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. Author manuscript; available in PMC: 2016 Mar 30.
Published in final edited form as: Cell Rep. 2016 Mar 17;14(12):2774–2783. doi: 10.1016/j.celrep.2016.02.069

Figure 1. KOR activation inhibits glutamate transmission in the BNST.

Figure 1

(A) Representative experiment demonstrating KOR-mediated inhibition of eEPSC amplitude. Inset, eEPSC trace from the same neuron showing pre (black) and post (red) 1μM U69,593 application. Scale bar represents 200pA by 20msec. (B) KOR activation by U69,593 inhibited eEPSC amplitude (red circles, paired t-test, baseline v. min 21-25, t4 = 30.70, P < 0.001) and was blocked by continuous application of the KOR antagonist norBNI, 100nM (yellow circles, paired t-test, baseline v. min 21-25, t4 = 0.003, P > 0.05); the KOR agonist U69,593 significantly inhibited eEPSC amplitude as compared to the norBNI block effect (unpaired t-test, acsf v. norBNI, min 16-20, t8 = 10.22, P < 0.001). (C) Representative experiment demonstrating KOR inhibition by 300nM Dynorphin-A. (D) KOR activation by 300nM Dynorphin-A produces a robust inhibition of eEPSCs (red circles, paired t-test, baseline v. min 21-25, t4 = 18.65, P < 0.001) that is blocked by the KOR antagonist norBNI (yellow circles, paired t-test, baseline v. min 21-25, t4 = 2.783, P = 0.05) mimicking the results seen with U69,593 (Fig. 1B). Both U69,593 (E) and Dynorphin-A (F) activation of KORs are non-reversible forms of inhibition. Post U69,593 application of the KOR antagonist norBNI (100nm) failed to reverse the inhibition by either KOR agonist (U69,593, paired t-test, baseline v. min 21-25, t4 = 13.88, P < 0.001; Dynorphin-A, paired t-test, baseline v. min 21-25, t4 = 14.30, P < 0.001). (G) The p38 inhibitor SB203580 (10μM) but not the MEK/ERK inhibitor SL-327 (20μM) blocked KOR-mediated inhibition of eEPSCs (SB203580 effect, baseline v. min 16-20, t4 = 2.619, P > 0.05; SL-327 effect, baseline v. min 16-20, t4 = 14, P < 0.0001). (H) The PKA inhibitor RpCamps (5μM) does not alter eEPSCs (paired t-test, baseline v. min 16-20, t4 = 10, P = 0.0004) (I) Representative mEPSC traces (top and bottom) pre (left) and post (right) U69,593 application, conducted in 500nM TTX and 5μM picrotoxin. No significant changes in mEPSC decay kinetics were seen, (not shown, paired t-test, t6 = 0.8170, P > 0.1). (J) mEPSC frequency (paired t-test, t5 = 5.567, P < 0.001) but not amplitude (K) (paired t-test, t5 = 0.2141, P > 0.1) was reduced following application of the KOR agonist U69,593 Raw values for mEPSC frequency (not shown, baseline frequency mean = 3.406, SEM = 3.475; post-U69,593 frequency mean = 2.179, SEM = 1.118) and amplitude (not shown, baseline amplitude mean = -29.36, SEM = 1.944, post-U69,593 amplitude mean = -25.71, SEM = 1.465). This inhibition is abolished in zero calcium aCSF, where both frequency (L) (paired t-test, t5 = 1.959, P > 0.05) and amplitude (M) (paired t-test, t5 = 2.017 P > 0.05) of mEPSCs remain unaltered by U69,593.