Neuropharmacology of Pontine Control of Breathing Frequency

描述（由申请人提供）： μ阿片受体（莫尔）激动剂如吗啡、芬太尼和瑞芬太尼（remi）是用于急性重度术后疼痛和慢性重度疼痛状态的最有效的围手术期镇痛药。最严重的，危及生命的副作用，这限制了他们的剂量，是呼吸频率（呼吸缓慢）和潮气量的深度抑制。这种风险在其他镇静剂如苯二氮卓类（BZD）和酒精的存在下增加。我们的研究表明，脑桥的臂旁/ K？liker-KF核（PB-KF复合体）中的神经元对与呼吸缓慢相关的低临床阿片类药物浓度非常敏感，并有可能导致呼吸骤停。我们还发现，小臂旁亚区（PBSR）的神经元控制呼吸频率（fB），似乎是介导阿片类药物诱导的呼吸缓慢的门户。局部兴奋的PBSR神经元增加fB，而那些减少神经元活动减少fB甚至到呼吸暂停的点。因此，任何影响PBSR神经元活动的药物都会对呼吸产生重大影响。我们的工作假设是PBSR作为fB的主要控制器，通过向产生阶段性吸气（I）和呼气（E）神经元放电模式的节律性前B tzinger复合体（preBC）提供兴奋性输入。通过单独或与镇静剂组合全身施用阿片类药物抑制PBSR神经元活性导致重度呼吸缓慢或呼吸骤停。PBSR还调节由慢适应肺牵张受体（PSR）介导的I时程（TI）和E时程（TE）的反射控制的增益。为了解决上述假设，将追求以下具体目标：1）如初步发现所建议的，在控制正常fB的PB-KF区域附近的背侧脑桥中精确定位PBSR，2）识别PBSR神经元亚型，确定它们的轴突是否投射到preBC/BC区域并量化它们对PSR输入的响应，3）确定各种PBSR神经元亚型的放电模式是如何通过A）NMDA和非NMDA受体介导的谷氨酸能内源性兴奋和B）GABA能和甘氨酸能内源性抑制产生和控制的，4）确定A）该区域内的神经元上的GABAA受体是否被BZD调节（例如，咪达唑仑），B）全身给药的BZD是否作用于PBSR内的神经元（被微量注射的氟马西尼拮抗），和C）微量注射的BZD是否调节静脉内瑞芬太尼诱导的 呼吸过缓，5）通过微量注射选择性激动剂和拮抗剂确定GABAB受体在PBSR神经元和TI和TE调节中的作用，6）鉴定介导由AMPA刺激和高度局部化的电刺激在PBSR内诱发的fB增加的前BC神经元亚型，和7 A）表征由PBSR介导的TI和TE的PSR反射控制的调节，和B），并确定这种调节是否是由于到孤束核（NTS）的PBSR输入改变到二级神经元的PSR神经传递。将使用超短效瑞芬太尼的全身静脉输注在体内去大脑犬模型中产生呼吸徐缓。将记录膈神经活动以测量TI和TE。将使用多管微量移液器和16电极探针（NeuroNexus）同时记录PBSR神经元的放电，同时皮可喷射神经活性剂。对PSR输入和轴突投射到前BC区域的响应将用于进一步分类PBSR神经元。PBSR似乎是控制呼吸频率的入口。因此，该离散区域的神经生理学和神经药理学特征的研究将提供对药物不利地影响呼吸频率的机制的主要见解，并提出减轻不良副作用的治疗措施。这些研究也将提供重要的新信息的功能作用的PBSR神经元和特定的神经递质/调制器的贡献PBSR神经元亚型在体内的放电模式和新的见解控制呼吸机制。

DESCRIPTION (provided by applicant): Mu-opioid receptor (MOR) agonists such as morphine, fentanyl and remifentanil (remi) are the most effective perioperative analgesics for both acute severe postoperative pain and chronic severe pain states. The most serious, life-threatening side effect, which limits their dosage, is profound depression of breathing rate (bradypnea) and tidal volume. This risk is increased in the presence of other sedatives such as benzodiazepines (BZDs) and alcohol. Our studies indicate that neurons in the parabrachial/ K�lliker-Fuse nuclei (PB-KF complex) of the pons are very sensitive to low clinical opioid concentrations associated with bradypnea and have the potential to cause respiratory arrest. We have also discovered that neurons in a small parabrachial subregion (PBSR) control breathing frequency (fB) and appear to be the portal that mediates the opioid-induced bradypnea. Localized excitation of PBSR neurons increase fB, while those that decrease neuronal activity decrease fB even to the point of apnea. Thus, any drugs that affect the activity of PBSR neurons will have a major impact on breathing. Our working hypothesis is the PBSR functions as the major controller of fB by providing excitatory inputs to the rhythmogenic preB�tzinger Complex (preBC) that produces phasic inspiratory (I) and expiratory (E) neuronal discharge patterns. Depression of PBSR neuronal activity by systemically administered opioids alone or combined with sedatives leads to severe bradypnea or arrest. The PBSR also modulates the gain of the reflex control of I-duration (TI) and E-duration (TE) mediated by slowly adapting pulmonary stretch receptors (PSRs). To address the above hypotheses, the following specific aims will be pursued: 1) Precisely locate the PBSR in the dorsal pons near the PB-KF area that controls eupneic fB as suggested by preliminary findings, 2) Identify the PBSR neuron subtypes, determine if their axons project to the preBC/BC region and quantify their responses to PSR inputs, 3) determine how the discharge patterns of the various PBSR neuron subtypes are generated and controlled by A) NMDA and nonNMDA receptor mediated glutamatergic endogenous excitation and B) by GABAergic and glycinergic endogenous inhibition, 4) determine A) whether the GABAA receptors on neurons within this region are modulated by BZDs (e.g., midazolam), B) whether systemically-administered BZDs act on neurons within the PBSR (antagonized by microinjected flumazenil), and C) whether microinjected BZDs modulate the effects of iv remifentanil-induced bradypnea, 5) determine the role of GABAB receptors in the modulation of PBSR neurons and of TI and TE via microinjections of selective agonists and antagonists, 6) identify preBC neuron subtypes that mediate increases in fB evoked within the PBSR by AMPA stimulation and highly localized electrical stimuli, and 7 A) characterize the modulation of the PSR reflex control of TI and TE mediated by the PBSR, and B) and determine whether this modulation is due to PBSR inputs to the nucleus of the solitary tract (NTS) that alter the PSR neurotransmission to the second order neurons. Systemic i.v. infusions of ultra short-acting remifentanil will be used to produce bradypnea in an in vivo decerebrate canine model. Phrenic nerve activity will be recorded to measure TI and TE. Multibarrel micropipettes and a 16-electrode probe (NeuroNexus) will be used to simultaneously record the discharge of PBSR neurons while picoejecting neuroactive agents. Responses to PSR inputs and axon projections to the preBC region will be used to further classify the PBSR neurons. The PBSR appears to act as the portal through which breathing rate is controlled. Thus, the study of the neurophysiological and neuropharmacological characteristics of this discrete region will provide major insight into the mechanisms by which drugs adversely affect breathing frequency and suggest therapeutic measures to alleviate undesirable side effects. These studies will also provide important new information on the functional roles of PBSR neurons and the contribution of specific neurotransmitters/modulators to the discharge patterns of PBSR neuron subtypes in vivo and new insights into control of breathing mechanisms.