Watanave Masashi, Kawachi Mika, Konno Ayumu, Aoki Ryo, Fukai Yuuki, Matsuzaki Yasunori, Kaneko Ryosuke, Hirai Hirokazu
Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Japan.
Viral Vector Core, Gunma University, Initiative for Advanced Research, Maebashi, Japan.
Front Cell Neurosci. 2024 Feb 16;18:1349878. doi: 10.3389/fncel.2024.1349878. eCollection 2024.
Protein kinase C γ (PKCγ), a neuronal isoform present exclusively in the central nervous system, is most abundantly expressed in cerebellar Purkinje cells (PCs). Targeted deletion of PKCγ causes a climbing fiber synapse elimination in developing PCs and motor deficit. However, physiological roles of PKCγ in adult mouse PCs are little understood. In this study, we aimed to unravel the roles of PKCγ in mature mouse PCs by deleting PKCγ from adult mouse PCs of PKCγ mice via cerebellar injection of adeno-associated virus (AAV) vectors expressing Cre recombinase under the control of the PC-specific L7-6 promoter. Whole cell patch-clamp recording of PCs showed higher intrinsic excitability in PCs virally lacking PKCγ [PKCγ-conditional knockout (PKCγ-cKO) PCs] than in wild-type (WT) mouse PCs in the zebrin-negative module, but not in the zebrin-positive module. AAV-mediated PKCγ re-expression in PKCγ-deficient mouse PCs in the zebrin-negative module restored the enhanced intrinsic excitability to a level comparable to that of wild-type mouse PCs. In parallel with higher intrinsic excitability, we found larger hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents in PKCγ-cKO PCs located in the zebrin-negative module, compared with those in WT mouse PCs in the same region. However, pharmacological inhibition of the HCN currents did not restore the enhanced intrinsic excitability in PKCγ-cKO PCs in the zebrin-negative module. These results suggested that PKCγ suppresses the intrinsic excitability in zebrin-negative PCs, which is likely independent of the HCN current inhibition.
蛋白激酶Cγ（PKCγ）是一种仅存在于中枢神经系统的神经元亚型，在小脑浦肯野细胞（PCs）中表达最为丰富。靶向删除PKCγ会导致发育中的PCs中攀缘纤维突触消除和运动缺陷。然而，PKCγ在成年小鼠PCs中的生理作用却鲜为人知。在本研究中，我们旨在通过在PC特异性L7-6启动子控制下，经小脑注射表达Cre重组酶的腺相关病毒（AAV）载体，从PKCγ小鼠的成年小鼠PCs中删除PKCγ，从而揭示PKCγ在成熟小鼠PCs中的作用。对PCs进行全细胞膜片钳记录显示，在zebrin阴性模块中，病毒缺失PKCγ的PCs[PKCγ条件性敲除（PKCγ-cKO）PCs]比野生型（WT）小鼠PCs具有更高的内在兴奋性，但在zebrin阳性模块中则不然。在zebrin阴性模块中，AAV介导的PKCγ在PKCγ缺陷小鼠PCs中的重新表达将增强的内在兴奋性恢复到与野生型小鼠PCs相当的水平。与较高的内在兴奋性并行，我们发现位于zebrin阴性模块的PKCγ-cKO PC中，超极化激活环核苷酸门控（HCN）通道电流比同一区域的WT小鼠PCs中的更大。然而，对HCN电流的药理学抑制并未恢复zebrin阴性模块中PKCγ-cKO PC中增强的内在兴奋性。这些结果表明，PKCγ抑制zebrin阴性PCs中的内在兴奋性，这可能与HCN电流抑制无关。
