Modulation of extracellular noradrenaline in rat cortex by selective reuptake inhibitors (SSRIs)

by Zoë A. Hughes

A thesis presented for the degree of Doctor of Philosophy to the University of London 1998

Department of Pharmacology University College London IT Trf^lT Street London WC1E6BT ProQuest Number: U642926

All rights reserved

INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest.

ProQuest U642926

Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author.

All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC.

ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ■ M :

So ?

l L' Abstract

This research project has investigated the modulation of central noradrenergic function in rat cortex by drugs. Particular attention has been paid to the type of known as the selective serotonin (5-HT) reuptake inhibitors (SSRIs). The therapeutic effects of these drugs are presently attributed exclusively to their inhibition of neuronal uptake of 5-HT. The aim of this work was to investigate whether the SSRIs, and citalopram, modify central noradrenergic function and, if so, was their site of action on noradrenergic neurones? In order to achieve this two techniques were used: the first used microdialysis to measure the concentration of extracellular noradrenaline in vivo, the second looked at the inhibition of ["^H]noradrenaline uptake into rat cortical synaptosomes in vitro.

In this thesis changes in the concentration of extracellular noradrenaline caused by local infusion of antidepressants in the frontal cortex of rats, were monitored using microdialysis. Fluoxetine and citalopram, as well as the noradrenaline uptake inhibitor, , increased noradrenaline efflux. To investigate whether inhibition of noradrenaline uptake could contribute to this increase in efflux, the effects of these drugs on synaptosomal [^H]noradrenaline uptake were studied. Because these drugs inhibit [^H]noradrenaline uptake, it is likely that inhibition of noradrenaline uptake contributes to the increase in noradrenaline efflux in vivo.

In view of this finding, the possible site(s) of action of these drugs were investigated. An uptake site targeted by SSRIs could be located on neurones. The 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT), was used as a tool to investigate this possibility. Because a selective lesion of 5-HT neurones did not affect the inhibition of [^H]noradrenaline uptake by any of the drugs, it is unlikely that fluoxetine, citalopram or desipramine were acting at a site on serotonergic neurones.

The second possible site of action investigated was one located on noradrenergic neurones. Pretreatment of rats with the noradrenergic neurotoxin, A-(2-chloroethy 1 )- A- ethyl-2-bromobenzylamine (DSP-4), modified the effects of these antidepressants on noradrenergic function. Low concentrations of desipramine inhibited a smaller proportion of [^H]noradrenaline uptake than in control rats. In contrast, a greater proportion of uptake in the cortex of DSP-4 lesioned rats was sensitive to inhibition by low concentrations of fluoxetine in vitro. The effects of these drugs on noradrenaline efflux in vivo were also changed after DSP-4 pretreatment. The increase in noradrenaline efflux induced by desipramine was greater in DSP-4 treated rats, whereas the fluoxetine-induced increase, apparent in control rats, was not evident after DSP-4. The effects of citalopram were unaffected by DSP-4 treatment, suggesting that SSRIs do not all have the same pharmacological profile and that fluoxetine, but not citalopram, could act at a site on noradrenergic neurones.

One additional, but important finding to emerge from work described in this thesis was that, although DSP-4 treatment caused a 70% reduction in tissue noradrenaline content, the concentration of extracellular noradrenaline was increased nearly 2-fold. Results suggest that neurones which survive DSP-4 treatment have a greater rate of noradrenaline release, moreover, released noradrenaline is taken up via low affinity uptake sites.

Overall, the experiments in this thesis have exposed marked effects of SSRIs on central noradrenergic function which could contribute to the efficacy of antidepressants hitherto regarded as acting selectively on serotonergic neurones. Acknowledgements

I would like to thank my supervisor Dr Clare Stanford for her guidance, support and encouragement throughout my three years.

I must also thank Kathryn Mason for being a great friend and for teaching me microdialysis.

My thanks also go to the rest of ‘the team’, Doreen Gettins for her technical help,

Katherine Wortley for her friendship and Richard M^Quade for his handy thesis writing tips.

Thanks to The Dickenson Lab for being fun and providing lots of tea and glossies. I would also like to thank Jo Leaney, Alison Reeve and Lisa Broad for being good friends throughout.

I would like to thank Roger Allman for putting up with me and lending me his Fanzines (for weeks & weeks).

Thanks as well to Nick Hayes for his help with all sorts throughout my time at UCL.

My thanks also go to Bob Muid for his help with computer problems and Kerry Rawlings for making slides and for scanning all the photos and diagrams for this thesis.

And last but not least, I must thank my family, and especially Ian, for their constant support and encouragement, and for looking after me.

I have to say that my three years at UCL were fun! Contents

page

Title...... 1 Abstract ...... 2 Acknowledgements...... 4 Contents...... 5 List of figures...... 11 List of tables ...... 13 List of abbreviations ...... 14 Publications arising from this thesis ...... 16

Chapter 1

General Introduction 17

1.1 The role of noradrenaline in depression (pre-1965)...... 19 1.2 The role of 5-HT in depression...... 21 1.3 Drug treatments of depression...... 22 1.4 Selective serotonin reuptake inhibitors (SSRIs)...... 25 1.5 Monoamine transporters in general...... 28 1.5.1 The noradrenaline transporter ...... 31 1.5.2 The 5-HT transporter ...... 32 1.5.3 The transporter ...... 33 1.5.4 Similarities between the noradrenaline and dopamine transporters 34 1.6 The noradrenergic innervation of rat brain...... 35 1.6.1 The locus coeruleus ...... 37 1.6.2 The lateral tegmental noradrenergic system ...... 38 1.7 The serotonergic innervation o f the cortex...... 40 1.8 Interactions between the noradrenergic and serotonergic systems...... 41 1.9 The noradrenergic neurotoxin, DSP-4...... 45 1.9.1 The selectivity of DSP-4 for locus coeruleus noradrenergic neurones...... 46 1.9.2 Changes in central noradrenergic neurones after treatment with DSP-4...... 49

Chapter 2

Materials and methods ______^

2.1 Introduction...... 55 2.1.1 In vivo sampling techniques ...... 56 2.1.1.1 What microdialysis measures...... 58 2.1.1.2 The perfusate...... 60 2.1.2 Measurement of monoamine uptake in vitro...... 62 2.2 Techniques...... 65 2.2.1 Intracerebral microdialysis ...... 65 2.2.1.1 Animals...... 65 2.2.1.2 Surgery & Anaesthesia...... 65 2.2.1.2.1 Non-recovery animals (“anaesthetised rats”) ...... 65 2.2.1.2.2 Recovery animals (‘freely-moving rats’) ...... 66 2.2.1.3 Verification of probe placement...... 67 2.2.1.4 Microdialysis procedure...... 69 2.2.1.4.1 Construction of microdialysis probes ...... 69 2.2.1.4.2 Collection of dialysates (anaesthetised rats) ...... 71 2.2.1.4.3. Collection of dialysates (freely-moving rats) ...... 71 2.2.1.4.4. Local administration of compounds via the dialysis probe (‘reverse dialysis’) ...... 74 2.2.1.5. Measurement of dialysate noradrenaline content...... 74 2.2.1.5.1 High pressure liquid chromatography coupled to electrochemical detection (HPLC-ECD)...... 74 2.2.1.5.2 The mobile phase ...... 75 2.2.1.5.3. Drugs and chemicals ...... 78 2.2.1.6 Statistical Analysis...... 78 2.2.1.7 Characterisation of noradrenaline efflux in vivo...... 78 2.2.2 Measurement of [^H]noradrenaline uptake into rat cortical synaptosomes in vitro...... 82 2.2.2.1 Animals...... 82 2.2.2.2 Preparation of cortical synaptosomes...... 82 2.2.2.3 Measurement of [^HJnoradrenaline uptake...... 83 2.2.2.4 Protein determination...... 84 2.2.2.5 Calculation of H]noradrenaline uptake...... 86 2.2.2.6 Statistical Analysis...... 86 2.2.2.?Drugs and chemicals...... 86 2.2.3 Lesions...... 87 2.2.3.1 Lesion of noradrenergic neurones with DSP-4...... 87 2.2.3.2 Lesion of 5-HT containing neurones...... 87 2.2.3.3 Lesion of dopamine containing neurones...... 88 2.2.3.4 Drugs and chemicals...... 89 2.2.4 Measurement of monoamine content of cortical tissue ...... 89 2.2.4.1 Detection of monoamines using HPLC-ECD...... 89 2.2.4.2 Statistical Analysis...... 90 2.2.4.3 Drugs and chemicals...... 90

Chapter 3

The effects of fluoxetine and desipramine on noradrenaline efflux and [^H]noradrenaline uptake ______91

3.1 Introduction...... 91 3.2 Methods ...... 96 3.2.1 Microdialysis in vivo ...... 96 3.2.2 Inhibition of [^H]noradrenaline uptake by fluoxetine, desipramine or 5-HT ...... 96 3.2.3 Statistical analysis ...... 97 3.3 Results...... 98 3.3.1 Microdialysis in vivo...... 98 3.3.1.1 Noradrenaline efflux during infusion of desipramine...... 98 3.3.1.2 Noradrenaline efflux during infusion of fluoxetine...... 98 3.3.1.3 Comparison of the effects of fluoxetine and desipramine on noradrenaline efflux...... 98 3.3.2 Inhibition of [^H]noradrenaline uptake in vitro...... 100 3.3.2.1 Inhibition of [^H] nor uptake by fluoxetine, desipramine or 5-HT...... 100 3.3.2.2 Biphasic inhibition of [^H]noradrenaline uptake by desipramine 100 3.3.2.3 Inhibition of [^H] nor adrenaline uptake fluoxetine...... 100 3.4 Discussion...... 106

Chapter 4

The effect of selective lesions of noradrenaline, 5-HT or dopamine on synaptosomal [^H] noradrenaline uptake ______116

4.1 Introduction...... 116 4.2 Methods...... 120 4.2.1 Selective lesion of neurones in the cerebral cortex 120 4.2.1.1 Lesion of serotonergic neurones with 5,7-DHT...... 120 4.2.1.2 Lesion of noradrenergic neurones with DSP-4...... 120 4.2.1.3 Lesion of dopaminergic neurones with 6-OHDA...... 120 4.2.2 Synaptosomal [^H]noradrenaline uptake ...... 121 4.2.3 Verification of the lesion ...... 121 4.2.4 Statistical Analysis ...... 121 4.3 Results...... 122 4.3.1 Inhibition of [^H]noradrenaline uptake by fluoxetine, citalopram or desipramine ...... 122 4.3.2 [^H]noradrenaline uptake into cortical synaptosomes from 5,7-DHT lesioned rats ...... 124 4.3.2.1 Cortical monoamine content after a 5,7-DHT lesion...... 124 4.3.2.2 Specific [^H]nor adrenaline uptake into synaptosomes from sham- or 5,7-DHT lesioned rats...... 124 4.3.2.3 Inhibition of H]noradrenaline uptake by desipramine...... 125 4.3.2.4 Inhibition of [^H]noradrenaline uptake by fluoxetine...... 125 43.2.5 Inhibition of [^H]noradrenaline uptake by citalopram...... 128 4.3.3 [^H]noradrenaline uptake into cortical synaptosomes from DSP-4 lesioned rats ...... 130 4.3.3.1 Cortical monoamine content after a DSP-4 lesion ...... 130 4.3.3.2 Specific HJnoradrenaline uptake into synaptosomes from DSP-4 lesioned rats...... 130 4.3.3.3 Inhibition of [^HJnoradrenaline uptake by desipramine...... 131 4.3.3.4 Inhibition of H]noradrenaline uptake by fluoxetine...... 131 4.3.3.5 Inhibition of [^HJnoradrenaline uptake by citalopram...... 132 4.3.4 [^H]Noradrenaline uptake into cortical synaptosomes from 6-OHDA lesioned rats ...... 136 4.3.4.1 Cortical monoamine content after a 6-OHDA lesion...... 136 4.4 Discussion...... 137

Chapter 5

The effects of fluoxetine, citalopram and desipramine on extracellular noradrenaline in DSP-4 or sham-lesioned rats ______143

5.1 Introduction...... 143 5.2 Methods...... 146 5.2.1 DSP-4 lesion of noradrenergic neurones ...... 146 5.2.2 Intracerebral microdialysis ...... 146 5.2.3 Measurement of monoamine content of cortical tissue...... 146 5.2.4 Calculation of “net” noradrenaline efflux ...... 147 5.2.5 Statistical analysis ...... 147 5.3 Results...... 148 5.3.1 The effects of fluoxetine, citalopram or desipramine or 5-HT on noradrenaline efflux ...... 148 5.3.2 Cortical monoamine content after a DSP-4 lesion ...... 148 5.3.3 Noradrenaline efflux during local infusion of fluoxetine ...... 151 5.3.4 Noradrenaline efflux during local infusion of citalopram ...... 154 5.3.5 Noradrenaline efflux during local infusion of desipramine ...... 154 5.3.6 A comparison of the effects of fluoxetine, citalopram and desipramine on noradrenaline efflux ...... 154 5.4 Discussion...... 159

Chapter 6

The concentration of extracellular noradrenaline in rat frontal cortex after DSP-4 treatment ______170

6.1 Introduction...... 170 6.2 Methods...... 172 6.2.1 DSP-4 lesion of noradrenergic neurones ...... 172 6.2.2 Microdialysis in vivo...... 172 6.2.3 Monoamine content of frontal cortex ...... 172 6.2.4 Changes in noradrenaline efflux induced by desipramine ...... 172 6.2.5 Ca^^-dependence of noradrenaline efflux ...... 173 6.2.6 K^-sensitivity of noradrenaline efflux ...... 173 6.2.7 Statistical analysis ...... 173 6.3 Results...... 174 6.3.1 Cortical monoamine content ...... 174 6.3.2 Desipramine-induced increase in noradrenaline efflux in DSP-4 or sham-lesioned rats ...... 175 6.3.3 The dependence of noradrenaline efflux on extracellular Ca^^ ...... 175 6.3.4 Depolarisation-evoked noradrenaline release ...... 178 6.4 Discussion...... 180

Chapter 7 General Discussion 186

References 198

10 List of figures

Figure 1.1: Schematic representation of the basic features of the Na'^ / CV-dependent monoamine transporters...... 30 Figure 1.2: The chemical structure of DSP-4...... 47

Figure 2.1: Position of the microdialysis probe in rat frontal cortex...... 68 Figure 2.2: Step-by-step construction of a microdialysis probe...... 70 Figure 2.3: The anaesthetised rat set-up...... 72 Figure 2.4: The freely-moving rat set-up...... 73 Figure 2.5: Schematic diagram of the HPLC-ECD set-up...... 76 Figure 2.6: Typical chromatograms showing noradrenaline, DOPAC and 5-HIAA 77 Figure 2.7: Effect of a depolarising iC pulse on noradrenaline efflux...... 80 Figure 2.8: Dependence of noradrenaline efflux on extracellular ...... 81

Figure 3.1: The effects of local infusion of fluoxetine or desipramine on noradrenaline efflux in the frontal cortex of anaesthetised rats...... 99 Figure 3.2: Inhibition of synaptosomal [^HJnoradrenaline uptake by fluoxetine and desipramine...... 102 Figure 3.3: Inhibition of HJnoradrenaline uptake by 5-HT...... 103 Figure 3.4: Biphasic inhibition of [^H]noradrenaline uptake by desipramine...... 104 Figure 3.5: Inhibition of [^HJnoradrenaline uptake by fluoxetine...... 105

Figure 4.1: Inhibition of synaptosomal [^H]noradrenaline uptake by desipramine, fluoxetine and citalopram...... 123 Figure 4.2: Uptake of [^HJnoradrenaline into synaptosomes from 5,7-DHT or sham-lesioned rat cortex in the presence of desipramine...... 126 Figure 4.3: Uptake of f^H] noradrenaline into synaptosomes from 5,7-DHT or sham-lesioned rat cortex in the presence of fluoxetine...... 127 Figure 4.4: Uptake of HJnoradrenaline into synaptosomes from 5,7-DHT or

11 sham-lesioned rat cortex in the presence of citalopram...... 129 Figure 4.5: Uptake of H] noradrenaline into synaptosomes from DSP-4 or sham-lesioned rat cortex in the presence of desipramine...... 133 Figure 4.6: Uptake of [^HJnoradrenaline into synaptosomes from DSP-4 or sham-lesioned rat cortex in the presence of fluoxetine...... 134 Figure 4.7: Uptake o f [^H] noradrenaline into synaptosomes from DSP-4 or sham-lesioned rat cortex in the presence of citalopram...... 135

Figure 5.1: Effects of fluoxetine, citalopram or desipramine on noradrenaline efflux in saline pre-treated (control) rats...... 149 Figure 5.2: Effect of 5-HT on noradrenaline efflux in freely-moving rats...... 150

Figure 5.3: Infusion of fluoxetine (5 pM) in sham- or DSP-4 lesioned rat frontal cortex...... 152 Figure 5.4: Infusion of fluoxetine (50 pM) in sham- or DSP-4 lesioned rat frontal cortex...... 153

Figure 5.5: Infusion of citalopram (50 pM) in sham- or DSP-4 lesioned rat frontal cortex...... 156 Figure 5.6: Infusion of desipramine (5 pM) in sham- or DSP-4 lesioned rat frontal cortex...... 157 Figure 5.7: Noradrenaline efflux during local infusion of desipramine, fluoxetine or citalopram...... 158 Figure 5.8: Hypothetical mechanism for changes in noradrenaline uptake after DSP- 4 ...... 168

Figure 6.1: Effects of local infusion of desipramine (5 pM) on noradrenaline efflux in the frontal cortex ...... 176 Figure 6.2: The dependence of noradrenaline efflux in the frontal cortex on extracellular Ca^'^...... 177 Figure 6.3: The effects on noradrenaline efflux of repeated depolarising pulses of Ringer's containing 80 mM ...... 179

12 List of tables

Table 4.1: Concentrations of 5-HT, noradrenaline and dopamine in the cerebral cortex of sham- or 5,7-DHT lesioned rats...... 124 Table 4.2: Concentrations of noradrenaline, dopamine and 5-HT in the cerebral cortex of sham- or DSP-4-injected rats...... 130 Table 4.3: Concentrations of 5-HT, noradrenaline and dopamine in the cerebral cortex of sham- or 6-OHDA lesioned rats...... 136

Table 5.1: Cortical monoamine content after a DSP-4 lesion...... 151

Table 6.1. Concentrations of noradrenaline, dopamine and 5-HT in the cerebral cortex of sham- or DSP-4 lesioned rats...... 174

13 Abbreviations

5,7-DHT 5,7-dihydroxytryptamine 5-HIAA 5-hydroxyindole-3-acetic acid 5-HT 5-hydroxytryptamine, or serotonin 6-OHDA 6-hydroxydopamine ATP adenosine triphosphate

Pmax total no. of binding sites aCSF artificial cerebrospinal fluid ANOVA analysis of variance AP anterior-posterior cDNA complementary deoxyribosenucleic acid c.p.m. counts per minute DpH dopamine-p-hydroxylase DOPAC 3,4,-dihydroxypheny 1| acetic acid d.p.m. disintegrations pre minute DSP-4 N-(2-chloroethyl)-A-ethyl-2-bromobenzylamine DV dorsal-ventral BCD electrochemical detection ECF extracellular fluid g gram GABA y-aminobutyric acid h hour [-^H] tritium HPLC high pressure liquid chromatography HVA homovanillic acid

I C 5 0 concentration causing 50 % inhibition of effect i.c.v. intra cerebroventricular i.d. inner diameter i.p. intra peritoneal

14 kD kilo Dalton K. inhibitor constant

Km affinity constant 1 litre m metre mol moles MAO monoamine oxidase MAGI monoamine oxidase inhibitor min minute M molar ML medial-lateral mRNA messenger ribonucleic acid n sample size NMDA A^-methyl-D-aspartate o.d. outer diameter P probability REDOX oxidation-reduction potential s.c. subcutaneous SEM standard error of the mean SSRI selective serotonin reuptake inhibitor TCA V Volt

P^max maximum rate (of transport)

W / V weight per volume

Prefixes m milli (X 10'^)

micro (X 10'^) n nano (X 10-') f femto (X id '^)

P pico (X i d 'd k kilo (X 10’)

15 Publications arising from this thesis

Hughes, Z. A. & Stanford, S. C. (1997) A partial noradrenergic lesion induced by DSP-4 increases extracellular noradrenaline concentration in rat frontal cortex: a microdialysis study in vivo. Psychopharmacol. (in press)

Hughes, Z. A. & Stanford, S. C. (1997) A microdialysis study of the effects of fluoxetine infusion on noradrenaline (NA) efflux in the frontal cortex of saline or DSP-4 treated rats. Br. J. Pharmacol, (abstract in press)

Hughes, Z. A & Stanford, S. C. (1997) The effect of a DSP-4 lesion on spontaneous and desipramine-increased noradrenaline efflux in the frontal cortex of rats. J. Psychopharmacol. 11 (suppl.): 160, A40

Hughes, Z. A. & Stanford, S. C. (1997) A serotonergic lesion does not affect inhibition of [^H]noradrenaline uptake into rat cortical synaptosomes by fluoxetine, citalopram or desipramine. Br. J. Pharmacol. 122: 99?

Hughes, Z. A. & Stanford, S. C. (1996) Increased noradrenaline efflux induced by local infusion of fluoxetine in the rat frontal cortex. Ear. J. Pharmacol. 317: 83-90.

Hughes, Z. A. & Stanford, S. C. (1996) A microdialysis study of the effects of the selective serotonin reuptake inhibitor, fluoxetine, on noradrenaline efflux in rat frontal cortex. Br. J. Pharmacol. 118: 77?

Hughes, Z. A. & Stanford, S. C. (1995) Lack of effect of a lesion of cortical noradrenergic neurones on inhibition of synaptosomal ['^H]noradrenaline uptake by serotonin uptake inhibitors ex vivo. Br. J. Pharmacol. 116: 236?

16 Chapter 1

Chapter 1

General Introduction

Little is known about the cause of depressive illness. It is widely thought that depression involves a dysfunction or imbalance of central noradrenergic and / or serotonergic neurotransmission. This is partly because many antidepressant drugs have primary effects on monoaminergic neurones. The therapeutic success of one class of antidepressant drug, the tricyclic antidepressants, is attributed to the ability of these drugs to inhibit the reuptake of noradrenaline and / or 5-HT. Another group of antidepressant drugs, the selective serotonin reuptake inhibitors (SSRIs), are presumed to be effective in the treatment of depression because of their selective inhibition of 5-

HT uptake. More recently, antidepressants which are selective for noradrenergic neurones have been developed.

The aim of this study was to reappraise the selectivity of the SSRIs, fluoxetine or citalopram, for inhibition of 5-HT uptake. The SSRIs are generally assumed to inhibit

17 Chapter 1 exclusively the reuptake of 5-HT. Experiments in this thesis were designed to determine whether, and how, these drugs affect noradrenergic transmission in rat frontal cortex.

This was achieved using two techniques: microdialysis in vivo and a preparation of cortical synaptosomes to measure [^H]noradrenaline uptake in vitro. The effects of the two SSRIs are compared throughout with those of the selective noradrenaline uptake inhibitor, desipramine. These experiments have led to the overall conclusion that the

SSRIs do not selectively inhibit the reuptake of 5-HT, but have considerable effects on noradrenaline uptake both in vitro and in vivo. Moreover, this study indicates the existence of at least two sites of noradrenaline uptake in rat cortex.

In this General Introduction the background to the present work is described.

Because of the fact that effective antidepressants increase the extracellular concentration of noradrenaline and / or 5-HT, the roles of noradrenaline and 5-HT in depression will first be discussed. Next, the drug treatments for depression, from the first antidepressants in the late 1950s to the antidepressants of the 1990s, in particular the

SSRIs, will be described. The sites of action of antidepressants which inhibit the reuptake of monoamines i.e. the monoamine transporters, will then be reviewed in brief.

Following this, the noradrenergic and serotonergic innervation of the cortex, as well as the complex interactions between these two monoaminergic systems will be described.

Finally, as experiments presented in this thesis have relied heavily on the use of the noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), the effects of this neurotoxin on noradrenergic function are also detailed.

18 Chapter 1

1.1 The role of noradrenaline in depression (pre-1965)

The aetiology of depressive illness remains unknown to date. It is agreed though, that dysfunction or imbalance of noradrenergic and/or serotonergic neurotransmission are likely causes. One of the first theories explaining the cause of depression was described by Schildkraut in 1965. The “catecholamine hypothesis of affective disorders” suggested that changes in central nervous system catecholamine metabolism could be the cause of disorders including depression and mania (then referred to as ‘elation’)- The theory was based on the findings that drugs which altered central function, improved or depressed the mood of healthy humans.

Noradrenaline was implicated in depression by the effects of drugs which either increased or decreased its synaptic availability. Reserpine, a drug which depletes intraneuronal stores of noradrenaline (as well as 5-HT and dopamine), was found to cause a reduction in locomotor activity and sedation of animals (Brodie & Shore, 1957;

Carlsson, 1961) as well as causing symptoms of depression in healthy humans (Harris,

1957). Furthermore, the effects of reserpine were found to be reversed by, or prevented by pretreatment with, known antidepressant agents such as iproniazid or

(see below) which increase the availability of noradrenaline (Chessin et a l, 1957; Sulser et a l, 1962 & 1964).

At the time of Schildkraut’s seminal paper (1965) there were two types of antidepressant drug available: the monoamine oxidase (MAO) inhibitor (MAGI), iproniazid, and imipramine. Iproniazid was ‘known’ to cause behavioural stimulation and be effective in the treatment of depression because of its inhibition of the metabolic enzyme, MAO (Kiloh et a l, 1960; Spector et a l, 1960). In contrast little was known about the mechanism of action of imipramine. This drug is now known to inhibit the

19 Chapter 1 reuptake of noradrenaline and 5-HT; in 1965 evidence for the inhibition of noradrenaline uptake by this drug, as well as by desipramine and amitryptiline, was just emerging (Glowinski & Axelrod, 1964; Hertting e ta l, 1961).

A further drug which implicated a role for noradrenaline in depression in 1965 was . Amphetamine was believed to cause the release of noradrenaline and the inhibition of noradrenaline uptake (Glowinski & Axelrod, 1965). The effects of amphetamine in the treatment of depression were mixed, with an immediate stimulant effect often being followed by a period of ‘rebound’ depression (Goodman & Gilman,

1955).

The importance of noradrenaline in the aetiology of depressive illness was further demonstrated by the reversal of the above mentioned ‘depressant’ effects of reserpine. The reduction in locomotor activity and sedation observed in reserpinized animals were associated with a reduction in brain levels of noradrenaline, dopamine and

5-HT (Brodie & Shore, 1957; Carlsson, 1961). Intracerebroventricular administration of noradrenaline, the precursor of noradrenaline and dopamine, DOPA, or the precursor of

5-HT, 5-hydroxytryptophan, were shown to reverse the behavioural deficits caused by reserpine treatment (Carlsson, 1961; Glowinski et a l, 1966). In so far as a reduction in locomotor activity resembles depression, these findings supported the proposed role of monoaminergic function underlying depressive illness.

Despite more than 30 years of research into depressive illness and treatments of depression since the work described by Schildkraut in 1965, our understanding of the cause of depressive illness has progressed very little. The next sections of this

20 Chapter 1

Introduction will be devoted to the developments in the field in the time since 1965.

This includes advances in research into the treatment of the symptoms of depression.

1.2 The role of 5-HT in depression

At the time when noradrenaline was first suggested to be involved in depression

(see section 1.1) a role for 5-HT was also being postulated. The first indications of the involvement of 5-HT in depression came from patients who were being treated with drugs known to affect the availability of 5-HT, e.g. reserpine. Although drugs like reserpine, iproniazid and imipramine were used to implicate noradrenaline in depression

(see above) these drugs also affect 5-HT in the same manner. Consequently, the effects of these drugs also served to implicate a role for 5-HT in depression.

Evidence specific for 5-HT includes the finding that administration of the precursor of 5-HT, /-, increased the clinical efficacy of MAOIs and tricyclic antidepressants (TCAs) (see Coppen et a l, 1963; Paykel & Hale, 1986). The reversal of the antidepressant effects of MAOIs and TCAs, by pretreatment with the inhibitor of

5-HT synthesis, p-chlorophenylalanine, was further support for the role of 5-HT in depression (Shopsin et a l, 1976).

Delgado and colleagues (1990) showed that when patients who were being treated with antidepressants were given a tryptophan-free amino acid mixture

(restricting 5-HT synthesis), the therapeutic effects of the drugs were reversed. These findings suggest that 5-HT plays an important role in the mechanism of therapeutic action of many antidepressant drugs. More recently, it has been shown that tryptophan

21 Chapter 1 depletion only causes a relapse in patients treated with serotonergic drugs (fluoxetine or sertraline) and conversely, only those patients treated with noradrenergic drugs

(desipramine or mazindol) experience a return to depressive symptoms following depletion of catecholamines with the hydroxylase inhibitor, a-methyl- paratyrosine (AMPT) (Delgado et al, 1993; Miller et ai, 1996).

1.3 Drug treatments of depression

The first treatment of depression came in the late 1950’s with the discovery of iproniazid, later to be identified as an MAOI (Crane, 1957; Kiloh et a l, 1960). The next drug found to possess antidepressant efficacy was imipramine, an inhibitor of 5-HT and noradrenaline reuptake (Kuhn, 1958). What these drugs had in common was that they increased the synaptic availability of noradrenaline and 5-HT though through different mechanisms.

MAO is an enzyme involved in the breakdown of monoamines which exists in two forms, MAG a and MAO b (Johnston, 1968). The former deaminates noradrenaline, adrenaline and 5-HT, the latter, 2-phenylethylamine. The disadvantage of the first

MAOIs, including pargyline and phenelzine, is that they cause irreversible inhibition of both MAO a and MAO b. Inhibition of MAO b can have profound hypertensive effects when tyramine-rich food is eaten (this is referred to as the ‘cheese effect’). For this reason the MAOIs had to be refined to achieve selective inhibition of MAO a. The second problem with these drugs was that their effects were irreversible; consequently if a patient was not responding to MAOI treatment there had to be a delay before alternative drugs could be prescribed to allow the MAOI to be metabolized and removed

22 Chapter 1

from the body and new MAO protein to be synthesised (see Frazer, 1997). The two

problems associated with the original MAOIs were overcome by the development of

reversible inhibitors of MAO a (RIMA), the first RIMA discovered was brofaromine

(Waldmeier & Baumann, 1983). This was superseded by the more potent RIMA,

moclobemide (Keller et al., 1987).

Imipramine is now classified as a TCA. The TCAs, which include drugs such as

desipramine and amitryptiline, are very effective and widely prescribed antidepressants

(Olfson & Klerman, 1993) which inhibit the reuptake of noradrenaline and / or 5-HT

(Glowinski & Axelrod, 1964). These drugs are plagued by their peripheral

anticholinergic effects which can frequently lead to serious adverse effects including

cardiotoxicity and orthostatic hypotension (Tollefson, 1991).

The TCAs were refined in the 1970’s to produce antidepressants, such as

, which had a better side-effect profile. Because of the antidepressant efficacy of the TCAs, drug discovery was directed at finding/designing drugs which could inhibit the reuptake of noradrenaline without affecting the cholinergic system. An example of these drugs are the noradrenaline selective reuptake inhibitors (NARIs),

such as , which were found to be effective antidepressants (Lauritsen &

Madsen, 1974).

In the 1970’s the development of antidepressants concentrated on enhancing

serotonergic transmission. The SSRIs have comparable efficacy {e.g. Bech, 1990;

Rickels & Schweizer, 1990) in the treatment of depression as the TCAs with the

important advantages of increased tolerability and a lack of anticholinergic side-effects

23 Chapter 1

(reviewed by Leonard, 1992). Five SSRIs are now available for the treatment of depression: fluoxetine, fluvoxamine, paroxetine, citalopram and sertraline. The SSRIs will be described in more detail in section 1.4 of the General Introduction.

More recent research into the development of antidepressants has moved away from drugs which act selectively on 5-HT neurotransmission. Two of the most recent antidepressants are the noradrenergic and specific serotonergic antidepressant (NASSA), and the mixed serotonin and noradrenaline reuptake inhibitor (SNRI), venlafaxine. Mirtazapine is an ai-adrenoceptor antagonist which increases locus coeruleus noradrenergic cell firing which in turn activates of tti-adrenoceptors causing an increase in the firing rate of dorsal raphe 5-HT neurones (Mongeau et a l, 1997).

Mirtazapine also has very low affinity for muscarinic, cholinergic and dopaminergic receptors, although it does have a high affinity for Hi histamine receptors (this could account for the side effects of drowsiness and sedation) (reviewed by de Boer, 1996). In contrast, venlafaxine causes direct inhibition of both noradrenaline and 5-HT reuptake but has no affinity for either muscarinic, histaminergic or receptors (Bolden-

Watson & Richelson, 1993).

The different types of antidepressant agent are numerous and their modes of action are quite varied: agents which inhibit enzyme activity, inhibit monoamine reuptake (noradrenaline and / or 5-HT), or act at adrenergic receptors can be effective antidepressants. The wide range of different compounds thought to have antidepressant effects is evidence of the complexity of the affective disorders. The success rate of currently available treatments of clinical depression is relatively low, only -10% of patients suffering from depression respond to treatment and in only 50% of these

24 Chapter 1 patients is the treatment a complete success (Broekkamp et al, 1995). There is a continuous search for a new pharmacological target for the development of novel and effective antidepressant drug treatments.

Future developments in the treatment of depression are likely to involve more diverse targets rather than monoamine uptake. Recent work has shown that drugs which reduce transmission at NMDA receptors, such as the competitive antagonist, AP-7 or the NMDA receptor-coupled channel blocker, MK-801, reduce immobility in the forced-swim test, a test generally predictive of antidepressant efficacy (Trullas &

Skolnick, 1990). Calcium channel antagonists are compounds which have also been investigated recently as potential antidepressants. Inhibitors of calcium channels have shown considerable effects on animals’ behaviour under conditions of environmental stress, suggesting potential antidepressant and antimanic properties (see Pucilowski et a l, 1990).

1.4 Selective serotonin reuptake inhibitors (SSRIs)

The SSRIs available in the clinic include, citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline. The SSRIs are considered to be of comparable efficacy in the treatment of depression as TCAs (e.g. Bech, 1990; Rickels & Schweizer, 1990). A major advantage of the SSRIs over the TCAs is their more favourable side effect profile

(discussed above) and their relative toxicity and safety in overdose. A lethal dose of

TCA may be as low as 10-15 times the normal daily therapeutic dose (deJonghe &

Swinkels, 1992). With SSRIs the therapeutic window is larger such that an SSRI- overdose generally does not seem to have fatal consequences (deJonghe & Swinkels,

1992). In his review of the SSRIs, Leonard (1992) reports that between 1985-1989 not a

25 Chapter 1 single death due to overdose with fluoxetine was reported. There are reports that SSRIs have been taken in doses of up to 13 times the maximum recommended dose without any cardiotoxic or epileptogenic effects being observed (paroxetine: Dechant &

Clissold, 1991; sertraline: Murdoch & McTavish, 1992).

The SSRIs are generally regarded as selective inhibitors of 5-HT reuptake. Their selectivity is based largely on the ratio of their affinity for inhibition of 5-HT uptake versus that for inhibition of noradrenaline uptake. The affinity of antidepressants for receptors, such as dopamine D2 receptors, 5-HT receptors, a- and p-adrenoceptors and histamine Hi receptors is also taken into consideration as this will affect the side effect profile of these drugs.

Citalopram is considered the most selective 5-HT uptake inhibitor as its affinity for receptors is morq than 200-fold lower than its affinity for inhibition of 5-HT uptake.

Fluoxetine however, shows appreciable binding to 5-HT%A receptors, will also inhibit noradrenaline uptake {K\ 0.14-10 |liM) and displaces [^H]desipramine binding with an

IC50 of 4.5 p.M (Hyttel, 1994; see Stanford, 1996).

A problem which becomes apparent on scrutiny of the literature is that the selectivity of SSRIs for inhibition of 5-HT over noradrenaline uptake is based largely on data from studies in vitro. These studies frequently compare uptake of monoamines into synaptosomes or brain slices prepared from different brain regions chosen according to which monoamine was being measured. For example, noradrenaline uptake in the hippocampus has been compared with 5-HT uptake in the cortex and with dopamine uptake in the striatum (Richelson & Pfenning, 1984; Bolden-Watson & Richelson,

26 Chapter 1

1993; Cheng et a l, 1993). This could well lead to spurious results as the uptake kinetics

vary considerably depending on which brain region is studied (Snyder & Coyle 1969;

Wong et al., 1975). In the study by Wong and colleagues (1975) the effects of systemic

fluoxetine pretreatment on [^H]5-HT uptake in different brain regions was compared.

This study found that the rate of [^H]5-HT uptake in the five brain regions differed

dramatically. Not surprisingly, the inhibition of [‘^H]5-HT uptake by fluoxetine also

varied according to the brain region.

A further problem with the classification of drugs as SSRIs is that despite their preferential inhibition of 5-HT uptake,when these drugs are administered systemically,the concentration of drug reached in the brain is well within the range of the concentration

of drug which inhibits the uptake of noradrenaline. The concentration of antidepressant

in rat brain has been measured by a number of groups and is consistently found to be in

the micromolar range {e.g. Dailey et a l, 1992; Melzacka et al., 1984). These references and others are discussed in detail in Chapter 3. Estimates for the K\ for inhibition of noradrenaline uptake by fluoxetine range between 0.1-10 |iM (see Stanford, 1996).

Indeed, in a study by Hyttel and colleagues (1984) it is pointed out that at 'clinical' plasma drug levels, the brain level of citalopram is 1 0 0 0 -fold higher than the concentration required to inhibit 5-HT uptake in vitro. Despite the brain concentrations of antidepressants being in the range of the K[ for inhibition of noradrenaline uptake by these drugs, the effects of SSRIs on the noradrenergic system are generally disregarded and hence, the antidepressant effects are attributed exclusively to the inhibition of 5-HT

reuptake.

