of the newest and currently most discussed topics regardingMD. The close interplay between the immune system and the HPA axis supports the idea of a heterogeneous disease and, equally to the HPA axis, the immune system is very sensitive to stressful events. Cytokines are small proteins secreted by cells and can be described as immuno- modulating agents, having an effect on the interaction and communication between cells. Associated withMD are the so called proinflammatory cytokines including interleukin (IL)-1,IL-6, tumor necrosis factor- α (TNF-α) and interferon-α (IFN-α) and IFN-γ. Elevated levels of these cytokines have been found in post-mortem tissue of the frontal cortex of patients withMD (Shelton et al. 2011). People who suffered from severe infections or autoimmune diseases have an increased risk of developping MD (Benros et al. 2013) and patients who are treated withILs or IFN- α against diseases like cancer or hepatitis c often develop depressive symptoms (Myint et al. 2009). Furthermore, experiments in rodents revealed that exposure to chronic stress like forced swim test (FST) and restrain elevated levels ofILs, TNF- α and IFN-γ (Himmerich et al. 2013). When administering cytokines or cytokine-inducers like lipopolysaccharides to rodents, a depressive-like behaviour is induced, becoming apparent in FST and tail-suspension test (TST) (Kaster et al. 2012). Moreover, bone marrow transplants from mice susceptible to stress to stress-naïve mice induces a stress susceptibility in the latter, while transgenic mice lackingIL-6 (IL-6 −/−) show a robust resilience to stress (Hodes et al. 2014).

1.2.1.6 Gut Microbiota Theory

The gut microbiota theory focuses on the connection between brain and gut, the so-called brain-gut axis. This network results from several pathways, including the immune system, the HPA axis and and nerve connections (for example the vagus nerve). Interestingly, there are not only top-down effects (from brain to gut), but also down-top effects: Alterations of the gut microbiota influence the brain and behaviour and an altered brain can change the regulation of the gut and the composition of the 9 1 Introduction

microbiota (Collins and Bercik 2009; Liang et al. 2018). Thus, the capacity of the microbes influencing directly the HPA axis and the immune system, both systems that are altered inMD, links it to having a potential role in the pathophysiology of the disease. Additionally, patients suffering fromMD often develop symptoms related to dysfunction of the brain-gut axis, like appetite disturbance, gastrointestinal disorders and alterations in the gut microbiome (Evrensel and Ceylan 2015; Jiang et al. 2015). Jiang and colleagues could show, that the diversity and richness of the gut microbiota declined in patients and that this effect was significantly different from healthy controls. Animal studies supported these findings and confirmed differences in the microbiome in several models ofMD, including olfactory bulbectomy, maternal separation and chronic restraint (Liang et al. 2015; O’Mahony et al. 2009; Park et al. 2013). Furthermore, transplantation of the fecal microbiota of depressed patients to either germ free or microbiota-depleted rodents resulted in both cases in an increase in depressive-like behaviour, such as anxiety and anhedonia (Kelly et al. 2016; Zheng et al. 2016). The treatment with probiotics including Lactobacillus ca- sei, Lactobacillus helveticus, and Bifidobacterium bifidum alleviates depression and depression-like symptoms both in animal and in double-blind, randomized, placebo- controlled human studies (Liang et al. 2015; Wallace and Milev 2017).

1.2.1.7 Dysregulation of the Reward Circuitry

The last component being discussed here, that seems to play an important role in the pathophysiology ofMD, is the dysregulation of the reward circuitry. Modulation of this circuit is in the focus of this thesis and is an important approach to ﬁnd new therapy options forMD, especially for treatment-resistant patients. As mentioned before,MD cannot be seen as a single disease, but more as a syndrome covering a spectrum of symptoms arising from diﬀerent areas of the brain and their networks. Classically, research focused on the serotonergic andNAergic systems and the areas of frontal cortex and hippocampus received most attention hereby (Dranovsky and Hen 2006). Nevertheless, it is unlikely that those areas account for all of the symptoms ofMD, as the hippocampus is associated with declarative memory and spatial learning and the frontal areas of the cortex mainly with working memory, impulse control and attention (Nestler et al. 2002a). Emotional and mood regulatory symptoms are not explained by these conventional targets, so the focus of interest has expanded to neural circuits involved in emotional behaviour. Abnormalities inMD were found in several brain areas, including thalamus, amygdala, striatum, hypo- 10 1 Introduction

thalamus and brainstem (Drevets 2001; Klimek et al. 2002; Mayberg 2003). One hallmark ofMD is anhedonia, the reduced ability to experience reward or pleasure and further symptoms are loss of motivation and abnormalities in vegetative functions, such as appetite, sleep circadian rhythm and energy level. Studies from drug addiction identified the NAc and its DA input from the ventral tegmental area (VTA) as the neural substrate for drug reward as well as for natural rewards like food, social interactions and sex (Koob and Le Moal 2001; Wise 1998). Also, the lateral habenula and the amygdala play a role in aversive and reward oriented behaviour, as it is crucial for learned associations linking environmental cues to aversive/rewarding stimuli (Davis and Whalen 2001; Namboodiri et al. 2016). Of course, the hypothalamus is known for its implication in vegetative functions, as the ones described above that are disturbed inMD, but additionally, this brain area seems also to contribute to reward. This is due to several neuropeptides being expressed in hypothalamic neurons and regulating eating behaviour, and these peptides have been shown to be implicated in reward as response to drugs (Fulton 2000). The ventromedial and lateral nuclei of the hypothalamus, the amygdala, the lateral habenula, the VTA and the NAc are all interconnected via the MFB. The anatomy of this system is described in detail in Section 1.2.2 on page 12. Passing through the MFB, is the VTA to NAc or mesolimbic pathway, which is thought to be dysregulated in anhedonia (Russo and Nestler 2013). This is supported by clinical findings that show a reduced activity in the NAc of depressed patients (Mayberg et al. 2000) and the limbic DA transmission is also altered in animal models like the Flinder’s Sensitive Line (FSL) rat (Friedman et al. 2005). Chronic stress is one of the causes for developingMD in humans and it is also used for generating a depressive-like phenotype in rodent models of the disease. Such stress models, including chronic restraint and social defeat increase the spontaneous and burst firing of VTA DA neurons in vivo and ex vivo in a brain slice (Anstrom and Woodward 2005; Cao et al. 2010; Krishnan et al. 2007). In social defeat experiments, only mice susceptible to stress showed these alterations in electrophysiology, resilient mice did not have alterations. Furthermore, the changes in the firing properties of VTA DA neurons were reversible by chronic administration of SSRIs (Cao et al. 2010). VTA DA neurons show two in vivo patterns of firing, low frequency tonic and high frequency phasic firing, whereby the phasic firing codes for reward (Grace et al. 2007). Two optogenetic studies support the regulation of depressive-like behaviour by DA midbrain neurons. Chaudhury and colleagues published that optogenetic activation of phasic firing of VTA DA neurons elicited mice with a depressive-like phenotype 11 1 Introduction

and an increased susceptibility to stress (Chaudhury et al. 2013). On the contrary, Tye and colleagues showed that optogenetic activation of the same neurons reversed a depressive-like phenotype induced by stress, whereas specific inhibition of VTA DA neurons induced a depressive-like phenotype (Tye et al. 2013). Both studies could show, that DA release in the terminals in the NAc was crucial for the effects. To explain these conflicting results, it should be considered that the groups used different stress models, chronic stress versus social defeat. Additionally, the timing of the optogenetic stimulation differed, as in one experiment it occurred during the stress protocol, while in the other experiment the stimulation took place during the behavioural testing. Finally, different types of VTA DA neurons might have been activated, as the VTA contains several subpopulations of DA neurons, depending on the exact location (core / medial or lateral shell) (Lammel et al. 2011). However, both studies underline a potential implication of alterations in the neural encoding of the limbic circuitry inMD. The mesolimbic and mesocortical DA pathways together with the MFB, which they pass, are also the neural substrate for the so called SEEKING system, one of at least seven primary process emotional systems, also including FEAR, LUST, GRIEF, PAIN, RAGE, CARE and PLAY (Alcaro and Panksepp 2011; Panksepp 2010). The SEEKING system is overlapping and very similar, but not identical to the reward system. It goes beyond the latter, as it regulates (i) overt behavioural responses such as exploration, seeking and approaching, (ii) memory and cognition, more specific the reinforcement of associative learning, activation of contextual memories and an- ticipatory predictions and (iii) positive affective feelings in a reward state (Alcaro and Panksepp 2011). Two of the key symptoms ofMD, anhedonia and hopelessness or helplessness, can be explained by a hypo-activity of the SEEKING system and modulating this system might be a promising target for further therapeutics (Alcaro and Panksepp 2011). It has already been described in clinical studies, that DBS of the supero-lateral MFB or the NAc evokes eagerness feelings and desire (Coenen et al. 2011; Schlaepfer et al. 2008). Furthermore, these human trials with bilateral DBS of the supero-lateral MFB showed promising results with significantly reduced symptoms in otherwise treatment-resistantMD patients (Bewernick et al. 2017; Schlaepfer et al. 2013). 12 1 Introduction

1.2.2 The Neuroanatomy of the Reward Circuitry

First steps in recognizing the neuroanatomy of the reward system were made with developing the intracranial self-stimulation (ICSS) experiments in the 1950s (Olds and Milner 1954). The experiments led to the discovery of several brain areas that, when activated, produce rewarding effects, and increase the likelihood of repetition of a given operant behaviour that was associated with the stimulation. In some ICSS studies, the rats even ignored aversive shocks or neglected biological needs over the self-stimulation and pressed levers until physical exhaustion (Olds 1958; Valenstein and Beer 1962). At first, the repetitive behaviour of the animals was thought to be due to a sensation of pleasure and satisfaction, but during other experiments it was seen, that the ICSS also evoked other behaviour like exploration and arousal. Therefore, it was suggested that the stimulation reinforces the drive to approach environmental stimuli, but rather in a motivational context than in an energizing context (Glickman and Schiff 1967; Trowill et al. 1969). Especially two sites being involved in reward processes were discovered, the septal areas being more linked to sexual pleasure and the lateral hypothalamus and MFB regions being linked to incentive motivation (Alcaro and Panksepp 2011). Lesion studies then revealed, that no single structure is the neural substrate for reward, but that several brain areas and their network are implicated (Ikemoto 2010). For recognizing rewards and for initiating their consumption, especially the VTA to NAc or mesolimbic pathway has been shown to be crucial (Koob and Le Moal 2008). For a schematic representation of the pathway see Figure 1.1.

Besides the NAc, the VTA DA neurons innervate also other brain regions that count for the reward system, including several areas of the PFC, the central and basolateral amygdala and the hippocampus and all of these regions are interconnected, also via the MFB. The NAc receives glutamatergic input from the PFC, amygdala and hippocampus, while PFC, amygdala and hippocampus form reciprocal glutamatergic connections amongst one another. Several types of GABAergic and cholinergic (in the NAc) interneurons modulate the functional output of the reward system and all of the regions also receive serotonergic as well asNAergic input from the midbrain raphe nuclei and the pontine locus coeruleus, respectively. Via neuropeptides, also the hypothalamus innervates the reward brain areas. In the NAc, DA is released by DAergic cells that originating from the A10 area, with the majority in the VTA. The NAc can be divided into two major sub-areas, the shell and the core, having diﬀerent connections. The core has eﬀerent projections to the dorsolateral 13 1 Introduction

Figure 1.1: Schematic representation of the mesolimbic and mesocortical DAergic pathway. Schematic representation of the mesolimbic and mesocortical neural pathway (green circles) in a rodent brain in sagittal plane. DAergic projections are shown in green, 5-HT projections in red. The DAergic projections originate from the VTA and pass through the MFB (black-bordered box) to brain regions including the PFC, hippocampus and lateral habenula. Grey-shaded boxes show brain areas that have been targeted in pre-clinical studies as treatment options for MD, addiciton or obsessive compulsive disorder (OB: olfactory bulb, OT: olfactory tubercle, vmPFC: ventromedial prefrontal cortex, NAC: nucleus accumbens, CC: corpus callosum, Hipp: hippocampus, DMT: dorso-medial thalamus, LHb: lateral habenula, DRN: dorsal raphe nucleus, CB: cerebellum, VTA: ventral tegmental area, SNr: substantia nigra pars reticulata, LH: lateral hypothalamus, MFB: medial forebrain bundle). Figure courtesy of Prof. Dr. med. Volker A. Coenen, reprinted from Döbrössy et al. 2015, with permission from Elsevier. ventral pallidum, the entopeduncular nucleus, the lateral part of the VTA and the substantia nigra (SN), while the shells’ efferents project to the ventromedial ventral pallidum, amygdala, lateral hypothalamus, entopeduncular nucleus, VTA,SN pars compacta, lateral preoptic area, lateral habenula, mesopontine reticular formation and the periaqueductal grey. The NAc receives afferent input from forebrain regions, including medial PFC, amygdala, hippocampus and thalamus as well as from mesopontine areas including VTA, dorsal raphe, and mesopontine reticular formation. The VTA receives afferent projections from several forebrain regions, including the PFC, NAc, bed nucleus of the stria terminalis, diagonal band of Broca, substantia innominata, lateral preoptic area, and lateral hypothalamus. Addition- ally, there are brainstem connections to the VTA from the superior colliculus,SN, dorsal raphe, parabrachial nucleus and the dentate nucleus of the cerebellum (Ike- moto and Panksepp 1999). In the VTA, not only DAergic neurons are found, but also GABAergic and glutamatergic neurons. The majority though is the DAergic population with around 60 % (Margolis et al. 2006), GABAergic neurons make up 14 1 Introduction

25 % (Margolis et al. 2012) and approximately 15 % are supposed to be glutamatergic (Yamaguchi et al. 2007). Furthermore, research has shown that VTA DA neurons are capable of co-releasing GABA and also glutamate, which contributes to their function. The co-release of GABA happens via the VMAT2, being independent of the presence of the vesicular GABA transporter (VGAT) (Tritsch et al. 2012). The DAergic neurons seem to be able to co-release glutamate because many catecholaminergic neurons, including VTA DA neurons express the vesicular glutamate transporter 2 (VGluT2) (Hnasko et al. 2010). Historically, the population of DAergic neurons in the VTA was seen as homogeneous, but electrophysiological recordings led to confusing results. One prominent characteristic to identify DA neurons in slice recordings is the so called Ih current, the hyperpolarization-activated current generated by hyperpolarization-activated cyclic nucleotide-gated cation (HCN)-channels.

This Ih current is not present in all VTA DA neurons (Zhang et al. 2010) and besides it has been shown that some of the neurons do not react to application of DA (Lammel et al. 2008), or that they are smaller then usual (Zhang et al. 2010). More recent studies then began to classify the neurons by their projections (Ike- moto 2007; Lammel et al. 2008; Margolis et al. 2006). The VTA DA neurons with classic electrophysiological properties including Ih-current project to the NAc lateral shell and are mainly present in lateral posterior and anterior VTA, whereas the "unconventional" DAergic neurons innervate the medial shell and core of the NAc, the medial PFC and the basolateral amygdala. They are located in medial posterior VTA (Lammel et al. 2014). The projections from VTA DA neurons to limbic and cortical regions are running through a huge ﬁber bundle, the MFB (Wise 2005), making the bundle a promising target for treatment ofMD via modulation of the reward system (Döbrössy et al. 2015).

1.2.3 Therapy for Major Depression

Therapies for MD are as various, as are the theories for its aetiology. Classic treatment is pharmacotherapy with antidepressants like MAO inhibitors and SSRIs. A problem is that 50 – 60 % of patients are treatment-resistant, which is deﬁned as a not adequate response to at least one antidepressant drug (McIntyre et al. 2014). For those patients, treatment options include high doses of antidepressants, combination of several antidepressants or combination with other pharmacological agents, combination with psychotherapy and ECT. Other emerging therapy options are TMS, DBS and VNS. 15 1 Introduction

1.2.3.1 Pharmacotherapy

Classic antidepressant therapies focus on the three monoaminergic neurotransmitter systems 5-HT, DA andNA. They include MAO inhibitors, cyclic antidepressants, SSRIs and serotonin-noradrenaline reuptake inhibitor (SNRI)s. Cyclic antidepressants are named after their chemical structure containing three (tricyclic) or four (tetracyclic) rings of atoms. They block the reuaptake of 5-HT andNA by binding to their transporter and so increasing their levels in the synaptic cleft. Especially tricyclic antidepressants used to be the gold standard treatment after their discovery in the 1950s, but today they are no longer the first choice due to high rates of antich- olinergic and antihistaminergic side effects, possible cardiotoxicity and seizures and due to their lethality at ten times or even less of the daily dose (Block and Nemer- off 2014; Holtzheimer and Nemeroff 2006). Similar to tricyclic antidepressants, also SSRIs and SNRIs inhibit the reuptake of either selectively 5-HT or of both 5-HT andNA by blocking their transporters. Paradoxically, it takes weeks until the full antidepressant response is reached, thus the biochemical changes in the neurotransmitters cannot be the whole explanation. Already in the 1980s, studies suggested, that it is rather an adaptation of the monoamine system that contributes to the effect, by downregulating receptors such as 5-HT2 and β-adrenoreceptor (Ögren et al. 1985). Alternative ideas emerged in the 1990s, focusing on a desensitization of 5-HT autoreceptors following antidepressant treatment (Blier and Montigny 1994). This receptor desensitization was thought to lead to a maintained increase in 5-HT levels in the synaptic cleft and so to the antidepressant effect. More recent evidence suggests that the monoamine feedback system might also play a role in the effect of the drugs. 5-HT neurons are feedback controlled via postsynaptic 5-HT receptors, including 5-HT2c-receptors. Mongeau and colleagues have shown that both restraint stress and agonists of this receptor type increase anxiety in social interaction tests (SITs) in mice and that this effect is prevented by the SSRI paroxetine (Mongeau et al. 2010). Furthermore, it has been proposed, that antidepressant medication leads to adaptations in the downstream signalling cascades. There are several genes being altered by the drugs, they increase for example BDNF levels and cAMP response element- binding protein (CREB), a transcription factor, both playing an important role in neuroplasticity (Lindefors et al. 1995; Thome et al. 2000). MAO inhibitors also el- evate the levels of monoamines in the synaptic cleft, by inhibiting the enzyme being responsible for degrading 5-HT, DA andNA. Disadvantages of the MAO inhibitors 16 1 Introduction

are severe drug-drug interactions, the need for a tyramine-free diet and lethality in overdose. Another heterogeneous group of drugs, the so called atypical antidepressants, also act on the monoamine neurotransmitter system. It includes 5-HT, DA andNA reuptake inhibitors, substances that increaseNA release and blockers / antagonists of different 5-HT-receptor subtypes. Besides the classic antidepressant drugs that act on the monoamine neurotransmitter system, there are also drugs acting on other targets inMD. Another neurotransmitter being targeted is glutamate, being present in over 80 % of the neurons and binding to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-d-aspartate (NMDA) and kainate receptors. In studies using proton magnetic resonance spectroscopy, it has been found that glutamate and glutamine levels were decreased in several brain areas such as the PFC, anterior cingulate cortex, amygdala and hippocampus in patients suffering fromMD (Yük- sel and Öngür 2010). Glutamate is also implicated in neurogenesis, being necessary for the development of dendritic branching, linking alterations in the hippocampal neurogenesis inMD to possible changes in the glutamate neurotransmitter system (Gorman and Docherty 2010). The substance (pre-) clinical trials were focusing on when targeting the glutamatergic system is ketamine, an NMDA receptor antagonist, that was originally developed and used as an anaesthetic. Murrough and colleagues showed in a two-site, randomized controlled trial a significant, rapidly on-setting response ofMD patients to a single, sub-anaesthetic, intravenous (i.v.) infusion of ketamine, that lasted up to seven days (Murrough et al. 2013). Other studies inMD and as well in bipolar disorder confirmed the effect of the drug. Due to severe side effects including liver and kidney damage and due to the high risk of abuse, also other NMDA receptor antagonists are under examination as potential drugs targeting the glutamatergic neurotransmitter system inMD (Mathews et al. 2012). Other potential pharmacological medication target the hyperactive HPA axis and try to reduce the inMD elevated levels of corticotropin releasing factor and cortisol. Therefore, several antiglucocorticoides, including mifepristone, an antagonist of the glucocorticoid receptor, have been studied and significant reduction of psychotic symptoms could be seen in psychotic depression (Block et al. 2018). As discussed above, also inflammatory cytokines seem to be linked toMD, so that anti-inflammatory agents have been suggested as a novel therapy strategy. Inhibi- tion of TNF-α with blockers and antagonists has been examined in animal models 17 1 Introduction

and human studies. Rats treated with a TNF-α antagonist displayed decreased immobility in a rat model of despair (Bayramgürler et al. 2013) and humans treated with a TNF-α antibody increased their Hamilton Depression Rating Scale (HDRS) score signiﬁcantly compared to controls, when comparing patients with high baseline values in biomarkers for inﬂammation with controls with low baseline levels (Raison et al. 2013). The last group of pharmaceuticals being discussed here is the one of the so called atypical antipsychotics, with lithium being the most prominent agent. Classical atypical antipsychotics target the DA2-receptor and the newer generations also target the

5-HT1A- and 5-HT2-receptors. Already in the 1980s, lithium was successfully used as an augmenting agent for patients that failed to respond to tricyclic antidepressants alone and also meta-analysis of several placebo-controlled studies conﬁrmed the eﬀectiveness of the drug as an augmentation agent (Bauer et al. 2003).

1.2.3.2 Psychotherapy

Psychotherapy includes several forms of therapy such as interpersonal therapy, cognitive-behavioural therapy, behavioural activation therapy, psychodynamic therapy, mindfulness-based therapy and problem-solving therapy. Especially cognitive- behavioural and interpersonal therapy have been shown to be effective in multiple controlled MD trials (Beck 2005; Feijo De Mello et al. 2005). Interpersonal therapy helps patients to identify and solve problems in relationships and social roles, while cognitive-behavioural therapy teaches them to identify their negative thinking patterns contributing to the disease and to challenge and replace them with positive, more accurate thoughts (Otte et al. 2016). Especially the latter mentioned therapy is also effective in treatment-resistant patients and also the combination of psychotherapy and antidepressant medication might be more effective than the respective ones alone (Friedman et al. 2004).

1.2.3.3 Electroconvulsive Therapy

ECT induces a seizure in the patient during a short anaesthesia and is thought to be the most effective, non-pharmacological treatment forMD until today (The UK ECT Review Group 2003). Right unilateral ECT seems to be as effective as bilateral treatment, but the latter shows a faster onset (The UK ECT Review Group 2003). Because of its fast onset, ECT is the first-line treatment for severe cases ofMD with 18 1 Introduction

high suicidal risk. Problematic side eﬀects concern cognitive performance of the patients such as anterograde and retrograde amnesia and another issue of ECT is the high rate of relapse, especially in formerly treatment-resistant patients (Sackeim et al. 2001).

1.2.3.4 Transcranial Magnetic Stimulation

TMS is the less invasive stimulation method being used for treatment ofMD. An electromagnetic coil is placed on the scalp and an electromagnetic field, which passes through the skull to the cortex, is applied. For treatingMD, repetitive TMS is used, applying multiple pulses rapidly to the dorsolateral PFC (Holtzheimer and Nemeroff 2006). The parameter of frequency can vary widely and TMS inMD is typically divided into high frequency with pulses above 5 Hz and low frequency with pulses under 1 Hz. Meta-analysis confirmed effectiveness of this treatment both as monotherapy and augmentation therapy (Berlim et al. 2013). Problematic is the lack of standardization in the TMS procedure, with wide varying frequencies, number of pulses and number of treatments (Block and Nemeroff 2014).

1.2.3.5 Vagus Nerve Stimulation

VNS was already used in the 1980s for treating epilepsy. The surgery includes the placement of a pacemaker in the chest which is connected to a stimulation electrode being wrapped around the vagus nerve. The nerve projects directly and indirectly to dorsal raphe, locus coeruleus, amygdala, hypothalamus, and cortical regions (Nem- eroff et al. 2006). The exact mechanism of action is still unclear, but several studies suggest that the effect comes from alterations in the monoamine system and in hippocampal neurogenesis (Rizvi et al. 2011). However, data from clinical studies is conflicting and there is a lack of randomized controlled trials to prove efficacy (Block and Nemeroff 2014).

1.2.3.6 Deep Brain Stimulation

As many patients do not respond to conventional therapies such as antidepressants, psychotherapy and ECT, there is an urgent need for the development of new treatment options forMD. DBS was originally developed for treating Parkinson’s 19 1 Introduction

disease and involves the magnetic resonance imaging (MRI) and electrophysiologic- ally guided, stereotactic implantation of electrodes, uni- or bilaterally to the target region. The electrodes are connected to an implanted neurostimulator, which electrically stimulates the targeted brain area (Schlaepfer and Lieb 2005). After the network model forMD came to the fore, the attention of researchers and clinicians was shifted towards the stimulation of the cortico-limbic network (Mayberg 1997), to hopefully generate more complete and appropriate treatment options. A ﬁrst target region built on an observation in patients suﬀering from obsessive compulsive disorder, which were stimulated in the ventral striatum / aterior limb of the internal capsule (ALIC), showing improvements in mood (Schlaepfer et al. 2010). A clinical study with 15 treatment-resistantMD patients resulted from this and a response rate of 40 % was reported after six months (Malone et al. 2009). As Mayberg and colleagues demonstrated a prominent role of the subgenuale cingulate cortex /

Brodman Area 25 (Cg25) in mediating the cortico-limbic networking, it was chosen as target for another trial (Mayberg 1997). A trial with 20 treatment-resistant patients resulted in a response rate of 60 % after six months that was sustained at one year (Lozano et al. 2008). A third target region is the NAc, which is thought to play an important role in mediating the anhedonic symptoms inMD (Schlaepfer et al. 2008). A study with 10 patients could show a response rate of 50 % after one year (Bewernick et al. 2010; Schlaepfer et al. 2008). This study originally was planned to be double-blind and sham-controlled, but the patients did not tolerate off-phases due to severe worsening of the symptoms. However, first larger clinical trials including a placebo control stimulating the Cg25 and the ALIC failed to prove efficacy (Dougherty et al. 2015; Morishita et al. 2014). A more recent clinical trial stimulated the supero-lateral branch of the medial forebrain bundle (slMFB), a bundle that projects to and interacts with all of the previously chosen targets for DBS in

MD, the NAc, the Cg25 and the ALIC (Coenen et al. 2011; Schlaepfer et al. 2013). Many structures shown in the pre-clinical, experimental studies to be implicated in reward or SEEKING arousal are inter-connected via the slMFB, which projects mainly to the ALIC, ventral striatum and NAc (Coenen et al. 2011). The stimulation of the slMFB in the clinical study resulted in a rapid antidepressant response with a response rate of 85 % after three months (Schlaepfer et al. 2013). Recently, long-term results of this DBS study have been published. After 12 months, six of eight patients (75 %) were responders (reduction of 50 % in the Montgomery-Åsberg Depression Rating Scale (MADRS)) and four patients reached remission (MADRS < 10) (Bewernick et al. 2017). The results remained stable with only few side-eﬀects, 20 1 Introduction

such as strabismus, until four years after the onset, rendering the slMFB an attract- ive target for future studies to treat treatment-resistantMD patients. Since the ﬁrst cohort, Coenen and Schlaepfer have continued the trial with new cohorts, including a group of 16 patients (Coenen et al. 2019), and they have recently started implant- ing a novel group of 50 patients. Additionally, the safety and eﬃcacy of MFB DBS has been supported by several preclinical rodent studies, showing improvements in stress coping and cognitive behaviours (Döbrössy et al. 2015; Thiele et al. 2018).

1.3 Animal models of Depression

A wide variety of animal models forMD is available to date, though a "complete" depression model is non-existing, due toMD not being a single-cause disease but more a heterogeneous syndrome. Additionally, some of the key symptoms like sad- ness, guilt and suicidality cannot be modelled in animals. The available models rely mostly on three principles: genetically modiﬁed or selectively bred animals, actions of known antidepressants and responses to stress. A selection of the models is described in the following sections.

1.3.1 The Chronic Mild Unpredictable Stress Protocol

The Chronic Mild Unpredictable Stress (CMUS) protocoll was developed in the late 1980s and is a model that reflects one of the core symptoms ofMD, anhedonia. It is thought to be relatively realistic, as stress increases the risk for developingMD in humans (Kessler 1997). The procedure is based on an observation, that showed that rats reduce their intake of sweet fluids when being exposed to stressors over a pro- longed period of time, possibly by decreasing the drive of their reward system (Katz 1981, 1982). Willner and colleagues modified the protocol, decreasing the intensity of the stressors for ethical reasons and to render the protocol more realistic, compared to daily stress in humans (Willner et al. 1987). They showed that after several weeks of CMUS during which the animals were exposed to a series of unpredictable stressors lasting few hours every day, the rats reduced their intake and preference of sucrose and that this was reversible by treatment with tricyclic antidepressants. Gen- erally, the protocol’s duration varies from several weeks to months and mild stressors alternate every few hours. Typical stressors are disruption of the light-dark-cycle, 21 1 Introduction

food and water deprivation, tilted cages, restraint, changing cagemates and soiled or wet bedding. For behavioural readout, the sucrose preference test (SPT) is used together with other tests for depressive-like behaviour, like FST and SIT. A caveat concerning this model is that no consistent and standardised protocol is used, the protocol varies between the laboratories. Also, the protocol of the main readout, the SPT diﬀers between research groups. Additionally, the protocol is labour intensive, demanding of space and of long duration. On the other hand, the chronicity of it is one of it’s strengths. Another established stress model is the chronic social defeat model. Here, rodents are exposed to physical and non-physical cues from aggressive conspeciﬁcs. The model is well established, but a disadvantage is that it can only be used in male animals.

