Supported by NIH award R01 DK093525 (GFG). We greatly appreciate the scientific review and grammatical editing of the manuscript by Dr. Amber Shaffer (University of Pittsburgh) and thank Michael Burcham for assistance in preparation of figures.

注:该协议是审查和批准匹兹堡机构动物护理和使用委员会的大学。 1.准备修改克雷布斯解决方案和测试药物的等分<ol><li>使6升的改良的Krebs溶液包含（以mM计）:117.9氯化钠，氯化钾4.7，25的NaHCO 3，1.3的NaH 2 PO 4，1.2 MgSO 4干燥，2.5氯化钙2，11.1 D-葡萄糖，2-丁酸钠，乙酸钠20 ，0.004硝苯地平（阻止自发性肌肉收缩），以及0.003吲哚美辛（阻断内源性合成前列腺素）。用冰冷和温暖的克雷布斯解决方案的组织剥离和单纤维记录，分别为。 </li><li>准备任何化学溶液（ 如 IS，辣椒素，ATP）在分装时所需的浓度。 </li></ol> 2.解剖结直肠神经组织<ol><li>麻醉和安乐死雄性小鼠（6 - 8周龄，20 - 30克）的在CO 2室中的流速即位移10 -每分钟的腔室容积，直至小鼠的30％停止呼吸完全没有胸部运动所指示的。 </li><li>紧接安乐死，抽血通过切割打开胸腔，射孔右心房，并浸渍鼠标胎体在充足体积的冰冷的Krebs（〜500毫升）（4℃）溶液鼓入卡波金（95％O 2的，5％的CO 2）。 </li><li>小心地取出所有的内脏，但结肠和盆腔器官。断面跨T12脊髓节段鼠标的一半，略高于横膈膜和尾部半转移到含有冰冷的解剖腔，起泡克雷布斯解决方案。 </li><li>在立体显微镜下，通过横断他们的路口尿道取出膀胱和生殖器官，并删除降/腹主动脉，直至其分叉成髂总动脉。释放PN或从他们surroundin的LSN克组织钝性分离，并按照从髂嵴，直至其腹入口点L6和S1椎列（PN）或T13和L1椎列（LSN）以外的神经。 </li><li>切开耻骨联合和左，右髋关节，去除骨骼。仔细自由无论是PN或LSN从接近结直肠，直到那里的神经进入脊柱附着的肌肉和结缔组织。 </li><li>仔细切除髂骨，露出远端结肠直肠。解剖出远端结肠直肠与连续所附PN或LSN。 </li><li>转移带贴在组织腔的浴室中的神经结直肠。通过进一步解剖除去过量结缔组织，并纵向沿着反肠系膜边界打开结肠直肠。 </li><li>与粘膜的一面朝上，PIN结直肠的靠近记录室中的肠系膜边缘进入室使用的聚硅氧烷基r和附加到连接到一个力致动器挂钩的耙结肠直肠的系膜长度（示于图1和拍摄在图2A）。 </li><li>延长PN或LSN到记录室，它是由一鼠标孔和栅极连接到该浴室中。轻轻地将神经干到在录音室中，这为神经坚持亲水表面的小玻璃镜面。 Superfuse洗澡舱用温水（30 - 32°C），含氧克雷布斯解决方案，填补与矿物油的录音室中。 </li></ol> 3.单纤维记录和感受野的本地化<ol><li>小心剥离回从PN或LSN神经外膜（神经鞘）的体视显微镜，在高放大倍率（50 - 60X）下。用细镊子，挑逗神经干到5 - 〜100微米厚度的8神经束。 </li><li>将铂 - 铱参考电极与在所述组织腔Krebs溶液接触。