Modulation of Nociceptin/Orphanin FQ on Hypocretin Neurons in Pain and Stress

描述（由申请人提供）：伤害感受肽/孤啡肽 FQ (N/OFQ) 通过其受体 NOP 调节伤害感受、压力和焦虑。 N/OFQ 被认为是一种抗阿片肽，因为中枢 N/OFQ 会导致伤害感受和应激诱导镇痛 (SIA) 的逆转。然而，N/OFQ 可以影响非阿片类药物介导的镇痛，并且其抗焦虑作用不太可能通过与阿片系统的相互作用来介导。 N/OFQ 和 NOP 在下丘脑中含量丰富，下丘脑中选择性合成下丘脑分泌素/食欲素 (Hcrts)。 Hcrts 调节觉醒和警觉性，调节伤害感受处理，并有助于 SIA。由于 N/OFQ 和 Hcrts 对大多数行为和评估的细胞作用产生相反的调节，因此我们假设 N/OFQ 通过直接调节外侧下丘脑 (LH) 中的 Hcrt 神经元活动和/或在接收 N/OFQ 和 Hcrt 输入并共同表达其同源受体的预计位点的相互作用，对神经行为产生影响，主要是 SIA 和恐惧或压力诱发的焦虑样行为。我们的初步研究表明，含有 N/OFQ 的纤维接触 Hcrt 神经元，并且 N/OFQ 通过突触前和突触后机制抑制 Hcrt 神经元活动。 N/OFQ 还抑制 orexin/cameleon 2.1 小鼠下丘脑切片中大多数 Hcrt 神经元的细胞质 Ca2+。我们发现 orexin/ataxin-3 神经退行性 (orexin/ataxin-3) 小鼠在约束模型中没有表现出 SIA。相反，脑室内 (icv) 给予 Hcrt 可以模拟 orexin/ataxin-3 小鼠中的 SIA，但可以阻止野生型 (WT) 小鼠中的 SIA。此外，外源性 Hcrt 在接受集中施用 N/OFQ 治疗的动物中恢复了 SIA。这些初步结果支持我们的假设，并引导我们提出以下具体目标： 1. 检验含 N/OFQ 的纤维接触 Hcrt 神经元的假设，并使用标准解剖技术（例如免疫组织化学、原位杂交和逆行示踪）确定 N/OFQ 神经支配的解剖学来源。我们将使用多重标记定量电子显微镜 (EM) 技术来进一步确定含 N/OFQ 的纤维是否突触接触 Hcrt 神经元。 2. 通过使用来自 orexin/EGFP 小鼠的 Hcrt 神经元的膜片钳记录以及使用 orexin/cameleon 2.1 小鼠的 Ca2 成像来表征 N/OFQ 对 Hcrt 神经元的细胞生理调节。 3. 通过研究 N/OFQ 对 WT 和 orexin/ataxin-3 小鼠伤害感受处理的影响，检验 N/OFQ 通过调节 Hcrt 系统部分阻断 SIA 的假设。 4. 使用条件性和非条件性恐惧范式检验 N/OFQ 部分通过抑制小鼠 Hcrt 介导的应激和焦虑反应来发挥抗焦虑样作用的假设。拟议的研究将揭示是否存在连接 N/OFQ 和 Hcrt 系统的集成神经元回路，该回路提供双重调节来平衡压力反应，特别是与伤害性处理和压力适应相关的压力反应。

DESCRIPTION (provided by applicant): Nociceptin/orphanin FQ (N/OFQ), via its receptor NOP, modulates nociception, stress, and anxiety. N/OFQ is regarded as an anti-opiate peptide because central N/OFQ causes pronociception and reversal of stress- induced analgesia (SIA). However, N/OFQ can affect nonopioid-mediated analgesia, and its anxiolytic effect is unlikely to be mediated through interaction with the opiate system. Both N/OFQ and NOP are abundant in the hypothalamus, where hypocretins/orexins (Hcrts) are selectively synthesized. The Hcrts regulate wakefulness and alertness, modulate nociceptive processing, and contribute to SIA. Because N/OFQ and Hcrts produce opposite modulations for most behaviors and cellular actions assessed, we hypothesize that N/OFQ exerts its effects on neurobehavior, primarily SIA and fear or stress-induced anxiety-like behavior, through direct modulation of Hcrt neuronal activity in the lateral hypothalamus (LH) and/or interaction at the projected sites that receive both N/OFQ and Hcrt inputs and co-express their cognate receptors. Our preliminary studies show that N/OFQ-containing fibers contact Hcrt neurons and that N/OFQ inhibits Hcrt neuronal activity via both pre- and postsynaptic mechanisms. N/OFQ also depresses cytoplasmic Ca2+ in a majority of the Hcrt neurons in orexin/cameleon 2.1 mouse hypothalamic slices. We found that orexin/ataxin-3 neurodegenerative (orexin/ataxin-3) mice exhibit no SIA in the restraint model. Conversely, intracerebroventricular (icv) administration of Hcrt mimicked SIA in orexin/ataxin-3 mice animals but prevented SIA in wildtype (WT) mice. Furthermore, exogenous Hcrt restored SIA in animals treated with centrally administered N/OFQ. These preliminary results support our hypothesis and lead us to propose the following Specific Aims: 1. Test the hypothesis that N/OFQ-containing fibers contact Hcrt neurons and determine the anatomical source(s) of N/OFQ innervation by using standard anatomical techniques (e.g., immunohistochemistry, in situ hybridization, and retrograde tracing). We will use using multiple labeling quantitative electron microscopical (EM) techniques to further determine whether N/OFQ-containing fibers synaptically contact Hcrt neurons. 2. Characterize the cellular physiological modulation of N/OFQ on Hcrt neurons by using patch clamp recordings of Hcrt neurons from orexin/EGFP mice and by Ca2+ imaging using orexin/cameleon 2.1 mice. 3. Test the hypothesis that N/OFQ blocks SIA partly through the modulation of the Hcrt system by investigating N/OFQ effects on nociceptive processing in WT and orexin/ataxin-3 mice. 4. Test the hypothesis that N/OFQ exerts anxiolytic-like effects partly via inhibition of the Hcrt-mediated stress and anxiety responses in mice using both conditioned and unconditioned fear paradigms. The proposed research will reveal whether there is an integrated neuronal circuit linking the N/OFQ and Hcrt systems that provides a dual-modulation to balance stress responses particularly related to nociceptive processing and stress adaptation.