27 Chapter 1

1.5 Monoamine transporters in general

In order for neurotransmission to occur, neurones must synthesize neurotransmitter and then store it intracellularly in vesicles. On stimulation of the neurones, neurotransmitter is released by exocytosis into the synapse. Once released, the action of neurotransmitter in the synapse must be terminated. In the case of monoamines, this is achieved by their removal from the synapse by uptake mechanisms.

Released transmitter is taken up by pre- and post-synaptic elements as well as into surrounding glial cells (Iversen, 1965; Russ et al., 1996). The monoamine transporters are of great interest as they are the site of action of many drugs, including antidepressants and psychomotor stimulants. As mentioned above, one of the features of the majority of antidepressants is that they increase monoaminergic transmission.

One way of achieving this is to inhibit the monoamine transporters and thus reduce the uptake of neurotransmitter.

Neurotransmitter reuptake occurs via membrane-bound transporter proteins.

Specialized transporter proteins exist for each of the monoamines. These transporters all have twelve (or thirteen) putative membrane spanning domains, the suggested arrangement of the protein within the plasma membrane is shown in Figure 1.1 (Giros et a l, 1991).

Giros and co-workers (1991) used chimeras of the noradrenaline and dopamine transporters to delineate discrete regions of the proteins responsible for the different features of these transporters. From their work it was concluded that transmembrane sections 9-12, and the carboxyl terminal region of the transporters were responsible for

28 Chapter 1 substrate specificity, with the carboxyl terminal portion conferring the high affinity substrate specificity. The sensitivity to uptake blockers seems to be conveyed by the regions of the transporter comprising: extracellular loop 3, intracellular loop 4 and transmembrane domains 6 , 7 & 8 (Figure 1,1). Buck and Amara (1995) have also studied chimeras of noradrenaline and dopamine transporters and largely confirmed the findings of Giros and colleagues.

The transport of monoamines is Na"^ and Cl’-dependent. The ionic dependence of monoamine transport has been reviewed by Kanner & Schuldiner (1987) and

Rudnick & Clark (1993). Interestingly, when the extracellular concentration of either

Na"^ or Cr is reduced, the transporter reverses so that monoamines are pumped out of the neurone; this was demonstrated when the human noradrenaline or dopamine transporters were expressed in COS-7 cells (Pifl et a i, 1997). Transport is also energy dependent as it often occurs against a concentration gradient. The energy required is generated from

Na^ / K^-ATPase derived from ion gradients across the plasma membrane (Amara &

Kuhar, 1993).

It is proposed (see Borowsky & Hoffman, 1995) that Na"^ and Cl and the substrate {i.e. monoamine) bind to the extracellular side of the transporter causing a conformational change of the transporter. Once the substrate is released intracellular! y, the transporter returns to its original ‘inactive’ conformation. This ‘alternate access pore’, or ‘flip-flop’, model of monoamine transport suggests the presence of a central aqueous pore within the transporter. Within this pore are ‘gates’ which expose the binding sites for substrates and ions to either the intra- or extracellular surface (for the

5-HT transporter: Mager et al, 1994).

29 Chapter 1

Figure 1.1: Schematic representation of the basic features of the Na'^ / Cl-dependent monoamine transporters

The transporter protein has 12 membrane spanning domains; both the NH 2 and the COOH- termini are on the cytoplasmic side of the membrane. The arrows ‘a’ and ‘b’ separate the putative functional domains of the monoamine transporter: the section from the N- terminus to arrow ‘a’ is thought to determine the ionic dependence of the uptake mechanism. The section of the protein between arrows ‘a’ and ‘b’ is responsible for interaction of the transporter with antidepressants and cocaine. The region between arrow ‘b’ and the carboxyl terminus is thought to determine the substrate affinity and stereoselectivity of the transporter. Figure adapted from Borowsky & Hoffman (1995).

30 Chapter 1

For the noradrenaline transporter the stoichiometry appears to be

1 Na"^:l Cl': I noradrenaline (Ramamoorthy et al, 1992; Friedrich & Bonisch, 1986).

The cloned dopamine transporter is proposed to transport dopamine with a ratio of

2 Na"^:! Cl':! dopamine (McElvain & Schenk, 1992). Transport of 5-HT via the cloned

5-HT transporter is facilitated by transport of or H^ (from outside to inside) with the ratio of transport being 1 Na^:l Cl‘:l K^:l 5-HT (see Rudnick & Clark, 1993; Mager et al, 1994).

Recent advances in molecular biology have allowed the cloning of many neurotransmitter uptake carriers. This has enabled the different monoamine transporters to be characterized. As discussed earlier, antidepressant monoamine reuptake inhibitors generally selectively inhibit transport by one or more of the monoamine transporters.

The selectivity of SSRIs, for example, is based on their affinity for inhibiting the reuptake of 5-HT via the 5-HT transporter. The ability to express cloned transporters in cultured cells allows the pharmacological effects of a drug on a particular transporter to be investigated in isolation. An additional benefit of cloned transporters, is that the anatomical distribution of different transporters can be mapped by looking at the distribution of the mRNA coding for the transporter proteins.

1.5.1 The noradrenaline transporter

The human noradrenaline transporter was one of the first neurotransmitter transporter proteins to be cloned (Pacholczyk et a l, 1991). All of the following features of the human noradrenaline transporter were characterised and studied in detail by

Pacholczyk and colleagues (1991). Uptake of noradrenaline via the human noradrenaline transporter was shown to be Na'^-dependent and sensitive to known

31 Chapter 1 inhibitors of noradrenaline uptake. The amino acid sequence of the noradrenaline transporter protein predicted a protein with 12 or 13 hydrophobic regions, indicating membrane spanning domains. The 69-kiloDalton (kD) protein was found to be expressed in the brain stem corresponding to the location of noradrenergic cell bodies in the locus coeruleus. The cDNA encoding the noradrenaline transporter was expressed in HeLa cells where the uptake of ['^H]noradrenaline was studied.

The protein showed an affinity for noradrenaline transport {Km) of 457 nM, with uptake of [^H]noradrenaline sensitive to inhibition by desipramine {K\ 3.9 nM).

Interestingly, uptake of [^H]noradrenaline by this protein was also inhibited by the

SSRI, paroxetine {K\ 312 nM), and dopamine (Æ; 139 nM). The amino acid sequence of this noradrenaline transporter shows remarkable similarity (46%) to the rat and human

GAB A transporters (Guastella et al, 1990; Nelson et al, 1990). The sequence homology between neurotransmitter transporter proteins has been exploited in that cDNA libraries have been screened for protein sequences which might represent other neurotransmitter transporters. This led to the isolation of cDNAs encoding 5-HT {e.g.

Blakely et al., 1991; Hoffman et al, 1991) and dopamine {e.g. Giros et a l, 1991; Kilty et a l, 1991; Shimada et al, 1991) transporter proteins.

1.5.2 The 5-HT transporter

The cDNA coding for a functional 5-HT transporter was identified using the polymerase chain reaction with degenerate oligonucleotides derived from two highly conserved regions of the noradrenaline and GAB A transporter proteins (Blakely et a l,

1991). This method identified an amino acid sequence which coded for a 69 kD protein which, when expressed, formed a fully functional 5-HT transporter. This transporter has

32 Chapter J been studied extensively by Blakely and colleagues (1991). The cloned 5-HT transporter has an affinity of 320 nM for 5-HT uptake. Transport of 5-HT via the cloned transporter is inhibited by many classes of uptake blocker; tertiary amine tricyclic antidepressants e.g. imipramine (K\ 18.7 nM), secondary amine tricyclic antidepressants e.g. desipramine (K[ 567 nM) and SSRIs such as paroxetine {K\ 0.4 nM). The cloned rat 5-HT transporter shows greater amino acid sequence conservation with the human noradrenaline transporter (50%) than the rat GAB A transport protein (43%) (Blakely et al., 1991).

1.5.3 The dopamine transporter

Giros and co-workers (1991) used a partial cDNA clone from the substantia nigra to clone and characterize a full length rat cDNA which encoded a cocaine- sensitive dopamine transporter. Using in situ hybridization, they showed that the mRNA for this cloned transporter is located in areas A9 and A10, the substantia nigra and the ventral tegmental area. These are the areas of the brain which contain large numbers of dopaminergic cell bodies, consistent with the cloned transporter being a dopamine transporter. Giros and co-workers (1991) expressed the 2.2 kb cDNA clone in

COS-7 cells and demonstrated that uptake of dopamine was Na'^-dependent. The affinity of the expressed transporter for uptake of dopamine was in the region of 300 nM. This uptake was blocked by known inhibitors of dopamine uptake, such as GBR

12909 (^i 12 nM) and nomifensine {Kj 318 nM) (Giros et al., 1991). Dopamine uptake was inhibited only weakly by the noradrenaline uptake blocker, desipramine (K\ >10 pM). The cloned rat dopamine transporter showed 78% overall identity with the noradrenaline transporter (Giros et al, 1994).

33 Chapter 1

1.5.4 Similarities between the noradrenaline and dopamine transporters

Studies of the noradrenaline and dopamine transporters found that the noradrenaline transporter efficiently transports dopamine and vice versa (Giros et al,

1994). The noradrenaline transporter binds noradrenaline and dopamine with similar affinities, whereas the dopamine transporter binds dopamine with almost 1 0 -fold higher affinity than noradrenaline. Interestingly, the dopamine transporter is reported to have a more than 2 -fold greater rate of uptake (Vmax) of noradrenaline than the noradrenaline transporter (Giros et a i, 1994), and the noradrenaline transporter transports dopamine with 1.6 times the V"max of the dopamine transporter (Buck & Amara, 1995).

Despite this weak substrate selectivity, the catecholamine transporters can be distinguished pharmacologically by their sensitivity to inhibition by uptake blockers.

The difference in the sensitivity of synaptosomal monoamine uptake to inhibition by desipramine (Richelson & Pfenning, 1984; Thomas et al., 1987; Bolden-Watson &

Richelson, 1993) is likely to be due to its selectivity for the transporter rather than the substrate.

Using cloned transporters it was shown that desipramine inhibits ['^H]dopamine uptake via the noradrenaline transporter (K\ 10 nM) more potently than [^H]dopamine uptake via the dopamine transporter (K\ 24 p,M, Giros et al., 1991). Similarly, the selectivity of fluoxetine for inhibition of 5-HT uptake is thought to reside in its selectivity for the transporter rather than the substrate {i.e. 5-HT). This was demonstrated in rat pineal gland where 5-HT is taken up via a noradrenaline transporter, this uptake of 5-HT is not inhibited by fluoxetine, but is inhibited by desipramine

(Fuller & Ferry, 1977). Unfortunately, there is a void in the literature when it comes to

34 Chapter I transport of noradrenaline by the cloned 5-HT transporter. Experiments have not been performed to test whether the 5-HT transporter is capable of transporting noradrenaline or dopamine as are the dopamine or noradrenaline transporters.

Considering the weak substrate selectivity of the cloned transporters the criteria for identification of a protein as a transporter of a specific monoamine are unclear. It would be interesting to see whether, like the dopamine transporter, the 5-HT transporter also transports noradrenaline. The sensitivity of noradrenaline uptake into non- noradrenergic neurones, to inhibition by SSRIs and other antidepressants should be investigated in order to determine the selectivity of these drugs.

The results of experiments which will be described in this thesis suggest that there are at least three different sites of noradrenaline uptake, distinguished by their sensitivity to inhibition by desipramine and fluoxetine, in the rat cortex. Whether any of these sites represents uptake of noradrenaline via a transporter classified as the 5-HT or dopamine transporter remains to be seen.

1.6 The noradrenergic innervation of rat brain

The presence of noradrenergic neurones in rat brain was first discovered when the formaldehyde histofluorescence method of Falck and Hillarp (Falck et al., 1962) was used by Dahlstrom and Fuxe (1964). Dahlstrom and Fuxe (1964) used formaldehyde fluorescence methods to identify catecholamine- and 5-HT-containing neurones in the rat brain. This group classified nerve cells in the brain stem according to their localization and morphology. The catecholamine cell bodies were divided into the

35 Chapter 1 groups A1-7. These are divided into the two main subgroups of noradrenergic nuclei, the locus coeruleus complex (A4 & A 6 ) and the lateral tegmental system (Al-3, A5 &

A7). The locus coeruleus noradrenergic system is described in the next section. The lateral tegmental system is described in section 1 .6 .2 .

Early studies of the central noradrenergic system gave root to the opinion that noradrenaline release was uncontrolled and non-specific. The release of noradrenaline has been likened to an aerosol, with released noradrenaline acting diffusely in tissue distant from the site of release (Dismukes, 1977). Experiments which visualized noradrenergic axons using autoradiography claimed that these axon terminals lacked discrete synaptic connections and suggested that noradrenaline was acting like a neuromodulator, influencing the release of other transmitters, rather than a neurotransmitter (Descarries et al., 1977).

There is, however, much evidence for the highly organized topographical arrangement of noradrenergic neurones suggesting specialized or controlled functions of noradrenaline in rat brain. Work using dopamine-P-hydroxylase (DfH, noradrenaline synthesizing enzyme) immunocytochemistry utilizing the peroxidase-antiperoxidase method (Olschowka et at., 1981) demonstrated the presence of “highly restricted synaptic contacts” through which noradrenergic neurones exerted their effects on other neurones. This finding was supported by later work performed by Papadopoulos and co-workers (1987) who immunocytochemically stained ultrathin sections of cortex with antibodies to noradrenaline. [Although not stated in this particular publication, antibodies were normally raised, not to noradrenaline itself, but rather to the synthesizing enzymes or to noradrenaline complexed with a larger protein (see

36 Chapter 1

Papadopoulos & Parnavelas, 1991).] Papadopoulos’ group (1987) showed that the majority (-90%) of stained terminals formed ""synapses characterized by specialized junctional appositions"\

Foote and colleagues (1983) described the release of noradrenaline as follows:

""the effects of the locus coeruleus- system on target neurons are characterized by temporal and spatial discreteness. That is, the locus coeruleus- norepinephrine system acts on a discrete, specifiable set of neurons under a particular set o f circumstances to produce a defined set o f effects. ”.

Despite this evidence however, noradrenaline release is still described as:

"a neural aerosol - one press on the locus coeruleus button and large areas of the brain are diffusely sprayed with noradrenaline" (see Rang & Dale, 1987) in one of the leading textbooks of Pharmacology.

1.6.1 The locus coeruleus

The locus coeruleus consists exclusively of noradrenergic neurone cell bodies

(approximately 1600 cells in each side of rat brain; Swanson, 1976) and accounts for approximately 43% of all noradrenaline neurones in the rat brain (see Swanson 1976).

There are 4 main pathways projecting from the locus coeruleus 1) the dorsal catecholamine bundle, 2) the median forebrain bundle, 3) the dorsal longtitudinal fasciculus in the periaqueductal grey and 4) the medullary catecholamine bundle (Moore

& Bloom, 1979). Locus coeruleus neurones show extensive collaterization such that one neurone can innervate parts of the cortex as well as the cerebellum, for example

(Loughlin gf a/., 1982).

37 Chapter 1

The locus coeruleus was found not to be simply a homogenous collection of cell bodies, but different parts of the locus coeruleus project to discrete brain regions

(Waterhouse et a l, 1983). In 1986, Loughlin and colleagues described six morphologically distinct types of cell in the locus coeruleus. The noradrenergic innervation of the cortex, the hippocampus and the olfactory bulb all derive exclusively from the locus coeruleus (Ungerstedt, 1971; Shipley et al, 1985). Many brain regions receive noradrenergic innervation from both the locus coeruleus and the lateral tegmental system (the hypothalamus receives the majority of its noradrenergic innervation from the lateral tegmental system, see section 1.6.2). Interestingly, no brain region is exclusively innervated by noradrenergic neurones which derive from the lateral tegmental system.

1.6.2 The lateral tegmental noradrenergic system

Fluorescence histochemical data suggest that no neurones from the lateral tegmental system innervate the neocortex (Moore & Bloom, 1979). Areas including the spinal cord, brainstem, hypothalamus and basal telencephalon are innervated by noradrenergic neurones of the lateral tegmental system. The principal target of lateral tegmental noradrenergic neurones is the medial hypothalamus. The locus coeruleus innervation of the hypothalamus is limited such that the hypothalamus is the brain structure closest to being exclusively innervated by noradrenergic neurones from the lateral tegmental system (Olson & Fuxe, 1972). Each of the nuclei of the hypothalamus has been shown to contain noradrenaline (Palkowitz et al, 1974). Interestingly, even the hypothalamic nucleus with the lowest noradrenaline content ( 1 2 ng / mg protein) contains more noradrenaline than samples of tissue from the neocortex (4 ng / mg protein). In general, the tissue noradrenaline content of brain regions innervated

38 Chapter J primarily by noradrenergic neurones from the lateral tegmental system, such as the hypothalamus is roughly 1 0 -fold higher than that of brain regions innervated by locus coeruleus noradrenergic neurones such as the cortex or hippocampus (Zaczek & Coyle,

1982).

Noradrenergic axons arising from the lateral tegmental system can be distinguished from locus coeruleus neurones by their histological appearance (see

Moore & Bloom, 1979). Lateral tegmental neurones have larger varicosities which are irregular in both their size and their distribution along the axons. A further difference in noradrenergic neurones originating in the locus coeruleus, compared with those of the lateral tegmental system, is their affinity for noradrenaline uptake. Synaptosomes prepared from the hypothalamus have a higher affinity for [^HJnoradrenaline uptake

(40 nM) than those prepared from the cortex (100 nM; Zaczek et a l, 1990). Another difference between the noradrenergic neurones of these two systems is that the turnover rate of noradrenaline in the hypothalamus is slower than that in the cortex (Lidbrink &

Jonsson, 1971).

These findings suggest that noradrenergic neurones originating in the locus coeruleus and the lateral tegmental system differ pharmacologically, functionally and are anatomically distinct. These two types of noradrenergic neurones also differ in their sensitivity to the noradrenergic neurotoxin, DSP-4 (locus coeruleus neurones are affected at lower doses). The effects of this neurotoxin are discussed in section 1.9.

39 Chapter 1

1.7 The serotonergic innervation of the cortex

As is the case with the central noradrenergic system, the serotonergic innervation of the brain shows a high degree of neuroanatomical organisation. 5-HT neurones can be visualised using the formaldehyde induced fluorescence histochemical technique

(Falck et a l, 1962). The projections of 5-HT neurones in the brain were subsequently mapped by a number of research groups (Falck et a l, 1962; Carlsson et a l, 1962;

Dahlstrom & Fuxe, 1964; Ungerstedt, 1971).

Due to the poor fluorescence of 5-HT (c/noradrenaline) it is difficult to visualise

5-HT nerve axons, however a combination of techniques has improved the situation.

The retrograde transport of tracers from known terminal fields (Segal & Landis, 1974) or the anterograde axonal transport of radiolabelled amino acids injected into regions rich in 5-HT containing cells (Azmitia & Segal, 1978) have been followed. Also, autoradiography has been used to localise [^H]5-HT which had been injected into the ventricles of animals (Chan-Palay, 1975; Descarries et a l, 1982). In addition to these techniques, immunohistochemistry using antibodies raised either to 5-HT itself or to tryptophan hydroxylase, the 5-HT synthesising enzyme, has been used to map the serotonergic pathways of the brain (Steinbusch et a l, 1978; Steinbusch et a l, 1981;

Weissmann e ta l, 1987).

In 1964, using formaldehyde induced fluorescence, Dahlstrom & Fuxe identified nine groups of 5-HT containing cell bodies in rat brain (B1-B9). These groups of 5-HT cell bodies are divided into two divisions: the caudal division (B1-B4) and the rostral division (B5-B9). The caudal division includes the medulla oblongata and caudal pons.

40 Chapter 1 and projects to the brain stem and spinal cord. The rostral division consists of the midbrain and the rostral pons and projects to the forebrain (Tork, 1990). The rostral division is further subdivided into the dorsal raphe nucleus (B 6 & B7) and the median raphe nucleus (B5 & B8 ) (Tork, 1990).

The projections from the dorsal and median raphe nuclei innervate the forebrain via a number of fibre tracts, the most important being the median forebrain bundle. In the terminal fields the serotonergic projections branch extensively, such that most areas of the forebrain are innervated by 5-HT neurones of the dorsal and median raphe nuclei.

The cortex and hippocampus are innervated by 5-HT neurones from the median and dorsal raphe nucleus, whereas regions such as the striatum and nucleus accumbens are innervated predominantly by 5-HT neurones of the dorsal raphe nucleus (Azmitia &

Segal, 1978; Vertes & Martin, 1988).

1.8 Interactions between the noradrenergic and serotonergic systems

The different monoaminergic systems within the CNS are no longer considered to be isolated systems. Interactions between the noradrenergic and serotonergic systems, for example, have been demonstrated extensively. The innervation of dorsal raphe serotonergic neurones by locus coeruleus noradrenergic neurones has been shown using a variety of techniques including electron microscopy autoradiography (Baraban

& Aghajanian, 1981) and retrograde tracing with horseradish peroxidase (Sakai et a l,

1977).

41 Chapter 1

The presence of ai-adrenergic receptors in the dorsal raphe was demonstrated by radioligand binding of the oci-adrenoceptor ligand, [^Hjprazosin (Unnerstall et al,

1985). The reduction in 5-HT cell firing caused by microiontophoretic application of

Œ]-antagonists is reversed by activation of tti-adrenoceptors with ai-agonists (Baraban

& Aghajanian, 1980a & b). Conversely, ai-adrenoceptor activation decreases 5-HT release. Systemic administration of the ai-adrenoceptor agonist, , reduces, while a2-antagonists, e.g. , increase 5-HT cell firing, (Garratt et a i, 1991;

Svensson et al., 1975). These findings suggest that 5-HT neuronal firing is under inhibitory control through a 2-adrenoceptors and is facilitated by activation of ai- adrenoceptors.

Conversely, and more relevant to this thesis (w.r.t. the effects of SSRIs on noradrenaline efflux), there is much evidence for the modulation of noradrenaline release by 5-HT. This can be due to activation of receptors located either on nerve terminals or on cell bodies. In experiments where drugs are administered systemically it is sometimes not possible to determine the location of the receptors involved. When slices of hippocampus, or cortex etc. are used, the effects of drugs on release can only be due to activation of receptors in the terminal field.

Feuerstein & Hertting (1986) demonstrated that the 5-HT agonists, 5-HT,

2-methyl-5-HT and carboxamidotryptamine increased electrically evoked noradrenaline release from preloaded rabbit hippocampal slices. This modulation of noradrenaline release was mediated by 5 -HT3 receptors as the effect was blocked by the 5-HT 3 , (Feuerstein & Hertting, 1986).

42 Chapter 1

In contrast, K'^-evoked noradrenaline release from slices of rat hypothalamus was not affected by 5-HT on its own (Blandina et a l, 1991). In the presence of (S-HTi.uke / 5-HTi antagonist) or (5-HTic / 5-HT2 antagonist),

5-HT caused a reduction in K'^-evoked noradrenaline release (Blandina et a l, 1991).

The 5-HT-mediated decrease in noradrenaline release was blocked by the 5-HT^ antagonists, or ICS 205-930. These results suggest that in the rat hypothalamus, release of noradrenaline is modulated through activation of 5 -HT3 receptors (Blandina e ta l, 1991).

Facilitation of noradrenaline release by 5-HT was also demonstrated by

Mongeau and colleagues (1994). This group showed that electrically-evoked release of

['^H]noradrenaline from hippocampal slices in vitro was increased by the 5-HTs agonist,

2-methyl-5-HT. It was also shown that the SSRI, paroxetine as well as exogenous 5-HT facilitated noradrenaline release. The involvement of 5 -HT3 receptors was indicated as the increase due to each of the above was blocked by the 5 -HT3 antagonist, ondansetron

(Mongeau et a l, 1994). The facilitation of noradrenaline release was not affected by a lesion of 5-HT neurones with 5,7-DHT, suggesting that these receptors were not on

5-HT neurones (Mongeau et a l, 1994).

Using in vivo voltammetry, 5-HTia, 5-HTib and 5-HT% receptors have been shown to modulate the neuronal activity of locus coeruleus noradrenergic neurones

(Clement et a l, 1992). Systemically administered agonists of 5-HTia and 5-HTib receptors ( and CGS-12066B, respectively) were shown to increase the

DOPAC signal, a measure of noradrenaline synthesis, in the locus coeruleus.

43 Chapter 1

Ritanserin, an antagonist of 5-HT% receptors, increases the neuronal activity of locus coeruleus neurones (Clement a/., 1992).

The modulation of noradrenaline release has also been measured using microdialysis in vivo. Done & Sharp (1992) showed that noradrenaline efflux in the hippocampus of anaesthetised rats was reduced (by 50%) after systemic administration, but not local infusion, of 5-HT 2/5 -HT1C receptor agonists (DOI or DOB). This decrease was prevented by pretreatment with which is an antagonists at S-HTia receptors. The 5 -HT2 agonist and uptake blocker, , and the 5-HT releasing agent, p-chloroamphetamine, also reduced noradrenaline efflux in the hippocampus through a mechanism inhibited by ritanserin (Done & Sharp, 1992).

It is suggested that noradrenaline release in the hippocampus is inhibited by activation of 5 -HT2 (possibly 5-HT2a) receptors on noradrenergic neurones in the locus coeruleus; this inhibitory control can be removed by a decrease in 5-HT release, by activation of 5-HTia autoreceptors or by blocking postsynaptic 5 -HT2 receptors (Done

& Sharp, 1994). This role of 5 -HT2 receptors is supported by the findings of Rasmussen

& Aghajanian (1986) that the effects of hallucinogens ( or 6^-lysergic acid diethylamide) on locus coeruleus activity were reversed by systemic treatment with the selective 5 -HT2 antagonists, LY 53857 and ritanserin.

5-HT IA receptors also modulate noradrenaline release as and

8-OH-DPAT (5-HTiA agonists), administered systemically, increase noradrenaline

efflux in the hippocampus and hypothalamus (Done & Sharp, 1994; Suzuki et al.,

1995). The increase in noradrenaline efflux in the hippocampus, due to systemic 8-OH-

44 Chapter 1

DP AT, is blocked by treatment with the 5-H T| a antagonist, WAY 100635 (Hajos-

Korcsok & Sharp, 1996). Because WAY 100635 had no affect on its own, these findings indicate that noradrenaline release can be stimulated by activation of 5-HT| a receptors which are not tonically activated (Hajos-Korcsok & Sharp, 1996). Because the drugs were only administered systemically in this study, the location of the receptors involved cannot be determined.

Suzuki and colleagues (1995) showed that 5-HTia receptor activation by systemic treatment with a 5-HTia agonist (MKC-242) also increases noradrenaline efflux in the rat hypothalamus. This effect was unaltered when 5-HT neurones were lesioned with 5,7-DHT, suggesting that noradrenaline efflux can be increased through activation of postsynaptic 5-HTiA receptors (Suzuki et al., 1995). This study also investigated the effect of local infusion, into the hypothalamus, of 8-OH-DPAT or a

5 -HT3 antagonist, MDL 72222. As local application of these compounds had no effect on noradrenaline efflux, it is likely that noradrenaline release in the hypothalamus, at least, is not modified through 5-HT receptors in the terminal field (Suzuki et a l, 1995).

1.9 The noradrenergic neurotoxin, DSP-4

DSP-4 is a beta haloethylamine derivative of benzylamine which preferentially targets noradrenergic neurones arising from the locus coeruleus (Jonsson et at., 1981).

More recently however, it has been shown that noradrenaline is depleted in areas including the hypothalamus when the dose of DSP-4 is increased {e.g. 100 mg / kg: Heal etal., 1993).

45 Chapter 1

The long-term effects of DSP-4 on peripheral and central noradrenaline levels

and neuronal uptake were originally described by Ross and Renyi (1976). DSP-4 has been used extensively as a tool to investigate behaviour when noradrenergic function is compromised {e.g. Spyraki et a l, 1982; Delini-Stula et al., 1984; Zagrodzka et al.,

1994; Matsumoto et al., 1995a). DSP-4 is thought to be taken up into neurones via the

high affinity desipramine-sensitive noradrenaline uptake site (Lee et al., 1982). The ability of DSP-4 to lesion noradrenergic neurones relies on a functional noradrenaline transporter, the neurodegenerative actions of DSP-4 can be prevented by pretreatment

with the noradrenaline uptake blocker, desipramine (15 mg / kg: Zieher & Jaim-

Etcheverry 1980; Jonsson etal., 1981).

DSP-4 is used in preference to other noradrenergic neurotoxins because, 1) it is extremely selective for noradrenaline over other monoamines (unlike 6- hydroxydopamine (6-OHDA)), and 2) it can be administered systemically to adult rats

(c/i.c.v. route for 6-OHDA) (Zieher & Jaim-Etcheverry, 1980). When DSP-4 is in its tertiary amine form it crosses the blood-brain barrier freely (Zieher & Jaim-Etcheverry,

1980). As with the other beta-haloamine neurotoxin, xylamine, (Ransom et al., 1982), it

is the aziridinium ion which accounts for the activity of DSP-4 in vitro (Zieher & Jaim-

Etcheverry, 1980). DSP-4 is thought to cause its neurotoxic effects by forming

irreversible covalent bonds with electrophilic centres within the nerve terminal (see

Dudley et al., 1990; Figure 1.2).

1.9.1 The selectivity of DSP-4 for locus coeruleus noradrenergic neurones

Among noradrenergic neurotoxins there is a variable degree of selectivity.

DSP-4 (Jonsson et al., 1981), and to a lesser extent 6-OHDA (Jonsson & Sachs, 1973),

46 Chapter 1

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)

Aziridinium ion

Tissue-SH

S Tissue

Figure 1.2: The chemical structure of DSP-4

The aziridinium ion formed from DSP-4 is highly reactive towards nucleophilic groups such as thiols and amines. This results in the formation of irreversible covalent bonds between DSP-4 and tissue proteins. Figure adapted from Dudley et al., (1990).

47 Chapter 1 preferentially targets locus coeruleus neurones, and both 6-OHDA and DSP-4 act at nerve terminals rather than at the cell bodies. Many theories explaining this selectivity have been put forward with the general conclusion that 6-OHDA and DSP-4 are quite different neurotoxins. Briefly, differences in the concentration of noradrenaline (Sachs

& Jonsson, 1975a), the surface area to volume ratio of varicosities (Sachs & Jonsson,

1975b) or the density of noradrenergic neurones in the terminal field (Kostrzewa, 1988) have been suggested to determine the sensitivity of noradrenergic neurones to neurotoxins.

These suggestions are disputed by Fritschy & Grzanna (1991) who believe that the selectivity of DSP-4 is more likely to be due to a difference in the reuptake of noradrenaline into locus coeruleus and lateral tegmental system noradrenergic neurones.

This is borne out by studies of ['^H]noradrenaline uptake into synaptosomes prepared from either the cerebral cortex (locus coeruleus neurones) or the hypothalamus

(predominantly lateral tegmental system neurones) which showed different affinities for noradrenaline uptake in these two regions (Zaczek et al, 1990).

The study by Zaczek and colleagues (1990) showed that the noradrenaline uptake carriers in these two regions may be pharmacologically distinct. Noradrenaline transport into hypothalamic synaptosomes had a of \ 40 nM whereas in cortical synaptosomes the Km was only 100 nM. These experiments also showed that DSP-4 causes competitive inhibition of [^H]noradrenaline uptake in vitro. Interestingly, DSP-4 inhibits uptake of [^H]noradrenaline into cortical synaptosomes (Ki -180 nM) more potently than uptake into hypothalamic synaptosomes (Ki -460 nM). These findings

48 Chapter 1 suggest that DSP-4 may have more toxic effects on locus coeruleus neurones because these neurones accumulate more DSP-4 than lateral tegmental system neurones.

1.9.2 Changes in central noradrenergic neurones after treatment with DSP-4

Indices of a noradrenergic lesion which are widely used include: depletion of tissue noradrenaline content, loss of noradrenaline uptake sites, loss of noradrenaline immunoreactivity, and loss of the noradrenaline synthesising enzyme, dopamine-p- hydroxylase (DpH) immunoreactivity.

Depletion of noradrenaline is the most widely used index of a noradrenergic lesion after treatment with DSP-4. Nearly all studies using DSP-4 have measured the noradrenaline concentration in different brain regions using either HPLC {e.g. Zahniser et a i, 1981; Heal et a l, 1993; Hallman & Jonsson, 1984) or fluorometric techniques

{e.g. Ross, 1976; Dooley et al, 1983). Investigations of the time-course and dose- dependence of the effects of DSP-4 have shown that loss of transmitter occurs within 3 days and doses of 50-75 mg / kg result in maximal depletion of cortical noradrenaline content (Ross, 1976; Bennett et al., 1984; Cheethamet a l, 1996).

The presence of DpH, is also used to demonstrate a noradrenergic lesion. In

1976, Ross used enzymatic assays to show that the activity of DPH is decreased in rat brain and heart after treatment with DSP-4. In this paper the dose-dependence of the reduction in DPH activity by DSP-4 was shown. More recently, immunohistochemical methods have been employed to visualize the distribution of DpH in rat brain (Fritschy

& Grzanna, 1989; Fritschy et al., 1990). A reduction in DpH staining is interpreted as

49 Chapter 1 being indicative of degeneration of noradrenaline neurones. The later report by Fritschy and colleagues (1990) studied the time course of the reduction in DpH- and noradrenaline immunoreactivity.

Unlike noradrenaline, there is a delay in the effect of DSP-4 on DpH staining. A marked reduction in noradrenaline staining was apparent 6 h after DSP-4 treatment, whereas DPH was not affected until day 4 after DSP-4 treatment. The effect of DSP-4 on DpH, as evidenced by the rapid and substantial loss of DpH protein, was most prominent between 4 and 5 days after treatment with DSP-4. DpH staining was only reduced in brain regions where NA staining was also diminished.

A reduction in the uptake of [^H]noradrenaline into synaptosomes or slices prepared from DSP-4 treated rats has often been found to be reduced (Ross, 1976; Lee et al., 1982; Hallman & Jonsson, 1984). This is thought to be due to binding of DSP-4 to the high-affinity desipramine-sensitive noradrenaline transporter (Lee et a l, 1982).

Binding of markers for the noradrenaline uptake site(s) is also reduced in brain tissue from DSP-4 lesioned rats. Lee and colleagues (1982) used [^HJdesipramine binding as a marker of the noradrenaline transporter(s). 3 days after DSP-4 treatment, high affinity

[^H]desipramine binding to cortical membranes was reduced by approximately 80%.

Hallman and Jonsson (1984) reported similar findings 1 week after DSP-4 treatment.

More recently, ['^H]nisoxetine was demonstrated to be a radioligand which labels neuronal noradrenaline uptake sites (Tejani-Butt et at., 1990). Cheetham and co­ workers (1996) found a reduction in [^H]nisoxetine binding after DSP-4. Treatment

50 Chapter 1 with DSP-4 caused a dose-dependent reduction in the number of ['^H]nisoxetine binding sites (20-97%) 3 days later (Cheetham et al., 1996).

Changes in noradrenaline turnover in regions affected by treatment with DSP-4 have also been reported. Hallman and Jonsson (1984) found that in DSP-4 treated rats, the pargyline-induced increase in noradrenaline concentration was greater in locus coeruleus-innervated brain regions. This suggests that in lesioned brain regions, noradrenergic neurones which survive DSP-4 treatment have an increased rate of noradrenaline turnover. This finding is supported by the work of Logue and colleagues

(1985) who showed that noradrenaline turnover, measured by the ratio of MHPG-SO 4 (a metabolite of noradrenaline) to noradrenaline concentration, was increased in surviving neurones in DSP-4 treated rats. Again, this increase was only evident in brain regions where noradrenaline was depleted {i.e. cortex and hippocampus).

Changes in the density and affinity of adrenergic receptors after treatment with

DSP-4 are the final issues which I will discuss. Results from radioligand binding studies of p-adrenergic receptors after DSP-4 treatment are generally in agreement with each other. An increase in the Bmax of P-adrenoceptor binding is reported by four studies which have used either [^H] ([^H]DHA), or [^^^I]

(['^^I]CYP) binding (Jonsson et al., 1981; Dooley et al., 1983; Zahniser et al., 1986;

Theron et al, 1993). A change in binding affinity has been found only by Theron’s group. This group found a reduced affinity of [*^^I]CYP binding in the cortex and hippocampus 1, 3 ,7 and 14 days after DSP-4 treatment. It is suggested that the changes in Bmax and Kd represent changes in binding to Pi-adrenoceptors. This is supported by

51 Chapter 1 results from a study by Dooley and colleagues (1986) which reported an increase in pi, but not p 2, receptor number after treatment with DSP-4. In the case of a-adrenergic receptors the effects of a DSP-4 lesion are less consistent. In 1981, Jonsson and co­ workers reported no change in the binding of the a-adrenoceptor ligand, ['^H]WB4101 after treatment with DSP-4.

Similarly, no change in the binding of the aradrenoceptor ligand [^^^I]BE2254 to cortical and hippocampal membranes prepared from DSP-4 treated rats was observed

(Zahniser et a i, 1986). Using the ligand [^H] however, Nowak and colleagues

(1991) reported an increase in the Bmax of cortical ai-adrenoceptors 20 days after treatment with DSP-4. Dooley and colleagues (1983) reported an increase in the affinity of [^H]p-aminoclonidine for ai-adrenoceptors in all brain regions, but no significant changes in receptor density were observed. The authors speculate that the extent of the

DSP-4 lesion is sufficient to cause an increase in agonist affinity, but insufficient to cause an increase in receptor number. The Bmax of ['^HJidazoxan binding to presynaptic

Œz-adrenoceptors in cortex, hippocampus, cerebellum and hypothalamus was reduced 3 days after treatment with DSP-4 (Heal et a l, 1993). After 15 days these changes were no longer evident (presumably due to postsynaptic az-adrenoceptors proliferation). This could explain why Dooley’s group (1983) failed to detect any changes in a 2-adrenoceptor number.

