Astrocyte swelling underlying high extracellular K+ induced intrinsic optical signals

我们研究了参与产生内源性光信号(IO)的细胞类型以及Na-K2Cl共转运体在激活海马片细胞肿胀中的作用。我们监测了内源性光信号(IOS)和星形胶质细胞与高外源性[K+]([K+]EXT)中神经元肿胀的关系。TTX(12μM)、AP-5(100μM)和NBQX(20μM)阻断兴奋性突触传递和兴奋性突触传递时，[K+]ext从4.5mM增加到10 mM可逆地增加海马片透光率。选择性Na-K-2Cl共转运体拮抗剂布美他尼(100μM)可逆地抑制高[K+]EXT诱发的IOS的幅度，为对照组的50.2%。识别Na-K-2Cl共转运蛋白NKCC1亚型的单抗T4在海马蛋白免疫印迹中检测到一条约180 kDa的条带。在海马区，NKCC1在CA1区锥体神经元的树突和胞体中均有较强的免疫染色。星形胶质细胞的免疫染色在组织切片中难以明确确定。因此，我们检测了急性分离的星形胶质细胞，发现GFAP+VE星形胶质细胞也对NKCC1呈免疫反应。为了确定星形胶质细胞和/或神经元是否在高[K+]EXT中肿胀，我们使用双光子激光扫描显微镜(TPLSM)来测量已识别的细胞中的肿胀，这些细胞被贴片并填充钙黄绿素。在高[K+]EXT组，星形胶质细胞突起直径增大至126.5%，而树突内未见肿胀(对照的98.0%)。这些结果表明，高[K+]EXT可主要通过激活NKCC1诱导海马区胶质细胞肿胀，神经元在高[K+]EXT时保护其细胞体积。

We investigated the cell types involved in generating intrinsic optical signals (IOS) and the roles for the Na K 2Cl cotransporter in activating cellular swelling in hippocampal slices. We monitored intrinsic optical signal (IOS) and astrocyte versus neuronal swelling in high external [K+] ([K+]ext). Increasing [K+]ext from 4.5 to 10 mM reversibly increased light transmittance through hippocampal slices even when spiking and excitatory synaptic transmission was blocked by TTX (12μM), AP-5 (100μM) and NBQX (20μM). Bumetanide (100μM), a selective Na-K-2Cl cotransporter antagonist, reversibly depressed the magnitude of the high [K+]ext-induced IOS to 50.2% of control. The monoclonal antibody, T4 which recognizes the NKCC1 isoform of the Na-K-2Cl cotransporter, detected a band of 〜180 kDa in western blots of hippocampal proteins. In the hippocampus robust immunostaining for NKCC1 was detected in both dendrites and somata of CA1 pyramidal neurons. Immunostaining of astrocytes was difficult to unequivocally ascertain in tissue sections. Therefore we examined acutely isolated astrocytes and found that GFAP +ve astrocytes were also immunoreactive for NKCC1. To determine whether astrocytes and/or neurons swelled in high [K+]ext we used two-photon laser scanning microscopy (TPLSM) to measure swelling in identified cells that were patched and filled with calcein. In high [K+]ext astrocyte processes increased their diameter to 126.5% whereas swelling was not observed in dendrites (98.0% of control). These results demonstrate that increased [K+]ext can induce glial swelling in the hippocampus principally by activation of the NKCC1 and that neurons defend their cell volume in high [K+]ext.