依次将各个神经束到由相同材料制成的记录电极。 </li><li>使用软画笔轻轻抚摸上下结表面，以唤起从大肠传入的AP。找到神经束（S），通过检测AP（动作电位）录音支配结肠。 注:PN和LSN也支配膀胱等盆腔器官。 </li><li>使用一对为30g针尖的神经束，以进一步分成10微米厚的556肌束长丝和放置一个单独的长丝到记录电极。 </li><li>将圆尖同心电极垂直于粘膜表面，以电激发传入神经末梢在阈上刺激强度（10毫安幅度，0.5毫秒持续时间@ 0.3赫兹），其产生的电流扩散一〜半径2毫米。将电极系统（1〜1.5毫米间距）沿着所述扁平大肠的长度和宽度来本地化接受的结局。 </li><li>当传入结局被激发，调整电极位置精确定位的激活（感受野，RF）的一个需要的最小刺激强度（刺激阈值）的位点。丢弃的结局与刺激的阈值&gt;3毫安19。 </li><li>计算刺激伪迹和动作电位的发生之间的传导速度（CV）从1）在感受野（RF）的刺激电极和记录位点和2）的导通延迟（ 例如， 图2B之间的距离） 。 CV（米/秒）=距离（mm）/传导延迟（毫秒）。 </li></ol>机械敏感性结传入放电4.功能分类<ol><li>定位一个射频电刺激后，取以下三个机械刺激到RF: <ol><li>通过按压CALIBRA的前端进行探测刺激特德·冯·弗雷，如尼龙单丝（0.4和1克力）朝垂直的扁平结直肠的RF。 </li><li>轻轻抚摸着大肠黏膜用细尼龙丝股（10毫克力）产生的RF小面剪应力进行抚摸刺激。 </li><li>用计算机控制的力致动器，它提供了一个倾斜的拉伸力进行周向拉伸（0 - 170 MN在5分钟/秒）沿大肠经由在步骤2.8中所述钩的耙抗肠系膜边缘圆周方向。 </li></ol></li><li>分类传入为浆膜（只响应钝探测），粘膜（回应粘膜抚摸和钝探测），肌肉（回应周向拉伸和钝探测）肌肉/粘膜（回应周向拉伸，粘膜抚摸和钝探测），或MIA（未响应于任何三个机械刺激的）。 </li><li>对于肠系膜传入（仅在该LSN支配），很难通过电刺激选择性激活，找到自己接受的结局由机械轻抚/探测肠系膜。 </li><li>对于拉伸敏感传入（肌肉和肌肉粘膜），确定响应的阈值，其被定义为，唤起的第一个AP期间倾斜拉伸的力。 </li><li>对于浆膜传入，记录他们的反应，上升点状探测的感受野由计算机控制的力致动器驱动的水平。 </li></ol>感受结尾5.化学应用/调制<ol><li>记录基线响应机械刺激（ 即响应斜坡舒展，点状探测，或粘膜行程）。 </li><li>外套一块管（黄铜或不锈钢，高10毫米和4×4 平方毫米的方形或4 -直径5mm）的底部边缘与凡士林，并将其放置在对结直肠感受野。 </li><li>拆除克雷布斯溶液的管内，并露出接受结束3 - 5分钟到170微升含有化学（S）以被测试的溶液中。 </li><li>监测过程中的化学应用传入的响应（有些传入的化疗敏感）。 </li><li>除去化学溶液和油管终止化学的作用。在4 - 6分钟，测试传入响应于相同的机械刺激如在基线响应。 </li><li>后洗出足够的时间（&gt; 15分钟）再重新应用机械刺激。 </li></ol> 6.记录和判别AP尖峰<ol><li>数字化记录在从轴突在20千赫的电信号并将数据保存到计算机。通过音频监视器监控上线的信号。 </li><li>分析AP尖峰离线和歧视基于个人尖峰波形25的主成分分析单个单位。 注意:一个记录不应包含更多次一个2轻松地辨别现役部队。 </li></ol>

