To determine if dynorphin decreased inhibitory transmission by acting at a presynaptic site in CeA, we used a paired-pulse facilitation (PPF) protocol of IPSPs evoked by delivering two stimuli of the same intensity separated by 100 ms. At this interval, the second IPSP (P2) is larger than the first IPSP (P1), and an increase of the paired-pulse ratio (P2/P1) reflects a decrease of transmitter release (see Siggins et al., 2005). Analysis of PPF revealed that dynorphin (1 μM) significantly increased the paired-pulse ratio to 128 ± 6% of control (n = 11, t = 4.372; Fig. 2B), from 1.11 ± 0.06 before dynorphin to 1.42 ± 0.08 in the presence of the peptide. In 4 of 4 experiments where washout of the peptide was performed, PPF values returned to control level (not shown). We also conducted a PPF protocol during superfusion of U69. U69 (0.5–1 μM) significantly increased the paired-pulse ratio of IPSPs to 126 ± 8% of control (n = 8, t = 3.409; Fig. 2B). These data indicate that KOR agonists decreased GABAA receptor-mediated inhibitory transmission by decreasing the release of GABA at a presynaptic site.
