Neuropeptides are an important class of neurotransmitters that has received relatively little attention in comparison to other neuromodulators such as acetylcholine and the monoamines. Because it has been difficult to selectively stimulate neuropeptide release from distinct cell types (however see (Ludwig and Leng, 2006
)), our understanding of neuropeptide signaling dynamics is limited. Photoactivatable molecules enable spatiotemporally precise delivery of endogenously occurring ligands in relatively intact brain tissue preparations. We were able to generate photoactivatable opioid neuropeptides that are sufficiently inert to allow large responses to be generated with a brief uncaging stimulus. The caged LE analogue CYLE provided robust, rapid and graded delivery of LE in acute brain slices. The ability to spatially restrict release allowed us to selectively evoke currents from regions of neurons that can be effectively voltage clamped in order to accurately measure the reversal potential of the mu opioid receptor mediated K+
current, which was not previously possible in brain slices of LC. These features further enabled us to quantitatively characterize the mechanisms governing peptide clearance and delineate the spatial profile of enkephalinergic volume transmission for the first time.
Based on extensive prior pharmacology, we identified the N-terminal Tyrosine side chain as a caging site where the relatively small CNB chromophore sufficiently attenuates potency on both LE and Dyn-8. Peptides may be inherently more difficult to `cage' than small molecules as the caging group will only interfere with one of multiple interaction sites with receptors. In particular, hydrophobic interactions contribute greatly to peptide-receptor binding and hydrophobic side chains lack functional handles for attaching caging groups. For these reasons the full length Dyn-17 or beta-endorphin may be more difficult to cage by the same approach.
CNB-tyrosine photolysis occurs with microseconds kinetics following a light flash (Sreekumar et al., 1998
; Tatsu et al., 1996
). Thus the time-course of activation we observed in slices likely reflects the time required for ligand binding and engagement of the G-protein mediated signaling pathway that activates GIRK channels. Indeed, we observed a 50–100 ms delay from the flash to the current onset and a peak response within 1–2 seconds, which closely matches the rates observed for GABAB
R-mediated GIRK activation in dissociated cells using rapid perfusion techniques (Ingram et al., 1997
; Sodickson and Bean, 1996
). However, the offset kinetics we observed are orders of magnitude slower than those measured in dissociated cells, where currents cease within 1–2 s of agonist washout. Instead, photorelease produced deactivation kinetics that were only two-fold faster than those obtained with local perfusion, likely reflecting slow diffusion of released peptide away from the recorded cell in neural tissue and concomitant proteolytic cleavage. Indeed, addition of a protease inhibitor cocktail slowed deactivation of the response to photolysis over large areas.
Prompted by the ability to spatially confine LE release, we revisited previous studies into opioid actions on rat LC neurons that were unable to unambiguously identify a K+
current using reversal potential measurements in brain slices (Osborne and Williams, 1996
; Travagli et al., 1995
). The reversal potential of the LE-dependent current that we measured is accounted for by a pure K+
current and 80% of the outward current was blocked by a high concentration of Ba2+
, consistent with a dominant role of GIRKs. It has been also been proposed that down-regulation of a cAMP-dependent standing Na+
current contributes 50% of the opioid response in rat LC (Alreja and Aghajanian, 1993
; Alreja and Aghajanian, 1994
). Although we cannot rule out that this component mediates the remaining 20% of the current not sensitive to Ba2+
or that this Na+
permeable channel may be enriched in the dendritic regions not activated by our somatodendritically-restricted uncaging stimulus, our results clearly demonstrate that the majority of the somatodendritic current is carried by K+
channels and thus cannot reflect the closing of Na+
channels. Consistent with previous work that suggests that poor voltage clamp of K+
currents originating in LC dendrites may underlie the apparent negative shift in K+
reversal potential (Ishimatsu and Williams, 1996
; Travagli et al., 1995
), photorelease of LE in the distal dendrites evoked currents that reversed at membrane potentials much more negative than somatically-evoked currents. This shifted membrane potential can arise from poor space-clamp of the large LC dendrites, K+ currents activated in unclamped and gap junction-coupled neighboring cells, or from other dendritically localized and opioid modulated conductances.
By varying the laser power and uncaging area used to photorelease LE, we found a correlation between the amplitude and duration of the outward current in voltage clamp and the duration of the pause in spontaneous firing recorded in current clamp (). The responses to the smallest stimuli demonstrate that small outward currents near the detection limit are sufficient to prevent just a few action potentials without causing significant hyperpolarization. At the opposite extreme, strong uncaging causes a large somatic hyperpolarization and pauses action potential firing for 30 s or longer. Thus, the effect of enkephalin on LC firing can be subtle or dramatic, highlighting that neuropeptides are capable of temporally precise actions in addition to volume transmission.
We found that LE could generate opioid-receptor mediated currents when released ~150 μm from the recorded cell. The slower onset kinetics observed when LE was released at locations distant from the soma suggest that the photolyzed peptide diffused from the release site to activate receptors on the soma and proximal dendrites. These distances are large compared to those over which fast-acting neurotransmitters such as glutamate (Carter et al., 2007
) and GABA (Chalifoux and Carter, 2011
) can spread, as clearance mechanisms for these neurotransmitters are present at high density in neural tissue. Under the conditions of our experiments, LE was nearly inactive when released 300 μm from the soma, which reflects the limit of detection by mu opioid receptors due to dilution of the peptide as it diffuses away from the release site. Assuming a diffusion-limited process, this absolute boundary depends not only on the initial quantity released, but also on the affinity of the receptor for the ligand. Our results may overestimate the mobility of LE in LC due to activation of receptors on dendrites that are closer to the release site than the soma and contributions from currents originating in gap junction-coupled neurons. Nonetheless, our results indicate that enkephalin can indeed function as a volume transmitter in LC and define the spatial profile of the spread of enkephalinergic signaling from a single release site. The spatial profile of signaling may be different in other brain regions due to variations in the densities and identities of proteases and possible differences in diffusional mobility.
Although we obtained similar results using two differently shaped photolysis beams, UV light scatters extensively in brain tissue. Studies in which similar spot sizes (10–25 μm) were employed for UV-uncaging of glutamate in brain slices report 25–50 μm lateral resolution (Katz and Dalva, 1994
; Kim and Kandler, 2003
). Below the surface of the brain slice, light scattering enlarges the photolysis spot by approximately two-fold in the x-y dimensions (Sarkisov and Wang, 2007
), consistent with these observations. Because 1-photon uncaging provides poor spatial control in the z-dimension, it is most practical to consider our results in terms of area of photolysis in the plane of the recorded cell. Thus we estimate that the 10 μm diameter collimated uncaging stimulus illuminates an area of ~300 μm2
at the depth of our recordings. At the light intensity employed, LE evoked substantial currents when released 150 μm from the uncaging site within 1–2 s of the light flash, indicating that the enkephalin signal rapidly spreads through at least 70,000 μm2
of tissue, which is ~200-fold greater than the area of origin. To more accurately address peptide mobility in brain tissue, two-photon sensitive caging groups must be employed so that release will be restricted to μ3
Here we have described novel 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 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 2-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 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 fiber-optic coupled carbon fibers with perfusion lines for peptide delivery and may thus enable spatiotemporal studies into opioidergic modulation of behavior with unprecedented precision.