<ol><li>Naliboff, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+two+distinct+perceptual+alterations+in+irritable+bowel+syndrome%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Evidence for two distinct perceptual alterations in irritable bowel syndrome.</a> Gut. 41, 505-512 (1997).</li>
<li>Verne, G. N., Robinson, M. E., Vase, L., Price, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversal+of+visceral+and+cutaneous+hyperalgesia+by+local+rectal+anesthesia+in+irritable+bowel+syndrome+%28IBS%29+patients%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Reversal of visceral and cutaneous hyperalgesia by local rectal anesthesia in irritable bowel syndrome (IBS) patients.</a> Pain. 105, 223-230 (2003).</li>
<li>Verne, G. N., Sen, A., Price, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrarectal+lidocaine+is+an+effective+treatment+for+abdominal+pain+associated+with+diarrhea-predominant+irritable+bowel+syndrome%5BTitle%5D)+AND+%22Journal+of+Pain%22%5BJournal%5D)">Intrarectal lidocaine is an effective treatment for abdominal pain associated with diarrhea-predominant irritable bowel syndrome.</a> Journal of Pain. 6, 493-496 (2005).</li>
<li>Chey, W. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linaclotide+for+irritable+bowel+syndrome+with+constipation%3A+a+26-week%2C+randomized%2C+double-blind%2C+placebo-controlled+trial+to+evaluate+efficacy+and+safety%5BTitle%5D)+AND+%22Am+J+Gastroenterol%22%5BJournal%5D)">Linaclotide for irritable bowel syndrome with constipation: a 26-week, randomized, double-blind, placebo-controlled trial to evaluate efficacy and safety.</a> Am J Gastroenterol. 107, 1702-1712 (2012).</li>
<li>Rao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+12-week%2C+randomized%2C+controlled+trial+with+a+4-week+randomized+withdrawal+period+to+evaluate+the+efficacy+and+safety+of+linaclotide+in+irritable+bowel+syndrome+with+constipation%5BTitle%5D)+AND+%22Am+J+Gastroenterol%22%5BJournal%5D)">A 12-week, randomized, controlled trial with a 4-week randomized withdrawal period to evaluate the efficacy and safety of linaclotide in irritable bowel syndrome with constipation.</a> Am J Gastroenterol. 107, 1714-1724 (2012).</li>
<li>Busby, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pharmacologic+properties%2C+metabolism%2C+and+disposition+of+linaclotide%2C+a+novel+therapeutic+peptide+approved+for+the+treatment+of+irritable+bowel+syndrome+with+constipation+and+chronic+idiopathic+constipation%5BTitle%5D)+AND+%22J+Pharmacol+Exp+Ther%22%5BJournal%5D)">Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.</a> J Pharmacol Exp Ther. 344, 196-206 (2013).</li>
<li>Mei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+chemosensitivity%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Intestinal chemosensitivity.</a> Physiol Rev. 65, 211-237 (1985).</li>
<li>Mei, N., Lucchini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+data+and+ideas+on+digestive+sensitivity%5BTitle%5D)+AND+%22J+Auton+Nerv+Syst%22%5BJournal%5D)">Current data and ideas on digestive sensitivity.</a> J Auton Nerv Syst. 41, 15-18 (1992).</li>
<li>Su, X., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanosensitive+pelvic+nerve+afferent+fibers+innervating+the+colon+of+the+rat+are+polymodal+in+character%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat are polymodal in character.</a> J Neurophysiol. 80, 2632-2644 (1998).</li>
<li>McMahon, S. B., Morrison, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spinal+neurones+with+long+projections+activated+from+the+abdominal+viscera+of+the+cat%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Spinal neurones with long projections activated from the abdominal viscera of the cat.</a> J Physiol. 322, 1-20 (1982).</li>
<li>Cervero, F., Sann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanically+evoked+responses+of+afferent+fibres+innervating+the+guinea-pig%27s+ureter%3A+an+in+vitro+study%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Mechanically evoked responses of afferent fibres innervating the guinea-pig's ureter: an in vitro study.</a> J Physiol. 412, 245-266 (1989).</li>
<li>Sengupta, J. N., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanosensitive+properties+of+pelvic+nerve+afferent+fibers+innervating+the+urinary+bladder+of+the+rat%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Mechanosensitive properties of pelvic nerve afferent fibers innervating the urinary bladder of the rat.</a> J Neurophysiol. 72, 2420-2430 (1994).</li>
<li>Sengupta, J. N., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+mechanosensitive+pelvic+nerve+afferent+fibers+innervating+the+colon+of+the+rat%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Characterization of mechanosensitive pelvic nerve afferent fibers innervating the colon of the rat.</a> J Neurophysiol. 71, 2046-2060 (1994).</li>
<li>Habler, H. J., Janig, W., Koltzenburg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+unmyelinated+afferent+fibres+by+mechanical+stimuli+and+inflammation+of+the+urinary+bladder+in+the+cat%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Activation of unmyelinated afferent fibres by mechanical stimuli and inflammation of the urinary bladder in the cat.</a> J Physiol. 425, 545-562 (1990).</li>
<li>Habler, H. J., Janig, W., Koltzenburg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+type+of+unmyelinated+chemosensitive+nociceptor+in+the+acutely+inflamed+urinary+bladder%5BTitle%5D)+AND+%22Agents+Actions%22%5BJournal%5D)">A novel type of unmyelinated chemosensitive nociceptor in the acutely inflamed urinary bladder.</a> Agents Actions. 25, 219-221 (1988).</li>
<li>Brierley, S. M., Jones, R. C. 3rd, Gebhart, G. F., Blackshaw, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Splanchnic+and+pelvic+mechanosensory+afferents+signal+different+qualities+of+colonic+stimuli+in+mice%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Splanchnic and pelvic mechanosensory afferents signal different qualities of colonic stimuli in mice.</a> Gastroenterology. 127, 166-178 (2004).</li>
