Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22903.
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22903
J Neurosci. 2018 Jan 31;38(5):1232-1248. doi: 10.1523/JNEUROSCI.1345-17.2017. Epub 2017 Dec 22.
Maintenance of a low intracellular Cl concentration ([Cl]) is critical for enabling inhibitory neuronal responses to GABA receptor-mediated signaling. Cl transporters, including KCC2, and extracellular impermeant anions ([A]) of the extracellular matrix are both proposed to be important regulators of [Cl] Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl] remains relatively low in RT neurons. Although we identify a small contribution of [A] to a low [Cl] in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy. Precise regulation of intracellular Cl levels ([Cl]) preserves appropriate, often inhibitory, GABAergic signaling within the brain. However, there is disagreement over the relative contribution of various mechanisms that maintain low [Cl] We found that the Cl transporter KCC2 is an important Cl extruder in the reticular thalamic (RT) nucleus, despite this nucleus having remarkably low KCC2 immunoreactivity relative to other regions of the adult brain. We also identified a smaller contribution of fixed, impermeant anions ([A]) to lowering [Cl] in RT neurons. Inhibitory signaling among RT neurons is important for preventing excessive activation of RT neurons, which can be responsible for generating seizures. Our work suggests that KCC2 critically restricts the spread of activity within the RT nucleus.
维持细胞内氯离子浓度 ([Cl]) 低水平对于使 GABA 受体介导的信号转导产生抑制性神经元反应至关重要。氯离子转运体，包括 KCC2，以及细胞外基质中的不可渗透的阴离子 ([A])，都被认为是调节 [Cl] 的重要因素。网状丘脑 (RT) 核的神经元表达降低的 KCC2 水平，表明 GABA 能信号转导可能在 RT 神经元中产生兴奋。然而，通过在大鼠 (雄性和雌性) 中进行穿孔贴片记录和钙成像实验，我们发现 RT 神经元中的 [Cl] 仍然相对较低。虽然我们确定 [A] 对 RT 神经元中的低 [Cl] 有较小的贡献，但我们的结果也表明，降低的 KCC2 水平仍然足以维持低 Cl 水平。然而，降低的 KCC2 水平限制了 RT 神经元在 GABA 能信号转导升高后快速排出 Cl 的能力。在一个具有类似于我们实验中发现的缓慢 Cl 外排动力学的局部 RT 网络的计算模型中，模型 RT 神经元容易发生从 GABA 介导的抑制到兴奋的活性依赖性转换。通过降低产生兴奋性 GABA 能信号所需的活动阈值，较弱的刺激能够在模型 RT 核内传播活动。我们的结果表明，即使 KCC2 水平降低，也能在 RT 核内维持抑制性信号传递的重要性，并表明在癫痫等疾病中，这个重要的活动瓶颈可能很容易被克服。细胞内氯离子浓度 ([Cl]) 的精确调节可维持大脑内适当的、通常是抑制性的 GABA 能信号传递。然而，对于维持低 [Cl] 的各种机制的相对贡献存在分歧。我们发现，氯离子转运体 KCC2 是网状丘脑 (RT) 核中的重要 Cl 外排器，尽管与成年大脑的其他区域相比，该核具有明显较低的 KCC2 免疫反应性。我们还确定了固定的、不可渗透的阴离子 ([A]) 对降低 RT 神经元中 [Cl] 的较小贡献。RT 神经元之间的抑制性信号传递对于防止 RT 神经元过度激活很重要，过度激活 RT 神经元可能导致癫痫发作。我们的工作表明，KCC2 严格限制了 RT 核内活动的传播。
