Photoactivatable Neuropeptides for Spatiotemporally Precise Delivery of Opioids in Neural Tissue

Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.
Neuron (Impact Factor: 15.05). 01/2012; 73(2):249-59. DOI: 10.1016/j.neuron.2011.11.016
Source: PubMed


Neuropeptides activate G protein-coupled receptors to acutely modulate cellular excitability and synaptic transmission. However, due to the lack of reagents for precise delivery of peptides within dense brain tissue, the spatiotemporal scale over which neuropeptides act is unknown. To achieve rapid and spatially delimited delivery of neuropeptides in mammalian brain tissue, we developed photoactivatable analogs of two opioids: [Leu⁵]-enkephalin (LE) and the 8 amino acid form of Dynorphin A (Dyn-8). These peptides are functionally inactive prior to photolysis, and exposure to ultraviolet (UV) light causes clean release of LE and Dyn-8. Recordings from acute slices of rat locus coeruleus (LC) demonstrated that photorelease of LE activates mu opioid receptor-coupled K+ channels with kinetics that approach the limits imposed by G protein-mediated signaling. Temporally precise and spatially delimited photorelease revealed the kinetics and ionic nature of the mu opioid response and the mechanisms that determine the spatial profile of enkephalinergic volume transmission in LC.

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Available from: Matthew R Banghart, Apr 02, 2014
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    • "The use of photosensitive compounds that act as photo-switches or ligands that bind to channels or receptors upon photo-conversion (Kramer et al., 2013) could exploit the high spatiotemporal control of both fluid and light delivery afforded by these optofluidic probes. Optopharmacological agents have been enthusiastically received in neuroscience for in vitro applications (Banghart and Sabatini, 2012; Callaway and Katz, 1993; Carter and Sabatini, 2004; Matsuzaki et al., 2001), but their use in vivo has been limited. Although compelling findings exist addressing the external visual nervous system and the surface of the cortex (Mourot et al., 2012; Noguchi et al., 2011; Polosukhina et al., 2012; Tochitsky et al., 2014), optopharmacological application in the deep brain remains a significant challenge. "
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    ABSTRACT: In vivo pharmacology and optogenetics hold tremendous promise for dissection of neural circuits, cellular signaling, and manipulating neurophysiological systems in awake, behaving animals. Existing neural interface technologies, such as metal cannulas connected to external drug supplies for pharmacological infusions and tethered fiber optics for optogenetics, are not ideal for minimally invasive, untethered studies on freely behaving animals. Here, we introduce wireless optofluidic neural probes that combine ultrathin, soft microfluidic drug delivery with cellular-scale inorganic light-emitting diode (μ-ILED) arrays. These probes are orders of magnitude smaller than cannulas and allow wireless, programmed spatiotemporal control of fluid delivery and photostimulation. We demonstrate these devices in freely moving animals to modify gene expression, deliver peptide ligands, and provide concurrent photostimulation with antagonist drug delivery to manipulate mesoaccumbens reward-related behavior. The minimally invasive operation of these probes forecasts utility in other organ systems and species, with potential for broad application in biomedical science, engineering, and medicine. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Jul 2015 · Cell
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    • "further purified by high-performance liquid chromatography as described in Banghart et al. (2012). Data Analysis. "
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    ABSTRACT: Desensitization of μ-opioid receptors (MORs) develops over 5-15 min following application of some but not all opioid agonists and lasts for 10s of min following agonist removal. The decrease in function is receptor selective (homologous) and could result from (1) a reduction in receptor number or (2) a decrease in receptor coupling. The present investigation used photolysis of two caged opioid ligands in order to examine the kinetics of MOR-induced potassium conductance before and following MOR desensitization. Photolysis of a caged antagonist, caged-naloxone (CNV-NLX), blocked the current induced by a series of agonists and the time constant of decline was significantly decreased following desensitization. The increase in the rate of current decay was not observed following partial blockade of receptors with the irreversible antagonist, β-CNA. The time constant of current decay following desensitization was never more rapid than 1s, suggesting an increased agonist off rate rather than an increase in the rate of channel closure downstream of the receptor. The rate of GIRK current activation was examined using photolysis of a caged agonist, [Leu](5)enkephalin (CYLE). Following acute desensitization or partial irreversible block of MORs with β-CNA there was an increase in the time it took to reach a peak current. The decrease in the rate of agonist-induced GIRK conductance was receptor selective and dependent on receptor number. The results indicate that opioid receptor desensitization reduced the number of functional receptors and the remaining active receptors have a reduced agonist affinity.
    Preview · Article · Apr 2014 · Molecular pharmacology
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    • "The traditional view is that G protein-linked signaling is restricted to the plasma membrane and based on rapid diffusion of downstream mediators. However, it is increasingly clear that even classical ''diffusible'' mediators such as cAMP are spatially restricted through local synthesis and destruction (Willoughby et al., 2006), and neuromodulators such as opioid neuropeptides exhibit a limited range of action in neural tissue (Banghart and Sabatini, 2012). Accordingly, the precise subcellular location of 7TMR activation is likely to be an important parameter in neuromodulation, particularly for projection neurons and neurons with extensive dendritic arbors. "
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    ABSTRACT: Cellular responsiveness to many neuromodulators is controlled by endocytosis of the transmembrane receptors that transduce their effects. Endocytic membrane trafficking of particular neuromodulator receptors exhibits remarkable diversity and specificity, determined largely by molecular sorting operations that guide receptors at trafficking branchpoints after endocytosis. In this Review, we discuss recent progress in elucidating mechanisms mediating the molecular sorting of neuromodulator receptors in the endocytic pathway. There is emerging evidence that endocytic trafficking of neuromodulator receptors, in addition to influencing longer-term cellular responsiveness under conditions of prolonged or repeated activation, may also affect the acute response. Physiological and pathological consequences of defined receptor trafficking events are only now being elucidated, but it is already apparent that endocytosis of neuromodulator receptors has a significant impact on the actions of therapeutic drugs. The present data also suggest, conversely, that mechanisms of receptor endocytosis and molecular sorting may themselves represent promising targets for therapeutic manipulation.
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