Protein kinase inhibitors reduce GABA but not glutamate release in the nucleus accumbens

Abstract

We investigated the role of endogenous protein kinase activity on synaptic transmission in the rat nucleus accumbens slice. The isoquinolinesulfonamide H-7 (50muM), a non-selective serine/threonine protein kinase inhibitor, had no effect on pharmacologically isolated glutamatergic EPSCs. However, it reduced GABA release in a dose-dependent manner. This effect of H-7 was not mimicked by the selective cAMP-dependent protein kinase inhibitor H-89, the PKC inhibitor Bisindolylmaleimide-1, or the cGMP-dependent protein kinase inhibitor KT5823. However, bath application of the myosin light chain kinase (MLCK) inhibitor, ML-7, significantly reduced IPSC amplitudes and partially occluded the reduction in IPSCs observed following bath application of H-7. These results suggest that endogenous protein kinase activity, specifically MLCK activity, regulates GABA, but not glutamate release, onto medium spiny neurons in the nucleus accumbens.

Figure 1

Inhibiting protein kinase activity reduces IPSC but not EPSC amplitudes. A. Bath application of 50 μM H-7 has no effect on EPSC amplitudes. Averaged EPSCs from a representative experiment for control and H-7 conditions are displayed above. B. Bath application of 50 μM H-7 produces a rapid reduction in IPSC amplitudes. Averaged EPSCs from a representative experiment are shown. C. Dose response curve of the H-7 effect on IPSCs. The dotted line shows a sigmoid fit to the data. The experimental n for each dose are provided in parenthesis. D. Bath application of 1 μM staurosporine inhibited IPSC amplitudes but had no effect on EPSC amplitudes * indicates p < 0.01

Figure 2

H-7 reduces the frequency of mIPSCs. A. Representative traces of mIPSCs recorded either before (Control) or after bath application of H-7 from an individual experiment. B. Cumulative probability distribution of mini amplitudes in control (solid line) and H-7 (dashed line) for the same experiment. C. Cumulative distribution of the inter-event intervals in control and H-7 for the same experiment. D. Average of four experiments shows a significant reduction in frequency but not in the mean amplitude of mIPSCs. * indicates p < 0.005.

Figure 3

Intracellular application of H-7 does not inhibit IPSCs. IPSC amplitude (normalized to the first two minutes) recorded for 10 min with 50 μM H-7 present in the internal solution. Subsequent bath application of 50 μM H-7 (solid bar) inhibited the IPSCs.

Figure 4

The MLCK inhibitor, ML-7 inhibits IPSC amplitudes. A. Bath application of the PKA inhibitor H-89 (5 μM) had no effect on IPSC amplitudes. B. Bath application of the PKC inhibitor Bis (1 μM) had no effect on IPSC amplitudes. C. Bath application of the PKG inhibitor KT5823 (1 μM) had no effect on IPSC amplitudes. D. Bath application of the MLCK inhibitor ML-7 (10 μM) significantly reduced IPSC amplitudes.

Figure 5

Preincubation with ML-7 partially occludes the H-7 mediated inhibition of IPSCs. Slices were incubated for 1 hour in control, 1 μM Bis, 5 μM H-89, 1 μM KT5823 or 10 μM ML-7. IPSC amplitudes before and after bath application of 50uM H-7 are compared and % reduction for each pretreatment is shown. * indicates p < 0.01 compared to control (one way ANOVA with Holms-Sidak posthoc test).