To conclude, there are numerous changes which occur to noradrenergic neurones after treatment with DSP-4. The different time courses of the various effects suggest that the response of noradrenergic neurones to DSP-4 treatment has at least two phases.

53 Chapter 1

There is an acute phase (up to 3-4 days) where transmitter and presynaptic adrenoceptors are lost followed by a neurodegenerative phase (day 4 onwards) where there is loss of DpH signifying the destruction of noradrenaline neurones as well as changes in adrenoceptor function. After 1 week post-DSP-4, adaptive changes seem to take place with possible extensive regrowth of noradrenaline neurones (Fritschy &

Grzanna, 1992).

54 Chapter 2

Chapter 2

Materials and Methods

2.1 Introduction

Over the past 40 years many methods have been developed to monitor the release and uptake of central monoamines. Early techniques relied upon measurement of tissue monoamine content, monoamine synthesis or measurement of monoamine metabolites to indicate the underlying changes in neurotransmission. The accuracy of such techniques is less than desired because the actual extracellular concentration of monoamines can only be inferred from these measurements; it cannot be measured directly. Synaptosomes prepared from homogenates of brain tissue, or brain slices are used widely to measure the uptake or release of radiolabelled monoamines. The use of synaptosomes and brain slices for measurement of monoamine uptake will be discussed in Methods section 2.1.2. More advanced methods have now been developed to enable

55 Chapter 2 the direct measurement of the concentration of extracellular monoamines themselves in vivo', these will be described briefly in the next section.

2.1.1 /w vivo sampling techniques

One of the first in vivo sampling techniques used was the cortical cup

(Macintosh & Oborin, 1953). This involved drilling a hole in the skull to expose the surface of the brain and then placing a cylinder, or 'cup', containing artificial cerebrospinal fluid (aCSF) on the surface of the cortex. Monoamines and other compounds of interest pass from the surface of the cortex into the aCSF in the cup which can then be sampled and analysed {e.g. Milby et a i, 1981) An obvious disadvantage of this method is that sampling is only possible from the surface of the cortex. However, this technique can be used in awake rats and causes very little tissue damage.

The next development was the open-ended perfusion probe, for example the push-pull cannula (Gaddum, 1961; Delgado, 1962). Such probes consist of two tubes: the inlet drips the perfusion fluid directly into the tissue, the outlet withdraws samples from the extracellular fluid (ECF). This was used by Stadler and colleagues (1975) to measure noradrenaline, dopamine and acetylcholine in limbic areas of the cat. The application of such perfusion methods is marred by the tissue damage caused by the perfusion of relatively large volumes (a flow rate of 100-200 pi / min) directly into the tissue. In an attempt to overcome this problem, the next development was microdialysis.

56 Chapter 2

Microdialysis involves the use of a semi-permeable membrane to separate the perfusate from the tissue of interest. The membrane allows free diffusion of water and solutes down their concentration gradient between the ECF and the solution within the dialysis membrane. The first application of this technique was by Bito and colleagues

(1966). This group chronically implanted a dialysis membrane sack containing 6% dextran into the brain of dogs. 10 weeks later the bag was removed and the concentration of solutes {e.g. amino acids, Na^ and K^) in dialysates measured.

This technique was later refined by Delgado and colleagues (1972) who developed a method which allowed repeated sampling of brain dialysates. The dialytrode consisted of a push-pull cannula attached to a small dialysis bag which could be implanted into any brain region. The presence of the dialysis membrane prevented tissue damage due to direct perfusion of the brain associated with the original push-pull cannulae. The dialytrode was refined further to create probes with smaller dimensions but greater dialysing areas, thus increasing efficiency and reducing tissue damage caused by implantation.

Zetterstrom et al. (1982) described a loop probe which consisted of two cannulae joined by a piece of dialysis tubing which was bent into a tight loop. This was suitable for implantation into any brain region. Tossman and Ungerstedt (1986) used a probe which had a concentric arrangement of two tubes (outer diameter 520 |im). Hernandez and co-workers (1986) described a similar concentric dialysis probe with a tip diameter of only 200 pm, which they used to measure dopamine.

57 Chapter 2

Sandberg et a l (1986) opted for an equally small dialysis probe with a side-by- side design. This type of probe consisted of two glass capillaries serving as inlet and outlet tubes inserted into a piece of dialysis tubing (o.d. 300 p.m). This design reduces the dead-volume between the area of tissue being dialysed and dialysate collection compared with concentric probes. The design of microdialysis probe used in all experiments in this thesis is a modification of the probe described by Sandberg et a l

(1986) (see section 2.2.1.4.1)

A different method for measuring the concentration of central monoamines developed was in vivo voltammetry (reviewed by Stamford, 1989). This involves the implantation of a carbon working electrode into a discrete brain region. Electroactive species at the electrode surface are oxidised and the ensuing current recorded.

Voltammetry has been used to measure catecholamines in rat brain (e.g. Gonon et a l,

1983). The advantages of voltammetry include the small size of the electrode (6-12 |im) compared to 300-500 |im for microdialysis probes, and the much better time resolution

(ms). Because the electrodes are so small they cause very little tissue damage when they are implanted in the brain. A disadvantage of using in vivo voltammetry is that detection is performed in situ, without prior separation of solutes, so it can be difficult to identify the peaks as different compounds can oxidise at similar potentials.

2.1.1.1 What microdialysis measures

Microdialysis is used to measure changes in the extracellular concentration of particular substances within the brain e.g. monoamines. Microdialysis does not measure the concentration of monoamine in the synapse, but rather the amount of monoamine

58 Chapter 2 which overflows into the extracellular space. Monoamines diffuse down their concentration gradient through the dialysis membrane into the probe. The concentration of monoamine in dialysates is often referred to as ‘efflux’ and is expressed as amount of monoamine per unit time (or per volume of dialysate). The amount of monoamine in the dialysates reflects the concentration of monoamine in the extracellular space. The extracellular concentration of monoamines will be determined by the net effects of many processes including uptake by and release from neurones.

Because of the constant flow of dialysate through the probe, the concentration of monoamine in the probe will not equilibrate with the extracellular concentration.

Consequently, the amount of monoamine in dialysates will be lower than the actual extracellular concentration. To go some way to correcting for this, the performance of probes can be assessed in vitro. This is done following these steps:

1) perfuse probe with the normal perfusion medium (aCSF or Ringer’s) at the

normal flow rate {e.g. 1 |il / min)

2) make up Ringer’s (or aCSF) containing the monoamine of interest {e.g.

noradrenaline 5 nM)

3) place the microdialysis probe in the Ringer’s containing monoamine and leave

for -20 min

4) collect dialysates at the normal sampling interval {e.g. 20 min)

5) measure the concentration of monoamine in dialysates as well as the

concentration in the Ringer’s

6) probe recovery is calculated as: concentration of monoamine in dialysate xlOO concentration of monoamine in Ringer’s

59 Chapter 2

The recovery of probes used to measure noradrenaline in this thesis, at a flow rate of 1 jil / min, was calculated as -46% (when probes were placed in 5 nM noradrenaline). In this thesis, the concentration of noradrenaline in dialysates is referred to as ‘noradrenaline efflux’. Both of these terms refer to the concentration of noradrenaline in dialysates and have not been corrected for probe recovery.

2.1.1.2 The perfusate

An advantage of microdialysis over other sampling methods is that there is no direct contact between the perfusate and the ECF because of the presence of the dialysis membrane. This ensures that brain tissue in the vicinity of the probe is not bathed in the artificial perfusate but will remain, to a large extent, in its normal extracellular fluid

(ECF).

The exact composition of the ECF in the brain is unknown hence it is impossible to replicate it. The perfusates used for microdialysis range from simple salt solutions

(Abercrombie et a l, 1988; van Veldhuizen et a l, 1990; Finberg et al., 1993;

Vahabzadeh & Fillenz, 1994) to buffered artificial cerebrospinal fluids (aCSFs) containing glucose and magnesium (Routledge & Marsden, 1987; Tanaka et al., 1991;

Done & Sharp, 1994). There is no general consensus as to which type of perfusate is preferable for measurement of brain monoamine levels. In all microdialysis experiments in this thesis a modified Ringer's solution comprising (mM): NaCl 145,

KCl 4, CaCl: 1.3 (pH 6.6) was used (from now on this will be referred to simply as

'Ringer's').

60 Chapter 2

The concentration of the perfusate is very important as it can alter both basal and drug-evoked changes in monoamine concentrations (Moghaddam & Bunney,

1989). The concentration of extracellular Ca^^ included in the perfusate varies between groups (Finberg et a l, 1993; 3.4 mM; Jordan et a l, 1994: 2.3 mM; Done and Sharp,

1994:1.2 mM). Perfusate Ca^^ concentrations between 0.075-1.3 mM affect greatly the concentration of extracellular noradrenaline. Increasing Ca^'^ concentration beyond

1.3 mM causes a smaller, more gradual, increase in noradrenaline levels (van

Veldhuizen et al, 1990). Attempts to measure the extracellular Ca^^ concentration in the brain using ion selective electrodes have estimated the concentration of free Ca^^ to be 1-2 mM (Silver and Erecinska, 1990). Throughout my experiments I have used

Ringer's containing 1.3 mM Ca'^'^.

Many microdialysis studies have included monoamine uptake blockers in the perfusate to raise the levels of extracellular monoamines in order to make them more readily detectable (for example: Abercrombie et a l, 1988; Sharpet al, 1989; Kask et a l, 1997). In experiments performed in this laboratory noradrenaline was readily detectable with basal concentrations being consistently above 15 fmol / 20 min; this is approximately 3-fold higher than the limit of detection for our HPLC set-up. The inclusion of an uptake blocker was therefore not necessary.

61 Chapter 2

2.1.2 Measurement of monoamine uptake in vitro

The first experiments investigating uptake of monoamines in the periphery involved systemic injection of radiolabelled monoamines into whole animals in vivo and then identifying organs or tissues which had accumulated radioactivity (Axelrod et a i,

1959; Whitby et a i, 1961). Similar experiments have been performed to investigate central monoamine uptake by injecting radiolabelled monoamines into the lateral ventricles of rats (Aghajanian & Bloom, 1967; Reivich & Glowinski, 1967; Fuxe et al.,

1968). In order to study the central noradrenaline uptake process in more detail it is advantageous to use in vitro preparations where a specific brain region can be isolated and the extracellular environment can be manipulated.

Monoamine uptake can be studied either using brain slices or brain synaptosomes. Using crude sucrose gradient centrifugation of homogenates of brain tissue, nerve terminals which form synaptosomes can be isolated (the preparation of synaptosomes is described in section 2.2.2.2). The preparation of synaptosomes from brain tissue has provided a useful tool for studying the reuptake and release of neurotransmitters. Using synaptosomes has the drawback that one is looking at arguably non-physiological 'pinched-off nerve terminals rather than intact neurones.

However, synaptosomes prepared in isotonic sucrose are, in important respects, capable of functioning like whole cells: they produce lactate and amino acids, generate ATP and have an active Na"^ / pump (Ling & Abdel-Latif, 1968; Bradford, 1969; Bradford &

Thomas, 1969). A further benefit of using synaptosomes is that the whole of the preparation will be equally and consistently exposed to both noradrenaline and test drugs.

62 Chapter 2

This is not the case with tissue slices; the problem arises here that it is very difficult to ensure that the incubation medium penetrates all parts of the slice. For a valid measurement of uptake it is necessary that the whole of the slice is exposed equally to the incubation medium so that one can be certain that the whole of the tissue is oxygenated and in contact with the correct concentration of ligand (and test-drug).

The situation when measuring uptake (compared to release) is made worse by the fact that it is impossible to prevent outer layers of tissue taking up the labelled monoamine and hence reducing the concentration of ligand penetrating the deeper layers of the slice.

A further advantage of synaptosomes over slices is that interactions between nerve terminals can be ruled out such that effects of drugs on release will not interfere with measurements of uptake.

In this thesis the uptake of noradrenaline has been studied using synaptosomes prepared from homogenates of rat cerebral cortex. The results of uptake experiments have been interpreted in conjunction with results from microdialysis experiments to determine whether any changes in noradrenaline efflux in vivo could be attributed to, at least in part, a change in noradrenaline uptake.

In the experiments in this thesis, non-specific [^H]noradrenaline uptake was defined as the accumulation of [^H]noradrenaline by synaptosomes incubated at 0-4 °C.

This non-specific uptake accounted for approximately 20-30% of total uptake at 37 °C.

There are many ways of correcting for non-specific (passive) uptake. These include addition of a high concentration of monoamine uptake blocker (Richelson & Pfenning,

1984; Cheethamet a i, 1996), measuring uptake at 0-4 °C (Thomas et ai, 1987), replacing Na"^ in the incubation medium with either Li^ or sucrose (Wood & Wyllie,

63 Chapter 2

1983), or measuring uptake after only 5 s (Wood & Wyllie, 1983). Uptake at 0-4 °C

was the method of choice for defining non-specific uptake as this was preferable to

using a high concentration of a noradrenaline uptake blocker as it was intended to examine the effects of the noradrenaline reuptake inhibitor, desipramine, over a wide

range of concentrations. Also, when Na"^ is excluded from the incubation medium it cannot be confirmed that the concentration of Na"^ in the direct vicinity of the

synaptosomes is zero as the synaptosomes should actively extrude Na^ in the absence of external Na"^ (Tissari et al., 1969). It has been shown recently, that noradrenaline transport via the human noradrenaline transporter is Na^-dependent; when the Na"^ concentration is reduced, the transporter reverses and noradrenaline is actively extruded from the cell (Pifl et al., 1997). Therefore using Na^-free buffer to determine non­ specific uptake is likely to give a misleading result, as under these conditions the transporter will be reversed.

The monoamine oxidase (MAO) inhibitor, pargyline, was included in the incubation buffer to prevent [^H]noradrenaline which has been taken up by synaptosomes from being broken down. If [^H]noradrenaline is broken down by MAO to 3,4-dihydroxyphenylglycol (DOPEG) this could diffuse out of the synaptosomes and hence reduce the estimation of [^H]noradrenaline accumulation.

64 Chapter 2

2.2 Techniques

2.2.1 Intracerebral microdialysis

2.2.LI Animals

Outbred male Sprague-Dawley rats (260-320 g), derived from a colony at

University College London, were used throughout. Subjects were housed in groups of 4 and maintained on a 12 h light/dark cycle (lights on at 07.00 h) with unlimited access to food and water. Drug-naive animals were used for every experiment and all procedures complied with the U.K. Scientific Procedures (Animals) Act, 1986.

2.2.1.2 Surgery & Anaesthesia

2.2.1.2.1 Non-recovery animals (“anaesthetised rats”)

Rats were placed in an induction chamber where anaesthesia was induced by inhalation of 5% mixed with 95% O 2 / 5% CO2 at a flow rate of 1000 ml / min. Once the righting reflex was lost, anaesthesia was maintained temporarily via a face mask (2.5% halothane mixed with 95% O2 / 5% CO2 at 1000 ml / min).

Throughout the surgery and subsequent experiment the rat’s core temperature was maintained at 37 °C using a homeothermic heating pad and rectal probe. Once the pedal reflex was lost, an incision was made and the trachea exposed using blunt dissection. A small incision was made in the trachea and a polythene cannula (2 cm; i.d. 1.67 mm, o.d. 2.42 mm, Portex) was inserted. The cannula was sewn in place using cotton thread and attached to a Y-piece for the delivery of anaesthetic (1.5% halothane in 95% O 2 /

5% CO 2 500 ml / min) and removal of expired gases.

The rat was then placed in a stereotaxic frame in the flat skull position (incisor bar set at 3.3 mm below the interaural line). An incision was made and the surface of

65 Chapter 2 the cranium cleared to reveal bregma. A small hole (approx. 2 mm diameter) was then drilled in the skull (AP +3.5; ML ±1.5 mm from bregma) using a trepanning drill burr.

The duramater was then broken using a needle and the Ringer-primed microdialysis probe implanted. The probe was slowly lowered (over 40-50 s) vertically into the frontal cortex AP +3.5; ML ±1.5; DV -5 mm from bregma (Paxinos & Watson, 1986).

The microdialysis probe was then perfused with a modified Ringer’s comprising (mM):

NaCl 145; KCl 4; CaCl% 1.3 (pH 6.6) at a rate of 1 )il / min for the duration of the experiment.

2.2.1.2.2 Recovery animals (‘freely-moving rats’)

Rats were placed in an induction chamber where anaesthesia was induced by inhalation of 5% halothane mixed with 95% 0% / 5% CO2 at a flow rate of 1000 ml / min. Once the righting reflex was lost, rats were transferred to a stereotaxic frame which incorporated a face mask which delivered the anaesthetic (1.5-2.0% halothane mixed with 95% 0% / 5% CO2 at 500 ml / min). Throughout the surgery the rat’s core temperature was maintained using a homeothermic heating pad and rectal probe. The rat was held in the flat skull position (incisor bar set at 3.3 mm below the interaural line) using a nose bar and blunted non-rupture ear bars.

Using sterile equipment an incision was made and the surface of the cranium cleared to reveal bregma. A hole 2 mm in diameter was then made in the skull (AP

+3.5; ML ±1.5 mm from bregma) using a trepanning drill burr. Two small wood screws were attached to the skull (positioned close to the implantation site) so that the microdialysis probe could be cemented in place once implanted. The duramater was then broken and the Ringer-primed microdialysis probe implanted. The probe was

66 Chapter 2 slowly lowered (over 40-50 s) vertically into the frontal cortex AP +3.5; ML ±1.5; DV -

5 mm from bregma (Paxinos & Watson, 1986). A small amount of bone wax was put around the probe at the surface of the brain. The probe was then secured to the cranium and wood screws using dental cement. Once secure, the inlet and outlet of the probe were sealed using bone wax and the rat was removed from the stereotaxic frame and allowed to recover.

Approximately one hour after recovery from surgery the rat was put in the cage, in which the experiment was to be carried out the following day, and returned to the animal house. Metabolic cages are well suited for this purpose because: 1) they have a high solid plastic ‘ceiling’ which reduces the chances of the rat knocking the dialysis probe (c/the bars in a normal cage). 2) these cages are made of plastic so that the rat can be observed from a distance. 3) the cage lids are removable so that the rat is accessible during experiments, yet cannot escape. The set-up used for freely-moving dialysis experiments is pictured in Figure 2.4.

2.2.1.3 Verification of probe placement

The position of the microdialysis probe was verified histologically. After completion of the experiment, brains were removed and stored in 10% formal saline.

Brains were sectioned on a freezing microtome and the position of the dialysis probe determined. The coronal brain slice was compared to the atlas of Paxinos & Watson

(1986). Figure 2.1 shows an example brain slice and the corresponding diagrammatic representation from Paxinos & Watson (1986).

67 Chapter 2

a)

b)

Figure 2.1 : Position of the microdialysis probe in rat frontal cortex

a) Photograph of a brain slice showing the tract left by a microdialysis probe implanted in the frontal cortex, b) Diagrammatic representation of the coronal section of rat brain 3.7 mm from bregma. The position of the probe in the frontal cortex is illustrated. The white area of the schematic drawing of the probe represents the dialysing area.

68 Chapter 2

2.2.1.4 Microdialysis procedure

2.2.1.4.1 Construction of microdialysis probes

The design of the microdialysis probes was a modification of the single-cannula probe described by Sandberg et al. (1986). The steps involved in the construction of

these probes are listed below.

Figure 2.2 shows a schematic representation of the step-by-step construction of a typical microdialysis probe. Two pieces of vitrous silica glass tubing (i.d. 75 }xm ,o.d.

150 |Lim; Scientific Glass Engineering) were cut to 3 cm in length and inserted into a

2 cm needle barrel (25 gauge, Coopers Needleworks). The coating of the silica glass was burnt off to reduce the outer diameter. The two pieces of glass were glued together with a 4 mm stagger. These were then inserted into a piece of semi-permeable dialysis membrane (i.d. 240 |im, o.d. 300 |im; molecular weight cut-off 40,000; Filtrai 12,

AN69; Hospal Industrie, France).

The membrane was secured to the glass and the needle barrel with fast setting epoxy resin (Araldite) and cut to approximately 6 mm in length (so that it protruded

2 mm over the end of the longer piece of glass). The end of the membrane at the tip of the probe was then sealed with a small amount of epoxy resin. The ensuing dialysing zone of the probe was 5 mm in length. The 2 lengths of silica glass tubing protruding from the other end of the needle barrel were each inserted into a 4 cm piece of polythene tubing (i.d. 0.28 mm, o.d. 0.61 mm; Portex) designated to become the probe inlet and outlet. These were secured to the needle barrel using epoxy resin. Probes were kept in an airtight container and used within 2 weeks of construction.

69 Portex outlet

Portex inlet

resin

Silica Needle glass barrel

- > - >

Epoxy resin

Epoxy 4 mm resin 5 mm Hospal / membrane

Figure 2.2: Step-by-step construction of a microdialysis probe

Diagram shows the steps involved in the construction of a microdialysis probe. Details of construction are given in Q I section 2.2.1.4.1. Drawings are not to scale. o (NJ Chapter 2

2.2.1.4.2 Collection of dialysates (anaesthetised rats)

Collection of dialysis samples was started 2 h after implantation and onset of perfusion of the dialysis probe. Dialysates were collected every 20 min into polyethyene tubes (Hughes & Hughes) containing 5 p.1 0.01 M perchloric acid. Samples were collected directly from the outlet of the probe (see Figure 2.3). The first samples (2 h after probe implantation) of dialysate were collected to establish the resting

(unstimulated) concentration of extracellular noradrenaline. This is referred to as

'spontaneous', or 'basal', 'noradrenaline efflux'. Once four dialysates with stable levels of basal efflux had been collected, the experiment was begun. The noradrenaline content of dialysis samples was analysed using HPLC-ECD (see section 2.2.1.5). Samples were refrigerated and analysed on the day of collection; if it was not possible to analyse samples on the same day, the complete set of dialysates were frozen at -70 °C until the following day.

2.2.1.4.3. Collection of dialysates (freely-moving rats)

The day after probe implantation the dialysis probe was reconnected and perfused with modified Ringer's comprising (mM): NaCl 145; KCl 4; CaCli 1.3 (pH

6.6) at 1 pi / min. A 20 cm length of small volume tubing (1.2 pi / 100 mm; Biotech

Instruments Ltd) was attached to the outlet of the probe to collect dialysates at a distance from the rat to cause as little disturbance as possible when changing samples. Figure

2.4. After allowing the rats to acclimatise to the new laboratory after transport from the overnight holding room (90 min), dialysates were collected every 20 min. 20 pi dialysates were collected into polyethyene tubes (Hughes & Hughes) containing 5 pi

0.01 M perchloric acid.

71 Chapter 2

a)

b)

as

Figure 2.3: The anaesthetised rat set-up

Photographs show the experimental set-up for microdialysis experiments in anaesthetised rats, a) A photograph of the overall set-up and b) A close-up showing the implanted dialysis probe.

72 Chapter 2

!

Figure 2.4: The freely-moving rat set-up

Photograph showing the experimental set-up for a freely-moving microdialysis experiment.

73 Chapter 2

First, samples of dialysate were collected to establish the resting (unstimulated) concentration of extracellular noradrenaline. This is referred to as 'spontaneous', or

'basal', 'noradrenaline efflux'. Once four dialysates with stable levels of basal efflux had been collected the experiment was begun. Dialysis samples were analysed using HPLC-

ECD (see section 2.2.1.5). If there was a delay between collection and analysis of samples, dialysates were either refrigerated or, for longer delays, stored at -70 °C.

2.2.1.4.4. Local administration of compounds via the dialysis probe (‘reverse dialysis’)

Test drugs were freshly dissolved in the modified Ringer's described earlier.

Gas-tight syringes (Hamilton) were filled with the drug solution required. A length of inlet tubing was primed with drug at least 20 min before the start of infusion. To infuse drugs, the original inlet was disconnected at the probe and the drug primed inlet connected. Drugs were infused at a rate of 1 pi / min.

2.2.1.5. Measurement of dialysate noradrenaline content

2.2.1.5.1. High pressure liquid chromatography coupled to electrochemical detection

(HPLC-ECD).

Noradrenaline content of dialysates was measured using high pressure liquid chromatography coupled to electrochemical detection (HPLC-ECD). The mobile phase was delivered at a constant rate of 1.275 ml / min by a solvent delivery system (ESA).

Pump noise was reduced by the use of an in-line pulse dampener (ESA). The mobile phase was conditioned using a guard cell (ESA) set at +350 mV. The mobile phase then passed through the injection port and a 2.5 cm cartridge guard column (7 pm cut-off,

Brownlee) to protect the analytical column. Dialysates were injected into the system via

74 Chapter 2 an injection port (Rheodyne 7125) fitted with a 25 |il stainless steel loop (Anachem).

Solutes were separated using a Hypersil: 5 ODS 250 x 4.6 mm analytical column

(Hypersil). Next the mobile phase passed through a high performance analytical cell

(Coulochem 5014, ESA) controlled by an electrochemical detector (Coulochem n,

ESA). The first channel of the cell was set at -180 mV, the second at +180 mV.

Electrical current was recorded by an on-line integrator (Chrom Jet, Spectra-Physics).

Peaks in chromatograms from dialysis samples were identified and quantified by the retention time and peak height of external standards. A schematic diagram of the set-up is shown in Figure 2.5. Figure 2.6 shows examples of chromatograms obtained from external standards and a sample of dialysate.

2.2.1.5.2 The mobile phase

Detection of noradrenaline used a mobile phase comprising: 83 mM sodium dihydrogen orthophosphate, 2.77 mM sodium octane sulphonic acid (Sigma), 0.85 mM

EDTA, 12 % methanol (Hypersolv grade, BDH). The mobile phase was adjusted to pH

3.4 with orthophosphoric acid. The mobile phase was degassed and filtered using FTK membrane filters with a removal rating of 0.1 p.m (Pall Process Filtration). One litre of mobile phase was recycled and used for 2 weeks, after this a fresh mobile phase was made up.

75 Chapter 2

coulometric , cell

mobile phase

analytical column

integrator — guard solvent delivery system (pump) electro­ chemical detector sample \ pulse- injection dampener guard port cell DIALYSATE

Figure 2.5: Schematic diagram of the HPLC-ECD set-up.

Diagram shows the components of the HPLC-ECD set-up. Thick grey lines indicate tubing which carries the mobile phase, thick black lines represent electrical connections. See text section 2.2.1.5.1 for more details.

76 Chapter 2

Noradrenaline Noradrenaline

DOPAC

5-HIAA 5-HIAA

DOPAC

Figure 2.6: Typical chromatograms showing noradrenaline, DOPAC and 5-HIAA

a) is a typical chromatogram obtained from HPLC-ECD analysis of an external standard containing 50 fmol noradrenaline, 500 fmol DOPAC and 2000 fmol 5-HIAA. b) is a representative chromatogram obtained from analysis of a dialysis sample from the frontal cortex of an anaesthetised rat. Peaks in chromatograms from dialysis samples were identified and quantified by the retention time and peak height of external standards.

77 Chapter 2

2.2.1.5.3 Drugs and chemicals

Unless otherwise stated, all constituents of the mobile phase and Ringer's were

AnalaR grade obtained from BDH. Noradrenaline for making up standards was the bitartrate salt and was purchased from Sigma. Fluoxetine HCl and desipramine HCl were also purchased from Sigma. Citalopram HBr was a generous gift from Lundbeck,

Denmark.

2.2.1.6 Statistical Analysis

Statistical tests used to analyse changes in noradrenaline efflux were performed on orthonormalised raw data using SPSS PC^ for Windows. Data were analysed using repeated-measures analysis of variance (ANOVA) with 'time' as the 'within-subjects' factor. Where a significant effect of time was found, and when appropriate, data were divided into bins of four consecutive samples and a split-plot ANOVA performed. This test used 'time' and 'bin' as 'within subjects' factors. To compare the effects of different drug treatments, 'group' was included as the 'between-subjects' factor. The level of statistical significance was set as P<0.05 for all tests.

2.2.7.7 Characterisation of noradrenaline efflux in vivo

The sensitivity of noradrenaline efflux to depolarisation was investigated by infusion, via the probe, of a depolarising pulse of K^. The high [K"^] Ringer's comprised

(mM): NaCl 71, KCl 80, CaCli 1.3 (pH 6.6); the concentration of NaCl was reduced to maintain the ionic tonicity. Infusion of 80 mM (Figure 2.7) caused a transient increase in noradrenaline efflux (anaesthetised: F|,6=12.0; P=0.01; freely-moving:

Fi,6=24.1; P=0.003). When the perfusate was switched back to normal Ringer's, noradrenaline efflux returned towards basal levels (c/basal: Fi,6=0.33; P=0.59).

78 Chapter 2

The Ca^'^-dependence of noradrenaline efflux was demonstrated by removal of

Ca"^^ from the perfusion medium. The Ca^^-free Ringer's containing 145 mM NaCl and

4 mM KCl was perfused via the probe for 60 min. This reduced noradrenaline efflux to approximately 35-40% of the mean basal level (anaesthetised: F],g=10.6; P=0.01; freely- moving: Fi,6=12.8; P=0.01) (Figure 2.8). When normal Ringer's was then perfused, noradrenaline efflux returned to basal levels (c/basal, anaesthetised: Fi,g=0.3; P=0.86; freely-moving: Fij=4.2; P=0.08).

These findings indicate that spontaneous noradrenaline efflux in the frontal cortex of anaesthetised or freely-moving rats derives largely from Ca'*“^-dependent exocytotic release. This efflux is also sensitive to a depolarising pulse of K^, consistent with noradrenaline being released from neurones in response to stimulation {e.g. by the arrival of an action potential).

79 Chapter 2

a) 200 1

180 -

160 -

140 -

80 -

60 -

T3 40 -

20 -

0 20 40 60 80 100 120 140 160 180 time (min)

b) 180 -]

160 - c E o 140 - CM Ô 120 - E

X 100 - 3 $= 0) 80 - Q) .s TO 60 - C 2 T3 40 - 2 o C 20 -

0 - 20 40 60 80 100 120 140 160 180

time (min)

Figure 2.7: Ejfect of a depolarising pulse on noradrenaline efflux

Graph shows noradrenaline efflux in the frontal cortex of a) anaesthetised rats and b) freely-moving rats. Horizontal bar represents duration of 80 mM infusion. Data are expressed as mean ± SEM (n=4-5) noradrenaline efflux.

80 Chapter 2

a)

55 1 c 50 E o 45 c\j _ 40 o E 35

X 30 = 3 1 25 CD C 20 « c 15 ■O2 E 10 o c 5 0 40 80 120 160 200 240

time (min)

b) 40 -|

35 -

25

20 -

5

0

c 5

40 80 120 160 200 240 280

time (min)

Figure 2.8: Dependence of noradrenaline efflux on extracellular Ca"^^

Graph shows noradrenaline efflux in the frontal cortex of a) anaesthetised rats and b) freely-moving rats. Horizontal bar represents duration of Ca^^ removal from the perfusate. Data are expressed as mean ± SEM (n=4-5) noradrenaline efflux. Chapter 2

2.2.2 Measurement of [^H]noradrenaline uptake into rat cortical synaptosomes in

vitro

2.2.2.1 Animals

Outbred male Sprague-Dawley rats (300-400 g), derived from a colony at

University College London, were used throughout. Subjects were housed in groups of

four and maintained on a 12 h light/dark cycle (lights on at 07.00 h) with unlimited

access to food and water. Drug-naive animals were used for every experiment and all

procedures complied with the U.K. Scientific Procedures (Animals) Act, 1986.

2.2.2.2 Preparation of cortical synaptosomes

Rats were killed by stunning and cervical dislocation. Crude synaptosomes were

prepared essentially as described by Gray & Whittaker (1962). The brain was removed

and the cerebral cortex was dissected on ice The cortex was homogenised gently with a

Teflon pestle in 2 ml 0.32 M sucrose. The homogeniser was washed twice with 2 ml

sucrose and the wash added to the homogenate. This was then centrifuged at 650 g for

10 min at 4 °C. The supernatant was then poured off and the pellet discarded. The

supernatant was then spun at 12,000 g under vacuum at 4 °C for 20 min. The ensuing crude mitochondrial pellet (?%) was resuspended in 6 ml 0.32 M sucrose while the

supernatant was discarded. The resuspended pellet was then re-spun at 12,000 g (as

above). The final pellet was then resuspended in incubation buffer (for composition see

section 2.2.2.3) at 50 % w/v.

82 Chapter 2

2.2.23 Measurement of [^H] nor adrenaline uptake

A modification of the method of Kilpatrick et al. (1986) was used to measure synaptosomal noradrenaline uptake. The incubation buffer used for the uptake assay was a modified Tris-Krebs buffer comprising (mM): NaCl 136, KCl 5, MgCl] 1.2,

CaCli 2.5, J-glucose 10, /-ascorbic acid 1, Tris base 20, pargyline 0.25. The buffer was gassed with 95% O 2 / 5% CO2 for 30 min and adjusted to pH 7.4 at 37 °C with either

Tris base or HCl. All additions to the uptake assay (test drug and [^H]noradrenaline) were made up in this incubation buffer.

Each tube in the assay contained a 100 |Ll1 aliquot of synaptosomes and 200 p.1 incubation buffer. These were incubated in a shaking water bath for 5 min at 37 °C. An aliquot of synaptosomes plus 300 p.1 buffer was incubated at 4 °C for estimation of non­ specific uptake. 100 pi test drug (or buffer for drug-free controls) was then added and incubated at 37 °C for a further 3 min. All samples were prepared in duplicate. Uptake was then initiated by addition of 100 pi [^Hjnoradrenaline (specific activity

13.3 Ci / mmol diluted 1 in 10 with cold noradrenaline) to give a final concentration of

50 nM. An overview of the additions to tubes in the assay and the timing thereof is given in Figure 2.9.

After 3 min the assay was terminated by filtration over 25 mm Whatman GF/B filters in a 12-well cell harvester (Millipore). Filters were washed twice with 2 ml ice- cold buffer and put into 20 ml scintillation vials. Once the filters were dry, 9 ml scintillation fluid (Ultima Gold, Canberra Packard) was added to each vial. The vials were left in the liquid scintillation counter (Beckman) overnight to reduce chemi- and

83 Chapter 2 photoluminescence and counted the following day. Each vial was counted for [^H] for

5 min. The scintillation counter automatically subtracted background and calculated disintegrations per minute (d.p.m.) from counts per minute (c.p.m.). The counting efficiency was approximately 60%.

2.2.2.4 Protein determination

The protein content of an aliquot of synaptosomes was determined using the method of Lowry et al. Two 100 p.1 aliquots of synaptosomes were taken for each brain used, these were diluted 1 in 10 with 0.5 M NaOH. To 200 p.1 of diluted synaptosomes were added 6 ml reagent A (NaiCO] 2%, CUSO 4 0.01%, sodium potassium tartrate 0.02 %). Samples were incubated in a shaking water bath set at 45 °C for 15 min. After this, 0.6 ml 20% Folin & Ciocalteau's Phenol reagent was added and the samples left at room temperature for at least 30 min. Samples were then measured in a colorimeter (Gallenkamp) with a red Ilford 608 filter (670 nm) against a blank of

0.5 M NaOH. A standard curve was plotted from bovine serum albumin standards to quantify protein content of synaptosomes. Protein content was then multiplied by 5 to give mg protein / 1 0 0 pi aliquot of synaptosomes.

84 Chapter 2

Time

100 pj synaptosom es 0 200 |iJ buffer 5 min 100 )il test drug 8 min 100 |ul [‘Hjnoradrenaline 11 min

b)

1 2 3 4 5 6 o o o o o o

o o o o o o 12 II 10 9 8 7

37 °C 4 °C

Figure 2.9: Schematic representation of the uptake assay

a) Diagram shows the additions made to each tube in the assay and the intervals at which they are made. The assay is terminated by filtration at 14 min (i.e. 3 min after addition of ['^Hjnoradrenaline). b) Diagram shows the 12 tubes in each assay. The arrow indicates the order in which tubes were filtered. Tubes 12-7 are duplicates of tubes 1-6.

Tubes 1-5 & 8-12 are incubated at 37 °C, tubes 6 & 7 at 4 °C. Tubes 1 & 12 and 6 & 7 are ‘drug-free’ controls: 100 pi buffer was added in the place of 100 pi test drug.

85 Chapter 2

2.2.2.5 Calculation of [^Hjnoradrenaline uptake

Uptake was calculated following these steps:

1) the average counts of duplicate samples was calculated

2) d.p.m. from samples at 4 °C subtracted from all other samples to correct for non­

specific uptake

3) d.p.m. multiplied by 10 to account for 10-fold dilution of [^H]noradrenaline with

'cold' noradrenaline

4) specific uptake multiplied by 0.45 to give pCi. (1 d.p.m. = 0.45 pCi)

5) pCi divided by specific activity (13.3 Ci / mmol) to give pmol of tritiated

noradrenaline

6) this divided by the protein content of 100 jil synaptosomes gives:

pmol noradrenaline / mg protein

2.2.2.6 Statistical analysis

Raw uptake data were analysed using 1-way analysis of variance (ANOVA) with post-hoc Duncan or Tukey tests (SPSS PC^ for Windows).

2.2.2.5 Drugs and chemicals

All drugs and chemicals were obtained from Sigma, except the following: HCl,

NaOH and sucrose British Drug Houses (BDH) and /-[7-^H]norepinephrine (New

England Nuclear). All constituents of incubation buffer were obtained from BDH.

Citalopram HBr was a generous gift from Lundbeck, Denmark.

86 Chapter 2

2.2.3 Lesions

2.2.3.1 Lesion of noradrenergic neurones with DSP-4

Rats were injected intraperitoneally with DSP-4 (40 mg / kg) dissolved in 0.9%

sterile saline. Control rats received an injection of saline vehicle 2 ml / kg. Rats

destined for synaptosomal uptake experiments were used 5 days later; those destined for

microdialysis experiments underwent surgery to implant the dialysis probe 4 days after

the lesion (see section 2.2.1.2.2) so that the experiment could be performed the

following day, i.e. 5 days after the injection of DSP-4.

