Farbman Albert I, Brann Jessica H, Rozenblat Alexander, Rochlin M William, Weiler Elke, Bhattacharyya Mitra
Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA.
J Neurocytol. 2004 May;33(3):331-43. doi: 10.1023/B:NEUR.0000044194.71426.ee.
Individual neurons dissected from immunohistochemically stained paraffin sections of the developing rat geniculate (VIIth cranial) ganglion were assayed for their content of mRNA of the neurotrophin receptor genes, p75 , trkA , trkB and trkC. Fetal and postnatal rats, from the 13th embryonic day (E13) until the 20th postnatal day (P20), were used. Single cells were subjected to RNA amplification, followed by treatment with reverse transcriptase and DNA amplification by the polymerase chain reaction (PCR). The identity of the PCR products was verified by subcloning and sequencing. A total of 227 neurons were examined, of which 212 (93%) gave a PCR signal for at least one neurotrophin receptor. We found: (1) Approximately half of the neurons expressed more than one receptor. (2) A truncated version of trkB , possessing the ligand-binding region but lacking the tyrosine kinase domain, occurred quite frequently, often in combination with the full-length trkB, with trkA or both. (3) The pattern of staining for trkB-like immunoreactivity was usually predictive that either its full length or truncated mRNA would be present. This was not the case for trkC-like immunoreactivity. Western blots on E15 brain tissue showed no band for full-length trkC ( approximately 150 kDa), suggesting the antibody may have been immunoreactive with a truncated ( approximately 120 kDa) but not a full-length version of the trkC receptor. (4) The pattern of neurotrophin receptor gene expression changed during development. (5) p75 expression occurred infrequently--in only 7 of the 212 neurons that gave a signal for any receptor.
从发育中的大鼠膝状（第七脑神经）神经节免疫组织化学染色石蜡切片中分离出单个神经元，检测其神经营养因子受体基因p75、trkA、trkB和trkC的mRNA含量。使用了从胚胎第13天（E13）到出生后第20天（P20）的胎儿和新生大鼠。对单个细胞进行RNA扩增，随后用逆转录酶处理并通过聚合酶链反应（PCR）进行DNA扩增。通过亚克隆和测序验证PCR产物的身份。共检查了227个神经元，其中212个（93%）至少对一种神经营养因子受体产生PCR信号。我们发现：（1）约一半的神经元表达一种以上的受体。（2）一种截短形式的trkB经常出现，它具有配体结合区域但缺乏酪氨酸激酶结构域，常与全长trkB、trkA或两者同时出现。（3）trkB样免疫反应性的染色模式通常可预测其全长或截短的mRNA是否存在。trkC样免疫反应性则并非如此。对E15脑组织进行的蛋白质免疫印迹分析未显示全长trkC（约150 kDa）的条带，这表明该抗体可能与截短形式（约120 kDa）而非全长形式的trkC受体发生免疫反应。（4）神经营养因子受体基因的表达模式在发育过程中发生变化。（5）p75的表达很少见——在212个对任何受体产生信号的神经元中仅7个出现。
