molecular mechanism underlying depression: the relationship between serotonin system and glutamate...
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Molecular mechanism underlying Depression: The relationship between serotonin system and glutamate system in mouse cortical neurons and HT22 cells
Adiba SHABNAM !2 September 2014!!
Overview
• Depression: Definition !
• Causes!
• Affected Brain areas!
• Antidepressant: Mechanism !
• Serotonin Theory!
• Glutamate Theory !
• Objective !
• Experimental Method!
• Results!
• Conclusion
DEPRESSION
A common psychiatric disorder characterized by low mood, loss of pleasure, sleep and appetite changes, poor concentration and suicidal tendency
Depression
Why to study depression?• Most common psychiatric
disorder • All age group affected (max.
40-59 yrs) • Women are more at risk • Lifetime prevalence 16.2 %
(USA) • Only 20 % people experience
the symptoms of depression. • Maximum global burden of
disease in 2030 (WHO, 2008)
Causes of depression
• Genetic or hereditary • Biochemical - monoamine • Endocrine factors • psychological factors
Involved Brain Parts
Involvement of Cortex in depression
Abnormal metabolism in the prefrontal cortex in various mood disorders
Involvement of Hippocampus in depression
Reduced Hippocampal volume (Bremner et al, 2000,Lange et al, 2004; Macmaster et al, 2007;Brien et al 2004, Sheline et al 1995 )
Chronic stress selectively reduces hippocampal volume in rats: a longitudinal MRI study (Lee, 2009)
Antidepressant SEROTONIN
Sero
toni
n Th
eory
of De
pres
sion
SSRI blocks 5-HT reuptake
Serotonin theory• Most widely accepted hypothesis • Depletion of 5-HT in depressed patients • Antidepressants like SSRI inhibits its re-uptake and
increases its concentration in the synaptic cleft. !
LIMITATIONS • Not all depressed patients (50%) responds to 5-HT
agonists and SSRI. (Mahar et al, 2014) • Delayed onset of effect (2 weeks) • A reduction in 5-HT level does not precipitate in
depressive phenotype in healthy individuals
What is the reason for the lag?
Glut
amat
e Th
eory
of De
pres
sion
Involvement of glutamate system in depression
The rapid response of ketamine (Berman et al, 2000)
Glutamatergic theoryMechanism of action of
Ketamine! AMPA-R activation
mTOR signalling pathway activated in prefrontal cortex
GluR1, Synapsin, spine density Synaptogenesis BDNF translation
Kavalali, 2012
Glutamate system also has antidepressant effect
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An6depressant�
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Glutamate Receptor: classification
Glutamate system is related to serotonin system
• Dosing of fluoxetine (SSRI) changed AMPA mRNA expression and AMPA receptor phosphorylation in frontal cortex and hippocampus (Barbon et al, 2006)
• AMPA knockout mice displayed decreased serotonin levels.
• Serotonin may mediate the antidepressant effects of ketamine.
5HT1A & 5HT2A/2C receptors in depression
5HT1A R • 5HT-1a R agonists (8-OH-DPAT) produce antidepressant
like effect (Cryan, 2005). • 5HT-1A auto-receptor desensitize with antidepressant drugs. • Presynaptic 5HT1A R (autoreceptor): risk for depression • Postsynaptic 5HT1A R : produce antidepressant effect • 5HT-1A R mediates ketamine effect (Fukumoto et al, 2014) 5HT2A R • Antidepressant drugs block 5-HT2A R mediated responses
(Celada et al, 2004)
Objective of the experiment
Investigation of the effect of 5-HT1A agonist and 5-HT 2A/2C agonist on m-RNA expression of AMPA-R (GluR1, GluR2, GluR3, GluR4), and BDNF
5HT 2A/2C agonist
5HT,BDNF
Group II mGluR antagonist
NMDA-R antagonist
AMPA-R activation mTOR signaling BDNF, Synaptogenesis
Antidepression
5HT1A agonist
Mechanism of Depression
Experiment
m-RNA expression of GluR1, GluR2, GluR3, GluR4, BDNF
5-HT 1A agonist !(8-OH-DPAT)
5-HT 2A/2C agonist!(DOI)
Experimental methodology
Cortical cell & HT-22 cells culture
5-HT 1A & 5-HT 2A/2C agonists addition
Total RNA Isolation
Real time PCR Analysis
Cell culture and TrypsinizationCells +S-DMEM
HBSS + 0.05% trypsin-EDTA
Drug Addition: Serotonin Agonist
8-OH-DPAT and DOI at 4 different concentrations
!8-OH DPAT
(0 nM) 8-OH DPAT
(1nM)8-OH DPAT
(10nM)8-OH DPAT
(100nM)
DOI (0 nM)
DOI (1 nM)
DOI (10 nM)
DOI (100 nM)
After 4 hours (HT22 cells)
RNA IsolationSample!
+ RNAiso Plus!
+ Chloroform!
+ Isopropanol!
+ RNase free water
Absorption Spectrometry
Total RNA concentration measurement
DNA Removal
Thermal cycler
Isolated RNA!+!
RNase free water !+!
7 * gDNA wipeout buffer
Reverse Transcription
Template RNA !+!
RT Primer mix !+!
Quantiscript Reverse Transcriptase!+!
Quantiscript RT Buffer!+!
Placed in thermal cycler!
REAL TIME PCR
Amplification Plot
Melting curve
RESULTS
Relative expression: 8-OH-DPAT on cortical cells
GluR2
Rela
tive
Expl
essio
n0.0
15.0
30.0
45.0
60.0
8OH_0nM 8OH_1nM 8OH_10nM 8OH_100nM
GluR1
Rela
tive
Expr
essio
n
0.0
0.4
0.8
1.2
1.6
8OH_0nM 8OH_1nM 8OH_10nM 8OH_100nM
Decreased m-RNA expression of GluR1!&!
Increased m-RNA expression of GluR2
Relative expression: 8-OH-DPAT on HT-22 cells
GluR3
Rel
ativ
e Ex
pres
sion
0.00E+00
2.50E-03
5.00E-03
7.50E-03
1.00E-02
8OH 0nM 8OH 1nM 8OH 10nM 8OH 100nM
BDNF
Rel
ativ
e Ex
pres
sion
0.00E+00
1.25E-02
2.50E-02
3.75E-02
5.00E-02
8OH 0nM 8OH 1nM 8OH 10nM 8OH 100nM
Decreased m-RNA expression of GluR3 & BDNF
Rel
ativ
e Ex
pres
sion
0.00E+00
2.50E+00
5.00E+00
7.50E+00
1.00E+01
DOI 0nM DOI 1nM DOI 10nM DOI 100nM
Relative expression: DOI on HT-22 cells
Increased mRNA expression of BDNF
Conclusion • Decreased mRNA expression of GluR1 and increased mRNA expression of GluR2 by 8-OH-DPAT (Cortex)!
• Decreased mRNA expression of GluR3 and BDNF by 8-OH-DPAT(HT22 cells)!
• Increased mRNA expression of GluR3 and BDNF by DOI (HT22 cells)!
• No amplification of GluR1, GluR2, GluR4,(HT22 Cells)!!SHORTFALLS!
• Manual error ? • defective primers ? (>1 year) • cDNA of HT-22 cells got
modified.Interaction between GluR and cDNA of HT22 cells
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