<li>Meyer, R. A., Davis, K. D., Cohen, R. H., Treede, R. D., Campbell, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanically+insensitive+afferents+%28MIAs%29+in+cutaneous+nerves+of+monkey%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Mechanically insensitive afferents (MIAs) in cutaneous nerves of monkey.</a> Brain Res. 561, 252-261 (1991).</li>
<li>Handwerker, H. O., Kilo, S., Reeh, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unresponsive+afferent+nerve+fibres+in+the+sural+nerve+of+the+rat%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Unresponsive afferent nerve fibres in the sural nerve of the rat.</a> J Physiol. 435, 229-242 (1991).</li>
<li>Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+silent+afferents+in+the+pelvic+and+splanchnic+innervations+of+the+mouse+colorectum%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Characterization of silent afferents in the pelvic and splanchnic innervations of the mouse colorectum.</a> Am J Physiol Gastrointest Liver Physiol. 300, G170-G180 (2011).</li>
<li>Feng, B., La, J. H., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Irritable+bowel+syndrome%3A+methods%2C+mechanisms%2C+and+pathophysiology.+Neural+and+neuro-immune+mechanisms+of+visceral+hypersensitivity+in+irritable+bowel+syndrome%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Irritable bowel syndrome: methods, mechanisms, and pathophysiology. Neural and neuro-immune mechanisms of visceral hypersensitivity in irritable bowel syndrome.</a> Am J Physiol Gastrointest Liver Physiol. 302, G1085-G1098 (2012).</li>
<li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+sensitization+of+mechanosensitive+and+-insensitive+afferents+in+mice+with+persistent+colorectal+hypersensitivity%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Long-term sensitization of mechanosensitive and -insensitive afferents in mice with persistent colorectal hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 302, G676-G683 (2012).</li>
<li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Altered+colorectal+afferent+function+associated+with+TNBS-induced+visceral+hypersensitivity+in+mice%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Altered colorectal afferent function associated with TNBS-induced visceral hypersensitivity in mice.</a> Am J Physiol Gastrointest Liver Physiol. 303, G817-G824 (2012).</li>
<li>Feng, B., Brumovsky, P. R., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+roles+of+stretch-sensitive+pelvic+nerve+afferents+innervating+mouse+distal+colon+and+rectum%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Differential roles of stretch-sensitive pelvic nerve afferents innervating mouse distal colon and rectum.</a> Am J Physiol Gastrointest Liver Physiol. 298, G402-G409 (2010).</li>
<li>Feng, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+guanylate+cyclase-C+attenuates+stretch+responses+and+sensitization+of+mouse+colorectal+afferents%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents.</a> J Neurosci. 33, 9831-9839 (2013).</li>
<li>Jolliffe, I. T. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aJolliffe%2C+IT+%22Principal+component+analysis%22">Principal component analysis</a>. , 2nd, Springer. New York, NY. (2002).</li>
<li>La, J. H., Feng, B., Schwartz, E. S., Brumovsky, P. R., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Luminal+hypertonicity+and+acidity+modulate+colorectal+afferents+and+induce+persistent+visceral+hypersensitivity%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Luminal hypertonicity and acidity modulate colorectal afferents and induce persistent visceral hypersensitivity.</a> Am J Physiol Gastrointest Liver Physiol. 303, G802-G809 (2012).</li>
<li>Christianson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((plasticity+and+modulation+of+visceral+afferents%5BTitle%5D)+AND+%22Brain+Research+Reviews%22%5BJournal%5D)">plasticity and modulation of visceral afferents.</a> Brain Research Reviews. 60, 171-186 (2009).</li>
<li>Kiyatkin, M. E., Feng, B., Schwartz, E. S., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+genetic+and+pharmacological+inhibition+of+TRPV1+and+P2X3+attenuates+colorectal+hypersensitivity+and+afferent+sensitization%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Combined genetic and pharmacological inhibition of TRPV1 and P2X3 attenuates colorectal hypersensitivity and afferent sensitization.</a> Am J Physiol Gastrointest Liver Physiol. 305, G638-G648 (2013).</li>
<li>Brumovsky, P. R., Feng, B., Xu, L., McCarthy, C. J., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cystitis+increases+colorectal+afferent+sensitivity+in+the+mouse%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Cystitis increases colorectal afferent sensitivity in the mouse.</a> Am J Physiol Gastrointest Liver Physiol. 297, G1250-G1258 (2009).</li>
<li>Shinoda, M., Feng, B., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+and+central+P2X+receptor+contributions+to+colon+mechanosensitivity+and+hypersensitivity+in+the+mouse%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Peripheral and central P2X receptor contributions to colon mechanosensitivity and hypersensitivity in the mouse.</a> Gastroenterology. 137, 2096-2104 (2009).</li>
<li>Tanaka, T., Shinoda, M., Feng, B., Albers, K. M., Gebhart, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+visceral+hypersensitivity+by+glial+cell+line-derived+neurotrophic+factor+family+receptor+%CE%B1-3+in+colorectal+afferents%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Modulation of visceral hypersensitivity by glial cell line-derived neurotrophic factor family receptor α-3 in colorectal afferents.</a> Am J Physiol Gastrointest Liver Physiol. 300, G418-G424 (2011).</li>
</ol>