1.3.2 Learned Helplessness

Like CMUS and social defeat, the learned helplessness model also belongs to the environmental stress models ofMD. The model is based on the observation that when rodents are exposed to inescapable and uncontrolled stress factors such as electric foot shocks, they develop a helplessness, so that when re-exposed to the stressors but now with an easy escape option, the animals will either show an increased escape latency or completely fail to escape (Seligman and Maier 1967). Other experiments revealed that rats developed persistent alterations following one or more foot shock sessions, including weight loss, in HPA axis activity, sleep patterns and loss of spine synapses in the hippocampus (Nestler et al. 2002b). The latency to escape and the number of animals that learn this helplessness is reduced by antidepressants and ECT (Nestler et al. 2002b). The learned helplessness model is ethically problematic as it includes extreme regimens of stress and it is discussed whether the procedure might not be a better model for post-traumatic stress disorder.

1.3.3 Early Life Stress

Also included in the stress models ofMD are the early life stress models, such as prenatal stress, early post-natal handling and maternal separation (Stepanichev et al. 2014). Early life stress reﬂects the early life neglect or loss of parents in humans. Restraint of pregnant rat dams in the last week of pregnancy increases depressive- like behaviour in the male oﬀspring (Morley-Fletcher et al. 2003). Early handling 22 1 Introduction

has been suggested as a model for resilience to stress, as the adult animals display an attenuated response of the HPA axis to stress, reduced emotional arousal and an increased level of glucocorticoid receptors in the PFC and hippocampus (Levine 1957; Meaney et al. 1988). In contrast to that, early maternal separation results in animals being more vulnerable to developing a depressive-like syndrome. Rats showed for example an increased immobility in the FST, corticosterone levels were elevated and HPA axis activity was higher in response to stress (Daniels et al. 2009; Kikusui and Mori 2009; Kuramochi and Nakamura 2009). The models are generally well reproducible and many of the induced symptoms are reversible by treatment with antidepressants (El Khoury et al. 2006).

1.3.4 Olfactory Bulbectomy

An injury model ofMD is the olfactory bulbectomy. The surgery results in behavioural, endocrine, neurotransmitter and immune system changes, which are also seen in the human disease (Hellweg et al. 2007). The olfactory system of rodents is part of the limbic region that also includes the hippocampus and the amygdala. These regions, implicated in emotion, are altered inMD. The bulbectomy results not only in a degeneration of neurons in the olfactory bulbs, amygdala and hippocampus, but also in cortex, locus coeruleus and in the raphe nuclei, leading to dysfunctions in 5-HT andNA neurotransmitter systems (Jancsàr and Leonard 1984). The most prominent change in rodent behaviour following an olfactory bulbectomy is an hyperactive response in a brightly illuminated open ﬁeld, which is reversed by chronic, but not acute treatment with antidepressants (Kelly et al. 1997).

1.3.5 Genetically Modiﬁed Rodents

Traditional genetic mouse models focus on monoamines or silence or overexpress candidate genes, that are thought to contribute to the pathophysiology ofMD. Examples are the 5-HT andNA transporter or receptor genes. Since SSRIs block the 5-HTT, it’s gene is one of the traditional candidate genes. Several laboratories developed 5-HTT knockout mice, with Bengel and colleagues being the ﬁrst ones (Bengel et al. 1998). An antidepressive phenotype following the knockout of the 5-HTT seems to be dependent on the background of the used mouse lines. A knockout in 129S6/SvEv mice resulted in an increase in FST immobility, but a decrease in TST immobility 23 1 Introduction

(Lira et al. 2003). In contrast to that, in C57Bl/6 mice immobility was increased in both TST and FST (Wellman et al. 2007; Zhao et al. 2006). Interesting here is the opposing effect of blocking the 5-HTT with antidepressants versus the knockout of the gene, receiving either anti- or prodepressive-like phenotypes. Especially, as SNRIs and knockout of theNA transporter both result in an antidepressant effect (Perona et al. 2008). This might be explained by the different influence of 5-HTT and NA transporter in brain development. When SSRIs and SNRIs are administered to mice shortly after their birth, mimicking the life-long blockade of a knockout, mice receiving SSRIs showed persistent behavioural changes, whereas SNRIs treatment did not show life-long changes (Ansorge et al. 2008). Thus, the life-long alterations after SSRI treatment might lead to (over-) compensatory mechanisms, which might help to explain the opposing effects of a persistant knockout of the 5-HTT versus a SSRI treatment during adulthood (Cowen and Editors 2013). Beneath theNA transporter, also adrenoreceptors are targeted for creating knockout mice. When tested for depressive-like phenotype, knockout of α2A adrenoreceptors resulted in an increase in immobility in the FST, mediated by a decreased climbing, but no changes in swimming (Schramm et al. 2001). In contrast to that, α2C knockout mice displayed a decreased immobility in the FST (Sallinen et al. 1999).

Additionally, the role of α1 adrenoreceptors has been examined, with α1A knockout showing decreased immobility in the FST and TST and the α1B knockout showing opposing eﬀects with an increase in immobility in both tests (Doze et al. 2009). Genetic models that do not target the monoamine system include mice heterozygous for the vesicular glutamate transporter 1 gene, which show depressive-like behaviour and neurochemical changes associated with depression and anxiety (Tordera et al. 2007). Furthermore, mice with a disruption of the gene coding for the cannabinoid 1 receptor exhibit a depressive-like phenotype with a reduced responsivness to reward (Maldonado et al. 2006). As alterations in HPA axis function also seem to play a role in the aetiology ofMD, other genetic mouse models focus on knocking out the genes for corticotrophin- releasing factor (CRF) receptors. CRF1 and CRF2 receptors seem to have opposing roles, as a knockout of CRF1 leads to an decrease in anxiety-like behaviour (Smith et al. 1998), whereas a knockout of CRF2 leads to an increase in anxiety-like behaviour as well as in immobility during the FST (Bale and Vale 2003). Another candidate gene is the one encoding for BDNF, as the neurotrophic factor is also implicated in the pathophysiology ofMD, as described in previous sections. 24 1 Introduction

Assessment of behaviour was done in heterozygote BDNF knockout mice, as ho- mozygote knockout was shown to be perinatally lethal. These mice show normal baseline behaviour with no or only subtle effects in emotional behaviour including FST (Chourbaji et al. 2004). However, mice overexpressing BDNF show an antidepressant behavior in the FST, but also increased anxiety during the elevated plus maze (EPM) (Govindarajan et al. 2006). Finally, there is one genetic rat model existing, a 5-HTT knockout rat (Wistar SLC6A4 ). Both hetero- and homozygous knockouts have been used in studies. A study comparing the homozygous knockout to the wild-type found significant differences in anxiety / locomotor behaviour, exploratory pattern (immobility), cocaine- induced locomotion and subcutaneous and abdominal fat (Homberg et al. 2010). A more recent work examined gene / environment interactions inMD and combined the hetero- and homozygous 5-HTT knockout with early life stress. Especially in the homozygous knockout rat the combination with the stress paradigm affected DNA methylation of the CRF gene promotor in the central nucleus of the amygdala. Additionally, they showed that this DNA methylation correlated with the level of CRF messenger ribonucleic acid (mRNA) in the amygdala and that this level again correlated with stress coping behaviour of the rats in a learned helplessness task (Doelen et al. 2015).

1.3.6 Selectively Bred Animals - The Flinder’s Sensitive Line Rat

There are several selectively bredMD models existing in rodents, they are summed up in Table 1.2. These rodents are bred for depressogenic and/or anxiogenic behaviour, such as increased immobility in the FST. Additionally, they have several physiological depressive-like features like elevated rapid eye movement (REM) sleep and altered 5-HT system function and they respond to, or at least to a certain de- gree, antidepressant medication. This section focuses on the description of the FSL model, as it is one of the models used in this work.

Originally, it was not intended to create a model for MD with the FSL rat, but to create a rat strain being resistant to the anticholinesterase agent diisopropyl ﬂuoro- phosphate. However, the selective breeding program did not result in a resistant rat strain, but in a strain that became more and more sensitive to diisopropyl ﬂuoro- phosphate and other cholinergic agonists and having elevated muscarinic receptor levels (Overstreet et al. 1984). When describing the features of the rat strain in 25 1 Introduction

Table 1.2: Selectively Bred Depression Models Model Species Reference FSL Rat Overstreet 1986 Wistar-Kyoto Rat Lahmame et al. 1997 Swim Low-Active Rat West and Weiss 1998 Low Reaction to Stress Mouse Touma et al. 2008 Inbred Learned Helplessness Rat Vollmayr et al. 2001 Fawn Hooded Rat Rezvani et al. 2007 the 1980s it was noticed that they resemble human depressive symptoms, including increased REM sleep, greater hormonal response to a cholinergic agonist challenge and increased immobility in the FST (Overstreet et al. 1988). Other key behavioural deficits are reduced appetite and weight, increased passive response to stress and increased aggression (Overstreet and Wegener 2013). The validity of the FSL model is also increased by expressing several physiological features that are also associated with clinicalMD, including reduced 5-HT synthesis (Hasegawa et al. 2006), reduced baseline levels of 5-HT, DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the NAc (Yadid et al. 2001), decreased BDNF levels in the hippocampus (Neumann et al. 2011), altered neuropeptide Y levels (Jiménez-Vasquez et al. 2000) and a hyperactive response of the HPA axis (Zambello et al. 2008). FSL rats do not show anhedonia-like behaviour in the SPT under basal conditions, but the sucrose preference compared to controls is decreased when they are exposed to a CMUS paradigm prior to the SPT (Pucilowski et al. 1993). A more recent study by Thiele and colleagues indicated that certain behaviour changes are transient with time / age of the animal, such as anxious behaviour in the EPM and immobility in the FST, whereas other impaired traits, like memory performance in the double- H maze (DH), seemed to be stable (Thiele et al. 2016). Additionally, a positron emission tomography (PET) scan revealed a significant, bilateral hypo-metabolism in the temporal lobes as well as possible alterations in the entorhinal cortex metabolism. Finally, DBS of the MFB in this rodent depression model seems to affect certain types of behaviour, including speed and precision of memory recall in the DH. However, other behaviours like ultrasonic vocalization (USV) and exploration remained unaltered (Thiele et al. 2018). 26 1 Introduction

1.3.7 Testing Depressive-like Phenotype

In the following sections is brieﬂy described, how the above mentioned animal models can be behaviourally tested for their depressive-like phenotype.

1.3.7.1 Forced Swim and Tail Suspension Test

One of the most frequently used tests to investigate the efficacy of new antidepressant drugs is the FST, which was first used by Porsolt and colleagues (Porsolt et al. 1977). The test is based on the effect of behavioural despair, with the animal being exposed to an inescapable situation, swimming in a cylinder without exit possibilities. The behavioural despair manifests in the rodent being immobile, so floating on the water instead of swimming, struggling or climbing the walls. Animals with a depressive- like phenotype exhibit an increased immobility compared to healthy controls. This immobility is reduced by antidepressant treatment. Similar to the FST, the TST measures the animals behavioural despair (Steru et al. 1985). The test is designed for mice, as rats are too heavy to be lifted at the tail. The test design comprises a mouse that is suspended by the tail from a lever for typically 6 min. The movement is recorded and distinguished in immobility and agitation. Antidepressant drugs, such as imipramine and desipramine, decrease the immobility during the test.

1.3.7.2 Open-space Swimming Test

The open-space swimming test (OSST) tracks the animals’ swimming (or immobility) behaviour in a big water pool over four trials à 15 min on four consecutive days. It was introduced in the early 2000s as an alternative for the FST, claiming to have several advantages over the latter. First, results of the OSST are more objective compared to those of the FST, as scoring is not done manually by the researcher, but a tracking software is used. This improves reproducibility across laboratories. Second, the test in a water pool does not limit the animals’ movement due to space restriction, as it does in the cylinder during the FST. Hence, the OSST might mimic the human disease better, displaying lack of motivation rather than lack of space, that restricts the movement. Animals with a depressive-like phenotype swim less / ﬂoat more, thus the measured tracklength is shorter. This immobility is reduced by 27 1 Introduction

tricyclic and tetracyclic antidepressants, MAO inhibitors and SSRIs (Sun and Alkon 2003).

1.3.7.3 Sucrose Preference Test

The SPT mimics another key feature ofMD, namely anhedonia (Katz 1982). The intake of a sucrose solution compared to water is measured in this test. Animals with a depressive-like phenotype, for example following a CMUS protocol, decrease their intake of sucrose compared to healthy controls, indicating a disturbance in the reward system. The deﬁcit is reversible by antidepressant medication (Willner et al. 1987).

1.3.7.4 Others

A stress reaction in rodents can also be measured and evaluated using the open field test (OF) and the EPM (Hall 1934; Handley and Mithani 1984). Both tests display exploration versus anxiety-like behaviour by measuring the time the animal spends exploring (open arms in the EPM / central zone in theOF) or showing anxious behaviour (closed arms in the EPM, corners in theOF). Furthermore, the emotional state of rodents is reflected by their vocalization, and this can be measured by equipement sensitive to ultrasonic vocalization. Negative or aversive behaviour is associated with calls in lower frequency bands around 20 kHz, whereas excitement and rewarding behaviour is reflected in higher frequency bands, ranging from 40 – 60 kHz (Burgdorf et al. 2000; Furlanetti et al. 2016; Sadananda et al. 2008). Moreover, inMD, the social behaviour can be impaired. It is possible to measure the social behaviour of rodents using the three chamber SIT (Kaidanovich-Beilin et al. 2011). The test measures the social behaviour towards familiar and strange animals and animals with a depressive-like phenotype, for example the FSL rats, show an altered social behaviour compared to controls (Cook et al. 2017). Cognitive symptoms such as memory deficits are also common inMD (Lee et al. 2012). These can be measured in rodents using several behaviour tests, including DH and object recognition test (OR). TheDH is a water-escape based learning and memory test and measures time and errors the animal needs / makes until finding the escape platform in the maze (Pol-Bodetto et al. 2011). Rats with a depressive- like phenotype, like the FSL rats, show robust and time stable deficits in theDH 28 1 Introduction

(Thiele et al. 2016). TheOR tests the rodents’ ability to distinguish between familiar and novel objects (Berlyne 1950). Here again, FSL rats show a signiﬁcantly altered behaviour when examining the novel object compared to control animals, indicating again deﬁcits in learning and memory (Thiele et al. 2016).

1.4 Optogenetics

To understand the pathophysiology of diseases likeMD, it is important to recognize which neurocircuits play a role, how they function and how they are altered in disease. Prior to the invention of optogenetics, no technique achieved the high temporal and cellular precision needed to examine those circuits in mammalian neural tissue (Fenno et al. 2011). Already in the late 1970s, Francis Crick found that the major challenge to understand the brain would require "a method by which all neurons of just one type could be inactivated, leaving the others more or less unaltered" (Crick 1979, p.222), which cannot be achieved by the use of electrodes for example. Per definition, optogenetics is the combination of optical and genetic methods to cause or inhibit defined events in distinct cell types in vitro and in behaving animals (De- isseroth 2011). For using this technology, three key features are needed: (i) microbial opsins, light-sensitive proteins originating from algae or archaebacteria, (ii) methods for targeting distinct cell populations and for genetically engineering them with the opsins and (iii) methods for guiding sufficient strong and precise light to the regions of interest in the brain, that contain the genetically engineered neurons (Deisseroth 2011).

1.4.1 Microbial Opsins

Microbial opsins come from microbes that developed such light-sensitive proteins in the absence of eyes. For halobacteria living in a highly saline environment they help for example to maintain the organisms’ osmotic balance (Stoeckenius 1985). These opsin genes are divided into two families, the microbial opsins (type I), which are found in archae, bacteria, algae and fungi and the animal opsins (type II), present in higher eukaryotes. Opsin proteins from both families require the vitamin-A-related organic cofactor retinal to absorb photons. When retinal is bound covalently to the opsin protein, the functional protein is called rhodopsin. Type II opsins encode G 29 1 Introduction

protein-coupled receptors that bind retinal in the 11-cis configuration, which upon illumination isomerize to the all-trans configuration. In contrast to that, type I opsins usually encode for proteins that use the retinal in the all-trans configuration, which, after photon absorption, photoisomerizes to the 13-cis configuration. The step after photoisomerization also differs between the two families of opsins. In the type II family, the linkage of opsin and retinal hydrolyses and the free all-trans retinal is transported out of the cell, being replaced by a fresh 11-cis retinal (Hofmann et al. 2009). In the type I family, the retinal does not dissociate from the opsin, but is thermally turned back into all-trans retinal (Haupts et al. 1997). The unique nature of microbial opsins is the combined ability of light sensation and ion flux in one gene (Fenno et al. 2011). The three most studied classes of microbial opsins are bacteriorhodopsin (BR), halorhodopsin (HR) and channelrhodopsin (ChR). The haloarcheal proton pumpBR was first identified by Oesterhelt and Stoeckenius (Oes- terhelt and Stoeckenius 1971). The pump is expressed in the membrane under low oxygen conditions and transports protons from the intracellular cytoplasm to the extracellular medium. The result is a proton-motive force to drive adenosine triphosphate (ATP) synthesis (Racker and Stoeckenius 1974). The second class of microbial opsins, theHRs, are light-activated chloride-pumps, which were first discovered in archaebacteria (Matsuno-Yagi and Mukohata 1977). Their function comprises of pumping chloride ions from the extracellular to the intracellular space. The today mainly usedHR for inhibition comes from Natronomonas pharaonis (NpHR, La- nyi and Oesterhelt 1982). The third class of microbial opsins encodes for ChR and comes from the green algae chlamydomonas reinhardtii. ChR1 was first discovered by Nagel and colleagues in 2002 (Nagel 2002) and is a proton-pump, being highly homologous toBR. ChR2 was discovered shortly after that (Nagel et al. 2003) and differs from ChR1 with being not only a proton-pump, but also being permeable for cations (Feldbauer et al. 2009). Proof of concept of ChR2 being able to be expressed stably and safely in mammalian neurons and to drive the neurons depolarization upon light-stimulation succeeded Boyden and colleagues in 2005 in cultured rat hippocampal neurons (Boyden et al. 2005). The ChR used in the work of this thesis is a ChR2 with a H134R mutation. This ChR2H134R is also maximally excited at 470 nm and differs from the normal ChR2 in improving currents 2-fold, but also slowing down channel-closure kinetics by 2-fold, which makes the excitation faster but less temporally precise (Gradinaru et al. 2007). 30 1 Introduction

1.4.2 Opsin Targeting Strategies

For the use of optogentic stimulation in in vivo behaving animals, a toolbox is needed to deliver the opsin to a speciﬁc target region in the brain. To achieve this, several targeting strategies can be used, including i) viral (promoter) targeting, ii) projection targeting and iii) transgenic animal targeting. Furthermore it is also possible to combine these methods.

1.4.2.1 Targeting with Viruses

The most popular way to introduce opsins into neural systems is via viral vectors. The advantages of this way are a fast implementation and a robust expression due to high infection rates (Fenno et al. 2011). For introducing opsins into the brain of rodents and primates, especially adeno-associated viral and lentiviral vectors have been used (Zhang et al. 2010). The use of both of these vectors result in a stable and long-term expression in the central nervous system (CNS) (Mohanty and Lak- shminarayananan 2015). They differ in their cloning capacity, as lenti viruss (LVs) can take up up to 10 kb compared to adeno-associated viruss (AAVs) with a maximum packaging capacity of 5 kb. It is possible to produce very high titers of AAV that result in larger transduced tissue volumes compared to LV (Yizhar et al. 2011). Furthermore, AAV vectors do not integrate into the genome compared to LV, which does integrate, which leads to AAVs being considered safer and being rated with biological safety level (BSL) 1, whereas LVs are rated with BSL 2+. Due to those advantages and because the limited packaging capacity was sufficient for this work, the AAV vector was chosen as delivering option. Of the AAVs, more than 100 variants have been identified so far, differing in their tropism and also their diffusion behaviour in the tissue. Following injection into CNS tissue, the serotypes 1, 2, 5, 7, 8, 9 and rh. 10 all show a strong neural tropism (Castle et al. 2016). Of these serotypes, AAV2 mediates the less widespread gene expression (Burger et al. 2004), which is why it was chosen for this work, as a very local gene expression in the VTA without lateral spreading to theSN was needed. Additionally the cell-type specificity of the viral vector is dependent on the used promoter. Examples of different promoters and their tropism is shown in Table 1.3 on the following page. For this work human synapsin I (hSynI) with its panneural tropism at higher titers was used for wild-type (WT) animals and eukaryotic translation elongation factor 1 alpha (Ef1α) for the Cre/loxP recombination system (described in detail in Section 1.4.2.4 on the 31 1 Introduction

next page), having been designed for recombinase-dependent opsin expression (Kim et al. 2017).

Table 1.3: Viral Promoters in AAVs for Speciﬁc Optogenetic Targeting Promoter Size Cell-type Speciﬁcity Reference CaMKIIα 1.3 Kb Excitatory CaMKIIα neurons in Tye et al. 2011 cortex, amygdala hSynI 0.5 Kb Panneural, but tropism for inhib- Nathanson et al. 2009 itory neurons at low titers Ef1α 1.2 Kb Strong, general promoter Kim et al. 2017 fSST 2.6 Kb Inhibitory neurons Nathanson et al. 2009 hGFAP 2.2 Kb Astrocytes Lawlor et al. 2009

1.4.2.2 Projection Targeting

Microbial opsins are not only expressed in cell bodies, but a strong expression can also be observed in the cells’ dendrites and axons (Gradinaru et al. 2010), creating light-sensitive projections. This property is opening up the possibility of transducting the cell bodies in one brain area and illuminating their projections in another (Tye et al. 2011). Like this distinct cell populations, defined by their anatomical connections, can be addressed. Another but very similar approach for projection-specific targeting uses viruses that either can infect axon terminals or are able to travel retrogradely along the projections. Viruses with these properties are for example rabies and herpes simplex virus (Callaway 2008). This allows to examine distinct roles of neurons of the same type, being located in the same area, differing by their connections to other brain areas.

1.4.2.3 Transgenic Animal Targeting

Difficulties like exact targeting of brain areas with stereotactic injections and limitations in packaging capacity of viruses can be overcome with the use of transgenic animals. The microbial opsin is expressed in distinct, genetically defined populations of neurons of the transgenic animals. Methods to develop those include (i) plasmid transgenic approaches, (ii) bacterial artificial chromosome (BAC) and (iii) knock-in approaches (Ting and Feng 2013). An example for a transgenic mouse strain that 32 1 Introduction

was developed using the plasmid transgenic approach is the Thy1.2-ChR2-EYFP, of which the lines 9 and 18 seemed to be the most broadly useful ones (Ting and Feng 2013). Both lines show a robust expression of ChR2 in the pyramidal cells of the cortical layer V, CA1 and CA3 pyramidal neurons of the hippocampus and in several nuclei of the midbrain, thalamus and brainstem. Optogenetic experiments using these transgenic mice for example brought new ﬁndings on cortical circuit mapping (Wang et al. 2007) and cortical information processing (Sohal et al. 2009). An example for a transgenic line produced with BAC is the VGluT2-ChR2(H134R)- EYFP mouse. It expresses ChR2 in the glutamatergic neurons of the hindbrain and spinal cord. It could be shown that optogenetic stimulation of these neurons in the lumbar region of the spinal cord is suﬃcient to drive a locomotor output (Hägglund et al. 2010). A knock-in mouse strain example is the mas-related G-protein-coupled receptor D (Mrgprd)-ChR2(H134R)-Venus, which is used to research the spinal cord connectivity of Mrgprd-expressing neurons. Mrgprd is a marker for non-peptidergic spinal cord sensory neurons that transmit polymodal pain information (Wang and Zylka 2009).

1.4.2.4 The Long Evans TH::Cre Rat and the Cre/loxP Recombination System

Another possibility for specific cell targeting is the combined use of transgenic recombinase driver lines and the injection of recombinase-dependent viruses, which will be explained here based on the used Cre/loxP recombination system in Long Evans (LE) tyrosine hydroxylase (TH)::Cre rats. By using the Cre (for "causes recombination") recombinase, which originally was isolated from the bacteriophage P1, it is possible to induce genetic recombination of specific sequences that are flanked by the so called loxP (for "locus of cross-over P1") recognition sites. The system was first used in in vitro experiments by Sauer and Henderson in the late 1980s (Sauer and Henderson 1988) and the first demonstra- tion of its’ functionality in the mouse succeeded in the early nineties (Lakso et al. 1992). As shown in Figure 1.2 on the following page the Cre recombinase mediates different genetic modifications depending on the orientation of the recognition sites and the number of different sequences involved. Having the loxP sites pointing into the same direction causes an excision, when they are flanking a sequence on the same chromosome. Having the recognition sites on two different chromosomes either 33 1 Introduction

causes an insertion or a translocation of the sequence of interest. When the sequence of interest is ﬂanked by two bidirectional recognition sites, an inversion is caused.

Figure 1.2: Possible genetic modifications using the Cre recombinase. (A) When sequence X is flanked by two recognition sites pointing into the same direction, an excision is caused by the Cre recombinase. (B) A single recognition site in the genome can be used as an acceptor for inserting a gene of interest, which is also combined with a recognition site. (C) Placing single recognition sites on different chromosomes can cause interchromosomal translocation. (D) Bidirectional recognition sites are required for an inversion of sequence X. Scissors represent the Cre recombinase and the blue triangles the recognition sites (loxP). Sequence X is the sequence of interest, e.g. ChR2.

For the DA-specific optogenetic stimulation in this work, a Cre-dependent AAV2 containing the opsin ChR2 in reverse orientation (double-floxed inverse open reading frame (DIO)) under an Ef1α promoter was used. To flip the opsin sequence to the right and readable orientation, it was injected intoLE TH::Cre rats. This recombinase driver line was generated by Witten and colleagues using the BAC approach (Witten et al. 2011). An injection of a Cre-dependent viruses into DA regions, e.g. VTA orSN; orNA regions, e.g. locus coeruleus of this rat strain results in a highly specific expression of the chosen opsin in catecholaminergic neurons (Witten et al. 2011). In the case of an injection into the VTA, as in the present work, the expression of the ChR2 is limited to the TH-positive neurons and their processes and projections, allowing a DA-specific optogenetic stimulation. 34 1 Introduction

1.4.3 Light Delivery

After having chosen the right microbial opsin and targeting strategy for the optogenetic project, the last challenge that has to be met is the light delivery. The options vary widely and are dependent on the type of experiment that is planned. To choose the right light delivering option one has to take into account three major factors of light control: (i) spatial, (ii) temporal and (iii) spectral (Yizhar et al. 2011). The considerations for the light delivery are discussed in the following subsections.

1.4.3.1 Light Requirements

First and most important, the right wavelength has to be chosen, to be able to maximally excite the opsin. For ChR2 the peak activation wavelength is about 473 nm. The next important step is to choose the appropriate temporal parameters for the experiment. Pulse frequency and duration, duty cycle and epoch patterns are crucial parameters to select. For example, inhibitory pumps like NpHR are usually driven by continuous light, whereas in contrast the activation of ChR2 requires well-separated pulses of light. For ChR2, also pauses of minimally 5 s between the light-on epochs are important, as the opsin inactivates during sustained light exposure and needs to recover in the dark state (Boyden et al. 2005). With modifying the pulse frequency, it can be controlled how the neurons are excited. It has been shown for example that high-frequency stimulation of the VTA with brief pulses of light induces phasic patterns of DA release in the NAc, whereas low-frequency stimulation evokes a tonic DA release pattern (Bass et al. 2013). The intensity of the delivered light has to be chosen carefully and is dependent on the type of experiment (e.g. in vitro versus in vivo, including the readout (e.g. electrophysiology versus behaviour testing), the light delivery method and the properties of the brain tissue in the target area). For deep targets like the MFB in the present experiments, usually fibre optics are used to deliver the light to the target area. Here it has to be taken into account that parameters like the numerical aperture and the diameter of the fibre influence the spread of light and therefore its’ intensity. For optogenetics it is more convenient to report the light power density (usually measured in mW mm−2) than the total light power in Watts. For in vitro activation of ChR2 a light power density of 8 –12 mW mm−2 is needed (Boyden et al. 2005), but for in vivo experiments higher values of around 20 mW mm−2 (measured 0.5 mm below the tip of the fibre) have been used (Tye et al. 2013). Many research groups, like Tye and colleagues, state 35 1 Introduction

the light power density measured not directly at the tip of the fibre but measured 0.5 mm below the tip of the fibre, taking into account the optical properties of the brain tissue, to get a more realistic value of the intensity the neurons are activated with below the fibre. The optical properties of the brain tissue and how to calculate the light spread in it are described in more detail in the following Section 1.4.3.3 on the next page.

