气道关闭引起的迷走色反射

　　所有动物程序遵循《 NIH指南》中概述的实验动物护理和使用指南的道德准则，所有程序均由哈佛医学院的机构动物护理和使用委员会批准。在恒定的环境条件下（23±1°C，46±5％的相对湿度），食物和水在12小时的光周期中提供了多余的态度。所有研究都在混合遗传背景中使用成年男性和雌性小鼠（6-24周）。所有Creer小鼠和对照同窝仔接受了他莫昔芬（Sigma，T5648，100 mg kg – 1，腹膜内，向日葵油，相距48小时）至少在进一步实验之前10天。含有CRE依赖性和FLP依赖性DTR等位基因的小鼠是M. Goulding的礼物，并购买了Calca-EGFP小鼠（Gensat，RRID：MMRRC_011187-UCD）。对于PVALB-T2A-CRE小鼠，由于雄性育种者的种系重组，仅使用雌性Cre小鼠进行饲养（Jackson Laboratory，012358）。男性和女性后代用于实验。通过针对OLFR78基因座3'UTR的CAS9蛋白的核注射cas9蛋白和一个单链DNA模板，将OLFR78-P2A-CRE小鼠产生，该蛋白的CRISPR SGRNA和含有P2A-CRE基因盒，其中150 BP同源臂进入C57BL/6 Embryos。通过PCR分析筛选了敲击幼崽，并通过原位杂交验证了转基因的正确表达。所有使用的CRE驱动线都是可行且肥沃的，并且未检测到异常表型。OLFR78-P2A-CRE小鼠的基因分型引物为GgatggTaAgggTcacgTgTT（野生型等位基因引物），CCGTTTTTTGGAAACAGCCTGG（P2A-CRE 5'PRIMER）和TGCGAACCTCACTCATCACTCACTCGTT（PCRED PRIMEREL pRIMEREL 3 n'se se se se se se sas sys sys sys sa野生型等位基因（192 bp）和敲入等位基因（562 bp）在两个单独的反应中。所有其他老鼠都是从杰克逊实验室购买的或内部制造的，然后存放到杰克逊实验室：ASCL1-CREERT2（012882），nkx2.1-ires-flp（028577），piezo2-egfp-ires-cre（027719），Inter-GαQ-Dreadd（26942），LSL-SALSA（31968）（31968），LSL-TDOMATO（lsl-tdtomato（007914），007914），Snap25-chap25-chcamp25-gcAmp6s（255-gccamp6s（2512），012-111111）C57BL/6J（000664），LSL-GαQ-DreadD（026220），Loxp-Piezo2（027720），Loxp-Piezo1（029213），vglut2-ires2-ires2-ires2-flpo（030212），Inter-ai65（0212），Inter-ai65（021875）（016963），NPY1R-GFP-CRE（030544），P2RY1-IRES-CRE（29284），PVALB-T2A-CRE（012358），CRHR2-IRES-CRE（33728），NPY2R-IRES-CRE（NPY2R-IRES-CRE（33728）（16223），GLP1R-IRES-CRE（29283），MC4R-2A-CRE（030759）和GPR65-IRES-CRE（029282）。 　　用氨基甲酸酯（1.6–1.8 mg g – 1腹膜腹膜注射至少在手术前30分钟）或异氟烷吸入（1.5-2％），在加热的平台上加热小鼠。除扩展数据中的所有实验涉及麻醉的所有实验外，该实验明确描述了图1中的异氟烷的使用。A tracheostomy was performed by inserting a cannula (18 or 20 gauge) to the carina and attaching the cannula to multipronged tubing with three openings: one to the atmosphere, one to a pressure transducer and one to an in-line gas and nebulized aerosol delivery port through which the animals are exposed to constant low-level flow rate (40 ml min–1, which creates a tracheal pressure of 2–4 mmH2O,由SAR-1000呼吸机，CWE，图1和100％氧气中的房间空气控制，以最大程度地减少低氧叹息）。测量了以下参数：使用在线压力传感器测量呼吸；心电图记录在爪子中置于皮下的三个针电极记录的心律；液体填充压力传感器的食道，咽和/或胸压；与肌电图记录的呼吸道肌肉收缩与耦合到放大器的同心双极针电极（1-2 kHz采样，MP150放大器系统，Biopac Acqknowledgeveledgeledge v.4.2，v.4.5或v.5.0）。如果指示，肌电图信号是数字整合（τ= 0.02 s）。使用带有小鼠爪传感器（Kent Scientific）的Mousestat JR进行脉搏血氧仪监测。 　　除非另有说明，否则除非另有说明，否则通过固定从前肢到Xiphoid工艺的肋骨笼子周围的袖口固定胸部压缩，除非另有说明，否则袖口在5 s上缓慢膨胀，以使每次呼吸峰值气管压力降低40-60％，除非另有说明。袖口压力根据尺寸和袖带拟合而变化，最大压力通常为5–30 cmh2o。通过将内线气体和雾化的气溶胶输送端口切换到数字控制的真空储层（SCIREQ）来应用气道吸力（5 s）。最终的施加吸气压力由气管中的压力传感器（低，-5 cm H2O；高，-10 cm H2O）确定。吸入气体（如数字和传说中，剩余百分比为n2）通过呼吸机上的进气口（40 mL min – 1，5分钟试验）在线交付。为了测量Hering -Breuer的灵感反射，通过增加通过呼吸机（10-25 ml min – 1 g – 1的体重，10 s）来实现肺通胀。