The authors have nothing to disclose.

这里所描述的在体外结直肠神经制剂已被证明是一个有效的方法来研究各个结传入，它很好的补充了对内脏感觉神经元等非功能性的方法（ 例如，细胞，分子和组织学研究）的神经编码功能（见综述27的详细信息）。促进伤害感受和长期结肠超敏反应神经机制已经揭示和药理操作已经执行了具有可以减轻内脏痛显示目标。成功实施本制剂的相关联的以下要点讨论如下:1）减少...

疼痛和过敏症是患者的主要抱怨从功能性胃肠疾病，包括肠易激综合征（IBS），在没有明显的病理生物学原因或组织损伤中存在的痛苦。例如，IBS患者表现出过敏症，包括在正常的肠功能提高反应直肠球囊扩张和增加的敏感性，以及体细胞介超敏反应（ 即，压痛到腹部的触诊）1。因为靶向结肠传入已被证明是有效的，以减轻疼痛和过敏症中IBS患者（ 例如，局部麻醉药2,3-直肠内滴注;鸟苷酸环化酶-C的利那洛肽激动剂4-6的口服摄取），改进的理解结直肠传入神经神经支配是很重要的。 内脏传入，包括结肠传入，能够应对化学/营养和热模式（ 如 7-9）的。然而，内脏传入响应于机械性刺激（ 即，机械敏感传入神经 ​​）一直是最彻底的研究，因为机械性刺激（ 例如，管腔腹胀，拉伸）是那些通常对感觉的，包括不适和疼痛10-16产生。此外，内脏也支配通过机械敏感传入神经 ​​（MIA中），通常被称为无声或睡眠伤害感受器17。在正常生理条件下，MIA中不回应机械性刺激或有非常高的反应阈值18，但可以成为积极的，并获得mechanosensitivity在病理情况下，有助于过敏。 使用体外准备和这里描述的协议，我们开发并采用电刺激策略，海<html> RCH为接受的结局，允许公正的鉴定两种机械敏感性和MIA的结局在结直肠19。大肠支配从腰内脏（LSN）和骨盆神经（PN）的途径获得，并且包括可以被分类成五个机械敏感类（浆膜，粘膜，肌肉，肌肉粘膜，肠系膜）和一个MIA类20大肠传入。使用这种在体外制备，我们发现，大肠癌MIA中取得mechanosensitivity（致敏）以下的感受域的短暂暴露于炎性汤（IS），这在LSN通路敏化在PN通路MIA中的71％和MIA中的23％的19。我们也记录MIA中的长期敏（28天）的长期行为内脏过敏的情况下（ 即在结肠内接受治疗与酵母多糖21或2,4,6-三硝基苯磺酸小鼠（TNBS）22） 。 jove_content"&gt;其中机械敏感性传入，肌肉发达，肌肉，黏膜传入是唯一的类tonically编码结直肠周向拉伸（ 即是拉伸敏感）和subserve性结直肠扩张23,24的编码，利用计算机控制力致动器，我们应用一个标准的，同质的，且可重复倾斜伸展的扁平大肠组织的圆周方向和进一步分类拉伸敏感传入神经 ​​作为低阈值和高阈值23，此外，拉伸的致敏的时间过程经过酵母多糖结肠内21或22 TNBS治疗敏感的传入对应于起病，持续性，和/或行为内脏敏感性的恢复，提示舒展敏感结传入内脏疼痛和感觉过敏的作用。