2.2.3.2 Lesion of 5-HT containing neurones

Rats were injected i.p. with desipramine 25 mg / kg i.p. (dissolved in sterile

saline 0.9%). Approximately 30 min later, rats were anaesthetised with halothane and

mounted in a stereotaxic frame (see section 2.2.1.2.2). An incision was made into the skin to expose bregma. In a position AP -0.8 and ML ±1.4 mm from bregma a small hole was drilled. 45 minutes after the desipramine injection a 27 G needle was lowered

3.9 mm into the left or right lateral ventricle (AP -0.8, ML ±1.4 and DV -3.9 mm;

Paxinos & Watson, 1986).

Once in position the intracerebroventricular injection was performed. The dose of 5,7-dihydroxytryptamine (5,7-DHT) used was 150 |ig / rat. This was dissolved in

10 |il 0.02% ascorbate in sterile saline to prevent oxidation of 5,7-DHT to a form which does not cross the blood brain barrier (Bjorklund et al., 1975). The injection was performed gradually over 10 min. Control rats received the desipramine pretreatment

followed by an i.c.v. injection of ascorbate saline vehicle. Once the injection was completed, the needle was left in place for at least 5 min before it was slowly withdrawn

87 Chapter 2

(over -60 s to prevent the drug seeping up the needle tract). When the needle was removed, the surface of the cranium was wiped with ethanol (95%) to minimise risk of infection and the incision was sutured. After recovery from anaesthesia rats were returned to the animal house to be used for experiments 7 days later.

22.3.3 Lesion of dopamine containing neurones

Rats were injected i.p. with desipramine 25 mg / kg i.p. and pargyline 50 mg / kg i.p.(dissolved in sterile saline 0.9%). Approximately 30 min later, rats were anaesthetised with halothane and mounted in a stereotaxic frame (see section 2.2.1.2.2).

An incision was made into the skin to expose bregma. In a position AP -0.8 and ML

±1.4 mm from bregma a small hole was drilled. 45 minutes after the desipramine and pargyline injections a 27 gauge needle was lowered 3.9 mm into the left or right lateral ventricle (AP -0.8, ML ±1.4 and DV -3.9 mm; Paxinos & Watson, 1986).

The dose of 6-hydroxydopamine (6-OHDA) used was 250 |ig / rat. This was dissolved in 0.15% ascorbate in saline to prevent the oxidation of 6-OHDA (the oxidised form of 6-OHDA does not cross the blood brain barrier) (Nowak et al., 1991).

A volume of 10 |Ll1 was injected gradually over 10 min. Control rats received the desipramine and pargyline pretreatment followed by an i.c.v. injection of ascorbate saline vehicle. Once the injection was completed the needle was left in place for at least

5 min before it was slowly withdrawn (over -60 s to prevent the drug seeping up the needle tract). When the needle was removed, the surface of the cranium was wiped with ethanol (95%) to minimise risk of infection and the incision was sutured. After recovery from anaesthesia rats were returned to the animal house to be used for experiments 7 Chapter 2 days later. During this period rat chow was supplemented with pieces of apple and condensed milk to minimise weight loss.

2.2.3.4 Drugs and chemicals

5,7-dihydroxytryptamine (5,7-DHT) creatinine sulphate, 6-hydroxydopamine

HBr (6-OHDA), desipramine HCl and pargyline HCl were purchased from Sigma.

DSP-4 HCl was purchased either from Sigma or RBI. /-ascorbic acid was obtained from

BDH.

2.2.4 Measurement of monoamine content of cortical tissue

2.2.4.1 Detection of monoamines using HPLC-ECD

Experiments where rats were treated with a neurotoxin required the monoamine content of cortical tissue to be measured. Aliquots of cortical tissue (from rats either used in microdialysis experiments or uptake experiments in vitro) were homogenised with a Teflon pestle in 0.1 M perchloric acid at a concentration of 1 mg / 10 |il.

Samples were then spun in a microcentrifuge at 9,500 g for 5 min. The noradrenaline, dopamine and 5-HT content of the supernatant was then measured using HPLC-ECD.

The mobile phase comprised (mM): sodium dihydrogen orthophosphate 100, sodium octanesulfonic acid 2.8, EDTA 0.7, 20% methanol and adjusted to pH 3.2 with orthophosphoric acid. The mobile phase was then filtered and degassed using FTK membrane filters with a removal rating of 0.1 |im (Pall Process Filtration). The mobile phase was delivered at 1 ml / min by a programmable pump (Beckman). Samples were injected onto the system via an injection port (Rheodyne 7125) with a 50 \i\ stainless steel loop (Anachem). The Hypersil analytical column (5 ODS, 250 x 4.6 mm,

89 Chapter 2

Hypersil) was protected by a 2.5 cm cartridge guard column (Brownlee). Monoamines were detected using a glassy carbon electrode (Bioanalytical Systems) set at an oxidising potential of +600 mV. Electrical current was recorded using a dual sensitivity chart recorder (Pantos). Peaks on the chromatogram were identified and quantified by the retention time and peak height of external standards.

2.2.4.2 Statistical analysis

Lesion-induced changes in monoamine content were analysed using the Mann-

Whitney U-test on SPSS PC^ for Windows.

2.2.4.3 Drugs and chemicals

All constituents of the mobile phase were AnalaR grade (methanol was

Hypersolv grade) and obtained from BDH. Noradrenaline, 5-HT and dopamine for making standards were obtained from Sigma.

90 Chapter 3

Chapter 3

The effects of fluoxetine and desipramine on noradrenaline efflux and [^Hjnoradrenaline uptake

3.1 Introduction

On the basis of the evidence described in the literature, it is claimed that fluoxetine acts by selectively inhibiting the neuronal reuptake of 5-HT. The efficacy of fluoxetine as an antidepressant has thus been attributed to this action. The most comprehensive evidence for the apparent selectivity of SSRIs comes from results of studies performed in vitro. These studies have compared the ability of antidepressant drugs to inhibit the reuptake of tritiated monoamines into rat brain synaptosomes or slices. A major flaw in these experiments is that uptake of noradrenaline, 5-HT and dopamine were often measured using synaptosomes prepared from different brain regions according to which monoamine was being studied; for instance, noradrenaline uptake in the hippocampus was compared with 5-HT uptake in the cortex and dopamine

91 Chapter 3 uptake in the striatum (Richelson & Pfenning, 1984; Bolden-Watson & Richelson,

1993; Cheng et al., 1993).

The problems with this approach become apparent when one compares uptake kinetics in different brain regions (Snyder & Coyle, 1969; Wong et a l, 1975). Wong et al. (1975) compared the uptake of [^H]5-HT in five different brain regions. Their results showed that the rate of uptake varied between 0.56 - 8.59 pmol / mg protein / 3 min depending on the brain region. In this same study the effects of pretreatment with fluoxetine on [^H]5-HT uptake in these brain regions was investigated. Fluoxetine was found to inhibit [^H]5-HT uptake by 70% in the cortex but by as little as 2% in the cerebellum. These findings provide an excellent example of how caution must be exerted when comparing the effects of a drug on uptake in different brain regions. To the best of my knowledge there are only two studies where uptake of [‘^H]5-HT and

[^Hjnoradrenaline were measured in the same brain region (cortex: Harms, 1983; hypothalamus: Thomas et a l, 1987). The study by Harms (1983) using slices of rat cortex, showed that the selectivity of fluoxetine for inhibition of [^H]5-HT over

[■^Hjnoradrenaline uptake was only 2-fold. In a study using synaptosomes prepared from rat hypothalamus (Thomas et al, 1987) the selectivity of fluoxetine for inhibiting

[^H]5-HT over [^Hjnoradrenaline uptake to was as low as 20-fold (AT,: 25 and 500 nM respectively). The results of these two studies indicate that when uptake of 5-HT and noradrenaline are both measured in the same brain region the selectivity of fluoxetine is considerably less than comparisons in different brain regions suggest.

It is generally accepted that the therapeutic effects of anti depress ants are a consequence of their ability to increase central noradrenergic and / or serotonergic

92 Chapter 3

transmission. Estimates of the selectivity of fluoxetine for inhibition of 5-HT uptake

over noradrenaline uptake vary between 2 and 192-fold. As the majority of evidence for

classifying fluoxetine as a selective inhibitor of 5-HT reuptake comes from experiments

in vitro, the present chapter investigates whether it is justified to ignore any effects of

fluoxetine on the extracellular concentration of noradrenaline in vivo. To determine

whether fluoxetine modifies central noradrenergic transmission its effect on the concentration of extracellular noradrenaline in rat frontal cortex is investigated.

Recent microdialysis experiments have shown that systemic (10 mg / kg) as well as local administration of fluoxetine (100 pM) increases extracellular noradrenaline (to

-180 % and -250% basal, respectively) in the rat medial prefrontal cortex (Jordan et al,

1994). The Jordan group presented their data as a percentage of pre-drug baseline and do not indicate the time-course of the effects they observed or whether these values represent a plateau or a maximum effect. The increase in noradrenaline efflux caused by fluoxetine infusion was significant at 100 pM, but not at 10 pM (despite an increase to -230% basal), suggesting that the threshold for fluoxetine to increase noradrenaline efflux lies between 10 and 100 pM. The present experiments investigate the concentration-dependence of the increase in noradrenaline efflux caused by local infusion of fluoxetine (0.5 - 50 pM) and compare this with that of the noradrenaline selective uptake inhibitor, desipramine, in the frontal cortex of anaesthetised rats.

In conjunction with these microdialysis experiments, a preparation of rat cortical synaptosomes was used to study ['^H]noradrenaline uptake and its inhibition by fluoxetine and desipramine in vitro. It should therefore be possible to determine

93 Chapter 3 whether any changes in extracellular noradrenaline in vivo could be attributed, at least in part, to drug-induced changes in noradrenaline uptake. To compare the effects of fluoxetine and desipramine, the inhibition of synaptosomal uptake of [^Hjnoradrenaline by a wide range of concentrations was studied. To investigate the possibility that the fluoxetine-induced increase in noradrenaline efflux could be secondary to an increase in the concentration of extracellular 5-HT (because fluoxetine inhibits 5-HT reuptake) the effects of 5-HT on [^Hjnoradrenaline uptake were also studied.

The literature indicates the presence of two desipramine binding sites in rat cortex (Backstrom et al, 1989; Lee et a l, 1982). These groups suggested that only the high affinity component of [^Hjdesipramine binding represented functional noradrenaline uptake sites on noradrenergic neurones. This suggestion is based partly on the finding that after a noradrenergic lesion, only high affinity [^Hjdesipramine binding is abolished (Backstrom et al, 1989; Lee et al, 1982). It remains to be determined whether low affinity [^Hjdesipramine binding sites could represent binding to a carrier capable of transporting noradrenaline which is not the noradrenaline transporter on noradrenergic neurones. The inhibition of [^Hjnoradrenaline uptake into cortical synaptosomes by desipramine was studied by Michel and colleagues (1984).

This group showed that inhibition of [^Hjnoradrenaline uptake by desipramine was biphasic: 66% total specific [^Hjnoradrenaline uptake was via a low affinity desipramine-sensitive site (IC 50 4 pM) and 34% was taken up via a high affinity site

(IC 50 0.4 nM). Only high affinity uptake was thought to represent uptake into noradrenergic neurones.

94 Chapter 3

Measuring noradrenaline uptake indicates the functional importance of the possible uptake sites defined by how much noradrenaline each site transports (as opposed to how many sites there are). The aim of the present synaptosomal uptake experiments was to investigate the concentration-dependence of the inhibition of

[^Hjnoradrenaline uptake by desipramine and to determine whether two distinct sites of noradrenaline uptake could be distinguished by their sensitivity to inhibition by desipramine. The proportion of noradrenaline transported by these sites was measured to give an indication of the amount of noradrenaline transported by each of these sites

(c/number of sites suggested from binding data). The concentration-dependence of the inhibition of [^Hjnoradrenaline uptake by fluoxetine is also studied as a comparison of the effects of antidepressant drugs of different classes.

95 Chapter 3

3.2 Methods

3.2.1 Microdialysis in vivo

Microdialysis experiments were performed in non-recovery, halothane- anaesthetised rats. For details of the procedures see Methods section 2.2.1 Once basal noradrenaline efflux was established and 4 samples with stable levels of noradrenaline efflux had been collected, test drugs were applied via the dialysis probe. Desipramine or fluoxetine were dissolved in fresh Ringer's and three syringes primed with concentrations of 0.5, 5 or 50 pM. After collecting the fourth basal sample, the perfusion medium was switched to one containing 0.5 p,M drug. This was infused for

80 min before switching to 5 |iM drug. After 80 min the perfusate was then switched to

Ringer's containing 50 |iM test drug for a further 80 min. Four dialysates were collected in the presence of each concentration of drug. The noradrenaline content of dialysates was measured using HPLC-ECD (see Methods section 2.2.1.5).

3.2.2 Inhibition of [^Hjnoradrenaline uptake by fluoxetine, desipramine or 5-HT

Synaptosomes were prepared from rat cerebral cortex and uptake of

['^Hjnoradrenaline was measured as described in Methods section 2.2.2. The effect of fluoxetine on [^Hjnoradrenaline uptake was investigated initially by including it in the incubation medium at final concentrations of 0.5, 5 or 50 pM. Experiments were repeated with desipramine as an active control. The inhibition of [^Hjnoradrenaline uptake by 5-HT was also investigated: 5-HT was included in the assay at concentrations between 0. 1 nM and 50 pM.

96 Chapter 3

Because the emerging results suggested the presence of more than one uptake

site sensitive to desipramine, in further experiments the range and number of

concentrations of test drug studied was increased. Twenty concentrations of

desipramine ranging between 1 nM-1 mM, and ten concentrations of fluoxetine between

100 nM-250 p.M were used. To enable ten concentrations of drug to be assayed in

duplicate (plus four ‘drug-free’ controls) in each run (as opposed to four concentrations

of drug using the original method, see Methods section 2.2.2.3) a cell harvester

(Brandel) capable of filtering twenty four samples simultaneously was used.

3.2.3 Statistical analysis

For dialysis experiments, data were spilt into bins of four consecutive samples.

The 4 basal samples represented the first bin and each of the subsequent 80 min periods

of drug infusion represented the other bins. Data were analysed using split-plot analysis

of variance (ANOVA) with 'bin' and 'time' as main factors. When effects of

desipramine and fluoxetine were compared, a 3-way ANOVA was used with 'drug' as

the ‘between subjects’ factor. Data from uptake experiments were analysed using 1-way

ANOVA with post-hoc Tukey test. Curves were fitted to the extended inhibition of

uptake data using the Origin 4 software package. This software was used to fit curves to

data with either a variable Hill coefficient or with the Hill coefficient constrained at

unity.

97 Chapter 3

3.3 Results

3.3.1 Microdialysis in vivo

3.3.1.1 Noradrenaline efflux during infusion of desipramine

Local infusion of desipramine significantly increased noradrenaline efflux

(F2,35 = 1 0 .6 ; P<0.001), Figure 3.1. During infusion of 0.5 jiM desipramine there was no main effect of bin (suggesting that the level of efflux in these four samples was not greater than that in the basal samples), but there was a significant bin x time interaction

(F2,i4=15.35; P<0.001) indicating that noradrenaline efflux was increasing over these four samples. During infusion of 5 and 50 p.M desipramine, noradrenaline efflux was significantly greater than the basal level (5 p.M: Fi,7=20.13; P=0.003; 50 |iM:

F|,7=22.48; P=0.002).

3.3.1.2 Noradrenaline efflux during infusion of fluoxetine

Noradrenaline efflux in the frontal cortex was increased by local infusion of fluoxetine (F 3,52=9.4; P < 0 . 0 0 1 ) . Figure 3 . 1 . The increase in noradrenaline efflux was significant at 5 and 5 0 |iM ( 5 |xM: Fijo=5.77; P=0.037; 50 |iM: Fi,io=19.48; P=0.001).

Fluoxetine did not affect noradrenaline efflux at 0 . 5 jiM.

3.3.1.3 Comparison of the effects of fluoxetine and desipramine on noradrenaline efflux

The effects of desipramine and fluoxetine at 0 . 5 pM approached the criterion for statistical significance (Fi,io=4.25; P=0.064). At 5 pM and 5 0 pM the increase due to desipramine was significantly greater than that due to fluoxetine ( 5 pM; F i , 9 = 1 6 . 3 ;

P=0.003; 50 pM: Fi,9=13.14; P=0.006).

98 Chapter 3

180 -

160 -

c 140 E o CM 120 Ô g 100 X _D

(D 80 0 C "côc 60 0

0 40 O C 50 |4M a 20 H 5 ] W\ 0.5 ïaM

50 100 150 200 250 300 350 time (min)

desipramine

fluoxetine

Figure 3.1: The ejfects of local infusion of fluoxetine or desipramine on noradrenaline efflux in the frontal cortex of anaesthetised rats

Once four basal samples had been collected, desipramine or fluoxetine were infused via the microdialysis probe. Periods of drug infusion at the different concentrations are indicated by the horizontal bars in the figure. Data are expressed as mean ± SEM noradrenaline efflux (fmol / 20 min) (n=5-6).

99 Chapter 3

3.3.2 Inhibition of [^HJnoradrenaline uptake in vitro

3.3.2.} Inhibition of [^H]noradrenaline uptake by fluoxetine, desipramine or 5-HT

Fluoxetine, desipramine and 5-HT all inhibited synaptosomal [^H]noradrenaline uptake (fluoxetine: F 3,25 =3 5 .5; P<0.001; desipramine: F 3,39= 1 9 .62; P<0.001; 5-HT:

Fg,56=12.6; P<0.001). Figure 3.2. Inhibition of uptake by fluoxetine was significant at 5 and 50 pM, whereas desipramine caused significant inhibition at all 3 concentrations

(P<0.05 post-hoc Tukey test). Inhibition of [^H]noradrenaline uptake by 5-HT was significant at concentrations of 0.01 pM and above (P<0.05, Figure 3.3).

3.3.2.2 Biphasic inhibition of [^H]noradrenaline uptake by desipramine

Desipramine inhibited synaptosomal [^H] noradrenaline uptake when included in the incubation medium over the range of concentrations: 1 nM-1 mM (F2o,33=14.5;

P<0.001). Post-hoc tests showed inhibition by concentrations of desipramine of 0.5 pM and above to be significant (P<0.05). Data were also calculated as % inhibition of maximum (‘drug-free’) uptake. The % data were calculated for each subject with drug- free uptake representing 100%. These data were fitted to a 2-site displacement curve with A^iS estimated at 34 nM and 22 pM. Figure 3.4. The high affinity desipramine- sensitive site accounted for approximately 2 0 % of total uptake inhibited by desipramine.

3.3.2.3 Inhibition of [^H]noradrenaline uptake by fluoxetine

Fluoxetine inhibited synaptosomal [^H]noradrenaline uptake over the range of concentrations studied (100 nM-250 pM: Fio,33=79.3; P<0.001). Inhibition of uptake by fluoxetine was significant at concentrations of 1 pM and above {Post-hoc Tukey test:

P<0.05). Data were also calculated as % maximum (‘drug-free’) uptake for each subject.

100 Chapter 3

Drug-free uptake was set at 100%; the % data for all rats was then meaned and a curve of best-fit drawn. The Hill coefficient of this curve was 1.5, suggesting that fluoxetine could be acting at more than one site. For this reason a K\ could not be calculated

(Figure 3.5a), the IC 50 for inhibition of [^H]noradrenaline uptake by fluoxetine however, was estimated to be 14 |iM. Figure 3.5b shows the same data fitted to a curve where the

Hill coefficient was constrained at unity. From this curve the K\ for inhibition of

[■^HJnoradrenaline uptake by fluoxetine was estimated at 12 \xM.

101 Chapter 3

a) c CD 1 .0 P Cl O) E 0 .8

Ô E 0 .6 CD CD Q. 3 0.4 CD C CD C 2 0 . 2 T3 P O c X 0 . 0 -n—//- D rug- -7 -6 -5 fre e log^o drug concentration (M)

b)

CD

1 7 0 -

Ç 60 - m 2 50 - •oCD S 40

^ qn -

Ic c

■7 ■6 •5 ■4

log^o drug concentration (M)

desipramine fluoxetine

Figure 3.2: Inhibition of synaptosomal H]noradrenaline uptake by desipramine and fluoxetine

Graphs show the effects of fluoxetine or desipramine on [^H]noradrenaline uptake into rat cortical synaptosomes. Data are mean ± SEM with n=7-l 1. a) Raw data are expressed as ['^Hjnoradrenaline uptake (pmol / mg protein). 'Drug-free' indicates the amount of specific uptake in the absence of test drugs, b) Data are expressed as percentage inhibition of specific (‘drug-free’) ['^H]noradrenaline uptake.

102 Chapter 3

100 CO Q. 80 - CD _c 10 c 60 H CD -o g o 40 - c T CO 20 - g -I—' ig 'sz 0 - _c

-10 -9 -8 -7 -0 -5 -4 Log,0 [5-HT] (M)

Figure 3.3: Inhibition of H]noradrenaline uptake by 5-HT

Data are expressed as %inhibition of [^Hjnoradrenaline uptake where specific uptake in the absence of 5-HT represents 0% inhibition. Data are mean ± SEM (n=4-7).

103 Chapter 3

100 -1

80 -

0

Q . 6 0 - 3 ■ E 3 I 4 0 - CO E

20 -

H

9 8 7 6 5 4 3 log^^[desipramine] (M)

Figure 3.4: Biphasic inhibition of [^H]noradrenaline uptake by desipramine

Graph illustrates the inhibition of [^H]noradrenaline uptake by an extended range of concentrations of desipramine ( 1 nM-1 mM). Data are mean ± SEM with n=6-8.

104 Chapter 3 a)

100 n Î . 80 - 0) CC R3 60 E3 I

I03 "" E

2 0 -

log^^[fluoxetine] (M) b)

100-1

80 - (D

i5 60 - 3Q. E 3 E 4 0 - X 03 E \

2 0 -

■4

log^^[fluoxetine] (M)

Figure 3.5: Inhibition of[^H] noradrenaline uptake by fluoxetine Graph illustrates the inhibition of [^Hjnoradrenaline uptake by an extended range of concentrations of fluoxetine (100 nM-250 pM). Data are mean ± SEM with n=5-6. a) Curve of best-fit (Hill coefficient =1.5). b) Curve with Hill coefficient constrained at unity.

105 Chapter 3

3.4 Discussion

The results of the microdialysis experiments indicate that fluoxetine increases the concentration of extracellular noradrenaline when it is infused locally into the frontal cortex of anaesthetised rats. The effect of fluoxetine on noradrenaline efflux was concentration-dependent, the increase in noradrenaline efflux during fluoxetine infusion was significant at probe concentrations of 5 |liM and 50 |iM. The increase in noradrenaline efflux induced by desipramine infusion was also concentration-dependent.

During infusion of 5 |iM desipramine there was a profound increase in noradrenaline efflux (-230% basal) which was significantly greater than that caused by the same concentration of fluoxetine. Fluoxetine appears to be -10-fold less potent at increasing noradrenaline efflux than desipramine, an increase in the same order as that caused by

5 \xM desipramine was caused by infusion of 50 p,M fluoxetine (-220% basal).

Although the concentrations at which fluoxetine increases noradrenaline efflux in vivo may appear rather high, they are well within the range of the concentration of fluoxetine attained in rat brain after single systemic injection of fluoxetine. After administration of a single anticonvulsant dose of fluoxetine (45 mg / kg), Dailey and colleagues (1992) measured the extracellular concentration of fluoxetine in the thalamus using microdialysis as 2 - 3 |iM, and the brain concentration using gas-chromatography as 82 nmol / g tissue.

The concentration of fluoxetine in homogenates of rat cortex after chronic treatment of rats with fluoxetine (10 mg / kg / day for 14 days) has also been measured.

106 Chapter 3

Goodnough & Baker (1994), using acétylation with gas chromatography, measured the brain concentration of fluoxetine to be in the region of 10 )iM.

More recently, the plasma concentration of fluoxetine of patients using this drug in the treatment of depression was measured using gas chromatography (Amsterdam et al., 1997). The mean plasma concentration of fluoxetine in patients who had been taking fluoxetine for 8 weeks (20 mg / day) was 97 ng / ml (-0.3 |LiM). The plasma concentration of fluoxetine varies with dosage and duration of treatment, however there is no definite relationship between plasma concentration and the therapeutic response

(see Baumann, 1996). In the review by Baumann (1996), the plasma concentrations of fluoxetine from a number of studies in depressed patients are listed; these lie between

0.2- 1.5 pM. All this data indicates that it is quite possible that micromolar concentrations of fluoxetine will be attained in the brains of patients being treated with fluoxetine for the symptoms of depression. These findings support the physiological (if not clinical) relevance of the concentrations of fluoxetine used in the present study.

It is important to emphasise that the concentrations of drug stated for dialysis experiments are the concentrations within the probe. These will be appreciably higher than the concentration of drug in the brain tissue surrounding the probe. The concentration of drug in the extracellular fluid in the vicinity of the probe will not be as high as that inside the probe because of the presence of the semi-permeable dialysis membrane. Measurements of microdialysis probe efficiency for fluoxetine are estimated at 10 - 25% (Dailey et al, 1992; Chen & Reith, 1994). During infusion of

107 Chapter 3

50 |iM fluoxetine, the concentration in the vicinity of the probe could be as low as

5 - 13 ^iM.

An additional consideration is that the concentration of drug will decrease with distance away from probe. The concentration of fluoxetine in the frontal cortex was not measured in this study because fluoxetine was infused locally into a restricted area and thus it was not evenly distributed through the brain as would presumably be the case had it been administered systemically. Also, even before correcting for diffusion of fluoxetine from the probe and diffusion through the tissue, the concentrations used were within a relevant physiological range. The concentrations of fluoxetine infused in the present study are also within the range of the K\ for inhibition of noradrenaline uptake by fluoxetine (0.1 - 10 p.M, see below), suggesting that inhibition of noradrenaline uptake could contribute to the increase in efflux.

This possibility is supported by results of experiments in the second section of this chapter showing that fluoxetine inhibited synaptosomal uptake of [^H]noradrenaline in vitro. The concentration of fluoxetine required to inhibit [^H]noradrenaline uptake was only 10-fold higher than effective concentrations of desipramine in vitro {i.e. fluoxetine was only ~ 10-fold less potent than desipramine at inhibiting

[^H]noradrenaline uptake). The finding that fluoxetine inhibits ['^H]noradrenaline uptake in vitro indicates that inhibition of noradrenaline uptake could contribute to the increase in noradrenaline efflux induced by infusion of micromolar concentrations of fluoxetine in vivo.

108 Chapter 3

Alternatively, as fluoxetine is an SSRI, the increase in noradrenaline efflux could

be secondary to an increase in extracellular 5-HT. Interestingly, the present experiments

showed that 5-HT itself was a potent inhibitor of ['’H]noradrenaline uptake. However,

inhibition of noradrenaline uptake by 5-HT cannot explain the effects of fluoxetine on

noradrenaline uptake in vitro as, in the synaptosomal preparation, the concentration of

5-HT will not accumulate because the pseudo extracellular space is infinite. The

inhibition of [^H]noradrenaline uptake by fluoxetine in vitro therefore is most likely due to a direct effect of fluoxetine on noradrenaline uptake.

Fluoxetine inhibited [^H]noradrenaline uptake with an estimated IC 50 of 14 p,M

(or Ki of 12 )xM, see Results section 3.3.2.3). Because the data for inhibition of

['^H]noradrenaline uptake by fluoxetine was fitted to a curve with a Hill coefficient of

1.5, this suggests that fluoxetine could be acting at more than one site. Fluoxetine appears to inhibit [^H]noradrenaline uptake via multiple sites which, unlike for desipramine, cannot be distinguished by their sensitivity to inhibition by fluoxetine. In a paper by DePaermentier and colleagues (1989) the authors refer to an earlier reference which states that it is not possible to distinguish more than one binding site when the difference in the affinity of the drug for these sites is less than 5- to 7-fold (Biirgisser,

1983). It is possible therefore, that fluoxetine inhibits uptake of [^H]noradrenaline by two sites with similar K\S. One of these sites could be a desipramine-sensitive uptake

site as Hrdina (1981) showed that fluoxetine displaces [^H]desipramine binding (IC 50 3

P-M). The sites of action of fluoxetine are investigated further in Chapters 4 and 5 of this thesis.

109 Chapter 3

The concentration of fluoxetine required to inhibit 50% [‘^H]noradrenaline uptake was 14 pM. This value is in line with, albeit somewhat higher than, other estimates of the K\ for inhibition of [^H]noradrenaline uptake by fluoxetine (Rubinstein-

Kawamoto et al., 1984: 6 pM; Thomas et a l, 1987: 0.5 pM; Bolden-Watson &

Richelson, 1993: 0.1 pM). The K\ for inhibition of 5-HT uptake by fluoxetine is reported to be between 12-55 nM {e.g. Richelson & Pfenning, 1984: 12 nM; Bolden-

Watson & Richelson, 1993: 14 nM; Thomas et a l, 1987: 25 nM). This data suggests that fluoxetine could inhibit 5-HT uptake with anything between 10-192-fold selectivity over noradrenaline uptake.

To get an accurate selectivity ratio uptake of [^H]noradrenaline and ['^H]5-HT must be studied in the same brain region and under the same conditions, preferably performed by the same experimenter. One study where this was the case was by

Thomas and colleagues (1987). As mentioned in the Introduction, when comparing the inhibition of [^H]noradrenaline uptake with [^H]5-HT uptake into synaptosomes prepared from the hypothalamus, Thomas and co-workers (1987) obtained a selectivity ratio for fluoxetine of 20. Using slices of rat cortex. Harms (1983) calculated a selectivity ratio of 2 for inhibition of [^H]5-HT over [^H]noradrenaline uptake by fluoxetine. These values are considerably lower than the selectivity ratio quoted when synaptosomes prepared from different brain regions are used for different monoamines

(can be as high as 200-fold, see Stanford, 1996). To the best of my knowledge there is no study which has measured both [^HJnoradrenaline and [^H]5-HT uptake into synaptosomes prepared from rat cortex.

110 Chapter 3

The final finding of the present experiments confirmed that the inhibition of

noradrenaline uptake by desipramine is biphasic (Michel et ai, 1984). TheK\s for

inhibition of [^H]noradrenaline uptake by desipramine in the present study were 34 nM

and 22 p,M. As little as -20% of [^H]noradrenaline was taken up via the higher affinity

site. Interestingly, [^H]desipramine binding to cortical membranes is also biphasic

(Hrdina et ai, 1981; Lee et a i, 1982; Bâckstrôm et a i, 1989). These groups have

shown that there are high and low affinity components of [^H]desipramine binding.

Based on the relationship between affinity of compounds to inhibit

[^H]noradrenaline uptake and to inhibit [^H]desipramine binding, it is presumed that

[^H]desipramine binds to neuronal noradrenaline uptake sites (Hrdina, 1981). However, after lesioning of noradrenergic neurones with DSP-4, only low affinity [^H]desipramine binding remains, suggesting that high, but not low, affinity [^Hjdesipramine binding represents binding to noradrenaline uptake sites on noradrenergic neurones (Lee et a i,

1982; Bâckstrôm et al., 1989). The results of experiments described in Chapter 4 of this thesis using synaptosomes prepared from DSP-4 treated rats indicate that only

[^H]noradrenaline uptake sensitive to low concentrations of desipramine is reduced after a noradrenergic lesion. Uptake of [^H]noradrenaline inhibited by higher concentrations of desipramine was intact (see Chapter 4). This result questions the validity of the argument that [^H]desipramine binding, present after DSP-4 treatment, does not represent binding to functional noradrenaline uptake sites (Lee et a i, 1982; Bâckstrôm et a i, 1989).

It is possible that low affinity [^H]desipramine binding could represent binding

to sites of noradrenaline uptake which are not on noradrenergic neurones. In the present

111 Chapter 3 study, the majority of [^H]noradrenaline uptake was not inhibited by low concentrations of desipramine. This could suggest that -80% of released noradrenaline is taken up by sites which are not on noradrenergic neurones. This finding is somewhat surprising and raises the question as to where the majority of released noradrenaline is sequestered?

There are two strong possibilities: 1) uptake by non-neuronal sites e.g. into glial cells, and / or 2) uptake by sites on non-noradrenergic neurones e.g. sites on dopaminergic neurones.

Support for the first possibility comes from studies of noradrenaline uptake in the periphery carried out more than 30 years ago. In 1965, Iversen demonstrated the uptake of monoamines into non-neuronal tissue (uptakez). Since then, it has been shown that noradrenaline is sequestered by glial cells {e.g. Henn & Hamberger, 1971).

Glial cells have been isolated using differential centrifugation or glial cell cultures

(Henn & Hamberger, 1971; Pelton et al, 1981). These glial cells were shown to accumulate [^H] noradrenaline, [^H] dopamine and [^t]5-HT; the uptake of

[^H]noradrenaline was sensitive to desipramine (35-50% inhibition by 5 fxM desipramine) (Henn & Hamberger, 1971). Further evidence for the uptake of catecholamines by glial cells comes from studies using primary astrocyte cultures

(Pelton et a l, 1981; Semenoff & Kimelberg, 1985). These showed that accumulation of noradrenaline by astrocytes was temperature-, and Na'^-dependent as well as sensitive to inhibition by desipramine (0.1 |iM). The study by Semenoff and Kimelberg (1985) used autoradiography to demonstrate the cellular localization of accumulated

[^H]noradrenaline and [^Hjdopamine. More recently, Russ and co-workers (1996) have demonstrated the presence of an non-neuronal monoamine transporter in cells derived from glial cells of the human CNS using radiolabelled 1-methyl-4-phenylpyridinium

112 Chapter 3

(['^H]MPP^), a substrate for uptakc 2 . These findings suggest that uptake into glial cells contributes to the clearance of extracellular noradrenaline in the brain and that this uptake can be blocked by desipramine.

Uptake of noradrenaline by a site on dopaminergic neurones is a second possibility. The strength of this possibility is based largely on the extensive (78%) overall sequence homology in the amino acid sequences of and a similarity in the substrate selectivity of the noradrenaline and dopamine transporters (see Chapter 1 section 1.5.4). The cloned noradrenaline transporter protein transports dopamine with high affinity and high Vmax and vice versa for the cloned dopamine transporter (Buck &

Amara, 1995; Giros et a i, 1994). Michel and colleagues (1984) suggested that a low- affinity desipramine-sensitive portion of noradrenaline uptake into cortical synaptosomes represented uptake of noradrenaline into dopaminergic cells. This hypothesis was based on the finding that only 34% of noradrenaline uptake in rat cerebral cortex was inhibited by nanomolar concentrations of desipramine. The remaining 6 6 % was blocked more potently by inhibitors of dopamine uptake {e.g. nomifensine) than desipramine. Also, the IC 50 value for high affinity desipramine inhibition of [^H] noradrenaline uptake was in the same range as that of desipramine in the hypothalamus (predominantly noradrenergic innervation), whereas the IC 50 for low affinity inhibition by desipramine was similar to that of desipramine in the striatum

(predominantly dopaminergic innervation). The location of the sites through which fluoxetine and desipramine inhibit noradrenaline uptake will be investigated in the next chapters (Chapters 4 & 5).

113 Chapter 3

To summarise, the results of the experiments described in this chapter show that

fluoxetine increases the concentration of extracellular noradrenaline when it is infused

locally in the frontal cortex. As fluoxetine was found to inhibit the uptake of

[^H]noradrenaline in vitro, it is likely that the increase in noradrenaline efflux in vivo is

due, at least in part, to inhibition of noradrenaline uptake. Because fluoxetine will cause

an increase in the extracellular concentration of 5-HT (as it is an SSRI) which was itself

shown to be a potent inhibitor of [^H]noradrenaline uptake, it is possible that inhibition

of noradrenaline uptake by 5-HT could also contribute to the increase in noradrenaline efflux during infusion of fluoxetine in vivo. Based on the results of experiments using

microdialysis in vivo described in Chapter 5, an increase in noradrenaline efflux secondary to an increase in extracellular 5-HT does not seem likely.

A further finding of the work in the present chapter was that desipramine

inhibited [^H]noradrenaline uptake via two sites which could be distinguished by their sensitivity to inhibition by desipramine. Only -20% of total noradrenaline uptake was transported via the high affinity desipramine-sensitive site. As only high affinity desipramine-sensitive noradrenaline uptake is thought to represent uptake into noradrenergic neurones (Michel et a i, 1984) this would suggest that the majority of released noradrenaline is sequestered by sites not on noradrenergic neurones. Uptake into dopaminergic neurones or glial cells are strong possibilities (Michel et a i, 1984;

Iversen, 1965). The location of these sites remains to be determined and will be discussed further at a later stage in this thesis (Chapters 4 & 5).

The results of microdialysis as well as synaptosomal uptake experiments raise

the question as to whether the clinical efficacy of fluoxetine should be attributed

114 Chapter 3 exclusively to inhibition of 5-HT reuptake? Despite the preferential inhibition of 5-HT uptake by fluoxetine, it is likely that, even following a single dose, the concentration of fluoxetine in the brain will be in the region of its K\ for inhibition of noradrenaline uptake (see Dailey et a i, 1992 and Baumann, 1996), and is therefore likely to have profound effects on central noradrenergic function in vivo.

