Korean J Physiol Pharmacol.  2010 Jun;14(3):127-132. 10.4196/kjpp.2010.14.3.127.

Modulation of Presynaptic GABA Release by Oxidative Stress in Mechanically-isolated Rat Cerebral Cortical Neurons

• Hahm ET
• Seo JW
• Hur J
• Cho YW

• KMID: 2285392
• DOI: http://doi.org/10.4196/kjpp.2010.14.3.127

Abstract

Reactive oxygen species (ROS), which include hydrogen peroxide (H2O2), the superoxide anion (O2-.), and the hydroxyl radical (OH.), are generated as by-products of oxidative metabolism in cells. The cerebral cortex has been found to be particularly vulnerable to production of ROS associated with conditions such as ischemia-reperfusion, Parkinson's disease, and aging. To investigate the effect of ROS on inhibitory GABAergic synaptic transmission, we examined the electrophysiological mechanisms of the modulatory effect of H2O2 on GABAergic miniature inhibitory postsynaptic current (mIPSCs) in mechanically isolated rat cerebral cortical neurons retaining intact synaptic boutons. The membrane potential was voltage-clamped at -60 mV and mIPSCs were recorded and analyzed. Superfusion of 1-mM H2O2 gradually potentiated mIPSCs. This potentiating effect of H2O2 was blocked by the pretreatment with either 10,000-unit/mL catalase or 300-micrometer N-acetyl-cysteine. The potentiating effect of H2O2 was occluded by an adenylate cyclase activator, forskolin, and was blocked by a protein kinase A inhibitor, N-(2-[p-bromocinnamylamino] ethyl)-5-isoquinolinesulfonamide hydrochloride. This study indicates that oxidative stress may potentiate presynaptic GABA release through the mechanism of cAMP-dependent protein kinase A (PKA)-dependent pathways, which may result in the inhibition of the cerebral cortex neuronal activity.

Keyword

Gamma-aminobutyric acid; Hydrogen peroxide; Oxidative stress; Inhibitory postsynaptic potentials

MeSH Terms

Adenylyl Cyclases
Aging
Animals
Catalase
Cerebral Cortex
Cyclic AMP-Dependent Protein Kinases
Forskolin
gamma-Aminobutyric Acid
Hydrogen Peroxide
Hydroxyl Radical
Inhibitory Postsynaptic Potentials
Membrane Potentials
Neurons
Oxidative Stress
Parkinson Disease
Presynaptic Terminals
Rats
Reactive Oxygen Species
Superoxides
Synaptic Transmission
Catalase
Cyclic AMP-Dependent Protein Kinases
Forskolin
Hydrogen Peroxide
Hydroxyl Radical
Reactive Oxygen Species
Superoxides
gamma-Aminobutyric Acid

Figure

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  Fig. 1. GABAergic mIPSCs recorded from mechanically dissociated cerebral cortical neurons. In the presence of 300 nM TTX, 3 μM CNQX, and 10 μM AP5, superfusion of 50 μM bicuculline completely and reversibly blocked mIPSCs in isolated cerebral cortical neurons. Holding voltage was –60 mV. Intracellular and extracellular Cl– concentrations were 140 mM and 161 mM, respectively. Asterisks represent a statistically significant difference (∗∗p<0.01).

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  Fig. 2. Effect of H2O2 on GABAergic mIPSCs. Each recording trace is the representative current trace of GABAergic mIPSCs recorded before (control), during and after the superfusion of 1 mM H2O2. The bar histograms show the mean±S.E.M. of the relative frequency and amplitude of mIPSCs. All frequencies and amplitudes are normalized to those of control mIPSCs. ∗∗p<0.01.

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  Fig. 3. Effect of NAC on the potentiating of H2O2 on GABAergic mIPSCs. Each recording trace shows the control current trace, the current trace recorded during the pretreatment with 300 μM NAC, and the current trace recorded during the superfusion of 1 mM H2O2 in the presence of NAC. Bar histograms show the mean± S.E.M. of the relative frequency and amplitude of mIPSCs recorded in the above conditions. The pretreatment with NAC suppressed the potentiating effect of H2O2 on the frequency and amplitude of mIPSCs.

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  Fig. 4. Effect of catalase on the potentiating of H2O2 on GABAergic mIPSCs. Each recording trace shows the control current trace, the current trace recorded during the pretreatment with 1,000-unit catalase, and the current trace recorded during the superfusion of 1 mM H2O2 in the presence of catalase. Bar histograms show the mean±S.E.M. of the relative frequency and amplitude of mIPSCs recorded in the above conditions. The pretreatment with catalase suppressed the potentiating effect of H2O2 on the frequency and amplitude of mIPSCs.

• 

  Fig. 5. Effect of forskolin on the potentiating of H2O2 on GABAergic mIPSCs. Each recording trace shows the control current trace, the current trace recorded during the superfusion of 10 μM forskolin, and the current trace recorded during the superfusion of 1 mM H2O2 in the presence of forskolin. Bar histograms show the mean± S.E.M. of the relative frequency and amplitude of mIPSCs recorded in the above conditions. Forskolin significantly potentiated the frequency of mIPSCs without any alteration of amplitude. In the presence of forskolin, H2O2 did not significantly alter the frequency and amplitude of mIPSCs. ∗p<0.05.

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  Fig. 6. Effect of H-89 on the potentiating of H2O2 on GABAergic mIPSCs. Each recording trace shows the control current trace, the current trace recorded during the superfusion of 100 nM H-89, and the current trace recorded during the superfusion of 1 mM H2O2 in the presence of H-89. Bar histograms show the mean±S.E.M. of the relative frequency and amplitude of mIPSCs recorded in the above conditions. H-89 did not significantly alter the frequency and amplitude of mIPSCs. In the presence of H-89, H2O2 did not significantly alter the frequency and amplitude of mIPSCs.

Reference

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