Journal List > Korean J Physiol Pharmacol > v.13(6) > 1025643

Lee: Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons

Abstract

The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3′,5′-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethy-limidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K+ channels by 4-aminopyridine, a K+ channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca2+ channels by Cd2+, a general voltage-dependent Ca2+ channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca2+ channels in the presynaptic nerve terminals of A1 neurons.

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Fig. 1.
GABAergic sIPSCs recorded from mechanically dissociated A1 neurons. (A) A representative trace of sIPSCs recorded before, during, and after the application of 50 μM bicuculline at a VH of –60 mV. The external solution contained 300 nM TTX, 3 μM CNQX and 10 μM AP5. Insets represent typical traces with an expanded time scale. (B) Traces of sIPSCs recorded at VH of –60, –40, 0, and +40 mV. Intracellular and extracellular Cl concentrations were 140 mM and 161 mM, respectively. I-V curve for the mean amplitude of sIPSCs recorded at various VHs. Each point is the mean of four neurons.
kjpp-13-461f1.tif
Fig. 2.
SNAP decreases GABAergic sIPSCs by presynaptic mechanisms. (A) A representative trace of sIPSCs recorded before, during, and after the application of 100 μM SNAP. Insets, represent typical traces with an expanded time scale (lower) and SNAP-induced changes in the rate of rise and decay time constant (upper). (B) Cumulative probability distributions for inter-event interval (left) and amplitude (right) of GABAergic sIPSCs recorded from the same neuron. p values indicate the results of K-S tests for frequency and amplitude. (C) Each column is the mean of 20 neurons. All frequencies and amplitudes are normalized to those of control sIPSCs. Asterisks represent a statistically significant difference (p<0.05, paired two-tailed t-test).
kjpp-13-461f2.tif
Fig. 3.
SNAP-induced presynaptic inhibition of GABAergic sIPSCs is coupled to the cGMP-dependent signal transduction pathway. (A) A representative trace of sIPSCs recorded before, during, and after the application of 100 μM 8-Br-cGMP. Insets, represent typical traces with an expanded time scale (lower) and 8-Br-cGMP-induced changes in the rate of rise and decay time constant (upper). (B) Cumulative probability distributions for inter-event interval (left) and amplitude (right) of GABAergic sIPSCs recorded from the same neuron. p values indicate the results of K-S tests for frequency and amplitude. (C) Each column is the mean of 6 neurons. All frequencies and amplitudes are normalized to those of control sIPSCs. Asterisks represent a statistically significant difference (p<0.05, paired two-tailed t-test).
kjpp-13-461f3.tif
Fig. 4.
Effect of carboxy-PTIO on the SNAP-induced inhibition of GABAergic sIPSCs. (A) Representative recording traces of sIPSCs observed before and during the application of 100 μM SNAP in the absence or presence of 1 μM carboxy-PTIO. Insets represent typical traces with an expanded time scale. (B) Cumulative distributions for inter-event interval (left) and amplitude (right) of sIPSCs recorded from the same neuron. p values indicate the results of K-S tests for frequency and amplitude. (C) Each column is the mean of 5 neurons. All frequencies and amplitudes are normalized to those of control sIPSCs.
kjpp-13-461f4.tif
Fig. 5.
Effect of the 4-aminopyridine on the SNAP-induced inhibition of GABAergic sIPSCs. (A) Representative recording traces of sIPSCs show the change in the SNAP effect in the presence of 100 μM 4-AP, a K+ channel blocker. Insets represent typical traces with an expanded time scale. (B) Cumulative distributions for inter-event interval (left) and amplitude (right) of sIPSCs in the same neuron. p values indicate the results of K-S tests for frequency and amplitude. (C) All amplitudes and frequencies are normalized to the control sIPSCs. Each column is the mean of 9 neurons. Asterisks represent a statistically significant difference (p<0.05, paired two-tailed t-test).
kjpp-13-461f5.tif
Fig. 6.
SNAP-induced inhibition of GABAergic sIPSCs is related to the presynaptic VDCCs. (A) Representative recording traces of sIPSCs shows the change in the SNAP effect in the external solution containing 100 μM Cd2+, a non-selective Ca2+ channel blocker. Insets represent typical traces with an expanded time scale. (B) Cumulative distributions for inter-event interval (left) and amplitude (right) of sIPSCs in the same neuron. p values indicate the results of K-S tests for frequency and amplitude. (C) All amplitudes and frequencies are normalized to the control sIPSCs. Each column is the mean of 6 neurons. Asterisks represent a statistically significant difference (p<0.05, paired two-tailed t-test). n.s indicates p>0.05.
kjpp-13-461f6.tif
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