115 Chapter 4

Chapter 4

The effect of selective lesions of noradrenergic, serotonergic or dopaminergic neurones on the synaptosomal uptake of [^H]noradrenaline

4.1 Introduction

Evidence discussed in Chapter 3, suggests that fluoxetine increases the concentration of extracellular noradrenaline in vivo and inhibits [^H]noradrenaline uptake into rat cortical synaptosomes in vitro. Because fluoxetine is generally considered to be a selective inhibitor of 5-HT reuptake (but see Stanford, 1996), the question arose as to how fluoxetine inhibits noradrenaline uptake and increases noradrenaline efflux? Fluoxetine could inhibit noradrenaline uptake via a transporter on

5-HT neurones or a transporter on noradrenergic neurones. Experiments went on to investigate the involvement of noradrenaline, dopamine or 5-HT neurones in the inhibition of [^H] noradrenaline uptake by fluoxetine. In order to see the extent to which inhibition of noradrenaline uptake might be generalised to other SSRIs, experiments were extended to include the more potent and selective SSRI, citalopram {K\ inhibition

116 Chapter 4

5-HT uptake: fluoxetine 25 nM; citalopram 2.6 nM, Thomas et al, 1987; selectivity ratio (5-HT : noradrenaline) fluoxetine 10-192; citalopram 1500-3000, see Stanford,

1996). Experiments in this chapter compare the effects of a serotonergic or a noradrenergic lesion on the inhibition of [^H]noradrenaline uptake by fluoxetine or citalopram into rat cortical synaptosomes in vitro. The effects of the lesions on the inhibition of [^H]noradrenaline uptake by desipramine served as active controls.

To investigate whether fluoxetine and citalopram could be exerting their effect on noradrenaline through an action at 5-HT neurones, 5-HT neurones of the cortex were lesioned by intraventricular injection of the neurotoxin, 5,7-dihydroxytryptamine,

(5,7-DHT). 5,7-DHT is preferentially taken up into neurones via the 5-HT uptake carrier, but, as it discriminates poorly between 5-HT and noradrenaline neurones, it causes some depletion of noradrenaline (Baumgarten et a i, 1973a). However, noradrenergic neurones can be protected by systemic pretreatment with desipramine

(Bjorklund et al, 1975). Once inside the neurones, 5,7-DHT oxidises to form quinone- like metabolites which are thought to alkylate essential proteins such as membrane transporters and enzymes. The depletion of brain 5-HT by 5,7-DHT is dose-dependent up to 200 |ig (Baumgarten et a l, 1975) with maximal reductions not being reached until

4-12 days after injection (Baumgarten et a l, 1973b). 5,7-DHT caused a reduction in tryptophan hydroxylase activity in all brain areas studied by Baumgarten and colleagues

(1973a), indicating widespread degeneration of 5-HT neurones. Depletion of transmitter (5-HT and noradrenaline) is also paralleled by a reduction in [^H]5-HT and

[^H]noradrenaline uptake in vitro (Baumgarten et a l, 1975).

117 Chapter 4

Because, in the present experiments, a 5,7-DHT lesion did not affect the

inhibition of [^H]noradrenaline uptake by fluoxetine or citalopram, further experiments

investigated whether a site on noradrenergic neurones could be involved. A lesion of

noradrenergic neurones was induced by treatment of rats with the noradrenergic

neurotoxin, DSP-4. DSP-4 preferentially targets noradrenergic neurones arising from the locus coeruleus (Jonsson et a i, 1981). Treatment of rats with DSP-4 causes a reduction in noradrenaline content, a loss of D(3H immunoreactivity, a loss of binding to noradrenaline uptake sites, an upregulation of ^-adrenoceptors and a loss of a-adrenoceptors (Ross, 1976; Dooley et a i, 1983; Fritschy et ai, 1990; Heal et al.,

1993; Cheetham et a i, 1996). The effects of DSP-4 treatment are discussed in greater detail in the General Introduction (Chapter 1).

The dose of DSP-4 used (40 mg / kg i.p.) was chosen to induce a partial lesion of noradrenergic neurones in the cortex without compromising the survival of the rats. The normal range of doses of DSP-4 used is between 40 - 100 mg / kg (e.g. Bennett et al.,

1984; Hallman & Jonsson, 1984). As the dose is increased the extent of the lesion is increased, however, in line with this, the rate of mortality increases dramatically (Mishra et ai, 1994). At 40 mg / kg the lesion was incomplete but the mortality rate in this study was zero. The effect of an incomplete lesion is of interest as some noradrenergic neurones survive, thus giving an indication of whether noradrenergic function is compromised in DSP-4 lesioned rats or whether surviving neurones are able to compensate for the loss. In comparing ['*H]noradrenaline uptake into synaptosomes prepared from DSP-4 lesioned rats with saline injected (sham lesioned) rats, it should also be apparent whether desipramine, fluoxetine or citalopram inhibit noradrenaline uptake through sites on noradrenergic neurones. Chapter 4

Finally, a selective lesion of dopaminergic neurones was attempted using the neurotoxin, 6-hydroxydopamine (6-OHDA). Intraventricular administration of

6-OHDA in rats causes a selective depletion of catecholamine neurones (Jacks et al,

1972; Bloom et al, 1969). It is another neurotoxin which is accumulated in nerve terminals where its concentration builds up to toxic levels. 6-OHDA is oxidised intraneuronally, forming a pam-quinone of 6-OHDA (which is thought to bind to nucleophilic centres of essential proteins), hydrogen peroxide and free radicals. The selectivity of 6-OHDA derives from its affinity for inhibition of catecholamine uptake

(Iversen, 1970). A selective lesion of dopaminergic neurones should be possible when uptake via the noradrenaline transporter is blocked by systemic pretreatment with desipramine (Sachs & Jonsson, 1975a; Samanin et a l, 1975).

119 Chapter 4

4.2 Methods

4.2.1 Selective lesion of monoaminergic neurones in the cerebral cortex.

Full details of the procedures involved in the induction of the three different lesions are given in Methods section 2.2.3.

4.2.1.1 Lesion of serotonergic neurones with 5,7-DHT

5-HT neurones of the cortex were lesioned using the neurotoxin 5,7-DHT.

Noradrenergic neurones were protected by systemic pretreatment with desipramine

(45 min prior to 5,7-DHT; 25 mg / kg, i.p.). 5,7-DHT (150 )ig) was injected i.c.v. into the right or left lateral ventricle. The control group of rats received desipramine pretreatment followed by an i.c.v. injection of ascorbate-saline vehicle. Rats were killed

7 days later and the cerebral cortex removed for the preparation of synaptosomes.

4.2.1.2 Lesion of noradrenergic neurones with DSP-4

The neurotoxin DSP-4 was used to induce a noradrenergic lesion of the innervation of the cortex. Rats were injected systemically with DSP-4 (40 mg / kg i.p.).

A group of control rats were injected with saline vehicle. Rats were killed and their brains removed, 5 days after pretreatment.

4.2.1.3 Lesion of dopaminergic neurones with 6-OHDA

A selective lesion of dopaminergic neurones was attempted using 6-OHDA.

After pretreatment (45 min prior to 6-OHDA) with desipramine (25 mg / kg, i.p.) and pargyline (50 mg / kg, i.p.), 6-OHDA (250 jig) was injected into either lateral ventricle.

Control rats were pre-treated with desipramine and pargyline and then injected i.c.v.

120 Chapter 4

with ascorbate vehicle. 7 days later, rats were killed and the cerebral cortex used to

prepare synaptosomes for uptake experiments.

4.2.2 Synaptosomal [^H]noradrenaline uptake

Crude cortical synaptosomes were prepared from the cortex of the lesioned or control rats. For full details of this procedure see Methods 2.2.2.2. Synaptosomes were

used to measure the uptake of [^H]noradrenaline uptake in control and lesioned tissue.

The inhibition of uptake by fluoxetine, citalopram and desipramine was studied by

including these drugs in the synaptosome incubation medium at either 0.5, 5 or 50 |iM.

A complete description of the processes involved in the measurement of

[■^H]noradrenaline uptake is given in Methods section 2.2.2.3. Uptake into synaptosomes from lesioned rats was compared to that in rats from the respective control (sham lesioned) group.

4.2.3 Verification of the lesion

The monoamine content of lesioned cortical tissue was compared with its respective control. Noradrenaline, dopamine and 5-HT content of an aliquot of tissue was measured using HPLC-ECD (see Methods 2.2.4) to confirm the selectivity and extent of the lesions.

4.2.4 Statistical analysis

Raw uptake data were analysed using 1-way ANOVA with post-hoc Tukey test.

When differences in uptake between treatment groups were compared, a 2-way ANOVA

121 Chapter 4

was used with ‘lesion’ as the additional between subjects factor. Cortical monoamine

content was analysed using the Mann-Whitney U-test.

4.3 Results

4.3.1 Inhibition of [^H]noradrenaline uptake by fluoxetine, citalopram or

desipramine

Preliminary uptake experiments were performed using synaptosomes prepared

from the cortex of dmg-naive, untreated rats. [^H]noradrenaline uptake was inhibited by

fluoxetine, citalopram and desipramine (Figure 4.1). Desipramine caused substantial

inhibition of uptake (F 3,39= 1 9 .6 ; P<0.001) which was significant at 0.5, 5 and 50 |iM

(P<0.05; post-hoc Tukey test). Fluoxetine also caused appreciable inhibition of

noradrenaline uptake (F3,2s=35.5; P<0.001), this was significant at 5 and 50 pM

(P<0.05; post-hoc Tukey test). Citalopram inhibited [^Hjnoradrenaline uptake at higher concentrations (F3,42=9.14; P<0.001) with significant inhibition at 25 and 50 pM

(P<0.05; post-hoc Tukey test).

122 Chapter 4

Q) 80 B Q. =3 0) C 60 "cô c "O 2 o 40 c

20

_c

0 6 5 4

Log^Q drug concentration (M)

desipramine fluoxetine citalopram

Figure 4.1: Inhibition of H]noradrenaline uptake by desipramine, fluoxetine or citalopram

Graphs illustrate the inhibition of [^H]noradrenaline uptake into rat cortical synaptosomes by test drugs. Data are expressed as % inhibition of 'drug-free' uptake.

Data are mean ± SEM with n=4-10.

23 Chapter 4

4.3.2 [^H]noradrenaline uptake into cortical synaptosomes from 5,7-DHT lesioned rats

4.3.2.1 Cortical monoamine content after a 5,7-DHT lesion

The 5-HT content of cortical tissue from 5,7-DHT lesioned rats was 54% lower

(see Table 4.1) than that of sham lesioned controls (P<0.001). Neither noradrenaline

(P=0.68) nor dopamine (P=0.33) content of lesioned tissue was significantly different from sham lesioned cortex (see Table 4.1).

Table 4.1: Concentrations of 5-HT, noradrenaline and dopamine in the cerebral cortex o f sham- or 5,7-DHT lesioned rats.

Data are expressed as mean ± SEM (ng / g wet tissue weight). Sample sizes are shown in parentheses. * P<0.001 (c/ sham lesioned group)

Concentration (ng / g wet tissue weight)

Sham 5,7-DHT

5-HT 379.1 ±50.7 (14) 174.7 ±16.2 (17)*

Noradrenaline 447.4 ±41.5 (14) 416.8 ±36.0 (17)

Dopamine 197.0 ±32.0 (12) 304.1 ±57.8 (17)

4.3.2.2 Specific [^H] nor adrenaline uptake into synaptosomes from sham- or 5,7-DHT lesioned rats •

[^Hjnoradrenaline uptake in the absence of test drugs, when the 'drug-free' data from all experiments were pooled, was the same in both treatment groups (pmol / mg protein, sham: 1.11 ±0.1 (n=10); 5,7-DHT: 1.02 ±0.1 (n=ll); P=0.53, Mann-Whitney

U-test).

124 Chapter 4

4.3.23 Inhibition of [^HJnoradrenaline uptake by desipramine

Desipramine caused profound inhibition of [^H]noradrenaline uptake into synaptosomes prepared from sham-lesioned rats (F3,23=14.3; P<0.001) (Figure 4.2).

This inhibition was statistically significant at all 3 concentrations tested. Desipramine also caused a significant inhibition of [^H]noradrenaline uptake into synaptosomes from

5,7-DHT lesioned rats (F3,23=18.0; P<0.001). Inhibition of uptake by all concentrations of desipramine was significant in both groups (P<0.05, post-hoc Tukey test). The effect of desipramine on uptake did not differ between the 2 groups (main effect of lesion:

Fj,47=0.002; P=0.97).

4.3.2.4 Inhibition of [^HJnoradrenaline uptake by fluoxetine

Fluoxetine significantly inhibited uptake of [^H]noradrenaline into synaptosomes prepared from sham (F 3J 3=3 7 .6 ; P<0.001 ) and 5,7-DHT lesioned (F 3J 5=9.2 P=0.002) rats (Figure 4.3). In the sham lesioned group, this inhibition was significant at 50 ]liM only (P<0.05, post-hoc Tukey test). In synaptosomes prepared from 5,7-DHT lesioned rats, the inhibition of uptake by 5 and 50 )liM fluoxetine was significant (P<0.05, post- hoc Tukey test). However, there was no difference between the amount of uptake in the presence of any concentration of fluoxetine between the sham- and the 5,7-DHT group

(main effect of lesion: F 1,29=3.5; P=0.08).

125 Chapter 4

c B o 1.2 ΠO) E 1.0 -

"Ô E 0.8

0)

CO 0.6 Q- 3 CD C 0.4 1Ô c E 0.2 TD E O C 0.0 - COI Drug- -7 -6 -5 -4 free

Log^Q [desipramine] (M)

sham lesion

5,7-D H T lesion

Figure 4.2: Uptake of['^H] noradrenaline into synaptosomes from5,7-DHT or sham- lesioned rat cortex in the presence of desipramine

'Drug-free' refers to the amount of uptake in the absence of test drug. Data are expressed as mean ± SEM uptake (pmol / mg protein) n=4-6.

126 Chapter 4

g '(D 1.6 2 Q. D) 1.4 E 1.2 Ô E 1.0

s 0.8 S Q. =3 0.6 CD _C 0.4 c 2 "O 0.2 E o c 0.0 Drug- -7 -6 -5 -4 free Log^Q [fluoxetine] (M)

— sham lesion

-V - 5,7-DHT lesion

Figure 4.3: Uptake of [ H]noradrenaline into synaptosomes from 5,7-DHT or sham- lesioned rat cortex in the presence of fluoxetine

'Drug-free' refers to the amount of uptake in the absence of test drug. Data are expressed as mean ± SEM uptake (pmol / mg protein) n=4.

127 Chapter 4

4.3.2.5 Inhibition of [^H]noradrenaline uptake by citalopram

Citalopram significantly reduced ['^H]noradrenaline uptake into synaptosomes prepared from sham- (F 3,21=3 .0 7 ; P=0.05) and 5,7-DHT lesioned (p3,25=9.1; P<0.001) rats (Figure 4.4). Inhibition of uptake was significant in both groups at 25 and 50 p.M

(P<0.05, post-hoc Duncan test), but not at 2.5 p,M citalopram. There was no significant

difference in the inhibition of uptake by citalopram between the two groups (F 1,4 7 = 8 .2;

P=0.42).

128 Chapter 4

c B o 1.2 Œ O) E 1.0 ■ Ô E 0.8 - 3 0 B 0.6 - Q- Z3 0 _C 0.4 - c 0 0.2 - -O 0 o c 0.0 - i h X CO Drug- -7 -6 -5 -4 free

Log [citalopram] (M)

- V “ sham lesion y 5,7-DHT lesion

Figure 4.4: Uptake of [^H]noradrenaline into synaptosomes from 5,7-DHT or sham-

lesioned rat cortex in the presence of citalopram

'Drug-free' refers to the amount of uptake in the absence of test drug. Data are expressed as mean ± SEM uptake (pmol / mg protein) n=5-7.

129 Chapter 4

4.3.3 [^H]noradrenaline uptake into cortical synaptosomes from DSP-4 lesioned rats.

4.3.3.1 Cortical monoamine content after a DSP-4 lesion

The noradrenaline content of cortical tissue from DSP-4 lesioned rats was 45% lower (see Table 4.2) than that of sham lesioned controls (P=0.004). Neither dopamine

(P=0.53) nor 5-HT (P=0.92) content of lesioned tissue was significantly different from sham lesioned cortex (see Table 4.2).

Table 4.2: Concentrations of noradrenaline, dopamine and 5-HT in the cerebral cortex o f sham- or DSP-4-injected rats.

Data are expressed as mean ± SEM (ng / g wet tissue weight). Sample sizes are shown in parentheses. * P<0.001 (c/ sham lesioned group)

Concentration (ng / g wet tissue weight)

Sham DSP-4

Noradrenaline 412.4 ±36.0 (22) 224.4 ±37.4 (21)*

Dopamine 328.0 ±82.0 (8) 252.0 ± 52.0 (8)

5-HT 398.0 ±22.0 (8) 398.0 ±46.0 (8)

4.3.3.2 Specific [^H]noradrenaline uptake into synaptosomes from DSP-4 lesioned rats

[■^H]noradrenaline uptake in the absence of test drugs, when the 'drug-free' data from all experiments were pooled, was the same in both treatment groups (pmol / mg protein, sham: 0.96 ± 0.06 (n=18); DSP-4: 0.81 ± 0.03 (n=18); P=0.15, Mann-Whitney

U-test).

130 Chapter 4

4.3.33 Inhibition of [^H]noradrenaline uptake by desipramine

In synaptosomes prepared from sham-lesioned rats, desipramine caused

substantial inhibition of [^H]noradrenaline uptake (F3j5=59.0; P<0.001) (Figure 4.5).

This inhibition was statistically significant at all 3 concentrations tested. Desipramine also caused a significant inhibition of [^H]noradrenaline uptake into synaptosomes from

DSP-4 lesioned rats (F3,2o=14.9; P=0.001). In lesioned rats however, the effect of

0.5 p,M desipramine was not statistically significant. A 2-way ANOVA identified a drug concentration x lesion interaction (F3,37=4.3; P=0.01). Uptake in the presence of

0.5 |iM (Fij=7.2; P=0.04) and 5 pM (F|j=17.1; P=0.004) desipramine was greater in synaptosomes prepared from sham-lesioned rats than from the DSP-4 lesioned group

{i.e. the inhibition of uptake by 0.5 and 5 pM desipramine was less in synaptosomes prepared from DSP-4 treated rat cortex).

4.3.3.4 Inhibition of [^H]noradrenaline uptake by fluoxetine

Fluoxetine significantly inhibited uptake of [^H] noradrenaline into synaptosomes prepared from saline (F3,ig=29.9; P=0.001) and DSP-4 pretreated (F3,2i=H.21 P<0.001) rats (Figure 4.6). In the saline-pretreated group, this inhibition was significant at 5 and

50 pM (P<0.05, post-hoc Tukey test). In synaptosomes prepared from DSP-4 lesioned rats, the uptake of [^H]noradrenaline in the presence of 0.5 pM fluoxetine was significantly less than that into synaptosomes prepared from sham-lesioned control rats

(Fi,8=6.7; P=0.04) i.e. the inhibition of uptake by 0.5 pM fluoxetine was greater in synaptosomes prepared from DSP-4 treated rat cortex.

131 Chapter 4

4.3.3.5 Inhibition of [^H]noradrenaline uptake by citalopram

Citalopram significantly reduced [^HJnoradrenaline uptake into synaptosomes prepared from saline-pretreated (p3,42=9.14; P<0.001) and DSP-4 lesioned (F 3,4 2 = 1 4 .4 ;

P<0.001) rats (Figure 4.7). Inhibition of uptake was significant in both groups at 25 and

50 p.M (P<0.05, post-hoc Tukey test), but not at 2.5 p,M citalopram. There was no significant difference in the inhibition of uptake by 2.5 p.M between the two groups

(Fi,,9=3.7; P=0.07).

132 Chapter 4

g 0.9 B o Œ 0.8 CD E ~ô E

CD 03 0.5

(D 0.4 _C c 0.3 E ■a E 0.2 o c coX 0.1 -7 -6 -5 -4 Drug- free Log^o [desipramine] (M)

sham lesion DSP-4 lesion

Figure 4.5: Uptake of [^H]noradrenaline into synaptosomes from DSP-4 or sham- lesioned rat cortex in the presence of desipramine

'Drug-free' refers to the amount of uptake in the absence of test drug. Data are expressed as mean ± SEM uptake (pmol / mg protein) n=4. | P<0.05 compared with the same concentration of desipramine in the sham lesioned group.

33 Chapter 4

c 1.0 - B o o . CD 0.8 - E

"ô E Q. 0.6 1

-2 Q. 3 0.4 - CD C m c 0 "O 0.2 - ce O c X co 0.0 ih -7 -6 -5 -4 Drug- free LoQio [fluoxetine] (M)

sham lesion DSP-4 lesion

Figure 4.6: Uptake of [ H]noradrenaline into synaptosomes from DSP-4 or sham- lesioned rat cortex in the presence of fluoxetine

'Drug-free' refers to the amount of uptake in the absence of test drug. Data are expressed as mean ± SEM uptake (pmol / mg protein) n=5. t P<0.05 compared with the same concentration of fluoxetine in the sham lesioned group.

34 Chapter 4

c 0 1.2 2 Q. e n 1.0 E

ô E 0.8 - * Q. 0

0 0.6 -

0 C 0.4 "cô c 2 ■D 0 0.2 - O C % co 0.0 -n- -7 -6 -5 -4 Drug- free Log^o [citalopram] (M)

sham lesion DSP-4 lesion

Figure 4.7: Uptake of H]noradrenaline into synaptosomes from DSP-4 or sham- lesioned rat cortex in the presence of citalopram

'Drug-free' refers to the amount of uptake in the absence of test drug. Data are expressed as mean ± SEM uptake (pmol / mg protein) n=10.

135 Chapter 4

4.3.4 [^H]Noradrenaline uptake into cortical synaptosomes from 6-OHDA lesioned

rats

4.3.4.1 Cortical monoamine content after a 6-OHDA lesion

Treatment of rats with 6-OHDA did not produce a selective lesion of dopaminergic neurones. Dopamine (P=0.03), noradrenaline (P=0.03) and 5-HT

(P=0.03) contents of cortical tissue were all lower in 6-OHDA lesioned rats than in sham-lesioned controls (see Table 4.3).

Table 4.3; Concentrations of 5-HT, noradrenaline and dopamine in the cerebral cortex o f sham- or 6-OHDA lesioned rats.

Data are expressed as mean ± SEM (ng / g wet tissue weight). Sample sizes are shown in parentheses. * P<0.05 (c/ sham lesioned group)

Concentration (ng / g wet tissue weight)

Sham 6-OHDA

Dopamine 106.5 ± 67.9 (6) 12.4 ± 6.5 (4)*

Noradrenaline 884.9 ± 79.8 (6) 541.7 ± 87.0 (4)*

5-HT 1082.5 ± 117.5(6) 822.0 ± 113.9 (4)*

136 Chapter 4

4.4 Discussion

The results show that fluoxetine and citalopram cause appreciable inhibition of

[^H]noradrenaline uptake into rat cortical synaptosomes, although fluoxetine is considerably more effective than citalopram.

7 days after treatment of rats with 5,7-DHT, the 5-HT content of cortical tissue was reduced by 50%. The inhibition of synaptosomal [^H] noradrenaline uptake by fluoxetine, citalopram or desipramine was not affected by the partial lesion of serotonergic neurones with 5,7-DHT: uptake in the presence of each of these drugs was the same into cortical synaptosomes prepared from sham lesioned as it was into those prepared from 5,7-DHT lesioned rats. The lack of effect of a 50% reduction in 5-HT content suggests that none of the above drugs causes appreciable inhibition of noradrenaline uptake through a site on 5-HT neurones. Because 5-HT neurones (or

5-HT content) were not completely abolished, it cannot be ruled out that surviving 5-HT neurones compensate for the lesion. Despite this, if the site of action of these drugs were predominantly on 5-HT neurones some change in the inhibition of

[^H] noradrenaline uptake would be expected. It is unlikely therefore, that fluoxetine or citalopram inhibit the uptake of noradrenaline through an action on 5-HT neurones.

The next possible site of action of the SSRIs to be investigated was noradrenergic neurones. The involvement of noradrenergic neurones in the inhibition of

[^H]noradrenaline uptake by fluoxetine, citalopram and desipramine was investigated using synaptosomes prepared from DSP-4 lesioned rat cortex. In the present experiments, inhibition of [^H]noradrenaline uptake by 0.5 and 5 |iM, but not 50 }iM,

137 Chapter 4 desipramine was reduced after DSP-4 treatment. This suggests that, of the two desipramine-sensitive noradrenaline uptake sites described in Chapter 3, only a higher affinity (uptake) site is lost after treatment with DSP-4.

In experiments described in Chapter 3 the high affinity desipramine-sensitive component of noradrenaline uptake represented only -20% of total specific uptake.

DSP-4 abolishes only the high affinity desipramine-sensitive component of

noradrenaline uptake (Chapter 3). Therefore, after DSP-4 treatment -80% of noradrenaline uptake should remain intact. This is consistent with the results of the present study: there was no statistically significant reduction in specific

[^H]noradrenaline uptake (-16%), after DSP-4. As only high affinity desipramine- sensitive noradrenaline uptake is thought to represent uptake into noradrenergic nerve terminals (Michel et al, 1984), the present work would suggest that the majority

(-80%) of noradrenaline released in the cortex is taken up by non-noradrenergic sites.

As mentioned in Chapter 3, there is much evidence to suggest that noradrenaline uptake by dopaminergic neurones in the cortex is a strong possibility. A study by

Michel and colleagues (1984) demonstrated that low affinity desipramine-sensitive noradrenaline uptake in the cortex was likely to represent uptake via a dopamine transporter (on dopaminergic neurones). This suggestion was based on two findings.

First, low affinity desipramine-sensitive [^H]noradrenaline uptake was inhibited more potently by dopamine uptake blockers, like nomifensine, than desipramine. Second, the

IC 50 for low affinity desipramine inhibition of noradrenaline uptake (4 p,M) was

138 Chapter 4

similar to the IC50 for inhibition of [^H]noradrenaline uptake in the striatum (3.8 |iM), a brain region innervated predominantly by dopaminergic neurones, but considerably higher than the IC50 for inhibition of [^H]noradrenaline uptake in the hypothalamus

(0.1 nM).

Further support for noradrenaline uptake by a site on dopaminergic neurones comes from studies of the cloned noradrenaline and dopamine transporters. These catecholamine transporters have 78% amino acid sequence identity and discriminate poorly between dopamine and noradrenaline: both transport noradrenaline and dopamine with similar affinity (Giros et a i, 1994; Buck & Amara, 1995; see also

General Introduction section 1.5.4).

The disproportionately small reduction in [^H]noradrenaline uptake (non­ significant -16% reduction in uptake c/~45% depletion of noradrenaline stores) after

DSP-4 treatment cannot be explained by surviving noradrenergic neurones compensating for the lesion. If surviving noradrenergic neurones could compensate by taking up more [^H]noradrenaline no change in the inhibition caused by desipramine would be detected. The results of the present experiments show that this is not the case because the inhibition of [^H]noradrenaline uptake by low concentrations (0.5 & 5 |iM) of desipramine was reduced, while total inhibition at higher concentrations of desipramine was unchanged. This result is consistent with noradrenaline uptake by this site being reduced due to a loss of desipramine-sensitive uptake sites on noradrenergic nerve terminals.

139 Chapter 4

After treatment with DSP-4, a greater proportion of [^H]noradrenaline uptake was inhibited by a low concentration of fluoxetine (0.5 p.M). This is consistent with low concentrations of fluoxetine inhibiting noradrenaline uptake via a site which is not affected by DSP-4. This is because such a site would assume greater importance after a lesion due to the loss of DSP-4-sensitive noradrenaline uptake sites. Whether this site of action of fluoxetine is on surviving noradrenergic neurones or elsewhere cannot be distinguished from these experiments. One conclusion that can be made is that the site at which fluoxetine inhibits noradrenaline uptake is not the same as the high affinity desipramine-sensitive site as this is diminished after treatment with DSP-4 (see above).

The inhibition of [^H] noradrenaline uptake by higher concentrations of fluoxetine (5 &

50 |iM) was not affected by treatment with DSP-4. Whether this indicates a non­ specific action of fluoxetine at a number of different uptake sites unaffected by DSP-4, possibly on dopaminergic neurones, remains to be determined.

The inhibition of [^Hjnoradrenaline uptake by citalopram was not affected by the

DSP-4 lesion at any of the concentrations used. This would suggest that citalopram inhibits noradrenaline uptake via a site which is neither a target for DSP-4, nor capable of compensating for the lesion by increasing its uptake of noradrenaline. Present experiments suggest that this site is neither on noradrenergic nor serotonergic neurones, the location of this site cannot be determined as yet. These preliminary results indicate a possible difference between the sites of noradrenaline uptake inhibited by fluoxetine and citalopram. This will be pursued further in Chapter 5 where the effects of these drugs on noradrenaline efflux in vivo are investigated.

140 Chapter 4

The obvious next experiment was to investigate the effect of a selective

dopaminergic lesion on the inhibition of [^H]noradrenaline uptake by fluoxetine, citalopram and desipramine. This was attempted using 6-OHDA. However, these experiments were unsuccessful due to problems with the selectivity of this toxin.

Despite protection of noradrenergic neurones with desipramine, 6-OHDA caused a significant depletion of cortical noradrenaline and 5-HT content. It was very difficult to cause a selective lesion of dopaminergic neurones in the cortex even when protecting noradrenergic neurones with desipramine. Attempts to increase the selectivity of 6-

OHDA for dopamine neurones by increasing the dose of desipramine invariably reduced the extent of the dopaminergic lesion (D. J. Heal, personal communication). The poor discrimination of 6-OHDA between dopaminergic and noradrenergic neurones is likely to be due to the fact that the catecholamine transporters are very similar such that noradrenaline and dopamine are transported by each other’s transporter (see General

Introduction section 1.5.4 and above). Indeed, Buck & Amara (1995) showed that the cloned noradrenaline transporter takes up 60% more dopamine than does the dopamine transporter. An additional problem with using 6-OHDA was that the rats suffered severe weight loss such that their survival was compromised. Because of the ethical implications, and the difficulty in increasing the selectivity of 6-OHDA, experiments using 6-OHDA to selectively lesion dopamine neurones were abandoned.

To summarise, the results of the experiments described in this chapter show that the SSRIs, fluoxetine and citalopram, inhibit the uptake of [^H]noradrenaline into rat cortical synaptosomes. The lack of effect of a 5,7-DHT lesion, on the inhibition of

[^H]noradrenaline uptake by fluoxetine, citalopram or desipramine, suggested that inhibition of [^H]noradrenaline uptake by these drugs does not involve serotonergic

141 Chapter 4 neurones in vitro. Using synaptosomes prepared from DSP-4 treated rats it was apparent that desipramine inhibited uptake via 2 sites, with only high affinity desipramine-sensitive uptake being reduced after a lesion of noradrenergic neurones.

Inhibition of noradrenaline uptake by fluoxetine or citalopram was not reduced after treatment with DSP-4, suggesting that these drugs could inhibit noradrenaline uptake via a site (or sites) which is (or are) unlikely to be on noradrenergic neurones. The disproportionately small reduction in total specific [^Hjnoradrenaline uptake when tissue noradrenaline content was depleted suggests that the majority of noradrenaline uptake in the cortex is not into noradrenergic nerve terminals; uptake into dopaminergic neurones is a strong possibility.

In light of the interesting findings of experiments using synaptosomes prepared from DSP-4 lesioned rats, further experiments concentrated on investigating the effects of a noradrenergic lesion in vivo. Chapters 5 & 6 describe experiments investigating the effects of a DSP-4 lesion on the concentration of extracellular noradrenaline in vivo.

142 Chapter 5

Chapter 5

The effects of fluoxetine, citalopram and desipramine on extracellular noradrenaline in DSP-4 or sham- lesioned rats

5.1 Introduction

The SSRIs, fluoxetine and, albeit at higher concentrations, citalopram both inhibit the uptake of [^HJnoradrenaline into rat cortical synaptosomes (Chapters 3 & 4;

Hughes & Stanford, 1996). Using microdialysis in vivo, it was found that local infusion of fluoxetine into the frontal cortex of anaesthetised rats caused an increase in noradrenaline efflux (Chapter 3). Experiments in this chapter investigate the effect of infusion of fluoxetine on noradrenaline efflux in the frontal cortex of freely-moving rats. In order to see whether the increase in noradrenaline efflux caused by fluoxetine was a common feature of SSRIs, the effect of the more selective SSRI, citalopram, was also investigated. The effect of the selective noradrenaline uptake inhibitor,

143 Chapter 5 desipramine, served as an active control. Each drug (one drug per rat) was infused via the microdialysis probe for 3 h to monitor the time course of any changes in noradrenaline efflux in the terminal field. To determine whether the SSRI-induced increase in noradrenaline efflux could be secondary to an increase in the concentration of extracellular 5-HT (due to inhibition of 5-HT reuptake by SSRIs) the effect of infusion of 5-HT via the probe on noradrenaline efflux in the frontal cortex was investigated.

The second objective of this study was to determine whether the increase in noradrenaline efflux induced by fluoxetine, citalopram or desipramine involved an action on noradrenergic neurones. Experiments described in this chapter investigated whether a lesion of noradrenergic neurones modifies the drug-induced increase in noradrenaline efflux in vivo. Noradrenergic neurones of the cortex were lesioned using

DSP-4. The effects of this noradrenergic neurotoxin and its mode of action are discussed in the General Introduction (Chapter 1). Among other effects, DSP-4 depletes noradrenaline, reduces the number of noradrenaline uptake sites (measured by

[^Hjnisoxetine or [^H]desipramine binding), causes an upregulation of p-adrenoceptors and a loss of dopamine-p-hydroxylase (DPH) protein (evidenced by reduced staining with antibodies to DpH) (Ross, 1976; Lee et al., 1982; Dooley et ai, 1983; Hallman &

Jonsson, 1984; Fritschy et al., 1990; Cheethamet al., 1996). At doses of DSP-4

<100 mg / kg, these effects are largely restricted to brain regions such as the cortex and hippocampus (Jonsson et al., 1981), where the noradrenergic innervation is predominantly, if not exclusively, derived from the locus coeruleus (Moore & Bloom,

1979).

144 Chapter 5

Previous experiments (Chapter 4) measured [^H]noradrenaline uptake into synaptosomes prepared from the cortex of rats pretreated with DSP-4. Results from these experiments showed that the inhibition of [^H]noradrenaline uptake by fluoxetine was not reduced when synaptosomes were prepared from DSP-4 treated rats. This suggested that the site for inhibition of [^H]noradrenaline uptake by fluoxetine was unlikely to be on noradrenergic neurones as it was not targeted by DSP-4 (Chapter 4).

Inhibition of [^HJnoradrenaline uptake into cortical synaptosomes from 5,7-DHT lesioned rats was not different from sham-lesioned controls. This suggested that the inhibition of noradrenaline uptake by either fluoxetine or citalopram did not involve a site on 5-HT neurones (Chapter 4).

In order to investigate the role of noradrenergic neurones in the action of these drugs in vivo, the effects of local infusion of fluoxetine, citalopram or desipramine, were studied in rats which had been pretreated with either DSP-4 or saline, 5 days earlier. The earlier finding that 5-HT was a potent inhibitor of [^H]noradrenaline uptake (Chapter 3), raised the question as to whether the increase in noradrenaline efflux induced by local infusion of fluoxetine could be secondary to an increase in the extracellular concentration of 5-HT. In an attempt to test this in the present experiments, the effect of local infusion of 5-HT on noradrenaline efflux was also investigated.

145 Chapter 5

5.2 Methods

5.2.1 DSP-4 lesion of noradrenergic neurones

One group of rats was injected systemically with DSP-4 (40 mg / kg i.p.); a second with saline vehicle (2 ml / kg). Rats from both groups were used for microdialysis experiments 5 days after this pretreatment. More details of the methodology are given in Materials and Methods section 2.2.3.

5.2.2 Intracerebral microdialysis

Experiments were performed in freely-moving rats on the day after probe implantation (for details of surgery see Materials and Methods 2.2.1) and 5 days after pretreatment with either saline or DSP-4. Once a stable level of spontaneous efflux was obtained, 4 samples of dialysate were collected in order to establish basal noradrenaline efflux. After collection of 4 stable basal samples, a perfusion medium containing either desipramine (5 |iM), fluoxetine (5 or 50 p-M) or citalopram (50 |iM) was infused. Noradrenaline efflux was measured in the presence of test drug for 3 h.

The effect of local infusion of 5-HT was studied in untreated rats. 5-HT (dissolved in

Ringer’s) was infused via the probe at a concentration of 5 pM for 80 min.

5.2.3 Measurement of monoamine content of cortical tissue

A sample of tissue from the frontal cortex (contralateral to position of probe) was analysed for noradrenaline, dopamine and 5-HT content using HPLC-ECD (details of the procedures involved are given in Methods 2.2.4). The monoamine content of cortical tissue of DSP-4 lesioned rats was compared with that of saline-injected controls.

146 Chapter 5

5.2.4 Calculation of “net” noradrenaline efflux

To take into account any differences in the basal levels between the two treatment groups "net" efflux of noradrenaline was calculated. This represents the change in efflux induced by drug infusion with respect to the basal level for each subject. This calculation involves subtracting the mean basal noradrenaline efflux for each rat from each point of the time course. The mean net efflux ± SEM for the whole group was then calculated from these values.

This is an example of the calculation:

sample no. raw data (fmol / 20 min) mean basal "net" efflux

basal 1 56.5 55.7 0.8 basal 2 51.9 -3.8 basal 3 59.9 4.2 basal 4 54.5 -1.2

test 1 83.8 28.1 test 2 69.8 14.1 test 3 93.8 38.1 test 4 46.6 -9.1 test 5 47.9 -7.8

5.2.5 Statistical analysis

Drug induced changes in the concentration of noradrenaline in cortical microdialysates were analysed by split-plot analysis of variance (ANOVA) with ‘time’ and ‘bin’ as ‘within-subjects’ factors and ‘treatment’ as the ‘between-subjects’ factor.

The Mann-Whitney U-Test was used to analyse changes in the monoamine content of cortical tissue after DSP-4 treatment.

147 Chapter 5

5.3 Results

5.3.1 The effects of fluoxetine, citalopram, desipramine or 5-HT on noradrenaline efflux

In saline pretreated (control) rats, local infusion of fluoxetine, citalopram or desipramine increased noradrenaline efflux. The order of potency was desipramine>fluoxetine>citalopram>5-HT. The increase due to fluoxetine was significant at 5 |iM (F2,i3=9.25; P=0.003) and 50 pM (F 2,14= 10.9; P=0.001) (Figure

5.1).