1.4.3.2 Light Sources

In surface targets that can easily be accessed with light, e.g. cultured neurons or brain slices, the light is usually delivered via a microscope illumination path. For deep brain targets, fibre optics are typically coupled to either lasers or light-emitting diodes (LEDs). As already mentioned, important for the decision are characteristics including high power, high temporal precision and sharp spectral tuning. Especially the last point is important, as some opsins get inactivated by wavelengths near their activation peak (Berndt et al. 2009) and sharp tuning is also critical in multiple- opsin experiments. Here, lasers have a clear advantage over LEDs, as they have a tighter spectral bandwith (< 1 nm versus 10 - 50 nm at half maximum). The other huge advantage of using lasers is the very efficient coupling between light source and fibre, which allows efficient illumination of the target area despite the loss of power due to brain tissue, connections between fibres, the use of rotary joints or even the splitting of beams. In contrast, the poor light-fibre coupling in LEDs makes it difficult to obtain a light power high enough to drive behavioural responses in in vivo experiments. A disadvantage in using lasers is that lasers of some wavelengths (e.g. yellow) are not able to produce pulses in a millisecond timescale (Warden et al. 2014). Using LEDs as a light source has some advantages, as this light source is much less expensive compared to lasers (approximately 300 € versus 1000 – 10,000 €). Additionally, LEDs are a lot more flexible in their configuration, they can be coupled to fibre optics for deep brain targets, similar to lasers, but they can also be used wireless with mounting the LED directly above the skull or even with inserting miniature diodes directly into the brain (Kim et al. 2013). Though, a disadvantage of using them directly on or even in the brain is the resulting local heat. Moreover, compared to lasers, LEDs are smaller, more stable and more reliable (Warden et al. 2014). Nevertheless, for the present work the focus lay on obtaining a power high enough for in vivo experiments using a split beam for bilateral optogenetic 36 1 Introduction

stimulation with a behavioural readout, which is why a laser was chosen as light source.

1.4.3.3 Optical Properties of Brain Tissue

Light propagation in biological tissue is aﬀected by three photophysical processes:

1. refraction,

2. scattering and

3. absorption.

The refraction index describes the linear optical properties of a tissue. When a light wave propagates through a tissue type A with a distinct refraction index, which bor- ders on a tissue type B with another refraction index, the light wave is redirected (refraction or reflection). Scattering in biological tissue depends on the morphology, the size and the structure of the components, like cell organelles. A source of scattering can be the mismatch- ing refraction indices of these organelles and the surrounding cytoplasm. Absorption is a process, where part of the energy of the light wave is extracted by molecular species. This shift from one energy level to another is called transition. The most common situation of dissipating the lights’ energy in a biological tissue is a combination of two processes, (i) optical dissipation by emitting a photon (e.g. fluorescence) and (ii) nonradiatively dissipation via kinetc energy (e.g. heating of the surrounding medium, Vo-Dinh 2003). Another factor that influences the transmission of light through a tissue is the wavelength, with longer wavelengths scattering less and therefore propagating deeper into the tissue (Yizhar et al. 2011). To be able to estimate the light power density in the tissue at a certain point below the fibre tip, several research groups performed measurements in blocks of unfixed brain tissue for a range of thicknesses (Aravanis et al. 2007; Yizhar et al. 2011). The resulting data was fit to mathematical standard equations for modelling light propagation in scattering medium (Kubelka-Munk model, Vo-Dinh 2003). Comparison of the results with electrophysiological measurements and immunohistological methods including c-fos staining validated the data (Aravanis et al. 2007; Gradinaru et al. 2009; Tye et al. 2011). Based on the collected data, an online calculator1 was generated that estimates the light power density as a

1https://web.stanford.edu/group/dlab/cgi-bin/graph/chart.php 37 1 Introduction

function of depth in tissue. Here the user can enter wavelength, numerical aperture of the fibre, fibre core radius and the light power measured at the tip of the fibre and then gets estimated the irradiance in mW mm−2 as a function of depth in mm.

1.4.4 Validation/Readouts

Optogenetics has been shown to be compatible with various readout methods. The three primarily used, (i) behavioural readout, (ii) electrical readout and (iii) optical readout are discussed in this section. Behavioural readouts in optogenetic experiments have been used widely and in a broad range of animal models, from worms over fish to rodents and primates (for a review see Fenno et al. 2011). A lot of the tests can be performed using fibre optics and changes in behaviour could be observed in tests like the EPM, sucrose or ethanol drinking behaviour or locomotion/rotation in models includingMD, parkinson’s disease and addiction (Bass et al. 2013; Gradinaru et al. 2009; Tye et al. 2013). Newer wireless technology allows the animals to move more freely and undisturbed, rendering the behavioural readout even more precise (Berndt et al. 2009; Kim et al. 2013). If the research focus is more on the neurological circuits level, electrical recordings or imaging techniques are more suitable. Patch-clamp electrophysiology is the key methodology to measure single cell activity like synaptic input and spiking in high-speed, but the method is limited in linking these results with the actual in vivo behaviour of the animal (Kim et al. 2017). This disadvantage can be overcome by combining electrophysiology with optogenetics in in vivo experiments. Like this, circuit physiology effects can be linked to the input of a specific cell type. Using this approach shed more light onto behaviours and their underlying circuits including reward (Stuber et al. 2011), anxiety (Padilla-Coreano et al. 2016) and food consumption (Wu et al. 2015). The very fast readout that can be achieved by combining optogenetics with electrophysiology also enables the possibility of closed-loop optogenetic interventions. Paz and colleagues used this elegant approach to optogenetically silence seizure activity in a rodent model of epilepsy after having detected the initiation of the seizure using electrophysiological recordings in thalamus and cortex of the animal (Paz et al. 2013). Patch-clamp electrophysiology might be the gold-standard for the recording of single- cell activity, but it is not suitable for a global activity readout. For investigating the activity of a local or even global neuron populations, functional MRI has been used 38 1 Introduction

for a long time and has also been combined with optogenetics (Lee et al. 2010). The obvious limitations are the slow temporal dynamics and the incompatibility with freely moving behaviour testing. A better solution might be oﬀered by the advances made in Ca2+ imaging with using so called genetically encoded Ca2+ indicators (GECIs). This technique allows to record the activity of hundreds of genetically deﬁned neurons on a local or even global level following optogenetic stimulation (Akerboom et al. 2013).

1.4.5 Optogenetics and Major Depression

A search for "optogenetics" and "Major Depression" in the PubMed database 2 reveals only 32 entries. Combining the two terms with "dopamine" results in 12 items (search performed on 30.06.2019). A summary of the publications (excluding reviews and studies regarding synaptic depression) is listed in Table 1.4 on page 40. Since 2012, depression in general, pain- and stress-induced depression,MD and treatment-resistant depression were in focus of optogenetic research. Optogenetic stimulation of the medial PFC via its’ projection in the dorsal raphe nucleus showed an increase in mobility in the FST, while stimulation via its’ projections in the lateral habenula showed the opposite effect on the escape-related behaviour during the FST (Warden et al. 2012). Pain-induced depression has been investigated using a sciatic nerve lesion model in mice (Barthas et al. 2015). Using optogenetic stimulation, a critical role for the anterior cingulate cortex in this context was revealed. Specifically treatment-resistant depression has been looked at once using optogenetic stimulation in a model named negative cognitive state rat (Clemm von Hohenberg et al. 2018). Using functional MRI as a readout a decrease in the default-mode network could be observed following optogenetic inhibition of the lateral habenula. Stress-induced depression models were used four times, twice a CMUS paradigm, once chronic social defeat and once chronic immobilization stress. Tye and colleagues could show that specific DA stimulation or silencing in the VTA directly seems to influence depressive-like behaviour in a stress model ofMD (Tye et al. 2013). In another study, optogenetic alteration of NAc medium spiny neurons provoked changes in the depressive-like behaviour in a rat model of social defeat (Francis et al. 2015). Another very recent study regarding stress-induced depression had a specific look into the role of the 5-HT system (Zhang et al. 2018). An optogenetic stimulation

2https://www.ncbi.nlm.nih.gov/pubmed/ 39 1 Introduction

of the dorsal raphe-lateral habenula projections alleviated depressive-like symptoms in the CMUS model, whereas optogenetic inhibition of these projections induced depressive-like symptoms. The last study using a stress induced depression model exposed C57BL/6 mice to chronic immobilization stress (Son et al. 2018). Son and colleagues specifically examined the role of glutamate, glutamine and glutamatergic activity in the medial PFC. Through glutamate-specific optogenetic stimulation of the medial PFC, the stress-induced depressive-like behaviour could be decreased. Two very recent studies are also worth mentioning, although not using an animal model ofMD, but examining behaviour and neural circuits that potentially are impaired in MD inWT animals and therfore might be a therapeutic target. Collins and colleagues researched the role of NAc cholinergic interneurons, and their photostimulation indeed showed an effect on reward-seeking behaviour (Collins et al. 2019). The other study shed more light onto the possible mechanism of action of novel rapd-acting antidepressants like ketamine (Hare et al. 2019). Using optogenetics in Cre-mice they could show that especially photostimulation of DA receptor D1 drives antidepressant responses. The combination of "optogenetics", "Major Depression" and "medial forebrain bundle" results in 0 entries, only searching for "optogenetics" combined with "medial forebrain bundle" shows 4 publications (search performed on 30.06.2019). Nevertheless, there is no study targeting the MFB for optogenetic stimulation (Gigante et al. 2016; Hernández et al. 2017; Kolodziej et al. 2014; Scardochio et al. 2015). The four studies are listed under that term because lesions or viruses were injected into the MFB or the MFB was electrically stimulated. Target region for optogenetic stimulation in these publications was the VTA. In conclusion, no publication about optogenetic stimulation of the MFB is found in PubMed. Optogenetic studies specifically dealing with the role of the DA-system inMD are only found twice (Francis et al. 2015; Tye et al. 2013). As a third DA- researching study, Clemm von Hohenberg and colleagues might be mentioned, as the examined connectivity of the default-mode network seems to be modulated primarily by DA and 5-HT (Clemm von Hohenberg et al. 2018). 40 1 Introduction electro- electro- in vivo in vivo in vitro in vitro in vitro in vitro Behaviour, electrophysiology inphysiology, behaviour, vitro ELISA PET , physiology, behaviour electrophysiology electrophysiology electrophysiology Behaviour, electrophysiology medial ,PFC basolateral amygdala NAc medial PFC Lateral habenulations in Functional lateralMRI habenula NAc Behaviour, Anterior cingulate cortex Behaviour, VTA Medial PFC viations projec- in dorsallateral raphe habenula or Behaviour, mice, no MD model MD model tion stress mice Negativestate rats cognitive stress in mice Sciatic nerve lesions in mice rats MD model Treatment-resistant depression sion 2015 Pain-induced depres- 2012 Depression Wild-type rats, no 2015 Depression Chronic social defeat 2019 Depression, Addiction ChAT::Cre rats, no 2018 Major Depression CMUS in rats Dorsal raphe via projec- 2019 Depression Drd1::Cre/Drd2::Cre 2018 2013 Major Depression CMUS in mice and 2018 Depression Chronic immobiliza- Optogenetic studies in Depression et al. et al. et al. et al. et al. et al. et al. et al. et al. ReferenceHare Disease Animal Model Stimulation Area Readout Collins Son Clemm vonberg Hohen- Zhang Francis Barthas Tye Warden Table 1.4: 41 2 Aims 2 Aims

The aim of this thesis was to examine the optogenetic stimulation of the MFB as a potential target for treatment ofMD. For this purpose, two different rat models of depression were used, the FSL rat and the CMUS protocol. Furthermore, the specific role of the DA-system in this context was examined using lesioning techniques and genetically modified animals. The following specific aims were addressed:

• The establishment of the optogenetic system in the laboratory,

• The establishment of the optogenetic viruses and their injection protocol, to reach a maximum possible transfection rate in the VTA,

• The establishment of a genetically modiﬁed rat line, theLETH::Cre rat,

• The behavioural characterization of theLE rat per se,

• The establishment of the CMUS protocol as a stress-induced depression model,

• To investigate the eﬀect of an optogenetic stimulation of the MFB in the FSL depression model and to analyse the role of the DA-system examining animals with a 6-Hydroxydopamine hydrochloride (6-OHDA) lesion or an intact DA- system,

• To study the eﬀect of a DA-speciﬁc stimulation of the MFB in a stress-induced depression model. 42 3 Materials and Methods 3 Materials and Methods

3.1 Chemicals and Equipment

The equipment and the used chemicals are listed with their source of supply in the methods part below. Consumables and standard equipment are not shown separ- ately.

3.1.1 Solutions for Immunohistochemistry

Solutions for immunohistochemistry were freshly prepared at the beginning of each staining and were stored at 4 ◦C until further usage. The stock solution of phosphate– buﬀered saline (PBS) was diluted 1:10 and the pH-value was adjusted to 7.0 – 7.5.

Table 3.1: Solutions for Histology Water aqua dest.: Milli-Q Wasseraufbereitungssysteme BS DAB: 3 % BSA, Sigma-Aldrich/Merck KGaA, Darmstadt, DE), 0.3 % Triton X-100 (Sigma-Aldrich/Merck KGaA, Darm- stadt, DE) in 1 x PBS BS fluo.: 5 % goat serum, 0.3 % Triton X-100 in 1 x PBS CS fluo.: 0.3 % Triton X-100 in 1 x PBS PBS 10 x 1 l: 80 g NaCl, 2 g KCl (Carl Roth GmbH & Co. KG, Karlsruhe DE), 7.7 g NaH2PO4 (Merck, Darmstadt, DE), 2 g KH2HPO4 (Merck, Darmstadt, DE), in aqua dest., pH = 6.8 Antifreeze sol. 1 l: 400 ml 1 x PBS, 300 ml Glycerin (Merck, Darmstadt, DE), 300 ml Ethylenglycol Sucrose sol.: 25 % sucrose (Calbiochem/Merck KGaA, Darmstadt, DE), 1 x PBS Fixing sol.: 4 % PFA, Merck KGaA, Darmstadt, DE) in 1 x PBS ABC Complex: 1 drop of Vectastain solution A and B (Biozol Diagnostica Vertrieb GmbH, Eching, DE) respectively per 5 ml of 1 x PBS DAB solution: 1 tablet of DAB and 1 µl H 2O 2 (Merck KGaA, Darmstadt, DE) per 10 ml of 1 x PBS TAE buffer 50x: 242 g Tris (Applichem GmbH, Darmstadt, DE), 100 ml 0.5 M Na2-EDTA (pH 8.0, Sigma-Aldrich/ Merck KGaA), 57.1 ml glacial acetic acid (Carl Roth GmbH & Co. KG) in 1500 ml Millipore water 43 3 Materials and Methods

3.1.2 Antibodies

The following tables show the used primary and secondary antibodies. For detailed description of the immunohistochemistry see the sections of the distinct projects below.

Table 3.2: Primary Antibodies Antibody Species Source of supply Dilution anti–TH Mouse Sigma–Aldrich/Merck KGaA, 1:2000 Darmstadt, Germany anti–GFP Rabbit Invitrogen/Thermo Fisher Sci- 1:1000 entiﬁc, Waltham, MA, USA

Table 3.3: Secondary Antibodies Antibody Species/Host Source of supply Dilution Alexa Fluor®488 Goat anti life technologies/Thermo Fisher 1:200 rabbit Scientiﬁc, Waltham, MA, USA, Alexa Fluor®568 Goat anti Invitrogen/Thermo Fisher Sci- 1:200 mouse entiﬁc, Waltham, MA, USA DAPI Sigma-Aldrich/Merck KGaA, 1:1000 Darmstadt, DE anti-Mouse IgG. Horse Vector laboratories, Burlingame, 1:200 biotinylated CA; USA

3.1.3 Substances Applied to the Animals

The following table shows substances and their concentrations that are applied to the animals throughout the diﬀerent projects, e.g. anaesthesia, pain medication, substances for lesioning etc. 44 3 Materials and Methods

Table 3.4: Substances Applied to the Animals Solution Application Concentration Source of supply Ketamine 10 % Anaesthesia 100 µl Ketamine and Ceva, Düsseldorf, Xylazine 2 % 40 µl xylazine per 100 g DE/Bayer AG, bodyweight Leverkusen, DE Isoﬂurane Anaesthesia 4 % induction phase, 1.8– Abbott, Chicago, IL, 2.5 % surgery, O2 0.8 l per USA minute Temgesic Analgesia 0.1 ml kg−1 in NaCl Essex pharma, München, (Bupren- DE orphin) Amphetamine Activity 2.5 mg kg−1 in NaCl Sigma-Aldrich/Merck Test KGaA, Darmstadt, DE 6-OHDA Lesions 3.6 mg ml−1 in Sigma-Aldrich/Merck 0.2 mg ml−1 ascorbic KGaA acid in NaCl Desipramin Protection 25 mg kg−1 Sigma-Aldrich/Merck Noradren- KGaA alin

3.1.4 Viruses for Optogenetics

The following viruses (kindly provided by Dr. R. Jude Samulski and the UNC Vector Core, Chapel Hill, NC, US) were used for unspeciﬁc and DA-speciﬁc optogenetic stimulation throughout the projects:

Table 3.5: Viruses for Optogenetics Virus Titer Source of supply AAV2-hSyn-hChR2(H134R)-EYFP 3.1 x 1012vg ml−1 UNC Vector Core AAV2-Ef1α-DIO-hChR2(H134R)- 3.6 x 1012vg ml−1 UNC Vector Core EYFP

3.1.5 Kits used for Molecular Analysis

The following kits were used for molecular analysis: 45 3 Materials and Methods

Table 3.6: Kits for Molecular Analysis Kit Application Source of supply DNeasy Blood and Tissue kit Genotyping Quiagen FCM ELISA kit FCM measurements Enzo

3.1.6 Primer

The following primer were used for genotyping of the Cre transgene:

Table 3.7: Primers Primer Sequence Source of supply Cre-5 5’–GCG GCA TGG TGC AAG TTG AAT–3’ Metabion Interna- tional AG, Planegg, DE Cre-3 5’–CGT TCA CCG GCA TCA ACG TTT–3’ Metabion Interna- tional AG, Planegg, DE

3.1.7 Mastermix for PCR

The following table shows the composition of the mastermix for the genotyping polymerase chain reaction (PCR).

Table 3.8: Mastermix for PCR Component Manufacturer Concentraion µl per rxn Extract-N-Amp Sigma 2 x 10 Cre-5 Sigma 25 µM 0.3 Cre-3 Sigma 25 µM 0.3 Sterile water 5.4 46 3 Materials and Methods

3.2 Animals

For this thesis different rats strains were used for the different experiments. For pilots and behavioural characterization, Sprague Dawley (SD) rats (Charles River, Germany) andLE rats (Charles River, Italy) were used. As depression model, FSL (own colony) were used and dopamine-specific stimulation was done in Long-Evans TH::Cre rats (own colony, first animals kindly provided by Karl Deisseroth / Rat Resource & Research Center, Columbia, MO, US). Both genders were used throughout the experiments; the specific gender for each experiment is found in the detailed description of the projects below. Animals were housed in the animal facility in type IV transparent acrylic cages in groups up to four animals (males) or up to five animals (females), depending on the weight of the animal. They were maintained on a 12:12 hours light/dark cycle with lights on at 6 a.m. and egg cartons, paper towels and wooden sticks served as cage enrichment. The rats were maintained at a temperature of 21 ± 1 ◦C and a relative humidity of 50 – 60 %, they had ad lib- itum access to tap water and standard diet (Provimi Kliba AG, Kaiseraugst, CH). Twice a week, animals were changed over to a new cage with fresh bedding. Some experiments required single housing of the animals in type III cages and restricted access to food and water, which is described in detail below. Experiments started when the animals reached an age of 11 - 12 weeks and took up to three months. All animals experiments were performed according to the approved applications G14-40, G16-61, G10-110 and G10-124 (Regierungspräsidium Freiburg).

3.2.1 Breeding and Genotyping of Long EvansTH::Cre Rats

To obtain the hemizygous animals that were needed for the experiments, hemizygous TH::Cre males were bred with wild-typeLE females. The breeding resulted in 50 % LE TH::Cre and 50 % wild-type littermates. The mean size of the litter was 15 animals per dam. Around post-natal day (PND) 14, samples for genotyping were collected, cutting oﬀ 5 mm of the pups’ tail. The samples were immediately put on ice and then stored at −20 ◦C until further processing. The pups were chipped with microchips for identiﬁcation on the same day. They were weaned between PND 21 and 27 and put into groups of 4–5 animals, sorted by gender. The TH::Cre strain carries a transgene in which Cre recombinase is inserted immediately before the start codon of a mouseTH gene. To distinguish transgene positive animals from transgene negative ones, they were tested for this Cre gene. For the DNA 47 3 Materials and Methods

isolation the DNeasy®Blood & Tissue Kit (Quiagen, Hilden, DE) was used. First, the samples were lysed using proteinase K and then buffering conditions were adjusted to an optimum environment for DNA. The lysate was loaded on the DNeasy mini spin column and centrifuged to bind the DNA selectively to the membrane. The membrane was washed two times to purify the DNA, then the DNA was eluted in buffer and ready to use for the PCR. For the Cre recombinase PCR the mastermix was prepared according to table 3.8 on page 45, the primers and their sequences are shown in table 3.7 on page 45. For the final reaction, 16 µl of the mastermix and 4 µl of the sample were put into 200 µl reaction tubes and the reaction was run in the T Gradient Thermocycler (Biometra GmbH, Jena, DE) with the following cycle parameters:

1. 94 ◦C 3 minutes 2. 94 ◦C 1 minute 3. 60.8 ◦C 1 minute 4. 72 ◦C 1 minute 5. Repeat steps 2–4 34 times for a total number of 35 cycles 6. 72 ◦C 10 minutes 7. 4 ◦C hold until refrigerate product.

For further analysis of the amplified DNA a 1.2 % agarose gel was prepared. The agarose (Sigma Aldrich/Merck KGaA) was diluted in tris-acetate-EDTA (TAE) buffer and brought briefly to boil in the microwave. The gel solution was cooled to 60 ◦C on the shaker and GelRed nucleic acid gel stain (Biotium, Fremont, CA, US) was added in a concentration of 1:10.000. Then it was poured into a Compact M gel chamber (Biometra GmbH) including one or two combs for 24 samples respectively and let to set for 45 min. The samples were prepared in a 96-well plate adding 15 µl of each sample and 3 µl of DNA loading dye (Thermo Fisher Scientific GmbH, Dreieich, DE). The combs in the gel were removed and the gel was placed into the electrophoresis chamber. The chamber was filled with TAE buffer and the pockets were filled with 5 µl of a 50 bp ladder (Thermo Fisher Scientific) and 15 µl of the sample mix, including blank, positive an negative control. 120 V were applied and the electrophoresis was run for 30 - 40 minutes. A positive Cre transgene was found at 232 bp, Cre negative samples showed no product. 48 3 Materials and Methods

3.3 Establishment of the Virus Injection

The used viruses for the following projects are listed in table 3.5 on page 44 and were obtained from the vector core of the University of North Carolina. For experiments withWT or FSL rats the AAV2-hSyn-ChR2-EYFP (hereafter referred to as "unspecific virus" or "unfloxed virus") was used, for experiments withTH::Cre rats the dopamine-specific AAV2-Ef1α-DIO-ChR2-EYFP (hereafter referred to as "DA- specific virus" or "floxed virus") was used. First, the injection of the virus needed to be established, which included finding the right coordinates for hitting the target in the best possible way and finding the right volume to transfect an adequate number of neurons. In four pilot experiments, two for the floxed virus and two for the unfloxed one, the coordinates and volumes were defined. Coordinates were chosen according to the atlas "The Rat Brain In Stereotaxic Coordinates" (Paxinos and Watson 2007). Due to problems with injections that ended up too posterior, the anterior-posterior (AP) coordinate was adjusted with the following formula, if the bregma-lambda-distance was not equal to 9 mm, as given in the atlas:

y xadjusted = x · (3.1) 9mm x = original x coordinate y = bregma-lambda-distance

The target site for injecting the virus in all projects described here was the VTA. As the AAV tends to spread to the neighbouringSN, a virus with a low serotype (2) was chosen instead of higher ones (e.g. 5), which tend to spread more.

3.3.1 Stereotactic Surgery - Virus Injection

Between four and six animals underwent surgery for the different protocols, respectively. The rats were placed inside the anaesthesia induction box (Workshop) and isoflurane anaesthesia (Abbott, Ludwigshafen, DE) was induced with 4 % and an oxygen flow of 1–2 l min−1. Then, they were fixed in the stereotactic frame (Stoelt- ing Co., Wood Dale, IL, US) and anesthesia was maintained at approximately 1.8– 2.5 %. The head was shaved (Wahl Supertrim, Unterkirnach, DE) and disinfected 49 3 Materials and Methods

with Softasept (B. Braun AG, Melsungen, DE), and the eyes were protected with Bepanthen ointment (Bayer AG, Leverkusen, DE). To provide the animals with fluid throughout the surgery, they received 1–2 ml 0.9 %NaCl or 5 % glucose solution (B. Braun Melsungen AG) subcutaneous (s.c.) and to keep the body temperature con- stant, they were lying on a heating plate (Havard Apparatus, Holliston, MA, US) with closed loop temperature control. After checking again for reflexes pinching the hind legs, a skin incision was made along the mid line of the skull using a number 10 scalpel (Feather Safety Razor Co., Ltd, Osaka, JPN). Clips were attached to the skin to flap it away from the surgery field. The reference points bregma and lambda were identified and the head was brought to flat skull position with adjusting the incisor bar until the heights of bregma and lambda were equal. Additionally, the distance between bregma and lambda was measured and theAP coordinate was adjusted with formula 3.1 on the preceding page if necessary. The drill was fixed onto the stereotactic arm and the coordinates of the target site were set according to Table 3.9 for the pilots and according to Table 3.10 on the following page for the final experiments. Holes were drilled carefully to avoid damaging the dura with resulting bleeding and the dura was removed cautiously using a needle.

Table 3.9: Pilot Coordinates Virus Coordinates [mm] Volume [µl] AAV2-hSyn-ChR2-EYFPAP -6.0 0.5 / 1 ML -0.4/-0.5 DV -7.5 AP -6.0 0.5 / 1 ML -1.4 DV -8.0 AAV2-Ef1α-DIO-ChR2-EYFPAP -6.0 0.5 / 1 ML ±0.7 DV -7.0 AP -5.4/-6.0 0.5 / 0.8 ML ±0.7 DV -7.5/-8.2

Next, a 2 µl Hamilton syringe ("Neuros", Hamilton, Reno, NV, US) was assembled to the pump of a micropump system ("Ultra Micro Pump II", World Precision Instru- ments, Berlin, DE), mounted to a steretactic arm. A small amount more virus than needed was drawn up into the syringe. The pump was tested before each injection by releasing a small amount of solution to verify that it was not clogged. Then, it 50 3 Materials and Methods

was lowered into the hole and down to the desired dorso-ventral (DV) coordinate. Injection speed always was very low with 100 nl min−1 to avoid tissue damage. After injection, the syringe was left in place for approximately 10 minutes to allow the virus solution to diﬀuse into the tissue and to avoid sucking up the solution, which would result in contaminations of other brain regions. When the syringe was with- drawn, it was checked again for clogging. The incision was closed using staples (B. Braun Melsungen AG) and the animal received buprenorphin ("Temgesic", Reckitt Benckiser, Slough, UK) as analgesia according to table 3.4 on page 44. It was placed into a recovery cage until it woke up, then the rat was put back in its home cage. Post-operative care, including weighing the animals and applying further pain medication, was done regularly.

Table 3.10: Final Coordinates Virus Coordinates [mm] Volume [µl] AAV2-hSyn-ChR2-EYFPAP -5.4/-6.0 0.8 ML ±0.7 DV -7.7 AAV2-Ef1α-DIO-ChR2-EYFPAP -5.4/-6.0 0.8 ML ±0.7 DV -7.5/8.2

Per virus type, two diﬀerent protocols were tried. The medio-lateral (ML) coordinate was varied due to bleeding of the sinus near the mid line. Also an approach with going in in an angle (ML = -1.4 mm) was tested. The DV coordinate had to be adjusted also. Additionally, one track/one deposit approaches were compared with two track/two deposits approaches. Diﬀerent volumes were tested between 0.5–1 µl.