气溶胶在盐水（PBS）中施用，并通过雾化器（来自Scireq的ANP-1100，其占空比为50％）。在随后的5分钟内监测反射。Nebulized aerosols were methacholine (10 mg ml–1, PBS, Cayman, 23092), citric acid (30% w/v, Sigma, C1909), KCl (Sigma, 12636) and microbeads (Thermo Scientific, 0.2 mm F8811, 2.0 mm F8827).与先前的呼吸相比，喘气的定义为单呼吸的到期，幅度率> 50％，如肌电图和气管或食管测量值所推断的。对于刺激引起的呼吸变化，通过与刺激引入前10 s基线的值相比，通过比较数据进行了标准化。 　　使用屈曲计算机控制的活塞呼吸机（SCIREQ）测量呼吸力学（扩展数据图1E，8C和10F）。将动物麻醉，气管静态（插入了18或20 g的套管）并附着在呼吸机上。在扩展数据中，然后将闭合胸动物瘫痪（1 mg kg – kg – 1 pancuronium，腹膜内，Sigma-Aldrich，P1918）；扩展数据图中显示的测量值。使用开胸动物进行8C和10F。除非另有说明，否则以每分钟150次呼吸通风，潮汐体积为10 ml kg – 1和3 cmh2o阳性末端呼气压力，除非另有说明。使用强制振荡技术评估呼吸力学。强制呼油量和压力卷环动作由Flexivent Software（Flexiware v.8.2）控制。 　　如先前所述的20,56，对迷走神经节的体内成像进行了较小的修饰。简而言之，如上所述，用尿电烷麻醉小鼠，并在稳态支撑手术中给定PBS（300μL，腹膜内）。左迷走神经节在外科手术中暴露在分支上，其分支高于转移的神经节，并固定在连接到操纵器的玻璃成像平台上。在大多数实验（7只小鼠中的4个）中，通过两光子显微镜（Olympus fvmpe谐振谐振两光子显微镜）在大多数实验中进行钙成像，并使用压电z-Stepper（P-915，Physik仪器）和×25，×25，NA1.0水上及时配置apper apper apper air-immersion apper apper air apper apper air apper airer airair a miair airair airair airair a。EHP DeepSee，Spectraphysics），激发调谐到940至975 nm，并用570 nm长的二细球和495-540 nm Bandpass Filter过滤tdttomato信号的575-645 NM Bandpass Filter滤波。通常在1.5–3 Hz的情况下，相距40-60 µm（Olympus Fluoview Software VFV31S-SW）收集体积图像。对于某些实验（7只小鼠中的3个），如前所述，通过共聚焦显微镜（具有×20的Leica SP5 II，Na1.0，Na1.0的leica SP5 II）进行钙成像。 　　使用斐济ImageJ（v.1.52p）中的“图像稳定器”插件对两通道图像进行运动校正。将红色荧光通道图像取平均值以描绘单个细胞并划定感兴趣的区域（ROI）。相对于基线，不健康的细胞通常表现出明显的强大和不变的GCAMP荧光，被排除在外。基线荧光（F0）是从刺激发作前的20 s周期中计算出来的，并计算了比率ΔF/F0强度，并在每个ROI处将其归一化为TDTOMATO荧光强度，以控制光漂白，运动和GCAMP6表达。如果刺激引起的ΔF/F0中的刺激诱发的增加至少为3 s.d。在整个成像会话中的平均荧光高于平均荧光。对于每个响应性细胞，计算了反应（RC/RI）（ΔF/F0）与压缩和通胀的比率；如果RC/RI> 2，将细胞归类为压缩选择性，如果RC/RI，则为通货膨胀选择性< 0.5 or as polymodal if 0.5 < Rc/Ri < 2. In Extended Data Fig. 2c, cells that did not respond to either airway inflation or airway closure were subsequently separated based on responsiveness to methacholine. Only some non-responsive neurons were selected for inclusion in indicated heatmaps based on computer randomization. 　　Vagus nerve optogenetics were performed as previously described6,14 using a DPSS laser light source (473 nm, 150 mW, Ultralaser) with software actuated illumination (10 s, 5–40 Hz, 10 ms dwell, 65–95 mW mm–2 Prizmatix Pulser v.2.3.1 TTL software). 　　Vagal sensory neurons were ablated as previously described6,34 with DT (Sigma, D0564) solution (2–5 ng DT, PBS with 0.05% Fast Green FCF dye) injected (10 × 10 nl, serially) into surgically exposed vagal ganglia using a Nanoject III injector (Drummond). NEB ablation was achieved by intranasal administration (daily for 4 days) of solution containing 10 ng DT in 30 μl PBS. Cell ablation controls involved DT-administered Cre-negative littermates. Animals were allowed to recover for at least 2 weeks before subsequent experiments. 　　