A SEMINAR BASED ON A RESEARCH ARTICLE

1. PARTICIPATION OF THE GABAERGIC SYSTEM ON THE
GLUTAMATE RELEASE OF FRONTAL CORTEX SYNAPTOSOMES
FROM WISTAR RATS WITH EXPERIMENTAL AUTOIMMUNE
ENCEPHALOMYELITIS
Ankit A. Gilani
Department of pharmacology & toxicology
NIPERA1113PC03

2. Flow of seminar
• INTRODUCTION
• ARTICLE INFORMATION
• MATERIALS AND METHODS
• RESULTS AND DISCUSSION
• CONCLUSION
• REFERENCES

3. MULTIPLE SCLEROSIS
• Multiple sclerosis (MS) known as disseminated
sclerosis or encephalomyelitis disseminata) is an
autoimmune inflammatory disease in which the
fatty myelin sheaths around the axons of
the brain and spinal cord are damaged, leading
to demyelination and scarring as well as a broad
spectrum of signs and symptoms.

4. MS
• Disease onset usually occurs in young adults, and
it is more common in women.
• It has a prevalence that ranges between 2 and
150 per 100,000.*
• MS was first described in 1868 by Jean-Martin
Charcot.
* Rosati G (April 2001). "The prevalence of multiple sclerosis in the world: an
update". Neurol. Sci. 22 (2): 117–39

6. Classification
1. progressive relapsing,
2. secondary progressive,
3. primary progressive, and
4. relapsing remitting.

9. Causes
• Genetics
• Environmental factors
1. Decreased sunlight exposure (decreased vit. D production)
2. severe stress
3. smoking
4. occupational exposure and toxins
• Infections

10. Pathophysiology
• Blood-brain barrier breakdown
• Autoimmunology
 Lesions
 Inflammation

11. Treatment
There is no known cure for multiple sclerosis at this time.
However, there are therapies that may slow the disease.
Medications used to slow the progression of multiple
sclerosis are taken on a long-term basis, they include:
• Interferons
• glatiramer acetate (Copaxone)
• mitoxantrone (Novantrone)
• natalizumab (Tysabri)
• Fingolimod (Gilenya )
• Methotrexate, azathioprine (Imuran), intravenous
immunoglobulin (IVIg) and cyclophosphamide (Cytoxan)

12. GLUTAMATE
• Glutamate is the major excitatory amino acid
transmitter within the CNS, with its signaling
being mediated by a number of postsynaptic
ionotropic and metabotropic receptors.
• The central role played by glutamate receptors
in mediating excitotoxic neuronal death in
stroke, epilepsy, trauma, and MS has been
well established.

13. GABA (γ-aminobutyric acid)
• GABA is the major inhibitory neurotransmitter
balanced with glutamate in the CNS.
• GABAA receptors, a large and diverse family of
Cl- permeable ion channels, mediate fast
transmission at inhibitory GABAergic synapses
and are critical for the development and
coordination of the neuronal activity
underlying the majority of physiological and
behavioral processes in the brain.

15. AIM
To investigate the possible impairment of the
GABAergic system and whether it regulates
the glutamate release and phosphorylation of
synapsin I in nerve terminals isolated from the
frontal cortex of sick and recovered EAE rats.

16. MATERIALS
• Myelin (purified from bovine spinal cords)
• Complete Freund’s adjuvant (CFA)
• GABA
• glutamate dehydrogenase (EC 1.4.1.3)
• NADP+
• 4-aminopyridine (4AP)
• [3H]-flunitrazepam
• Percoll
• Diazepam
• Picrotoxin
• Synapsin I-specific antibody (AB1543P) and synapsin
phosphorylation state P-site 1 (Ser-9) antibody (AB5881)

17. METHODS FOLLOWED BY
RESULTS AND DISCUSSION

18. Animals and EAE induction
Wistar rats
intradermal inoculation
in both hind feet
0.5 ml of an 0.5 ml of the
emulsion same emulsion
consisting of 0.25 Without any
ml saline solution antigenic
and 0.25 ml CFA preparation
containing 8 mg (CFA group/
bovine myelin Control group)
(EAE group)

19. • About 85% of the animals from the EAE group developed a
monophasic course (acute stage, 11–13 dpi)
• Animals were assessed daily for clinical signs of EAE and
scored as follows:
0 - no clinical expression of the disease
1 - flaccid tail
2 - hind limb weakness
3 - complete hind leg paralysis
accompanied by urinary incontinence
4 - quadriparesis, moribund state, or
death.

20. • Control and sick EAE animals were decapitated
at 24–36 h after onset of the disease.
• Also, the CFA and EAE rats completely
recovered from any clinical signs (EAErec)
were sacrificed between 20 and 22 dpi.

21. Preparation of cerebrocortical synaptosomes
• The frontal cortex was isolated
• synaptosomes were purified on discontinuous Percoll
gradients
• Synaptosomes which sedimented between the 10 and 23%
Percoll bands were collected
• Diluted in a final volume of 30 ml of HEPES buffer medium, pH
7.4.
• centrifugation at 27,000 g for 10 min at 4 °C.
• pellets thus formed were resuspended in 5 ml of HEPES buffer
medium
• the protein content was determined by the Bradford assay

22. Glutamate release assay
• Glutamate release from cerebrocortical
synaptosomes was monitored online, using an
assay employing exogenous glutamate
dehydrogenase and NADP+ to couple the
oxidative decarboxylation of the released
glutamate.
• Then, the generated NADPH was detected
fluorometrically.