3.3.2 Immunohistochemistry

After four weeks of transfection time, the animals were transcardially perfused, the brains were coronally cut and the sections were stained forTH, GFP and DAPI. For compositions of all solutions used in immunohistochemistry see table 3.1 on page 42. The used antibodies are shown in table 3.2 on page 43 and 3.3 on page 43. The enhanced yellow fluorescent protein (EYFP) marker was additionally stained with a GFP and an Alexa Fluor®488 antibody, as the available epifluorescent microscope did not have any filters for EYFP. 51 3 Materials and Methods

3.3.2.1 Transcardial Perfusion

Fixation of the tissue disrupts processes of metabolism and avoids post-mortal symptoms of decline. The current state of the tissue needed to be fixed by cross-linking proteins and binding amino acids to aldehyde groups. The immunohistochemical staining of the cells is allowed by preservation of their antigen characterization. The animals received an intraperitoneal (i.p.) injection of 10 % ketamine and 2 % xylazine. For terminal anaesthesia the concentration from table 3.4 on page 44 was doubled. Once the areflexic state was reached, the rat was placed on a styrofoam plate and the extremities were fixed with needles. After opening the thorax and exposure of the heart, the aorta descendens was clamped to avoid perfusion of the caudal body parts. The left ventricle of the heart was opened with a small cut and a 13-G hypodermic blunt needle was inserted and carefully pushed upwards until it could be visualized at the beginning of the ascending aorta. To fix the needle, a clamp was attached around needle and heart. The right atrium was opended with a small incision to allow venous drainage. The blunt needle was connected to a tubing system and a pump (neoLab Migge GmbH, Heidelberg, DE), which allowed continuous flow of the solutions with a speed of approximately 60 ml min−1. First, cold PBS was pumped through for three minutes, followed by three minutes of cold 4 % PFA solution. A good perfusion was indicated by stiffening of the head and the upper extremities. After decapitation and dissection of the brain, the brain was post-fixed in 4 % PFA over night and transferred to 25 % sucrose solution for storage until further processing. The purpose of sucrose infiltration was to prevent freezing artefacts in the tissue during sectioning.

3.3.2.2 Immunoﬂuorescent Stainings

Using a sliding microtome ("SM 2010R", Leica Biosystems Nussloch GmbH, Nuss- loch, DE), the frozen brain was cut into coronal slices with a thickness of 40 µm. The slices were collected into a series of 12 wells to allow a correct anatomical reconstruc- tion. They were stored in antifreeze solution at −20 ◦C until further processing. For immunofluorescent staining, secondary antibodies coupled to fluorescent molecules are used, which can be visualized under a epifluorescence or confocal laser scanning microscope. The staining procedure started with washing the free-floating sections (3 x 10 minutes), then the blocking solution (BS) (see table 3.1 on page 42) was added to the vials for one hour for blocking unspecific binding sites. Afterwards, the 52 3 Materials and Methods

sections were incubated with the primary antibodies forTH and GFP (see table 3.2 on page 43 on the shaker at 4 ◦C over night. The following day, they were rinsed thoroughly (3 x 10 minutes) and then incubated with the secondary antibodies, including DAPI (see table 3.3 on page 43) at room temperature (RT) for one hour in the dark, which was followed again by three washing steps with PBS for 10 minutes, respectively. For the projects of this thesis,TH was always combined with Alexa Fluor®568 and GFP with Alexa Fluor®488. Following the staining process, the brain sections were mounted onto glass slides ("Superfrost Plus", R. Langenbrinck Labor- und Medizintechnik, Emmendingen, DE) and covered with ﬂuorescent mounting medium (Dako,Glostrup Kommune, DK) and cover slips (R. Langenbrinck Labor- und Medizintechnik). They were allowed to dry at RT over night in the dark and then were stored at 4 ◦C until microscopical analysis took place.

3.3.2.3 Microscopical Analysis - Epiﬂuorescent Microscopy

Following the staining and mounting of the sections, they were analysed with an epiﬂuorescent microscope ("AX70", Olympus, Tokio, JPN) and pictures were taken using a XC10 camera (Olympus) and CellP software (Olympus). Image processing was done in Adobe Photoshop CC 2018, Microsoft Image Compositor and ImageJ Fiji (Schindelin et al. 2012). Fiji was also used for cell counting and ﬁgures were made with Inkscape software.

3.4 Behavioural Characterization of Long Evans vs. Sprague Dawley Rats

In order to be able to target specifically DA neurons with the AAV, the breeding and experimental use ofLETH::Cre rats was established in the laboratory. The background of this genetically modified strain is theLE rat strain. As the laboratory exclusively usedSD and FSL rat strains in previous projects, a behavioural characterization of theLE rat in comparison to theSDs was the first step for future projects using this strain.LE males (n=7) andLE females (n=7) were compared withSD males (n=7) andSD females (n=7) in the following behaviour tests: SPT, USV,DH, FST, EPM andOF. After testing was completed, the animals were sac- rificed using CO2 gas. 53 3 Materials and Methods

3.4.1 Sucrose Preference Test

The SPT assesses differences in sucrose solution consumption or rather the preference of sucrose solution over water across groups. The results reflect the animals hedonic behaviour. A low intake of sucrose solution or no distinct preference compared to water indicates a decreased sensitivity to experiencing reward, which is understood as a pre-clinical correlate of anhedonia (Katz 1982). One day before the test, the rats were seperated into single cages. On the following day, they received two bottles, one containing 400 ml of tap water and the other containing 400 ml of 4 % sucrose solution for 24 hours. The position of the bottles in the cages alternated from animal to animal. After 24 hours, the bottles were removed and weighed to determine the amount of consumed fluids. After the end of the test, the rats were put back into their groups and homecages.

3.4.2 Ultrasonic Vocalization

Experiments have shown before, that vocalization of rodents varies with their affective state. Negative or aversive behaviour is reﬂected by calls in lower frequency bands (20 Hz), whereas excited behaviour or rewarding experiences are associated with calls in higher frequency bands (40–60 Hz) (Burgdorf et al. 2000; Sadananda et al. 2008; Thiele et al. 2016). The subjects were placed into individual round cages (40 cm x 40 cm, diameter x height) and microphones were installed 60 cm above them. USV was recorded with Sonotrack software (Metris, Hoofddorp, NL) for 20 minutes, then the animals were put back into their homecages. The number of calls in a frequency range of 15 – 90 kHz was assessed and analysis was done comparing the events in the speciﬁc frequency ranges of 20 – 30 and 40 – 60 kHz.

3.4.3 Double-H Maze

TheDH is a water-escape based learning and memory test (Kirch et al. 2013; Pol- Bodetto et al. 2011; Thiele et al. 2016). The maze has the shape of two contiguous "H"s and is made of transparent plexiglas. It is placed on a table (80 cm high) in a room with well-contrasted cues on the wall. The apparatus consists of three arms (160 cm in length, 20 cm wide), which are connected by a central corridor (also 160 cm in length, 20 cm wide, see Figure 3.1 on the next page). 54 3 Materials and Methods

Figure 3.1: Double-H Maze. (A) shows theDH set up during the training with the starting point in the south (S)-arm and the platform in the south-east (SE)-arm with the north (N)-arm being blocked. (B) shows the set up for the probe trial on day six with the starting point switched to theN-arm and the S-arm being blocked.

The height of the walls is 35 cm and the maze is ﬁlled with 22 ±1 ◦C warm water up to 15 cm. The water is coloured with an opaque white, innocuous and odourless dye, so that the animals are unable to see the escape platform (14 cm high, 11 cm in 55 3 Materials and Methods

diameter). The middle arms are defined asN andS, their corresponding neighbouring arms as north-east (NE)/SE and north-west (NW) /south-west (SW), respectively. The middle arms serve as start point, the escape platform is placed in one of the lateral arms. The task was to learn to swim from the start point in theS-arm to the target location in theSE-arm with theN-arm being blocked. On day one animals were habituated to the maze, the water and the escape platform, then 4 consecutive training days followed. Each day consisted of 4 trials per animal, with a maximum duration of 60 seconds, respectively. Between trials, the rats were allowed to rest for 30 seconds. The trials were recorded with a digital video camera (Sony Corporation, Japan) connected to the workstation with a tracking software ("Viewer2", Biobserve, Sankt Augustin, DE) and the latency to find the platform was measured in seconds. Additionally, the initial (first entry into a wrong arm) and repetitive (repeated entry into a wrong arm) errors the animals made were counted. On day six, the learning and memory of the animals was tested in a probe-trial with changing the position of the start point from theS-arm to theN-arm, with theS-arm now being blocked. This revealed whether the rats learned the way by going south-east using the cues on the wall (hippocampus-dependent learning) or whether they learned to turn right twice (striatum-dependent learning).

3.4.4 Forced Swim Test

The FST is used for testing depressive-like behaviour in rodents (Porsolt et al. 2001; Slattery and Cryan 2012; Thiele et al. 2016). The animal is placed in a cylinder filled with water without any possibility to escape. In the beginning, it will try to escape but after a while it will become more and more immobile, reflecting a behaviour of despair. The cylinder was made out of transparent plexiglas and measured 65 cm in height and 20 cm in diameter. It was filled with 22 ±1◦C warm water up to 45 cm to avoid the animals being able to touch the bottom with their tails. On day one, the rats were habituated to the set-up for 15 minutes. On day two, they were tested in a single trial for 5 minutes with the activity being recorded. The time during which the rat was immobile was measured with immobility being defined as floating behaviour with no movement of three out of four paws and no struggling or climbing. 56 3 Materials and Methods

3.4.5 Elevated Plus Maze

The EPM assesses the animals’ anxiety level (Furlanetti et al. 2015; Handley and Mithani 1984; Thiele et al. 2016). The apparatus was made out of grey PVC and consisted of two opposite open arms (50 x 12 cm) and two opposite closed arms with the same measurements, but surrounded by 50 cm high walls (see Figure 3.2). The center zone (12 x 12 cm) allowed the animal to transit from one arm to the other. The maze was elevated 1 metre above the ground and experiments were carried out under low light conditions (30 lux). The animals were tested in a single trial with a duration of 5 minutes, during which they were allowed to explore the maze freely. They were recorded and tracked ("Viewer2", Biobserve) to measure the time spent in each arm and the maze was disinfected thoroughly between animals, to remove any odours.

Figure 3.2: Elevated Plus Maze. The apparatus for the EPM consists of two closed arms and two open arms, with the maze being elevated 1 m above the ground. The rats are allowed to explore the maze freely for 5 min.

3.4.6 Open Field Test

TheOF can be used to assess the locomotor and exploration activity as well as anxiety-like behaviour in rodents (Belzung 1999; Hall 1934; Thiele et al. 2016). The rats were placed into square boxes (75 x 75 x 80 cm) and were allowed to explore these freely for 30 minutes. The space was divided into a central zone (30 x 30 cm) 57 3 Materials and Methods

and a peripheral zone. The animals were recorded and tracked using the Viewer2 software. Subsequent analysis included the track length and the time spent in each zone.

3.5 Establishment of the Chronic Mild Unpredictable Stress Protocol

The CMUS protocol is an animal model of depression (Willner et al. 1987, 1992) as described in Section 1.3.1 on page 20. The protocol is suitable when the use of genetic MD models is not possible, as in the use ofTH::Cre rats for DA-speciﬁc stimulation. The CMUS protocol has not been used for theLE strain in the laboratory, therefore a ﬁrst experiment served as establishment of the protocol. MaleLEWTs were used for this experiment, with an age of 12 weeks at the beginning. According to the results of the baseline SPT and USV, they were sorted into two equal groups, control (CTRL) (n=8) and CMUS (n=7). One animal of the stress group had to be excluded before the start of the CMUS due to health problems. The experiment started with the baseline SPT and USV, followed by seven weeks of CMUS protocol with weekly SPTs, according to Table 3.12 on the following page.

Table 3.11: Applied Stressors I Applied stressor Duration of exposition 17 hours food deprivation followed by 1 hour of reduced 18 hours amount of food (5 g per animal) 45 ◦ tilted cage 16 hours Continuous lighting 24 hours Stroboscopic lighting (100 pulses per minute) 8 hours Wet bedding (2 ml g−1 bedding) 14 hours Food and water deprivation in course of SPT 20 hours White noise (www.simplynoise.com, 70 dB) 2 hours 17 hours water deprivation followed by 1 hour of expos- 18 hours ition to empty bottle

The CTRL group did not undergo the stress protocol, but the animals’ stress level was monitored during the weekly SPT. The CMUS group was exposed to seven weeks of CMUS protocol with weekly SPTs. The stress protocol included eight 58 3 Materials and Methods

diﬀerent stressors which are listed in Table 3.11 on the previous page. Once a week, the stressing was paused for at least 24 hours. After the end of the CMUS protocol, both groups were tested in the following behavioural tests:OF, USV, EPM,OR, SIT, FST and SPT. The procedure of the tests is described in Section 3.4 on page 52, new tests are described below. Additionally, the weight of the animals was monitored and their corticosterone levels were measured.

Table 3.12: CMUS protocol I Group Day Stressor Time period CMUS 1 White noise 14:00 – 18:00 CMUS 2 Tilted cage 17:00 – 09:00 CMUS 3 Continuous lighting 09:00 – 09:00 CMUS, CTRL 4 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL5 SPT 16:00 – 17:00 CMUS 6 No stressor CMUS 7 Water deprivation / empty bottle 17:00 – 11:00 CMUS 8 Stroboscopic lighting 11:00 – 19:00 CMUS 9 Wet bedding 19:00 – 09:00 CMUS 10 Tilted cage 17:00 – 09:00 CMUS, CTRL 11 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 12 SPT 16:00 – 17:00 CMUS 13 Continuous lighting 09:00 – 09:00 CMUS 14 No stressor CMUS 15 Food deprivation /reduced food 17:00 – 11:00 CMUS 16 Stroboscopic lighting 11:00 – 19:00 CMUS 17 White noise 14:00 – 18:00 CMUS, CTRL 18 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 19 SPT 16:00 – 17:00 CMUS 20 No stressor CMUS 21 Continuous lighting 09:00 – 09:00 CMUS 22 White noise 14:00 – 18:00 CMUS 23 Tilted cage 17:00 – 09:00 CMUS 24 Wet bedding 19:00 – 09:00 CMUS, CTRL 25 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 26 SPT 16:00 – 17:00 CMUS 27 No stressor CMUS 28 Water deprivation / empty bottle 17:00 – 11:00 59 3 Materials and Methods

Table 3.12: CMUS protocol I - continuation Group Day Stressor Time period CMUS 29 White noise 14:00 – 18:00 CMUS 30 Wet bedding 19:00 – 09:00 CMUS 31 Tilted cage 17:00 – 09:00 CMUS, CTRL 32 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 33 SPT 16:00 – 17:00 CMUS 34 Continuous lighting 09:00 – 09:00 CMUS 35 No stressor CMUS 36 Wet bedding 19:00 – 09:00 CMUS 37 White noise 14:00 – 18:00 CMUS 38 Tilted cage 17:00 – 09:00 CMUS, CTRL 39 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 40 SPT 16:00 – 17:00 CMUS 41 No stressor CMUS 42 Continuous lighting 09:00 – 09:00 CMUS 43 Food deprivation /reduced food 17:00 – 11:00 CMUS 44 Stroboscopic lighting 11:00 – 19:00 CMUS 45 Wet bedding 19:00 – 09:00 CMUS, CTRL 46 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 47 SPT 16:00 – 17:00 CMUS 48 White noise 14:00 – 18:00 CMUS 49 No stressor CMUS 50 Tilted cage 17:00 – 09:00 CMUS 51 Stroboscopic lighting 11:00 – 19:00 CMUS 52 Wet bedding 19:00 – 09:00 CMUS, CTRL 53 Food and water deprivation SPT 20:00 – 16:00 CMUS, CTRL 54 SPT 16:00 – 17:00 CMUS 55 Continuous lighting 09:00 – 09:00 CMUS 56 No stressor

3.5.1 Weight Measurements

Weight development is an important readout to monitor the animals’ condition. Stressed animals often show a drop in weight or a slower increase in weight compared 60 3 Materials and Methods

to unstressed CTRLs (Matthews et al. 1995). Rats of both groups were weighed weekly and the data was normalized by setting the baseline value to 100 % and calculating the other values according to that.

3.5.2 Corticosterone Measurements

Corticosterone is a main glucocorticoid in rodents, which is secreted by the cortex of the adrenal gland. It is produced in response to stimulation of the adrenal cortex by adrenocorticotropic hormones. Since stress increases the production of corticosterone, the measurement of its’ concentration is a major indicator of stress (Palme 2019; Thiele et al. 2016). Usually, the hormone is extracted from blood plasma. As the process of drawing blood from the tail vein of the awake rat itself evokes stress in the animal, a protocol for a non invasive extraction of corticosterone from faeces was established in the laboratory. Faeces of each individual were collected before and after the CMUS protocol and frozen at −20 ◦C until further processing. Then, the faeces were ground with liquid nitrogen using a pestle and mortar and 50 mg of the homogenized faeces were filled into an Eppendorf tube and 1 ml 0f 80 % methanol was added. The mixture was vortexed at high speed for 30 minutes and then centrifuged at 2.500 x g for 20 minutes. The supernatant was transferred to a new tube and frozen −20 ◦C until further analysis. To ensure that the extraction of protein worked, a Bradford protein essay was performed. A BSA standard was calculated at 0, 125, 250, 50, 750, 1500 and 2000 µg ml−1 to produce a standard curve. 1 ml of Quick Start Bradford Dye was pipetted into each cuvette, followed by 20 µl of standard or sample and vortexted briefly. Then the standards and samples were incubated at RT for at least 5 minutes and not longer than 1 hour. After incubation, the absorbance of blank samples, standard and samples was measured using an Eppendorf Photometer at a wavelength of 595 nm. To obtain the protein levels in mg ml−1 the standard factor was calculated. The mean absorbence of the standards was divided by the concentration (0 – 2000 µg ml−1) and the standard factor was created by averaging this value for each concentration. Finally, the sample absorbence was multiplied by the standard factor. For the quantitative determination of fecal corticosterone metabolite (FCM) in the faeces extract, an ELISA was performed using a corticosterone ELISA kit (Enzo Life Sciences Inc., Farmingdale, NY, US). An ELISA is an antibody-based assay, using enzymatic colour change to detect the presence of a substance. Briefly, 100 µl of FCM samples were pipetted into a 96-well plate (Greiner Bio-One, Kremsmünster, AUT) and a blue solution of alkaline phos- 61 3 Materials and Methods

phatase conjugated with FCM and a yellow solution of sheep poly-clonal antibody to FCM were added to the vials, according to the Enzo protocol. An FCM standard is added in concentrations of 20000, 4000, 800, 160 and 32 pg ml−1 to allow extraction eﬃciency to be accurately determined. Control wells were also added for blank, total activity (TA), maximum binding wells (BO) and non-speciﬁc binding (NSB). The plate was incubated for 2 hours, washed and a stop solution was added to stop the reaction. The plate was read immediately at 405 nm using a Tecan plate reader (Tecan, Männedorf, CH) with the following parameters:

Table 3.13: ELISA Parameter Shaking (orbital) duration: 5 s Shaking (orbital) amplitude: 1 mm Mode : Absorption Wavelength: 405 nm Number of ﬂashes: 5 Rest period: 0 ms

The concentration of FCM was calculated by taking the average net optical density (OD) bound for each standard and sample and subtract the average NSB:

AverageNet OD = AverageBound OD − Average NSB OD (3.2)

Then, the binding of the standards was calculated as a percentage of theB O, using the following formula:

Net OD P ercentBound = · 100 (3.3) Net BO

The percent bound was plotted versus the concentration of FCM for the standards and a linear regression line was added to the graph. The concentration of FCM now could be calculated by interpolation of the values into the formula of the linear regression. 62 3 Materials and Methods

3.5.3 Sucrose Preference Test - New Protocol

The protocol of the SPT was changed to possibly enhance differences between groups. According to protocols in literature (Matthews et al. 1995; Willner et al. 1987) a habituation and food and water deprivation was added. The protocol started with habituating the animal to the two bottle paradigm, providing them with one bottle filled with tap water and one bottle filled with 4 % of sucrose solution for 24 hours. This was followed by a food and water deprivation for 20 hours and subsequently the test was carried out. Here, the animals again were provided with 2 bottles containing 200 ml of tap water or 4 % sucrose solution for 1 hour. The position of the bottles varied from cage to cage. Afterwards the bottles were weighed to determine the amount of consumed fluids.

3.5.4 Social Interaction Test

For testing the animals’ social behaviour, a three-chamber set up was used (Cook et al. 2017; Kaidanovich-Beilin et al. 2011). The apparatus was made of transparent acrylic glass and measured 1 m x 0.5 m. It was divided into three equal chambers (50 cm x 33.3 cm) that were connected through 10,cm wide openings in the walls. The center zone was empty, whereas the left and right zones contained a small cage, respectively. The cage, 20 cm in diameter and 15 cm high was made off acrylic glass bars with gaps of 0.5 cm in between, allowing a social contact between the animals by sniffing, but avoiding possible biting. The test consisted of a five minute habituation, during which the animal was allowed to explore the empty apparatus freely. Then two trials of ten minutes testing time followed. In trial one, one strange animal (Stranger 1, age and gender matched) was placed into one of the cages, the other cage was left empty. In trial two, the familiar animal was left in its cage and a new strange animal (Stranger 2, also age and gender matched) was placed into the former empty cage (see Figure 3.3 on the next page). The positioning of stranger one and two varied between animals. Habituation and both trials were recorded and tracked using the Viewer2 software. The time the rat spent in each compartment was taken from the software’s results and number and time of social interactions was scored manually. 63 3 Materials and Methods

Figure 3.3: Social Interaction Test. The SIT consists of a habituation, during which the animal explores the empty apparatus. In trial one, the interest in a strange animal (Stranger 1) versus an empty cage is measured and in trial two the social interaction with the familiar animal (Stranger 1) versus a strange animal (Stranger 2) is tested. 64 3 Materials and Methods

3.5.5 Object Recognition Test

With the object recognition test the rats’ ability to distinguish between familiar and novel objects was tested (Berlyne 1950; Leger et al. 2013; Thiele et al. 2016). The subjects were habituated to the square boxes (75 x 75 x 80 cm) by letting them explore their environment freely for 30 minutes the day prior to testing. On testing day, the rats underwent two trials. In trial one, they were placed in the box, which now contained two similar objects in the center, placed with the same distances to the walls. The animals were allowed to explore them for ﬁve minutes, being recorded and tracked using the Viewer2 software. Trial two was carried with a 4 hour delay and now, one of the objects was switched to a new one, diﬀering from the familiar one in shape and colour. Again, the rats explored the box and objects for 5 minutes.

3.6 Optogenetic Stimulation of the Medial Forebrain Bundle in the Flinder’s Sensitive Line Rat Depression Model - 6-OHDA Lesioned vs. Unlesioned Rats

Figure 3.4: 6-OHDA-Stim Project Design. This arrow illustrates the experimental groups and the project design for the 6-OHDA-Stim project.

The aim of this project with the short title "6-OHDA-Stim" was to test, whether a stimulation of the MFB leads to an improvement of the depressive-like phenotype in 65 3 Materials and Methods

the female FSL rat. To examine the role of the DA-system in this question, animals with a lesioned DA-system were compared to ones with an intact system. The animals were sorted into four equal groups according to the SPT baseline testing: Two control groups without stimulation, one with an intact DA-system (DA+) and one with a NAc 6-OHDA lesion (DA-). Two groups underwent optogenetic stimulation of the MFB, one with intact DA-system (DA+Stim) and one with a NAc 6-OHDA lesion (DA-Stim) (see Figure 3.4 on the preceding page). Experiments were planned with n = 8 animals per group, but due to surgery side-eﬀects and misplacement of light cannulas, some of the animals had to be excluded from the experiments. The ﬁnal group sizes were:

• DA+: n = 5

• DA+Stim: n = 4

• DA-: n = 6

• DA-Stim: n = 7.

3.6.1 Stereotactic Surgery - 6-OHDA-Lesion

Stereotactic surgery was performed as described in Section 3.3.1 on page 48. In brief, rats received an i.p. injection of desipramin (25 mg kg−1) to protect noradrenergic neurons 30 min prior to surgery. Then, they were anaesthetized and fixed into the stereotactic frame. The scalp was opened and bregma and lambda were identified. The head was brought to flat skull position and holes were drilled at the target coordinates (Table 3.14).

Table 3.14: NAc Coordinates Coordinates [mm] 6-OHDA conc. Volume / deposit AP + 0.8 / + 1.4 / + 2.1 ML ±1.8 / ±1.6 / ±1.4 DV -7.0 3.6 mg ml−1 1 µl

The animals received injections of 6-OHDA or NaCl (control animals with intact DA- system) at three sites bilaterally to obtain a maximum lesion of the VTA neurons projecting to the NAc. The Hamilton syringe was left in space for at least 5 min to 66 3 Materials and Methods

avoid contamination of the dorsal striatum. Finally, the incision of the scalp was closed with staples and the rats received buprenorphin as analgesia.

3.6.2 Stereotactic Surgery - Virus Injection and Cannula Implantation

Virus injection was performed in all groups as described in Section 3.3.1 on page 48 three weeks after the 6-OHDA lesion surgery. Coordinates and volume of the injected AAV2-hSyn-ChR2-EYFP are described in Table 3.10 on page 50. During the same surgery, stim groups were implanted with the light cannulas (core diameter 200 µm, outer diameter 230 µm, 0.48 NA, cleaved to 9 mm of length, Doric Lenses, Ville de Québec, CAN). After the virus injection was completed, four holes for the anchoring screws were drilled around the target site. Flat tip screws (1 mm in length, 0.75 mm in diameter, Bürklin Elektronik, Oberhaching, DE) were screwed into the holes carefully without touching the brain using a micro screw driver. Then, two holes for the cannulas were drilled at the following coordinates: AP: -2.8 mm, ML: ± 2.3 mm. The dura was carefully removed using a needle. The cannula holder (Doric Lenses) was attached to the stereotactic arm and the cannula was inserted. Then it was lowered to the brain and the DV coordinate was measured. The cannula was lowered carefully to the desired depth at DV: -7.5 mm in an angle of 3 ◦. It was necessary to go in in an angle to have enough space in between both cannulas for connecting them to the patch cords for stimulation. Glue ("Loctite 401", Henkel, Düsseldorf, DE) was added on the skull bone around the cannula and after it was dry, cement ("Palacos R+G", Heraeus, Hanau, DE) was added around the cannula to ﬁx it to the skull. The cannula was carefully removed from the holder and the second cannula was implanted to the other hemisphere. Cement was added around both cannulas, leaving at least 0.5 mm free at the top for connection to the patch cords, and around the screws. Finally, the surface of the cement was smoothed to avoid painful irritations of the skin. If necessary, the scalp margin in front and behind the implant was closed with staples. The animal was supplied with analgesia (buprenorphin according to Table 3.4 on page 44) and placed into a recovery cage for waking up. Post-operative care, including weighing the rats and providing them with additional pain medication, was done regularly. 67 3 Materials and Methods

3.6.3 Laser Set Up and Light Parameters

Light stimulation took place directly prior to each behaviour test for 30 min. One week before the ﬁrst stimulation started, the animals were trained daily for getting connected to the patch cords and habituated to the stimulation cage. For stimulation, the chronically implanted light cannulas were connected via their ferrule to the metal jacket protected patch cords (core diameter 200 µm, outer diameter 230 µm, numerical aperture 0.48, Doric Lenses) by a zirconia mating sleeve (Doric lenses). The optic ﬁbres were attached to a rotary joint (numerical aperture 0.5, Doric Lenses) in the cage lid, which allowed the animal to move freely in the cage.

Figure 3.5: Laser Set Up. This picture illustrates the set up of the laser system. The rats’ implanted cannulas are attached to two patch cords, which are connected to a rotary joint beam splitter. From the joint, one cord runs to the laser. The laser itself is connected with the pulse generator and the laptop with the controlling software.

The rotary joint also served as beam splitter. Another patch cord connected the joint with the 100 mW, 473 nm laser ("LuxX", Omicron-Laserage Laserprodukte GmbH, Rodgau, DE) via FC/PC plugs (see Figure 3.5). The Laser was controlled by the Omicron control center software (Omicron-Laserage Laserprodukte GmbH) and light pulses were generated through a pulse generator (Workshop). The light paradigm was 8 pulses at 30 Hz every 5 s and light on epoch was 30 min. On each day before starting stimulations, the laser beams were aligned to get a maximum power output and all patch cords were tested with a power meter ("PM130D", Thorlabs Newton, NJ, US). 68 3 Materials and Methods

3.6.4 Behaviour Testing

The FSL rats’ behaviour was tested in the following behaviour tests: SPT, USV, homecage activity under stimulation, amphetamine-induced activity, OF, FSTand SIT as described in Section 3.4 on page 52 and in Section 3.5 on page 57. The SPT was performed twice according to the new protocol, once as baseline test to sort groups and once under stimulation.

