Depression has a major effect on the hippocampus. Studies have beyond doubt associated depression with reduced level of serotonin in the brain. It is for this reason that most of the treatments for depression are directed towards serotonin concentration enhancement in synaptic clefts. However, only 50% of the patients receiving the treatment responds and in the responding patients, although the rise in serotonin level is rapid, the complete evasion of the depressive symptoms takes weeks to months. However, Ketamine, an N-methyl-D-aspartate (NMDA) receptor antag- onist which leads to activation of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors has a fast and sustained antidepressive eect. So, research towards understanding the mechanism behind depression is now targeting gluta- matergic system too. Clinical evidence suggests decreased -aminobutyric acid (GABA) level in plasma, Cerebro Spinal Fluid (CSF) and brain of depressed patients. The proposed hypothesis is that these agents function together to reverse the biochemical changes due to depression. This experiment involves addition of 5-HT 1A agonist (8-OH DPAT) and 5-HT 2A/2C agonist (DOI) to cultured HT-22 cells and observing changes in m-RNA expression of AMPA receptors (GluR1, GluR2, GluR3 and GluR4), BDNF and GABAAalpha1 receptors.

1. Molecular mechanism underlying Depression: The
relationship between serotonin system and glutamate
system in mouse cortical neurons and HT22 cells
Adiba SHABNAM !
2 September 2014!
!

2. Overview
• Depression: Definition !
• Causes!
• Affected Brain areas!
• Antidepressant: Mechanism !
• Serotonin Theory!
• Glutamate Theory !
• Objective !
• Experimental Method!
• Results!
• Conclusion

4. DEPRESSION
Depression
A common psychiatric
disorder characterized
by low mood, loss of
pleasure, sleep and
appetite changes, poor
concentration and
suicidal tendency

5. 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)

6. Causes of depression
• Genetic or hereditary
• Biochemical - monoamine
• Endocrine factors
• psychological factors

7. Involved Brain Parts

8. Involvement of Cortex in depression
Abnormal metabolism in the prefrontal cortex in various mood disorders

9. Involvement of Hippocampus in depression
Chronic stress selectively reduces
hippocampal volume in rats: a
longitudinal MRI study (Lee, 2009)
Reduced Hippocampal volume (Bremner et al, 2000,Lange et al, 2004; Macmaster et al, 2007;Brien et
al 2004, Sheline et al 1995 )

10. Antidepressant SEROTONIN

11. Serotonin
Theory of Depression
SSRI blocks
5-HT reuptake

12. 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

13. What is the reason for the lag?

14. Glutamate
Theory of Depression
The rapid response of ketamine (Berman et al, 2000)
Involvement of glutamate system in depression

15. Glutamatergic theory
Mechanism of action of
Ketamine!
AMPA-R activation
mTOR signalling pathway
activated in prefrontal cortex
GluR1, Synapsin, spine density
Synaptogenesis
BDNF translation
Kavalali, 2012

16. Glutamate system also has antidepressant effect
Group&I&and&II&mGluR&antagonists&
NMDAR&antagonists&
5HT1A&agonist&
5HT2A/2C&antagonist&
Ac6va6on&of&AMPAR
BDNF
mTORsignaling
Synapseforma6on
5HT
BDNF(hippo,cortex)
An6depressant

17. Glutamate Receptor: classification

18. 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.

19. 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)

20. 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

21. Mechanism of Depression
5HT 2A/2C agonist
5HT,BDNF
NMDA-R antagonist
Group II mGluR
antagonist
AMPA-R activation
Antidepression
mTOR signaling BDNF,
Synaptogenesis
5HT1A agonist

22. Experiment
m-RNA expression of GluR1, GluR2,
GluR3, GluR4, BDNF
5-HT 1A agonist !
(8-OH-DPAT)
5-HT 2A/2C agonist!
(DOI)

23. Experimental methodology
Cortical cell HT-22 cells culture
5-HT 1A 5-HT 2A/2C agonists addition
Total RNA Isolation
Real time PCR Analysis

24. Cell culture and Trypsinization
Cells +S-DMEM
HBSS + 0.05% trypsin-EDTA

25. 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)

26. RNA Isolation
Sample!
+
RNAiso Plus!
+
Chloroform!
+
Isopropanol!
+
RNase free water

27. Absorption Spectrometry
Total RNA
concentration
measurement

28. DNA Removal
Isolated RNA!
+!
RNase free water !
+!
7 * gDNA wipeout buffer
Thermal cycler

29. Reverse Transcription
Template RNA !
+!
RT Primer mix !
+!
Quantiscript Reverse Transcriptase!
+!
Quantiscript RT Buffer!
+!
Placed in thermal cycler!

30. REAL TIME PCR

31. Amplification Plot
Melting curve

32. RESULTS

33. Relative expression: 8-OH-DPAT on cortical cells
GluR2 60.0
Explession
45.0
30.0
Relative 15.0
0.0
8OH_0nM 8OH_1nM 8OH_10nM 8OH_100nM
GluR1
Relative Expression
1.6
1.2
0.8
0.4
0.0
8OH_0nM 8OH_1nM 8OH_10nM 8OH_100nM
Decreased m-RNA expression of GluR1!
!
Increased m-RNA expression of GluR2

34. Relative expression: 8-OH-DPAT on HT-22 cells
GluR3
Relative Expression
1.00E-02
7.50E-03
5.00E-03
2.50E-03
0.00E+00
8OH 0nM 8OH 1nM 8OH 10nM 8OH 100nM
BDNF
Relative Expression
5.00E-02
3.75E-02
2.50E-02
1.25E-02
0.00E+00
8OH 0nM 8OH 1nM 8OH 10nM 8OH 100nM
Decreased m-RNA expression of GluR3 BDNF

35. Relative expression: DOI on HT-22 cells
Relative Expression
1.00E+01
7.50E+00
5.00E+00
2.50E+00
0.00E+00
DOI 0nM DOI 1nM DOI 10nM DOI 100nM
Increased mRNA expression of BDNF

36. 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