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

描述(由申请人提供)：伤害素/孤儿FQ(N/OFQ)，通过其受体NOP，调节伤害性感受、压力和焦虑。N/OFQ被认为是一种抗阿片肽，因为中枢N/OFQ可引起原痛觉和应激诱导的镇痛(SIA)的逆转。然而，N/OFQ可影响非阿片介导的镇痛作用，其抗焦虑作用不太可能通过与阿片系统的相互作用来实现。下丘脑富含N/OFQ和NOP，选择性地合成下丘脑中的下丘脑和食欲素。HCRT调节觉醒和警觉，调节伤害性处理，并对SIA起作用。由于N/OFQ和Hcrt对所评估的大多数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Hcrt神经元在下丘脑外侧(LH)的活动和/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(主要是SIA和恐惧或应激诱导的焦虑样行为)产生影响。我们的初步研究表明，含有N/OFQ的纤维接触Hcrt神经元，N/OFQ通过突触前和突触后机制抑制Hcrt神经元的活动。N/OFQ还能抑制食欲素/Cameleon 2.1小鼠下丘脑脑片中大多数Hcrt神经元的胞浆钙离子。我们发现，在束缚模型中，食欲素/阿塔克辛-3神经退行性变(食欲素/阿塔克辛-3)小鼠没有表现出SIA。相反，侧脑室(Icv)注射Hcrt可模拟食欲素/ataxin-3小鼠的SIA，但可阻止野生型(WT)小鼠的SIA。此外，外源性Hcrt可恢复中枢N/OFQ治疗动物的SIA。这些初步结果支持我们的假说，并引导我们提出以下具体目标：1.用标准的解剖学技术(如免疫组织化学、原位杂交和逆行追踪)验证N/OFQ纤维接触Hcrt神经元的假说，并确定N/OFQ神经支配的解剖来源(S)。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N/OFQ的纤维是否与Hcrt神经元突触接触。2.用膜片钳记录和钙离子成像的方法研究N/OFQ对Hcrt神经元的生理调节作用。3.通过研究N/OFQ对WT和oresin/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的假说。4.使用条件性和非条件性恐惧范式对N/OFQ部分通过抑制Hcrt介导的应激和焦虑反应发挥类焦虑作用的假设进行检验。这项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，提供双重调制来平衡应激反应，特别是与伤害性加工和应激适应有关的应激反应。

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.