3.6.4.1 Activity measurements

Activity measurements were performed in square cages (36 x 36 x 42 cm) that were equipped with light beams (Workshop). Crossings of the beams were counted auto- matically. Amphetamine-induced activity measurements were performed two weeks after the lesion surgery, as it is possible to indicate the extend of the lesion through the results. As an indirect sympathomimetic, amphetamine increases locomotor activity but with an increasing extent of a DA lesion, this locomotor activity decreases. The acitivity test was performed twice with half of the animals receiving an amphetamine injection i.p.( 2.5 mg kg−1 in saline) and the other half receiving an injection of NaCl and vice versa. Activity was measured for 90 min. Homecage activity was also measured comparing light stimulation oﬀ with light stimulation on. Beam crossings were counted for 30 min without stimulation and subsequently for 30 min under light stimulation with the parameters described in Section 3.6.3 on the previous page.

3.6.5 Immunohistochemistry

Animals were transcardially perfused as described in Section 3.3.2.1 on page 51 and sectioning of the brains and immunoﬂuorescent staining was performed according to the description in Section 3.3.2.2 on page 51. Additionally, a DABTH staining was performed to evaluate the extent of the DA lesion and the position of the light cannulas in the MFB. For this, the coronal brain sections were rinsed three times in

PBS for 5 min, respectively. This was followed by a 10 min incubation with 3 % H2O2 and 10 % methanol. Again, the slices were washed in PBS for three times, which was followed by the blocking of the unspeciﬁc binding sites with an incubation of 2 hours inBS (for composition see Table 3.1 on page 42). Then, the sections were incubated 69 3 Materials and Methods

with the primary antibody (anti-TH mouse, see Table 3.2 on page 43) over night and the next day washed again in PBS for three times. Now, they were incubated with the secondary antibody (anti-mouse IgG. biotinylated, see Table 3.3 on page 43) for one hour and afterwards again rinsed in PBS for three times. The next step was a one hour incubation with the avidin-biotin-complex (ABC), which had to be prepared 30 min prior to use. Finally, after rinsing three times in PBS, the DAB solution was added to the slices for approximately 5 min. After washing the brain sections again thoroughly in PBS, they were ready for being mounted. The sections were mounted on Superfrost glass slides and dried over night. The next day they were dehydrated by immersing them for 5 min each in 70 %, 95 % and 100 % ethanol (Sigma-Aldrich/Merck KGaA), two times respectively. Then the slices were cleared by immersing them into xylol (VWR International, Radnor, PA,US) twice for 5 min each. Mounting medium (Paul Marienfeld GmbH Co. KG, Lauda-Königshofen, DE) was added onto the slides and they were covered with cover slips.

3.6.6 Microscopical Analysis

The DABTH staining was analysed using the Olympus AX70 microscope, and pictures were taken using the XC10 camera and CellP software. CellP was also used for counting theTH positive cells in the VTA to evaluate the extend of the lesion. Three consecutive slices with the most pronounced VTA (between -5.0 – - 6.0 mm from bregma) were chosen for counting theTH positive neurons from each brain. A composite image was created from three images at 4 x magnification. For the verification of the position of the light cannulas, a picture was taken at 1.25 x magnification. The fluorescent TH-GFP double staining was analysed with a confocal laser scanning microscope ("LSM 880 with Airyscan", Carl Zeiss AG, Oberkochen, DE) with 405, 488 and 561 nm lasers. Again, pictures were taken from the three slices with the most pronounced VTA at a magnification of 20 x. Composite images of 3 x 2 pictures were created at an angle of ±13 ◦ for left and right VTA, respectively. The laser and scanning properties are shown in Table 3.15 on the next page and Table 3.16 on the following page. Laser gain was slightly adjusted in case of varying intensity of the stainings.TH and GFP positive cells were counted using ImageJ Fiji. Counts were partially automated using ImageJ particle analyser. Fluorescent images were linearly brightened for print using Adobe Photoshop. 70 3 Materials and Methods

Table 3.15: Laser Properties Laser Laser Power [%] Laser Gain 405 nm 0.2 980 488 nm 0.2 880 561 nm 0.1 800

Table 3.16: Scanning Properties Scan Setting Value Pinhole 31.1 Pixel Dwell 2.05 µm Scan Time 40.27 s Speed 6 Picture Resolution 1024 x 1024 px

3.7 Dopamine-speciﬁc Optogenetic Stimulation of the Medial Forebrain Bundle in a Stress-induced Rat Depression Model

In this project with the short title "DA-Stim", the effect of a DA-specific stimulation of the MFB in a stress-induced depression model was examined. 28 femaleLE TH::Cre underwent an SPT baseline test according to the new protocol at an age of 11 weeks. 4 animals with the lowest scores in sucrose consumption were excluded from the project, while the others were divided into two groups with similar results. Both groups underwent surgery and were injected with the same virus (AAV2-Ef1α- DIO-hChR2(H134R)-EYFP), but only the DA-stim group was implanted with light cannulas, controls underwent a sham surgery. Both groups were exposed to the CMUS protocol, but only one of the groups, DA-stim, was stimulated prior to behaviour testing, while the others, DA-CTRL, did not receive any treatment (see Figure 3.6 on the next page). Due to misplacement of cannulas and very low transfection rates (under 30 %), two animals had to be excluded, leading to a final n of 11 animals per group. TheTH::Cre reats were kept in pairs of two animals per cage, until recovery phase from surgery was over. For the CMUS protocol and the behaviour testing period they were single housed in type III cages. Figure 3.6 on the following page shows the design and time schedule of this project. 71 3 Materials and Methods

Figure 3.6: DA-stim Project Design. This arrow illustrates the experimental groups and the project design for the DA-stim project.

3.7.1 Stereotactic Surgery - Virus Injection and Cannula Implantation

Stereotactic surgery was performed as described in Section 3.3.1 on page 48 and Section 3.6.2 on page 66. The double-ﬂoxed virus (AAV2-Ef1α-DIO-hChR2(H134R)- EYFP) was injected into the VTA according to the coordinates in Table 3.10 on page 50 in 2 tracks with two deposits, respectively, per hemisphere.

3.7.2 CMUS Protocol

After two weeks of recovery, all animals underwent the CMUS protocol for five weeks. The protocol of the pilot (see Section 3.5 on page 57) was adjusted to render the stress effects more pronounced. Stroboscopic light was changed to no / soiled bedding, during which the animals were kept in a cage without bedding for eight hours. The removed bedding of all animals was mixed and distributed to all cages after the eight hours were over. The rats spent the rest of the days until cleaning day in soiled bedding. Furthermore, two other stressors were added to the protocol: Paired housing and restrain. During paired housing, the rats were paired with another rat from their group for four hours. The pairs differed every time. For restrain, the animals were kept in a small box (17 x 10 x 8 cm) with air holes for one hour, in which they could hardly move. Stressors were applied more frequently compared to the old protocol with most of the time applying one stressor during the day and another one over night. One day of the week was stressor-free for at least 24 hours. For all applied stressors see Table 3.17 on the next page and for the complete protocol Table 3.18 on the following page. 72 3 Materials and Methods

Table 3.17: Applied Stressors II Applied stressor Duration of exposition 17 hours food deprivation followed by 1 hour of reduced 18 hours amount of food (5 g per animal) 45 ◦ tilted cage 16 hours Continuous lighting 24 hours No / Soiled Bedding 8 hours Wet bedding (2 ml g−1 bedding) 14 hours White noise (70 dB) 2 hours 17 hours water deprivation followed by 1 hour of expos- 18 hours ition to empty bottle Restrain 1 hour Paired Housing 4 hours

Table 3.18: CMUS protocol II Day Stressor Time period 1 No / soiled bedding 09:00 – 17:00 Wet bedding 19:00 – 09:00 2 White noise 14:00 – 16:00 Tilted cage 17:00 – 09:00 3 No stressor 4 Continuous lighting 09:00 – 09:00 Restrain 14:00 – 15:00 5 White noise 14:00 – 16:00 Tilted cage 17:00 – 09:00 6 Continuous lighting 09:00 – 09:00 7 White noise 14:00 – 16:00 Food deprivation /reduced food 17:00 – 11:00 8 Paired Housing 13:00 – 17:00 9 No / soiled bedding 09:00 – 17:00 Wet bedding 19:00 – 09:00 10 Continuous lighting 09:00 – 09:00 Restrain 14:00 – 15:00 11 No stressor 12 Paired Housing 13:00 – 17:00 Water deprivation / empty bottle 17:00 – 11:00 13 White noise 14:00 – 16:00 73 3 Materials and Methods

Table 3.18: CMUS protocol II - continuation Day Stressor Time period 14 No / soiled bedding 09:00 – 17:00 Tilted cage 17:00 – 09:00 15 Paired Housing 13:00 – 17:00 Wet bedding 19:00 – 09:00 16 No stressor 17 Continuous lighting 09:00 – 09:00 Restrain 14:00 – 15:00 18 No / soiled bedding 09:00 – 17:00 19 Continuous lighting 09:00 – 09:00 Restrain 14:00 – 15:00 20 White noise 14:00 – 18:00 Food deprivation /reduced food 17:00 – 11:00 21 Paired Housing 13:00 – 17:00 Continuous lighting 17:00 – 17:00 22 Restrain 09:00 – 10:00 Tilted cage 17:00 – 09:00 23 Paired Housing 13:00 – 17:00 Wet bedding 19:00 – 09:00 24 No stressor 25 No / soiled bedding 09:00 – 17:00 Tilted cage 17:00 – 09:00 26 Paired Housing 13:00 – 17:00 Water deprivation / empty bottle 17:00 – 11:00 27 White noise 14:00 – 18:00 28 No / soiled bedding 09:00 – 17:00 Wet bedding 19:00 – 09:00 29 No stressor 30 Paired Housing 13:00 – 17:00 Tilted cage 17:00 – 09:00 31 Continuous lighting 09:00 – 09:00 Restrain 09:00 – 10:00 32 No / soiled bedding 09:00 – 17:00 33 White noise 14:00 – 18:00 Food deprivation /reduced food 17:00 – 11:00 74 3 Materials and Methods

Table 3.18: CMUS protocol II - continuation Day Stressor Time period 34 Paired Housing 13:00 – 17:00 35 Continuous lighting 17:00 – 17:00 Restrain 09:00 – 10:00

3.7.3 Behaviour Testing

The TH::Cre rats’ behaviour was examined in the following tests: SPT, USV, homecage activity under stimulation,OF, FST, SIT and EPM as described in Section 3.4 on page 52, Section 3.5 on page 57 and Section 3.7 on page 70. The SPT was performed three times, once as baseline test, once after the CMUS protocol was completed and once at the end of behaviour testing, combined with stimulation. Moreover, a new test was added to the portfolio, the OSST. All rats from the DA-stim group were stimulated for 30 min prior to each behaviour test with 8 pulses at 30 Hz every 5 s, as described in Section 3.7 on page 70.

3.7.3.1 Open Space Swimming Test

The OSST is another test to detect a depressive-like phenotype in rodents. The diﬀerence to the FST is, that there is no restriction to the movement of the animal like in a narrow cylinder and that the analysis is objective due to the use of a tracking system. The test is performed on four consecutive days with one trial of 15 min each day. On day one, habituation took place, which also served as baseline measurement without any stimulation. Prior to trial two, three and four, the stim group received optogenetic stimulation. For performing the OSST, the rats were placed into a round water pool with a diameter of 132 cm and a height of 60 cm. The pool was ﬁlled with water (22 ±1◦C) to a depth of 40 cm, which was rendered white with odourless, innocuous color to improve tracking of the animals. The subjects were allowed to swim (or not to swim) freely for 15 min. The swimming or drifting behaviour was tracked and recorded using the Viewer2 software. For analysis the track length of the animals was compared, with the track being longer, the longer the animal was actively swimming and shorter when the rat was immobile. 75 3 Materials and Methods

3.7.4 Immunohistochemistry and Microscopy

Perfusion, sectioning of the brains, immunohistochemistry and microscopy was performed as described before (Section 3.7 on page 70). Briefly, a DABTH staining was done to verify the position of the cannulas and fluorescentTH-GFP double stainings were performed for determining the transfection rate of the VTA DA neurons. DAB stainings were analysed with the Olympus AX70 microscope, fluorescent stainings with the Zeiss LSM 880. Cells were counted using Fiji ImageJ software. Again, fluorescent images were linearly brightened for print using Adobe Photoshop.

3.8 Final Analysis and Statistics

All data was collected in tables using Microsoft Excel software (Microsoft Cor- poration, Redmond, WA, US) and means and standard error of the means were calculated. SigmaPlot (Systat Software GmbH, Erkrath, DE) was used to create graphs and statistics were performed with TIBCO Statistica software (Palo Alto, CA, US). The Shapiro-Wilk test was used to test for normal distribution. Normally distributed data was analysed further using t-tests (t) or (repeated measurements) ANOVA (F) combined with Newman-Keuls post-hoc test, depending on the data. Not normal distributed data was tested with Man-Whitney-U (U ), Wilcoxon (T) or Kruskal-Wallis (H ) tests. Speciﬁc tests for each experiments are listed in the results part below. Data are presented as mean ± SEM and statistical signiﬁcance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001. 76 4 Results 4 Results

The focus of this thesis was to examine the effect of optogenetic stimulation of the MFB on a depressive-like phenotype in the rat. Starting with the establishment of the virus injection, the characterization of theLE rat and the evaluation of the CMUS protocol, the work resulted in two final main projects, the optogenetic stimulation in 6-OHDA lesioned FSL rats and the DA-specific stimulation in theTH::Cre rat.

4.1 Establishment of the Virus Injection

The injection of the specific and the unspecific AAV needed to be established in terms of target coordinates and injected volume. Volumes between 0.5 – 1 µl were used throughout the different pilot surgeries. One track injections were compared to two track injections and single deposits with two deposits per track. Coordinates were adjusted to hit the VTA in the best possible way with avoiding bleeding from the mid line sinus and spreading of the virus lateral to theSN. For the DA-specific virus a two track, two deposits approach with 0.8 µl per deposit turned out to be ideal (see Figure 4.1 on the following page). The developed final coordinates were:AP- 5.4/-6.0,ML ±0.7 and DV -7.5/8.2 mm. With this approach a mean transfection rate of 76.2 ±5.3 % of the DA positive neurons in the VTA was reached. Figure 4.2 on page 78 shows an example of the injected DA specific virus in the VTA. A high number of transfected DA neurons can be seen (double labelling ofTH in red and GFP in green) with hardly any contamination to theSN. For the unspecific virus a two track, one deposit approach with 0.8 µl per deposit was used with the following final coordinates:AP -5.4/-6.0,ML ±0.7 and DV -7.7 mm. Here, less virus had to be injected to avoid a too pronounced transfection rate of the non-DA neurons in and around the injection site. This compromise resulted in a lower transfection rate of 62.8 ±3.3 % of the DA-positive neurons. 77 4 Results

Figure 4.1: Virus Injection Site – AAV2-Ef1α-DIO-ChR2-EYFP. (A) shows the plates with the final coordinates:AP -5.4/-6.0,ML ±0.7, DV -7.5/8.2 mm (modified from Paxinos and Watson 2007). The blue circles indicate the injection sites in the VTA. (B) shows a confocal image of the injection site of an exampleTH::Cre rat with an overview of the VTA andSN in 10 x magnification (red: TH, green: GFP). 78 4 Results

Figure 4.2: Transfected Midbrain DA Neurons – AAV2-Ef1α-DIO-ChR2-EYFP. An example of transfected midbrain DA neurons in the VTA in 20 x magniﬁcation (red:TH, green: GFP, blue: DAPI).

4.2 Behavioural Characterization of Long Evans vs. Sprague Dawley Rats

LE is the genetic background of theTH::Cre rats which were used in later optogenetic studies of this thesis. This strain had not been used in the laboratory, so a ﬁrst pilot aimed to characterize the behaviour of theLE rat compared to theSD rat. The main ﬁndings are summarized in Table 4.1 on the following page.

4.2.1LE andSD Rats Diﬀer in Weight But Not in Weight Growth Dynamics

When comparing the weight of theLE and theSD strain between an age of 12 to 19 weeks, there is no signiﬁcant diﬀerence seen (repeated measurements (rm) ANOVA, group, F(1,26) = 2.24, n.s.), althoughLE rats tended to be a little bit heavier (Figure 4.3 (A)). The weight of theLE rats rose from 381 ± 21.9 g to 461.2 ± 33.0 g, 79 4 Results

Table 4.1: LE vs. SD – Summary of Results Test Main findings - Strain Main findings - Gender Weight No difference in weight, no differ- LE females weigh sig. more than ence in growth dynamics their counterparts over all weeks, LE males weigh sig. more thanSD males in week 4 and 7, no difference in growth dynamics OF Track length sig. longer inLE, no Track length sig. longer in both difference in time spent in theCZ female and maleLE compared to their counterparts, no difference in time spent in theCZ DHSDs need sig. longer to find es- BothSD genders need sig. longer cape platform,SDs make more to find escape platform compared IE, no difference inRE to their counterparts,SD females make sig. moreIE compared to fe- maleLEs, do difference in males, no difference inRE EPM No differences No differences FST No differences No differences SPTSD rats consume sig. more SD females consume sig. more sucrose, no difference in sucrose sucrose thanLE females, no dif- preference ference in sucrose preference USVSDs emit sig. more calls in the SD males emit sig. more calls in high band thanLEs, no difference the high band thanLE rats, no in low band difference in females, no difference in low band whereas the weight of theSD rats increased from 318 ± 33.9 g to 385 ± 39.0 g. Having a closer look at the genders during the distinct time points revealed that there were significant differences (rm ANOVA, gender x time, F(12,96) = 161.61, p > 0.001). Newman-Keuls post-hoc testing displayed a significant difference between males in week 4 (LE males: 517 ± 8.0 g,SD males: 473 ± 9.5 g, p < 0.01) and week 7 (LE males: 576 ± 11.8 g,SD males: 524 ± 10.5 g, p < 0.001) and between females over all weeks withLE females being heavier and increasing their weight from 305 ± 9.7 g to 346 ± 12.1 g compared to an increase from 200 ± 14.5 g to 246 ± 7.0 g inSD females (p < 0.001 for each time point, Figure 4.3 (B)). Weight dynamics, as measured as gained weight over time with normalized data did neither show a difference in strain (rm ANOVA, group, F(1,26) = 0.84, not significant (n.s.)), nor in gender (rm ANOVA, gender, F(3,24) = 1.05, n.s.). 80 4 Results

Figure 4.3: LE vs. SD – Weight Development. (A) Weight development of the two strains (LE: black, SD: white) over 7 weeks, (B) weight development divided into strain and gender. There was no general strain eﬀect (rm ANOVA, F (1,26)=2,24, n.s.), but female LE rats were signiﬁcantly heavier than female SD rats over all weeks and male LE rats were heavier compared to SD males during week four and seven (F (12,96)=161,61, p < 0.001).

4.2.2SD Rats Show a Decreased Exploratory Behaviour Compared toLE Rats

TheOF is used to measure the exploratory behaviour in rodents. After the 30 min trial theLE rats had a significant longer track length with 6411 ± 392.9 cm than the SD rats with 4599 ± 389.0 cm (ind. t-test, t(24) = 3.26, p < 0.01, Figure 4.4 (A)). This effect was seen in both genders (one-way (o.w.) ANOVA, gender, F(3,22) = 12.77, p < 0.001, Figure 4.4 (B)) with post-hoc assessing a significant difference of p < 0.01 between females (LE: 7425 ± 418.1 cm,SD: 5589 ± 436.5 cm) and p < 0.05 between males (LE: 5398 ± 389.5 cm,SD: 3891 ± 429.6 cm). The measurement of the time the animals spent in theCZ during the 30 min trial did not show any significant differences, neither between strain (LE: 87.6 ± 14.58 ,SD: 116 ± 24.5 s, U (14,12) = 77, z= -0.33, n.s., Figure 4.4 (C)), nor between genders (LE males: 56.8 ± 15.92 ,s, SD males: 130 ± 38.6 s,LE females: 118 ± 18.8 s,SD females: 96.7 ± 25.81 s, H (3) = 5.95, n.s., Figure 4.4 (D)). 81 4 Results

Figure 4.4: LE vs. SD – Open Field Test. (A) LE rats show a significant increase in track length compared to SD rats (t(24)=3.26, p < 0.01). (B) This effect is seen in both genders (F (3,22)=12.77, p < 0.001). There is no significant difference in time the animals spent in theCZ of the OF, neither in strain ((C), U(14,12)=77, z=-0.33, n.s.), nor in gender ((D), H(3)=5.95, n.s.). 82 4 Results

4.2.3SD Rats Show Deﬁcits in Spatial Learning Compared to LE Rats

Figure 4.5: LE vs. SD – Double H – Latency. SD rats took significantly longer to find the escape platform compared to LE rats as shown in the overall means ((A), U(238,221) = 20177.5, z = -4.31, p < 0.001) and broken down to the single trials (B). There are significant differences in trials D1.1 (p < 0.001), D2.1 (p < 0.05) and the test trial (p < 0.01). (C) This effect of the overall mean is seen when comparing genders (H(3) = 24.53, p < 0.001), but is only pronounced in female animals (female p < 0.001, male n.s.). (D) When broken down to single trials, there is a significant difference in male animals in trial D1.1 (p < 0.001).

TheDH maze is used to examine spatial learning in rodents. The latency until the animals find the escape platform is measured and errors are counted. Rats of theSD strain needed significantly more time to find the escape platform with 16.3 ± 2.60 s compared to those of theLE strain with 11.6 ± 1.71 s (overall mean, U (238,221) = 20177.5, z = -4.31, p < 0.001, Figure 4.5 (A)). When broken down to the single trials, significant differences were seen twice and additionally the test trial was significantly different (rm ANOVA, group x trial, F(16,400) = 2.09, p < 0.01, Figure 4.5 (B)). 83 4 Results

The significant differences were seen particularly on the first trials of day one ((D1.1: LE: 32.4 ± 3.69 s,SD: 47.3 ± 4.77 s, p < 0.001) and two (D2.1:LE: 7.29 ± 0.39 s,SD: 21.1 ± 4.47 s, p < 0.05).SD animals were also significantly slower in finding the platform during the test trial on the last day ((LE: 17.1 ± 1.52 s,SD: 28.7 ± 4.68 s, p < 0.01).

Figure 4.6: LE vs. SD – Double H – Initial Errors. Comparing the strains, there was no significant difference present in the overall mean of the IEs (U(238,221) = 23910, z = -1.68, n.s.)and when splitting the data to single trials ((B), F (15,375)=0,76, n.s.). Gender-wise data showed a significant difference in the number ofIEs ((C), H(3) = 27.28, p < 0.001), specifically in female animals (LE

When dividing the results up gender-wise, this significant effect was seen in the overall mean (H (3) = 24.53, p < 0.001) with SD females taking significantly longer to find the platform compared to LE females (SD females: 17.3 ± 2.00 s,LE females: 11.3 ± 1.93 s, U (119,102) = 3995.5, z = -4.38, p < 0.001), but with no significant difference when comparing the males (SD males: 15.5 ± 3.27 s, U (119,119) = 6252.5, z = -1.56, n.s., Figure 4.5 (C)). In the single trials (rm ANOVA, gender x trials, 84 4 Results

F(48,368) = 1.92, p < 0.001), the most pronounced effect was the significant difference between males in trial one on day one (SD males: 56.4 ± 3.57 s,LE males: 27.3 ± 5.73 s, p < 0.001, Figure 4.5 (D)). Furthermore, there was no significant difference inIEs when comparing the strains withLEs having a mean of 0.50 ± 0.15IEs andSDs having a mean of 0.85 ± 0.17IEs (overall mean, U (238,221) = 23910, z = -1.68, n.s., Figure 4.6 (A)), but a significant difference was revealed when comparing genders (H (3) = 27.28, p < 0.001, Figure 4.6 (C)). This was due to a pronounced significant difference in female animals (LE female: 0.48 ± 0.18IEs,SD females: 1.32 ± 0.21IEs, U (119,102) = 4636, z = -3,02, p < 0.01), male animals did not show a significant difference (LE males: 0.48 ± 0.15IEs,SD males: 0.45 ± 0.19IEs, U (119,119) = 6865 z = 0,41, n.s.). The siginificant differences were not present, when the data was broken down to single trials, neither in strain (rm ANOVA, group x trial, F(15,375) = 0.76, n.s., Figure 4.6 (B)), nor in gender (rm ANOVA, gender x trial, F(45,345) = 0.80, n.s., Figure 4.6 (D)).

4.2.4 No Strain and Gender Diﬀerences are Detected in EPM and FST

Figure 4.7: LE vs. SD – Elevated Plus Maze. (A)LE andSD rats spent similar times in the closed arm of the EPM( t(26) = 1.51, n.s.) and also in gender there were no signiﬁcant diﬀerences (F (3,24) = 2.97, n.s. (B)).

In the EPM, a measure of anxiety,LE rats spent 62.0 ± 3.44 % of the time in the closed arms of the apparatus,SDs a little less with 55.1 ± 2.98 %. Splitting up the data gender-wise,LE males spent slightly more time in the closed arms with 69.4 ± 5.37 % compared toSD males with 55.0 ± 4.99 %. In females, no diﬀerence 85 4 Results

could be observed withLE females spending 54.5 ± 1.94 % andSD females spending 55.3 ± 3.69 % in the closed arms. Signiﬁcant diﬀerences could neither be detected in strain (t(26) = 1.51, n.s., Figure 4.7 (A)), nor in gender (o.w. ANOVA, F(3,24) = 2.97, n.s., Figure 4.7 (B)).

Figure 4.8: LE vs. SD – Forced Swim Test. (A) Both strains showed low levels of immobility that increased in a similar manner over time (F (9,225) = 1.81, n.s.) and also the total immobility (B) was not significant different (U(14,13) = 81, z = 0.46, n.s.). A significant difference was present in gender-wise, single bin data ((C), F (27,207) = 1.64, p < 0.05), but post-hoc testing revealed no significant difference betweenLE andSD males or females, respectively. (D) Total immobility was not significant different in males or in females (H(3) = 6.26, n.s.).

BothLE andSD animals were hardly showing any immobility in the FST.LEs showed a maximum immobility in the last bin of 8.21 ± 1.47 s,SDs showed their maximum immobility during the second last bin with 6.79 ± 1.83 s. No signiﬁcant diﬀerences between strains were present when looking at the single time bins (rm ANOVA, group x bins, F(9,225) = 1.81, n.s., Figure 4.8 (A)) or the total immobil- 86 4 Results

ity (LE: 10.7 ± 2.03 %,SD: 10.6 ± 2.59 %, U (14,13) = 81, z = 0.46, n.s., Figure 4.8 (B)). However, gender-wise data showed a significant difference in the single bins (rm ANOVA, gender x bins, F(27,207) = 1.64, p < 0.05, Figure 4.8 (C)), but post-hoc testing revealed no significant differences when comparingLE andSD males and females, respectively. Gender-wise total immobility confirmed no significant difference within the distinct gender, although females showed a tendency to a higher immobility (LE females: 14.7 ± 3.34 %,SD females: 15.5 ± 3.61 %,LE males: 6.66 ± 1.19 %, SD males: 7.39 ± 2.81 %, H (3) = 6.26, n.s., Figure 4.8 (D)).

4.2.5 Sucrose Preference is Equally Pronounced in Both Strains

Figure 4.9: LE vs. SD – Sucrose Preference Test. (A)SD rats consumed significantly more sucrose solution per bodyweight compared toLE rats t(26) = -4.47, p < 0.001). (B)This effect was due to a pronounced difference in female animals, but not males (F (3,24) = 10.89, p < 0.001, post-hoc:SD female >LE female p < 0.001). (C) Both strains preferred sucrose over water in the same manner (U(14,14) = 58, z = -1.82, n.s.), and also in gender, there was no significant difference in sucrose preference ((D), H(3) = 4.73, n.s.). 87 4 Results

With 497 ± 48.5 ml kg−1,SD rats consumed significantly more of the 4 % sucrose solution in 24 h compared toLE rats with 232 ± 34.0 ml kg−1 (t(26) = -4.47, p < 0.001, Figure 4.9 (A)). This effect resulted from the pronounced significant difference in female animals (SD females: 602± 72.1 ml kg−1,LE females: 254 ± 64.6 ml kg−1), which was not present in male animals (SD males: 393 ± 36.4 ml kg−1,LE males: 212 ± 26.0 ml kg−1, o.w. ANOVA, gender, F(3,24) = 10.89, p < 0.001, post-hoc:SD female >LE female p < 0.001, Figure 4.9 (B)). Regarding the sucrose preference over water, there was no significant difference between strains, asLE animals consumed a mean of 87.6 ± 3.85 % andSD animals a mean of 91.7 ± 2.18 % of the sucrose solution (U (14,14) = 58, z = -1.82, n.s., Figure 4.9 (C)). No significant differences were found in gender,LE males drank 91.6 ± 0.96 % andSD males 94.0 ± 1.29 %, LE females drank 83.6 ± 7.62 % andSD females 89.3 ± 4.14 % (H (3) = 4.73, n.s., Figure 4.9 (D)).

4.2.6LE Males Emit Less Calls in the High Band Compared to SD Males

Recording of USV showed thatLE animals emitted 46.4 ± 12.02 % of calls in the high band (40 – 60 kHz) and rats of theSD strain emitted 82.3 ± 3.32 % in this band, indicating a tendency that theLEs tend to have less calls in the high band (U (14,14) = 65, z = -1.49, n.s., Figure 4.10 (A)).

