m.sc presentation

Study leader: Prof. B.H. HarveyAssisting study leader: Dr. M. Möller-WolmaransCollaborators: Dr. G. WegenerM.Sc. Student: Dewald E. Coutts

Behavioral, neuroendocrine and neurochemical

studies on agomelatine in social isolation reared rats

Depression

INTRODUCTION

Depression – not a single neurotransmitter disorder, but represents a continuum of environmental, genetic and neurochemical determinants that occupy a distinct role.

Chronic psychosocial & environmental stressors play a role in development.

Many overlapping symptoms as well as biological mechanisms characterize mood disorders.

There are many promising hypothesis of depression and antidepressant action:

Harvey et al., 2008; Fone & Porkess, 2008

INTRODUCTION

Hypothesis

Monoamines Neurotrop

hic factors

HPA-axis & corticoster

oneGABA-glutam

ate signalin

g

Circadian

rhythms

Oxidative stress & Immune-inflammat

ory dysfunctio

n

INTRODUCTION

The monoamine hypothesis:

Postulates that depression is caused by deficits in monoamine function (5-HT, NA & DA).

Deficits may be restored by MAOI’s, TCA’s and SSRI’s.

Clinical evidence suggests that serotonergic ADs are less effective. SSRI’s induce cognitive and emotional blunting with improvements in anhedonia often the last group of symptoms to remit.

Berton et al., 2006; Krishnan et al., 2008; Machado-Vieira et al., 2009

Watson & Breedlove

INTRODUCTION

Neurotrophin hypothesis: BDNF

Stress decreases BDNF in the hippocampus and dentate gyrus partly due to glucocorticoid release and partly other mechanisms such as an increase in serotonin and glutamate transmission.

Evidence linking BDNF and depression has been obtained from studies

Nestler et al., 2002; Harvey et al., 2003; Savitz & Drevets 2009; Naert et al., 2011

INTRODUCTION

BDNF

Stress Increased activity of the HPA-axis

Production of inflammatory cytokines

Increased secretion of glucocorticoids

Atrophy & death of neurons

Nestler et al., 2002; Harvey et al., 2003

STRESS

BDNF

Sprouting of 5-HT fibers, accelerate regrowth

Modifies HPA-axisNaert et al., 2011; Souêtre et al., 1989Hsiao et al., 2010; Doane et al., 2013

INTRODUCTIONCircadian rhythm hypothesis:

80 % of depressed patients suffer from insomnia.

It clear that altered circadian rhythm is a core biological manifestation of depression and neuroendocrine abnormalities.

These alternations in cortisol secretion have been related to HPA-axis hyper-activity.

The nucleus accumbens, amygdala and certain hypothalamic nuclei are critical in regulating circadian rhythms and are abnormal in depressed patients

Soria & Urretavizcaya, 2009; McClung, 2013

Recovered ControlDepressed

Sleepperiod

7 911131517192123 7 9 1113531

37.2

37.0

36.8

36.6

36.4

36.2

36.0

BODY TEMPERATURE (°C)

20

40

60

80

100

6 9 12 15 18 21 24 3 6 9

Sleep

PLASMA MELATONIN(pg/ml)

Clock Time

PLASMA CORTISOL(ng/ml)

Sleep

20

70

120

170

220

6 9 12 15 18 21 24 3 6 9

Circadian rhythms in depression

Souetre E. et al Am J Psychiatry. 1988; 145:1133-7

Reduced amplitude

Phase shifted

Clock Time Clock Time

Melchitzky et al., 2010

INTRODUCTION

HPA-axis hypothesis:

Activation of the HPA-axis is a mechanism by which the brain reacts to acute and chronic stress and is controlled by the hippocampus and amygdala.

What is especially relevant is that elevated levels of glucocorticoids under prolonged and severe stress may damage hippocampal neurons and explains hippocampal shrinkage

Nestler et al., 2002; Savitz & Drevents, 2009

INTRODUCTION

Hippocampus Hypothalamus

Anterior Pituitary

Adrenal Cortex

Cortisol

CRH

ACTH

Immune system

Thyroid

T3

T4

TRH

TSH

BDNF reducedNeurogenesis

reduced

Nestler et al., 2002; Savitz & Drevets, 2009

INTRODUCTION Immune-inflammatory hypothesis: Immune-inflammatory dysfunction and oxidative stress

are important components in depression and are known to evoke monoaminergic changes.

Changes in nitric oxide-cyclic guanosine monophosphate, altered kynurenine metabolism are known pro-oxidative mechanisms.