Vagal anatomical tracing was performed as previously described6,14 and involved AAV-eGFP (AAV9.CB7.Cl.eGFP.WPRE.rBG, 105542-AAV9, Addgene) and AAV-flex-TdTomato (pAAV-FLEX-tdTomato, 28306-AAV9, Addgene). Animals recovered for at least 2 weeks before tissue collection. 　　Whole-body plethysmography was performed in freely behaving animals using a VivoFlow chamber system (SCIREQ). Chamber airflow was measured by a pneumotach at constant temperature and humidity with 0.5–0.6 l min–1 bias flow, and respiratory measurements were amplified, digitized and recorded using the VivoFlow-usbAMP and lox2 software (v.2.10.5.28, SCIREQ). Gas challenges involved hypoxia (12% O2), hypercapnia (5% CO2, 21% O2) and normoxia (21% O2) balanced with nitrogen (Airgas). Animals were acclimated in the plethysmography chamber for 40–60 min, and then baseline respiratory data were recorded for 30 min. CNO injections involved brief removal of the animal from the chamber for administration of CNO (3 mg kg–1, intraperitoneally, 100 μl PBS), and animals were immediately returned to the chamber for further recordings (30 min). Breaths were assigned and respiratory parameters (tidal volume, breaths per minute (BPM), minute volume) were calculated using Iox2 software (v.2.10.5.28 SCIREQ). Gasp-like breaths were manually identified from pneumotachographs and defined as a 50% increase in both inspiration and expiration compared with preceding and subsequent breaths. For quantification of eupneic breathing parameters, data were filtered to exclude respiratory events outside typical adult mouse breathing (tidal volume >2 ml or <0.05 ml; BPM > 400), averaged over the recording period (with a 7 min delay after CNO introduction), and breathing measures dependent on airway volume were normalized to the body weight of the animal. 　　For immunochemistry in tissue cryosections, tissues were collected from animals after transcardial perfusion of fixative (PBS followed by 4% paraformaldehyde in PBS), immersed in fixative (4% paraformaldehyde, PBS, overnight, 4 °C), cryopreserved (30% sucrose, PBS, overnight, 4 °C) and embedded in OCT. Tissue cryosections were obtained, washed (2 × 5 min, PBS, room temperature), permeabilized (0.3% Triton X-100, PBS, 10 min, room temperature), blocked (5% donkey serum, 0.3% Triton X-100, 0.05% Tween-20, PBS, 1 h, room temperature) and incubated with primary antibody diluted in blocking buffer (overnight, 4 °C; anti-NCAM1, 1:250, Cell