Figure 4.10: LE vs. SD – Ultrasonic Vocalisation. (A) In the high band, 40 – 60 kHz,LE rats showed a tendency to emitting less calls compared toSD rats, but the difference was not significant ( U(14,14) = 65, z = -1.49, n.s.). (B) Female rats emitted nearly the same amount of calls in the high band, but in males there was a significant difference withLE males emitting less calls ( U(7,7) = 6.5, z = -2.24, p < 0.05). 88 4 Results

In comparing the gender, there was no difference in female animals,LE females emitted 78.5 ± 6.29 % of calls in the high band andSD females 77.9 ± 3.83 % (U (7,7)=22, z=0.26, n.s.), but a significant difference was detected in male animals, withLE males emitting less calls (LE males: 14.3 ± 10.10 %,SD males: 86.7 ± 2.45 %, U (7,7) = 6.5, z = -2.24, p < 0.05, Figure 4.10 (B)). In the low band (20 – 30 kHz), the rats did not emit or hardly emitted any calls (LE: 1.21 ± 0.37,SD: 0.50 ± 0.20). There was no significant difference, neither in group (U (14,14) = 69,50, z = 1.29, n.s.), nor in gender (H (3) = 4.93, n.s.).

4.3 Establishment of the CMUS Protocol

For the CMUS pilot, one group ofLE rats underwent a seven week stress protocol, whereas the control group was kept under normal conditions. After these weeks, a battery of behavioural testing followed, to read out the eﬀects of the chronic stress. See Table 4.2 for a summary of the results.

Table 4.2: CMUS – Summary of Results Test Main findings CMUS vs. CTRL Weight Slight decrease in CMUS animals between week 2 – 7, no sig. differences ELISA No sig. differences between CMUS and CTRL rats, but sig. increased values post-CMUS in both groups EPM No differences SPT No sig. differences, but slight drop in sucrose consumption in CMUS group after week 4 OF No differences SIT No differences FST No differences USV No differences OR CMUS animals spent sig. less time with novel object 89 4 Results

4.3.1 CMUS Had no Signiﬁcant Eﬀect on the Weight of the Animals

The CMUS protocol did not have a significant effect on the weight of the stressed animals. After eight weeks, CTRL rats reached 138 ± 2.7 % of their baseline weight and CMUS rats reached 137 ± 1.6 %. The curve of the normalized data looks similar to the one of the control animals, except for the time between stress week two and seven, where stressed animals showed a small but non-significant decrease in weight (rm ANOVA, group x time, F(8,104) = 1.85, n.s., Figure 4.11 (A)).

Figure 4.11: CMUS – Physiological Measurements. (A) The weight development was not significantly different between groups. The normalized data increased in a similar manner in CTRL and CMUS animals (rm ANOVA, group x time, F (8,104) = 1.85, n.s.), a small but not significant decrease in weight could be observed between stress week two and seven in the CMUS group. (B) Levels of FCM were not significantly different between groups (rm ANOVA, group x time point, F (1,13) = 1.06, n.s.), but the overall data increased significantly post-CMUS(rm ANOVA, time point, F (1,13) = 20.60, p < 0.001) compared to pre-CMUS.

4.3.2 Levels of FCM Increased Signiﬁcantly in Both Groups After CMUS

Measures of FCM were performed to read out the stress level of the rats. Baseline FCM values were 171 ± 31.5pg ml−1 in CTRL rats and 292 ± 41.9pg ml−1 in CMUS rats. In the second measurement post-CMUS, those levels increased in both groups, more specifically up to 1027 ± 244.8pg ml−1 in the CTRL group and up to 832 ± 176.0pg ml−1 in the CMUS group. No significant differences between groups 90 4 Results

could be detected (rm ANOVA, group x time point, F(1,13) = 1.06, n.s., Fig- ure 4.11 (B)). However, there was an overall eﬀect, comparing the data pre-CMUS with post-CMUS, with the post-CMUS data being signiﬁcantly increased (pre: 228 ± 29.7pg ml−1, post: 936 ± 151.1pg ml−1, rm ANOVA, time point, F(1,13) = 20.60, p < 0.001).

4.3.3 CMUS Did Not Have an Eﬀect on the Rats Performance in EPM, SPT,OF, SIT, FST and USV

The EPM did not show signiﬁcant diﬀerences concerning anxiety-related behaviour between groups (ind. t-test, t(13) = -0.78, n.s., Figure 4.12 (A)). However, CMUS animals spent a slightly increased time in the closed arm of the apparatus (59.7±7.41 % vs. 52.4 ± 5.86 %).

Figure 4.12: CMUS – Elevated Plus Maze and Sucrose Preference. (A) Both CTRL and CMUS rats spent a similar amount of time in the closed arms of the EPM (ind. t-test, t(13) = -0.78, n.s.). (B) Over the time course of nine weeks, no signiﬁcant diﬀerence between groups could be detected in sucrose consumption during the weekly SPT (rm ANOVA, F (8,104) = 1.19, n.s.).

A SPT was performed as baseline before the start of the CMUS protocol and then animals were tested once a week for indication of change during the stress period. Baseline values were 35.2 ± 4.08ml kg−1 in one hour for the CTRL group and 33.3 ± 4.35ml kg−1 for the CMUS group. The rats did not show a diﬀerence in sucrose consumption over the length of the experiment (rm ANOVA, F(8,104) = 1.19, n.s., Figure 4.12 (B)), but a small decrease in the stressed group could be observed after 91 4 Results

stress week four with reaching the most pronounced difference after stress week six with 37.2 ± 4.75ml kg−1 of consumption in CTRL rats and 30.8 ± 3.49ml kg−1 in CMUS rats. After ending the CMUS protocol, levels went back to an equal value in both groups. During theOF, rats of the CMUS group had a slightly shorter track length with 5175 ± 557.5 cm compared to CTRL rats with 5791 ± 587.4 cm, but this difference was not significant (U (8,7) = 21, z = 0.75, n.s., Figure 4.13 (A)). Both groups spent a very similar amount of time in theCZ of theOF(CMUS: 58.0 ± 10.43 s, CTRL: 60.8 ± 9.15 s, ind. t-test, t(13) = 0.20, n.s., Figure 4.13 (B)).

Figure 4.13: CMUS – Open Field Test. (A) Rats of the CMUS group had a slightly, but not signiﬁcantly shorter track length in the OF( U(8,7) = 21, z = 0.75, n.s.). (B) The time both groups spent in theCZ of theOF was very similar (t(13) = 0.20, n.s.).

In the SIT, the rats’ social behaviour towards age- and gender-matched, strange or familiar animals was examined. During the 5 min habituation, the animals did not show any preference for one of the two empty cages, the time spent with the cages was approximately 50 : 50 (time spent with cage S1, CTRL: 50.3 ± 2.93 %, CMUS: 50.7 ± 2.97 %). In trial 1, both groups spent clearly more time with the strange rat (Stranger 1) compared to the empty cage (percent of time spent with Stranger 1 of total time spent with the cages, CTRL: 85.8 ± 2.53 %, CMUS: 73.1 ± 10.76 %). Thus, CMUS rats spent a little less time with Stranger 1 than CTRL rats, but this diﬀerence was not signiﬁcant (U (8,7) = 22, z = 0.64, n.s., Figure 4.14 (A)). During trial 2, the duration of social contacts with the new stranger (Stranger 2) was increased compared to the familiar animal (Stranger 1). Rats of the CTRL group spent 67.4 ± 4.82 % of the total time spent with the two rats with Stranger 2, rats of the CMUS group 64.6 ± 5.08 %, which was a very similar behaviour (t(13) = 0,39, n.s., Figure 4.14 (B)). 92 4 Results

Figure 4.14: CMUS – Social Interaction Test. (A) CTRL and CMUS rats spent more time with Stranger 1 than with an empty cage. In CMUS this was a little less pronounced, but the difference between groups was not significant (U(8,7) = 22, z = 0.64, n.s.). (B) Both groups spent more time with a new strange rat (Stranger 2) than with the now familiar rat Stranger 1, there was no significant difference between groups regarding this (t(13) = 0.39, n.s.).

In the FST, which is a classical test for depressive-like behaviour, the animals hardly showed any immobility. The curves of both groups rose in a similar manner until they reached their maximum value in the last bin, with a maximum immobility of 14.9 ± 2.69 s in CTRL rats and 9.49 ± 3.04 s in CMUS rats. No significant differences could be detected in the data broken down to single bins (rm ANOVA, group x bins, F(13,169) = 0.70, n.s., Figure 4.15 (A)). The total immobility was slightly lower in CMUS animals with 13.1 ± 2.26 % compared to 15.6 ± 2.79 % in the CTRL group, but this effect was not significant (ind. t.test, t(13) = 0.70, n.s., Figure 4.15 (B)).

USVs can reflect the animals’ emotional state, with the high band, 40 – 60 kHz, being linked to excitement and the low band, 20 – 30 kHz, being linked to negative or aversive behaviour. In these recordings, the majority of the rats did not emit calls in the low band, the mean number of calls per 20 min was 0.25 ± 0.25 in CTRL rats and 1.0 ± 0.69 in CMUS rats. The vast majority of calls was emitted in the high band in both pre-CMUS and post-CMUS recordings. In CTRL animals, the percent of calls in the high band was 73.9 ± 11.86 % of the total calls pre-CMUS and 71.1 ± 11.33 % post-CMUS. In CMUS rats those values were marginally less with 57.3 ± 15.92 % pre-CMUS and 59.4 ± 15.52 % post-CMUS. Neither in the pre-, nor in the post-CMUS recordings could be detected any significant differences between groups or pre and post time point (rm ANOVA, group x time point, F(1,13) = 0.03, n.s., Figure 4.16 (A)). 93 4 Results

Figure 4.15: CMUS – Forced Swim Test Test. (A) The immobility, here shown in 30 s bins, rises in a similar manner in both groups, no significant differences could be detected (rm ANOVA, group x bins, F (13,169) = 0,70, n.s.). (B) The total immobility in percent of the total time is somewhat lower in CMUS rats compared to the immobility of the CTRL rats, but this difference was not significantly different (ind. t.test, t(13) = 0.70, n.s.).

4.3.4 Rats That Underwent CMUS Spent Signiﬁcantly Less Time with Novel Objects

Figure 4.16: CMUS – Ultrasonic Vocalization and Object Recognition. (A) CMUS rats emitted marginally less calls in the high band, 40 – 60 kHz, compared to CTRL rats, but this difference was not significant. Furthermore there was no difference in emitted calls pre- and post-CMUS(rm ANOVA, group x time point, F (1,13) = 0.03, n.s.). (B) CTRL and CMUS rats spent approximately the same amount of time with the novel object in the OR (ind. t-test, t(13) = 0.67, n.s.), but the number of contacts with the novel object was significantly different with being lower in CMUS animals (ind. t-test, t(13) = 3.61, p < 0,01).

With theOR the rats’ ability to distinguish between familiar and novel objects was tested. The time the rats spent with the novel object was measured as well as the 94 4 Results

number of contacts. In the duration of contacts with the novel object, no difference was seen between groups. CTRL animals spent 46.4 ± 6.62 % of the total time spent with the objects with the novel object, CMUS animals slightly less with 40.3 ± 6.07 % (ind. t-test, t(13) = 0.67, n.s., Figure 4.16 (B) Duration). This difference is more pronounced when looking at the number of contacts with the novel object. Here, the number of contacts reached 51.8 ± 3.23 % in CTRL rats, whereas it only reached 36.8 ± 2.48 % in CMUS rats, which was significant different (ind. t-test, t(13) = 3.61, p < 0.01, Figure 4.16 (B) Number).

4.4 Optogenetic Stimulation of the Medial Forebrain Bundle in the Flinder’s Sensitive Line Rat Depression Model - 6-OHDA Lesioned vs. Unlesioned Rats

The project 6-OHDA-stim examined the eﬀect of optogenetic stimulation of the MFB in the FSL depression model. To research the role of DA regarding this question, animals with a lesioned DA-system (DA-) were compared to animals with an intact system (DA+). Half of the group received stimulation as treatment prior to every behaviour test, respectively (DA+stim, DA-stim). The rats were tested in activity, FST,OF, SIT, SPT and USV. The results are summarized in Table 4.3.

Table 4.3: 6-OHDA-Stim – Summary of Results Test Main ﬁndings Lesion DA- groups had sig. lessTH+ neurons in the VTA Amph.-induced Activity increased more in DA+ animals, but not sig. activity Stim.-induced Activity under stim. was sig. increased activity SPT Less consumption / preference in DA-stim, but not sig. OF Track length in stim rats slightly increased, but not sig. SIT Slightly increased in DA+stim rats, stim rats spent more time in zone2, but not sig. FST No sig. diﬀerences USV Tendency to more calls in DA- rats in high band, but not sig. 95 4 Results

To evaluate the amount of the lesion,TH-positive neurons in the VTA were counted. Cell numbers do not relate to the VTA in total, but to the three counted slices, where the VTA was most pronounced. DA- rats with an overall mean of number of 52.7 ± 6.82 and DA-stim rats with 55.9 ± 8.00 had signiﬁcantly lessTH-positive neurons compared to DA+ rats with 129 ± 15.1 and DA+stim rats with 113 ± 10.7 (o.w. ANOVA, group, F(3,18) = 14.80, p < 0.001, post-hoc: DA- < DA+, p < 0.001, DA-stim < DA+stim, p < 0.01, Figure 4.17 (A).

Figure 4.17: 6-OHDA-Stim – Histology and Ultrasonic Vocalization. (A) DA-lesioned rats had significantly lessTH-positive neurons in the VTA compared to rats with an intact system (o.w. ANOVA, group, F (3,18) = 14.80, p < 0.001, post-hoc: DA- < DA+, p < 0.001, DA-stim < DA+stim, p < 0.01). For assessing these numbers, the three brain slices with the most pronounced VTA were counted. (B) In USV recordings, animals of the DA- groups showed tendencies to emit more calls in the high band (40 – 60 kHz), but the differences were not significant between groups (H(3) = 5.584, n.s.).

4.4.1 Mean Transfection Rate of VTA DA neurons Was Low

In the 6-OHDA-stim project, the unspecific AAV-hSyn-ChR2(H134)-EYFP was injected into the VTA, which resulted not only in transfected DA neurons, also other neuron types were transfected. Figure 4.18 (A) shows a schematic representation of the injection and stimulation protocol with different transfected neurons (triangles, circles and squares) in and around the VTA and the stimulation of the MFB. In Figure 4.18 (B), a confocal microscope picture of the VTA in 20 x magnification is shown. It can clearly be seen that the virus is not exclusively expressed in TH- positive neurons (red) but also in other neuron types in and around the VTA. In pilot experiments, this injection protocol resulted in transfection rates of over 60 %. However, in this project, the rates were much lower. In DA+ rats, 24.6 ± 3.21 % of 96 4 Results

theTH-positive neurons also were GFP-positive, in DA+stim rats 27.9 ± 6.57 %, in DA- rats 39.4 ± 7.81 % and in DA-stim rats 25.0 ± 4.32 %.

Figure 4.18: 6-OHDA-Stim – Evaluation of Transfection Rate. (A) Schematic representation of the injection and stimulation protocol. The unspecific AAV was injected into the VTA, where it transfected not only DA neurons (triangles) but also other present neuron types (circles / squares). Optogenetic stimulation happened in the area of the MFB. (B) Confocal microscopy pictures in 20 x magnification of the VTA of one example animal.TH is shown in red, GFP in green, DAPI in blue. Clearly to see is the unspecific transfection of all present neuronal types in the GFP staining. 97 4 Results

Also the positioning of the light cannulas was veriﬁed using immunohistochemistry. Figure 4.19 shows the correct position of the light cannula in the MFB at approximately -2.8 mm from Bregma.

Figure 4.19: 6-OHDA-Stim – Evaluation of Cannula Position. The left side shows an example slice stained for TH, using a DAB staining. On the right side the respective slide from the Paxinos brain atlas is shown. The blue circle indicates the MFB, on the left side the position of the cannula inside this circle can be seen. Modiﬁed from Paxinos and Watson 2007.

4.4.2 Amphetamine-induced Activity Was Decreased in Lesioned Rats

Measurements of homecage-activity induced by i.p. injections of amphetamine are a readout possibility to evaluate the extent of the DA lesion. The less intact DA neurons are still present, the less the animals are activated in their locomotion. When comparing the activity levels after saline injection with the activity after amphetamine injection, activity increased in DA+ as well as in DA- rats. In DA+ rats, with their intact DA system, this increase was much more pronounced but not 98 4 Results

signiﬁcantly diﬀerent with a plus of 721 ± 288.1 % compared to 285 ± 78.8 % in DA- animals (U (9,13) = 31, z = 1.80, n.s., Figure 4.20 (A).

Figure 4.20: 6-OHDA-Stim – Activity Measures. (A) Homecage-activity increased less in DA- animals compared to DA+ animals after amphetamine injection, but the difference was not significant (U(9,13) = 31, z = 1.80, n.s.). (B) When comparing activity without optogenetic stimulation with activity under stimulation, there was no difference between DA+stim and DA-stim groups, but comparing the overall data without stimulation versus stimulation showed a significant increase of activity under stimulation (rm ANOVA, w/o stim vs. stim, F (1,9) = 6.20, p < 0.05).

4.4.3 Optogenetic Stimulation Increased Homecage-Activity

Furthermore, the homecage-activity under optogenetic stimulation was evaluated. 30 min without stimulation were compared with 30 min under stimulation. Without stimulation, the mean number of light barrier crossings in rats of the DA+stim group was 363 ± 63.3 and in DA-stim rats very similar with 355 ± 46.9. Under stimulation, these values increased in both groups, in DA+stim up to 417 ± 121.3 and in DA-stim up to 484 ± 54.2. There was no significant difference between groups, but comparing the overall data without stimulation versus stimulation revealed a significant increase of activity under stimulation (rm ANOVA, w/o stim vs. stim, F(1,9) = 6.20, p < 0.05, Figure 4.20 (B). 99 4 Results

4.4.4 Testing of a Depressive-like Phenotype Did Not Show Signiﬁcant Diﬀerences Between Groups

In the FST, as a measure of hopelessness and despair, no or hardly any immobility could be detected. DA+stim animals did not show any immobility, the other groups reached their maxima during the last or the second last bin with 2.26 ± 1.43 s in DA-, 2.11 ± 1.23 s in DA-stim and 0.69 ± 0.69 s in DA+. The data broken down to 30 s bins was not significantly different between groups (rm Anova, 7 last bins x group, F(12,90) = 0.77, n.s., Figure 4.21 (A)). Total immobility was 0.95 ± 0.60 % in the DA- group, 1.20 ± 0.76 % in DA-stim and 0.22 ± 0.22 % in DA+. No significant differences could be observed (H (3) = 2.72, n.s., Figure 4.21 (B)).

Figure 4.21: 6-OHDA-Stim – Forced Swim Test. (A) Single-bin-data did not show significant differences between the four groups (rm Anova, 7 last bins x group, F (12,90) = 0.77, n.s.) and animals generally showed hardly any immobility. (B) This is also seen in the total immobility, reaching only a maximum of 1.20 ± 0.76 % in DA-stim rats. Between groups, no significant differences were observed (H(3) = 2.72, n.s.).

Sucrose consumption during the one hour SPT was slightly lower in lesioned animals. DA+ rats consumed 64.6 ± 10.53 ml kg−1 of the 4 % sucrose solution, Da+stim rats 58.2 ± 6.30 ml kg−1, DA- rats 57.2 ± 3.49 ml kg−1 and DA-stim rats 44.6 ± 6.60 ml kg−1. The consumption rate was not significantly different between the four groups (o.w. ANOVA, groups, F(3,18) = 1.58, n.s., Figure 4.22 (A)). The sucrose preference over water was similarly high in all groups except for the DA-stim group, where it was somewhat lower. DA+ rats drank 80.8 ± 3.98 % of the sucrose solution, DA+stim 81.9 ± 0.57 %, DA- 81.2 ± 2.73 % and in DA-stim rats 72.5 ± 5.80 %. This difference between groups was not significant (H (3) = 1.90 n.s., Figure 4.22 (B)). 100 4 Results

Figure 4.22: 6-OHDA-Stim – Sucrose Preference Test. (A) Consumption of the 4 % sucrose solution in one hour did not diﬀer between groups (o.w. ANOVA, groups, F (3,18) = 1.58, n.s.), (B) neither did the sucrose preference over water (H(3) = 1.90, n.s.).

In USV recordings, the majority of the animals did not emit calls in the low band (20 – 30 kHz). Rats of the DA+ group emitted 1.50 ± 0.81 calls, DA+ stim 1.33 ± 1.15, DA- 0.43 ± 0.20 and rats of the DA-stim group emitted no calls in the low band. There were no significant differences between groups (H (3) = 5.84, n.s.). Calls in the high band (40 – 60 kHz) were 35.5 ± 17.52 % of the total calls emitted in DA+ animals, 36.9 ± 19.23 % in DA+stim, 83.9 ± 3.41 % in DA- and 57.0 ± 17.10 % in DA-stim. Although especially DA- rats seemed to emit more calls in the high band compared to the other groups, this difference was not significant (H (3) = 5.58, n.s., Figure 4.17 (B)).

4.4.5 Social Behaviour as Measured in the SIT Did Not Diﬀer Between Groups

During habituation, the rats did not show any preference for neither of the cages, DA+ rats spent 52.0 ± 8.94 % of the total time they spent with the cages with the cage of Stranger 1, DA+stim 49.7 ± 5.77 %, DA- 56.0 ± 4.14 % and DA-stim 53.5 ± 8.14 % (o.w. ANOVA, group, F(3,18) = 0.12, n.s.). In trial 1, all of the rats spent clearly more time with Stranger 1 than with the empty cage. The groups’ values were similar except for group DA-stim, in which the duration of contacts was a little lower. DA+ animals spent 88.8 ± 4.36 % of the total time they spent with the cages with Stranger 1, DA+stim 91.8 ± 3.34 %, DA- 86.5 ± 3.83 % and DA-stim 71.2 ± 12.03 %. This duration of contacts was not signiﬁcantly diﬀerent between the four groups (H (3) = 3.57, n.s., Figure 4.23 (A)). In trial 2, in which the rats could choose to visit the familiar rat from trial 1 or a new stranger (Stranger 2), all of the 101 4 Results

animals spent the majority of the time with Stranger 2. The duration of contacts was 56.5 ± 9.38 % in rats of the DA+ group, 71.9 ± 7.87 % in Da+stim, 60.1 ± 7.26 % in DA- and 62.5 ± 7.27 % in DA-stim rats. Comparing the four groups, this was not significantly different (o.w. ANOVA, group, F(3,18) = 0.54, n.s., Figure 4.23 (B)). Furthermore, stimulated rats of both lesioned and intact DA-system rats, seemed to spend more, but not significantly more time in the zone of the apparatus, where the cage with Stranger 2 was located. The difference of the time the rats spent in zone 2 and the time they spent in zone 1 were in the negative range for the unstimulated animals (DA+: -16.3 ± 94.82 s, DA-: -56.0 ± 82.77 s), whereas the difference was in the positive range for the stimulated groups (DA+stim: 174 ± 157.39 s, DA-stim: 72.7 ± 55.79 s). Nevertheless, these result were not significantly different between the groups (o.w. ANOVA, group, F(3,18) = 1.11, n.s., Figure 4.23 (C)).

Figure 4.23: 6-OHDA-Stim – Social Interaction Test. (A) In trial 1, all of the animals spent more time with Stranger 1 than with the empty cage. There was no significant difference between the groups (o.w. ANOVA, group, F (3,18) = 0.12, n.s.). (B) In trial 2, all groups spent the majority of the time with the new Stranger 2 than with the familiar animal Stranger 1, but there was no significant difference in this behaviour between the four groups (o.w. ANOVA, group, F (3,18) = 0.54, n.s.). (C) In trial 2, stimulated animals seemed to spend more time in the zone, where the cage of Stranger 2 was located (zone 2), compared to unstimulated rats, which seemed to spend more time in the zone where the familiar animal was located (zone 1). This difference was not significant between the groups (o.w. ANOVA, group, F (3,18) = 1.11, n.s.). 102 4 Results

4.4.6 No Diﬀerences Were Seen in the Animals’ Exploratory Behaviour

TheOF measures the exploratory behaviour of the rats in measuring the track length and the time, the rats spend in theCZ of the field. Stimulated animals showed a slightly longer track length compared to unstimulated rats with DA+ having walked 10799 ± 2706.0 cm, DA+stim 16610 ± 2480.4 cm, DA- 11417 ± 3114.2 cm and DA- stim 13498 ± 2231.40 cm. The track length was not significantly different between the groups (o.w. ANOVA, group, F(3,18) = 0.72, n.s., Figure 4.24 (A)). The time, the rats spent moving or sitting in theCZ of theOF varied little between groups, with DA- rats seeming to spend more time there with 242 ± 48.2 s, compared to DA+ with 121 ± 30.0 s, DA-stim with 175 ± 28.9 s and DA+stim with 165 ± 40.8 s. These slight differences between the groups were not significant (o.w. ANOVA, group, F(3,18) = 1.62, n.s., Figure 4.24 (B)).

Figure 4.24: 6-OHDA-Stim – Open Field Test. (A) The track length in theOF was not significantly different between the groups (o.w. ANOVA, group, F (3,18) = 0.72, n.s.), (B) neither was the duration the animals spent in the CZ of the field (o.w. ANOVA, group, F (3,18) = 1.62, n.s.).

4.5 Dopamine-speciﬁc Optogenetic Stimulation of the Medial Forebrain Bundle in a Stress-induced Rat Depression Model

The DA-Stim project focused on the role of the DA-system in stimulation of the MFB in a rat depression model. For the speciﬁc stimulation of VTA DA neurons a combination of genetically alteredTH::Cre rats and a ﬂoxed AAV2 carrying the 103 4 Results

ChR2 was used. A depressive-like phenotype was induced by a 5-week CMUS protocol. This was followed by a battery of behavioural tests including activity measurements, EPM,OF, SIT, FST, SPT and USV. Furthermore, a new test as an alternative for the FST was implemented, the OSST. As in the prior experiments, one of the groups (DA-stim) received 30 min of optogenetic stimulation before each test and was compared to a control group (DA-CTRL) not receiving any treatment. Additionally the weight and FCM levels of all animals were monitored. The results are summarized in Table 4.4.

Table 4.4: DA-Stim – Summary of Results Test Main findings Weight No sig. group effect FCM Levels in DA-stim were sig. higher, overall levels decreased sig. over time Stim.-induced No increase in activity under stimulation activity EPM DA-stim rats spent sig. less time in closed arms OSST Track length under stimulation was sig. increased OF DA-stim rats had a sig. shorter track length and spent sig. less time in theCZ SIT DA-stim rats had sig. more contacts with both stranger1 and stranger2 FST No sig. differences SPT No sig. differences USV No sig. differences

4.5.1 The Applied Virus Injection Parameters Led to a Transfection Rate of over 50 %

The applied virus injection protocol with two tracks and two deposits per track for each hemisphere with a total injected volume of 6.4 µl resulted in a mean transfection rate of over 50 %. In DA-CTRL animals, 50.7 ± 3.46 % of allTH-positive neurons were also GFP-positive and in DA-stim animals 56.1 ± 2.35 %. This transfection rate was not signiﬁcantly diﬀerent between the two groups (ind. t-test, t(20) = -1.39, n.s.). Confocal microscopy pictures of the VTA of an example animal and a schematic representation of the injection and stimulation protocol can be seen in Figure 4.25 on the following page. 104 4 Results

Figure 4.25: DA-Stim – Evaluation of Transfection Rate. (A) Schematic representation of the injection and stimulation protocol. The specific / floxed AAV was injected into the VTA, where it transfected only DA neurons (triangles). Optogenetic stimulation happened in the area of the MFB. (B) Confocal microscopy pictures in 20 x magnification of the VTA of one example animal.TH is shown in red, GFP in green, DAPI in blue. Clearly to see is the DA-specific transfection of neurons. 105 4 Results

4.5.2 The Weight Development in Stressed Animals Was Slightly Decreased

The weight development of the rats was an important read out for the stress protocol. Baseline weight for rats of the DA-CTRL group was 244 ± 5.4 g, for rats of the DA- stim group 249 ± 6.9 g. In control rats, the weight increased to 286 ± 6.8 g, in DA-stim rats to 285 ± 7.2 g. In the normalized data, it could be observed, that already after stress week 2 the weight of the animals in both groups raised slightly slower than before. The weight of both groups developed in a similar way, although statistically diﬀerences were detected (rm ANOVA, group x time point, F(7,140) = 3.10, p < 0.01, Figure 4.26 (A)). Nevertheless, in post-hoc testing, no group diﬀerences could be detected.