Ng et al., 2008; Garcia-Cazorla et al., 2008; Maes et al., 2011; Wegener et al., 2010; Dhir & Kulkarni, 2011; Von Gall et al., 2002

Monoamines

Trophic factors: (BDNF)

Exitotoxicit

y: Glutamate

Inflammation

IDOTRP

QUIN

KYNANeuro

degenerative Neuro

ProtectiveNMDA-

Ragonist NMDA-R

antagonist

Stressful

stimulus

Glutamate release

Pro-inflammat

ory cytokines

NOS-activation

Accumulation of nitrosactive/oxidative mediators

O-

H2O2 2H2

O

SOD

GSH-Px

NO ONNO-

GSH GSSHStructure damage

LPxMDA-relea

se

Ng et al., 2008; Garcia-Cazorla et al., 2008; Maes et al., 2011; Wegener et al., 2010; Dhir & Kulkarni, 2011; Von Gall et al., 2002

AGOMELATINE

Primary node of action

Mechanism

Anxiolytic

Devoid of 5-HT side effects

C Munoz, Servier; Banasr et al., 2006; Racagni et al., 2011

MT1MT2

5-HT2C

The Biological Clock

Mignot E. et al. Nat Neurosci. 2002; 5:1071-5    Turek FE, et al. Arch Neurol. 2001.

Biological, physiological, and behavioral parameters

Suprachiasmatic nuclei (SCN)(Anterior hypothalamus)

SCN 5HT PVN Pineal

Retina

Sleep regulating centreseg. RN, dl-hypothal

Peripheral clocks

Melatonin

Other zeitgebers, egwork schedules

AGOMELATINE These actions have a fundamental effect on frontal cortical

function and as a result on all contributing factors of depression.

Beneficial effects can be attributed to its fronto-cortical DAergic properties and lack of 5-HTergic actions.

Banasr et al., 2006; Racagni et al., 2011; C. Munoz, Servier; Sansone & Sansone. 2010

RECEPTORS The expression of 5-HT2C and MT1 receptors also show a diurnal

rhythmicity in the SCN & HPC. SCN targets in the hypothalamus modulates brain stem monoamine nuclei.

This implying that monoamines are indirectly affected by changes in the SCN.

MT1 & MT2

Hardeland et al., 2011; Millan et al., 2003; De Barardis et al., 2011; Tardito et al., 2012; Racagni et al., 2011; McClung, 2013; Harvey & Slabbert ,2014

Stahl SM. Circuits in Psychopharmacology. In: Stahl’s Essential Psychopharmacology. Neuroscientific Basis and Practical Application. C Cambridge University Press, Cambridge. Third Edition 2008.Chapter 7:195-222.

ANIMAL MODELS Exposing humans or animals to early-life adverse events, such

as maternal separation or isolation, profoundly affects brain development and leads to psychiatric disorders.

The use of animal models: Animal models are needed to identify new drug targets, and in

evaluating causative or mechanistic hypotheses regarding depression.

Early-life SIR of rat pups is known to produce late-life bahavioral and biological changes with the neurodevelopmental hypothesis of depression.Face

validityConstruct

validityPredictive

validityReproduces

clinical symptoms

Theoretical rational

regarding illness

Predict a given response

Fone & Porkess 2008; Pryce & Klause, 2013; Toua et al., 2010; Möller et al., 2011; Möller et al., 2013a

ANIMAL MODELS

SIR Model

Reversed with an AD and an antipsychotic

Behavioral level: Neurophobia Aggression Cognitive rigidity Impaired

sensorimotor + social interaction

Neuro-biological level: Reduced PFC volume Decreased cortical &

hippocampal synaptic plasticity

Monoaminergic deficits

Möller et al., 2013a,b; Giannantonio & Martinotti, 2012

ANIMAL MODELS

In our hands this model demonstrated excellent validity for modeling the neurobiology and behavioral profile for schizophrenia, while other laboratories have established its relevance for depression.

To the best of our knowledge, the ability of agomelatine to reverse SIR-induced bio-behavioral changes has not been undertaken.

Möller et al., 2013a, 2013b; Heidbreder et al., 2000

MY PROJECT

MY PROJECT

Biological rhythms

Immune-inflammatory

Redox-, Resilience

pathways

SIR Behavioral, Endocrine Neurochemi

cal analysisAgomelati

ne

RESEARCH PROBLEM

Antidepressant efficacy is disappointing – 55-60% effective

Treatments have restricted actions on monoaminergic systems.

Circadian rhythm plays a major role to ensure optimal functioning.

