Participation of the gabaergic system on the glutamate
PARTICIPATION OF THE GABAERGIC SYSTEM ON THEGLUTAMATE RELEASE OF FRONTAL CORTEX SYNAPTOSOMESFROM WISTAR RATS WITH EXPERIMENTAL AUTOIMMUNEENCEPHALOMYELITIS Ankit A. Gilani Department of pharmacology & toxicology NIPERA1113PC03
Flow of seminar• INTRODUCTION• ARTICLE INFORMATION• MATERIALS AND METHODS• RESULTS AND DISCUSSION• CONCLUSION• REFERENCES
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.
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
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)
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.
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.
AIMTo investigate the possible impairment of theGABAergic system and whether it regulatesthe glutamate release and phosphorylation ofsynapsin I in nerve terminals isolated from thefrontal cortex of sick and recovered EAE rats.
Animals and EAE induction Wistar rats intradermal inoculation in both hind feet0.5 ml of an 0.5 ml of theemulsion same emulsionconsisting of 0.25 Without anyml saline solution antigenicand 0.25 ml CFA preparationcontaining 8 mg (CFA group/bovine myelin Control group)(EAE group)
• 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.
• 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.
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
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.
• 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.
Reduction of glutamate release of frontalcortex synaptosomes in EAE animals 4-aminopyridine (4AP)-evoked glutamate release from rat frontal cortex synaptosomes.
Loss of GABAergic inhibition of theglutamate release of synaptosomes fromEAE animals
[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.
• 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.
The flunitrazepam-sensitive GABAA receptordensity was decreased in synaptosomes fromEAE animals There were lower binding sites for flunitrazepam in the EAE rats
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.
• 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.
Loss of GABAergic regulation on Ca2+-dependent phosphorylation of synapsin I innerve terminals from EAE animals CFA EAE EAErecGABA had no effect on the phosphorylation of synapsin I insynaptosomes from EAE and EAErec animals, but induced adecrease in the 4AP-evoked phosphorylation of synapsin I insynaptosomes from CFA rats with respect to control condition.
% activity of control GABA GABA/P CFA 64 ± 11% 80 ± 12% EAE 106 ± 13% 87 ± 10%EAErec 89 ± 19% 90 ± 10%
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.
• 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
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