Figure 4.26: DA-Stim – Physiological Measurements. (A) The weight development of the rats was very similar, although animals of the DA-stim group gained weight a little slower compared to DA-CTRL rats (rm ANOVA, group x time point, F (7,140) = 3.10, p < 0.01) No significant group differences were detected in post-hoc testing. (B) Levels of FCM were significantly higher in DA-stim rats compared to DA-CTRLs (rm ANOVA, group, F (1, 20) = 8,76, p < 0.01.) In both groups, the FCM levels were first dropping, but between the end of the CMUS protocol and the end of the behavioural testing phase, the levels of the stimulated animals kept further dropping, whereas the levels of the controls were increasing again.

4.5.3 Corticosterone Levels Decreased over the Length of the Experiment

For further examination of the animals’ stress level, their FCM levels were measured. For DA-CTRL rats the baseline value was 567 ± 89.0 pg ml−1, the postCMUS value 106 4 Results

was 378 ± 48.9 pg ml−1 and the post-stim value was 429 ± 65.9 pg ml−1. Those values for the DA-stim group were 998 ± 160.9 pg ml−1 for baseline, 582 ± 146.6 pg ml−1 for post-CMUS and 509 ± 48.4 pg ml−1 for post-stim. In the rm ANOVA only a group eﬀect could be detected (F(1, 20) = 8.76, p < 0.01, Figure 4.26 (B)). So in the overall mean, DA-stim rats had signiﬁcantly increased levels of FCM compared to controls. But when looking at the time period between the end of the stress protocol and the end of the behaviour testing phase, it could be observed that in stimulated rats the levels of FCM were slightly dropping, whereas in control rats, the FCM levels were increasing again.

4.5.4 Optogenetic Stimulation Did not Lead to an Increase in Home-Cage-Activity

To examine whether the optogenetic stimulation had an effect on the locomotion of the rats, the activity in their home cage was measured. Animals had a mean number of light barrier crossings of 359 ± 18.4 without stimulation and a mean number of 362 ± 18.8 under stimulation. This was not significantly different (T(11) = 26, z = 0.62, n.s., Figure 4.27 (A)).

Figure 4.27: DA-Stim – Activity and Anxiety Measurements. (A) In home cage activity measurements, the number of light barrier crossing was not increased by stimulation (T (11) = 26, z = 0.62, n.s.). (B) In the EPM, DA-stim rats spent signiﬁcantly less time in the closed arms of the apparatus compared to rats of the DA-CTRL group (t(20) = 2.69, p < 0.05). 107 4 Results

4.5.5 Anxiety-Behaviour was Decreased in DA-Stim Animals

The anxiety-related behaviour was examined in the EPM. DA-CTRL rats spent 72.3 ± 2.63 % of the total time in the closed arms of the apparatus and DA-stim animals 61.4 ± 3.09 %. The results showed that DA-stim rats spent signiﬁcantly less time in the closed arms of the apparatus (t(20) = 2.69, p < 0.05, Figure 4.27 (B)).

4.5.6 DA-Stim Rats Spent Less Time in the Center Zone of the OF Compared to CTRL Rats

Figure 4.28: DA-Stim – Open Field Test. (A) In theOF, control rats had a significantly longer track length compared to DA-stim rats (ind. t-test, t(20) = 2.20, p < 0.05). (B) Also, the time the animals spent in theCZ was significantly higher in DA-CTRLs compared to DA-stim rats (U(11,11) = 13, z = 3.07, p < 0.01). (C) Visit latency until the animals entered theCZ of the arena was not significantly different between the groups (U(11,11) = 46, z = -0.92, n.s.). (D) There was also no significant difference between DA-CTRL and DA-stim rats in the track length within theCZ when set in relation to the total track length (ind. t-test, t(20) = 1.90, n.s.).

TheOF is a test that examines the animals’ locomotion as well as their exploration or anxious behaviour. The track length in DA-CTRL rats was with 8897 ± 2682.4 cm 108 4 Results

significantly longer than the track length of the DA-stim rats with 6515 ± 1964.2 cm (ind. t-test, t(20) = 2.20, p < 0.05, Figure 4.28 (A)). Additionally, DA-stim animals spent significantly less time in theCZ of the arena with only 29.4 ± 8.87 s compared to 74.5 ± 22.47 s in DA-CTRLs (U (11,11) = 13, z = 3.07, p < 0.01, Figure 4.28 (B)). The visit latency until the rodents entered theCZ of theOF was also measured. Here, DA-CTRL rats needed 67.1 ± 20.21 s until they moved into theCZ, DA-stim rats needed 77.8 ± 23.45 s (Figure 4.28 (C)). Furthermore, the track length the animals moved inside theCZ was measured and set in relation to the total track length The value for the DA-stim group was 4.78 ± 1.44 %, the one for the DA-CTRLs 6.56 ± 1.98 % (Figure 4.28 (D)). Neither the visit latency (U (11,11) = 46, z = -0.92, n.s.), nor the track length in theCZ in relation to the total track length (ind. t-test, t(20) = 1.90, n.s.) was significantly different between the two groups.

4.5.7 Social Behaviour is Increased in DA-Stim Rats

Figure 4.29: DA-Stim – Social Interaction Test. (A) In trial 1 of the SIT, DA-stim rats had signiﬁcantly more social contacts (in % of total contacts) with Stranger 1 than the control animals (ind. t-test, t(20) = -4.37, p < 0.001). (B) In trial 2, both groups preferred the new Stranger 2 over Stranger 1, but again, DA-stim animals had signiﬁcantly more social contacts with Stranger 2 than DA-CTRL animals (ind. t-test, t(20) = -2.71, p < 0.05).

The social behaviour towards strange and familiar rats was measured in the SIT. During habituation, the rodents did not show any preference for one of the cages. DA-CTRL rats spent 49.7 ± 3.27 % of the total time spent with the cages with the cage of Stranger 1, DA-stim animals 52.6 ± 1.66 %. Statistically, this was not significantly different (ind. t-test, t(20) = -0.80, n.s.). In trial 1, the rats could choose to spent time with an age- and gender-matched conspecifics or an empty cage. Of the 109 4 Results

total contacts with both cages, DA-stim animals had 71.0 ± 1.88 % social contacts with Stranger 1. That was signiﬁcantly more than the control animals, which had 58.4 ± 2.19 % of the contacts with Stranger 1 (ind. t-test, t(20) = -4.37, p < 0.001, Figure 4.29 (A)). Although both groups preferred Stranger 2 over Stranger 1 in trial 2, the stimulated group of rats also had signiﬁcantly more contacts with Stranger 2, compared to the control animals (DA-stim: 62.0 ± 2.47 %, DA-CTRL: 52.8 ± 2.35 %, ind. t-test, t(20) = -2.71, p < 0.05, Figure 4.29 (B)).

4.5.8 Stimulation Rescued the Depressive-like Phenotype in the OSST

Figure 4.30: DA-Stim – Open-Space Swimming Test and Ultrasonic Vocalization. (A) In the OSST no group differences were seen statistically (rm ANOVA, group, F (1,20) = 1.91, n.s., but the trials within the groups were significantly different (rm ANOVA, group x trial, F (3,60) = 4.84, p < 0.01). In DA-CTRL animals, the track length in trials 1, 2 and 3 was significantly decreased compared to the habituation (p < 0.01, respectively). In DA-stim animals, the track length in trials 2 and 3 was significantly increased compared to the track length in trial 1 (p < 0.05 and p < 0.01). (B) In USV recordings, both DA-CTRL and DA-stim rats emitted a similar amount of calls in the high band in relation to the total calls. There was no significant difference between the groups (U(11,11) = 52.5, z = 0.49, n.s.).

The OSST is a test for detecting depressive-like phenotype in rodents, it detects immobility similar to the FST but without the restricting walls of the cylinder. On day one, a habituation without stimulation took place, in which the animals performed very similar. DA-CTRL rats had a track length of 13809 ± 1625.2 cm and DA-stim rats of 14138 ± 1406.5 cm. On the second day the DA-stim group received the usual stimulation protocol prior to the trial. Nevertheless, the track length in both groups decreased to 10892 ± 1145.6 cm in DA-CTRLs and to 12048 ± 1411.1 cm in DA-stim 110 4 Results

rats. On the following days, during trial 2 and 3, the track length of the control animals stayed more or less on the same level, in trial 2 they scored 10958 ± 1088.9 cm and in trial 3 10913 ± 946.6 cm. However, in DA-stim rats the track length increased again, even above the baseline value of the habituation. In trial 2 their track length was 14429 ± 1611.4 cm and in trial 3 15410 ± 1195.8 cm (Figure 4.30 (A)). The rm ANOVA did not show a group effect (F(1,20) = 1.91, n.s.), but a significant difference between the trials (group x trial, F(3,60) = 4.84, p < 0.01). Post-hoc testing showed, that in DA-CTRL rats the track length in all trials was significantly decreased compared to the habituation (p < 0.01, respectively). However, in the stimulated group, the track length in trial 2 and trial 3 was significantly increased compared to the track length in trial 1 (p < 0.05 and p < 0.01).

4.5.9 Immobility, Sucrose Consumption and USVs Did Not Diﬀer Between Groups

When recording the USVs of the rats, as before, they did not call or hardly called in the low frequency band (20 – 30 kHz). DA-CTRL rats emitted 0.55 ± 0.28 calls and DA-stim rats 0.45 ± 0.25 calls, which was not significantly different (U (11,11) = 56.5, z = 0.23, n.s.). In the high band (40 – 60 kHz) they emitted clearly more calls. In DA-CTRL animals, 64.7 ± 7.87 % of all calls were in the high band, in DA-stim rats 50.8 ± 11.68 % (Figure 4.30 (B)). There was no significant difference between the stimulated and the control animals (U (11,11) = 52.5, z = 0.49, n.s.).

Figure 4.31: DA-Stim – Forced Swim Test. (A) In the FST the animals did not show a signiﬁcantly diﬀerent immobility behaviour, neither in the single-bin data (rm ANOVA, group x bins, F (13,260) = 0.59, n.s.), (B) nor in the total immobility (U(11,11) = 43, z = 1.116, n.s.). 111 4 Results

Similar to the projects before, the rats hardly showed any immobility in the FST. When looking at the single-bin data, the curves of both groups rose in a similar manner, the maximum values were reached in the last and second-last bin with 4.04 ± 0.85 s of immobility in one 30 s bin in DA-CTRL rats and 4.59 ± 1.33 s in DA-stim rats (Figure 4.31 (A)). There was no significant difference between the two groups in the single-bin data (rm ANOVA, group x bins, F(13,260) = 0.59, n.s.). In addition, the total immobility also was not significantly different between the groups (U (11,11) = 43, z = 1.12, n.s.), DA-CTRL rats reached values of 25.2 ± 3.21 s and DA-stim rats of 30.4 ± 8.43 s (Figure 4.31 (B)).

Figure 4.32: DA-Stim – Sucrose Preference Test. (A) The sucrose consumption decreased over the course of the three SPTs, but there was no significant difference in consumption between the two groups (rm ANOVA, group x time point, F (2,40) = 0.46, n.s.). (B) Sucrose preference did not change during the three SPTs. In both groups, the preference of sucrose solution over water was at around 80 %, there was no significant difference (rm ANOVA, group x time point, F (2,40) = 0.77, n.s.).

The consumption of sucrose solution decreased in stimulated rats as well as in control rats from baseline to post-CMUS and again to the SPT under stimulation (Figure 4.32 (A)). Baseline value in DA-CTRL rats was 63.2 ± 4.66 ml kg−1 and it decreased to 50.3 ± 5.07 ml kg−1 in post-CMUS and even to 42.7 ± 2.62 ml kg−1. In stimulated rats, the baseline value was 59.7 ± 4.62 ml kg−1 and it decreased to 51.9 ± 2.99 ml kg−1 in post-CMUS and to 46.0 ± 4.47 ml kg−1 in the SPT under stimulation. There were no signiﬁcant diﬀerences between stimulated and control rats (rm ANOVA, group x time point, F(2,40) = 0.46, n.s.). When looking at the sucrose preference in percent of the total intake, there was no change over the course of the three SPTs. The baseline sucrose preference being 79.3 ± 2.35 % in DA-CTRL rats and 76.7 ± 2.34 % in DA-stim rats hardly changed in the following SPTs (Figure 4.32 112 4 Results

(B)). In addition, there was no signiﬁcant diﬀerence in sucrose preference between the groups (rm ANOVA, group x time point, F(2,40) = 0.767, n.s.). 113 5 Discussion 5 Discussion

With the present thesis "Optogenetic neuromodulation in a rodent model of depression", the effect of general and DA-specific stimulation of the MFB in two different rodent models ofMD has been examined for the first time. In the course of this project, the method of optogenetics was successfully established in the laboratory, including the development of the virus injection protocol. To be able to specifically address the DA-system using optogenetic stimulation, the breeding and use of a genetically modified rat line, theLETH::Cre, was established. For refining the behavioural readout, the background strain of thisTH::Cre rat, theLE rat, was behaviourally characterized and compared to theSD rat, a strain already established in use in the laboratory. Besides the model of the FSL rat, a second model of depression was introduced, the CMUS protocol. The two main projects investigated (i) the stimulation of the MFB in the FSL depression model with focusing on the role of the DA-system, comparing 6-OHDA lesioned animals to animals with an intact DA-system; and (ii) the DA-specific stimulation of the MFB in the CMUS-induced depression model. The results show that with the chosen virus injection protocol, a robust transfection rate could be achieved. The comparison of behaviour of the two rat strains displayed a very similar behaviour ofSD andLE rats, enabling to use the established behavioural readout following the optogenetic stimulation of theLETH::Cre rat. The results of the 6-OHDA-stim project need to be looked at carefully, due to low animal numbers and low virus transfection rates. No significant differences could be detected between the groups, but stimulated animals showed tendencies of increased activity and increased social behaviour. TH::Cre rats with DA-specific stimulation of the MFB in the CMUS-induced depression model showed a decrease in anxiety- and depression-related behaviour in tests including the EPM, OSST and SIT. Other tests displayed conflicting results, such as decreased time spent in theCZ of theOF. Overall the MFB seems to be a promising target for a potential treatment ofMD and the results, especially of the final project, suggest an important role of the DA-system. The following sections discuss the initial aims in detail. 114 5 Discussion

5.1 Establishment of optogenetics

The methodology of optogenetics has successfully been established in the laboratory. As stereotactic surgery has already been used frequently, the elaboration of the virus injection and cannula placement was straight forward. Histological results showed the spread of the virus being very limited to the area of the VTA, conﬁrming the choice of the AAV2. The slow infusion rate of 100 nl min−1 prevented tissue damage. Retrospectively, the amount of transfected DA neurons could have been higher, especially for behavioural readout only. Mean transfection rate was between 62 - 76 %, depending on the virus type used. For future experiments it might be desirable to aim for higher rates (> 80 %) by injecting an increased volume of virus (1 - 1.5 µl which might impact more the the animals’ behaviour.

Table 5.1: Controlled (+) Versus Non-Controlled (–) Potential Eﬀects of Genetic versus Light Control Groups Potential eﬀects Genetic control Light control Viral infection + + Exogenous opsin expression + – Visible light – + Tissue heating – +

For control groups in optogenetics, different approaches have been used. Mainly, one has to decide between two options with (i) either having the animal groups genetically altered in the exact same way or (ii) to infuse a control virus, not containing an opsin but only a fluorescent marker, and subsequently stimulate the control group exactly as the other group. Advantage of the first approach is the same genetic change in the groups, advantage of the second approach is to have the same effects of laser stimulation (e.g. heating of brain tissue) in both groups. All advantages and disadvantages are summarized in Table 5.1. For this thesis, the first approach has been used. However, for further experiments it is recommended to also use the second approach, to rule out behaviour effects that might be evoked by potentially visible light pulses or tissue heating during the experiment. DA is naturally released in two patterns: phasic and tonic. Tonic firing is a result of low frequency firing (around 5 Hz) and evokes a steady-state DA concentration being lower than 50 nM in the NAc (Parsons and Justice 1992). In contrast, DA neurons burst-firing in frequencies of more then 30 Hz (phasic firing) result in a transient in- 115 5 Discussion

crease in DA concentration of significantly more then 50 nM (Aragona et al. 2008). In addition to the greater DA release at the terminals, bursting activity of DAergic neurons also correlates with synaptic plasticity in the VTA during reward-learning related tasks (Schultz and Dickinson 2000). Interestingly, differences in burst firing of VTA DA neurons have been found when comparing control (SD rats) to rats displaying a depressive-like phenotype (FSL rats) (Friedman et al. 2005).SD rats show bursts with a large amount of spikes, whereas FSL rats do not show this pattern often. This alteration in electrophysiological burst properties is restored under treatment with the TCA desipramine (Friedman et al. 2008). As it has been shown before, that tonic DA release rather inhibits reward-related behaviour (Mikhailova et al. 2016) and that phasic firing can rescue CMUS-induced depressive-like behaviour (Tye et al. 2013), the latter was used in the experiments of this thesis. The effect of (partially) reversing a depressive-like behaviour could be repeated in the final experiment (see section 5.5 on page 123). For further experiments it would be also desirable to dissect the types of neurons even further, as it has been shown that the DA neuron population in the VTA is not a homogeneous, but rather a heterogeneous population (Lammel et al. 2014; Roeper 2013). In his review, Roeper listed 7 different types of DA neurons, i.e. mesostriatal, mesolimbic an mesocortical, with further subtypes respectively. These subtypes differ in their electrophysiological properties, including tonic and phasic (burst) firing and therefore are likely to have different effects on the animal’s behaviour. For the design of the light stimulation, two options are possible: (i) stimulation during the behaviour experiment or (ii) stimulation directly prior to the behaviour test. Because of the huge variety of behaviour tests used in this work, including very large or deep apparatuses, the second option was chosen for this project with the animals receiving 30 min of light stimulation prior to the testing. However, for future experiments it would be very interesting to compare the present data with animals that were stimulated right during the testing. An elegant option for allowing the animals to move more freely in a variety of apparatuses would be the use of wireless optogenetics. Using implantable microscale inorganic LEDs, several either head-mounted or fully-implantable devices have been introduced in the recent years (for a review see Qazi et al. 2018). One of the key questions in neuroscience is how neural activity results in behaviour. Answering this question requires the recording of neural activity while an animal is performing a behavioural task. However, a subsequent correlation does not necessarily prove a causal relationship. For examining this further, the neural activity 116 5 Discussion

would need to be manipulated, e.g. by stimulation or inhibition, and this is where optogenetics comes into play (Miyamoto and Murayama 2016). The combination of optogenetic manipulation of neurons with a behavioural readout, as performed in this thesis’ work, allows to carefully draw conclusions about which type of neuron or neural circuit evokes which kind of behaviour. Nevertheless, the exact mechanisms inside the brain remain unrevealed. For this, a combination of optogenetics plus behaviour and recording and/or imaging techniques would be required. Electrophysiological recordings can be used in freely-moving animals, simultaneously to optogenetic stimulation. This methodology allows for example to investigate potential alterations in neural encoding in areas downstream of the stimulation site. Tye and colleagues showed that optogentic stimulation of the VTA potently altered the neural encoding in the NAc, combined with changes in depression-related behaviour (Tye et al. 2013). This leads to the presumption of a similar alteration when stimulating the MFB, as in the present experiments. In terms of combining optogenetics with imaging techniques, a well-established method used is functional MRI. It has been shown that functional MRI can measure changes in blood oxygenation level-dependent signals downstream of stimulation sites (Lee et al. 2010). However, limitations occur in terms of slow temporal resolution and the incompatibility with freely-moving animal behaviour. Another imaging technique that can be combined with optogenetics is in vivo (one- or two-photon) microscopy. This method allows the observation of neural activity with high-spatial resolution, but again it cannot be applied to freely moving animals. A compromise might be the possible application in awake, head-restrained animals performing behaviour tasks on a spherical treadmill in a virtual environment (Dom- beck et al. 2010). Nevertheless this is a rather artificial approach not allowing to have a look into a number of behaviours including social behaviour and stress- or anxiety-related behaviour (Miyamoto and Murayama 2016). Furthermore, Ca2+ imaging can be combined with the in vivo microscopy. GECIs even allow the recording of genetically targeted neuron population, and when using independently addressable spectral channels, this technique can elegantly be combined with optogenetics (Akerboom et al. 2013). Again, this technique has been used primarily in head-fixed animals, but there have been recent attempts to adjust the tools to freely moving animals, for example with the use of micro-endoscopes in mice (Rehani et al. 2019). Apart from these fluorescent protein-based sensors that report calcium and also glutamate in high temporal resolution, recently additionally a sensor has been de- 117 5 Discussion

veloped that specifically targets DA (Patriarchi et al. 2018). This tool allows to measure specifically DA transients with high spatio-temporal resolution in freely moving animals. Combining this technique with optogenetics, Corre and co-workers could show that heroin increases DA levels in the NAc through the activation of DA neurons located in the medial VTA (Corre et al. 2018). Finally, to evaluate the optogenetic stimulation further, it could also be combined with a biochemical readout measuring DA fluctuations, e.g. microdialysis or voltammetry. Although generally microdialysis is a nice tool to reflect on neurotransmitter levels, it lacks the high temporal resolution to being able to reflect on changes evoked by neural bursts following optogenetic stimulation (Zhang et al. 2015). In contrast to that, fast-scan cyclic voltammetry provides high temporal resolution and is capable to reflect on rapid changes in neurotransmitter levels. It could have been shown for example, that phasic optogenetic stimulation of the VTA led to significantly higher DA concentrations in the NAc compared to tonic stimulation (Tsai et al. 2009). Summing up, several electrical or biochemical recording techniques and imaging techniques are available to be combined with optogenetics and free-moving animal behaviour. For future experiments it is strongly recommended to verify that the optogenetic stimulation with the properties chosen evokes spiking / changes in neural encoding / changes in neurotransmitter levels in downstream areas of the MFB (e.g. NAc, PFC) to further strengthen the potential correlation between stimulation and behaviour response.

5.2 The Long Evans TH::Cre Rat

In 2011, the optogenetic toolbox was extended with the introduction of recombinase- driven rat strains, includingTH::Cre rats (Witten et al. 2011). In combination with Cre-dependent opsin-expressing viral vectors, these rats allow the speciﬁc targeting of DAergic neurons with a precision of 98 % in the VTA (Witten et al. 2011). The background of theseTH::Cre rats is theLE strain, which has not been used before in the laboratory. Therefore the behaviour of the animals was characterized in a pilot experiment in comparison with the in the laboratory establishedWT rats of theSD strain. In general, both strains behaved relatively similar. There were no signiﬁcant differences in EPM, FST and sucrose preference. The track length in theOF was 118 5 Discussion

significantly longer inLE rats, but no difference was found in the time spent in the CZ of the arena, which can serve as a measure of anxiety. In USV recordings, male LE rats emitted significantly less calls in the high band. However, this seemed to be an unusual result, as in a following experiment (CMUS pilot) usingLE rats, the percentage of calls emitted in the high band was in the range of 57 - 74 %, which is comparable to the calls emitted by maleSD rats. However, in the final experiments female rats were used, and no differences could be observed. The most pronounced difference between theLE andSD strain could be observed in theDH maze.SD rats needed significantly longer to find the escape platform and they made significantly more IEs. These results were not surprising, as finding the way in this maze is supported by visual cues on the walls of the room.SD rats, as being albino rats with lower visual acuity, of course have more problems to see and use those cues to find their way through the maze. Taken together, except for theDH results, no huge differences could be found in the behaviour of the two strains, especially not in depression-like and anxiety-related behaviour. Thus, for the in the laboratory newly introduced strains ofLE rats and LETH::Cre rats, the established behavioural readout could be used in a similar way compared to the until now usedSD rats. Also, theLETH::Cre rat has been successfully used in a number of experiments, examining the DA system with the help of optogenetics, combined with behaviour and electrical or biochemical readouts (Lozano-Montes et al. 2019; McCutcheon et al. 2014; Tye et al. 2013). This made it, in combination with the CMUS protocol, a promising model for this thesis’ work.

5.3 The CMUS Protocol

The CMUS protocol is a model of depression that is meant to induce one of the core symptoms of the disease, anhedonia. The procedure is based on an experiment that showed that rats reduced their intake of sweet fluids after being exposed to stressors (Katz 1982). As the CMUS protocol was newly established in the laboratory, a pilot was conducted first. The protocol used was based on modifications of the original protocol that were introduced by Willner and colleagues (Willner et al. 1987). Male LE rats underwent CMUS for 7 weeks, being stressed with 8 different stressors, with 1 stressor per day and one 24 h stressing pause per week. The effect of the pro- 119 5 Discussion

cedure was monitored by a weekly SPT, where the animals’ drinking behaviour was compared to a non-stressed control group. After 7 weeks of stressing, the rats were tested behaviourally in a battery of tests. Over the course of the experiment, no significant differences between groups could be detected in the SPT. There was a slight, but not significant decrease in weight in CMUS animals compared to CTRLs between weeks 2 - 7, which is consistant with a weight loss between 0 - 10 % that has been reported in literature (Willner et al. 1996). Behaviour tests including EPM,OF, FST and USV did not show any differences between groups. Levels of FCM were significantly higher post-CMUS compared to pre-CMUS, nevertheless this effect was observed in both stressed and non-stressed animals. Changes in corticosteroid levels are not only evoked by stress, but the level is also influenced by many other factors including circadian / seasonal rhythm (Allen-Rowlands et al. 1980). Otherwise it could only be speculated if the control rats were exposed to stress in the animal facility. Finally, CMUS rats spent significantly less time with the novel object in theOR compared to CTRLs, indicating cognitive impairments. Cognitive deterioration is one of the symptoms ofMD in humans and it has been shown that exposure to stress can evoke this effect in theOR (Calabrese et al. 2017). There is no ’standard’ chronic mild stress procedure existing. The methodology of applying a series of mild stressors to laboratory animals has been published under a variety of names, including ’mild chronic stress’, ’chronic unpredictable mild stress’, ’unpredictable subchronic mild stress’, ’chronic ultramild stress’, and ’chronic variable, varied, or variate stress’, using a variety of different stressing approaches (Willner 2005). This makes the CMUS model difficult to establish. While the majority of research groups using the model reported decreases in hedonic behaviour, e.g. the SPT, or increases in other depression-related behaviours, e.g. immobility FST, also some ’anomalous’ effects have been published (Willner 2005). These comprise for example increases in sucrose intake (Murison and Hansen 2001) and decreases in immobility during the FST (Haidkind et al. 2003). Additionally it needs to be taken into account that a diurnal variation in the effect of CMUS has been observed, which could explain the results of the SPT (D’Aquila et al. 1997). This study showed decreased sucrose intake and preference only in rats tested during the dark phase and not during the light phase. Due to a not reversed light-dark cycle in the animal facility, all behaviour tests were performed during the light phase of the animals, 120 5 Discussion

which could complicate the readout. Nevertheless, the application of the CMUS protocol is a relevant and realistic model ofMD (Willner et al. 1992), which is why it was decided to further use it in the ﬁnal experiment of this thesis, especially also because the results of theOR were promising. However the protocol was adjusted to render the eﬀects more pronounced: 3 new stressors were introduced and the stressors were applied more frequently (see section 3.7.2 on page 71). The results of this experiment are discussed in section 5.5 on page 123.

5.4 Optogenetic Stimulation of the MFB in the FSL Depression Model - 6-OHDA Lesioned vs. Unlesioned Rats

This experiment was the ﬁrst of two examining the optogenetic stimulation of the MFB. Here the FSL rat was used as model ofMD and the role of the DA system was investigated by lesioning the animals with 6-OHDA. Comparing animals with an intact DA system with animals with a depleted DA system allows to carefully draw conclusions on the role this speciﬁc system plays inMD. The depressive-like phenotype was assessed behaviourally in untreated FSL rats versus FSL rats that were treated with photostimulation of the MFB. This approach was chosen because at that time point the approval of the local government (Regierungspräsidium) for the use of a stress-induced depression model inTH::Cre rats was not yet granted.

5.4.1 The FSL rat

The FSL rat has been used as a model of depression for more than 30 years now (Overstreet and Wegener 2013) and there are over 300 peer reviewd publications using that model. It displays several depressive-like features including altered REM sleep and increased immobility in the FST, which are reversible by antidepressant medication (Overstreet 1993). Thus, the FSL rat was considered a suitable depression model for the sub-project "Optogenetic stimulation of the MFB in the FSL rat depression model - 6-OHDA lesioned vs. unlesioned rats". 121 5 Discussion

It was decided to conduct the experiments using female FSL rats, as the prevalence to developMD is 1.5 – 2.5 times higher in women compared to men (Fava and Kendler 2000). The results of this experiment are discussed in more detail in section 5.4.2, but one issue that influenced these results was that the FSL rats used in this project hardly displayed a depressive-like phenotype. When comparing for example the results of the FST to the work of Thiele and colleagues from 2016 (Thiele et al. 2016), the values of immobility are clearly lower here, with the rats reaching a maximum of 1.20 ± 0.76 % in total immobility compared to an immobility of almost 65 % during the last 150 s in female FSL rats in Thiele’s work. There are two possible explanations for the lost phenotype in the animals. First, the FSL rat was developed and is maintained by systematic breeding of animals that display a robust depressive-like phenotype. Since the introduction to the laboratory, the own colony has not been bred selectively anymore, which might explain a less stringent expressioned or even a loss of the phenotype. The second possibility is that the depressive-like phenotype seems to be transient in the FSL rat (Thiele et al. 2016). As mentioned above, Thiele et al. showed a robust immobility in female FSL rats, however this was only significantly higher than in controls in young rats being 2 – 3 months old. The immobility in older rats of 6 – 7 months decreased and there was no difference observable between FSL and controls anymore. This transience was also true for other behaviours including EPM,OF, SPT andOR. A combination of both of these factors could be a conceivable reason for the weak depressive-like phenotype of the FSL in the present study.

