Department of Cellular and Molecular Neuroendocrinology, Institute of Physiology of the Academy of Sciences of the Czech Republic.
J Neurosci. 2013 May 1;33(18):8035-44. doi: 10.1523/JNEUROSCI.4682-12.2013.
The hypothalamic suprachiasmatic nuclei (SCN), the circadian master clock in mammals, releases ATP in a rhythm, but the role of extracellular ATP in the SCN is still unknown. In this study, we examined the expression and function of ATP-gated P2X receptors (P2XRs) in the SCN neurons of slices isolated from the brain of 16- to 20-day-old rats. Quantitative RT-PCR showed that the SCN contains mRNA for P2X 1-7 receptors and several G-protein-coupled P2Y receptors. Among the P2XR subunits, the P2X2 > P2X7 > P2X4 mRNAs were the most abundant. Whole-cell patch-clamp recordings from SCN neurons revealed that extracellular ATP application increased the frequency of spontaneous GABAergic IPSCs without changes in their amplitudes. The effect of ATP appears to be mediated by presynaptic P2X2Rs because ATPγS and 2MeS-ATP mimics, while the P2XR antagonist PPADS blocks, the observed enhancement of the frequency of GABA currents. There were significant differences between two SCN regions in that the effect of ATP was higher in the ventrolateral subdivision, which is densely innervated from outside the SCN. Little evidence was found for the presence of P2XR channels in somata of SCN neurons as P2X2R immunoreactivity colocalized with synapsin and ATP-induced current was observed in only 7% of cells. In fura-2 AM-loaded slices, BzATP as well as ADP stimulated intracellular Ca(2+) increase, indicating that the SCN cells express functional P2X7 and P2Y receptors. Our data suggest that ATP activates presynaptic P2X2Rs to regulate inhibitory synaptic transmission within the SCN and that this effect varies between regions.
下丘脑视交叉上核（SCN）是哺乳动物的昼夜节律主钟，以节律方式释放 ATP，但细胞外 ATP 在 SCN 中的作用尚不清楚。在这项研究中，我们检查了从 16-20 天大的大鼠脑分离的切片中 SCN 神经元中 ATP 门控 P2X 受体（P2XR）的表达和功能。定量 RT-PCR 显示 SCN 含有 P2X1-7 受体和几种 G 蛋白偶联 P2Y 受体的 mRNA。在 P2XR 亚基中，P2X2 > P2X7 > P2X4 mRNA 最为丰富。来自 SCN 神经元的全细胞膜片钳记录显示，细胞外 ATP 应用增加了自发性 GABA 能 IPSC 的频率，而不改变其幅度。ATP 的作用似乎是通过突触前 P2X2R 介导的，因为 ATPγS 和 2MeS-ATP 模拟物，而 P2XR 拮抗剂 PPADS 阻断观察到的 GABA 电流频率增强。两个 SCN 区域之间存在显著差异，即 ATP 的作用在外侧亚区更高，该亚区是由 SCN 外部密集神经支配的。由于 P2X2R 免疫反应性与突触素共定位，并且仅在 7％的细胞中观察到 ATP 诱导的电流，因此在 SCN 神经元的胞体中几乎没有发现 P2XR 通道的存在。在负载 fura-2 AM 的切片中，BzATP 以及 ADP 刺激细胞内 Ca（2+）增加，表明 SCN 细胞表达功能性 P2X7 和 P2Y 受体。我们的数据表明，ATP 激活突触前 P2X2R 来调节 SCN 内的抑制性突触传递，并且这种作用在不同区域之间存在差异。