<table><tbody><tr><td>Leica MZ16 stereo microscope</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Leica IC D camera</td><td>Leica Microsystems Inc.</td><td></td><td></td></tr><tr><td>Amplifier</td><td>World Precision instruments, Inc.</td><td>SYS-DAM80</td><td>Low-noise differential amplifier</td></tr><tr><td>Two-compartment tissue chamber</td><td>Custom made</td><td></td><td></td></tr><tr><td>Power1401</td><td>Cambridge Electronic Design Limited</td><td>Power1401</td><td>Data acquisition, analog signal input/out</td></tr><tr><td>Spike2 v5.02</td><td>Cambridge Electronic Design Limited</td><td></td><td>Software package that works with the Power1401</td></tr><tr><td>Audio monitor</td><td>Natus</td><td>Am 8</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus</td><td>S48</td><td>To deliver electrical stimuli</td></tr><tr><td>Photoelectric isolation unit</td><td>Natus</td><td>PSIU6</td><td>Stimulus isolation to reduce noise</td></tr><tr><td>Concentric bipolar microelectrode</td><td>FHC Inc.</td><td>CBFFG75</td><td>To deliver electrical stimuli</td></tr><tr><td>Dual-mode lever system</td><td>Aurora Scientific Inc.</td><td>Series 300C</td><td>To deliver mechanical stimuli</td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-00</td><td>Forceps with fine tips</td></tr></tbody></table>

in vitro functional characterization of mouse colorectal afferent endings

详细此视频演示了使用鼠标结直肠神经制剂体外单纤维电生理记录协议。该方法允许公正的鉴定和个人结传入的功能特性。传播动作电位（APS），从一个或几个传入神经 ​​起源的细胞外记录（ 即，单纤维）感受域（RFS）在结直肠从逗神经纤维束状制成。结直肠与任何骨盆（PN）或连接并打开纵向腰内脏（LSN）神经去除。该组织被放置在记录室中，钉扎平并灌注充氧的Krebs溶液。焦电刺激被用于定位大肠癌的传入神经末梢，它们由三个不同的机械刺激进一步测试（钝探测，粘膜划线和周向拉伸）到传入功能分类成五个mechanosensi略去类。结局回应没有这些机械的刺激被归类为机械不敏感的传入（MIA中）。两个机械敏感和MIA中可评估致敏（ 即，增强的响应，减小阈值，和/或获取mechanosensitivity的）通过射频对化学品的局部暴露（ 例如，炎性汤（IS），辣椒素，三磷酸腺苷（ATP））。我们描述的设备和结直肠神经录音准备，收获结直肠附加PN或LSN，在结直肠，单纤维从神经束记录射频识别的，和化学品的本地化应用的RF。此外，制备和应用的标准化机械刺激的挑战进行了讨论。

