Department of Pharmacology, Faculty of Pharmacy, University of Basque Country UPV/EHU, Paseo de Universidad 7, 01006 Vitoria-Gasteiz, Spain.
Neuroscience. 2012 Oct 11;222:239-68. doi: 10.1016/j.neuroscience.2012.06.039. Epub 2012 Jun 23.
The present study describes a complete and detailed neuroanatomical distribution map of the phospholipase C beta1 (PLCβ1) isoform along the adult rat neuraxis, and defines the phenotype of cells expressing PLCβ1, along with its subcellular localization in cortical neurons as assessed by double-immunofluorescence staining and confocal laser scanning. Immunohistochemical labeling revealed a considerable morphological heterogeneity among PLCβ1-positive cells in the cortex, even though there was a marked predominance of pyramidal morphologies. As an exception to the general non-matching distribution of GFAP and PLCβ1, a high degree of co-expression was observed in radial glia-like processes of the spinal cord white matter. In the somatosensory cortex, the proportion of GABAergic neurons co-stained with PLCβ1 was similar (around 2/3) in layers I, II-III, IV and VI, and considerably lower in layer V (around 2/5). Double immunofluorescence against PLCβ1 and nuclear speckle markers SC-35 and NeuN/Fox3 in isolated nuclei from the rat cortex showed a high overlap of both markers with PLCβ1 within the nuclear matrix. In contrast, there was no apparent co-localization with markers of the nuclear envelope and lamina. Finally, to assess whether the subcellular expression pattern of PLCβ1 involved specifically one of the two splice variants of PLCβ1, we carried out Western blot experiments in cortical subcellular fractions. Notably, PLCβ1a/1b ratios were statistically higher in the cytoplasm than in the nuclear and plasma membrane fractions. These results provide a deeper knowledge of the cellular distribution of the PLCβ1 isoform in different cell subtypes of the rat brain, and of its presence in the neuronal nuclear compartment.
本研究描述了磷脂酶 Cβ1（PLCβ1）同工型在成年大鼠神经轴突中的完整和详细的神经解剖分布图谱，并定义了表达 PLCβ1 的细胞表型及其在皮质神经元中的亚细胞定位，方法是通过双重免疫荧光染色和共聚焦激光扫描进行评估。免疫组织化学标记显示，皮质中 PLCβ1 阳性细胞存在相当大的形态异质性，尽管存在明显的锥体形形态占主导地位。作为 GFAP 和 PLCβ1 分布不匹配的一般情况的例外，在脊髓白质的放射状胶质样过程中观察到高度的共表达。在体感皮层中，与 PLCβ1 共染色的 GABA 能神经元的比例在 I 层、II-III 层、IV 层和 VI 层相似（约 2/3），在 V 层（约 2/5）显著降低。来自大鼠皮质的分离核中针对 PLCβ1 和核斑点标记物 SC-35 和 NeuN/Fox3 的双重免疫荧光显示，这两种标记物与核基质内的 PLCβ1 高度重叠。相比之下，与核膜和层的标记物没有明显的共定位。最后，为了评估 PLCβ1 的亚细胞表达模式是否特别涉及 PLCβ1 的两个剪接变体之一，我们在皮质亚细胞级分中进行了 Western blot 实验。值得注意的是，PLCβ1a/1b 比值在细胞质中明显高于核和质膜级分。这些结果提供了对 PLCβ1 同工型在大鼠大脑不同细胞亚型中的细胞分布及其在神经元核区室中的存在的更深入了解。
