To

more accurately address peptide mobility in brain tissue, two-photon sensitive caging groups must be employed so that release will be restricted to μm3-scale volumes. Here, we have described photoactivatable tools for the study of opioid signaling within the mammalian brain. By caging both LE and Dyn-8, we provide reagents that can be used to study mu, delta, and kappa receptors. Using UV-mediated photolysis of caged LE in brain slices, we demonstrated that somatic mu receptors in the LC generate an outward current mediated primarily by K+ channels. These reagents allowed us to probe the mechanisms that regulate the spread click here of opioid signaling in brain tissue and revealed that with graded, temporally precise, and spatially confined release, neuropeptides are capable of subtle and relatively short-lasting

modulation of neuronal function. This approach represents a general strategy for probing the spatiotemporal dynamics of neuropeptides and should be applicable to other peptide transmitters. These reagents are expected to interface well with two-photon Ca2+ and voltage imaging methods, as the CNB chromophore exhibits poor sensitivity to two-photon excitation. Similarly, one-photon excitation, with the visible wavelengths used to image fluorophores such as GFP and activate light-sensitive ion channels such as channelrhodopsin, is MK 8776 also compatible with our probes. However, the intense UV light used for uncaging can photobleach fluorophores and partially activate channelrhodopsin, so care must be taken to control the area of illumination and minimize the requisite UV

light intensity in these contexts. Extension to in vivo studies, including amperometry to measure the effects of opioids on monoamine release, should be possible by equipping optrodes and fiberoptic coupled carbon fibers with perfusion lines for peptide delivery and may thus enable spatiotemporal studies into opioidergic modulation of behavior with unprecedented precision. Custom chemical synthesis was carried out by Peptech Corporation (Burlington, MA) using standard Fmoc-based solid-phase peptide synthesis. The carboxynitrobenzyl-modified tyrosine was prepared by modifying established protocols (Sreekumar et al., 1998). most After arrival, CYLE and CYD8 were typically handled under lighting filtered using Rosculux #312 Canary optical filter paper to remove any traces of UV light that could lead to unintentional photolysis. It was essential to further purify the synthetic material by semipreparative reverse-phase high-pressure liquid chromatography (RP-HPLC, Agilent) to remove contaminating photolysis products, which typically included ∼1% LE or Dyn-8. Crucially, the UV (and VIS) lamps on the detector were turned off during the purification to prevent photolysis of the purified material.