设置示于图1，它包括一个特制的组织腔，其容纳在硅氧烷衬里的浴室并在一个连续的矿物油填充室中的附神经结直肠。两室室从丙烯酸塑料由CNC机床的固体块加工;两个隔室的底部，随后衬有牢固的硅氧烷，以允许容易销的大肠癌组织中向下。从梳理神经束外的AP使用低噪声，电池供电的差分放大器具有高共模抑制比（CMRR〜60 dB为单位）被记录。放大器的增益被设定为x10,000和带过滤器...

Watch this Scientific Journal Video about In vitro Functional Characterization of Mouse Colorectal Afferent Endings at JoVE.com

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

在小鼠大肠传入结尾的体外功能研究

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

in-vitro-functional-characterization-mouse-colorectal-afferent

Research

JoVE Journal

Medicine

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

9.5K 次观看.  University of Pittsburgh. This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation. 

视频: In vitro Functional Characterization of Mouse Colorectal Afferent Endings

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

灵活的结肠镜检查在小鼠使用经过验证的内镜评分系统来评估结肠炎和结肠直肠肿瘤的严重程度

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

科研

医学

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

神经科学

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

视频成像和时空地图来分析胃肠动力小鼠

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Combined Recording of Mechanically Stimulated Afferent Output and Nerve Terminal Labelling in Mouse Hair Follicle Lanceolate Endings

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The technique is very cost-effective and easily undertaken with materials commonly found in most electrophysiology laboratories, or easily purchased. The dissection is simple and fast, with the mechanical displacement provided by a generic electroceramic wafer controlled by proprietary software. The same software also records and analyses the electroneurogram output. The recording of the evoked nerve activity is through a commercial differential amplifier connected to fire-polished standard glass microelectrodes. Helpful tips are given for improving the quality of the preparation, the stimulation and the recording conditions to optimize recording quality. The system is suitable for assaying the electrophysiological and optical properties of lanceolate terminals of palisade endings of hair follicles, as well as the outcomes from their pharmacological and/or genetic manipulation. An example of combining electrical recording with mechanical stimulation and labeling with a styryl pyridinium vital dye is given.

A simple and novel technique for recording afferent discharge due to mechanical stimulation of lanceolate terminals of palisade endings innervating mouse ear skin hair follicles is presented.

机械刺激传入输出和神经末梢标签联合录制在小鼠毛囊披针形结局

A novel dissection and recording technique is described for monitoring afferent firing evoked by mechanical displacement of hairs in the mouse pinna. The ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

在鼠标空肠和结肠段肠系膜传入神经活性的体外记录

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory bowel diseases and diabetes. Here, we describe a protocol for the in vitro characterization of the electrophysiological properties of colonic afferents in rats. The colorectum, with the intact pelvic ganglion (PG) attached, is removed from the rat; superfused with carbogenated Krebs solution in the recording chamber; and cannulated at the oral and anal ends to allow for distension. A fine nerve bundle emanating from the PG is identified, and the multiunit afferent nerve activity is recorded using a suction electrode. Distension of the colonic segment elicits gradual increases in multiunit discharge. A principal component analysis is conducted to differentiate the low-threshold, the high-threshold, and the wide-dynamic range afferent fibers. Chemical sensitivity of colonic afferents can be studied through the bath or intraluminal administration of test compounds. This protocol can be modified for application to other species, such as mice and guinea pigs, and to study the differences in the electrophysiological properties of thoracolumbar/hypogastric and lumbosacral/pelvic afferents of the descending colon in normal and pathological conditions.