5.4.2 Optogenetic Stimulation of the MFB in the FSL Depression Model - Discussion of Results

To investigate the role of the DA-system in MFB stimulation, half of the animals were lesioned with 6-OHDA and the rats with impaired DA-system were compared to rats with an intact-DA system. The lesioning worked out well, as immunohistochemistry showed that lesioned rats (DA-) had significantly lessTH-positive neurons in the VTA compared to controls (DA+). DA- rats also displayed a decreased activity in the amphetamine-induced activity test compared to DA+ rats, although this difference was not significant. The de- 122 5 Discussion

creased locomotor activity is what would be expected according to literature, as amphetamine is a DA releaser (Fleckenstein et al. 2007) and with a reduced number of DA neurons, a reduced amount of DA would be anticipated. Additionally, the results of the immunohistochemistry revealed, that the mean transfection rate ofTH-positive cells in the VTA was rather low and that in some animals, cannulas were misplaced. Transfection rates of the unspecific AAV were expected to be a little lower compared to those of the DA-specific virus. Though in pilot experiments rates of over 60 % were reached, here the means ofTH and GFP double-stained neurons in the VTA only reached between 28 – 40 %. Reasons for this are unknown, it could be speculated that the virus titer decreased e.g. due to possible cold chain interruptions or too long storage, or during surgery the Hamilton syringe might have been clogged, or the target region might not have been hit very well. Due to misplacement of the cannula or surgery side effects, some animals had to be excluded from the experiment, so that the resulting groups were small with n = 5 (DA+), n = 4 (DA+Stim), n = 6 (DA-) and n = 7 (DA-Stim). Thus, there are three major compounds that need to be taken into account when interpreting the behavioural data of this experiment:

1. The small number of animals, which decreases the reliability of the statistical analysis,

2. The low transfection rate, which might not have been suﬃcient to evoke a behavioural response, and

3. The low expression of a depressive-like phenotype in the FSL rats, as discussed in the prior section.

Activity, as measured by light barrier crossings, was increased signiﬁcantly under photostimulation compared to without stimulation for both DA+ and DA- animals. Corresponding to that, also the track length during theOF was increased, though not signiﬁcantly, in stimulated groups. DA neurons are linked to locomotion and stimulation of these neurons, elevation of DA levels in the NAc or application of DA receptor agonists increase locomotor activity in theOF chamber (Ikemoto and Panksepp 1999). As this increase happens in the absence of any objects the animals can interact with, and as the same manipulations also increase other behaviours in other environments (e.g. operant tasks in operant chambers), it might be more appropriate to speak about increased exploratory behaviour or even novelty-seeking instead of just an increase of locomotor activity (Ikemoto and Panksepp 1999). A re- 123 5 Discussion

duced locomotor activity or exploratory behaviour also corresponds to a depressive- like phenotype and decreasing levels of DA in the NAc by optogenetic inhibition, 6-OHDA lesioning or injection of DA antagonists has led to a hypoactivity in animals (Ahlenius et al. 1987; Stephen Fink and Smith 1980; Tye et al. 2013). The low transfection rate probably is the reason why in the conducted behaviour tests (SPT, SIT, FST, USV) no significant differences could be detected when comparing stimulated and not stimulated animals. Only tendencies could be observed, for example in the SIT photostimulated rats seemed to spend more time in zone 2, which was the zone where the new, unfamiliar conspecific was located. Spending more time with Stranger 2 compared to Stranger 1, which is the familiar conspecific from trial 1, is an indicator for increased social novelty and is expected byWT rats, but rather not by depressive-like rats (Kaidanovich-Beilin et al. 2011). The data here suggest, that optogenetic stimulation of the MFB might have the ability to rescue a depressive-like phenotype in terms of social novelty behaviour, but due to the experimental complications listed above, this data would clearly need to be verified in a set up with bigger animal numbers, a higher transfection rate of VTA DA neurons and a stronger expressed depressive-like phenotype.

5.5 DA-speciﬁc Optogenetic Stimulation of the MFB in the CMUS-induced Depression Model

The final project of this thesis investigated the effects of a DA-specific optogenetic stimulation of the MFB in a stress-induced model ofMD. To achieve a DA specificity,TH::Cre rats were infused with a floxed virus into the target area in the VTA. Mean transfection rate again was lower as in pilot experiments with a maximum of 56 % compared to over 76 %. Possible reason for the lower rate were already discussed in the section of the lesion experiment (see section 5.4 on page 120). For achieving a better transfection rate, for future experiment a slightly bigger volume should be injected. To prevent tissue damage, a lower volume of 0.8 µl per deposit was used in the present experiment, this should be increased to at least 1.0 – 1.2 µl with maybe simultaneously lowering the injection speed a little. Compar- able experiments, like the one of Tye and colleagues, typically use 1.0 µl per deposit (Tye et al. 2013). Weight development in both groups was very similar, although DA-stim rats gained weight a little slower compared to controls, but that difference was not significantly 124 5 Discussion

different. During the beginning of the CMUS protocol, animals seemed to gain weight slower, but again in statistical tests this could not be confirmed to be significant. As discussed before, this is comparable with what has been reported in literature before, where researchers found weight decrease between 0 – 10 % following a stress protocol (Willner et al. 1996). To evaluate the stress level of the rats, FCM levels were examined again. Results are difficult to interpret, as baseline levels of corticosterone were very high in both groups. Animals might have still been stressed by surgery or by separation from group cages to single cages at the time point where the faeces were collected. Thus, no statement can be made regarding the effect of the CMUS protocol on FCM concentration in the rats. But interestingly, when comparing the post-CMUS data with the post-stim / post-behaviour data, it was observed that in photostimulated rats the FCM level kept decreasing, whereas in the untreated control group the levels rose again, indicating that those animals might have been more stressed. Statistic- ally this data was not significantly different though. Corticosterone levels are not only depended of circadian/diurnal rhythm (Allen- Rowlands et al. 1980), they also change in response to environmental cues, as husbandry-related disturbances (Cavigelli et al. 2006). So it is crucial that samples are time matched and that environmental stress factors are kept at a minimum level and that experimental and control groups are exposed to the same kind of stimuli. For future improvement of the readout of corticosterone concentrations in rat faeces, the collection of samples should occur under stricter conditions. Instead of collecting the faeces from the bedding of the home cage, wire-bottom cages should be used, so that the samples fall through the grid onto absorbent paper-lined trays. This might improve the ELISA results as the probability of urine contamination is minimized. Instead of collecting a sample of faeces from over night, a better defined time period should be used, for example as described by Cavigelli and colleagues (Cavigelli et al. 2006), and all samples of this time period should be used for analysis. DA-specific optogenetic stimulation of the MFB did not increase home cage activity. This is contradictory to the results of the 6-OHDA-stim experiment, where stimulation increased locomotor activity in the home cage significantly. The reasons for this could be divers, the used rat strains and depression models are different, the transfection rate differed and a DA-specific stimulation was used compared to an unspecific stimulation. DA-specific optogenetic stimulation of VTA DA neurons does not necessarily evoke increases in locomotor activity (Tye et al. 2013). But interestingly, electrical stimu- 125 5 Discussion

lation of the MFB has been shown to increase rat locomotor activity (Talwar et al. 2002). As this form of stimulation also is unspecific as in the 6-OHDA-stim project, it leads to the assumption that other neuron types than DA might play a role here. As a matter of fact, Guo and colleagues published work demonstrating elevated locomotor activity in rats upon optogenetic stimulation of calcium/calmodulin-dependent protein kinase type II alpha chain (CaMKIIα) expressing neurons in the VTA. Those neurons were shown to be partly DA neurons co-releasing glutamine and DA and non-DA neurons, probably being purely glutamatergic (Guo et al. 2014). This might explain why pure DAergic stimulation does not rise levels in activity, but unspecific stimulation does. Originally, theOF was developed to assess emotionality or timidity in rats (Hall 1934) and has since then been adapted to also measure locomotor or exploratory activity and anxiety-like behaviour (Prut and Belzung 2003). In this experiment, DA-stim rats even displayed a decreased track length compared to controls. It is rather doubtful that this is due to a lower locomotor activity, as this was not reflected in the measurments of the homecage activity. A difference however was that home cage activity was measured during stimulation, while theOF was performed right after the end of the 30 min stimulation period. Also, this does not automatic- ally mean that the exploratory behaviour of the photostimulated rats is impaired. Neither the track length in theCZ in relation to the total track length is significantly different in both groups, nor is the latency to visit theCZ for the first time, suggesting that exploratory behaviour does not differ between groups. What is displayed though is a significant difference in the time the animals spend in theCZ. DA-stim rats spend significantly less time in the middle of the arena compared to control rats. It has been shown before that MFB DBS stimulation decreases the time spent in theCZ of theOF (Thiele et al. 2018). This might be an indication for a normalized freezing response. Another reason for the increased time the DA-stim rats spent in the periphery of the arena could be the increased DA transmission after stimulation. Thigmotaxis, or wall-hugging, has shown to be more pronounced in rats that received treatment with drugs that increase the DA transmission, including indirect catecholaminergic agonists like amphetamine and anxiogenic drugs like Pentylenetetrazole (Simon et al. 1994). This links thigmotaxis to anxiety, with the data suggesting an increased anxiety-like behaviour in photostimulated animals compared to controls. However, this is contrary to the results that were found in the EPM, a classical test for assessing anxiety-related behaviour, which was performed because comor- 126 5 Discussion

bid anxiety disorders occur in 85 % of depression patients (Gorman 1996). In the test performed here, both groups of rats spent more time in the closed arms of the maze, which reflects the rats’ normal behaviour, avoiding open spaces (Valle 1970). However, optogenetic stimulation reduced the time the animals spent in the closed compartments significantly, or in other words, they showed an increase in exploratory, i.e. a decrease in anxiety-like behaviour. To further examine the contradictory results ofOF and EPM, additional behaviour tests might be required. Especially also because the use of theOF for assessing locomotor activity and anxiety-like behaviour is being doubted (Rodgers 2007). Anxiety and exploration could be further examined using the rats’ innate fear to novelty, as in theOR or in the novelty suppressed feeding test. TheOR for example displayed a significantly decreased exploratory behaviour in stressed animals in the here conducted CMUS pilot (see section 4.3 on page 88). It would be interesting to see if this effect is rescued by stimulation of the MFB. Novelty suppressed feeding examines two conflicting motivations: (i) the drive to eat and (ii) the fear to enter a novel, bright-lit open space. The latency to begin eating serves as a measure of anxiety- and depression-like behaviour and is altered by anxiolytic and chronically applied antidepressive drugs (David et al. 2009). Apart from anxiety- and exploratory-related behaviour, also the rats’ social behaviour was altered through optogenetic stimulation of the MFB. The social behaviour increased in stimulated rats, they had significantly more contacts with Stranger 1 in trial 1 and Stranger 2 in trial 2. In trial 1, the animals’ behaviour towards an empty cage versus an unfamiliar conspecific (Stranger 1) is examined. A wild-type rat would always spend more time with the rat than with the empty cage. This indicates normal sociability, social motivation and affiliation (Kaidanovich-Beilin et al. 2011). The stressed group showed a reduction in this behaviour, whereas stressed rats that subsequently underwent optogenetic stimulation spend significantly more time with Stranger 1, thus showing a normalized social behaviour. The second trial estimates social novelty and social memory, comparing the time the rats spent with the familiar conspecific (Stranger 1) with the time they spent with the novel Stranger 2 (Kaidanovich-Beilin et al. 2011). Non-treated rats did not spend more time with Stranger 2 compared with Stranger 1 (53 % versus 47 %), but stimulated rats spent more time with Stranger 2 (62 % versus 38 %), which was significantly different from controls. Thus, in depressive-like animals the sociability seems to be impaired, which has also been shown in other models ofMD, as in the FSL rat (Cook et al. 2017). This impairment seems to be normalized by DA-specific photostimulation of the 127 5 Discussion

MFB. A depressive-like phenotype and the effect of subsequent antidepressant treatment has been assessed using the FST for more than 40 years (Porsolt et al. 1977). This test examines behavioural despair by exposing the animal to an inescapable situation, a water filled cylinder, and measuring the time the animal spends swimming or struggling versus the time the animal displays immobility, which is defined as behavioural despair. It has been shown before that different depression models, including stress-inducedMD models and the FSL rat, exhibit increased immobility during the FST. This depressive-like phenotype can be rescued through DBS of the MFB in young but not old FSL rats (Thiele et al. 2018) and through DA-specific optogenetic stimulation of the VTA(Tye et al. 2013). Throughout the different projects for this thesis, readout of depressive-like phenotype using the FST appeared to be challenging. In contrast to what has been shown in literature, using the FST neither could display a difference in depressive-like phenotype between FSL or CMUS animals versus controls, nor between photostimulated animals versus controls. And again in this experiment, no significant difference could be found between non-stimulated and stimulatedTH::Cre rats. In fact, inconsistent data for the FST has been reported. One reason is that the method varies between laboratories and that there are many factors influencing this test. These include: (i) biological factors (e.g. strain, age, weight), (ii) precondi- tioning and treatment (e.g. handling, housing, type of drug), (iii) test design (e.g. equipment, settings, scoring) and (iv) environmental factors (e.g. noise, light) (Bog- danova et al. 2013). Due to those problems, an additional test for assessing depressive-like behaviour was introduced for this last experiment, the OSST. In this setup rats are allowed to freely swim (or not to swim) in a pool filled with water for 15 min on four consecutive days. To analyse the test the track length is measured with a tracking software. Antidepressants, including Imipramine, and SSRIs like Alaproclate reduce the time the animals are immobile, compared to controls (Sun and Alkon 2003). Sun and colleagues claim that this test might be a better readout for a depressive-like phenotype compared to the FST, asMD is rather characterized by a lack of motivation than by a lack of space, as is the case in the FST cylinder (Sun and Alkon 2003). Further advantages of this test are (i) that the analysis is more objective as it does not involve human scoring, (ii) that the extent of swimming (vigorous or mild) is directly reflected in the analysis, (iii) and that as said above, the animal is not restricted in space. 128 5 Discussion

In this experiment, on day one a habituation was performed with no treatment (no stimulation), resulting in a similar track length in both groups. For the following three trials on three consecutive days the DA-stim group received 30 min photostimulation directly prior to the OSST. This led to a significantly increased mobility of these animals compared to unstimulated controls. Thus, DA-specific optogentic stimulation of the MFB seems to rescue depressive-like behaviour in terms of immobility in the OSST. The stimulation neither showed an effect on the preference of sucrose in the SPT, nor on the number of calls in the USV. Tye and colleagues elegantly showed that optogenetic excitation of the VTA led to an increase in sucrose preference, whereas an inhibition led to a decrease (Tye et al. 2013). However, they only saw this effect under stimulation and it disappeared in the post-stimulation test period lasting 30 min after end of stimulation. This might explain why in this experiment the effect of the stimulation could not be detected, as the SPT was performed after the end of the stimulation. For future projects it would be interesting to have a look at data from an SPT that is performed during the optogenetic stimulation period. Rats emit calls in the range of 50 kHz during positive social interactions and appetitive situations and calls in the range of 22 kHz in aversive situations and in response to social or environmental threats (Alcaro and Panksepp 2011). There- fore the hypothesis was that depressive-like animals might emit more calls in the low band or less calls in the high band compared to controls. It has been shown for example, that Wystar-Kyoto rats that underwent a juvenile isolation protocol emitted significantly less call in the 50 kHz band compared to controls (Shetty and Sadananda 2017). During the course of experiments for this thesis, neither differences between depressive-like rats (both FSL and stress-induced) and controls were found nor between unstimulated and stimulated rats. Also, Thiele and colleagues could not show any effect of MFB DBS on the number of calls emitted in FSL rats (Thiele et al. 2018). There are several reasons that might clarify a lacking effect. First, the USV recordings were done during the light phase, so the sleeping phase of the rats. This might explain why the total number of calls was rather low (around 30 calls in the high band and only 0.5 calls in the low band in mean), as the animals tended to fall asleep during the conduction of the test. Second, the animals had been living isolated in single cages for several weeks prior to this experiment. This could be an explanation for a lack in social communication. Third, according to literature, calls in the low 22 kHz band are primarily present in acute aversive situations, e.g. when the animal is exposed to a foot shock (Cullen and Rowan 1994) 129 5 Discussion

or an anxiety-inducing environment like the EPM (D’Souza and Sadananda 2017). Thus, further experiments should be conducted during the dark phase and if measuring calls in the low band, either mild foot shocks could be used to evoke those or the USV recordings could be combined with another behaviour test like the EPM orOF. Taken together, DA-speciﬁc optogenetic stimulation of the MFB seems to have an effect on performance in EPM,OF, SIT and OSST, suggesting a role of the DA-system in reversing depressive-like, anxiety-like and impairments in social behaviour.

5.6 Conclusions

In conclusion, it can be said that (i) optogenetics is an appropriate tool to investigate the role of specific neural circuits in disease, that (ii)TH::Cre rats are valid models for examining specifically the DA system, and that (iii) FSL rats and CMUS seem to be appropriate models of depression, when biological factors like age and breeding, and protocol design for CMUS are taken into account carefully. The results of the 6-OHDA-stim project emphasize the importance of the sufficient number of animals with a pronounced depressive-like phenotype. The "dirty" approach of investigating the role of the DA-system by lesioning the rats involves risks, as the animals are undergoing a second surgery, and generally with increasing complexity of the experiment, the number of systemic errors increases. However, results of the SIT might indicate a slight improvement of sociability in stimulated rats. Finally, the DA-stim project indicates an important role of the DA-system in reversing a stress-induced depressive-like phenotype. Further effects were seen on social behaviour, which improved following stimulation, and on anxiety-like behaviour, although results regarding this were inconsistent.

5.6.1 Potential Mechanism of Action of the Optogenetic Stimulation of the MFB

The target for optogenetic stimulation in this work was the MFB. The anatomy of this important ﬁbre system in the forebrain has been studied for decades in rodents. It runs in a longitudinal pathway from the midbrain to the limbic forebrain, more 130 5 Discussion

specifically from the VTA to the olfactory tubercle (Nieuwenhuys et al. 1982). The fibre bundle consist of more than 50 fibre subcomponents and more than 13 neurotransmitters are associated with it (Nieuwenhuys et al. 1982; Veening et al. 1982). In contrast to the rodent MFB, the human MFB only has been described in the recent years (Coenen et al. 2011, 2012) and the slMFB was proposed as promising target for DBS in treatment-resistant patients (Schlaepfer et al. 2013; Schlaepfer et al. 2014). Short-term and long-term results of the slMFB DBS trial (85 % and 75 % response rate), as well as results from a follow-up gateway trial (100 % response rate in 61 % months) suggest an acute and sustained antidepressant effect of slMFB DBS (Bewernick et al. 2017; Coenen et al. 2019). The efficacy of the stimulation is further indicated, as discontinuation of the slMFB DBS led to rapid and significant worsening of the clinical symptoms ofMD (Kilian et al. 2019). Current hypothesis suggest that DBS might be normalizing a dysfunctional reward system with the DA-system playing at least a partial role (Döbrössy et al. 2015; Schlaepfer et al. 2013). The clinical data is underpinned by several experimental projects using DBS or optogenetics in animal models of depression. The majority of these also suggest a role of the DA-system (Edemann-Callesen et al. 2015; Klanker et al. 2017; Thiele et al. 2018; Tye et al. 2013). This hypothesis is strengthened by the effect of the stimulation on behaviour tests including FST (anti-despair), and SPT (anti-hedonic), in which the DA-system seems to play an important role. Thus it has been proposed that through electrical or optogenetic stimulation of the MFB, a change in neurotransmitter transmission is occuring that positively modu- lates the involved neural circuits (Thiele et al. 2018). However, DA-release following stimulation of the fibre bundle is controversial. Bregman and colleagues for example found a robust anti-depressant effect in the FST following MFB DBS, but using microdialysis, no DA release was detected in the NAc (Bregman et al. 2015). By contrast, another study using microdialysis showed elevated levels of DA and it’s metabolites in the NAc and medial frontal cortex following self-stimulation of the MFB (Nakahara et al. 1992). The big difference between these two studies was that the sample collection frequency differed, Bregman et al. collected samples every 30 min whereas Nakahara et al. collected the samples every 10 min or every 20 min, indicating that a 30 min frequency might be too long to be able to detect a difference in the DA levels. Additionally, the effect in Nakahara’s study might have 131 5 Discussion

been enhanced due to the animals receiving a DA reuptake inhibitor prior to the microdialysis. A more elegant way to detect changes in DA levels even in the sub-second range is to use fast-scan cyclic voltammetry. Using this technique it was reported that significantly enhanced and sustained DA release was found in the ventromedial striatum, at least during the initial 40 s of the measurement (Klanker et al. 2017). A role of the DA-system is further supported by the results of Dandekar and colleagues, who found, using high performance liquid chromatography, a significant increase in DA D2 receptors in the PFC following DBS of the MFB. Finally, the results of this thesis indicate an important role of the DA-system, partially reversing a depressive-like phenotype when using DA-specific optogenetic stimulation of the MFB. Taken together, the careful assumption can be made that stimulation of the MFB, either by indirect recruitment of glutamatergic axons (electrical stimulation) or by direct recruitment of DAergic axons (DA-specific optogenetic stimulation), leads to an increase in DA in ascending areas of the MFB, including the NAc. Addtitionally, MFB stimulation could also act via descending fibres, as effects of stimulation on learning and memory have been observed, as well as a hypometabolism in the ento- rhynal cortex (Thiele et al. 2018). In conclusion, stimulation of the MFB seems to normalize a dysfunctional reward system at least partially by increasing DAergic transmission in the midbrain. Nev- ertheless, asMD is a heterogenous disease and factors other than the reward system seem to play a role in the pathophysiology of the disease, further research is needed to shed more light onto this matter.

5.7 Outlook

As research on a multifactorial disease likeMD can be challenging, and as the pathophysiology of the disease still is not understood completely, further experiments are needed. Several suggestions for improvement have already been made throughout the discussion chapter. Summing up, it is crucial, especially in behaviour, to work with a suﬃcient amount of animals. The depressive-like phenotype, no matter which model ofMD is being used, should be clearly expressed. Control animals have to be chosen carefully, and for future experiments especially light controls should be used to rule 132 5 Discussion

out effects of tissue heating. To get a better idea of the role of the DA-system in MD, optogenetic experiments with inhibiting specifically the DA-system should be conducted and compared to those where the DA-system was specifically stimulated (excited). Moreover, the optogenetic technique should not only be combined with behaviour testing, but additional readouts including electronical, biochemical and imaging should be used to better understand the whole picture. Indeed, optogenetics already has and definitely will continue to add knowledge to the implication of neural circuits being involved in psychiatric diseases likeMD.

5.7.1 Application of Optogenetics in Humans

To date, optogenetics is no approved therapy option in humans. Searching clinicaltrials.gov1 for the term ’optogenetics’ only reveals two listings of phase I/II clinical trials using optogenetics to restore vision in Retinitis Pigmentosa (NCT02556736 and NCT03326336, search performed on 23.06.2019, Simunovic et al. 2019). Both trials are recruiting, data has not been published yet. Searching clinicaltrials.gov for the term ’gene therapy’ reveals nearly 4000 listings (search performed on 23.06.2019)and, in fact, already 17 gene therapies have been approved in the U.S.2. Back in 2017, already nearly 3000 clinical trials using gene therapy were conducted, most of them in cancer (Lundstrom 2018). Of those 3000 trials, almost 70 % have been using viral vectors for gene delivery, although the development of non-viral delivering options such as CRISPR are advancing quickly. Clinical trials using gene therapies delivered by viral vectors also already have targeted the CNS. For example in Parkinson’s disease, the transgene encoding glutamic acid decarboxylase has been introduced to patients and therapy was well-tolerated with no adverse reactions related to the gene therapy (Feigin et al. 2007). Anyhow gene therapy using viral vectors has some inherent risks including

• immune system reactions,

• recombination (can occur in older vectors),

• pleiotropy (due to low speciﬁcity),

1https://clinicaltrials.gov/ 2https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/ approved-cellular-and-gene-therapy-products, content current as of 29.03.2019 133 5 Discussion

• mutagenesis or carciogenesis (can occur in older generation of retroviral vectors Delbeke et al. 2017).

However, due to the number of clinical trials and the maturity of the technique of viral gene delivery, safety concerns should not be the obstacle for clinical application of optogenetics. Limiting could rather be the still big gap in general knowledge about the brain. The future clinical application of optogenetics might therefore be both direct and indirect. In already pre-clinically well-studied neural circuitries, like the visual circuit that is targeted in the Retinitis Pigmentosa trials, clinical trials should and must be performed to assess the safety and efficacy in humans further. On the other hand, the lessons learned from basic and pre-clinical research will definitely feed into treatment options for humans, and thus lead to an indirect human profit from this exciting methodology. 134 Bibliography Bibliography

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It would not have been possible to write this master thesis without the help and support of the kind people around me, to only some of whom it is possible to give particular mention here.

First and foremost I oﬀer my sincerest gratitude to my supervisor, Prof. Dr. med. Volker A. Coenen, for his guidance, caring, patience and for providing me with an excellent environment, equipment and materials for doing research. I also would like to thank Prof. Dr. Wolfgang Driever for agreeing to be the co- supervisor for my thesis.

I would like to thank the group leader PD Dr. Máté D. Doebroessy for his support, guidance, caring and patience. Thank you for always having an open door.

My work was partially funded by the "Fill in the gap" scholarship of the medical faculty of the university of Freiburg. I am very grateful for having received this scholarship.

Special thanks goes to Stephanie Thiele. Steﬃ, without your support this would not have been possible (and this is not just a saying). I cannot even list everything here that I am grateful for. So thank you for your help whenever needed and thank you for your friendship. Thank you also for making Freiburg a better and nicer place to stay!

I appreciate the help of our laboratory manager Johanna Wessolleck and our tech- nician Jasmin Weis very much. I thank them both for their support and for always being so helpful regarding any problem in the laboratories. Thank you guys also for your friendship and caring. I also would like to thank Marlene Löﬄer and Maria Elko for their technical assist- ance.

I would like to thank Prof. Dr. Ulrich Hofmann and lab members for their collabor- ation and support whenever needed.

I would like to thank the Prof. Dr. Ilka Diester and lab members for their support and guidance regarding the establishment of optogenetics in our laboratory. Thanks for lending me your power meter (again and again!). 172 Acknowledgements

Many thanks also to Manuela Schätzle, for administrative support and always a good chat.

I am very grateful for the helpfulness of my lab members Alex Cook, Hannah Abbott, Julian Jäger, Verena Martini, Luciano Furlanetti, Nadine Hoﬀmann, and Marie- Christine Pauly.

A big thank you also goes to our animal caretakers: Joshua Schäuble and Simon Junker. Thank you so much for your help and support with the animals, whenever needed. Special thanks goes to Jennifer Weigand for always having an open door, even if the pharmacy was closed.

I would like to thank the team of the neurocentre scientiﬁc workshop very much: Thomas Günter, Frank Huethe, Waldemar Schimpf, and Gerd Strohmeier. Without their incredible work, building and repairing the equipment, the projects could not have been conducted.

I would also like to thank my parents and my brother. They were always supporting me and encouraging me with their best wishes.

Finally, I would like to thank my husband Tobias Pfeiﬀer. He was always there cheering me up and stood by me through the good times and bad. Thanks for your optimism and for believing in me.

So long, and thanks for all the ﬁsh. 173 List of Publications List of Publications

1. Thiele S., L. Furlanetti, L. M. Pfeiﬀer, V. A. Coenen and M. D. Döbrössy (2018). ’The eﬀects of bilateral, continuous, and chronic Deep Brain Stim- ulation of the medial forebrain bundle in a rodent model of depression’. In: Experimental Neurology 303. February, pp. 153-161. DOI: 10.1016/j.expneurol.2018.02.002.

2. Cook, A.∗, L. M. Pfeiﬀer∗, S. Thiele, V. A. Coenen and M. D. Döbrössy (2017). ’Olfactory discrimination and memory deﬁcits in the Flinders Line Sensitive rodent model of depression’. In: Behavioural Processes 143. March, pp. 25-29. DOI: 10.1016/j.beproc.2017.08.006.

3. Prinz, A.∗, L. M. Selesnew∗, B. Liss, J. Roeper and T. Carlsson (2013). ’In- creased excitability in serotonin neurons in the dorsal raphe nucleus in the 6- OHDA mouse model of Parkinson’s disease’. In: Experimental Neurology 248. June, pp. 236-245. DOI: 10.1016/j.expneurol.2013.06.015.

∗Co-First Author