Molecular and Electrophysiological Characterization of Dorsal Horn Neurons in a GlyT2-iCre-tdTomato Mouse Line.
Molecular and Electrophysiological Characterization of Dorsal Horn Neurons in a GlyT2-iCre-tdTomato Mouse Line.
复制标题
DOI：
10.2147/jpr.s296940
复制
发表时间：
2021
影响因子：
2.7
通讯作者：
Lu Y
中科院分区：
文献类型：
--
作者：
He X;Liu P;Zhang X;Jiang Z;Gu N;Wang Q;Lu Y
脊髓甘氨酸能神经元作为疼痛和瘙痒的脊髓门的关键元件起作用。我们最近的研究表明，脊髓PKCγ+神经元接受Aβ初级传入驱动的前馈抑制性输入。甘氨酸能神经元控制PKCγ+神经元的兴奋性，因此门控机械性异常性疼痛。然而，从初级传入纤维的脊髓甘氨酸能中间神经元的突触驱动的动态或电生理分析在很大程度上是缺席的。本研究的目的是利用基因标记的动物模型分析脊髓甘氨酸能中间神经元与初级传入神经元之间的突触动力学。通过CRISPR/Cas9技术构建GlyT 2-P2 A-iCre小鼠。然后通过将GlyT 2-P2 A-iCre小鼠与荧光报告小鼠杂交产生GlyT 2-iCre-tdTomato小鼠。膜片钳全细胞记录用于分析GlyT 2-iCre-tdTomato小鼠中甘氨酸能神经元的动态突触输入。用免疫组织化学方法观察GlyT 2-tdTomato神经元在脊髓背角的分布。通过电生理记录和细胞内注射生物胞素的方法，观察了GlyT 2-tdTomato神经元的放电模式和形态学特征。成功构建GlyT 2-P2 A-iCre和GlyT 2-tdTomato小鼠。GlyT 2-tdTomato荧光与甘氨酸、GlyT 2和Pax 2在胞体中的免疫反应性广泛共定位，证实了转基因在甘氨酸能神经元中的选择性表达。GlyT 2-tdTomato神经元主要分布于脊髓Ⅲ ~ Ⅳ板层。GlyT 2-tdTomato神经元的放电模式和形态特征符合强直性中枢或胰岛型脊髓抑制性中间神经元的特征。大多数（72.1%）记录的GlyT 2-tdTomato神经元接受来自Aβ纤维的初级输入。本研究表明，脊髓GlyT 2阳性甘氨酸能神经元主要接受初级传入Aβ纤维的传入; GlyT 2-P2 A-iCre和GlyT 2-tdTomato小鼠为进一步研究GlyT 2 +-PKCγ+前馈抑制回路在生理和病理条件下的功能提供了一个有用的动物模型。
Spinal glycinergic neurons function as critical elements of a spinal gate for pain and itch. We have recently documented that spinal PKCγ+ neurons receive the feedforward inhibitory input driven by Aβ primary afferent. The glycinergic neurons control the excitability of PKCγ+ neurons and therefore gate mechanical allodynia. However, a dynamic or electrophysiological analysis of the synaptic drive on spinal glycinergic interneurons from primary afferent fibers is largely absent. The present study was aimed to analyze the synaptic dynamics between spinal glycinergic interneurons and primary afferents using a genetic labeled animal model. The GlyT2-P2A-iCre mice were constructed by the CRISPR/Cas9 technology. The GlyT2-iCre-tdTomato mice were then generated by crossing the GlyT2-P2A-iCre mice with fluorescent reporter mice. Patch-clamp whole-cell recordings were used to analyze the dynamic synaptic inputs to glycinergic neurons in GlyT2-iCre-tdTomato mice. The distribution of GlyT2-tdTomato neurons in the spinal dorsal horn was examined by the immunohistochemistry method. The firing pattern and morphological features of GlyT2-tdTomato neurons were also examined by electrophysiological recordings and intracellular injection of biocitin. The GlyT2-P2A-iCre and GlyT2-tdTomato mice were successfully constructed. GlyT2-tdTomato fluorescence was colocalized extensively with immunoreactivity of glycine, GlyT2 and Pax2 in somata, confirming the selective expression of the transgene in glycinergic neurons. GlyT2-tdTomato neurons were mainly distributed in spinal lamina IIi through IV. The firing pattern and morphological properties of GlyT2-tdTomato neurons met the features of tonic central or islet type of spinal inhibitory interneurons. The majority (72.1%) of the recorded GlyT2-tdTomato neurons received primary inputs from Aβ fibers. The present study indicated that spinal GlyT2-positive glycinergic neurons mainly received primary afferent Aβ fiber inputs; the GlyT2-P2A-iCre and GlyT2-tdTomato mice provided a useful animal model to further investigate the function of the GlyT2+-PKCγ+ feedforward inhibitory circuit in both physiological and pathological conditions.