Signaling Technology, 99746 S; anti-GFP, 5 μg ml–1, Aves Labs, GFP-1020; anti-mCherry/RFP, 3 μg ml–1, OriGene Technologies, AB0040-200; anti-HB-EGF (human), 1:250, R&D Systems, AF-259-NA; and anti-RFP, Rockland, 1:1,000, Rockland, 600-401-379). Slides were then washed (3 × 10 min, 0.3% Triton X-100, 0.05% Tween-20, PBS) and incubated with secondary antibodies in blocking buffer (4 h, room temperature, all 1:1,000, donkey polyclonal, Jackson Immunoresearch; anti-Chicken IgG-Alexa fluor 488, anti-rabbit IgG-Cy3, anti-rabbit IgG Cy5, anti-goat IgG Cy5 and anti-goat IgG Cy3; RRIDs: AB_2340375, AB_2307443, AB_2340607, AB_2340415 and AB_2307351, respectively). Samples were washed (3 × 10 min, 0.3% Triton X-100, PBS, room temperature), stained for nuclei visualization (5 min, 1:1,000 Hoechst 33342, PBS) and mounted (ProLong Glass Antifade; Thermo Fisher) for microscopy. RNA in situ hybridization for Piezo2 was performed on tissue cryosections using the probe and protocol involving hybridization chain reaction provided by the manufacturer (Molecular Instruments). Immunostained slides and native tissue fluorescence were imaged by either confocal microscopy (Leica SP5 II or Nikon Ti2) or by widefield microscopy (Zeiss AxioZoom or AxioObserver microscopes with Zen Blue software, v.2.6 and v.3.2, respectively). For whole-mount lung histology in Fig. 3h, tissue was stained and cleared using published iDisco methodology involving anti-mCherry/RFP primary antibody (6 μg ml–1) and Cy5-conjugated anti-goat IgG secondary antibody (1:500) and imaged by light sheet microscopy (UltraMicroscope II by LaVison, ImSpector v.7.1.4). 　　Whole lungs below the trachea were collected from 10 Calca-eGFP and 10 Ascl1-creER;lsl-tdTomato mice (5–7 weeks old, equal male and female, 10 days after tamoxifen injection), pooled by strain, minced and incubated (60 min, 37 °C) in oxygenated papain dissociation buffer (Worthington Biochemical, LK003150). Residual tissue was mechanically dissociated through a 100 μm cell strainer, pelleted by centrifugation (400g, 7 min, 4 °C), washed, resuspended in red blood cell lysis buffer (150 mM NH4Cl, 10 mM NaCHO3 and 0.1 mM EDTA) for 5 min, pelleted and resuspended in FACS buffer (0.5% BSA, 2 mM EDTA, PBS, 4 °C). Immune cells were depleted using anti-CD45 magnetic beads according to the manufacturer’s instructions (BioLegend, 480027), and the remaining cells were resuspended in viability buffer (TO-PRO-3 and CellTrace Violet, both 1:10,000, in RPMI 1640; Thermo Fisher, T3605, 65-0854-39 and 11835030, respectively). Cells were collected by FACS using a FACS Aria II (BD Bioscience) with gates to select for fluorescent reporter expression and viability (CellTrace Violet positive, TO-PRO-3 negative). Collected cells were individually encapsulated in nanodroplets using a 10x Genomics platform (v.3 chemistry). Single-cell cDNA was prepared according to the manufacturer’s protocol and sequenced at the Harvard Medical School Biopolymers Facility on a NextSeq 500 platform. For analysis, sequence reads were aligned to the mm10 reference transcriptome, and feature barcode matrices were generated using Cell Ranger (10x Genomics; pipeline v.3.1.0), and analysed in R (v.4.1.3) using Seurat (v.4.1.1) for quality control, pre-processing, normalization, clustering and differential expression analysis. Transformed matrices from both strains were integrated (nFeature = 3,000) before cluster identification and UMAP representation. Analysis used a standard process excluding cells with >15％线粒体读取或<500个独特功能。鉴定出神经内分泌细胞簇，以富集EPCAM，CALCA和ASCL1的表达。在扩展数据中描述了定义其他肺部细胞类型的基因图9a。经过差异表达分析，基因本体学富集分析使用了使用富集61（https://maayanlab.cloud/enrichr/），使用了以显着性（P值）排名的前50个最富集的基因。 　　ASCL1-CREER; LSL-SALSA; LSL-GαQ-DreadD小鼠先前注射了他莫昔芬（葵花籽油中的100 mg kg – 1，腹膜内，两次）被麻醉，并经过仔细地添加并用10 ml冷的冷，氧气的PBS经过仔细地灌注。通过在37°C下通过气管套管引入2％低熔融琼脂糖并在冰上迅速冷藏（30分钟），从而使肺膨胀。切除肺叶，并在冷的，含氧成像缓冲液中使用纤维仪（以MM：115 NaCl，5 KCl，25 Nahcl，25 Nahco3，1 MgCl2，2 Cacl2，2 Cacl2，2 Cacl2，10葡萄糖和10 HEPES，10 HEPES，pH 7.3）获得了200 µm的切片。将切片转移到新鲜的成像缓冲液（37°C； 5％CO2）中进行成像（通常30分钟后）。基于TDTomato表达鉴定NEB，并通过共聚焦显微镜测量莎莎荧光（Leica SP5 II，×20，Na1.0，Na1.0水浸水物镜，GCAMP6F，488 nm兴奋和495-535的发射； TDTOMATO；通过重力饲料连续灌注成像缓冲液，并应用CNO或KCL进行成像，如图6A所示。使用LAS AF软件（v.2.3.6 Leica）获取数据，并在ImageJ中进行了分析。 　　如先前所述的14,21,53，进行了整个迷走神经电生理记录，并进行了较小的修饰。简而言之，如上所述，为气道生理学测量所述，用手术准备了氨基烷 - 纳阿纳治疗的动物（1.6 mg g – 1）。然后将左迷走神经转移，将肺部连接的神经端放置并放在一对铂 - iridium电极上。将神经和电极浸入盐碳油中，并将地面电极放在附近的肌肉上。扩增多活性神经活性（CP511，草技术），数字化（MP150，Biopac），记录（ACQKNOWEGGEDEND软件，v.4.5，Biopac）和Integrated（Elenco，RS-400）。刺激诱导的反应是根据基线活性的百分比变化计算的，并在给药后100 s的100 s上对羟色胺（腹膜内，10 mm，400μlPBS）的反应进行了归一化。 　　在全身多生理图中，记录了动物（Logitech C920 HD Pro摄像头）。适应后（1 h）后，通过鲍里斯软件（v.8.20.4）62手动评分了行为，该基因型盲目的研究者在CNO给药前和第7-17分钟之前和分钟（3 mg kg kg – 1 – 1 – 1 – 1 – 1 – 1，intaperaineanealealeality）中测量了时间探索，饲养，修饰，嗅探或驼背的时间10分钟。弯曲是根据特征性的卧姿姿势定义的，通常与呼吸和荷叶毛皮有关。 　　除非另有说明，否则图中的数据表示为平均值±S.E.M.。使用Prism（GraphPad）进行统计分析，并在图和传说中报告了统计测试和样本量。除非另有说明，否则所有重复均为生物学，统计检验是双面的。所有代表性的图像均来自至少三个独立的实验。样本量是根据我们领域以前的专业知识和出版物确定的。研究人员对与图1和图1的相关的多个生理学，生理和行为实验的组分配视而不见。3–5和扩展数据图7；在其他实验中，组分配并未蒙蔽。在适当的情况下，在传说中报告了精确和调整的P值，并且具有显着性的星号定义如下：*p <0.05，** p <0.01，*** p <0.001，**** p <0.0001。 　　所有不市售的试剂将根据合理的要求提供。OLFR78-P2A-CRE小鼠将存入杰克逊实验室，并将在达成标准材料转移协议后可用。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得。