Mu-Opioid Effects on the Central Mechanisms that Control Breathing

描述(由申请人提供)： 吗啡和合成的5-阿片受体(5OR)类似物，如芬太尼和瑞芬太尼(REMI)是用于治疗严重急性和慢性疼痛的高效止痛剂。严重的呼吸抑制(呼吸迟缓、呼吸暂停)可发生在临床相关的血浆浓度。这些效应是由于高度阿片类药物敏感呼吸区的抑制，还是分散在许多突触上仍未解决。寻找阿片类药物高度敏感的靶点是设计预防阿片类药物镇痛期间呼吸抑制的策略的重要步骤。我们的研究表明，阿片类药物诱导的呼吸迟缓不是由前Bvtzinger复合体(PBC)中5ORs的激活引起的，PBC是可能的节律产生的基因座。初步研究表明，桥脑呼吸群(PRG)神经元位于臂旁核/Kvlliker-Fuse核团(PB-KF区)。我们的工作假设是，临床浓度的全身性5-阿片类药物作用于PB-KF区的50R，通过直接激活PRG神经元亚型上的50R和/或间接地通过其他阿片敏感的桥脑神经元的兴奋性和/或抑制性突触输入而产生呼吸迟缓。这些亚型的PRG神经元和肺伸展感受器(PSR)输入控制着延髓的PBC/Bvtzinger复合体(BC)神经元，这些神经元负责呼吸期的定时/切换。此外，激活PB-KF区的特异胺受体可能逆转阿片类药物引起的呼吸抑制。为了解决这些假设，将追求以下具体目标：目标1：通过DAMGO(5OR激动剂)微量注射，通过监测膈神经图(PNG)呼吸期的变化，定位PB-KF区产生阿片类药物诱导的呼吸暂停的区域。在静注REMI期间微量注射纳洛酮(NAL；阿片拮抗剂)将用于确定相同的50R是否会产生呼吸迟缓。将使用组织化学方法识别5OR免疫反应(IR)高密度区域，以确定功能定位区域。目的：确定哪些类型的PRG神经元最容易受到IVREMI和PB-KF区域DAMGO抑制的影响。目的：通过NAL在单个神经元上的翻转微射，确定PRG神经元放电的IV Remi抑制是否是由于突触后激活5ORs所致。3B：对于不可逆的PRG神经元，通过微射DAMGO来确定它们是否具有50R。目的：探讨NMDA和AMPA受体介导的谷氨酸能兴奋和/或GABA能和甘氨酸能抑制是否参与了PRG神经元的IV REMI抑制。在静注Remi慢呼吸之前和期间，将在单个PRG神经元上喷射选择性拮抗剂。目的5.确定PRG神经元亚型上是否存在5HT1A、D1多巴胺和12种肾上腺素能受体，作为对抗5OR诱导的抑郁的治疗靶点。系统静脉注射REMI将用于在去大脑的犬体内模型中产生呼吸迟缓。将使用膈神经活动来测量I相和E相的时程。多管微管将被用来记录单个PRG神经元在喷射神经活性物质时的放电活动。16电极阵列探头(NeuroNexus)将用于在REMI诱导的呼吸暂停之前和期间获得多个PRG神经元的同时记录。对逆行激活和PSR输入的反应将被用来对PRG神经元进行分类。这些研究将回答PB-KF区的5ORs是否介导了临床浓度的全身性5-阿片类药物对呼吸频率的抑制，将识别阿片类药物敏感的PRG神经元的亚型，并回答影响是否直接和/或间接。此外，这些研究将确定PRG神经元上的胺能神经调节剂受体是否提供了将阿片类药物诱导的抑郁效应降至最低的治疗靶点。这些研究还将为PRG神经元的功能作用以及特定的神经递质/调节剂在体内各种PRG神经元放电模式的产生中的作用提供重要的新信息，并对相位计时机制提供新的见解。

DESCRIPTION (provided by applicant): Morphine and synthetic 5-opioid receptor (5OR) analogs such as fentanyl and remifentanil (remi) are highly effective analgesics used to treat severe acute and chronic pain. Profound respiratory depression (bradypnea, apnea) can occur at clinically relevant plasma concentrations. Whether these effects are due to depression of highly opioid sensitive respiratory regions or dispersed over many synapses remains unresolved. Finding highly opioid sensitive targets is an important step in designing strategies to prevent respiratory depression during opioid analgesia. Our studies indicate that opioid-induced bradypnea does not result from activation of 5ORs in the preBvtzinger Complex (pBC), the putative locus for rhythm generation. Preliminary studies suggest that 5ORs on/near pontine respiratory group (PRG) neurons in parabrachial/ Kvlliker-Fuse nuclei (PB- KF region) are targets. Our working hypothesis is that clinical concentrations of systemic 5-opioids act at 5ORs in the PB-KF region to produce bradypnea by either direct activation of 5ORs on subtypes of PRG neurons and/or indirectly via excitatory and/or inhibitory synaptic inputs by other opioid-sensitive pontine neurons. These subtypes of PRG neurons and pulmonary stretch receptors (PSRs) inputs control the medullary pBC/Bvtzinger complex (BC) neurons responsible for respiratory phase timing/switching. Also, activation