Citalopram infusion (50 pM) caused an increase in noradrenaline efflux which was significant during the first hour of drug infusion only (Fij=6.25; P=0.04). During

the 2"^ (F i,7 = 3 .3 6 ; P=0.I1) and 3"^^ hours (Fij=5.72; P=0.08) of infusion, citalopram did not cause a significant increase in noradrenaline efflux (Figure 5.1).

Infusion of 5 pM desipramine (Figure 5.1) significantly increased noradrenaline efflux for the duration (3 h) of its infusion (F2,n=6.01; P=0.017).

Local infusion of 5-HT (5 pM) did not affect noradrenaline efflux in the frontal cortex of untreated rats (Fi, 4=0 .0 1 ; P=0.94) (Figure 5.2).

5.3.2 Cortical monoamine content after DSP-4 treatment

5 days after pretreatment, the cortical noradrenaline content of DSP-4 lesioned rats was 70% lower than that of sham-lesioned controls (P<0.001). 5-HT content

(P=0.66) dopamine content (P=0.98) were not significantly different from sham- lesioned controls (Table 5.1). There was a substantial increase in dopamine content after treatment with DSP-4, but this was not statistically significant (see section 5.4).

148 Chapter 5

c 80 ' E o 70 - C\J 60 - "5 g 50 - X 40 - 0) 30 - (D _C 20 -

C 0) 10 -

co 0 - O C -10 - o c 0 40 80 120 160 200 240 280 time (min)

desipramine 5 pM fluoxetine 5 pM fluoxetine 50 pM citalopram 50 pM

Figure 5.1: Effects of fluoxetine, citalopram or desipramine on noradrenaline efflux in saline pre-treated (control) rats.

Graphs show mean ± SEM (n=7-9) net noradrenaline efflux expressed as fmol /

20 min. Horizontal bar indicates duration of drug infusion.

149 Chapter 5

50 -

45 -

40 - o c\j 35 -

30 -

25 -

0 20 - 0 c m 15 - c

2 10 - "O 0 5 -

0 0 20 40 60 80 100 120 140 160 180

time (min)

Figure 5.2: Ejfect of 5-HT on noradrenaline efflux in freely-moving rats

Graph shows mean ± SEM (n=3) noradrenaline efflux expressed as fmol / 20 min. Horizontal bar indicates duration of 5-HT (5 pM) infusion.

150 Chapter 5

Table 5.1: Cortical monoamine content after DSP-4 treatment

The concentrations of noradrenaline, dopamine and 5-HT of the cerebral cortex

5 days after pretreatment with either saline vehicle (sham) or DSP-4. Data show mean

± SEM ng /g wet tissue weight with sample size in parentheses. * P<0.001 cf. sham- lesioned control group.

Concentration (ng / g wet tissue weight) sham DSP-4

noradrenaline 579.4 ± 77.9 (10) 174.7 ±11.9 (22)*

dopamine 25.8 ±7.2 (10) 62.3 ±26.2 (16) 5-HT 171.8 ±48.8 (10) 151.0 ±19.5 (21)

5.3.3 Noradrenaline efflux during local infusion of fluoxetine

In rats pretreated with DSP-4, local infusion of fluoxetine did not increase noradrenaline efflux at either concentration (5 |iM: p 2,8= 0.51; P=0.62, Figure 5.3;

50 |iM: Fi,15=0.48; P=0.631; Figure 5.4). Because the level of basal noradrenaline efflux was significantly greater in lesioned rats than in saline controls (P<0.001) "net" noradrenaline efflux was calculated; this involved subtracting the mean basal efflux for each rat from each point on the time course (see section 5.2.4). The net efflux induced by 50 |iM fluoxetine was significantly less in the DSP-4 lesioned group (Fi,g=17.31;

P=0.003; Figure 5.4b). Net efflux during infusion of fluoxetine (5 |LtM) was not different in the two treatment groups. This is likely to be because the size of the change was small in both groups making it more difficult to detect a significant difference.

151 Chapter 5

(a) 100

o CM 80

60

40

20

40 80 120 160 200 240 280

tim e (min)

sham DSP-4

(b) c 4 0 E o CM 30 Ô E 20

10 m c 1Ô 0 E ■o 2 -10 co 0) c 40 80 120 160 200 240 280

tim e (min)

Figure 5.3: Infusion of fluoxetine (5 pM) in sham- or DSP-4 lesioned rat frontal cortex.

Graphs show mean ± SEM (n=7-9) noradrenaline efflux expressed as fmol / 20 min. Horizontal bar indicates duration of drug infusion. * P<0.05 c/sham-lesioned controls.(a) Noradrenaline efflux (b) Data expressed as net noradrenaline efflux.

152 Chapter 5

(a) ^ 100

^ 80

6 0

4 0

20

0 40 80 120 160 200 240 280

tim e (m in)

sham DSP-4

(b) I 8 0

o c\j - 60 o E ^3 4 0 Ü) g 20 03 0)C T3 2 c:o 0) c 0 4 0 80 120 160 200 240 280

tim e (m in)

Figure 5.4: Infusion o f fluoxetine (50 fiM) in sham- or DSP-4 lesioned rat frontal cortex.

Graphs show mean ± SEM (n=6-8) noradrenaline efflux expressed as fmol / 20 min. The horizontal bar indicates duration of drug infusion. * P<0.05 c/sham-lesioned controls, (a) Noradrenaline efflux (b) Data expressed as net noradrenaline efflux.

53 Chapter 5

5.3.4 Noradrenaline efflux during local infusion of citalopram

In rats pretreated with DSP-4, citalopram caused a significant increase in noradrenaline efflux during the hour of infusion (Fij3=4.96; P=0.04, Figure 5.5).

During the 2nd and 3rd hours of infusion, the effect of citalopram was not significant

(Fi,14=1.58; P=0.23 and Fi,i4=3.11; P=0.10, respectively). Because the basal level of efflux was significantly greater in the lesioned group (P=0.02) net noradrenaline efflux was calculated for a clearer comparison of the drug-induced changes in the two treatment groups (Figure 5.5b). Net efflux was not different in the two groups

(F i,26=0.12; P=0.73) indicating that the effect of citalopram was not modified by treatment with DSP-4.

5.3.5 Noradrenaline efflux during local infusion of desipramine

Infusion of 5 pM desipramine produced an increase in noradrenaline efflux in

DSP-4 pretreated rats (Fi,22=6.02; P=0.008; Figure 5.6). The increase (as seen by net efflux. Figure 5.6b) was significantly greater in DSP-4 treated rats, when compared with sham controls, during the 2"^ and 3"^^ hours of desipramine infusion (Fi,i2=5.92;

P=0.03).

5.3.6 A comparison of the effects of fluoxetine, citalopram and desipramine on noradrenaline efflux

For an overview of the effects of fluoxetine, citalopram and desipramine on net noradrenaline efflux in saline pre-treated rats, the mean net efflux during 3 h drug infusion for each drug has been plotted as a barchart (Figure 5.7a). The magnitude of the increase in noradrenaline efflux over 3 h of desipramine (5 pM) infusion was not significantly different from that caused by fluoxetine (50 pM) {post-hoc Student

154 Chapter 5

Newman Keuls Test: P>0.05). The increase in efflux during fluoxetine (5 |iM) was the same as that during citalopram (50 pM) (P>0.05), but significantly smaller than that due to either desipramine 5 pM or fluoxetine 50 pM.(P<0.05).

In DSP-4 treated rats (Figure 5.7b), mean net noradrenaline efflux in the presence of fluoxetine (5 pM) was no different from efflux during fluoxetine (50 pM) or citalopram (50 pM) {Post-hoc Student Newman Keuls Test: P>0.05). In DSP-4 treated rats the increase due to infusion of desipramine (5 pM) was significantly greater than that due to any of the other drugs (Student Newman Keuls Test: P<0.05).

155 Chapter 5

(a) 1 70 1 o ^ 60 - Ô è 50 - X I 40 - (D Ç 30 - 03 I 20 - ■D 03 b 10 - c

0 40 80 120 160 200 240 280

tim e (m in

g sh am (b) E DSP-4 o CM Ô E 3X 20 c0) 1Ô 10 c E E 0 b c -10

40 80 120 160 200 240 280

tim e (m in)

Figure 5.5: Infusion of citalopram (50 pM) in sham- or DSP-4 lesioned rat frontal cortex.

Graphs show mean ± SEM (n=5-7) noradrenaline efflux expressed as fmol / 20 min. The horizontal bar indicates duration of drug infusion. * P<0.05 c/sham-lesioned controls, (a) Noradrenaline efflux, (b) Data expressed as net noradrenaline efflux.

156 Chapter 5

(a) Ç E o 175 C\J 0 150 E 125 X it: 100 CD 0) c 75 IB 1 50 ? o 25 c 0 0 40 80 120 160 200 240 280

tim e (min) sham DSP-4

(b) o CM 120

I 100

3X 80 CD 60 CD C 40 0)c "O 20 2 co CD c 0 4080 120 160 200 240 280

tim e (min)

Figure 5.6: Infusion of desipramine (5 pM) in sham- or DSP-4 lesioned rat frontal cortex.

Graphs show mean ± SEM (n=5-9) noradrenaline efflux expressed as fmol / 20

min. The horizontal bar indicates duration of drug infusion. * P<0.05 cf sham-

lesioned controls, (a) Noradrenaline efflux (b) Data are expressed as net noradrenaline efflux.

57 Chapter 5

a)

0) 40

m 30

desipramine^, fluoxetine . , 5 hM 50 <=i'aloprani 5 pM 50 pM

b)

Ï 40

desipramine^, . fluoxetine . , 5 u M flPO*e*'Pe 50 uM 5 pM 5 0 pM

Figure 5.7: Noradrenaline efflux during local infusion of desipramine, fluoxetine and citalopram.

Graphs illustrate mean net noradrenaline efflux during infusion of desipramine, fluoxetine or citalopram in the frontal cortex of a) saline-injected control, and b) DSP-4 lesioned rats. Data are mean ± SEM (n=4-9).

58 Chapter 5

5.4 Discussion

It has been shown previously that fluoxetine and citalopram inhibit the uptake of noradrenaline into rat cortical synaptosomes in vitro (Chapters 3 & 4). The results of microdialysis experiments described in this chapter show that fluoxetine and citalopram increase noradrenaline efflux in vivo. As these drugs both inhibit

['^H] noradrenaline uptake in vitro, it is likely that inhibition of noradrenaline uptake contributes to the increase in noradrenaline efflux in vivo.

Fluoxetine appears to be approximately 10-fold less potent than desipramine at increasing noradrenaline efflux in vivo. The mean increase in noradrenaline efflux during 5 |liM desipramine infusion was -32 fmol / 20 min, compared with

-39 fmol / 20 min during 50 pM fluoxetine. This is in line with their relative potencies for inhibition of noradrenaline uptake in vitro (Chapter 3; 50% uptake inhibited by

1 pM and 12 pM, respectively).

The mean net increase in noradrenaline efflux during 5 pM fluoxetine was

15 fmol / 20 min compared with 14 fmol / 20 min during 50 pM citalopram. Together with their relative potencies for inhibition of [^H]noradrenaline uptake in vitro

(Chapter 4 IC50 S: citalopram 40 pM, fluoxetine 12 pM;), this suggests that fluoxetine is in the region of 5-10 times more potent at increasing noradrenaline efflux and inhibiting [^H]noradrenaline uptake than citalopram. There appeared to be a good parallelism between the ability of drugs to increase noradrenaline efflux in vivo and inhibit [^H]noradrenaline uptake in vitro, thus supporting the concept that the increase

159 Chapter 5 in noradrenaline efflux induced by the SSRIs, fluoxetine and citalopram, is due, at least in part, to inhibition of noradrenaline uptake.

The substantial effect of fluoxetine and, to a lesser extent, citalopram on noradrenaline efflux questions the apparent selectivity of SSRIs for inhibition of 5-HT uptake in vivo. SSRIs are used routinely by a number of laboratories to raise levels of

5-HT so that they are more readily detectable using microdialysis (e.g. citalopram

1 |iM; Sharp et a i, 1989). Fluoxetine (10 |iM) increases the concentration of extracellular 5-HT nearly 2-fold when it is infused into hippocampus, for example

(Matsumoto et a i, 1995b). Citalopram (1 pM) increased 5-HT efflux from ~2 to

-18 fmol / 5 pi when infused into the frontal cortex (Romero et al., 1996). Although local infusion of SSRIs is known to increase the extracellular concentration of 5-HT, to the best of my knowledge, only one other group has investigated the effect of local infusion of fluoxetine on spontaneous noradrenaline efflux. Jordan et al. (1994) showed that infusion of fluoxetine increased the concentration of extracellular 5-HT to

500% basal in the medial prefrontal cortex at a concentration of 100 pM (no significant increase at 10 pM). The extracellular concentrations of noradrenaline (270% basal) and dopamine (600% basal) were also increased during infusion of fluoxetine (100 pM).

In support of the present findings, recent observations suggest that systemic fluoxetine causes an increase in the concentrations of extracellular dopamine and noradrenaline as well. Tanda and colleagues (1994) used microdialysis to measure the extracellular concentration of dopamine and 5-HT in the prefrontal cortex of rats.

After systemic injection of fluoxetine (10 mg / kg s.c.) dopamine efflux was increased

160 Chapter 5 to 230% of basal while the increase in the concentration of 5-HT reached a plateau at

170% of basal. The blunted effect of fluoxetine on extracellular 5-HT is most likely due to inhibition of raphe firing due to activation of 5-HT] A autoreceptors (see effects of paroxetine on the extracellular concentration of 5-HT and raphe 5-HT cell firing:

Garisidt et ai, 1995).

Another study measured the concentrations of extracellular 5-HT, dopamine and noradrenaline in the medial prefrontal cortex (Jordan et a i, 1994). Systemic injection of fluoxetine (10 mg / kg) increased extracellular concentrations of 5-HT, noradrenaline and dopamine. A recent study by Gobert et al. (1997) showed that fluoxetine (10 mg/kg s.c.) caused a substantial increase in the extracellular concentrations of 5-HT, noradrenaline and dopamine (percent basal: 210%, 190% and

160%, respectively) in the frontal cortex of freely moving rats.

These findings suggest that in addition to the effects of SSRIs on extracellular

5-HT described in section 1.4 of the General Introduction, in certain areas of the brain, a single systemic dose of fluoxetine has substantial effects on noradrenergic and dopaminergic function.

Interestingly, electrophysiological studies in vivo, showed that systemic fluoxetine, and to a lesser extent, citalopram, causes a dose-dependent reduction in the firing rate of ventral tegmental area dopaminergic neurones (Frisco & Esposito, 1995).

These drugs were without effect on dopaminergic neurones of the substantia nigra pars compacta. The effect of fluoxetine on dopaminergic neurone firing was abolished after treatment with 5,7-DHT or pretreatment with the mixed 5-HT2c/2b receptor antagonist,

161 Chapter 5

. Coupled with findings of Tanda and colleagues (1995) which

demonstrated that the increase in extracellular dopamine in the prefrontal cortex after

treatment with fluoxetine was prevented by either systemic treatment with or local

infusion of an antagonist of 5 -HT3 receptors, ICS 205930. These findings suggest the

effects of fluoxetine on dopaminergic neurones are secondary to an increase in the

concentration of extracellular 5-HT.

However, it is feasible that these drugs could have a direct effect on

noradrenaline and / or dopamine uptake as the concentration of antidepressant in rat

brain, after systemic administration, is reported to be within the range of the K\S for

inhibition of noradrenaline and dopamine uptake. The concentration of fluoxetine in

the brain, after a single systemic dose, is reported to be in the micromolar region

(Fuller et al, 1991; Dailey et a l, 1992; Goodnough & Baker 1994; see Chapter 3,

section 3.3). Also, the concentration of fluoxetine in the plasma of patients being

treated with this drug is in the micromolar range (Baumann, 1996). These concentrations are well within the range of the K\S for inhibition of noradrenaline and

dopamine uptake in vitro {e.g. fluoxetine: K\ dopamine uptake, 1-15 |xM;

noradrenaline uptake, 0.14-10 |XM; see Stanford, 1996). It is therefore likely that the

increase in the concentrations of dopamine and noradrenaline, after treatment with fluoxetine, are due, at least in part, to the direct inhibition of dopamine and

noradrenaline uptake by this drug in vivo.

By the same logic, the increase in noradrenaline efflux during local infusion of

fluoxetine (5 or 50 p,M) or citalopram (50 |iM) in this study is likely to be due to direct

inhibition of noradrenaline uptake. Moreover, the inhibition of [^H]noradrenaline

162 Chapter 5 uptake by neither fluoxetine nor citalopram was affected by a lesion of serotonergic neurones (Chapter 4), making inhibition of noradrenaline uptake by an action of fluoxetine and citalopram on 5-HT neurones unlikely.

The second aim of the experiments of this Chapter was to determine whether fluoxetine and / or citalopram could be acting directly at a site on noradrenergic neurones? Could these drugs inhibit noradrenaline uptake into noradrenergic neurones or noradrenaline uptake into other, possibly 5-HT, neurones? Possible differences in the mode of action of fluoxetine, citalopram and desipramine became apparent when their effects on noradrenaline efflux in DSP-4 lesioned rats were studied. 5 days after

DSP-4 treatment, the noradrenaline content of the frontal cortex was decreased by approximately 70%. 5-HT and dopamine contents were not significantly altered after

DSP-4 treatment. Although dopamine content appeared to be increased after DSP-4 treatment, this was not statistically significant. An increase in dopamine content could be explained by a reduction in the activity of DpH in noradrenergic neurones; in the absence of DpH, intraneuronal dopamine would accumulate as it would not be converted to noradrenaline. An increase in tissue dopamine content after a noradrenergic lesion has been reported previously (Reader & Brière, 1983; Harik,

1984).

An additional important finding was that the basal level of noradrenaline efflux was consistently higher in DSP-4 treated rats than in saline pretreated controls. This could be explained by a reduction in noradrenaline uptake and / or an increase in the rate of noradrenaline release. Interestingly, the desipramine-induced increase in noradrenaline efflux was greater in lesioned rats than in saline controls, indicating that

163 Chapter 5 noradrenaline uptake is in fact increased in DSP-4 lesioned rats (this finding is discussed in detail below). As noradrenaline uptake is not reduced, the rate of noradrenaline release from surviving neurones must be increased to account for the higher level of basal efflux in lesioned rats. Further investigation of the increased level of basal efflux in DSP-4 lesioned rats is described in Chapter 6.

After treatment with DSP-4, the desipramine-induced increase in noradrenaline efflux was greater than in saline controls. This indicates that surviving neurones have a large capacity for noradrenaline uptake. Experiments studying the inhibition of

['^H]noradrenaline uptake into synaptosomes prepared from DSP-4 lesioned rats

(Chapter 4) suggested that the high affinity desipramine-sensitive uptake site was disrupted by treatment with DSP-4. This is supported by the abolition of high affinity

[^H]desipramine binding to cortical membranes prepared from DSP-4 treated rats (Lee et a i, 1982; Bâckstrôm et a i, 1989). The desipramine-induced increase in noradrenaline efflux after DSP-4, must therefore be due to inhibition of a low affinity desipramine-sensitive noradrenaline uptake site.

Because desipramine had a greater effect in rats pretreated with DSP-4, it is suggested that the low affinity desipramine site assumes greater importance in the clearance of extracellular noradrenaline in vivo (i.e. it transports a greater absolute amount of noradrenaline) after a DSP-4 lesion. It is feasible that uptake via this site is driven by the elevated concentration of extracellular noradrenaline in vivo (see above) evident after DSP-4 treatment. One way in which an increase in the extracellular concentration of noradrenaline could increase uptake via the low affinity desipramine- sensitive site is if these uptake sites are distant from the site of noradrenaline release.

164 Chapter 5

Released noradrenaline would only reach these sites if the extracellular concentration was high enough. Such ‘spatial buffering’ has been shown in studies of the immunocytochemical localisation of dopamine transporters (Pickel et al, 1996). This could also explain why an increase in the inhibition of [^H]noradrenaline uptake by high concentrations of desipramine was not seen in vitro (Chapter 4). This is because in the synaptosomal preparation, the extracellular concentration of noradrenaline is artificially determined (and fixed at 50 nM) and will not be affected by changes in release of noradrenaline, if only because the extracellular space is infinite and thus noradrenaline will not accumulate in the vicinity of the uptake sites. Furthermore, because the synaptosomal preparation uses ‘pinched-off nerve terminals, the interactions between sites of noradrenaline release and uptake could be disrupted.

In contrast to desipramine, the increase in noradrenaline efflux induced by infusion of fluoxetine (5 or 50 |iM) in saline-injected rats was not evident after treatment with DSP-4. The absence of the fluoxetine-induced increase in noradrenaline efflux in DSP-4 lesioned rats cannot be explained by loss of the fluoxetine-sensitive uptake site. This is shown by findings of studies of the inhibition of [^H]noradrenaline uptake by fluoxetine in vitro described in Chapter 4. These experiments showed that the fluoxetine-sensitive noradrenaline uptake site was not disrupted by treatment of rats with DSP-4.

There are several possible explanations for the abolition of the fluoxetine- induced increase in noradrenaline efflux after treatment with DSP-4. A change in the modulation of noradrenaline release by activation of 5-HT heteroreceptors (see General

Introduction section 1.8) is one possibility. However, a number of pieces of evidence

165 Chap 1er 5 suggest that the increase in noradrenaline efflux induced by local inlusion of fluoxetine or citalopram is not secondary to an increase in the concentration of extracellular 5-HT.

1) Despite citalopram being a more potent inhibitor of 5-HT uptake, this drug was considerably less potent at increasing noradrenaline efflux than fluoxetine. 2) In contrast to fluoxetine, the increase in noradrenaline efflux caused by infusion of citalopram was still evident in DSP-4 lesioned rats, suggesting different sites of action.

3) Mimicking an increase in the concentration of extracellular 5-HT caused by infusion of an SSRI (by infusing 5-HT via the microdialysis probe) had no effect on noradrenaline efflux.

A second possible explanation for the lack of effect of fluoxetine after DSP-4 treatment is that the sites of noradrenaline uptake in DSP-4 treated rats are different to those in controls. Based on the assumption that fluoxetine increases noradrenaline efflux through inhibition of noradrenaline uptake, the following points could explain the lack of effect of fluoxetine after DSP-4.

Fluoxetine increases noradrenaline { efflux through inhibition of I- noradrenaline uptake (Chapters

0 40 80 120 160 200 240 280 320 3&4)

8 ; «> After DSP-4, fluoxetine does not increase i I " I noradrenaline efflux in vivo (Chapter 5) I ' 1 0 40 80 120 160 200 240 280

These findings suggest that noradrenaline uptake via the fluoxetine-sensitive site might be reduced after DSP-4.

166 Chapter 5

100

80 However in vitro, inhibition of [ fIfnoradrenaline s . i 60 40 uptake by fluoxetine into synaptosomes from DSP-4 1 g 20 treated rats is not reduced (Chapter 4)

lo g ,g [flu o x e tin e ]

This indicates that the fluoxetine site is functionally intact after DSP-4.

The disparity between the 2 measures {in vivo cf in vitro) of the fluoxetine effect after

DSP-4, might be due to changes in the extracellular conditions in vivo after DSP-4.

• Indeed, basal noradrenaline efflux was greater after DSP-4 treatment,

possibly due to increased noradrenaline release (Chapter 6).

This increased basal noradrenaline efflux could explain the apparent lack of effect of fluoxetine in vivo. Thus, when noradrenaline release is increased (after DSP-4), the relative importance of the fluoxetine site (w.r.t noradrenaline efflux) is diminished.

See Figure 5.8 for a schematic representation of this hypothetical explanation.

The results of the microdialysis experiments described in this chapter indicate that the antidepressant uptake blockers, desipramine and fluoxetine, could act at different sites of noradrenaline uptake. The results of the present dialysis experiments are consistent with there being at least 3 distinct noradrenaline uptake sites in the frontal cortex. These are: 1) a high affinity desipramine-sensitive site, 2) a low affinity desipramine-sensitive site, and 3) a fluoxetine sensitive noradrenaline uptake site. The findings of synaptosomal uptake studies (described in Chapters 3 & 4) together with the results of the present dialysis experiments indicate that after treatment with DSP-4, the high affinity desipramine-sensitive uptake site is lost, but a low affinity, high capacity, desipramine-sensitive site as well as a fluoxetine-sensitive site, remain.

167 Chapter 5

SALINE CONTROL DSP-4 TREATED

RELEASE RELEASE EFFLUX

-20 + 55 EFFLUX +90 UPTAKE - 30 UPTAKE

-10 FLU extracellular extracellular ?pqc9 HA s p a c e

Figure 5.8: Hypothetical mechanism for changes in noradrenaline uptake after DSP-4 Figures show the noradrenaline release into, and uptake from, the extracellular space. The square box is representative of the extracellular space. All numbers stated are arbitrary units of noradrenaline. Noradrenaline efflux is determined by release minus uptake. In saline-treated rats noradrenaline efflux = 30 [55 (release) - 25 (uptake)]. In DSP-4 treated rats noradrenaline efllux = 60 [90 (release) - 30 (uptake)].

There are 3 sites of noradrenaline uptake: 1) low affinity desipramine-sensitive site (LA), 2) high affinity desipramine-sensitive site (HA), and 3) fluoxetine-sensitive- site (FLU). In controls the LA site and the FLU site each transport 10 units of noradrenaline, and the HA site 5 units. After treatment with DSP-4, the HA site is abolished. The LA site transports more noradrenaline (20 units) due to the elevated level of noradrenaline efflux. Uptake via the FLU site remains the same (10 units).

Fluoxetine infusion blocks uptake via the FLU site. In saline-treated rats inhibition of the uptake of 10 units of noradrenaline represents a 33% increase in noradrenaline efflux, whereas in DSP-4 treated rats 10 units of noradrenaline represents only 16% noradrenaline efflux. Because a change in noradrenaline efflux of 16% is unlikely to be detected as a significant change from basal levels of efflux, after treatment with DSP-4 the increase in noradrenaline efflux induced by fluoxetine infusion is not evident.

168 Chapter 5

In summary, the present results show that the SSRIs fluoxetine and, to a lesser extent, citalopram increase extracellular noradrenaline when infused locally in the frontal cortex of freely-moving rats. Furthermore, the finding that the fluoxetine-, but not the desipramine- or citalopram-, induced increase in noradrenaline efflux was abolished after a DSP-4 lesion, suggests that these antidepressants modify the concentration of extracellular noradrenaline through different mechanisms, possibly involving different noradrenaline uptake sites.

169 Chapter 6

Chapter 6

The concentration of extracellular noradrenaline in rat frontal cortex after DSP-4 treatment

6.1 Introduction

The experiments described in Chapter 5 investigated the role of noradrenergic neurones in the increase in noradrenaline efflux induced by local infusion of antidepressants. Stores of noradrenaline were depleted by systemic injection of the neurotoxin, DSP-4 (40 mg / kg i.p.). It is generally accepted that a depletion of tissue noradrenaline content indicates a lesion of noradrenergic neurones. As discussed in detail in the General Introduction, the effects of treatment with DSP-4 are much more diverse than simply depleting transmitter stores. After treatment with DSP-4 there is a loss of staining with antibodies to DpH, a reduction in the number of presynaptic

Œi-adrenoceptors, an upregulation of p-adrenoceptors, a loss of [^Hjdesipramine and

[^H] nisoxetine binding sites as well as a reduction in the uptake of noradrenaline (see

Chapter 1, General Introduction & Chapter 6 for references).

170 Chapter 6

Although all these changes support the view that DSP-4 is a neurotoxin which

targets noradrenergic neurones in the brain, less is known about the impact of such a

lesion on noradrenergic transmission in vivo. This raises the question of how treatment with DSP-4 affects the extracellular concentration of noradrenaline in the brain?

Experiments described in Chapter 5 measured noradrenaline efflux in the frontal cortex of rats which had been treated with DSP-4, 5 days earlier. In the course of these experiments it became evident that the concentration of extracellular noradrenaline was increased nearly 2-fold after treatment with DSP-4. This finding was fascinating as the opposite is generally assumed (Daoust et al., 1990; Dudley et al., 1990; Wieczorek &

Romaniuk, 1994). The aim of the work described in this chapter was to determine whether the increase in basal noradrenaline efflux, after treatment with DSP-4, was due to a reduction in the uptake and / or an increase in the rate of noradrenaline release from surviving neurones. The experiments investigated the Ca^^-dependence of noradrenaline release and its sensitivity to depolarising pulses of K^. The results of the experiments involving the local infusion of desipramine, described in Chapter 5, have been reproduced in the present Chapter for ease of comparison, as they serve as a measure of the amount of noradrenaline uptake after treatment with DSP-4.

171 Chapter 6

6.2 Methods

6.2.1 DSP-4 lesion of noradrenergic neurones

Rats were treated with DSP-4 (40 mg / kg i.p.) as described in Methods (section

2.2.3.1). Control rats were injected with 0.9% saline vehicle (2 ml / kg).

6.2.2 Microdialysis in vivo

4 days after injection of DSP-4 or vehicle, microdialysis probes were implanted into the frontal cortex under halothane anaesthesia (see Methods 2.2.1). Rats were used for microdialysis experiments (freely-moving rats) the following day (i.e. 5 days after pretreatment). Each rat was used for one experiment only. Test compounds were administered locally via the microdialysis probe at a flow rate of 1 p,l / min, once four stable basal samples had been collected.

6.2.3 Monoamine content of frontal cortex

After completion of every experiment, rats were killed and a sample of the frontal cortex dissected from the side which was not used for microdialysis. This was to avoid detecting possible changes in monoamines due to the infused drug. These tissue samples were stored at -20°C for subsequent measurement of noradrenaline, 5-HT and dopamine content by HPLC-BCD (see Methods 2.2.4).

6.2.4 Changes in noradrenaline efflux induced by desipramine

Four basal samples of dialysate were collected in order to define spontaneous

(‘basal’) noradrenaline efflux. The perfusion medium was then changed to a modified

Ringer's (for composition see Methods 2.2.1.4.3) containing desipramine (5 pM).

172 Chapter 6

Thereafter, dialysis samples were collected every 20 min for 3 h and their noradrenaline content measured by HPLC-ECD (see Methods 2.2.1.5). Changes in noradrenaline efflux were compared in sham- and DSP-4 lesioned rats.

6.2.5 Ca^^-dependence of noradrenaline efflux

After collecting four basal dialysates, the normal perfusion medium was replaced with modified Ringer's from which CaCli had been omitted (KCl 4, NaCl 145 mM).

This Ca'^'^-free Ringer's was infused for 80 min after which the normal perfusion medium was reinstated. Subsequent noradrenaline efflux was monitored for a further

120 min. Noradrenaline efflux was compared in DSP-4 and sham-lesioned rats.

6.2.6 K^-sensitivity of noradrenaline efflux

Once stable basal noradrenaline efflux was established, the perfusion medium was changed to one containing an increased concentration of this modified Ringer’s solution comprised (mM): KCl 80, NaCl 71, CaCE 1.3. After 40 min, this was replaced with normal Ringer's. This cycle was repeated twice more with intervening recovery periods of 60 min. Experiments were performed in DSP-4 lesioned, as well as in sham- lesioned rats.

6.2.7 Statistical analysis

Statistics were performed on the raw data as well as on net efflux. Drug induced changes in the concentration of noradrenaline in cortical microdialysates were analysed by split-plot analysis of variance (ANOVA) as described in (Methods 2.2.1.6).

Data were split into bins of four consecutive samples with 'time' or 'bin' as the 'within- subjects' factors and 'lesion' as the 'between-subjects' factor. Where differences in

173 Chapter 6 noradrenaline efflux at particular time points were investigated {e.g. transient increase during high K"^) 1-way ANOVA on the pair of points of interest was performed.

Changes in tissue monoamine content were analysed using the Mann-Whitney U test.

6.3 Results

6.3.1 Cortical monoamine content

Treatment with DSP-4 caused a 75% reduction in the noradrenaline content of the frontal cortex when compared with that of sham-lesioned (saline-injected) rats

(?<0.001, Table 6.1). Neither the 5-HT (P=0.78) nor the dopamine (P=0.39) content of the cortex was affected by treatment with DSP-4.

Table 6.1. Concentrations of noradrenaline, dopamine and 5-HT in the frontal cortex of sham- or DSP-4 lesioned rats.

Data are pooled from all experiments and show mean ± SEM ng / g wet tissue weight with sample size in parentheses. * P<0.001 c/sham-lesioned control group.

Concentration (ng / g wet tissue weight)

oiiciiii ju/vji —r noradrenaline 854.7 ± 118.6 (9) 189.7 ±22.4 (20) * dopamine 24.4 ±7.0 (9) 65.4 ±17.5 (20) 5-HT 187.1 ±39.5 (9) 176.0 ±25.5 (20)

174 Chapter 6

6.3.2 Desipramine-induced increase in noradrenaline efflux in DSP-4 or sham- lesioned rats

Basal noradrenaline efflux was almost 2-fold greater in DSP-4 pretreated rats than in the sham-lesioned controls (sham: 27.2 ± 0.5 fmol / 20 min (n=5); DSP-4: 50.2

± 1.8 fmol / 20 min (n=6); F|,13=14.97; P=0.02) (Figure 6.1a).

Infusion of 5 pM desipramine caused a statistically significant increase in noradrenaline efflux in the frontal cortex of both DSP-4 lesioned (F 2,22=6.02; P=0.008) and sham-lesioned rats (F 2,27=5.57; P=0.006). Because of the difference in basal efflux in the two treatment groups, 'net efflux' was calculated (see Chapter 5 section 5.2.4;

Figure 6.1b). Statistics performed on this data showed that net noradrenaline efflux, during the 2"^ and 3"^^ h of desipramine infusion, was greater in the DSP-4 lesioned group than that in sham controls (Fi,i2=5.92; P=0.03).

6.3.3 The dependence of noradrenaline efflux on extracellular Ca^^

Noradrenaline efflux was again significantly greater in basal samples collected from DSP-4 lesioned rats than sham-lesioned controls (sham: 26.3 ± 2.5 fmol / 20 min

(n=4); DSP-4: 42.8 ± 7.3 fmol / 20 min (n=5); Fi,g=5.41; P<0.05) (Figure 6.2). In both groups, noradrenaline efflux was reduced to approximately 10 fmol / 20 min within

80 min of Ca"^"^ being excluded from the perfusate. This decrease was statistically significant in both groups (sham: Fi,6=12.8; P=0.01; DSP-4: Fi,g=10.7; P=0.01). The reduction in net efflux after 80 min of Ca'*”^'free Ringer’s was significantly greater in

DSP-4 treated rats than in saline controls (Fij=15.3; P=0.006), indicating that a greater amount of noradrenaline efflux was Ca'^'^-dependent in DSP-4 treated rats.

175 Chapter 6

(a) 175 O CM 150

125

X 100 0) 0) 75 c Id 50 § % 25 o c 0 0 40 80 120 160 200 240 280

tim e (min) sham DSP-4 (b) E 120 o CM - 100 o E

X =3 0) CD c 40 (d c 2 20 TD Cd cO 0) c 0 40 80 120 160 200 240 280

tim e (min)

Figure 6.1. Ejfects of local infusion of desipramine (5 flM) on noradrenaline efflux in the frontal cortex

Graphs show the effect of desipramine in sham- (n=5) and DSP-4 lesioned (n=9) rats. Horizontal bar indicates 3 h duration of drug infusion. Data are expressed as mean

± SEM noradrenaline efflux (fmol / 20 min). * P<0.05 cf sham-lesioned control.

Results show (a) absolute levels of noradrenaline efflux and (b) net noradrenaline efflux.

176 Chapter 6

(a)

o CvJ 5 0

3 0

20

10

0

0 40 80 120 160 200 240 280 time (min) (b) sham D S P - 4 I 20 o in o E

X ë -10 ■ CD (D Ç -2 0 - 03 C CD - 3 0 - E g - 4 0 - Q) C

0 4 0 80 120 160 200 240 280 time (min)

Figure 6.2. The dependence of noradrenaline efflux in the frontal cortex on extracellular

Effects were compared in sham-lesioned (n=4) and DSP-4 lesioned rats (n=5).

Horizontal bar indicates 80 min infusion of Ca^^-free Ringer's. Data expressed as mean

± SEM noradrenaline efflux (fmol / 20 min). *?<0.05 cf sham-lesioned control.

Results show (a) absolute levels of noradrenaline eftlux and (b) net noradrenaline efflux.

177 Chapter 6

6.3.4 Depolarisation-evoked noradrenaline release

In this experimental group basal noradrenaline efflux in DSP-4 treated rats was once again greater than that in sham-lesioned controls (sham: 32.2 ± 4.4 fmol / 20 min

(n=5); DSP-4: 52.9 ± 4.9 fmol / 20 min (n=4); F ,.27=29.8; P<0.01) (Figure 6.3a).

Because of this difference, net efflux of noradrenaline was calculated. In both DSP-4 and sham-lesioned rats, all three depolarising pulses of caused a rapid increase in noradrenaline efflux (Figure 6.3b).

The increase caused by the first pulse in the DSP-4 lesioned rats was considerably greater than that in the sham controls (F|.g=7.3; P=0.03). The second and third pulses caused the same increase in net noradrenaline efflux in both groups

(Fi.6=0.03; P=0.87 and Fi.6=0.82; P=0.40, respectively). Also, in the DSP-4 lesioned group, the increase in noradrenaline efflux induced by the first pulse

(-270 fmol / 20 min) was significantly greater than that due to the 2"^ (F|.6=24.1;

P=0.003) and 3"^^ (Fi.4=9.91; P=0.04) pulses (-120 fmol / 20 min;).

178 Chapter 6

(a) 400 c E 350 o CM 300 O E 250 3 200 9= CD CD 150 c c 100 E •a 50 2 o c 0

0 40 80 120 160 200 240 280 320 360 400

time (min)

(b) c 350 sham E DSP-4 o 300 CM Ô 250 I, 200 3X 150

CD C 100 £= E 50 E 0 co CD -50 c 0 40 80 120 160 200 240 280 320 360 400 time (min)

Figure 6.3: The ejfects on noradrenaline efflux of repeated pulses of 80 mM fC .

