We characterized these tools in brain slices and used them to define

the spatiotemporal dynamics of opioid signaling with unprecedented resolution. Enkephalins and dynorphins are the most prominent opioid peptides in the brain (Khachaturian et al., 1985). We chose to work with LE and Dyn-8 (Figure 1A) because they are the smallest and most chemically stable endogenous opioids from these peptide families. LE activates delta and mu opioid receptors with nanomolar affinity but is inactive at kappa receptors (Toll et al., 1998). The three additional C-terminal amino acids found in Dyn-8 confer nanomolar potency at kappa receptors in addition to mu and delta receptors (Toll et al., 1998). To render these peptides inactive until exposed to light, we produced analogs modified find more at the N-terminal tyrosine side chain with the carboxynitrobenzyl (CNB) chromophore,

which photoreleases tyrosine with high quantum yield (∼0.3) (Sreekumar et al., 1998) on the microsecond timescale (Tatsu et al., 1996) (see Supplemental Information, available online, for information on peptide production and handling). Extensive studies into the structure-activity relationships of enkephalins (Morley, 1980) and dynorphins (Chavkin and Goldstein, 1981) have revealed an essential role for their common N-terminal tyrosine (Y) in receptor activation. In particular, alkylation (Beddell et al., 1977) or removal (Terenius et al., 1976) of the phenolic OH group reduces the potency of enkephalin analogs, suggesting that modification of the tyrosine side chain of LE and Dyn-8 may be a viable caging strategy. HDAC inhibitor Based on these considerations, we designed Dipeptidyl peptidase CNB-Y-[Leu5]-enkephalin (CYLE) and CNB-Y-Dyn-8 (CYD8) (Figure 1A) to release LE and Dyn-8, respectively, in response to illumination with UV light. The chemical structure of CYLE is shown in Figure 1B. Reverse-phase high-pressure

liquid chromatography experiments confirmed that both peptides cleanly photorelease their parent peptides in pH 7.4 phosphate-buffered saline in response to 355 nm laser illumination (Figure S1) and that they are stable in the dark at room temperature for >48 hr (data not shown). To determine whether CYLE and CYD8 are inactive at opioid receptors prior to photolysis, we compared their activity on opioid receptors relative to that of LE and Dyn-8, respectively, using an in vitro functional cellular assay. To detect opioid receptor activation, we utilized HEK293 cells that stably express a Gαs-Gαi chimera (S.D. Liberles and L.B. Buck, personal communication). This chimeric protein allows GPCRs that normally do not signal through Gαs to stimulate adenylate cyclase and control the transcription of a cAMP-dependent reporter construct. Cells were cotransfected with the opioid receptor of interest and the reporter construct such that receptor activation leads to production of secreted alkaline phosphatase (SEAP).