Therapeutic target in mood disorders = disrupted circadian rhythm

Optimal AD treatment should act as a resynchronizer

Agomelatine's action involves resynchronizing of circadian rhythms (via regulation of 5HT vs. melatonergic activity in SCN) plus a direct effect on frontal NA/DA release (via 5HT2c antag) plus an indirect action by modifying monoamine release in brain stem (via re-entrainment of biological rhythms). However the exact actions still requires clarification

Melatonin is an antioxidant with recent clinical evidence suggesting the same for agomelatine.

Nestler et al., 2002; Mairesse et al., 2012; Morley-Fletcher et al., 2011; Molteni et al., 2013

AIMS & OBJECTIVES

Face Validity Construct validity

Predictive validity

Reversal of behavioral, neuroendocrine and neurochemical disturbances with antidepressant

• Agomelatine

Monoamine disturbances

Dyregulation of the HPA-axis (corticosterone)

Oxidative stress & Immune-inflammatory dysfunction

Altered kynurenine metabolism

Cognitive changes

Anhedonia

Anxiety Depressive-like

behaviour

How the above actions of agomelatine compares to that of melatonin (MT1 & MT2 agonist) and S32006 (5-HT2C antagonist). And reversing agomelatine’s effects with a melatonin antagonist or

worsening the manifestations.

RESEARCH METHODOLOGY:

ANIMALS

Male Sprague-Dawley rats (300-350g; Vivarium, North West University) randomly allocated to groups – 12 rats/group

PND 21 the animals will be randomized to SIR (1 animal/cage) or social rearing (3-4 animals/cage) for 8 weeks

Animals will be handled according to the code of ethics in research (ethical approval will be obtained for this study).

Möller et al., 2013a

RESEARCH METHODOLOGY:

DRUG TREATMENT

All drugs will be of the highest grade provided by Servier.

Drug treatment will take place during the final 14 days of rearing:

Treatment will be given s.c. at 16h00 in the afternoon.

Treatments

Chronic Agomeltine

40mg/kg

Chronic S32006 (5-HT2C

antagonist)10mg/kg

Chronic Melatonin40mg/kg

Chronic S22153 MT1 & MT2 antagonist20mg/kg

Möller et al ., 2012a; Möller et al., 2013a; Molteni et al., 2013; Schmelting et al., 2014; C Munoz, Servier; Norman et al., 2012; Papp et al., 2010; Mairesse et al., 2012

RESEARCH METHODOLOGY:

STUDY DESIGN

6 Weeks

11 12 13 14

PND 21

Treatment period: 14 days

Expt 1

Behavioral studies

SIR group

Social group

Behavioral Studies

FST & OFTSPT NO

R

RESEARCH METHODOLOGY:

STUDY DESIGN

14

Neurochemical and Neuroendocrine studies

12 Rats per groupThus 240 rats in total

PND 21

6 Weeks

Treatment period: 14 days

Expt 2Neuroendocrine & neurochemical studies

Social group

SIR group

RESEARCH METHODOLOGY:BEHAVIORAL STUDIES

Beh

avio

ral

Stud

ies

Sucrose Preference

TestAnhedonia

Novel Object Recognition

Cognitive impairment

Open Field Test

Forced Swim Test

Depressive-like

symptoms & Anxiety

Möller et al., 2013; Harvey et al., 2010; Lieberberg et al., 2010

RESEARCH METHODOLOGY:BEHAVIORAL STUDIES

Assessment of anhedonia:This is a significant component of depression. Anhedonia will be assessed using the sucrose preference test (SPT) on day 11 & 12.

• Beginning of test, animals will be singly housed for 48h. • 2 Bottles will be available in each cage (200ml sucrose &

200ml tap water)

Preferences will be measured as follows:• Sucrose consumption• Water consumption• Total liquid (sucrose & water)

At the end of the 48h, the bottles will be removed, consumption noted and the animals returned to their previous housing conditions.

Brenez Sáenz, Villagra & Fornaguera Trias, 2006

RESEARCH METHODOLOGY:BEHAVIORAL STUDIES

Cognitive assessmentNovel object recognition will be evaluated on day 13 as an expression of declarative recognition memory.

• Rats will be exposed to a 5-min habituation session in the NOR box, followed by experimental trails where each rat will be exposed to 2 identical objects for 5 min.

• Rats will be returned to their home cages for a 1.5h inter-trail interval.

• Box will be cleaned, both items removed and replaced with 1 identical familiar object and a novel unfamiliar object.