of specific amine receptors in the PB-KF region may reverse opioid-induced respiratory depression. To address these hypotheses, the following specific objectives will be pursued: Objective 1: To locate the region in the PB-KF area that produces opioid-induced bradypnea via DAMGO (5OR agonist) microinjections by monitoring changes in respiratory phase durations from the phrenic neurogram (PNG). Naloxone (NAL; opioid antagonist) microinjections during IV remi will be used to determine if the same 5ORs produce bradypnea. Histochemical methods will be used to identify regions with high densities of 5OR immunoreactivity (IR) to confirm the functionally localized regions. Objective 2: To determine which types of PRG neurons are most susceptible to IV remi and PB-KF regional DAMGO depression. Objective 3A: To determine if the IV remi depression of PRG neuronal discharge is due to postsynaptic activation of 5ORs, indicated by NAL reversal picoejected on single neurons. 3B: For nonreversed PRG neurons, to determine if they possess 5ORs via picoejection of DAMGO. Objective 4A: To determine if changes in glutamatergic excitation mediated by NMDA and AMPA receptors and/or GABAergic and glycinergic inhibition are involved in IV remi depression of PRG neurons. Picoejection of selective antagonists on single PRG neurons before and during IV remi bradypnea will be used. Objective 5. To determine if 5HT1A, D1 dopamine and 12 adrenergic receptors are present on subtypes of PRG neurons, to serve as therapeutic targets to counteract 5OR-induced depression. Systemic IV infusions of remi will be used to produce bradypnea in an in vivo decerebrate canine model. Phrenic nerve activity will be used to measure I- and E-phase duration. Multibarrel micropipettes will be used to record the discharge activity of single PRG neurons while ejecting neuroactive agents. A 16-electrode array probe (NeuroNexus) will be used to obtain simultaneous recordings of multiple PRG neurons before and during remi-induced bradypnea. Responses to antidromic activation and PSR inputs will be used to classify PRG neurons. These studies will answer whether 5ORs in the PB-KF region mediate the depression of breathing frequency produced by systemic 5-opioids at clinical concentrations, will identify subtypes of opioid sensitive PRG neurons and answer if effects are direct and/or indirect. In addition, these studies will determine whether receptors for aminergic neuromodulators on PRG neurons offer a therapeutic target to minimize opioid-induced depressant effects. These studies will also provide important new information on the functional roles of PRG neurons and the contribution of specific neurotransmitters/modulators in the generation of discharge patterns of various PRG neurons in vivo and new insights into phase-timing mechanisms.