Using this assay, we obtained dose-response curves for CYLE and CYD8 at mu, delta, and kappa opioid receptors

and compared those to the actions of the parent peptides (Figures 1C, 1D, and S2). The potency of CYLE was reduced SCH 900776 concentration 100- to 500-fold with respect to LE at both delta (LE EC50, 3.2 ± 0.8 nM; CYLE EC50, 1.7 ± 0.4 μM) and mu receptors [LE EC50, 90 ± 11 nM; CYLE EC50, 16 ± 1.7 μM (Figure 1D)]. Similar to LE, CYLE did not activate kappa receptors (Figure S2), and the presence of 100 nM CYLE did not reduce the affinity of LE at mu and delta receptors (Figure 1C) or that of Dyn-17 at kappa receptors (Figure S2), indicating that CYLE does not act as an antagonist. CYD8 exhibited similar reductions in potency in comparison to Dyn-8 at kappa (Dyn-8 EC50, 7.1 ± 0.8 nM; CYD8 EC50, 16 ± 1.7 μM), mu [Dyn-8 EC50, 63 ± PLX-4720 4.4 nM; CYD8 EC50, 23 ± 2.6 μM (Figure 1D)], and delta receptors [Dyn-8 EC50, 9.6 ± 2.3 nM; CYD8 EC50, 3.9 ± 0.6 μM (Figure S2)], with no indications of antagonism. Summary dose-response data are tabulated in Table S1. These

data reveal that CYLE and CYD8 possess no significant agonist or antagonist activity at concentrations that should strongly activate receptors following photolysis. In order to characterize the ability of these molecules to activate neuropeptide receptors in brain slices with spatiotemporal precision, we took advantage of the well-described opioid-receptor-mediated activation of K+ channels in neurons of the locus coeruleus (LC). The LC is heavily innervated by enkephalinergic afferents (Curtis et al., 2001 and Drolet et al., 1992), and LC neurons express a high density of mu opioid receptors in their

somata and dendrites (Van Bockstaele et al., 1996a and Van Bockstaele et al., 1996b), activation of which pauses spontaneous firing (Pepper and Henderson, 1980) by producing large outward currents (Travagli et al., 1995, Travagli et al., 1996 and Williams et al., 1982), Adenylyl cyclase at least in part through G protein-coupled inward rectifier K+ (GIRK) channels. We obtained whole-cell recordings from neurons in acute horizontal slices of rat LC (Figure 2A) and characterized the ability of CYLE to photoactivate mu receptors by releasing LE. In current-clamp recordings, these cells spontaneously fired action potentials at 2–8 Hz, while local perfusion of 10 μM LE caused a strong hyperpolarization and transient pause in spiking (Figure 2B), consistent with previous studies (Williams et al., 1982). In voltage-clamp recordings at a holding potential of −55mV, application of 10 μM LE via a perfusion pipette placed near the cell of interest evoked average (n = 6) outward currents of 199 ± 23 pA in amplitude (Figure 2C). In contrast, when applied to the same cells, 10 μM CYLE evoked an average outward current of 5.6 ± 4 pA, measured 3.