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

描述（由申请人提供）：痛觉肽/孤啡肽FQ （N/OFQ）通过其受体NOP调节痛觉、压力和焦虑。N/OFQ被认为是一种抗阿片肽，因为中枢N/OFQ可引起前觉性和应激性镇痛（SIA）的逆转。然而，N/OFQ可以影响非阿片类药物介导的镇痛，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在下丘脑中都很丰富，下丘脑是选择性合成下丘脑分泌/食欲素（Hcrts）的地方。Hcrts调节清醒和警觉性，调节伤害性加工，并有助于SIA。由于N/OFQ和Hcrt对大多数被评估的行为和细胞活动产生相反的调节，我们假设N/OFQ通过直接调节外侧下丘脑（LH）的Hcrt神经元活动和/或在接受N/OFQ和Hcrt输入并共同表达其同源受体的投射部位的相互作用，对神经行为产生影响，主要是SIA和恐惧或应激诱导的焦虑样行为。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神经元的活动。在食欲素/cameleon 2.1小鼠下丘脑切片中，N/OFQ也抑制大多数Hcrt神经元的细胞质Ca2+。我们发现，在抑制模型中，orexin/ataxin-3神经退行性（orexin/ataxin-3）小鼠未表现出SIA。相反，脑室内（icv）给药Hcrt模拟了食欲素/ataxin-3小鼠动物的SIA，但阻止了野生型（WT）小鼠的SIA。此外，外源性Hcrt可以恢复中央给药N/OFQ治疗的动物SIA。这些初步结果支持了我们的假设，并引导我们提出以下具体目标：验证含有N/OFQ的纤维接触Hcrt神经元的假设，并通过标准解剖技术（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）技术进一步确定含有N/ ofq的纤维是否与Hcrt神经元突触接触。2. 通过对orexin/EGFP小鼠Hcrt神经元的膜片钳记录和对orexin/cameleon 2.1小鼠的Ca2+成像，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小鼠伤害性加工的影响，验证N/OFQ通过调节Hcrt系统部分阻断SIA的假设。4. 在条件恐惧和非条件恐惧模式下，测试N/OFQ部分通过抑制hcrt介导的应激和焦虑反应来发挥抗焦虑样作用的假设。该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，该回路提供双调制来平衡应激反应，特别是与伤害性加工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.