1. Study leader: Prof. B.H. Harvey
Assisting study leader: Dr. M. Möller-Wolmarans
Collaborators: Dr. G. Wegener
M.Sc. Student: Dewald E. Coutts
Behavioral, neuroendocrine
and neurochemical studies on agomelatine
in social isolation reared rats
3. 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
5. 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
7. 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
8. 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
9. STRESS
BDNF
Sprouting of 5-HT fibers, accelerate
regrowth
Modifies HPA-axis
Naert et al., 2011; Souêtre et al., 1989
Hsiao et al., 2010; Doane et al., 2013
10. INTRODUCTION
Circadian 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
11. Recovered ControlDepressed
Sleep
period
7 9 11 13 1517 192123 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
13. 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
15. 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
17. AGOMELATINE
Primary node of action
Mechanism
Anxiolytic
Devoid of 5-HT side
effects
C Munoz, Servier; Banasr et al., 2006; Racagni et al., 2011
18. MT1 MT2
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 centres
eg. RN, dl-hypothal
Peripheral clocks
Melatonin
Other zeitgebers, eg
work schedules
19. 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
20. 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.
21. 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
22. 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.
23. 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 validity Construct validity Predictive validity
Reproduces
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
24. 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
25. 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
27. 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
28. 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.
29. 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
30. 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 Melatonin
40mg/kg
Chronic S22153
MT1 & MT2 antagonist
20mg/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
31. 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 NOR
32. RESEARCH METHODOLOGY:
STUDY DESIGN
14
Neurochemical and
Neuroendocrine studies
12 Rats per group
Thus 240 rats in total
PND 21
6 Weeks
Treatment period: 14 days
Expt 2
Neuroendocrine &
neurochemical
studies
Social
group
SIR
group
34. 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
35. RESEARCH METHODOLOGY:
BEHAVIORAL STUDIES
Cognitive assessment
Novel 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
36. 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.
37. 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 the
test 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 for
their final 5min swim test.
Immobility, climbing, diving and swimming behaviors
will be recorded.
38. 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.
Peripheral&
Neuroendocrine
Corticosterone ELISA Kit
Tryptophan,
Kynurenine, KYNA,
QA
Validated LCMS
method
Superoxide
dismutase ELISA Kit
Harvey et al., 2006; Möller et al., 2011; 2012b
39. 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
40. RESEARCH METHODOLOGY:
BRAIN DISSECTION
Braindissection
Reduced vs. oxidized
glutathione analysis
Quantification using a LCMS
method
Monoamine analysis
5-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 protein
Möller et al., 2011; 2013a; Harvey et al., 2008
41. 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
42. 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.
43. 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.
44. TIME LINE
June 2014
Presentation
of study and
approval
Sept/Oct 2014
Behavioral
study
commences
Jan/Feb 2015
Blood &
neurochemical
study commences
July-Oct 2015
Data analysis &
writing up of
dissertation
Nov 2015
Submit
dissertation
July 2014
Submission
of ethics
application
45. FUNDING
Study will be financed by:
NRF & MRC funding awarded to BHH
Cost of all drugs will be carried by Servier, Paris, France.
