Examination of co-release hypothesis of glutamate and GABA at the hippocampal mossy fiber synapse

最近的研究表明，幼年啮齿动物的海马苔藓纤维终末除了释放谷氨酸外，还释放GABA。在这项研究中，我们利用海马片的全细胞记录以及幼鼠(P14-P21)的免疫组织化学研究了GABA和谷氨酸共同释放的确切细胞机制。在谷氨酸受体拮抗剂10μM CNQX和25μM D-AP5存在下，对齿状回颗粒细胞层的强烈刺激可在CA3神经元中可靠地诱导IPSCs。进一步应用GABAA受体拮抗剂10 0μM印防己毒素可取消这些假定的单突触IPSCs。相反，在CNQX和D-AP5存在的情况下，对颗粒细胞层的弱刺激从未诱导出IPSCs。谷氨酸受体拮抗剂在强刺激和弱刺激下的不同作用表明，颗粒细胞层的强刺激招募了一些额外的细胞成分。我们还用免疫组织化学方法检测了GABA和GABA合成酶GAD的定位，发现GABA和GAD在苔藓纤维终末有微弱的表达。但在终末内未检测到囊泡状GABA转运体VGAT。这些结果表明，强烈刺激颗粒细胞层除刺激苔藓纤维外，还通过刺激抑制性中间神经元产生明显的单突触IPSCs，幼鼠海马苔藓纤维终末可能不释放GABA。

Recent studies suggested that hippocampal mossy fiber terminals of young rodents release GABA in addition to glutamate. In this study, we examined the exact cellular mechanisms underlying co-release of GABA and glutamate using whole cell recordings in hippocampal slices as well as immunohistochemistry in young mice (P14-P21). Strong stimulus to the granule cell layer of dentate gyrus reliably elicited IPSCs in CA3 neurons in the presence of glutamate receptor antagonists 10μM CNQX and 25μM D-AP5. These putative monosynaptic IPSCs were abolished by further application of GABA_A receptor antagonist 100μM picrotoxin. In contrast, weak stimulus to the granule cell layer never elicited IPSCs in the presence of CNQX and D-AP5. The differential effects of the glutamate receptor antagonists between strong and weak stimuli suggested that some additional cellular components were recruited by the strong stimulus of the granule cell layer. We also examined localization of GABA and the GABA-synthesizing enzyme GAD immunohistochemically, and found that GABA and GAD were expressed weakly at the mossy fiber terminals. However, vesicular GABA transporter VGAT was not detected within the terminals. These results suggested that strong stimulus to the granule cell layer causes apparent monosynaptic IPSCs by stimulating inhibitory interneurons in addition to mossy fibers, and the hippocampal mossy fiber terminals themselves may not release GABA in young mouse.