Experiments compared the effects of 3 consecutive pulses of 80 mM in sham-lesioned controls (n=5) and DSP-4 lesioned rats (n=4). Horizontal bar indicates 40 min periods of infusion of high-K"^ Ringer's, at all other times normal Ringer's was

infused. Data are expressed as mean ± SEM noradrenaline efflux (fmol / 20 min). *

P<0.05 cf sham-lesioned control. Results show (a) absolute levels of noradrenaline efflux, and (b) net noradrenaline efflux.

179 Chapter 6

6.4 Discussion

The noradrenaline content of the frontal cortex was reduced by approximately

75% in rats which had been treated with DSP-4 5 days previously. This depletion suggests an extensive, albeit not total, lesion of noradrenergic neurones in this brain region. This is in keeping with the degree of transmitter depletion reported by other groups {e.g. Ross, 1976; Bennett et al., 1984; Dooley et al., 1983). The 5-HT content of cortical tissue was not significantly affected after treatment with DSP-4. Although there was a substantial increase in tissue dopamine content, this was not statistically significant. An increase in cortical dopamine content has been reported in other studies using noradrenergic neurotoxins (Harik, 1984; Reader & Brière, 1983). The cause of this effect is unclear but the accumulation of dopamine due to reduced DpH activity is a possible explanation.

The degree of noradrenaline depletion in the present set of experiments and the experiments described in Chapter 5 (-70%) is somewhat greater than that observed in

Chapter 4. This may be due to the fact that in Chapter 4, where brains were used for the preparation of synaptosomes, a sample of tissue from chopped whole of the cortex was used. In experiments where microdialysis was used, the sample of tissue used for verification of the lesion was taken from the frontal cortex only, as this was the brain region which was being studied. The mean noradrenaline content of tissue samples from the frontal cortex was significantly greater than that of samples from the cerebral cortex (740 ± 326.8 (14) and 412.4 ± 36.0 (22) ng / g tissue, respectively; P=0.004). The difference between frontal and whole cerebral cortex noradrenaline levels may reflect a difference in the noradrenergic innervation of the frontal cortex. As it is generally

180 Chapter 6

accepted that DSP-4 is taken up into neurones via a desipramine-sensitive uptake site, it

is possible that the amount of DSP-4 accumulated, and therefore the apparent toxicity of

DSP-4 will vary between different brain regions according to the characteristics of

noradrenaline uptake.

A number of groups have shown that the neurotoxic effects of DSP-4 can be prevented by pretreatment of rats with desipramine prior to injection of DSP-4 (Zieher

& Jaim-Etcheverry, 1980; Jonsson et a l, 1981). Furthermore, DSP-4 treatment was shown to diminish high, but not low, affinity desipramine-sensitive [^H]noradrenaline uptake, suggesting that DSP-4 could target only high affinity desipramine-sensitive noradrenaline uptake sites (Chapter 4).

Zaczek and colleagues (1990) have shown that the selectivity of DSP-4 for locus coeruleus neurones over noradrenergic neurones from the lateral tegmental system may well reside in differences in noradrenaline uptake in different brain regions. This group showed that noradrenaline uptake in the hypothalamus was quite different from noradrenaline uptake in the cortex. Since DSP-4 is taken up by a noradrenaline uptake site it can be used as a competitive inhibitor of noradrenaline uptake in vitro. Uptake of

[^H]noradrenaline into synaptosomes prepared from the hypothalamus was less sensitive to inhibition by DSP-4 in vitro than uptake into those prepared from the cortex

(Ki 460 nM & 180 nM, respectively). These findings suggest that the neurotoxicity of

DSP-4 is determined by its uptake via desipramine-sensitive noradrenaline uptake sites.

If the proportion of neurones in the frontal cortex with high affinity desipramine- sensitive noradrenaline uptake sites is greater, or if neurones of the frontal cortex have a

181 Chapter 6 higher affinity for noradrenaline uptake, this might explain why the extent of the lesion caused by DSP-4 was greater in the frontal cortex than in whole cerebral cortex.

Despite the reduction in tissue noradrenaline content, the concentration of extracellular noradrenaline was nearly 2-fold (20 fmol / 20 min) greater in the lesioned rats, compared with sham-lesioned controls. Kask and colleagues (1997) did not detect a change in basal noradrenaline efflux in anaesthetised rats after a DSP-4 lesion.

However, this discrepancy could be attributed to the state of consciousness of the rats, or, perhaps more importantly, the inclusion of an uptake blocker (10 p,M nomifensine) in their perfusion medium. It is difficult to predict how either of these factors might affect spontaneous noradrenaline release after a lesion. In another study using the neurotoxin, 6-OHDA, a lesion of noradrenergic neurones in the hippocampus was not found to produce a reduction in the extracellular concentration of noradrenaline

(Abercrombie & Zigmond, 1989).

An increase in the concentration of extracellular noradrenaline after a DSP-4 lesion could be explained by either an increase in the amount of noradrenaline release from, and / or a decrease in the amount of noradrenaline uptake by, surviving neurones.

Because, in the present experiments, the increase in noradrenaline efflux during infusion of desipramine was greater in DSP-4 treated rats than in controls, this indicates that uptake of noradrenaline by neurones which survive the DSP-4 pretreatment is functionally intact.

This finding would seem to be at variance with results from experiments looking at synaptosomal uptake of [^H]noradrenaline in vitro. These experiments showed that

182 Chapter 6 the proportion of [^H]noradrenaline uptake inhibited by low, but not higher, concentrations of desipramine was reduced after DSP-4 treatment (see Chapter 4 section

4.3.3.3). This supports evidence that there could be two components of desipramine- sensitive uptake of noradrenaline (Michel et a l, 1984) and only one, a high affinity component, is affected by a lesion of noradrenergic neurones. It is likely, therefore, that the desipramine-induced increase in noradrenaline efflux, seen in the present experiments after DSP-4 treatment, is due to inhibition of noradrenaline uptake via the low affinity uptake site. Since microdialysis showed that the desipramine-induced increase in noradrenaline efflux was greater in lesioned rats than in the sham controls, this low affinity site evidently has a large capacity for noradrenaline uptake.

An increase in noradrenaline uptake however, will not cause an increase in its extracellular concentration, but rather a reduction. Because noradrenaline efflux is determined by the net effects of uptake and release, treatment with DSP-4 must also affect noradrenaline release. An elevation of the concentration of extracellular noradrenaline can only be explained if the increase in noradrenaline uptake is accompanied by an even greater increase in the amount of noradrenaline released from surviving neurones. This possibility was investigated by measuring the amount of noradrenaline released in response to a depolarising pulse of K^.

Approximately 75% of spontaneous noradrenaline efflux measured in the lesioned rats was dependent on the presence of extracellular Ca"^^. This suggests that extracellular noradrenaline in lesioned rats, as in saline-injected rats, is largely derived from Ca'^'^-dependent exocytotic release of transmitter from surviving neurones. The possibility that release rate is increased after DSP-4 treatment is supported by the effects

183 Chapter 6 of a depolarising pulse of on noradrenaline efflux; the increase in efflux in the

DSP-4 pretreated group was more than twice that caused by the same challenge in sham control rats. Interestingly, on repeating the K'^-challenge, the exaggerated noradrenaline response was no longer evident: i.e. the increase in efflux was the same as in the controls. It appears that neurones which survive the DSP-4 treatment cannot sustain a high rate of stimulated transmitter release.

An increase in noradrenaline release after a DSP-4 lesion is consistent with results from two early studies. These showed that noradrenaline turnover (measured either by monoamine oxidase inhibition or the ratio of the MHPG-SO 4 to noradrenaline concentrations) was increased, but only in brain regions where tissue noradrenaline content was greatly depleted (cortex and hippocampus: Hallman & Jonsson, 1984;

Logue et al, 1985). Also, Chiodo et al. (1983) found a 4-foId increase in the firing rate of locus coeruleus noradrenergic neurones in vivo in brain regions which survived a

6 -OHDA lesion.

The reason for the increased rate of release is undetermined. It is possible that release rate is increased as a consequence of the loss of regulation by autoreceptors after

DSP-4 treatment (reduction in B^ax of presynaptic ai-adrenoceptors: Heal et a l, 1993).

Alternatively, release of transmitter could be affected at the level of synaptic vesicles.

Treatment with DSP-4 might disrupt the storage, transport or supply etc. of synaptic vesicles within the presynaptic nerve terminal.

To summarise, these experiments suggest that an extensive, yet incomplete lesion of noradrenergic neurones, induced by DSP-4 treatment, causes a paradoxical

184 Chapter 6 increase in the concentration of extracellular noradrenaline in the rat frontal cortex. It seems that both uptake and release of noradrenaline are increased in the lesioned rats.

However, an imbalance in these changes, possibly arising from loss of the high affinity desipramine-sensitive noradrenaline uptake site, means that uptake cannot keep pace with release, thus resulting in an increased level of extracellular noradrenaline. These results challenge the widely held assumption that a DSP-4 lesion of central noradrenergic neurones invariably reduces noradrenergic transmission in the brain. The conclusions of behavioural studies which assume that the effects of drugs which affect behaviour after treatment with DSP-4 cannot involve changes in noradrenergic transmission should perhaps be reconsidered {e.g. anti immobility effect of desipramine:

Esposito et al., 1987).

185 Chapter 7

Chapter 7

General Discussion

Experiments presented in this thesis have investigated the effects of the SSRIs, fluoxetine and citalopram, on noradrenaline efflux in vivo and noradrenaline uptake in vitro in rat cortex. Using microdialysis in vivo it was shown that fluoxetine and citalopram, like the noradrenaline uptake blocker, desipramine, increase noradrenaline efflux when they are infused locally in the frontal cortex of freely-moving rats.

Noradrenaline efflux is an index of the concentration of extracellular noradrenaline and is determined by the balance between noradrenaline release and reuptake. An increase in noradrenaline efflux can be caused by an increase in release or a reduction in uptake of noradrenaline (and vice versa for a reduction in efflux). The

SSRIs, fluoxetine and citalopram, and the tricyclic antidepressant, desipramine which increased noradrenaline efflux also inhibited the uptake of [^Hjnoradrenaline into rat

186 Chapter 7 cortical synaptosomes in vitro. It is likely, therefore, that inhibition of noradrenaline uptake by these drugs contributes to the increase in noradrenaline efflux in vivo.

The finding that fluoxetine, and to a lesser extent, citalopram, increase noradrenaline efflux and inhibit [^H]noradrenaline uptake was interesting as these drugs are considered to selectively inhibit the uptake of 5-HT. It is conceded that the effects of citalopram (~50 |iM) and fluoxetine (~5 pM) on the noradrenergic system occurred at concentrations higher than their K\ s for inhibition of 5-HT reuptake in vitro (2.6 &

25 nM respectively; Thomas et al., 1987). However, the concentrations at which the

SSRIs affected noradrenergic transmission were within a physiologically relevant range.

As mentioned in earlier chapters, the extracellular concentration of fluoxetine after a single anticonvulsant dose (45 mg / kg) has been measured using microdialysis to be ~2-

3 pM (Dailey et a l, 1992). Other groups have also measured the brain concentration of fluoxetine to be -10 pM (Goodnough & Baker, 1994; see Chapter 3, Discussion). More recently, the plasma concentration of fluoxetine in depressed patients being treated with this drug has been reported to be between 0.2-1.5 pM (see Baumann, 1996). These findings suggest that the SSRIs, fluoxetine and citalopram at least, are merely preferential inhibitors of 5-HT uptake, which are unlikely to affect 5-HT uptake exclusively when these drugs are used clinically for the treatment of depressive illness.

It is also possible that the SSRI-induced increase in noradrenaline efflux could reflect 5-HT-stimulated noradrenaline release. Interactions between the noradrenergic and serotonergic systems have been demonstrated extensively (see General Introduction section 1.8). Briefly, activation of either 5-HT2 (a), 5-H Tia/ib or 5-HT] receptors

187 Chapter 7 increases noradrenaline release {e.g. Clement et al., 1992; Done & Sharp, 1994;

Mongeau et al., 1994). As discussed in Chapter 5 however, the SSRI-induced increase in noradrenaline efflux does not appear to be secondary to an increase in extracellular

5-HT. This is because: 1) citalopram was approximately 10-fold less potent at increasing noradrenaline efflux than fluoxetine despite being ~ 10-fold more potent at inhibiting 5-HT uptake than fluoxetine in vitro (Thomas et al, 1987). 2) The effect {fj fluoxetine, but not that of citalopram, was diminished by treatment with DSP-4, suggesting that these drugs are acting through different mechanisms. 3) Local infusion of 5-HT did not affect noradrenaline efflux in the frontal cortex. 4) Fluoxetine, and albeit at higher concentrations, citalopram inhibited [^H]noradrenaline uptake into rat cortical synaptosomes; this cannot be explained by an increase in the concentration of

5-HT, as 5-HT will not accumulate in the synaptosomal preparation. There was also a good parallelism between the potency of antidepressants to inhibit [ 'HJnoradrenaline uptake and increase noradrenaline efflux, suggesting that the former could contribute to the latter.

The use of neurotoxins selective for particular monoaminergic neurones served to determine whether noradrenaline is taken up by noradrenergic and/or serotonergic neurones and how inhibition of this uptake might contribute to the effects of SSRIs or desipramine on noradrenergic function. As the depletion of 5-HT stores after treatment with 5,7-DHT did not affect the inhibition of synaptosomal ["'H]noradrenaline uptake by fluoxetine or citalopram, the possibility that these SSRIs were inhibiting noradrenaline uptake through an action at a site on serotonergic neurones was considered unlikely.

Uptake of noradrenaline via a site on serotonergic neurones could not be mled out with complete certainty as the lesion of serotonergic neurones was only partial. It could be

188 Chapter 7

that surviving serotonergic neurones are capable of compensating for the lesion such

that a change in the inhibition of [^H]noradrenaline uptake is not detected. Evidence that

the 5-HT transporter is unable to transport noradrenaline (Paczkowski et al, 1996)

suggests that the effects of fluoxetine or citalopram on ['^H]noradrenaline uptake are not

explained by inhibition of sites on serotonergic neurones.

Desipramine was shown to inhibit [^H]noradrenaline uptake in the cortex via

two sites distinguished by their sensitivity to inhibition by this drug. Michel and co­

workers (1984) suggested that [^H] noradrenaline uptake inhibited by high concentrations of desipramine represented uptake into dopaminergic neurones. In this thesis, the involvement of noradrenergic neurones was investigated by causing a

noradrenergic lesion using DSP-4. In uptake experiments, pretreatment of rats with

DSP-4 diminished uptake via the high, but not the low, affinity desipramine-sensitive site. This supports Michel and colleagues’ (1984) suggestion that noradrenaline might be taken up by sites not located on noradrenergic neurones. This finding is also consistent with DSP-4 being transported into neurones via a desipramine-sensitive uptake site suggested by the finding that the toxic effects of DSP-4 can be prevented by pretreatment with desipramine (Zieher & Jaim-Etcheverry, 1980; Jonsson et a l, 1981).

A difference in noradrenaline uptake sites could well be a reason underlying the selectivity of DSP-4 for locus coeruleus neurones (rather than neurones from the lateral tegmental system). Zaczek and co-workers (1990) demonstrated that competitive inhibition of [^H]noradrenaline uptake by DSP-4 in vitro was different in brain regions innervated by the locus coeruleus from those innervated by the lateral tegmental system

(Æi hypothlamus: 460 nM; cortex: 180 nM). This suggests that the neurotoxicity of

189 Chapter 7

DSP-4 could be determined by the characteristics of noradrenaline uptake sites. If low and high affinity desipramine-sensitive noradrenaline uptake sites are present on separate neurones, or if some neurones have more high affinity desipramine-sensitive sites than others, this might explain the selectivity of DSP-4 for particular noradrenergic neurones. This might also account for the difference in the extent of the depletion of noradrenaline seen in the frontal cortex compared to whole cortex. Neurones with few or no high affinity desipramine-sensitive noradrenaline uptake sites, or low affinity for noradrenaline uptake would not accumulate enough DSP-4 to cause degeneration, and would survive treatment with DSP-4.

The finding that inhibition of [^H]noradrenaline uptake by fluoxetine was not reduced in synaptosomes prepared from DSP-4 pretreated rats, suggested that the sites of noradrenaline uptake inhibited by fluoxetine were not on neurones targeted by DSP-

4. The possibility that fluoxetine might inhibit noradrenaline uptake via more than one site comes from the results of two separate experiments. 1) The Hill coefficient for the inhibition of [^H]noradrenaline uptake by fluoxetine (in untreated rats) was 1.5 (a Hill coefficient of 1 indicates a single site of action). 2) Inhibition of [^H]noradrenaline uptake by 0.5 |iM fluoxetine, but not 5 or 50 |xM fluoxetine, was greater in synaptosomes prepared from DSP-4 pretreated rat cortex than in controls. This could suggest that the site of noradrenaline uptake inhibited by 0.5 |xM fluoxetine assumed greater importance after a noradrenergic lesion due to a greater proportion of noradrenaline being taken up by this site.

The results of dialysis experiments performed in DSP-4 treated freely-moving rats provide more information about the sites of noradrenaline uptake inhibited by

190 Chapter 7 fluoxetine and desipramine. The reduced affinity of low concentrations of desipramine for inhibiting ['^H]noradrenaline uptake in vitro suggested that uptake via the high affinity desipramine-sensitive site is diminished after DSP-4. However, as the effect of desipramine on noradrenaline efflux was greater after treatment with DSP-4 (Chapter 5), it is suggested that this is due to inhibition of the low affinity desipramine-sensitive uptake site.

A shift in uptake to a site with lower affinity for noradrenaline means that the

'threshold' for uptake will be higher. This is borne out by microdialysis experiments in vivo where the concentration of extracellular noradrenaline was elevated after treatment with DSP-4.

The increase in noradrenaline efflux induced by fluoxetine infusion in saline pretreated rats was not evident after treatment with DSP-4. This finding on its own might suggest that fluoxetine inhibits noradrenaline uptake via a site on neurones targeted by DSP-4, such as the high affinity desipramine-sensitive noradrenaline uptake site. The results of synaptosomal uptake experiments however, show that the fluoxetine-sensitive uptake site transports noradrenaline, even after treatment with

DSP-4. Taken together, these two results suggest that the lack of effect of fluoxetine in vivo, after DSP-4, might be explained by a reduction in the proportion of released noradrenaline taken up via a fluoxetine-sensitive site. Relative to the amount of noradrenaline transported by the low affinity desipramine-sensitive site, transport via the fluoxetine site is negligible, hence when it is inhibited {i.e. by infusion of fluoxetine) no increase in efflux is detected. If the fluoxetine site is blocked, excess noradrenaline will

191 Chapter 7

be ‘mopped up’ by the low affinity, large capacity, desipramine-sensitive site (see

Figure 5.9 for schematic representation of this hypothesis).

The site of action of citalopram is difficult to determine as the effects of this drug were somewhat smaller than those of either fluoxetine or desipramine. The site of noradrenaline uptake sensitive to citalopram does not appear to be on noradrenergic neurones, as pretreatment with DSP-4 did not affect the action of citalopram either in vitro or in vivo (Chapters 4 & 5). Whereas the fluoxetine-induced increase in

noradrenaline efflux was not evident after DSP-4, the increase in efflux due to citalopram was unaltered. This difference indicates that it is unlikely that the SSRIs, fluoxetine and citalopram, act through the same mechanism.

Results presented in this thesis indicate that there are at least three sites of noradrenaline uptake in the rat frontal cortex: 1) a high affinity desipramine-sensitive site, 2) a low affinity desipramine-sensitive site, and 3) a fluoxetine-sensitive site.

These have been identified by a combination of the results of uptake experiments in vitro, and dialysis experiments in vivo, after treatment with DSP-4.

The final experimental chapter of this thesis (Chapter 6) investigated the effect of a noradrenergic lesion on the extracellular concentration of noradrenaline in vivo.

Experiments were designed to determine whether the, nearly 2-fold, increase in noradrenaline efflux, after treatment with DSP-4, was due to an increase in noradrenaline release or a reduction in noradrenaline uptake. The finding that infusion of desipramine, via the microdialysis probe, caused a greater increase in noradrenaline

192 Chapter 7

efflux in DSP-4 pretreated rats than in saline pretreated controls indicated that, after

DSP-4, the total amount of desipramine-sensitive noradrenaline uptake was increased.

An increase in noradrenaline uptake, however, will not cause an increase in the concentration of extracellular noradrenaline, therefore noradrenaline release must also

be increased. An increase in depolarisation evoked noradrenaline release was demonstrated by the greater (c/saline controls) increase in noradrenaline efflux induced by infusion of a depolarising pulse of K"^. Extracellular noradrenaline in the frontal cortex was shown, by infusion of Ca'^^-free Ringer’s, to derive from Ca^'^-dependent exocytotic release. These findings suggested that, after treatment with DSP-4, the extracellular concentration of noradrenaline is increased due to uptake being predominantly via a low affinity site and an increase in noradrenaline release. Whether

DSP-4 exposes a population of neurones with a high rate of noradrenaline release, or whether treatment with DSP-4 causes noradrenaline release from surviving neurones to be increased {e.g. by alkylating Œz-autoreceptors or by affecting the synaptic transmitter release process) remains to be determined.

The increase in the concentration of extracellular noradrenaline after treatment with DSP-4 challenges the widely held assumption that a depletion of noradrenaline stores is invariably paralleled by a reduction in noradrenergic transmission {e.g. Daoust et al., 1990; Dudley et al., 1990). The conclusions of experiments performed after treatment with noradrenergic neurotoxins like DSP-4 which assume that there is no

noradrenaline release or uptake after a lesion should be reconsidered. An example of a behavioural study which falls into this category is that of Esposito and colleagues

(1987). This study reported that the antiimmobility effects of desipramine in the forced

193 Chapter 7 swim test were still evident after DSP-4 treatment. It was assumed that this indicated that the antiimmobility effect of desipramine was not due to its inhibition of noradrenaline uptake. Experiments discussed in this thesis have demonstrated clearly that desipramine does still increase the extracellular concentration of noradrenaline even after an extensive noradrenergic lesion.

The findings of experiments described in this thesis have implicated a substantial role of sites which are resistant to DSP-4 in the clearance of released noradrenaline. As little as 20% of total specific [^H]noradrenaline was inhibited by low (nanomolar) concentrations of desipramine. Only noradrenaline uptake inhibited with high affinity by desipramine is thought to represent uptake into noradrenergic neurones (Michel et a l, 1984). The remainder, and majority, of noradrenaline uptake could be into non­ neuronal elements or dopaminergic neurones (Iversen, 1965; Michel et a l, 1984; Russ et al, 1996). The latter is supported by studies of the cloned noradrenaline and dopamine transporters. As discussed in the General Introduction (section 1.5.4) a number of groups have demonstrated that the dopamine transporter has a similar affinity for noradrenaline uptake as it does for dopamine uptake (Giros et a l, 1994; Buck &

Amara, 1995; Pifl e ta i, 1996).

In summary, work presented in this thesis has revealed a number of interesting features about noradrenergic transmission in the cortex. The majority of noradrenaline in the cortex does not appear to be taken up by sites on noradrenergic neurones. The selective serotonin reuptake inhibitors, fluoxetine, and to a lesser extent, citalopram have profound effects on noradrenaline uptake in vitro and the concentration of extracellular noradrenaline in vivo. This is thought to be due to direct inhibition of

194 Chapter 7

noradrenaline uptake by these drugs. Their action is unlikely to be mediated by 5-HT

neurones although the precise location of their site(s) of action remain(s) to be determined. After treatment with the noradrenergic neurotoxin, DSP-4, the concentration of extracellular noradrenaline in the frontal cortex is increased nearly 2- fold. This increase appears to be due to a shift in noradrenaline uptake to a low affinity site as well as an increase in noradrenaline release.

The results discussed in this thesis raise several questions, including:

Why is the majority of noradrenaline released in the cortex taken up by uptake

sites not on noradrenergic neurones?

Why is a transporter which transports noradrenaline more efficiently than

dopamine a dopamine transporter?

How selective does a drug have to be in order for it to be classified as an SSRI?

Perhaps the term ‘preferential serotonin reuptake inhibitor’ would be a more

accurate description. If the therapeutic effects of fluoxetine cannot be attributed

exclusively to inhibition of 5-HT uptake, then selective serotonergic

antidepressants may not be the way forward.

195 Chapter 7

Future work related to this project will include further investigation of the following points.

Experiments in this thesis have shown that the fluoxetine-induced increase in noradrenaline efflux is unlikely to be secondary to an increase in the concentration of extracellular 5-HT. However, it is possible that the smaller, citalopram-induced, increase in noradrenaline efflux could be due to inhibition of 5-HT uptake by this drug. The contribution of 5-HT mediated noradrenaline release to the increase in noradrenaline efflux caused by citalopram should be investigated. This could be achieved by pretreating rats with antagonists of 5-HT receptors whose activation has been shown to increase noradrenaline release (i.e. 5 -H T ia/ib, 5-HT]A and 5 -HT3 , see

General Introduction section 1.8). If the increase in noradrenaline efflux were due to a 5-HT receptor-mediated effect, pretreatment with antagonists of these receptors should prevent the increase in noradrenaline efflux during citalopram infusion.

Identification of the fluoxetine-sensitive noradrenaline uptake sites would be beneficial. Because fluoxetine is an effective antidepressant, exploitation of its site of action may lead to the development of more effective antidepressants. To test whether fluoxetine could be acting at the noradrenaline transporter (like low concentrations of desipramine), the effects of a combination of desipramine and fluoxetine on noradrenaline efflux could be investigated. If the two drugs act at different sites their effects would be additive.

196 Chapter 7

Because of the clinical use of these drugs and the delay before the therapeutic effects of antidepressants become apparent, the effects of chronic (systemic) treatment with fluoxetine are of interest.

Another important point to consider is the cause of the increase in noradrenaline release after treatment with DSP-4. The possibility that this is due to loss of autoreceptor control of noradrenaline release could be tested. Systemic injection of an a%-antagonist, such as idazoxan or RX 821002 would increase noradrenaline efflux and conversely, an az-agonist, such as clonidine, would be expected to reduce efflux if noradrenaline release was under autoreceptor control.

Finally, it would be interesting to determine how a lesion of noradrenergic neurones with DSP-4 affects rats' behaviour in models of depression. It would be useful to see whether a noradrenergic lesion alters the effects of fluoxetine or desipramine on animals' behaviour in the social interaction test as this might throw light upon the mechanism of action of these drugs as well as the role of noradrenaline in depression.

197 References

Abercrombie E.D. & Zigmond MJ. (1989) Partial injury to central noradrenergic neurons: reduction of tissue norepinephrine content is greater than reduction of extracellular norepinephrine measured by microdialysis. J. Neurosci. 9: 4062-4067 Abercrombie E.D., Keller R.W. & Zigmond M.(1988) Characterization of hippocampal norepinephrine release as measured by microdialysis perfusion: pharmacological and behavioural studies. Neurosci. 27: 897-904 Aghajanian G.K. & Bloom F.E. (1967) Electron microscopic localization of tritiated norepinephrine in rat brain. J. Pharmacol. Exp. Ther. 156: 407-416 Amara S.G. & Kuhar M.J. (1993) Neurotransmitter transporters: recent progress. Anna. Rev. Neurosci. 16: 73-93 Axelrod J., Weil-Malherbe H. & Tomchick R. (1959) The physiological disposition of H^-epinephrine and its metabolite metanephrine. J. Pharmacol. Exp. Ther. 127: 251-256 Azmitia E C. & Segal M. (1978) An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Comp. Neurol. 179: 641-667 Backstrom. I.T., Ross S B. & Marcusson J.O.(1989) [^HjDesipramine binding to rat brain tissue: binding to both noradrenaline uptake sites and sites not related to noradrenaline neurones. J. Neurochem. 52: 1099-1106 Baraban J.M. & Aghajanian G.K (1980a) Suppression of serotonergic neuronal firing by a-adrenoceptor antagonists: evidence against GAB A mediation. Eur. J. Pharmacol. 66; 287-294 Baraban J.M. & Aghajanian G.K (1980b) Suppression of firing activity of 5-HT neurons in the dorsal raphe by a-adrenoceptor antagonists. Neuropharmacol. 19: 355-363 Baraban J.M. & Aghajanian G.K (1981) Noradrenergic innervation of serotonergic neurons in the dorsal raphe: demonstration by electron microscopic autoradiography. Brain Res. 204: 1-11

198 Baumann P. (1996) Pharmacokinetic-pharmacodynamic relationship of the selective serotonin reuptake inhbitors. Clin. Pharmacokinet. 31: 444-469 Baumgarten H.G., Bjorklund A, Nobin A., Rosengren B. & Schlossberger H.G. (1975) Neurotoxicity of hydroxylated : strucure-activity relationships: 1. Long­ term effects on monoamine content and fluorescence morphology of central monoamine neurones. Acta. Physiol. Scand. (Suppl.) 429: 7-25 Baumgarten H.G., Bjorklund A., Lachenmayer, L. & Nobin A. (1973b) The evaluation of the effects of 5,7-dihydroxytryptamine on serotonin catecholamine neurons in the rat CNS. Acta. Physiol. Scand. (Suppl.) 391: 3-19 Baumgarten H.G., Victor S.J. & Lovenberg W. (1973a) Effect of intraventricular injection of 5,7-dihydroxytryptamine on regional tryptophan hydroxylase of rat brain. J. Neurochem. 21: 251-253 Bech P. (1990) A meta-analysis of the antidepressant properties of serotonin reuptake inhibitors. Int. Rev. Psychiatry 2: 209-211 Bennett C., Hock P. & McGaugh J.L. (1984) Dose and time dependence of noradrenergic depletion after V-(2-chloroethyl)-V-ethyl-2-bromobenzylamine. IRCS Med. Sci. 12: 181-182 Bito L., Davson H., Levin P., Murray M. & Snider N. (1966) The concentrations of free- amino acids and other electrolytes in cerebrospinal fluid, in vivo dialysates of brain, and blood plasma of the dog. J. Neurochem. 13: 1057-1067 Bjorklund A., Baumgarten H.G. & Rensch A. (1975) 5,7-dihydroxytryptamine: improvement of its selctivity for serotonin neurones in the CNS by pretreatment with desipramine. J. Neurochem. 24: 833-835 Blakely R.D., Berson H.P., Fremeau R.T., Caron M.G., Peek M.M., Prince H.K. & Bradley C.C (1991) Cloning and expression of a functional serotonin transporter from rat brain. Nature 354: 66-70 Blandina P., Goldfarb J., Walcott J. & Green J.P. (1991) Serotonergic modulation of the release of endogenous norepinephrine from rat hypothalamic slices. J. Pharmacol. Exp. Ther. 256: 341-347 Bloom P.P., Algeri S., Groppetti A., Revuelta A. & Costa P. (1969) Lesions of central norepinephrine terminals 6-hydroxydopamine: biochemistry and fine structure. Science 166: 1284-1286

199 Bolden-Watson C. & Richelson E. (1993) Blockade by newly-developed antidepressants of biogenic amine uptake into rat brain synaptosomes. Life Set. 52: 1023-1029 Borowsky B. & Hoffman B.J. (1995) Neurotransmitter transporters: molecular biology, function, and regulation. Int. Rev. Neurobiol. 38:139-199 Bradford H.F. & Thomas A.J. (1969) Metabolism of glucose and glutamate by synaptosomes from mammalian cerebral cortex. J. Neurochem. 16: 1495-1504 Bradford H.F. (1969) Respiration in vitro of synaptosomes from mammalian cerebral cortex. J. Neurochem. 16: 675-684 Brodie B.B. & Shore P.A. (1957) A concept for a role of serotonin and norepinephrine as chemical mediators in the brain. Ann. NY Acad. Sci. 96: 279-288 Broekkamp C.L., Leysen D., Peeters B.W. & Pinder R.M. (1995) Prospects for improved antidepressants. J. Med. Chem. 38: 4615-4633 Buck K.I. & Amara S.G. (1995) Structural domains of catecholamine transporter chimeras involved in selective inhibition by antidepressants and psychomotor stimulants. Molec. Pharmacol. 48: 1030-1037 Biirgisser M.M. (1983) Model testing in radioligand / receptor interaction by Monte Carlo simulation. J. Rec. Res. 3: 248-281 Carlsson A. (1961) Brain monoamines and psychotropic drugs. Neuropsychopharmacol. 2:417 Carlsson A.B., Falck B. & Hillarp N.A. (1962) Cellular localisation of brain mono?CLmnQS. Acta Physiol. Scand. 56: 1-28 Chan-Palay V. (1975) Fine structure of labelled axons in the cerebellar cortex and nuclei of rodents and primates after intraventricular infusions with tritiated serotonin. Anat. Embryol. 148: 235-265 Cheetham S.C., Viggers J.A., Butler S.A., Prow MR. & Heal D.J. (1996) [^H]nisoxetine-a radioligand for noradrenaline reuptake sites: correlation with inhibition of [^Hjnoradrenaline uptake and effect of DSP-4 lesioning and antidepressant treatments. Neuropharmacol. 35: 63-70 Chen N-H. & Reith M.B.A. (1994) Effects of locally applied cocaine, lidocaine, and various uptake blockers on monoamine transmission in the ventral tegmental area of freely moving rats: a microdialysis study on monoamine interrelationships. J. Neurochem. 63: 1701-1713

200 Cheng C.H.K., Costall B., Naylor RJ. & Rudd J.A. (1993) The effect of 5-HT receptor ligands on the uptake of [^H]5-hydroxytryptamine into rat cortical synaptosomes. Eur. J. Pharmacol. 239: 211-214 Chessin M., Kramer E.R. & Scott C.T. (1957) Modifications of the pharmacology of reserpine and serotonin by iproniazid. J. Pharmacol. Exp. Ther. 119: 453-460 Chiodo L.A., Aceson A.L., Zigmond M.J. & Strieker E.M. (1983) Subtotal destruction of central noradrenergic projections increases the firing rate of locus coeruleus cells. Brain Res. 264: 123-126 Clark R.N., Ashby C.R. Jr., Dewey S.L., Ramachandran P.V. & Strecker R E. (1996) Effect of acute and chronic fluoxetine on extracellular dopamine levels in the caudate-putamen and nucleus accumbens of rat. Synapse 23: 125-131 Clement H.W., Gemsa D. & Wesemann W. (1992) Serotonin-norepinephrine interactions: a voltammetric study on the effectof serotonin receptor stimulation followed in the N. Raphe dorsalis and the locus coeruleus of the rat. J. Neural Transm. 88: 11-23 Coppen A.J., Shaw DM. & Farrell J.P. (1963) Potentiation of the anti depressive effect of a monoamine-oxidase inhibitor by tryptophan. Lancet 1: 79-81 Crane G.E. (1957) Iproniazid (Marsilid) phosphate, a therapeutic agent for mental disorders and debilitating diseases. Psychiat. Res. Rep. Amer. Psychiat. Ass. 8: 142- 152 Dahlstrom A. & Fuxe K. (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta. Physiol. Scand. (Suppl.) 232: 1-55 Dailey J.W., Yan Q.S., Mishra P.K., Burger R.L. & Jobe P C. (1992) Effects of fluoxetine on convulsions and on brain serotonin as detected by microdialysis in genetically epilepsy-prone rats. J. Pharmacol. Exp. Ther. 260: 533-540 Daoust M., Protais P. & Ladure P. (1990) Noradrenergic system: effect of DSP4 and FLA-57 on ethanol intake in ethanol preferring rats. Pharmacol. Biochem. Behav. 36: 133-137 deBoer Th (1996) The pharmacologic profile of mirtazapine. J. Clin. Psychiatry 57 Suppl. 4: 19-25

201 Dechant K.L. & Clissold S.P. (1991) Paroxetine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in depressive illness. Drugs 41: 225-253 deJonghe F. & Swinkels J.A. (1992) The safety of antidepressants. Drugs 43 Suppl. 2: 40-46 Delgado J.M.R. (1962) Pharmacological modifications of social behaviour, in Pharmacological Analysis of Central Nervous Action (Paton W.D.M., ed), pp. 265- 292. Pergamon Press, Oxford. Delgado J.M.R., DeFeudis F.V., Roth R.H., Ryugo D.K. & Mitruka B.M. (1972) Dialytrode for long term intracerebral perfusion in awake monkeys. Arch. Int. Pharmacodyn. 198: 9-21 Delgado P.L., Charney D.S., Price L.H., Aghajanian G.K., Landis H. & Heninger G.R. (1990) Serotonin function and the mechanism of antidepressant action. Arch. Gen. Psychiatry 47: 411-419 Delgado P.L., Miller H.L., Salomon R.M., Lieinio J., Heninger G.R., Gelenberg A.J. & Charney D.S. (1993) Monoamines and the mechanism of antidepressant action: effects of catecholamine depletion on mood of patients treated with antidepressants. Psychopharmacol. Bull. 29: 389-396 Delini-Stula A., Mogilnicka E., Hunn C. & Dooley D.J. (1984) Novelty-oriented behavior in the rat after selective damage of locus coeruleus projections by DSP-4, a new noradrenergic neurotoxin. Pharmacol. Biochem. Behav. 20: 613-618 DePaermentier F., Cheetham S.C., Crompton M R. & Horton R.W. (1989) Beta- adrenoceptors in human brain labelled with [^Hjdihydroalprenolol and [^H]CGP 12177. Eur. J. Pharmacol. 167: 397-405 Desearries L., Watkins K.C. & Lapierre Y. (1977) Noradrenergic axon terminals in the cerebral cortex of rat. III. Topometric ultrastructural analysis. Brain Res. 133: 197- 222 Descarries L., Watkins K.C., Garcia S. & Beaudet A. (1982) The serotonin neurons in nucleus raphe dorsalis of adult rat: a light and electron microscope radioautographic study. J. Comp. Neurol. 207: 239-254 Dismukes K. (1977) New look at the aminergie nervous system. Nature 269: 557-558 Done C.J. & Sharp T. (1992) Evidence that 5-HTi receptor activation decreases noradrenaline release in rat hippocampus in vivo. Br. J. Pharmacol. 107: 240-245