10cm

10cm 10cm

10cm 10cm

10cm 10cm

10cm

RESEARCH METHODOLOGY:BEHAVIORAL STUDIES

Open field test (OFT):Test will be used to measure locomotor and anxiety activity which is a parameter used to test the general ability of the animal to move and negotiate its surrounding.

On the day of testing, rats will be placed in the OFT arena and allowed to explore the arena for 5min under low light. During this time the rat is video-taped.

Scoring: Total number of lines crossed during the session will be used as a measure of general activity, while the number of entries into the central square of the arena will provide a measurement of anxiety-like behavior.

RESEARCH METHODOLOGY:BEHAVIORAL STUDIES

Assessment of depressive-like symptoms:These symptoms will be assessed using the forced swim test (FST) on day 14. On the penultimate day of treatment, 1.e. 24h prior to the final swim test, the rats will be placed in a room to habituate for 60min after which they will be subjected to 15min of pre-swimming in transparent Perspex cylinders (30cm (d) x 40cm (h)) containing 30cm of clean water (25 ˚± 2C).

Final day of treatment, after 20min habituation in thetest room the rats will be assessed in an open field arena to determine locomotor activity.

1h later the rats will be reintroduced to the cylinders fortheir final 5min swim test.

Immobility, climbing, diving and swimming behaviorswill be recorded.

RESEARCH METHODOLOGY:BLOOD COLLECTION

After decapitation, trunk blood will be collected in pre-chilled, 4ml vacutainer tubes, plasma stored at -80 ˚C until the day of analysis.

Peri

pher

al &

N

euro

endo

cri

ne

Corticosterone ELISA Kit

Tryptophan, Kynurenine, KYNA, QA

Validated LCMS

methodSuperoxide dismutase ELISA Kit

Harvey et al., 2006; Möller et al., 2011; 2012b

RESEARCH METHODOLOGY:BRAIN DISSECTION

The brain regions will be snap frozen in liquid

nitrogen and stored at -80 º C until the day of analysis.

Molteni et al., 2013; Toua et al., 2010; Racagni et al., 2011; Paxinos & Watson, 2010; Davidson et al., 2009

RESEARCH METHODOLOGY:BRAIN DISSECTION

Bra

in

diss

ectio

n Reduced vs. oxidized glutathione analysis

Quantification using a LCMS method

Monoamine analysis5-HT, DA, NA and

their metabolites will be analysed using a

HPLC method

Lipid peroxidation analysis

Will be determined by thiobarbituric acid

(TBA) assay.Data will be expessed

as pmol malonialdehyde

formed/mg proteinMöller et al., 2011; 2013a; Harvey et al., 2008

STATISTICAL ANALYSIS:

Statistical analysis will be done with Graphpad Prism 11; SPSS® Software, under supervision of NWU statistical consultation.

Three or two-way ANOVA with Bonferroni post-hoc test will be used to model the body weight, behavioral, blood and neurochemical measurements.

Möller et al., 2013

EXPECTED OUTCOMES

We propose the following outcomes:

1. SIR will induce profound depressive-like symptoms:• Deficits in cognition• Elevated Anxiety• Anhedonia Agomelatine will reverse these symptoms The melatonin agonist may reverse some manifestations

(maybe anxiety) while the melatonin antagonist will worsen the symptoms.

The 5-HT2C antagonist may improve congnition.

2. SIR will present with elevated plasma corticosterone levels

Agomelatine will reverse this effect. Melatonin and the 5-HT2C antagonist will not reverse this

effect. Melatonin antagonist may elevate corticosterone levels.

EXPECTED OUTCOMES

3. SIR will induce reduced regional brain monoamines akin to depression

That will be reversed by agomelatine. That will not be reversed by melatonin That may be partly reversed by the 5-HT2C antagonist. The melatonin antagonist will not reverse this effect, but

worsen this reduction.

4. SIR will induce immune-redox changes: elevated QUIN, regional brain lipid peroxidation and reduced KYNA, SOD activity, and GSH:GSSG ratio, akin to depression

That will be reversed by agomelatine That will not be reversed by melatonin or the 5-HT2C

antagonist. That will be worsened by a melatonin antagonist.

TIME LINE

June 2014 Presentati

on of study and approval

Sept/Oct 2014 Behaviora

l study commences

Jan/Feb 2015 Blood &

neurochemical study commences

July-Oct 2015 Data

analysis & writing up of dissertation

Nov 2015 Submit

dissertation

July 2014 Submissio

n of ethics application

FUNDING

Study will be financed by: NRF & MRC funding awarded to BHH Cost of all drugs will be carried by Servier, Paris,

France.