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

描述（由申请人提供）：孤啡肽/孤啡肽FQ（N/OFQ）通过其受体NOP调节伤害感受、应激和焦虑。N/OFQ被认为是一种抗阿片肽，因为中枢N/OFQ引起前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作用，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都在下丘脑中丰富，下丘脑选择性地合成下丘脑泌素/食欲素（Hcrts）。Hcrts调节觉醒和警觉，调节伤害性处理，并有助于SIA。由于N/OFQ和Hcrts产生相反的调制大多数行为和细胞的行动评估，我们假设，N/OFQ发挥其对神经行为的影响，主要是SIA和恐惧或压力诱导的焦虑样行为，通过直接调制Hcrt神经元的活动在外侧下丘脑（LH）和/或相互作用的预计网站，接收N/OFQ和Hcrt输入和共表达其同源受体。我们的初步研究表明，含N/OFQ的纤维接触Hcrt神经元和N/OFQ抑制Hcrt神经元的活动，通过突触前和突触后机制。N/OFQ还抑制食欲素/cameleon 2.1小鼠下丘脑切片中大多数Hcrt神经元的胞质Ca 2+。我们发现，食欲素/共济失调蛋白-3神经退行性（食欲素/共济失调蛋白-3）小鼠在束缚模型中没有表现出SIA。相反，脑室内（icv）给予Hcrt模拟食欲素/共济失调蛋白-3小鼠动物中的SIA，但防止野生型（WT）小鼠中的SIA。此外，外源性Hcrt恢复SIA的动物与中央管理的N/OFQ。这些初步结果支持了我们的假设，并使我们提出了以下具体目标：1。测试含N/OFQ的纤维接触Hcrt神经元的假设，并通过使用标准解剖技术（例如，免疫组织化学、原位杂交和逆行追踪）。我们将使用多重标记定量电子显微镜（EM）技术，以进一步确定是否N/OFQ含有纤维突触接触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和通过使用orexin/cameleon 2.1小鼠的Ca 2+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和食欲素/共济失调蛋白-3小鼠伤害性感受加工的影响，检验N/OFQ部分通过调节Hcrt系统阻断SIA的假设。4.使用条件性和非条件性恐惧范例，在小鼠中检验N/OFQ部分地通过抑制Hcrt介导的应激和焦虑反应而发挥抗焦虑样作用的假设。这项研究将揭示是否存在一个连接N/OFQ和Hcrt系统的集成神经元回路，该神经元回路提供双重调节以平衡应激反应，特别是与伤害性处理和应激适应相关的应激反应。

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.