In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats

Abnormal sensory function underlies visceral pain and other symptoms of functional and inflammatory bowel diseases. A protocol for the electrophysiological recording of the colonic afferent nerves in an ex vivo rat colorectum preparation is presented here.

体外大鼠结肠传入纤维的电生理特性

Dysfunction of the colonic sensory nerves has been implicated in the pathophysiology of several common conditions, including functional and inflammatory ...

Advanced Imaging of Dorsal Root Ganglion Neurons for Sensory Modality Research

In Vivo Calcium Imaging of Dorsal Root Ganglia Neurons' Response to Somatic and Visceral Stimuli

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the protocol for in vivo calcium imaging of the exposed DRG in response to various visceral and somatic stimuli. Pirt-GCaMP6s mice or C57BL6 mice intrathecally injected with AAV viruses packaged with GCaMP6s were utilized to capture Ca2+ transients. The amplitude of these transients indicates sensitivity to specific sensory modalities. Afferent fibers originate from internal organs, with primary neuronal cell bodies in spinal or vagal ganglia. Studies on visceral nociception and acupuncture analgesia can potentially be conducted on primary sensory neurons using advanced imaging technologies like in vivo calcium imaging, allowing for the recording of neuronal activity ensembles in the intact animal during stimulation or intervention. The responses of DRG neuron ensembles to somatic and visceral stimuli applied to their corresponding receptive fields were recorded. This technique illustrates how neuronal populations react to various types of somatic and visceral stimuli. It is possible to comprehensively compare neuronal ensemble responses to different stimuli, which is a particularly valuable approach in research on visceral pain and segmental mechanisms of somatic stimulation, such as acupuncture.

The present protocol outlines in vivo calcium imaging for measuring the responses of ensembles of lumbar-6 DRG neurons to somatic and visceral stimuli. Thorough comparisons can be made among neurons responding to different stimuli. This protocol is valuable for investigating mechanisms of visceral pain and somatic stimulation, such as acupuncture.

A technique is described for surgically exposing the dorsal root ganglion (DRG) of the lumbar-6 in a live, anesthetized laboratory mouse, along with the ...

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intraluminal Injection

Source: Boerner, K. et al. <a href="https://www.jove.com/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021).
In this video, we describe a surgical procedure to generate mouse proximal colon loop or pcLoop segment followed by intraluminal injection of fluorescent markers into the segment to study the intestinal epithelial permeability.

Generation of Mouse Proximal Colon Loop Model: A Surgical Procedure to Generate Proximal Colon Loop and Measure Intestinal Permeability by Detection of Fluorescent Markers in Serum Following Intralumi

小鼠近端结肠袢模型的生成:一种通过检测腔内注射后血清中的荧光标志物来生成近端结肠袢并测量肠道通透性的外科手术

Source: Boerner, K. et al. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized ...

JoVE Encyclopedia of Experiments

生物学

Rodent Models

Adult

Electrophysiological Recording from Colonic Afferent Nerves in an Ex Vivo Rat Colon

Source: Meng, Y. et al., <a href="https://app.jove.com/v/56090">In Vitro Characterization of the Electrophysiological Properties of Colonic Afferent Fibers in Rats.</a> J. Vis. Exp. (2017)
This video demonstrates a method to record the activity of colonic afferent nerves in an ex-vivo rat colon, cannulated at both ends and infused with saline to increase intraluminal pressure. As pressure increases, the nerve signal rises, indicating its stimulation.

离体大鼠结肠中结肠传入神经的电生理记录

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Neuroscience

Neurophysiology

Electrophysiology Techniques

Recording of Mesenteric Afferent Nerve Activity in an Ex Vivo Mouse Jejunum Segment

Source: Nullens, S., et al. <a href="https://app.jove.com/v/54576">In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments.</a> J. Vis. Exp. (2016).
This video shows the process of recording electrical signals from mesenteric afferent nerves in a dissected mouse jejunal segment to study the effects of tissue pressure and movement changes on afferent nerve activity.