23. • synaptosomal pellets were resuspended in HEPES buffer
medium and incubated in a stirred and thermostated cuvette
maintained at 37 °C Spectrofluorimeter .
• 1 mM NADP+, 50 units/ml glutamate dehydrogenase, and 1.2
mM CaCl2 were added after 3 min. After 5 min of
incubation, 3 mM 4AP was added to stimulate the glutamate
release.
• Where indicated, synaptosomes were incubated in the
presence of GABA (500 μM) for 4 min or GABA (500 μM) plus
picrotoxin (100 μM) for 10 min prior to the addition of 4AP.
• Data points were obtained at 1-s intervals.

24. Reduction of glutamate release of frontal
cortex synaptosomes in EAE animals
4-aminopyridine
(4AP)-evoked
glutamate
release from rat
frontal cortex
synaptosomes.

25. Loss of GABAergic inhibition of the
glutamate release of synaptosomes from
EAE animals

28. [3H]-flunitrazepam binding assay
• The specific binding of [3H]-flunitrazepam
was measured by a filtration technique .
• Binding was carried out in the presence of radioligand at
final concentrations of 0.5, 1, 2, 3, 4, 5, 8, and 9 nM, at 4 °C.
• Each assay was performed in triplicate using 1-ml aliquots
containing 0.3 mg of proteins from the synaptosomal
fractions.
• After 60 min of incubation, samples were filtered under
vacuum through Whatman filters using a Brandel M-24
filtering manifold.

29. • Samples were washed three times with 4 ml of ice-cold
Tris–HCl buffer (50 mM, pH 7.4) and the radioactivity
was measured using an LKB-1214-RackBeta counter.
• The values Kd and Bmax were obtained by using the
following equation:
Y = Bmax * X/ (Kd+X),
where Bmax = maximal binding, and
Kd = concentration of ligand required
to reach half-maximal binding.

30. The flunitrazepam-sensitive GABAA receptor
density was decreased in synaptosomes from
EAE animals
There were lower binding sites for
flunitrazepam in the EAE rats

31. Immunoblot analysis
• Synaptosomal samples were resuspended in HEPES
buffer medium, 1.2 mM CaCl2 was added, and samples
were incubated at 37 °C for 2 min with stirring.
• This was followed by a further incubation with 3 mM
4AP for 5 min in order to stimulate Ca2+- dependent
synapsin I phosphorylation concomitant to the
glutamate release.
• Where indicated, synaptosomes were incubated in the
presence of GABA (500 M) for 4 min or GABA (500 M)
plus picrotoxin (100 M) for 10 min prior to the addition
of 4AP.

32. • Aliquots were rapidly solubilized in sample buffer, and
subjected to SDS-PAGE and then electrotransferred onto
nitrocellulose membranes.
• Immunoblotting was performed at a 1:500 dilution of the
synapsin phosphorylation state-specific antibody to P-site
1 and at a 1:1000 dilution of synapsin I-specific antibody
for detected total synapsin I.
• The immunoreactive bands in the immunoblot were
detected by infrared probe-labeled secondary antibodies
and the fluorescence was then analyzed by the Odyssey
scanner with the fluorescence intensity being quantified
by the GelPro analyzer software.

33. Loss of GABAergic regulation on Ca2+-
dependent phosphorylation of synapsin I in
nerve terminals from EAE animals
CFA EAE EAErec
GABA had no effect on the phosphorylation of synapsin I in
synaptosomes from EAE and EAErec animals, but induced a
decrease in the 4AP-evoked phosphorylation of synapsin I in
synaptosomes from CFA rats with respect to control condition.

34. % activity of control
GABA GABA/P
CFA 64 ± 11% 80 ± 12%
EAE 106 ± 13% 87 ± 10%
EAErec 89 ± 19% 90 ± 10%

35. CONCLUSION
• The changes observed in the EAE frontal cortex
suggest a role plated by alterations of the
GABAergic system in the EAE cortical pathology.
• The decrease of the GABAA receptor density in
nerve terminals from this cortical region and the
failure of GABAergic regulation on glutamate
release and synapsin I phosphorylation in
synaptosomes from EAE rats may have
contributed to clinical symptoms and disease
progression.

36. • These findings could also have implications for
the neuronal and synaptic dysfunction in EAE
and possibly in MS cortex.
• Further studies, however, are needed to
determine whether GABAergic transmission
modulation could be successful in MS therapy

37. SUMMARY
Neuronal GABA
membrane
depolarization EAE
and Ca2+influx SYNAPTOSOMES
Binds to GABAA
receptor
Ca2+-dependent
phosphorylation of
synapsin 1
Excitotoxic
neuronal
damage
Glutamate release

38. References
• Bhat R, Axtell R, Mitra A, Miranda M, Lock C, Tsien RW, Steinman L (2010)
Inhibitory role for GABA in autoimmune inflammation. Proc Natl Acad Sci
U S A 107:2580–2585.
• Bhatt S, Zalcman S, Hassanain M, Siegel A (2005) Cytokine modulation of
defensive rage behavior in the cat: role of GABAA and interleukin-2
receptors in the medial hypothalamus. Neuroscience 133:17–28.
• Bolton C, Paul C (2006) Glutamate receptors in neuroinflammatory
demyelinating disease. Mediators Inflamm 2006:1–12. ID 93684.
• Calabrese M, Rinaldi F, Mattisi I, Grossi P, Favaretto A, Atzori M, Bernardi V,
Barachino L, Romualdi C, Rinaldi L, Perini P, Gallo P (2010) Widespread
cortical thinning characterizes patients with MS with mild cognitive
impairment. Neurology 74:321–328.

39. References
• Cesca F, Baldelli P, Valtorta F, Benfenati F (2010) The synapsins: key actors
of synapse function and plasticity. Prog Neurobiol 91:313–348.
• Chalifoux JR, Carter AG (2011) GABAB receptor modulation of voltage-
sensitive calcium channels in spines and dendrites. J Neurosci 31:4221–
4232.

40. Thank yo