202 Done C.J.G. & Sharp T. (1994) Biochemical evidence for the regulation of central

noradrenergic activity by 5-HTia and 5 -HT2 receptors: microdialysis studies in the awake and anaesthetised rat. Neuropharmacol 33: 411-421 Dooley D.J., Bittiger H., Hauser K.L., Bischoff S.F. & Waldmeier PC. (1983) Alteration of central alpha%- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin. NeuroscL 9: 889-898 Dooley D.J., Heal D.J. & Goodwin G.M. (1986) Repeated electroconvulsive shock prevents increased neocortical pi-adrenoceptor binding after DSP-4 treatment in rats. Eur. J. Pharmacol 134: 333-337 Dudley M.W., Howard B.D. & Cho A.K. (1990) The interaction of the beta-haloethyl bbenzylamines, xylamine, and DSP-4 with catecholaminergic neurons. Annu. Rev. Pharmacol Toxicol 30: 387-403 Esposito E., Ossowska G. & Samanin R. (1987) Further evidence that noradrenaline is not involved in the anti-immobility activity of chronic desipramine in the rat. Eur. J. Pharmacol 136; 429-432 Falck B., Hillarp N,-Â., Thieme G. & Torp A. (1962) Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem. 10: 348-354 Feuerstein T.J., Hertting G. & Jakisch R. (1986) Serotonin (5-HT) enhances hippocampal noradrenaline (NA) release: evidence for facilitatory 5-HT receptors within the CNS.Naunyn-Schmiedeberg’s Arch. Pharmacol 333: 191-197 Finberg J.P., Pacak K., Kopin I.J. & Goldstein D.S. (1993) Chronic inhibition of monoamine oxidase type A increases noradrenaline release in rat frontal cortex. Naunyn Schmiedeberg's Arch. Pharmacol 347: 500-505 Foote S.L., Bloom F.E. & Aston-Jones G. (1983) Nucleus locus coeruleus; new evidence of anatomical and physiological specificity. Physiol Rev. 63: 844-914 Frazer A. (1997) Anti depressants. J. Clin. Psychiatry 58 Suppl. 6: 9-25 Friedrich U. & Bonisch H. (1986) The neuronal noradrenaline transport system of PC- 12 cells: kinetic analysis of the interaction between noradrenaline, Na"^ and Cl' in transport. Naunyn Schmiedebergs Arch. Pharmacol. 333: 246-252 Fritschy J-M., Geffard M. & Grzanna R. (1990) The response of noradrenergic axons to systemically administered DSP-4 in the rat: an immunohistochemical study using

203 antibodies to noradrenaline and dopamine-p-hydroxylase. J. Chem. Neuroanat. 3: 309-321 Fritschy J.-M. & Grzanna R. (1989) Immunohistochemical analysis of the neurotoxic effects of DSP-4 identifies two populations of noradrenergic axon terminals. Neurosci. 30: 181-197 Fritschy J.-M. & Grzanna R. (1991) Selective effects of DSP-4 on locus coeruleus axons: are there pharmacologically different types of noradrenergic axons in the central nervous system? Prog. Brain Res. 88: 257-268 Fritschy J.-M. & Grzanna R. (1992) Restoration of ascending noradrenergic projections by residual locus coeruleus neurons: compensatory response to neurotoxin-induced cell death in the adult rat brain. J. Comp. Neurol. 321: 421-441 Fuller R.W, Wong D.T. & Robertson D.W. (1991) Fluoxetine, a selective inhibitor of serotonin uptake. Med. Res. Rev. 11: 17-34 Fuller R.W. & Perry K.W. (1977) Increase of pineal noradrenaline concentration in rats by desipramine but not fluoxetine: implications concerning the specificity of these uptake inhibitors. J. Pharm. Pharmac. 29: 710-711 Fuxe K., Hamberger B. & Hokfelt T. (1968) Distribution of noradrenaline nerve terminals in cortical areas of the rat. Brain Res. 8: 125-131 Gaddum J.H. (1961) Push-pull cannulae. J. Physiol. (Lond.) 155: 219-232 Garratt J.C., Crespi F., mason R. & Marsden C.A. (1991) Effects of idazoxan on dorsal raphe 5-hydroxytryptamine neuronal function. Eur. J. Pharmacol. 193: 87-93

Gartside S.E., Umbers V. & Sharp T. (1995) Interaction between a selective 5 -H T ia antagonist and an SSRI in vivo: effects on 5-HT cell firing and extracellular 5-HT. Br. J. Pharmacol. 115: 1064-1070 Giros B., El Mestikawy S., Bertrand L. & Caron M.G. (1991) Cloning and functional characterization of a cocaine-sensitive dopamine transporter. FEBS Lett. 295: 149- 154 Giros B., Wang Y-M., Suter S., McLeskey S B., Pifl C. & Caron M.G. (1994) Delineation of discrete domains for substrate, cocaine, and tricyclic antidepressant interactions using chimeric dopamine-norepinephrine transporters. J. Biol. Chem. 269:15985-15988 Glowinski J. & Axelrod J. (1964) Inhibition of tritiated noradrenalin in the intact rat brain by imipramine and structurally related compounds. Nature 204: 1318-1319

204 Glowinski J. & Axelrod J. (1965) The effect of drugs on the uptake, release and metabolism of rf-norepinephrine in the rat brain. J. Pharmacol. Exp. Ther. 149: 43- 49 Glowinski J., Iversen L.L. & Axelrod J. (1966) Storage and synthesis of norepinephrine in reserpinized rat brain. J. Pharmacol. Exp. Ther. 151: 385-399 Glowinski J., Kopin I.J. & Axelrod J. (1965) Metabolism of H^-norepinephrine in the rat brain. J. Neurochem. 12: 25-30 Gobert A., Rivet J.M., Cistarelli J.M. & Millan M.J. (1997) Buspirone enhances duloxetine- and fluoxetine-induced increases in dialysate levels of dopamine and noradrenaline, but not serotonin, in the frontal cortex of freely moving rats. J. Neurochem. 68: 1326-1329 Gonon F., Buda M., DeSimoni G. & Pujol J.F. (1983) Catecholamine metabolism in the rat locus coeruleus as studied by in vivo differential pulse voltammetry. U. Pharmacological and behavioral study. Brain Res. 273: 207-216 Goodman L.S. & Gilman A. (1955) The pharmacological basis of therapeutics. New York. Macmillan Co. vol II p 518

Goodnough D.B. & Baker G.B. (1994) 5-hydroxytryptamine2 and p-adrenergic receptoe regulation in rat brain following chronic treatment with desipramine and fluoxetine alone and in combination. J. Neurochem 62: 2262-2267 Gray E.G. & Whittaker V.P. (1962) The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96: 1-36 Guastella J., Nelson N., Nelson H., Czyzyk L., Keynan S., Miedel M.C., Davidson N., Lester H.A. & Kanner B.I. (1990) Cloning and expressionof a rat brain GAB A transporter. Science 249: 1303-1306 Hajos-Korcsok E. & Sharp T. (1996) 8-OH-DPAT-induced release of hippocampal noradrenaline in vivo: evidence for a role of both 5-HTia and dopamine D, receptors. Eur. J. Pharmacol. 314: 285-291 Hallman H. & Jonsson G. (1984) Pharmacological modifications of the neurotoxic action of the noradrenaline neurotoxin DSP4 on central noradrenaline neurons. Eur. J. Pharmacol. 103: 269-278

205 Hallman H., Sundstrôm E. & Jonsson G. (1984) Effects of the noradrenaline neurotoxin DSP 4 on monoamine neurons and their transmitter turnover in rat CNS. J. Neural. Transm. 60: 89-102 Harik S.I. (1984) Locus coeruleus lesion by local 6-hydroxydopamine infusion causes marked ans specific destruction of noradrenergic neurons, long-term deplertion of norepinephrine and the enzymes that synthesize it, and enhanced dopaminergic mechanisms in the ipsilateral cerebral cortex. J. Neurosci. 4: 699-707 Harms H.H. (1983) The antidepressant agents desipramine, fluoxetine, fluvoxamine and norzimelidine inhibit uptake of [^H]noradrenaline and ['^H] 5-hydroxytryptamine in slices of human and rat cortical brain tissue. Brain Res. 275: 99-104 Harris T.H. (1957) Depression induced by rauwolfia compounds. Amer. J. Psychiat. 113: 950 Heal D.J., Butler S.A., Prow M.R. & Buckett W.R. (1993) Quantification of presynaptic

a 2-adrenoceptors in rat brain after short-term DSP-4 lesioning. Eur. J. Pharmacol. 249:37-41 Henn F.A. & Hamberger A. (1971) Glial cell function: uptake of transmitter substances. Proc. Natl. Acad. Sci. 68: 2686-2690 Hernandez L., Stanley B.G.,& Hoebel B.C. (1986) A small, removable microdialysis probe. Life Sci. 39: 2629-2637 Hertting G., Axelrod J. & Whitby L.G. (1961) Effect of drugs on the uptake and metabolism of H^-noepinephrine. J. Pharmacol. Exp. Ther. 134: 146-153 Hoffman B.J., Mezey E. & Brownstein M.J. (1991) Cloning of a serotonin transporter affected by antidepressants. Science 254: 579-580 Hrdina P.D. (1981) Pharmacological characterization of [^H]desipramine binding in rat cerebral cortex. Prog. Neuro-Psychopharmacol. 5: 553-557 Hughes Z.A. & Stanford S.G. (1996) Increased noradrenaline efflux induced by local infusion of fluoxetine in the rat frontal cortex. Eur. J. Pharmacol. 317: 83-90 Hyttel J. (1994) Pharmacological characterization of selective serotonin reuptake inhibitors (SSRIs). Int. Clin. Psychopharmacol. 9 (Suppl. 1): 19-26 Hyttel J., Over0 K.F. & Arnt J. (1984) Biochemicaf effects and drug levels in rats after long-term treatment with the specific 5-HT-uptake inhibitor, citalopram. Psychopharmacol. 83: 20-27

206 Iversen L.L. (1965) Uptake of catecholamines at high perfusion concentrations in the rat isolated heart: a novel catecholamine uptake process. Br. J. Pharmacol 25: 18-33 Iversen L.L. (1970) Inhibition of catecholamine uptake by 6-hydroxydopamine in rat brain. Eur. J. Pharmacol 10: 408-410 Jacks B.R., de Champlain J. & Cordeau J.P. (1972) Effects of 6-hydroxydopamine on putative transmitter substances in the central nervous system. Eur. J. Pharmacol 18: 353-360 Johnston J.P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem. Pharmacol 17: 1285-1297 Jonsson G. & Sachs C. (1973) Pharmacological modifications of the 6-hydroxy-dopa induced degeneration of central noradrenaline neurons. Biochem. Pharmacol 22: 1709-1716 Jonsson G., Hallman H., Ponzio F. & Ross S. (1981) DSP4 (N-(2-chloroethyl)-N-ethyl- 2-bromobenzylamine) - a useful denervation tool for central and peripheral noradrenaline neurons. Eur. J. Pharmacol 72: 173-188 Jordan S., Kramer G.L., Zukas P.K. Moeller M. & Petty F. (1994) In vivo biogenic amine efflux in medial prefrontal cortex with imipramine, fluoxetine, and fluvoxamine. Synapse 18: 294-297 Kanner B.I. & Schuldiner S. (1987) Mechanism of transport and storage of neurotransmitters. CRC Crit. Rev. Biochem. 22: 1-38 Kask A., Harro J., Tuomainen, Rago L. & Mannisto P.T. (1997) Overflow of noradrenaline and dopamine in frontal cortex after [N-(2-chloroethyl)-N-ethyl-2- bromobenzylamine] (DSP-4) treatment: in vivo microdialysis study in anaesthetized rats. Naunyn-Schmiedeberg Arch. Pharmacol 355: 267-272 Keller H.H., Kettler R., Keller G. & DaPrada M. (1987) Short-acting novel MAO inhibitors: in vitro evidence for the reversibility of MAO inhibition by moclobemide and Ro \6-649\. Naunyn-Schmiedebergs Arch. Pharmacol 335: 12-20 Kiloh L.G., Child J.P. & Latner G.A. (1960) A controlled trial of iproniazid in the treatment of endogenous depression. J. Ment. Sci 106: 1139-1144 Kilpatrick A.T., Goodwin K. & Brown C.M. (1986) Characterisation of sodium- dependent uptake of 5-hydroxytryptamine into guinea-pig brain. Neuropharmacology 25: 1037-1041

207 Kilty J.E., Lorang D. & Amara S.G. (1991) Cloning and expression of a cocaine- sensitive rat dopamine transporter. Science 254: 578-579 Kostzrewa R.M. (1988) Reorganization of noradrenergic neuronal systems following neonatal chemical and surgical injury. Prog. Brain Res. 73: 405-423 Kuhar M.J. (1973) Neurotransmitter uptake: a tool in identifying neurotransmitter- specific pathways. Life Sci. 13: 1623-1634 Kuhn R. (1958) The treatment of depressive states with G22355 (imipramine hydrochloride). Amer. J. Psychiat. 115: 459-464 Lauritsen B.J. & Madsen H. (1974) A multinational, double-blindtrial with a new antidepressant maprotiline (Ludiomil) and amitryptiline. Acta Psychiatr. Scand. 50: 192-201 Lee C M., Javitch J.A. & Snyder S.H. (1982) Characterization of desipramine binding associated with neuronal norepinephrine uptake sites in rat brain membranes. J. Neurosci. 2: 1515-1525 Leonard B.E. (1992) Pharmacological differences of serotonin reuptake inhibitors and possible clinical relevance. Drugs 43 Suppl. 2: 3-10 Leonard B.E. (1997) The potential contribution of sigma receptors to antidepressant actions, in Antidepressants: new pharmacological strategies (Skolnick P. ed.), pp 159-172. Humana Press, New Jersey. Li M.Y., Yan Q.S., Coffey L.L. & Reith M.E. (1996) Extracellular dopamine, norepinephrine, and serotonin in the nucleus accumbens of freely moving rats during intracerebral dialysis with cocaine and other monoamine uptake blockers. J. Neurochem. 66: 559-568 Lidbrink P. & Jonsson G. (1971) Semiquantitative estimation of formaldehyde induced fluorescence of noradrenaline in central noradrenaline nerve terminals. J. Histochem. Cytochem. 19: 747-757 Ling C.M. & Abdel-Latif A.A. (1968) Studies on sodium transport in rat brain nerve ending particles. J. Neurochem. 15: 721-729 Logue M., Growdon J.H., Lopez IL. G. & Wurtman R.J. (1985) Differential effects of DSP-4 administration on regional brain norepinephrine turnover in rats. Life Sci. 37: 403-409

208 Loughlin S.E, Foote S.L. & Bloom F.E. (1986) Efferent projections of nucleus locus coeruleus: morphologic subpopulations have different efferent targets. NeuroscL 18: 307-319 Loughlin S.E., Foote S.L. & Fallon J.H. (1982) Locus coeruleus projections to cortex: topography, morphology and collaterization. Brain Res. Bull. 9: 287-294 Lowry O.H., Rosebrough N.J., Farr A.L. & Randall R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275 Macintosh F.C. & Oborin F.E. (1953) Release of acetylcholine from intact cerebral cortex. (Abstr.), in Abstracts of the XlXth International Physiology Congress, pp 580-581 Mager S., Min C., Henry D.J. Chavkin C., Hoffman B.J. Davidson N. & Lester H.A. (1994) Conducting states of a mammalian serotonin transporter. Neuron 12: 845-859 Matsumoto K., Ojima K. & Watanabe H. (1995a) Noradrenergic denervation attenuates desipramine enhancement of aggressive behavior in isolated mice. Pharmacol. Biochem. Behav. 50: 481-484 Matsumoto M., Yoshioka M., Togashi H., Tochihara M., Ikeda T. & Saito H. (1995b) Modulation of norepinephrine release by serotonergic receptors in the rat hippocampus as measured by in vivo microdialysis. J. Pharmacol. Exp. Ther. 272: 1044-1051 McElvain J. & Schenk J. (1992) A multisubstrate mechanism of striatal dopamine uptake and its inhibition by cocaine. Biochem. Pharmacol. 43: 2189-2199 Melzacka M., Rurak A., Adamus A. & Daniel W. (1984) Distribution of citalopram in the blood serum and in the central nervous system of rats after single and multiple dosage. Pol. J. Pharmacol. Pharm. 36: 675-682 Michel M., Hiemke C. & Ghraf R. (1984) Preferential uptake of norepinephrine into dopaminergic terminals of a synaptosomal preparation from rat cerebral cortex. Brain Res. 301: 149-152 Milby K.H., Mefford I.N., Chey W. & Adams R.N. (1981) In vitro and in vivo depolarization coupled efflux of ascorbic acid in rat brain preparations. Brain Res. Bull. 7: 237-242 Miller H.L., Delgado P.L., Salomon R.M., Berman R., Krystal J.H., Heninger G.R. & Charney D.S. (1996) Clinical and biochemical effects of catecholamine depletion on antidepressant-induced remission of depression. Arch. Gen. Psychiatry. 53: 117-127

209 Mishra P.K., Burger R.L., Bettendorf A.F., Browning R.A. & Jobe P C. (1994) Role of norepinephrine in forebrain and brainstem seizures: chemical lesioning of locus coeruleus with DSP-4. Exper. Neurol 125: 58-64 Moghaddam B. & Bunney B.S. (1989) Ionic composition of microdialysis perfusing solution alters the pharmacological responsiveness and basal outflow of striatal dopamine. J. Neurochem. 53: 652-654

Mogilnicka E. (1986) Increase in |3- and ai-adrenoceptor binding sites in the rat brain

and in the ai-adrenoceptor functional sensitivity after the DSP-4-induced noradrenergic denervation. Pharmacol Biochem. Behav. 25: 143-746 Mongeau R., Blier P. & deMontigny C. (1997) The serotonergic and noradrenergic systems of the hippocampus: their interactions and the effects of antidepressant treatments. Brain Res. Rev. 23: 145-195 Mongeau R., de Montigny C. & Blier P. (1994) Activation of 5-HT3 receptors enhances the electrically evoked release of [^Hjnoradrenaline in the rat brain limbic structures. Eur. J. Pharmacol 256: 269-279 Moore R.Y. & Bloom F.E. (1979) Central catecholamine neuron systems: anatomy and physiology of the norepinephrine and epinephrine systems. Ann. Rev. Neurosci 2: 113-168 Murdoch D. & McTavish D. (1992) Sertraline: a review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in depression and obsessive- compulsive disorder. Drugs 44: 604-624 Nelson H., Mandiyan S. & Nelson N. (1990) Cloning of the human brain GAB A transporter. FEBS Lett. 269: 181-184 Nowak G., Zak J. & Superata J. (1991) Role of dopaminergic neurons in denervation-

induced -adrenergic upregulation in the rat cerebral cortex. J. Neurochem. 56: 914- 916 Olfson M. & Klerman G.L. (1993) Trends in the prescription of antidepressants by office-based psychiatrists. Am. J. Psychiatry 150: 571-577 Olschowka J.A., Molliver M.E., Grzanna R., Rice F.L. & Coyle J.T. (1981) Ultrastructural demonstration of noradrenergic synapses in the rat central nervous system by dopamine-b-hydroxylase immunocytochemistry. J. Histochem. Cytochem. 29: 271-280

210 Olson L. & Fuxe K. (1972) Further mapping of central noradrenaline systems. Projections of the ‘subcoeruleus’ area. Brain Res. 43: 289-295 Pacholczyk T., Blakely R.D. & Amara S.G. (1991) Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 350: 350-354 Conclusive evidence for distinct transporters for 5-hydroxytryptamine and noradrenaline in pulmonary endothelial cells of the rat. Paczkowski N.J., Vuocolo H.E. & Bryan-Lluka L.J. (1996) Conclusive evidence for distinct transporters for 5-hydroxytryptamine and noradrenaline in pulmonary endothelial cells of the rat. Naunyn Schmiedebergs Arch. Pharmacol. 353: 423-430 Palkowitz M., Brownstein M. & Saavedra J.M. (1974) Norepinephrine and dopamine content of hypothalamic nuclei of the rat. Brain Res. 77: 137-149 Papadopolous G.C., Parnavelas J.G. & Buijs R. (1987) Monoamnergic fibres form conventional synapses in the cerebral cortex. Neurosci. Lett. 76: 275-279 Papadopoulos G.C. & Parnavelas J.G. (1991) Monoamine systems in the cerebral cortex: evidence for anatomical specificity. Prog. Neurobiol. 36: 195-201 Paxinos G. & Watson C. (1986) The rat brain in stereotaxic coordinates. Academic Press London, U.K. Paykel E.S. & Hale A.S. (1986) Recent advances in the treatment of depression, in: The biology of depression, ed. Deakin D.W. Gaskell, London Pel ton E.W., Kimelberg H.K., Shipherd S.V. & Bourke R.S. (1981) Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture. Life Sci. 28: 1655-1663 Perry K.W. & Fuller R.W. (1992) Effect of fluoxetine on serotonin and dopamine concentration in microdialysis fluid from rat striatum. Life Sci. 50: 1683-1690 Pickel V.M., Hirenberg M.J. & Milner T.A. (1996) Ultrastructural view of central catecholaminergic transmission: immunocytochemical localization of synthesizing enzymes, transporters and receptors. J. Neurocytol. 25: 843-856 Pifl C., Agneter E., Drobny H., Reither H. & Singer E.A. (1997) Induction by low Na-i- or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters. Br. J. Pharmacol. 121: 205-212 Pifl C., Hornkiewicz O., Giros B. & Caron M.G. (1996) Catecholamine transporters and 1 -methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity: studies comparing the

211 cloned human noradrenaline and human dopamine transporter. J. Pharmacol. Exp. Ther. 277: 1437-1443 Frisco S. & Esposito E. (1995) Differential effects of acute and chronic fluoxetine administration on the spontaneous activity of dopaminergic neurones in the ventral tegmental area. Br. J. Pharmacol. 116: 1923-1931 Pucilowski O., Overstreet D.H., Rezvani A.H. & Janowsky D.S. (1990) Effect of verapamil on submissive behavior in genetically bred hypercholinergic rats in a water competition test. Eur. J. Pharmacol. 187; 507-511 Ramamoorthy S., Leibach P., Mahesh V. & Ganapathy V. (1992) Active transport of dopamine in human placental brush-border membrane vesicles. Am. J. Physiol. 262: Cl 189-1196 Rang H P. & Dale M.M. (1987) Chemical transmission and drug action in the central nervous system. In Pharmacology. Churchill Livingstone pp 447-470 Ransom R.W., Kammerer R.C. & Cho A.K. (1982) Chemical transformation of xylamine (N-2'-chloroetyl-N-ethyl-2-methylbenzylamine) in solution. Mol. Pharmacol. 21: 380-386 Rasmussen K. & Aghajanian O.K. (1986) Effect of hallucinogens on spontaneous and sensory-evoked locus coeruleus unit activity in the rat: reversal by selective 5-HT2 antagonists. Brain Res. 385: 395-400 Reader T.A. & Brière R. (1983) Long-term unilateral noradrenergic denervation: monoamine content and ^H-prazosin binding sites in rat neocortex. Brain Res. Bull. 11: 687-692 Reivich M. & Glowinski J. (1967) An autoradiographic study of the distribution of C*'^- norepinephrine in the brain of the rat. Brain 90: 633-646 Richelson E. & Pfenning M. (1984) Blockade by antidepressants and related compounds of biogenic amine uptake into rat brain synaptosomes: most antidepressants selectively block norepinephrine uptake. Eur. J. Pharmacol. 104: 277-286 Rickels K. & Schweizer E. (1990) Clinical overview of serotonin reuptake inhibitors. J. Clin. Psychiatry 51: 9-12 Romero L., Bel N., Casanovas J.M. & Artigas F. (1996) Two actions are better than one: avoiding self-inhibition of serotonergic neurones enhances the effects of serotonin uptake inhibitors. Int. Clin. Psychopharmacol. 11 Suppl. 4: 1-8

212 Ross S.B. & Renyi A.L. (1976) On the long-lasting inhibitory effect of N-(2- chloroethyl)-N-ethyl-2-bromobenzylamine (DSP 4) on the active uptake of noradrenaline. J. Pharm. Pharmac. 28: 458-459 Ross S.B. (1976) Long-term effects of 7/-2-chloroethyl-A-ethyl-2-bromobenzylamine hydrochloride on noradrenergic neurones in the rat brain and heart. Br. J. Pharmacol. 58: 521-527 Routledge C. & Marsden C.A. (1987) Comparison of the effects of selected drugs on the release of hypothalamic adrenaline and noradrenaline measured in vivo. Brain Res. 426: 103-111 Rubinstein-Kawamoto J., Davis J.M. & Pandey G.N. (1984) Effects of antidepressants on the uptake and release of norepinephrine from rat cerebral cortex. Psychopharmacol. 84: 155-157 Rudnick G. & Clark J. (1993) From synapse to vesicle: the reuptake and storage of biogenic amine neurotransmitters. Biochim. Biophys. Acta 1144: 249-263 Russ H., Staudt K., Martel F., Gliese M. & Schomig E. (1996) The extraneuronal transporter for monoamine transmitters exists in cells derived from human central nervous system glia. Eur. J. Pharmacol. 8: 1256-1264 Sachs C. & Jonsson G. (1975b) Mechanisms of action of 6-hydroxydopamine. Biochem. Pharmacol. 24: 1-8 Sachs C. & Jonsson, G. (1975a) Effects of 6-hydroxydopamine on central noradrenaline neurons during ontogeny. Brain Res. 99: 277-291 Sachs C., Jonsson G., Heikkila R. & Cohen G. (1975) Control of the neurotoxicity of 6- hydroxydopamine by intraneuronal noradrenaline in rat iris. Acta. Physiol. Scand. (Suppl.) 93: 345-351 Sakai K., Sal vert D., Touret M. & Jouvet M. (1977) Afferent connections of the raphe dorsalis in the cat as visualized by the horseradish peroxidase technique. Brain Res. 137: 11-35 Samanin R., Bernasconi S. & Garattini S. (1975) The effect of nomifensine on the depletion of brain serotonin and catecholamines induced respectively by and 6-hydroxydopamine in rats. Eur. J. Pharmacol. 34: 377-380 Sandberg M., Butcher S.P. & Hagberg H. (1986) Extracellular overflow of neuroactive amino acids during severe insulin-induced hypoglycemia: in vivo dialysis of the rat hippocampus. J. Neurochem. 47: 178-184

213 Schildkraut J.J. (1965) The catecholamine hypothesis of affective disorders: a review of supporting evidence. Am. J. Psychiat. Schildkraut J.J., Gordon E.K. & Durell J. (1965) Catecholamine metabolism in affective disorders. I. Normetanephrine and VMA excretion in depressed patients treated with imipramine. J. Psychiatr. Res. 3: 213-228 Segal M. & Landis S. (1974) Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase. Brain Res. 78: 1-15 Semenoff D. & Kimelberg H.K. (1985) Autoradiography of high affinity uptake of catecholamines by primary astrocyte cultures. Brain Res. 348: 125-136 Sharp T., Bramwell S.R., Clark D. & Grahame-Smith D.G. (1989) In vivo measurement of extracellular 5-hydroxytryptamine in hippocampus of the anaesthetized rat using microdialysis: changes in relation to 5-hydroxytryptaminergic neuronal activity. J. Neurochem. 53: 234-240 Shimada S., Kitayama S., Lin C-L., Patel A., Nanthakumar E., Gregor P., Kuhar M. & Uhl G. (1991) Cloning and expression of a cocaine-sensitive dopamine transporter complementary DNA. Science 254: 576-578 Shipley M.T., Halloran F.J. & de la Torre J. (1985) Surprisingly rich projection from locus coeruleus to the olfactory bulb in the rat. Brain Res. 329: 294-299 Shopsin B., Friedman E. & Gershon S. (1976) Parachlorophenylalanine reversal of trancylpromine effects in depressed patients. Arch. Gen. Psychiatry 33: 811-819 Silver LA. & Erecinska M. (1990) Intracellular and extracellular changes of [Ca^'*'] in hypoxia and ischemia in rat brain in vivo. J. Gen. Physiol. 95: 837-866 Snyder S.H. & Coyle J.T. (1969) Regional differences in H^-norepinephrine and H^- dopamine uptake into rat brain homogenates. J. Pharmacol. Exp. Ther. 165: 78-86 Spector S., Shore P.A. & Brodie B.B. (1960) Biochemical and pharmacological effects of the monoamine oxidase inhibitors, iproniazid, 1-phenyl-2-hydrazinopropane (JB516) and l-phenyl-3-hydroazinobutane (JB835). J. Pharmacol. Exp. Ther. 128: 15-21 Spyraki C., Arbuthnott G.W. & Fibiger H.C. (1982) The effect of DSP-4 on some positively reinforced operant behaviors in the rat. Pharmacol. Biochem. Behav. 16: 197-202

214 Stadler H., Ciria M.G. & Bartholini G. (1975) "In vivo" release of endogenous neurotransmitters in cat limbic regions: effect of and of electrical stimulation. Naunyn-Schmiedebergs-Arch-Pharmacol. 288: 1-6 Stamford J.A. (1989) In vivo voltammetry - prospects for the next decade. TiNS 12: 407- 412 Stanford S.C. (1996) Prozac: panacea or puzzle? TiPS 17: 150-154 Steinbusch H.W., Nieuwenhuys R., Verhofstad A.A. & van der Kooy D. (1981) The nucleus raphe dorsalis of the rat and its projection upon the caudatoputamen. A combined cytoarchitectonie, immunohistochemical and retrograde transport study. J. Physiol Paris 11 \ 157-174 Steinbusch H.W., Verhofstad A.A. & Joosten H.W. (1978) Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. NeuroscL 3: 811-819 Sulser F., Bickel M.H. & Brodie B.B. (1964) The action of desmethylimipramine in counteracting sedation and cholinergic effects of reserpine-like drugs. J. Pharmacol Exp. Ther. 144: 321-330 Sulser P., Watts J. & Brodie B.B. (1962) On the mechanism of anti depressant action of imipramine-like drugs. Ann. NY Acad. Sci 96: 279-288 Suzuki M., Matsuda T., Asano S., Somboonthum P., Takuma K. & Baba A. (1995) Increase of noradrenaline release in the hypothalamus of freely moving rat by postsynaptic 5 -hydroxy tryptame % A receptor activation. Br. J. Pharmacol 115: 703- 711 Svensson T.H., Bunney B.S. & Aghajanian G.K. (1975) Inhibition of both NA and 5-HT neurons in brain by the a- clonidine. Brain Res. 92: 291-306 Swanson L.W. & Hartman B.K. (1975) The central adrenergic system. An immunofluorescence study of the location of cell bodies and their efferent connections in the rat utilizing dopamine-p-hydroxylase as a marker. J. Comp. N eurol 163: 467-506 Swanson L.W. (1976) The locus coeruleus: A cytoarchitectonie, Golgi and immunohistochemical study in the albino rat. Brain Res. 110: 39-56 Tanaka T., Yokoo H., Mizoguchi K., Yoshida M., Tsuda A. & Tanaka M. (1991) Noradrenaline release in the rat amygdala is increased by stress: studies with intracerebral microdialysis. Brain Res. 544: 174-176

215 Tanda G., Carboni E., Frau R. & Di-Chiara G. (1994) Increase of extracellular dopamine in the prefrontal cortex: a trait of drugs with antidepressant potential? Psychopharmacol Berl 115: 285-288

Tanda G., Frau R. & Di-Chiara G. (1995) Local 5 -HT3 receptors mediate fluoxetine but not desipramine-induced increase of extracellular dopamine in the prefrontal cortex. Psychopharmacol Berl 119: 15-19 Tejani-Butt S.M., Brunswick D.J. & Frazer A. (1990) [‘^Hjnisoxetine: a new radioligand for norepinephrine uptake sites in brain. Eur. J. Pharmacol 191: 239-243 Theron C.N., de Villiers A.S. & Taljaard J.J.F. (1993) Effects of DSP-4 on monoamine and monoamine metabolite levels and on beta adrenoceptor binding kinetics in rat brain at different times after administration. Neurochem. Res. 18: 1321-1327 Thomas D R., Nelson D R. & Johnson A.M. (1987) Biochemical effects of the antidepressant paroxetine, a specific 5-hydroxytryptamine uptake inhibitor. Psychopharmacol 93: 193-200 Tissari A.H., Schdnhdfer P.S., Bogdanski D.F. & Brodie B.B. (1969) Mechanism of biogenic amine transport. II. Relationship between Na"^ and the mechanism of ouabain blockade of the accumulatin of serotonin and noradrenaline by synaptosomes. Mol Pharmacol 5: 593-604 Tollefson G.D. (1991) Antidepressant treatment and side effect considerations. J. Clin. Psychiatry 52: 4-13 Tdrk I. (1990) Anatomy of the serotonergic system. Ann. NY. Acad. Sci 600: 9-34 Tossman U. & Ungerstedt U. (1986) Microdialysis in the study of extracellular levels of amino acids in the rat brain. Acta. Physiol Scand. 128: 9-14 Trullas R. & Skolnick P. (1990) Functional antagonists at the NMD A receptor complex exhibit antidepressant actions. Eur. J. Pharmacol. 185; 1-10 Ungerstedt U. (1971) Stereotaxic mapping of the monoamine pathways in the rat brain. Acta. Physiol. Scand. (Suppl) 367: 1-48 Ungerstedt U., Herrera-Marschitz M. & Zetterstrom T. (1982) Dopamine neurotransmission and brain function. Prog. Brain Res. 55: 41-49 Unnerstall J.R., Fernandez I. & Oresanz L.M. (1985) The alpha-: radiohistochemical analysis of functional characteristics and biochemical differences. Pharmacol. Biochem. Behav. 22: 859-874

216 Vahabzadeh A. & Fillenz M. (1994) Comparison of stress-induced changes in noradrenergic and serotonergic neurons in the rat hippocampus using microdialysis. Eur. J. Neurosci. 6: 1205-1212 van Veldhuizen M.J.A., Feenstra M.G.P., Boer G.J. & Westerink B.H.C. (1990) Microdialysis studies on cortical noradrenaline release: basic characteristics, significance of extracellular calcium and massive post-mortem increase. Neurosci. Lett. 119: 233-236 Vertes R.P. & Martin G.F. (1988) Autoradiographic analysis of ascending projections from the pontine and mesencephalic reticular formation and the median raphe nucleus in the rat. J. Comp. Neurol. 275: 511-541 Waldmeier P C. & Baumann P.A. (1983) Effects of CGP 11305 A, a new reversible and selective inhibitor of MAO A, on biogenic amine levels and metabolism in the rat brain. Naunyn-Schmiedebergs Arch. Pharmacol. 324: 20-26 Waterhouse B.D., Lin C.-S., Burne R.A. & Woodward D.J. (1983) The distribution of neocortical projection neurons in the locus coeruleus. J. Comp. Neurol. 217: 418-431 Weissmann D., Belin M.F., Aguera M., Meunier C., Maitre M., Cash C D., Ehret M., Mandel P. & Pujol J.F. (1987) Immunohistochemistry of tryptophan hydroxylase in the rat brain. Neurosci. 23: 291-304 Wieczorek M. & Romaniuk A. (1994) The effects of dorsal and ventral noradrenergic system lesions with DSP-4 on emotional-defensive behaviour and regional brain monoamines content in the cat. Behav. Brain Res. 63: 1-9 Whitby L.G., Axelrod J. & Weil-Malherbe H. (1961) The fate of H^-norepinephrine in animals. J. Pharmacol. Exp. Ther. 132: 193-201 Wong D.T., Bymaster F.P., Horng J.S. & Molloy B.B. (1975) A new and selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3-(p- trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine. J. Pharmacol. Exp. Ther. 193: 804-811 Wood M.D. & Wyllie M.G. (1983) Critical assessment of noradrenaline uptake in synaptosomal preparations. Naunyn-Schmiedeberg's Arch. Pharmacol. 322: 129- 135 Zaczek R. & Coyle J.T. (1982) Rapid and simple method for measuring biogenic amines and metabolites in brain homogenates by HPLC-electrochemical detection. J. Neural. Transm. 33: 1-5

217 Zaczek R., Fritschy J-M., Culp S., De Souza E.B. & Grzanna R. (1990) Differential effects of DSP-4 on noradrenaline axons in cerebral cortex and hypothalamus may reflect heterogeneity of noradrenaline uptake sites. Brain Res. 522: 308-314 Zagrodzka J., Wieczorek M. & Romaniuk A. (1994) Social interactions in rats: behavioural and neurochemical alterations in DSP-4-treated rats. Pharmacol. Biochem. Behav. 49: 541-548 Zahniser N.R., Heidenreich K.A. & Molinoff P.B. (1981) Binding of amino-6,7- dihydroxy-1,2,3,4-tetrahydro napthalene to rat striatal membranes. Mol. Pharmacol. 19: 372-378 Zahniser N.R., Weiner G.R., Worth T., Philpott K., Yasuda R.P., Jonsson G. & Dunwiddie T.V. (1986) DSP-4 induced noradrenergic lesions increase p-adrenergic receptors and hippocampal electrophysiological responsiveness. Pharmac. Biochem. Behav. 24: 1397-1402 Zetterstrom T., Vernet L., Ungerstedt U., Tossman U., Jonzon B. & Fredholm B.B. (1982) Purine levels in the intact rat brain. Studies with an implanted perfused hollow fibre. Neurosci. Lett. 29: 111-115 Zieher L.M. & Jaim-Etcheverry G. (1980) Neurotoxicity of N-(2-chloroethyl)-N-ethyl- 2-bromobenzylamine hydrochloride (DSP4) on noradrenergic neurons is mimicked by its cyclic aziridinium derivative. Eur. J. Pharmacol. 65: 249-256

218