Jacquin M F, Hu J W, Sessle B J, Renehan W E, Waite P M
Department of Anatomy and Neurobiology, St. Louis University School of Medicine, MO 63104.
J Neurosci Methods. 1992 Oct-Nov;45(1-2):71-86. doi: 10.1016/0165-0270(92)90045-f.
Currently available methods for studying the morphology of physiologically characterized primary afferents are limited by difficulties inherent in impaling thin fibers and by the limited distances over which conventional tracers move during the course of a recording session. We have encountered an alternative method that overcomes these limitations. Neurobiotin (NB; Vector) injections into rat trigeminal (V) primary afferents in the brain stem or V ganglion provided rapid, long-range staining with recording and electrophoretic parameters that are commonly used to eject horseradish peroxidase (HRP) or Phaseolus vulgaris leucoagglutinin (PHA-L). When NB was injected into brain stem fibers responsive to vibrissal deflection with A-beta conduction velocities, collaterals were darkly stained in each of the 4 V subnuclei, as well as the cervical dorsal horn. Labeled fibers were also seen in the V root and peripherally in the infra-orbital nerve for a distance up to 15 mm from the injection site (30 mm total). Cell bodies in the ganglion were never labeled. When NB was injected into V ganglion cells with low- or high-threshold receptive fields and A-beta or A-delta conduction velocities, parent axons were stained in the V spinal tract to the level of the obex, and collaterals were visible in each of the 4 V subnuclei. Such long-range staining occurred within 4 h of tracer injection. HRP never stained brain stem fibers following ganglion cell injections and, when injected centrally with the same survival intervals used with NB, dark staining was limited to within 4 mm of the injection site. Unlike NB or HRP, PHA-L injections rarely produced useful data, either because of the high mortality accompanying attempts to achieve a 1-2 week survival period or because injected neurons were not recovered. Due to its rapid and robust transport, NB is a more convenient and reliable tracer than PHA-L for producing long-range staining of the projections of identified ganglion cells. Intracellular injection of NB also produces rapid Golgi-like staining of fibers over much greater distances than HRP under equivalent staining parameters.
刺入细纤维存在固有困难，以及在记录过程中传统示踪剂移动的距离有限。我们遇到了一种克服这些限制的替代方法。将神经生物素（NB；Vector公司）注射到大鼠脑干或三叉神经节的三叉神经（V）初级传入纤维中，能以常用于注射辣根过氧化物酶（HRP）或菜豆白细胞凝集素（PHA-L）的记录和电泳参数进行快速、远距离染色。当将NB注射到对触须偏转有反应且具有A-β传导速度的脑干纤维中时，在4个三叉神经亚核以及颈背角中，侧支均被深染。在三叉神经根以及眶下神经外周距离注射部位达15毫米处（总共30毫米）也可见到标记纤维。神经节中的细胞体从未被标记。当将NB注射到具有低阈值或高阈值感受野以及A-β或A-δ传导速度的三叉神经节细胞中时，母轴突在三叉神经脊髓束中被染至闩平面，并且在4个三叉神经亚核中均可见侧支。这种远距离染色在示踪剂注射后4小时内即可出现。在注射神经节细胞后，HRP从未对脑干纤维进行染色，并且当以与NB相同的存活时间进行中枢注射时，深色染色仅限于注射部位4毫米范围内。与NB或HRP不同，PHA-L注射很少产生有用的数据，这要么是因为试图实现1至2周存活期会伴随高死亡率，要么是因为注射的神经元无法恢复。由于其快速且强大的运输能力，对于产生已识别神经节细胞投射的远距离染色而言，NB是比PHA-L更方便且可靠的示踪剂。在等效染色参数下，NB的细胞内注射也能在比HRP大得多的距离上对纤维产生快速的高尔基样染色。
