ASC transporters mediate D-serine transport into astrocytes adjacent to synapses in mouse brain

Background: D-serine is an important signaling molecule, which activates N-methyl D-aspartate receptors (NMDARs) in conjunction with its fellow co-agonist, the neurotransmitter glutamate.
Question: Despite its involvement in plasticity and memory related to excitatory synapses, its cellular source and sink remain a question.
Importance: This removal of synaptic D-serine and its subsequent metabolic degradation is expected to reduce its extracellular availability, influencing NMDAR activation and NMDAR-dependent synaptic plasticity.
Hypothesis: We hypothesise that astrocytes, a type of glial cell that surround synapses are likely candidates to control the extracellular concentration of D-Serine by removing it from the synaptic space.
Notable finding: We observed D-serine induced transport-associated currents upon puff-application of 10 mM D-serine on astrocytes. Further, O-benzyl-L-serine and trans-4-hydroxy-proline, known substrate inhibitors of the alanine serine cysteine transporters (ASCT) reduced D-serine uptake. These results indicate that ASCT is a central mediator of astrocytic D-serine transport and plays a role in regulating its synaptic concentration by sequestration into astrocytes. Similar results were observed in astrocytes of the somatosensory cortex and Bergmann glia in the cerebellum, indicative of a general mechanism expressed across a range of brain areas.
How was it gathered: We used patch clamping technique, to study the transmembrane electrical signals from individual identified astrocytes in the CA1 region of hippocampus, primary somatosensory cortex and Bergmann glia. It involved placing a tiny glass patch pipette, onto the surface of the astrocytic membrane, to record D-serine transporter currents.
Methods: Using in situ patch clamp recordings and pharmacological manipulation of astrocytes in CA1 region of the mouse hippocampal brain slices, we investigated transport of D-serine across the plasma membrane.
How to interpret data: The spread sheet contains all the mean astrocytic D-serine currents that were recorded from individual cells.
How to use it: The raw data traces are most compatible with Clampfit 10.7.0.3 (P Clamp - Molecular devices 2016).

背景：D-丝氨酸是一种重要的信号分子，可与其共同激动剂——神经递质谷氨酸协同激活N-甲基-D-天冬氨酸受体（N-methyl D-aspartate receptors, NMDARs）。
问题：尽管其参与了兴奋性突触相关的突触可塑性与记忆过程，但其细胞来源与清除通路仍有待阐明。
研究意义：这种突触间隙D-丝氨酸的清除及其后续代谢降解过程，会降低其细胞外可用浓度，进而影响NMDAR的激活以及依赖NMDAR的突触可塑性。
研究假设：我们提出如下假设：星形胶质细胞——一种环绕突触的胶质细胞——极有可能通过从突触间隙清除D-丝氨酸，来调控其细胞外浓度。
重要发现：我们观察到，向星形胶质细胞微喷射10 mM D-丝氨酸时，可诱导出与转运相关的电流。进一步实验显示，已知的丙氨酸-丝氨酸-半胱氨酸转运体（alanine serine cysteine transporters, ASCT）底物抑制剂O-苄基-L-丝氨酸和反式-4-羟基脯氨酸，可抑制D-丝氨酸的摄取。上述结果表明，ASCT是星形胶质细胞摄取D-丝氨酸的核心介导因子，并通过将D-丝氨酸转运至星形胶质细胞内，参与调控其突触间隙浓度。该现象在躯体感觉皮层星形胶质细胞以及小脑伯格曼胶质细胞中均得到验证，提示这一机制在多个脑区均广泛存在。
数据采集方式：我们采用膜片钳（patch clamping）技术，对小鼠海马CA1区、初级躯体感觉皮层以及小脑伯格曼胶质细胞中的单个已鉴定星形胶质细胞的跨膜电信号进行研究。该技术通过将微小的玻璃膜片钳电极置于星形胶质细胞膜表面，以记录D-丝氨酸转运体电流。
实验方法：我们借助小鼠海马脑片CA1区星形胶质细胞的原位膜片钳记录与药理学操控手段，探究了D-丝氨酸跨细胞膜的转运过程。
数据解读说明：本电子表格包含了从单个细胞中记录得到的所有星形胶质细胞D-丝氨酸电流的均值。
数据使用说明：原始数据轨迹最适配Clampfit 10.7.0.3（P Clamp - Molecular devices 2016）软件进行分析。